**Mutations of PARK Genes and Alpha-Synuclein and Parkin Concentrations in Parkinson's Disease**

Anna Oczkowska, Margarita Lianeri, Wojciech Kozubski and Jolanta Dorszewska

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/57107

## **1. Introduction**

Parkinson's disease (PD) is a chronic and progressive neurological disorder characterized by resting tremor, rigidity, and bradykinesia, affecting at least 2% of individuals above the age of 65 years. Parkinson's disease is a result of degeneration of the dopamine-producing neurons of the *substantia nigra*. Available therapies in PD will only improve the symptoms but not halt progression of disease. The most effective treatment for PD patients is thera‐ py with L-3,4-dihydroxy-phenylalanine (L-dopa) [Olanow, 2008].

It is now believed that the cause of PD, are both environmental and genetic factors. During the last two decades, there has been breakthrough progress in genetics of PD. It is known that genetic background of PD is in mutations a number of pathogenic genes PARK, e.g. *SNCA*, *PRKN*, *UCHL1, DJ-1, PINK1, ATP13A2*, and *LRRK2* (Polrolniczak et a., 2011, 2012). In 2001, Shimura et al. first described the presence in the human brain complex contain‐ ing Parkin with the glycosylated form of the alpha-synuclein (ASN, alpha-SP22). More‐ over, the study by Dorszewska et al. (2012) has been shown, that in the PD patients increased plasma level of ASN was associated by the decreased of Parkin plasma level. It has also shown that configuration: increased plasma level of ASN and decreased of Parkin concentra‐ tion was associated with earlier onset of PD. It seems that in PD genotypic testing of PARK mutations and analysis of their phenotypes (e.g. ASN, Parkin) may be diagnostic agents for these patients.

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **2. Mutations in** *PRKN***,** *SPR* **and** *HTRA2* **genes and polymorphism of NACP-Rep1 region of** *SNCA* **promoter in the patients with Parkinson's disease**

Ahn et al., 2008; Liu et al., 2004; Nishioka et al., 2009; Nuytemans et al., 2009). Therefore, it have been suspected that not only mutations in the *SNCA* gene, but also other factors affecting

Mutations of PARK Genes and Alpha-Synuclein and Parkin Concentrations in Parkinson's Disease

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3

The study by Chiba-Falek et al. (2006) has shown that the region NACP-Rep1 of *SNCA* gene promoter, there is the polymorphic region differenting in dinucleotide repeats count and affecting the level of ASN expression. Moreover, it has been shown that polymorphism of NACP-Rep1 region in promoter of *SNCA*, are associated with an increased risk of SPD in some population like: German, Australian, American and Polish, but the other multi-population studies have observed no association or reported an inverse association between the risk allele and PD (Farrer et al., 2001; Kruger et al., 1999; Maraganore et al., 2006; Polrolniczak et al., 2012;

Region NACP-Rep1 contains dinucleotide repeats (TC)x(T)2(TC)y(TA)2(CA)z, which may vary both the number of repeats, and include substitutions of nucleotides. However, it has been proven, that a change in the length of the NACP-Rep1 region more than substitutions, affects the regulation of the expression of ASN (Fuchs et al., 2008; Mellick et al., 2005; Tan et al., 2003). As the most common in humans it has been described five alleles of NACP-Rep1 of the *SNCA* gene promoter: -1, 0, +1, +2, +3. Generally in the European population the most frequently was allele +1 of NACP-Rep1. It has been also shown, that the allele 0 of NACP-Rep1 region in *SNCA* promoter is two pairs shorter than allele +1, allele -1 respectively, shorter by

Functional analysis on the two most common NACP-Rep1 alleles +1 and +2 suggested that the +2 allele is associated with an up-regulation of *SNCA* expression, whereas the +1 variant shows reduced gene expression (Chiba Falek & Nussbaum, 2001; Cronin et al., 2009). In addition, allele +1 of the region NACP-Rep1 of *SNCA* promoter, containing 259 bp, significantly reduces the risk of PD in the population of Europe, America and Australia (Fuchs et al., 2008; Mara‐ ganore et al., 2006; Tan et al., 2000) while another study failed to replicate the finding in

Nerveless, although protective effect of allele +1 rather not currently subject to discussion, but for alleles 0, +2 and +3 it has been suggested both no impact, as well as increasing the risk of PD, and even sometimes the protective action (Maraganore et al., 2006; Spadafora et al., 2003; Tan et al., 2000; Trotta et al., 2012). The following studies by Tan et al. (2000) and Myhre et al. (2008) observed a higher frequency of the +3 allele in PD cases compared with healthy controls while in the study by both Tan et al. (2003) and Spadafora et al. (2003) no significant differences of the various genotypes between PD and controls were found in population of Singapore and Italy. However, the study in Italy population, have also shown evidence of association for allele +2 on NACP-Rep1 (Trotta et al., 2012). In 2006, a meta-analysis of 11 study populations provided strong evidence that the 263bp allele was more frequent in PD cases increasing risk of this disease while the 261bp allele did not differ between PD cases and unaffected controls but the authors suggested, that the lack of association of the +2 allele in the meta-analysis could be due to the large fluctuation in its frequencies observed in the analyzed populations (Maraganore et al., 2006). Therefore the aim of the study was analysis of NACP-Rep1 region

population of Japan, Singapore and Italy (Spadafora et al., 2003; Tan et al., 2003).

the expression of ASN may contribute to the PD manifestation including, SPD.

4 bp however alleles 2 and 3 are longer by 2 and 4 bp.

in PD patients and in controls in Polish population.

Tan et al., 2003).

During the last two decades, there has been breakthrough progress in genetics of PD. Currently it is known that genetic background of PD is heterogeneous and mutations in a number of pathogenic genes (e.g. *SNCA*, *PRKN*, *UCHL1, DJ-1, PINK1, ATP13A2*, and *LRRK2*) have been described as associated with familial (FPD) or as genetic risk factors increasing the risk to develop of sporadic PD (SPD). Some of these genes (like *SNCA* and *PRKN)* are fairly well understood while the others (like *SPR* and *HTRA2*) are still little known (Corti et al., 2011).

Monogenic forms, caused by a single mutation in a dominantly or recessively inherited gene, are well-established. Nevertheless, they are relatively rare types of PD and account for about 30% of the FPD and 3–5% of the SPD cases. Although 18 specific chromosomal locus (called *PARK* and numbered in chronological order of their identification) have been reported as more or less convincingly related to FPD (Klein & Westenberger, 2012), the majority of PD cases are SPD (only about 10% of patients report a positive family history) [Thomas & Beal, 2007] while the results of the studies of SPD genetic are still ambiguous and divergent in different ethnic origin (Klein & Schlossmacher, 2007; Lesage & Brice, 2009). Few studies (e.g. Abbas et al., 1999; Gilks et al., 2005; Guo et al., 2010; Mellick et al., 2005; Trotta et al., 2012) suggest a strong correlation of genetic factors with an increased risk of SPD development, while the other reports are contradictory (Chung et al., 2011; Spadafora et al., 2003). However, genome-wide association studies have provided convincing evidence that polymorphic variants in some genes contribute to higher risk of SPD (Gao et al., 2009). Moreover, it is suggested that the etiology of PD is multifactorial, which probably results from coocurence of genetic and environmental factors (Klein & Westenberger, 2012).

Summarizing, from the existing studies reported, it is not yet clear how common mutations in few genes, including: *PRKN*, *HTRA2*, *SPR* and *SNCA* genes contribute to idiopathic PD (Nuytemans et al., 2010). Finally despite previous reports, significance of these genes mutation and polymorphism in pathogenesis of PD (especially SPD) is not clear and is still debated, mainly because of discrepancy of studies results and variance between different ethnic populations. To clarify these issues, more data of genetic analysis are needed while there were only a few reports of genetic studies of PD in Polish populations (Bialecka et al., 2005; Koziorowski et al., 2010). Moreover, little is understood about putative director functional interactions between the genes that cause PD, and a single pathway unifying these factors has not been confirmed (Bras et al., 2008; Brooks et al., 2009; Klein et al., 2005).

#### **2.1. Polymorphism of NACP-Rep1 region of** *SNCA* **promoter in the patients with Parkinson's disease**

*SNCA* gene, encoding ASN, was first gene describing as related with PD. Missence mutations and multiplications of this gene, generally have been described as related with FPD (Kruger et al., 1998), however *SNCA* duplications were also reported in SPD (Abeliovich et al., 2000; Ahn et al., 2008; Liu et al., 2004; Nishioka et al., 2009; Nuytemans et al., 2009). Therefore, it have been suspected that not only mutations in the *SNCA* gene, but also other factors affecting the expression of ASN may contribute to the PD manifestation including, SPD.

**2. Mutations in** *PRKN***,** *SPR* **and** *HTRA2* **genes and polymorphism of NACP-Rep1 region of** *SNCA* **promoter in the patients with Parkinson's**

During the last two decades, there has been breakthrough progress in genetics of PD. Currently it is known that genetic background of PD is heterogeneous and mutations in a number of pathogenic genes (e.g. *SNCA*, *PRKN*, *UCHL1, DJ-1, PINK1, ATP13A2*, and *LRRK2*) have been described as associated with familial (FPD) or as genetic risk factors increasing the risk to develop of sporadic PD (SPD). Some of these genes (like *SNCA* and *PRKN)* are fairly well understood while the others (like *SPR* and *HTRA2*) are still little known (Corti et al., 2011).

Monogenic forms, caused by a single mutation in a dominantly or recessively inherited gene, are well-established. Nevertheless, they are relatively rare types of PD and account for about 30% of the FPD and 3–5% of the SPD cases. Although 18 specific chromosomal locus (called *PARK* and numbered in chronological order of their identification) have been reported as more or less convincingly related to FPD (Klein & Westenberger, 2012), the majority of PD cases are SPD (only about 10% of patients report a positive family history) [Thomas & Beal, 2007] while the results of the studies of SPD genetic are still ambiguous and divergent in different ethnic origin (Klein & Schlossmacher, 2007; Lesage & Brice, 2009). Few studies (e.g. Abbas et al., 1999; Gilks et al., 2005; Guo et al., 2010; Mellick et al., 2005; Trotta et al., 2012) suggest a strong correlation of genetic factors with an increased risk of SPD development, while the other reports are contradictory (Chung et al., 2011; Spadafora et al., 2003). However, genome-wide association studies have provided convincing evidence that polymorphic variants in some genes contribute to higher risk of SPD (Gao et al., 2009). Moreover, it is suggested that the etiology of PD is multifactorial, which probably results from coocurence of genetic and

Summarizing, from the existing studies reported, it is not yet clear how common mutations in few genes, including: *PRKN*, *HTRA2*, *SPR* and *SNCA* genes contribute to idiopathic PD (Nuytemans et al., 2010). Finally despite previous reports, significance of these genes mutation and polymorphism in pathogenesis of PD (especially SPD) is not clear and is still debated, mainly because of discrepancy of studies results and variance between different ethnic populations. To clarify these issues, more data of genetic analysis are needed while there were only a few reports of genetic studies of PD in Polish populations (Bialecka et al., 2005; Koziorowski et al., 2010). Moreover, little is understood about putative director functional interactions between the genes that cause PD, and a single pathway unifying these factors has

not been confirmed (Bras et al., 2008; Brooks et al., 2009; Klein et al., 2005).

**2.1. Polymorphism of NACP-Rep1 region of** *SNCA* **promoter in the patients with**

*SNCA* gene, encoding ASN, was first gene describing as related with PD. Missence mutations and multiplications of this gene, generally have been described as related with FPD (Kruger et al., 1998), however *SNCA* duplications were also reported in SPD (Abeliovich et al., 2000;

environmental factors (Klein & Westenberger, 2012).

**Parkinson's disease**

**disease**

2 A Synopsis of Parkinson's Disease

The study by Chiba-Falek et al. (2006) has shown that the region NACP-Rep1 of *SNCA* gene promoter, there is the polymorphic region differenting in dinucleotide repeats count and affecting the level of ASN expression. Moreover, it has been shown that polymorphism of NACP-Rep1 region in promoter of *SNCA*, are associated with an increased risk of SPD in some population like: German, Australian, American and Polish, but the other multi-population studies have observed no association or reported an inverse association between the risk allele and PD (Farrer et al., 2001; Kruger et al., 1999; Maraganore et al., 2006; Polrolniczak et al., 2012; Tan et al., 2003).

Region NACP-Rep1 contains dinucleotide repeats (TC)x(T)2(TC)y(TA)2(CA)z, which may vary both the number of repeats, and include substitutions of nucleotides. However, it has been proven, that a change in the length of the NACP-Rep1 region more than substitutions, affects the regulation of the expression of ASN (Fuchs et al., 2008; Mellick et al., 2005; Tan et al., 2003). As the most common in humans it has been described five alleles of NACP-Rep1 of the *SNCA* gene promoter: -1, 0, +1, +2, +3. Generally in the European population the most frequently was allele +1 of NACP-Rep1. It has been also shown, that the allele 0 of NACP-Rep1 region in *SNCA* promoter is two pairs shorter than allele +1, allele -1 respectively, shorter by 4 bp however alleles 2 and 3 are longer by 2 and 4 bp.

Functional analysis on the two most common NACP-Rep1 alleles +1 and +2 suggested that the +2 allele is associated with an up-regulation of *SNCA* expression, whereas the +1 variant shows reduced gene expression (Chiba Falek & Nussbaum, 2001; Cronin et al., 2009). In addition, allele +1 of the region NACP-Rep1 of *SNCA* promoter, containing 259 bp, significantly reduces the risk of PD in the population of Europe, America and Australia (Fuchs et al., 2008; Mara‐ ganore et al., 2006; Tan et al., 2000) while another study failed to replicate the finding in population of Japan, Singapore and Italy (Spadafora et al., 2003; Tan et al., 2003).

Nerveless, although protective effect of allele +1 rather not currently subject to discussion, but for alleles 0, +2 and +3 it has been suggested both no impact, as well as increasing the risk of PD, and even sometimes the protective action (Maraganore et al., 2006; Spadafora et al., 2003; Tan et al., 2000; Trotta et al., 2012). The following studies by Tan et al. (2000) and Myhre et al. (2008) observed a higher frequency of the +3 allele in PD cases compared with healthy controls while in the study by both Tan et al. (2003) and Spadafora et al. (2003) no significant differences of the various genotypes between PD and controls were found in population of Singapore and Italy. However, the study in Italy population, have also shown evidence of association for allele +2 on NACP-Rep1 (Trotta et al., 2012). In 2006, a meta-analysis of 11 study populations provided strong evidence that the 263bp allele was more frequent in PD cases increasing risk of this disease while the 261bp allele did not differ between PD cases and unaffected controls but the authors suggested, that the lack of association of the +2 allele in the meta-analysis could be due to the large fluctuation in its frequencies observed in the analyzed populations (Maraganore et al., 2006). Therefore the aim of the study was analysis of NACP-Rep1 region in PD patients and in controls in Polish population.

## *2.1.1. Patients*

The studies were conducted on 90 patients with PD [SPD patients, 10 with early onset of PD, EOPD, and 80 with late onset of PD, LOPD patients), including 42 women and 47 men aging 34-82 years. Control group included 113 individuals, 79 women and 34 men, 39-83 years of age. Demographic data of all groups summarized in Table 1.

(5'-GACTGGCCCAAGATTAACCA-3'; 5'- CCTGGCATATTTGATTGCAA-3') under the conditions: (95°C 30'', 64°C 45'', 72°C 30'') [Tan et al., 2003]. One of the primers was labeled with fluorescent marker – FAM. Sizing of the PCR products was performed by capillary electrophoresis on the 3130xl Genetic Analyzer (Applied Biosystems HITACHI, USA) using GeneScan Size Standard 600LIZ (Applied Biosystems, USA) and controls. The results of electrophoresis were analyzed using Peak Scanner Software v.1.0 (Applied Biosystems, USA). Genotypes were differentiated according to the length of the PCR product. Designations of

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5

Moreover, random duplicate samples (10%) were genotyped for all assays for quality control

*Statistical analysis.* Statistical analysis was performed using Statistica for Windows Software. The level of significance was set at 5%. Chi-square test and Fisher's exact probability test, test for the two components of the structure, univariate odds ratio (ORs) and logistic regression analysis were used to compare the categorical variables and distribution of alleles and genotypes. The allele frequencies of PD patients and controls were evaluated with regards to

Screening for mutation c.88 G>C of *SNCA* gene in patients with PD and neurologically healthy controls detected no mutations in both group allow the exclusion of FPD determined by this

Using PCR amplification and capillary electrophoresis five previously described polymorphic alleles of NACP-Rep1 region in *SNCA* promoter were identified (designated -1, 0, +1, +2, +3) [Farrer et al., 2001; Xia et al., 2001]. Alleles and genotypes frequencies were in Hardy-Weinburg equilibrium in both groups: PD and controls with the exception of alleles +1 and +2, which frequencies in PD patients differed significantly from the expected frequencies calculated from Hardy-Weinburg equilibrium (exact test; p=0.032 and p=0.006 respectively). The frequency of allele +1 (Table 2) was significantly higher in healthy controls as compared to PD patients (p<0.001). In contrast to the allele +1, the frequency of alleles +2 and +3 were significantly higher in PD patients as compared to controls (p<0.01; p<0.05 respectively). However, the frequency of allele 0 was similar between PD and controls. Moreover, presence of allele -1 was detected

The frequency of +1/+1 genotype was almost fourfold higher in control group than in PD patients (p<0.001) whereas the frequency of the genotype +1/+2 was similar in both groups (Table 3). Comparisons of +2/+2 genotype frequencies between PD patients and control group revealed no significant differences but the frequency of this genotype was almost twofold higher in PD patients as compared to controls (p=0.056). It has been also detected, that the frequency of +2/+3 was significantly higher in PD patients compared to controls and was almost threefold higher in PD patients (p<0.05). Moreover, genotype +1/+3 has been detected only in

one PD patient while genotype -1/+1 occurred only in controls (Table 3).

alleles was followed those previously described (Farrer et al., 2001; Xia et al., 2001).

Hardy-Weinburg equilibrium using standardized formula.

only in control subjects (Polrolniczak et al., 2012).

with 100% reproducibility.

*2.1.3. Results*

mutation.

Patients with PD were diagnosed using the criteria of UK Parkinson's Disease Society Brain Bank (Litvan et al., 2003), however stage of disease according to the scale of Hoehn and Yahr (Hoehn & Yahr, 1967).

None of the control subjects had verifiable symptoms of dementia or any other neurological disorders. All subjects had negative family history of PD. All patients were recruited from the Neurology Clinic of Chair and Department of Neurology, University of Medical Sciences, Poznan in Poland. Only Caucasian, Polish subjects were included in this study. A Local Ethical Committee approved the study and the written consent of all patients or their caregivers was obtained.


**Table 1.** Demographic data of patients with PD and control subjects analyzed for NACP-Rep1 region in *SNCA* promoter. SD – standard deviation, F – female, M – male.

#### *2.1.2. Genetic investigations*

*Isolation of DNA.* DNA was isolated from peripheral blood lymphocytes by fivefold centrifu‐ gation in a lytic buffer, containing 155 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA, pH 7.4, in the presence of buffer containing 75 mM NaCl, 9 mM Na2EDTA, pH 8.0, and sodium dodecyl sulfate and proteinase K (Sigma, St. Louis, MO). Subsequently, NaCl was added, the lysate was centrifuged, and DNA present in the upper layer was precipitated with 98% ethanol. Extracted genomic DNA was stored at -80°C.

*Analysis of G88C mutation of SNCA gene.* For exon 3 of *SNCA* analysis, a total of 20 ng gDNA was amplified in 25 µl PCR reactions using specific primers (5'-AAGTGTATTT‐ TATGTTTTCC-3'; 5'-AACTGACATTTGGGGTTTACC-3') [Lin et al., 1999] and empirically defined reaction conditions. The PCR product was digested with MvaI (Fermentas, Canada) according to Kruger et al. (1998) method for screening c.88 G>C mutation in *SNCA* gene.

*Analysis of NACP-Rep1 polymorphism of SNCA promoter region.* For analysis of NACP-Rep1 region of *SNCA* promoter, analyzed region was amplified using described previously primers (5'-GACTGGCCCAAGATTAACCA-3'; 5'- CCTGGCATATTTGATTGCAA-3') under the conditions: (95°C 30'', 64°C 45'', 72°C 30'') [Tan et al., 2003]. One of the primers was labeled with fluorescent marker – FAM. Sizing of the PCR products was performed by capillary electrophoresis on the 3130xl Genetic Analyzer (Applied Biosystems HITACHI, USA) using GeneScan Size Standard 600LIZ (Applied Biosystems, USA) and controls. The results of electrophoresis were analyzed using Peak Scanner Software v.1.0 (Applied Biosystems, USA). Genotypes were differentiated according to the length of the PCR product. Designations of alleles was followed those previously described (Farrer et al., 2001; Xia et al., 2001).

Moreover, random duplicate samples (10%) were genotyped for all assays for quality control with 100% reproducibility.

*Statistical analysis.* Statistical analysis was performed using Statistica for Windows Software. The level of significance was set at 5%. Chi-square test and Fisher's exact probability test, test for the two components of the structure, univariate odds ratio (ORs) and logistic regression analysis were used to compare the categorical variables and distribution of alleles and genotypes. The allele frequencies of PD patients and controls were evaluated with regards to Hardy-Weinburg equilibrium using standardized formula.

#### *2.1.3. Results*

*2.1.1. Patients*

4 A Synopsis of Parkinson's Disease

(Hoehn & Yahr, 1967).

obtained.

The studies were conducted on 90 patients with PD [SPD patients, 10 with early onset of PD, EOPD, and 80 with late onset of PD, LOPD patients), including 42 women and 47 men aging 34-82 years. Control group included 113 individuals, 79 women and 34 men, 39-83 years of

Patients with PD were diagnosed using the criteria of UK Parkinson's Disease Society Brain Bank (Litvan et al., 2003), however stage of disease according to the scale of Hoehn and Yahr

None of the control subjects had verifiable symptoms of dementia or any other neurological disorders. All subjects had negative family history of PD. All patients were recruited from the Neurology Clinic of Chair and Department of Neurology, University of Medical Sciences, Poznan in Poland. Only Caucasian, Polish subjects were included in this study. A Local Ethical Committee approved the study and the written consent of all patients or their caregivers was

**Factor Controls Patients with PD**

**Individuals** 113 90 **Age** 39-83 34-82 **Mean age ±SD** 55.5±9.5 61.9±10.1 **F/M** 79/34 42/47

**Table 1.** Demographic data of patients with PD and control subjects analyzed for NACP-Rep1 region in *SNCA*

*Isolation of DNA.* DNA was isolated from peripheral blood lymphocytes by fivefold centrifu‐ gation in a lytic buffer, containing 155 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA, pH 7.4, in the presence of buffer containing 75 mM NaCl, 9 mM Na2EDTA, pH 8.0, and sodium dodecyl sulfate and proteinase K (Sigma, St. Louis, MO). Subsequently, NaCl was added, the lysate was centrifuged, and DNA present in the upper layer was precipitated with 98% ethanol.

*Analysis of G88C mutation of SNCA gene.* For exon 3 of *SNCA* analysis, a total of 20 ng gDNA was amplified in 25 µl PCR reactions using specific primers (5'-AAGTGTATTT‐ TATGTTTTCC-3'; 5'-AACTGACATTTGGGGTTTACC-3') [Lin et al., 1999] and empirically defined reaction conditions. The PCR product was digested with MvaI (Fermentas, Canada) according to Kruger et al. (1998) method for screening c.88 G>C mutation in *SNCA* gene.

*Analysis of NACP-Rep1 polymorphism of SNCA promoter region.* For analysis of NACP-Rep1 region of *SNCA* promoter, analyzed region was amplified using described previously primers

promoter. SD – standard deviation, F – female, M – male.

Extracted genomic DNA was stored at -80°C.

*2.1.2. Genetic investigations*

age. Demographic data of all groups summarized in Table 1.

Screening for mutation c.88 G>C of *SNCA* gene in patients with PD and neurologically healthy controls detected no mutations in both group allow the exclusion of FPD determined by this mutation.

Using PCR amplification and capillary electrophoresis five previously described polymorphic alleles of NACP-Rep1 region in *SNCA* promoter were identified (designated -1, 0, +1, +2, +3) [Farrer et al., 2001; Xia et al., 2001]. Alleles and genotypes frequencies were in Hardy-Weinburg equilibrium in both groups: PD and controls with the exception of alleles +1 and +2, which frequencies in PD patients differed significantly from the expected frequencies calculated from Hardy-Weinburg equilibrium (exact test; p=0.032 and p=0.006 respectively). The frequency of allele +1 (Table 2) was significantly higher in healthy controls as compared to PD patients (p<0.001). In contrast to the allele +1, the frequency of alleles +2 and +3 were significantly higher in PD patients as compared to controls (p<0.01; p<0.05 respectively). However, the frequency of allele 0 was similar between PD and controls. Moreover, presence of allele -1 was detected only in control subjects (Polrolniczak et al., 2012).

The frequency of +1/+1 genotype was almost fourfold higher in control group than in PD patients (p<0.001) whereas the frequency of the genotype +1/+2 was similar in both groups (Table 3). Comparisons of +2/+2 genotype frequencies between PD patients and control group revealed no significant differences but the frequency of this genotype was almost twofold higher in PD patients as compared to controls (p=0.056). It has been also detected, that the frequency of +2/+3 was significantly higher in PD patients compared to controls and was almost threefold higher in PD patients (p<0.05). Moreover, genotype +1/+3 has been detected only in one PD patient while genotype -1/+1 occurred only in controls (Table 3).


**Allele Heterozygous model Homozygous model Common odds ratio**

Mutations of PARK Genes and Alpha-Synuclein and Parkin Concentrations in Parkinson's Disease

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**p OR (95% CI) p OR p**

**-1** - >0,05 - >0.05 - >0.05 (F) **0** - >0,05 - >0.05 - >0.05 (C) **+1** 0.406 (0.210-0.785)\*\* **<0.01** 0.107 (0.035-0.322)\*\*\* **<0.001** 0.342\*\*\* **<0.001 (C) +2** 2.719 (1.292-5.719)\*\* **<0.01** 4.615 (1.774-12.009)\*\* **<0.001** 2.163\*\*\* **<0.001 (C) +3** 4.601 (1.445-14.647)\*\* **<0.01** - >0.05 4.601\*\* **<0.01 (F)**

**Table 4.** Modulation of PD risk manifestation by NACP-Rep1 variants measured by odds ratio. Logistic regression analysis, Fisher's exact test and Chi square test were used. OR – odds ratio; CI – confidence interval; F-Fisher's exact

Our results similarly to studies in the European, Australian and American populations indicated that, the presence of genotype +1/+1 may reduce PD risk while another study failed to replicate the finding in population of Italy (Fuchs et al., 2008; Maraganore et al., 2006; Mellick et al., 2005; Polrolniczak et al., 2012; Spadafora et al., 2003; Tan et al., 2003; Trotta et al., 2012). It is suggested, that reduction of PD risk by genotype +1/+1 may be related with decreasing ASN expression (Chiba-Falek et al., 2006; Fuchs et al., 2008). In the study in Polish population it has been also observed, in PD patient with genotype +1/+1 tendency to slower progression of the disease and better response to pharmacotherapy at using low doses of the L-dopa treatment compared the other genotypes of NACP-Rep1 (Polrolniczak et al., 2012). It seems, that in PD patients with genotype +1/+1 reduced ASN level, due to reduce ASN aggregation and maintenance of dopamine homeostasis in the central nervous system (CNS) probably leads to milder course of disease compared to patients with other genotypes of NACP-Rep1

Although the study in Singapore and Italian populations shown no association for alleles +2 and +3 with PD our results confirming the study in populations: German, Italian, Japanese, and multipopulation research detected higher frequency of those alleles in PD patients compared with controls and indicated association of genotypes +2/+2 and +2/+3 with increased risk of PD in Polish population (Maraganore et al., 2006; Mellick et al., 2005; Polrolniczak et al., 2012; Spadafora et al., 2003; Tan et al., 2003; Trotta et al., 2012). It is believed that the influence of genotype +2/+2 and +2/+3 on the risk of PD most likely may be associated with over-expression of ASN, leading to increased aggregation of ASN and the severity of the neurotoxic effect (Chiba-Falek et al., 2006; Cronin et al., 2009; Fuchs et al., 2008). Furthermore in our study in patients with genotypes +2/+2 and +2/+3 we observed tendency to faster progression of the disease but no association with response to therapy (Polrolniczak et al., 2012). This observations seems corresponding with the results of the study by Ritz et al. (2012) shoved that risk of faster decline of motor function was increased four-fold in carriers of the +3 allele of NACP-Rep1 promoter variant. Moreover, the study by Kay et al. (2008) have

test; C-Chi square test. Differences significant at: \*\*p<0.01; \*\*\*p<0.001, as compared to the controls.

**OR (95% CI)**

(Maguire-Zeiss et al., 2005).

**Table 2.** NACP-Rep1 alleles frequency in PD patients and controls. Results are expressed as a percentage. Test for two components of the structure was used. Differences significant at: \*p<0.05; \*\*p<0.01; \*\*\*p<0.001, as compared to the controls.


**Table 3.** NACP-Rep1 genotype frequencies in PD patients and in controls. Results are expressed as a percentage. Test for two components of the structure was used. Differences significant at: \*p<0.05; \*\*\*p<0.001, as compared to the controls.

Logistic regression analysis have shown, that PD risk (as measured by OR, Table 4) has been reduced in presence of allele +1 and reduces with increasing dose of +1 allele. Moreover, OR pointed to the association the presence of allele +2 with increased risk of PD manifestation in dose dependent manner. Influence of the presence of allele +3 of the increase PD risk has been detected only in heterozygous variant. Genotype +3/+3 have not been detected in any person in both control and PD patient group.


**Allele Controls Patients with PD**

**-1** 1% 0% **0** 5% 6% **+1** 53% 33%\*\*\* **+2** 40% 54%\*\* **+3** 2% 7%\*

**Table 2.** NACP-Rep1 alleles frequency in PD patients and controls. Results are expressed as a percentage. Test for two components of the structure was used. Differences significant at: \*p<0.05; \*\*p<0.01; \*\*\*p<0.001, as compared to the

**Genotypes Controls Patients with PD**

**Table 3.** NACP-Rep1 genotype frequencies in PD patients and in controls. Results are expressed as a percentage. Test for two components of the structure was used. Differences significant at: \*p<0.05; \*\*\*p<0.001, as compared to the

Logistic regression analysis have shown, that PD risk (as measured by OR, Table 4) has been reduced in presence of allele +1 and reduces with increasing dose of +1 allele. Moreover, OR pointed to the association the presence of allele +2 with increased risk of PD manifestation in dose dependent manner. Influence of the presence of allele +3 of the increase PD risk has been detected only in heterozygous variant. Genotype +3/+3 have not been detected in any person

113 90

**-1/+1** 2% 0% **0/+1** 7% 7% **0/+2** 3% 4% **+1/+1** 23% 6%\*\*\* **+1/+2** 50% 47% **+1/+3** 0% 1% **+2/+2** 12% 22% **+2/+3** 4% 13%\*

**Total subjects number** 226 180

controls.

6 A Synopsis of Parkinson's Disease

Total subjects number

in both control and PD patient group.

controls.

**Table 4.** Modulation of PD risk manifestation by NACP-Rep1 variants measured by odds ratio. Logistic regression analysis, Fisher's exact test and Chi square test were used. OR – odds ratio; CI – confidence interval; F-Fisher's exact test; C-Chi square test. Differences significant at: \*\*p<0.01; \*\*\*p<0.001, as compared to the controls.

Our results similarly to studies in the European, Australian and American populations indicated that, the presence of genotype +1/+1 may reduce PD risk while another study failed to replicate the finding in population of Italy (Fuchs et al., 2008; Maraganore et al., 2006; Mellick et al., 2005; Polrolniczak et al., 2012; Spadafora et al., 2003; Tan et al., 2003; Trotta et al., 2012). It is suggested, that reduction of PD risk by genotype +1/+1 may be related with decreasing ASN expression (Chiba-Falek et al., 2006; Fuchs et al., 2008). In the study in Polish population it has been also observed, in PD patient with genotype +1/+1 tendency to slower progression of the disease and better response to pharmacotherapy at using low doses of the L-dopa treatment compared the other genotypes of NACP-Rep1 (Polrolniczak et al., 2012). It seems, that in PD patients with genotype +1/+1 reduced ASN level, due to reduce ASN aggregation and maintenance of dopamine homeostasis in the central nervous system (CNS) probably leads to milder course of disease compared to patients with other genotypes of NACP-Rep1 (Maguire-Zeiss et al., 2005).

Although the study in Singapore and Italian populations shown no association for alleles +2 and +3 with PD our results confirming the study in populations: German, Italian, Japanese, and multipopulation research detected higher frequency of those alleles in PD patients compared with controls and indicated association of genotypes +2/+2 and +2/+3 with increased risk of PD in Polish population (Maraganore et al., 2006; Mellick et al., 2005; Polrolniczak et al., 2012; Spadafora et al., 2003; Tan et al., 2003; Trotta et al., 2012). It is believed that the influence of genotype +2/+2 and +2/+3 on the risk of PD most likely may be associated with over-expression of ASN, leading to increased aggregation of ASN and the severity of the neurotoxic effect (Chiba-Falek et al., 2006; Cronin et al., 2009; Fuchs et al., 2008). Furthermore in our study in patients with genotypes +2/+2 and +2/+3 we observed tendency to faster progression of the disease but no association with response to therapy (Polrolniczak et al., 2012). This observations seems corresponding with the results of the study by Ritz et al. (2012) shoved that risk of faster decline of motor function was increased four-fold in carriers of the +3 allele of NACP-Rep1 promoter variant. Moreover, the study by Kay et al. (2008) have indicated a trend of decreasing onset age with increasing allele size while the other study have shown, that age at onset of carriers of at least one allele +2 was earlier compared to noncarriers (Hadjigeorgiou et al., 2006).

population in 11% (Hattori et al., 1998). In Italian population mutations of *PRKN* occurred in frequency 8-13%, in French in 16%, in German in 9% and in Americans in 4% while in North African in 21%, and in Brazilian in about 8% (Chen et al., 2003; Klein et al., 2005; Lucking &

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The observation, that mutations in the *PRKN* gene are common in juvenile- (JPD) and EOPD and increasing evidence supporting a direct role for Parkin in LOPD make this gene a particularly compelling candidate for intensified investigation. However, despite previous reports, significance of *PRKN* mutation and polymorphism in pathogenesis of PD is still not

The aim of the study was to estimate the frequency of *PRKN* mutation in Polish PD patients

According to the inclusion and exclusion criteria a total of 199 subjects were included in this study: 87 SPD patients (10 EOPD patients, and 77 sporadic LOPD patients), including 41 women and 45 men aging 34-82 years. Control group included 112 individuals, 78 women and 34 men, 39-83 years of age. Demographic data of all groups summarized in Table 5. Patients with PD were diagnosed using the criteria of UK Parkinson's Disease Society Brain Bank (Litvan et al., 2003), however stage of disease according to the scale of Hoehn and Yahr (Hoehn & Yahr, 1967). None of the control subjects had verifiable symptoms of dementia or any other neurological disorders. All subjects had negative family history of PD. All patients were recruited from the Neurology Clinic of Chair and Department of Neurology, University of Medical Sciences, Poznan in Poland. Only Caucasian, Polish subjects were included in the study. A Local Ethical Committee approved the study and the written consent of all patients

**Factor Controls Patients with PD**

**Table 5.** Demographic data of patients with PD and control subjects analyzed for *PRKN* mutation. SD – standard

*Analysis of deletion of exon 2 and 4 of PRKN gene.* Exon deletion in *PRKN* was examined by amplifying exon 2 and 4 using internal and external specific primers previously described (5'- ATGTTGCTATCACCATTTAAG-3'; 5'-AGATTGGCAGCGCAGGCGGCA-3' for exon 2)

**Individuals** 112 87 **Age** 39-83 34-82 **Mean age ±SD** 55.6±9.5 61.4±9.9 **F/M** 78/34 41/45

Briece, 2000; Periquet et al., 2003).

or their caregivers was obtained.

deviation, F – female, M – male.

*2.2.2. Genetic investigations*

*Isolation of DNA*. See point 2.1.2

clear.

and controls.

*2.2.1. Patients*

However, in contrast to the results of Kay et al. (2008) in Polish population it has not indicated any association of allele 0 with risk of PD, however presence of this genetic variant was correlated in Spearman correlation test (p=0,019; r=-0,507) with decrease in stage of disease in patients suffering for PD over 10 years compared patients with the other genotypes of NACP-Rep1 (Polrolniczak et al., 2012).

It seems that examination of genotypes of region NACP-Rep1 of *SNCA* promoter may help to explain the pathogenesis of PD, as well as facilitate early diagnosis and determine the degree of risk for this neurodegenerative disease.

#### **2.2. Mutations in** *PRKN* **in the patients with Parkinson's disease**

Mutations of *PRKN* were first identified in Japanese families with autosomal recessive juvenile Parkinsonism and since then more than 100 mutations in this gene have been found. Mutation in *PRKN* gene encoding Parkin, have been found both in the EOPD (<40 years) and in the LOPD (>40 years) forms of PD (Bardien et al., 2009; Kitada et al., 1998). Although *PRKN* mutations have been identified in all 12 exons of this gene, the most common seem to be mutations in exons 2, 4, 7, 8, 10 and 11. The vast majority, FPD conditioned by *PRKN* mutation is inherited as an autosomal recessive but it has been also reported heterozygous mutations related with PD manifestation.

Furthermore, it has been shown that mutations in the gene *PRKN* occur at different frequencies both in Caucasians and in populations of African and Asian countries (Kitada et al., 1998; Lucking & Briece, 2000). However, the literature on the prevalence of mutations in *PRKN* and their involvement in the modulation of PD risk are very diverse and have a wide variation depending on the studied population, and the age of subjects included in the study.

It is suggested, that mutations in *PRKN*, including homo- and heterozygous mutations are detected in about 40-50% of early-onset FPD and in about 1.3-20% of SPD patients (Choi et al., 2008; Herdich et al., 2004; Kann et al., 2002; Mellick et al., 2009; Sironi et al., 2008).

The study by Abbas et al. (1999), point mutations of *PRKN* in the European population were approximately twice as common as homozygous exonic deletions. In the European population it has been reported *PRKN* mutations in about 19% of SPD and 50% of early-onset FPD (Lucking & Briece, 2000). Further the study by Lucking et al. (2001) in the sporadic cases revealed that 77% with age of disease onset below 20 years had mutations in *PRKN* gene, but in cases with age of disease onset between 31 and 45 years mutations were found only in 3% in European population. The larger cases studies have confirmed reports of Lucking et al. (2001) and it has shown *PRKN* mutations in 67% of cases with age of onset below 20 years and in 8% of cases with an age of onset between 30–45 years. In another study involving 363 affected subjects from 307 families it has identified *PRKN* mutations in 2% of all late-onset families screened, thereby directly implicating the *PRKN* gene in LOPD (Oliveira et al., 2003). In population of Korea it has been detected *PRKN* mutations in EOPD in 5% frequency while in Japanse population in 11% (Hattori et al., 1998). In Italian population mutations of *PRKN* occurred in frequency 8-13%, in French in 16%, in German in 9% and in Americans in 4% while in North African in 21%, and in Brazilian in about 8% (Chen et al., 2003; Klein et al., 2005; Lucking & Briece, 2000; Periquet et al., 2003).

The observation, that mutations in the *PRKN* gene are common in juvenile- (JPD) and EOPD and increasing evidence supporting a direct role for Parkin in LOPD make this gene a particularly compelling candidate for intensified investigation. However, despite previous reports, significance of *PRKN* mutation and polymorphism in pathogenesis of PD is still not clear.

The aim of the study was to estimate the frequency of *PRKN* mutation in Polish PD patients and controls.

#### *2.2.1. Patients*

indicated a trend of decreasing onset age with increasing allele size while the other study have shown, that age at onset of carriers of at least one allele +2 was earlier compared to noncarriers

However, in contrast to the results of Kay et al. (2008) in Polish population it has not indicated any association of allele 0 with risk of PD, however presence of this genetic variant was correlated in Spearman correlation test (p=0,019; r=-0,507) with decrease in stage of disease in patients suffering for PD over 10 years compared patients with the other genotypes of NACP-

It seems that examination of genotypes of region NACP-Rep1 of *SNCA* promoter may help to explain the pathogenesis of PD, as well as facilitate early diagnosis and determine the degree

Mutations of *PRKN* were first identified in Japanese families with autosomal recessive juvenile Parkinsonism and since then more than 100 mutations in this gene have been found. Mutation in *PRKN* gene encoding Parkin, have been found both in the EOPD (<40 years) and in the LOPD (>40 years) forms of PD (Bardien et al., 2009; Kitada et al., 1998). Although *PRKN* mutations have been identified in all 12 exons of this gene, the most common seem to be mutations in exons 2, 4, 7, 8, 10 and 11. The vast majority, FPD conditioned by *PRKN* mutation is inherited as an autosomal recessive but it has been also reported heterozygous mutations related with

Furthermore, it has been shown that mutations in the gene *PRKN* occur at different frequencies both in Caucasians and in populations of African and Asian countries (Kitada et al., 1998; Lucking & Briece, 2000). However, the literature on the prevalence of mutations in *PRKN* and their involvement in the modulation of PD risk are very diverse and have a wide variation

It is suggested, that mutations in *PRKN*, including homo- and heterozygous mutations are detected in about 40-50% of early-onset FPD and in about 1.3-20% of SPD patients (Choi et al.,

The study by Abbas et al. (1999), point mutations of *PRKN* in the European population were approximately twice as common as homozygous exonic deletions. In the European population it has been reported *PRKN* mutations in about 19% of SPD and 50% of early-onset FPD (Lucking & Briece, 2000). Further the study by Lucking et al. (2001) in the sporadic cases revealed that 77% with age of disease onset below 20 years had mutations in *PRKN* gene, but in cases with age of disease onset between 31 and 45 years mutations were found only in 3% in European population. The larger cases studies have confirmed reports of Lucking et al. (2001) and it has shown *PRKN* mutations in 67% of cases with age of onset below 20 years and in 8% of cases with an age of onset between 30–45 years. In another study involving 363 affected subjects from 307 families it has identified *PRKN* mutations in 2% of all late-onset families screened, thereby directly implicating the *PRKN* gene in LOPD (Oliveira et al., 2003). In population of Korea it has been detected *PRKN* mutations in EOPD in 5% frequency while in Japanse

depending on the studied population, and the age of subjects included in the study.

2008; Herdich et al., 2004; Kann et al., 2002; Mellick et al., 2009; Sironi et al., 2008).

(Hadjigeorgiou et al., 2006).

8 A Synopsis of Parkinson's Disease

Rep1 (Polrolniczak et al., 2012).

PD manifestation.

of risk for this neurodegenerative disease.

**2.2. Mutations in** *PRKN* **in the patients with Parkinson's disease**

According to the inclusion and exclusion criteria a total of 199 subjects were included in this study: 87 SPD patients (10 EOPD patients, and 77 sporadic LOPD patients), including 41 women and 45 men aging 34-82 years. Control group included 112 individuals, 78 women and 34 men, 39-83 years of age. Demographic data of all groups summarized in Table 5. Patients with PD were diagnosed using the criteria of UK Parkinson's Disease Society Brain Bank (Litvan et al., 2003), however stage of disease according to the scale of Hoehn and Yahr (Hoehn & Yahr, 1967). None of the control subjects had verifiable symptoms of dementia or any other neurological disorders. All subjects had negative family history of PD. All patients were recruited from the Neurology Clinic of Chair and Department of Neurology, University of Medical Sciences, Poznan in Poland. Only Caucasian, Polish subjects were included in the study. A Local Ethical Committee approved the study and the written consent of all patients or their caregivers was obtained.


**Table 5.** Demographic data of patients with PD and control subjects analyzed for *PRKN* mutation. SD – standard deviation, F – female, M – male.

#### *2.2.2. Genetic investigations*

#### *Isolation of DNA*. See point 2.1.2

*Analysis of deletion of exon 2 and 4 of PRKN gene.* Exon deletion in *PRKN* was examined by amplifying exon 2 and 4 using internal and external specific primers previously described (5'- ATGTTGCTATCACCATTTAAG-3'; 5'-AGATTGGCAGCGCAGGCGGCA-3' for exon 2) [Choi et al., 2008] or generated using the online software Pimer3 (http://www-ge‐ nome.wi.mit.edu/cgibin/primer/primer3\_www.cgi) based on the published genomic se‐ quence of the *PRKN* gene (5'-TTTCCCAAATATTGCTCTA-3'; 5'- GCAGTGTGGAGTAAAGTTCAAGG-3' for exon 2 and 5'- GCATTATTAGCCACTTCTTCTGC-3'; 5'-TGCTGACACTGCATTTCCTT-3'; 5'- AGATTTCACTCTTGGAGCATAAA-3'; 5'-CAAAGGCGCATAAACGAAA-3' for exon 4). PCR cycling conditions were empirically defined (Polrolniczak et al., 2012).

of *PRKN* substitution increased risk of PD over six-fold (p<0.001; OR=6.059). All substitutions

*PRKN mutations* 8% 31%\*\*\* 6.059 2.188-11.207 <0.001 (C)

**Table 6.** Total *PRKN* point mutations frequencies in PD patients and controls. Results are expressed as a percentage. Chi square test was used. OR – odds ratio; CI – confidence interval; C - Chi square test. Differences significant at:

In exon 4 of *PRKN* two mutations were detected: c.500 G>A transition leads to S167A substi‐ tution (with frequency sevenfold higher in PD than in control group; p<0.05) and a novel heterozygous mutation c.520 C>T resulting L174F substitution and occurring only in PD patients. Furthermore, first time in Polish population we detected c.823 C>T (exon 7, R275T; only in PD) and c.930 G>C (exon 8, E310D) substitutions (over threefold more frequently in PD than in controls; p<0.01). Moreover, we detected also a transition c.1180 G>A in exon 11 of *PRKN*. It has been also shown, that c.500 G>A, c.930 G>C and c.1180 G>A substitutions significantly increased PD risk (Table 7). Simultaneously analysis of the amino acid sequence of the Parkin (encoded by *PRKN* gene) revealed that the substitution E310D and L174F are located in conserved region whereas substitution R275T and D394N in a limited conserved

**Controls Patients with PD OR 95% CI p**

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112 87 - - -

**c.500 G>A c.520 C>T c.823 C>T c.930 G>C c.1180 G>A**

**Controls** 1% 0% 0% 5% 2% **PD patients** 7%\* 2% 1% 18%\*\* 11%\*\* **OR** 8.000 - - 3.926 6.938 **95% CI** 0.945-67.712 - - 1.436-10.735 1.480-32.528 **p** <0.05 (F) >0.05 (F) >0.05 (F) <0.01 (C) <0.01 (C)

**Table 7.** *PRKN* point mutations frequencies in PD patients and controls. Results are expressed as a percentage. Logistic regression analysis, Fisher's exact test and Chi square test were used. OR – odds ratio; CI – confidence interval; F - Fisher's exact test; C - Chi square test. Differences significant at: \*p<0.05; \*\*p<0.01 as compared to the controls.

Additionally in 5% PD patients it has been detected more than one mutation in *PRKN* gene while all control subjects who had substitution in *PRKN*, had only one mutation (Table 8).

were non-synonymous and were in heterozygous state.

**Total subjects number**

**Mutation/ polymorphism**

\*\*\*p<0.001, as compared to the controls.

region of this protein (Polrolniczak et al., 2012).

*Analysis of exon 4, 7 and 11 of PRKN gene.* High resolution melting (HRM) were used for mutation screening in exon 4, 7 and 11 of *PRKN*. HRM was performed with the LightCycler 480 Real-Time PCR system (Roche, USA) and High Resolution Master Mix (Roche, USA). Reactions were performed on 96-well plates, using 5 ng of template DNA, 1x Master Mix, 2.5 mM MgCl2, and 10 pmol primers (on request) in a 10 µl reaction volume. PCR cycling conditions comprised of an initial denaturation step of 95°C for 5 min, 30 cycles of denaturation at 95°C for 15 s, annealing at 64°C (or 63°C for exon 4) for 15 s, extension at 72°C C for 15 s, and a final extension step of 72°C for 7 min. HRM analysis was performed from 55°C to 95°C (Polrolniczak et al., 2012). Melting curves and difference plots were analyzed by 3 investigators blinded to phenotype. For the samples with shifted melting curves, PCR products were cleaned and sequenced in the forward and reverse directions. Sequencing was performed using the 3130xl Genetic Analyzer (Applied Biosystems HITACHI, USA) and reads were aligned to the human reference genome with BioEdit Software (Tom Hall Ibis Biosciences, Canada). Coding DNA mutation numbering is relative to NM\_004562.2.

*Analysis of c.930 G>C substitution in exon 8 of PRKN gene.* For exon 8 of *PRKN* analysis 20 ng gDNA was amplified in 25 µl using PCR reaction with specific primers (5'- CTAAA‐ GAGGTGCGGTTGGAG-3'; 5'- GGAGCCCAAACTGTCTCATT-3') generated using the online software Pimer3 based on the published genomic sequence of the *PRKN* gene. PCR cycling conditions were empirically defined. Screening for the c.930 G>C mutation of *PRKN* was performed by a RFLP analysis on 2% agarose gels using Mva I (Fermentas, Canada) as restriction enzyme. All detected mutations were confirmed by sequencing of PCR product.

Moreover, random duplicate samples (10%) were genotyped for all assays for quality control with 100% reproducibility.

*Statistical analysis.* Statistical analyses were performed using Statistica for Windows Software. The level of significance was set at 5%. Chi-square test and Fisher's exact probability test, univariate odds ratio (ORs) and logistic regression analysis were used to compare the catego‐ rical variables and distribution of alleles. The allele frequencies of PD patients and controls were evaluated with regards to Hardy-Weinburg equilibrium using standardized formula.

#### *2.2.3. Results*

Analysis of deletions of exons 2 and 4 *PRKN* has detected no genetic changes both in PD patients and control group. However, point mutation screening in patients with PD and healthy controls identified 5 missence substitutions which were almost fourfold more frequent in PD patients as compared with controls (p<0.001) [Table 6]. We also showed, that the presence


of *PRKN* substitution increased risk of PD over six-fold (p<0.001; OR=6.059). All substitutions were non-synonymous and were in heterozygous state.

[Choi et al., 2008] or generated using the online software Pimer3 (http://www-ge‐ nome.wi.mit.edu/cgibin/primer/primer3\_www.cgi) based on the published genomic se‐ quence of the *PRKN* gene (5'-TTTCCCAAATATTGCTCTA-3'; 5'- GCAGTGTGGAGTAAAGTTCAAGG-3' for exon 2 and 5'- GCATTATTAGCCACTTCTTCTGC-3'; 5'-TGCTGACACTGCATTTCCTT-3'; 5'- AGATTTCACTCTTGGAGCATAAA-3'; 5'-CAAAGGCGCATAAACGAAA-3' for exon 4).

*Analysis of exon 4, 7 and 11 of PRKN gene.* High resolution melting (HRM) were used for mutation screening in exon 4, 7 and 11 of *PRKN*. HRM was performed with the LightCycler 480 Real-Time PCR system (Roche, USA) and High Resolution Master Mix (Roche, USA). Reactions were performed on 96-well plates, using 5 ng of template DNA, 1x Master Mix, 2.5 mM MgCl2, and 10 pmol primers (on request) in a 10 µl reaction volume. PCR cycling conditions comprised of an initial denaturation step of 95°C for 5 min, 30 cycles of denaturation at 95°C for 15 s, annealing at 64°C (or 63°C for exon 4) for 15 s, extension at 72°C C for 15 s, and a final extension step of 72°C for 7 min. HRM analysis was performed from 55°C to 95°C (Polrolniczak et al., 2012). Melting curves and difference plots were analyzed by 3 investigators blinded to phenotype. For the samples with shifted melting curves, PCR products were cleaned and sequenced in the forward and reverse directions. Sequencing was performed using the 3130xl Genetic Analyzer (Applied Biosystems HITACHI, USA) and reads were aligned to the human reference genome with BioEdit Software (Tom Hall Ibis Biosciences, Canada). Coding DNA

*Analysis of c.930 G>C substitution in exon 8 of PRKN gene.* For exon 8 of *PRKN* analysis 20 ng gDNA was amplified in 25 µl using PCR reaction with specific primers (5'- CTAAA‐ GAGGTGCGGTTGGAG-3'; 5'- GGAGCCCAAACTGTCTCATT-3') generated using the online software Pimer3 based on the published genomic sequence of the *PRKN* gene. PCR cycling conditions were empirically defined. Screening for the c.930 G>C mutation of *PRKN* was performed by a RFLP analysis on 2% agarose gels using Mva I (Fermentas, Canada) as restriction enzyme. All detected mutations were confirmed by sequencing of PCR product.

Moreover, random duplicate samples (10%) were genotyped for all assays for quality control

*Statistical analysis.* Statistical analyses were performed using Statistica for Windows Software. The level of significance was set at 5%. Chi-square test and Fisher's exact probability test, univariate odds ratio (ORs) and logistic regression analysis were used to compare the catego‐ rical variables and distribution of alleles. The allele frequencies of PD patients and controls were evaluated with regards to Hardy-Weinburg equilibrium using standardized formula.

Analysis of deletions of exons 2 and 4 *PRKN* has detected no genetic changes both in PD patients and control group. However, point mutation screening in patients with PD and healthy controls identified 5 missence substitutions which were almost fourfold more frequent in PD patients as compared with controls (p<0.001) [Table 6]. We also showed, that the presence

PCR cycling conditions were empirically defined (Polrolniczak et al., 2012).

mutation numbering is relative to NM\_004562.2.

with 100% reproducibility.

10 A Synopsis of Parkinson's Disease

*2.2.3. Results*

**Table 6.** Total *PRKN* point mutations frequencies in PD patients and controls. Results are expressed as a percentage. Chi square test was used. OR – odds ratio; CI – confidence interval; C - Chi square test. Differences significant at: \*\*\*p<0.001, as compared to the controls.

In exon 4 of *PRKN* two mutations were detected: c.500 G>A transition leads to S167A substi‐ tution (with frequency sevenfold higher in PD than in control group; p<0.05) and a novel heterozygous mutation c.520 C>T resulting L174F substitution and occurring only in PD patients. Furthermore, first time in Polish population we detected c.823 C>T (exon 7, R275T; only in PD) and c.930 G>C (exon 8, E310D) substitutions (over threefold more frequently in PD than in controls; p<0.01). Moreover, we detected also a transition c.1180 G>A in exon 11 of *PRKN*. It has been also shown, that c.500 G>A, c.930 G>C and c.1180 G>A substitutions significantly increased PD risk (Table 7). Simultaneously analysis of the amino acid sequence of the Parkin (encoded by *PRKN* gene) revealed that the substitution E310D and L174F are located in conserved region whereas substitution R275T and D394N in a limited conserved region of this protein (Polrolniczak et al., 2012).


**Table 7.** *PRKN* point mutations frequencies in PD patients and controls. Results are expressed as a percentage. Logistic regression analysis, Fisher's exact test and Chi square test were used. OR – odds ratio; CI – confidence interval; F - Fisher's exact test; C - Chi square test. Differences significant at: \*p<0.05; \*\*p<0.01 as compared to the controls.

Additionally in 5% PD patients it has been detected more than one mutation in *PRKN* gene while all control subjects who had substitution in *PRKN*, had only one mutation (Table 8).


increased risk LOPD probably in combination with other genetic or environmental factors, as evidenced by Bardien et al. reports (2009). The other two identified *PRKN* mutations (c.823 C> T, c.520 C> T) were detected only in PD patients, what may indicate a high penetration of these substitutions (Sinha et al., 2005), while novel mutation c.520 C>T was identified in two patients

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It is suggested, that haploinsufficiency may be considered as a reduction of normal gene expression accompanied by a loss of normal protein activity. Moreover, a lot of reports indicate to the existence of a second, undetected mutation in these patients, perhaps in the promoter

Our results, also suggests that the presence more than one heterozygous mutation in the *PRKN* gene may be necessary to PD manifestation. This hypothesis was first proposed by Abbas et al. (1999) moreover, later reviews generally assume the existence of a second, undetected mutation (Giasson & Lee, 2001). In our study also it is probably that patient who had one mutation in *PRKN* may have more genetic changes in not tested region of the gene so extension the studies of the other region of *PRKN* gene is necessary to clarify this issue. On the other hand it can not be ruled that one heterozygous mutation in *PRKN* may be sufficient to increase

Finally, it seems that clinically, PD patients with *PRKN* substitution generally are characterized by slower progression of the disease compared with PD patients without mutation. Moreover, it has been also observed, that in PD patients with *PRKN* mutations response to L-dopa therapy has been better than in PD patients without substitutions. This observation are generally consistent with the typical descriptions of *PRKN* patients which present slow disease progres‐ sion (Abbas et al., 1999; Lucking & Briece, 2000) and good response to L-dopa treatment although it have been showed that patients with *PRKN* mutation were more likely to develop treatment-induced motor complications earlier in the treatment (Khan et al., 2005; Lucking &

It seems, that point mutation in *PRKN* gene may be involved in the pathogenesis of LOPD and modulate clinical futures in this disease. It is also probably, that analysis of mutations in *PRKN*

It seems that presence of mutation in the other genes involved in the pathogenesis of PD like *SPR* (involved in dopamine biosynthesis) and *HTRA2* (involved with mitochondrial pathway of PD) probably may additionally affect the levels of ASN and Parkin through interaction with these proteins (Bogaerts et al., 2008; Karamohamed et al., 2003; Sharma et al., 2006; Sharma et al., 2011; Strauss et al., 2005). However, role of those genes in pathogenesis of PD is not enough known. The serine protease HTRA2 is localized to the inner membrane space of mitochondria (Suzuki et al., 2001). Mitochondrial dysfunction as well as ubiquitin–proteasome system damage has been proposed as possible mechanisms leading to dopaminergic neuronal degeneration (Lin & Beal, 2006; Malkus et al., 2009; Rubinsztein, 2006). Therefore *HTRA2*

risk of PD and induce preclinical changes in *substantia nigra* (Khan et al., 2005).

gene may be useful for diagnostic and prognostic process in PD.

**2.3. Mutations in** *HTRA2* **and** *SPR* **in the patients with Parkinson's disease**

and led to a relatively early onset of disease before age 40.

or intronic regions (Giasson & Lee, 2001).

Briece, 2000).

**Table 8.** Coexistence more than one *PRKN* point mutations in PD patients in Polish population (Polrolniczak et al., 2012).

It is suggested, that single or multiple exon deletions and duplications occur with a frequency of 15.8% and account for about 50% of all mutations of *PRKN* gene (Nuytemans et al., 2010). Nevertheless, althought many reports indicated important role of *PRKN* exons 2 and 4 deletions in pathogenesis of idiopatic PD (Choi et al., 2008; Guo et al., 2010; Macedo et al., 2009; Pankratz et al., 2009) in the study in Polish population it has not detected any deletion of exon 2 and 4 in *PRKN* gene as opposed to the German and Japan population, as well as the results obtained in the multipopulation study (Cookson et al., 2008; Nishioka et al., 2009; Polrolniczak et al., 2011; 2012; Shapira et al., 2002). On the other hand, our results were consistent with the study by Kruger et al. (1999), Sinha et al. (2005), as well Barsottini et al. (2011). However, it not be ruled out, that Polish patients have deletion of other not tested exons. Oliveri et al. (2001) suggested that deletion mutations of *PRKN* were not as common in LOPD as in EOPD. Therefore, it seems that copy number variation of *PRKN* is most probably related with EOPD (Wang et al., 2004).

However, point mutations in *PRKN* gene although they are characteristic for EOPD, currently it is suggested that it can be also involved in the pathogenesis of LOPD. However, studies utilizing common mutations and polymorphisms in tests for association with LOPD have produced mixed results (Hu et al., 2000; Oliveri et al., 2001; Satoh & Kuroda, 1999; Wang et al., 1999).

Furthermore there is no question that Parkin-associated parkinsonism is recessive; that is, both alleles are mutant, but despite previous reports whether a heterozygous mutation can cause or increase the risk for PD remains an issue of debate (Farrer et al., 2001; Klein et al., 2000; Lucking et al., 2001; Maruyama et al., 2000).

In the German population the frequency of *PRKN* mutations was 9% (Kann et al., 2002), in Brazilian population 8% (Periquet et al., 2001), and in the American population reached value of less than 4% (Chen et al., 2003) while in the Japanese population reached 66% (Hattori et al., 1998). In Polish population it has been showed small share of *PRKN* mutation in the patho‐ genesis of EOPD (Dawson & Dawson, 2003) while our study in LOPD have shown, that *PRKN* mutation in Polish population occurred with frequency 20,6% (Polrolniczak et al., 2011; 2012) what was similar to SPD in European population (Nishioka et al., 2009).

Moreover, we showed, that in the Polish population the most frequently were polymorphisms c.500 G>A, c.1180 G>A and c.930 G>C of *PRKN*. Simultaneously, it appears that these poly‐ morphisms may have incomplete penetration or lead to preclinical changes in the CNS and increased risk LOPD probably in combination with other genetic or environmental factors, as evidenced by Bardien et al. reports (2009). The other two identified *PRKN* mutations (c.823 C> T, c.520 C> T) were detected only in PD patients, what may indicate a high penetration of these substitutions (Sinha et al., 2005), while novel mutation c.520 C>T was identified in two patients and led to a relatively early onset of disease before age 40.

**Coexistence of substitutions in** *PRKN* **gene Percentage of PD patients** c.823 C>T , c.1180 G>A 1% c.500 G>A, c.520 C>T 1% c.930 G>C, c.1180 G>A 2% c.500 G>A, c.930 G>C, c.1180 G>A 1%

**Table 8.** Coexistence more than one *PRKN* point mutations in PD patients in Polish population (Polrolniczak et al.,

It is suggested, that single or multiple exon deletions and duplications occur with a frequency of 15.8% and account for about 50% of all mutations of *PRKN* gene (Nuytemans et al., 2010). Nevertheless, althought many reports indicated important role of *PRKN* exons 2 and 4 deletions in pathogenesis of idiopatic PD (Choi et al., 2008; Guo et al., 2010; Macedo et al., 2009; Pankratz et al., 2009) in the study in Polish population it has not detected any deletion of exon 2 and 4 in *PRKN* gene as opposed to the German and Japan population, as well as the results obtained in the multipopulation study (Cookson et al., 2008; Nishioka et al., 2009; Polrolniczak et al., 2011; 2012; Shapira et al., 2002). On the other hand, our results were consistent with the study by Kruger et al. (1999), Sinha et al. (2005), as well Barsottini et al. (2011). However, it not be ruled out, that Polish patients have deletion of other not tested exons. Oliveri et al. (2001) suggested that deletion mutations of *PRKN* were not as common in LOPD as in EOPD. Therefore, it seems that copy number variation of *PRKN* is most probably related

However, point mutations in *PRKN* gene although they are characteristic for EOPD, currently it is suggested that it can be also involved in the pathogenesis of LOPD. However, studies utilizing common mutations and polymorphisms in tests for association with LOPD have produced mixed results (Hu et al., 2000; Oliveri et al., 2001; Satoh & Kuroda, 1999; Wang et al.,

Furthermore there is no question that Parkin-associated parkinsonism is recessive; that is, both alleles are mutant, but despite previous reports whether a heterozygous mutation can cause or increase the risk for PD remains an issue of debate (Farrer et al., 2001; Klein et al., 2000;

In the German population the frequency of *PRKN* mutations was 9% (Kann et al., 2002), in Brazilian population 8% (Periquet et al., 2001), and in the American population reached value of less than 4% (Chen et al., 2003) while in the Japanese population reached 66% (Hattori et al., 1998). In Polish population it has been showed small share of *PRKN* mutation in the patho‐ genesis of EOPD (Dawson & Dawson, 2003) while our study in LOPD have shown, that *PRKN* mutation in Polish population occurred with frequency 20,6% (Polrolniczak et al., 2011; 2012)

Moreover, we showed, that in the Polish population the most frequently were polymorphisms c.500 G>A, c.1180 G>A and c.930 G>C of *PRKN*. Simultaneously, it appears that these poly‐ morphisms may have incomplete penetration or lead to preclinical changes in the CNS and

what was similar to SPD in European population (Nishioka et al., 2009).

2012).

12 A Synopsis of Parkinson's Disease

1999).

with EOPD (Wang et al., 2004).

Lucking et al., 2001; Maruyama et al., 2000).

It is suggested, that haploinsufficiency may be considered as a reduction of normal gene expression accompanied by a loss of normal protein activity. Moreover, a lot of reports indicate to the existence of a second, undetected mutation in these patients, perhaps in the promoter or intronic regions (Giasson & Lee, 2001).

Our results, also suggests that the presence more than one heterozygous mutation in the *PRKN* gene may be necessary to PD manifestation. This hypothesis was first proposed by Abbas et al. (1999) moreover, later reviews generally assume the existence of a second, undetected mutation (Giasson & Lee, 2001). In our study also it is probably that patient who had one mutation in *PRKN* may have more genetic changes in not tested region of the gene so extension the studies of the other region of *PRKN* gene is necessary to clarify this issue. On the other hand it can not be ruled that one heterozygous mutation in *PRKN* may be sufficient to increase risk of PD and induce preclinical changes in *substantia nigra* (Khan et al., 2005).

Finally, it seems that clinically, PD patients with *PRKN* substitution generally are characterized by slower progression of the disease compared with PD patients without mutation. Moreover, it has been also observed, that in PD patients with *PRKN* mutations response to L-dopa therapy has been better than in PD patients without substitutions. This observation are generally consistent with the typical descriptions of *PRKN* patients which present slow disease progres‐ sion (Abbas et al., 1999; Lucking & Briece, 2000) and good response to L-dopa treatment although it have been showed that patients with *PRKN* mutation were more likely to develop treatment-induced motor complications earlier in the treatment (Khan et al., 2005; Lucking & Briece, 2000).

It seems, that point mutation in *PRKN* gene may be involved in the pathogenesis of LOPD and modulate clinical futures in this disease. It is also probably, that analysis of mutations in *PRKN* gene may be useful for diagnostic and prognostic process in PD.

#### **2.3. Mutations in** *HTRA2* **and** *SPR* **in the patients with Parkinson's disease**

It seems that presence of mutation in the other genes involved in the pathogenesis of PD like *SPR* (involved in dopamine biosynthesis) and *HTRA2* (involved with mitochondrial pathway of PD) probably may additionally affect the levels of ASN and Parkin through interaction with these proteins (Bogaerts et al., 2008; Karamohamed et al., 2003; Sharma et al., 2006; Sharma et al., 2011; Strauss et al., 2005). However, role of those genes in pathogenesis of PD is not enough known. The serine protease HTRA2 is localized to the inner membrane space of mitochondria (Suzuki et al., 2001). Mitochondrial dysfunction as well as ubiquitin–proteasome system damage has been proposed as possible mechanisms leading to dopaminergic neuronal degeneration (Lin & Beal, 2006; Malkus et al., 2009; Rubinsztein, 2006). Therefore *HTRA2* likewise *PRKN* may be included in mitochondrial pathway of PD independently of *PRKN* but that does not exclude the effects of dysfunction of *HTRA2* and *PRKN* may be additive.

**Factor Controls Patients with PD**

Mutations of PARK Genes and Alpha-Synuclein and Parkin Concentrations in Parkinson's Disease

http://dx.doi.org/10.5772/57107

15

**Table 9.** Demographic data of patients with PD and control subjects analyzed for *HTRA2* and *SPR* mutations. SD –

*Analysis of HTRA2 gene.* Exons 1 and 7 of *HTRA2* were amplified in 25 µl by PCR under the empirically defined conditions. Primers to amplify (on request) were generated using the online software Pimer3 based on the published genomic sequence of the *HTRA2* gene. PCR products were digested with MboII, MvaI and MspI restriction enzymes (Fermentas, Canada) for screening for c.421 G>T, c.1195 G>A and c.1210 C>T mutations (respectively) and analyzed

*Analysis of SPR gene*. Exon 3 of *SPR* gene were amplified using PCR with specific primers (5'- TCCATGTTCAGTGGGCTTTT-3'; 5'- TTTCTGGGCTGACACCTTG-3') generated with Primer3 software under the empirically defined conditions. Screening for the c.637 T>A and c.637 T>G mutations of *SPR* was performed by a RFLP analysis on 2.5% agarose gels using TaaI and SsiI (Fermentas, Canada) as restriction enzymes. All detected mutations were confirmed by sequencing of PCR product. Moreover, random duplicate samples (10%) were

In Polish population the presence of *HTRA2* point mutation was detected in 3% of PD patients (in 2% - c.1195 G>A resulting A141S substitution and in 1% c.421 G>T leads to G399S substi‐ tution) and none of controls (Table 10). However, c.1210 C>T mutation of *HTRA2* has not

**Mutation/polymorphism c.421 G>T c.1195 G>A c.1210 C>T Total substitutions Controls** 0% 0% 0% 0% **PD patients** 1% 2% 0% 3% **OR** - - - 9.080 **95% CI** - - - **p** >0.05 (F) >0.05 (F) >0.05 (F) =0.05 (F)

**Table 10.** *HTRA2* point mutations frequencies in PD patients and in controls. Results are expressed as a percentage. Logistic regression analysis and Fisher's exact test were used. OR – odds ratio; CI – confidence interval; F-Fisher's exact

genotyped for all assays for quality control with 100% reproducibility.

**Individuals** 113 89 **Age** 39-83 34-82 **Mean age ±SD** 55.5±9.5 62.0±10.1 **F/M** 79/34 41/47

standard deviation, F – female, M – male.

*Isolation of DNA*. See point 2.1.2

*Statistical analysis.* See point 2.2.2.

occurred both in PD patients and controls.

*2.3.2. Genetic investigations*

on 2.5% agarose gels.

*2.3.3. Results*

test.

Locus of *HTRA2* gene was recently assigned the PARK13 name, but the association of HTRA2 mutations and PD has not been confirmed in independent studies yet. In the study by Strauss et al. (2005) a single heterozygous *HTRA2* substitution (c.1195 G>A, Gly399Ser) was detected in four German patients with sporadic PD while another substitution (c.421 G>T, Ala141Ser) was more frequently found in PD than in controls. However, in a recent study by Simin-Sanchez & Singleton (2008) both variations were not associated with PD while in Belgian population it have been detected another substitution of *HTRA2* (c.1210 C>T, R404W) in sporadic PD (Bogaerts et al., 2008). These inconsistent findings raise a question about the role of mitochondrial *HTRA2* in PD susceptibility.

*SPR* gene is located in region covered by the locus PARK3 on chromosome 2p13 (Gasser et al., 1998), but the gene responsible for PD in PARK3 families has not yet been identified. One of the candidates is *SPR* gene. The study of Karamohamed et al. (2003) refined association to a region containing the *SPR* gene with PD, and it have been confirmed in further reports, but the study by Sharma et al. (2011) have not shown the association *SPR* and PD risk (Karamo‐ hamed et al., 2003; Sharma et al., 2006; 2011). However, authors emphasize varied genetic distributions between different populations (Sharma et al., 2011). It is known, that SPR is involved in dopamine synthesis and likewise ASN probably may be responsible for distur‐ bances in methabolisme of dopamine. The study of Tobin et al. (2007) has shown that expres‐ sion of *SPR* was significantly increased in PD patient compared with controls. However mutations in *SPR* in PD have not been analyzed so far. Moreover, it is known that phosphor‐ ylation of *SPR* increase sensitivity for protease activity and that in human SPR protein phosphorylated is only Ser213 (Fujimoto et al., 2002). Therefore, we decided to search for mutation in codon 213 of *SPR* in PD cases.

#### *2.3.1. Patients*

The studies were conducted on 89 patients with PD (10 EOPD patients, and 79 sporadic LOPD patients), including 41 women and 47 men aging 34-82 years. Control group included 113 individuals, 79 women and 34 men, 39-83 years of age. Demographic data of all groups summarized in Table 9.

Patients with PD were diagnosed using the criteria of UK Parkinson's Disease Society Brain Bank (Litvan et al., 2003), however stage of disease according to the scale of Hoehn and Yahr (Hoehn & Yahr, 1967).

None of the control subjects had verifiable symptoms of dementia or any other neurological disorders. All subjects had negative family history of PD. All patients were recruited from the Neurology Clinic of Chair and Department of Neurology, University of Medical Sciences, Poznan in Poland. Only Caucasian, Polish subjects were included in the study. A Local Ethical Committee approved the study and the written consent of all patients or their caregivers was obtained.


**Table 9.** Demographic data of patients with PD and control subjects analyzed for *HTRA2* and *SPR* mutations. SD – standard deviation, F – female, M – male.

#### *2.3.2. Genetic investigations*

likewise *PRKN* may be included in mitochondrial pathway of PD independently of *PRKN* but that does not exclude the effects of dysfunction of *HTRA2* and *PRKN* may be additive.

Locus of *HTRA2* gene was recently assigned the PARK13 name, but the association of HTRA2 mutations and PD has not been confirmed in independent studies yet. In the study by Strauss et al. (2005) a single heterozygous *HTRA2* substitution (c.1195 G>A, Gly399Ser) was detected in four German patients with sporadic PD while another substitution (c.421 G>T, Ala141Ser) was more frequently found in PD than in controls. However, in a recent study by Simin-Sanchez & Singleton (2008) both variations were not associated with PD while in Belgian population it have been detected another substitution of *HTRA2* (c.1210 C>T, R404W) in sporadic PD (Bogaerts et al., 2008). These inconsistent findings raise a question about the role

*SPR* gene is located in region covered by the locus PARK3 on chromosome 2p13 (Gasser et al., 1998), but the gene responsible for PD in PARK3 families has not yet been identified. One of the candidates is *SPR* gene. The study of Karamohamed et al. (2003) refined association to a region containing the *SPR* gene with PD, and it have been confirmed in further reports, but the study by Sharma et al. (2011) have not shown the association *SPR* and PD risk (Karamo‐ hamed et al., 2003; Sharma et al., 2006; 2011). However, authors emphasize varied genetic distributions between different populations (Sharma et al., 2011). It is known, that SPR is involved in dopamine synthesis and likewise ASN probably may be responsible for distur‐ bances in methabolisme of dopamine. The study of Tobin et al. (2007) has shown that expres‐ sion of *SPR* was significantly increased in PD patient compared with controls. However mutations in *SPR* in PD have not been analyzed so far. Moreover, it is known that phosphor‐ ylation of *SPR* increase sensitivity for protease activity and that in human SPR protein phosphorylated is only Ser213 (Fujimoto et al., 2002). Therefore, we decided to search for

The studies were conducted on 89 patients with PD (10 EOPD patients, and 79 sporadic LOPD patients), including 41 women and 47 men aging 34-82 years. Control group included 113 individuals, 79 women and 34 men, 39-83 years of age. Demographic data of all groups

Patients with PD were diagnosed using the criteria of UK Parkinson's Disease Society Brain Bank (Litvan et al., 2003), however stage of disease according to the scale of Hoehn and Yahr

None of the control subjects had verifiable symptoms of dementia or any other neurological disorders. All subjects had negative family history of PD. All patients were recruited from the Neurology Clinic of Chair and Department of Neurology, University of Medical Sciences, Poznan in Poland. Only Caucasian, Polish subjects were included in the study. A Local Ethical Committee approved the study and the written consent of all patients or their caregivers was

of mitochondrial *HTRA2* in PD susceptibility.

14 A Synopsis of Parkinson's Disease

mutation in codon 213 of *SPR* in PD cases.

*2.3.1. Patients*

summarized in Table 9.

(Hoehn & Yahr, 1967).

obtained.

#### *Isolation of DNA*. See point 2.1.2

*Analysis of HTRA2 gene.* Exons 1 and 7 of *HTRA2* were amplified in 25 µl by PCR under the empirically defined conditions. Primers to amplify (on request) were generated using the online software Pimer3 based on the published genomic sequence of the *HTRA2* gene. PCR products were digested with MboII, MvaI and MspI restriction enzymes (Fermentas, Canada) for screening for c.421 G>T, c.1195 G>A and c.1210 C>T mutations (respectively) and analyzed on 2.5% agarose gels.

*Analysis of SPR gene*. Exon 3 of *SPR* gene were amplified using PCR with specific primers (5'- TCCATGTTCAGTGGGCTTTT-3'; 5'- TTTCTGGGCTGACACCTTG-3') generated with Primer3 software under the empirically defined conditions. Screening for the c.637 T>A and c.637 T>G mutations of *SPR* was performed by a RFLP analysis on 2.5% agarose gels using TaaI and SsiI (Fermentas, Canada) as restriction enzymes. All detected mutations were confirmed by sequencing of PCR product. Moreover, random duplicate samples (10%) were genotyped for all assays for quality control with 100% reproducibility.

*Statistical analysis.* See point 2.2.2.

#### *2.3.3. Results*

In Polish population the presence of *HTRA2* point mutation was detected in 3% of PD patients (in 2% - c.1195 G>A resulting A141S substitution and in 1% c.421 G>T leads to G399S substi‐ tution) and none of controls (Table 10). However, c.1210 C>T mutation of *HTRA2* has not occurred both in PD patients and controls.


**Table 10.** *HTRA2* point mutations frequencies in PD patients and in controls. Results are expressed as a percentage. Logistic regression analysis and Fisher's exact test were used. OR – odds ratio; CI – confidence interval; F-Fisher's exact test.

In 213 codon of *SPR* gene novel mutation c.637 T>A was identified in 4% patients with PD and 2% controls (Table 11). This substitution is non-synonymous and leads to S213T changes in amino acid chain. However, we did not detected the second analyzed substitution c.637 C>G *SPR* in any of the subjects.

in patients with PD (Fig. 1). It seems that co-occurrence of point mutation of *PRKN* and polymorphism of *SNCA* promoter region may in additive manner increase risk of PD mani‐

Mutations of PARK Genes and Alpha-Synuclein and Parkin Concentrations in Parkinson's Disease

**Figure 1.** The frequency of NACP-Rep1 region of *SNCA* promoter variants in subjects with *PRKN* mutations (PD pa‐

Furthermore, in the patients with PD we demonstrated coexistence of point mutations in *PRKN* and *SPR* or *PRKN* and *HTRA2* genes (Table 12). However, in controls, coexistence of mutations *PRKN* and *SPR* have been observed also in one person. Therefore it seems that in patients with mutation of *PRKN* and *HTRA2* genes, simultaneous incorrect function of two proteins involved in the mitochondria proper functioning (HTRA2 and Parkin) may additionally increase risk

25 PD patient c.500 G>A - c.637 T>A 114 PD patient c.930 G>C c.1195 G>A - 202 PD patient c.930 G>C c.421 G>T - 18 Control c.930 G>C - c.637 T>A

**Table 12.** Coexistence of mutations in more than one analyzed gene in PD patients and controls.

**3. Role of alpha-synuclein in pathogenesis of Parkinson's disease**

Alpha-synuclein is a protein composed of 140 amino acids and is a part of family of proteins with the β- and γ-synuclein (Clayton & George, 1998). For many years, the structure of ASN was determined as the,,not-folded" chain of amino acids, taking the helical form only in conjunction with the lipids of cell membranes. It was thought that the ASN is a monomer form but the recent studies have shown that under physiological conditions ASN largely takes the

**Substitutions**

*PRKN HTRA2 SPR*

0/+1 +1/+1 +1/+2 +2/+2 +2/+3

http://dx.doi.org/10.5772/57107

17

festation.

tients and controls).

of PD manifestation.

**number Group**

**Probe**


**Table 11.** *SPR* point mutations frequencies in PD patients and in controls. Results are expressed as a percentage. Logistic regression analysis and Fisher's exact test were used. OR – odds ratio; CI – confidence interval; F-Fisher's exact test.

In PD patients with substitutions in *HTRA2* gene it have been observed slower progression of the disease wherein there was statistically significant association (Spearman correlation test) transition c.1195 G>A with decrease stage of disease (p=0.029; r=-0.237) but association of c.421 G>T substitution have not been significant and have remained at the level of trend. Further‐ more it have been shown, that using in PD patients with *HTRA2* mutations doses of L-dopa were lower than in patients without mutations and the response to therapy was better in presence of substitution. Finally, it seems that identified *HTRA2* mutations may be one of PD risk factor, especially since Strauss et al. (2005) demonstrated the presence of olfactory dysfunction in asymptomatic *HTRA2* mutation carrier.

Moreover, it seems that mutation c.637 T>A, because of localization, probably may affect phosphorylation of SR and thereby its activity and finally regulate biosynthesis of DA and serotonin (5-HT). However, analysis of expression and functional testing are necessary to explain importance and role of this mutation. Nevertheless, what is important, in our study c. 637 T>A *SPR* mutation has been significantly associated in Spearman correlation test, with the presence of depressive symptoms in PD patients (p<0.0001; r=0.371) probably by regulating the level of 5-HT (McHugh et al., 2009). Simultaneously, the presence of c.637 T>A of *SPR* mutation in PD patients have not been associated with differences in progression of the disease, response to L-dopa therapy, amount using L-dopa dose or presence of dementia compared to PD patients without *SPR* mutation.

#### **2.4. Coexistence of mutations in more than one gene (***SNCA, PRKN***,** *HTRA2* **and** *SPR)* **in the patients with Parkinson's disease**

Our study indicated, that in PD patients as well as in controls in the Polish population, *PRKN* mutations most frequently accompanied by the presence of genotype +1/+2. Interesting the coexistence of mutations *PRKN* with genotypes +2/+2 and +2/+3 have been demonstrated only in patients with PD (Fig. 1). It seems that co-occurrence of point mutation of *PRKN* and polymorphism of *SNCA* promoter region may in additive manner increase risk of PD mani‐ festation.

In 213 codon of *SPR* gene novel mutation c.637 T>A was identified in 4% patients with PD and 2% controls (Table 11). This substitution is non-synonymous and leads to S213T changes in amino acid chain. However, we did not detected the second analyzed substitution c.637 C>G

> **Mutation/polymorphism c.637 T>A c.637 C>G Controls** 2% 0% **PD patients** 4% 0% **OR** - - **95% CI** - -

**Table 11.** *SPR* point mutations frequencies in PD patients and in controls. Results are expressed as a percentage. Logistic regression analysis and Fisher's exact test were used. OR – odds ratio; CI – confidence interval; F-Fisher's exact

dysfunction in asymptomatic *HTRA2* mutation carrier.

PD patients without *SPR* mutation.

**patients with Parkinson's disease**

In PD patients with substitutions in *HTRA2* gene it have been observed slower progression of the disease wherein there was statistically significant association (Spearman correlation test) transition c.1195 G>A with decrease stage of disease (p=0.029; r=-0.237) but association of c.421 G>T substitution have not been significant and have remained at the level of trend. Further‐ more it have been shown, that using in PD patients with *HTRA2* mutations doses of L-dopa were lower than in patients without mutations and the response to therapy was better in presence of substitution. Finally, it seems that identified *HTRA2* mutations may be one of PD risk factor, especially since Strauss et al. (2005) demonstrated the presence of olfactory

Moreover, it seems that mutation c.637 T>A, because of localization, probably may affect phosphorylation of SR and thereby its activity and finally regulate biosynthesis of DA and serotonin (5-HT). However, analysis of expression and functional testing are necessary to explain importance and role of this mutation. Nevertheless, what is important, in our study c. 637 T>A *SPR* mutation has been significantly associated in Spearman correlation test, with the presence of depressive symptoms in PD patients (p<0.0001; r=0.371) probably by regulating the level of 5-HT (McHugh et al., 2009). Simultaneously, the presence of c.637 T>A of *SPR* mutation in PD patients have not been associated with differences in progression of the disease, response to L-dopa therapy, amount using L-dopa dose or presence of dementia compared to

**2.4. Coexistence of mutations in more than one gene (***SNCA, PRKN***,** *HTRA2* **and** *SPR)* **in the**

Our study indicated, that in PD patients as well as in controls in the Polish population, *PRKN* mutations most frequently accompanied by the presence of genotype +1/+2. Interesting the coexistence of mutations *PRKN* with genotypes +2/+2 and +2/+3 have been demonstrated only

**p** >0.05 (F) >0.05 (F)

*SPR* in any of the subjects.

16 A Synopsis of Parkinson's Disease

test.

**Figure 1.** The frequency of NACP-Rep1 region of *SNCA* promoter variants in subjects with *PRKN* mutations (PD pa‐ tients and controls).

Furthermore, in the patients with PD we demonstrated coexistence of point mutations in *PRKN* and *SPR* or *PRKN* and *HTRA2* genes (Table 12). However, in controls, coexistence of mutations *PRKN* and *SPR* have been observed also in one person. Therefore it seems that in patients with mutation of *PRKN* and *HTRA2* genes, simultaneous incorrect function of two proteins involved in the mitochondria proper functioning (HTRA2 and Parkin) may additionally increase risk of PD manifestation.


**Table 12.** Coexistence of mutations in more than one analyzed gene in PD patients and controls.

## **3. Role of alpha-synuclein in pathogenesis of Parkinson's disease**

Alpha-synuclein is a protein composed of 140 amino acids and is a part of family of proteins with the β- and γ-synuclein (Clayton & George, 1998). For many years, the structure of ASN was determined as the,,not-folded" chain of amino acids, taking the helical form only in conjunction with the lipids of cell membranes. It was thought that the ASN is a monomer form but the recent studies have shown that under physiological conditions ASN largely takes the

form of tetramers, and may take the helical form without connection to the lipid membrane (Bartels et al., 2011).

**3.1. Alpha-synuclein concentration in Parkinson's disease**

Demographic data of all groups summarized in Table 13.

and in control group.

any other neurological disorders.

included in the study.

caregivers was obtained.

deviation, F – female, M – male.

*3.1.1. Patients*

It has been shown that aggregation of the ASN may be caused among others by multiplication of *SNCA* gene. Furthermore, it has been shown that triplication of *SNCA* gene leads to twofold increase of ASN level, while duplication of *SNCA* gene increases the level of this protein one and a half-fold (Farrer et al., 2001; Singleton et al., 2003). Therefore it is believed that increased level of ASN may be related with PD manifestation (Farrer et al., 2001; Mata et al., 2004). It is also known that over-expression of ASN in neuron facilitates aggregation of this protein even in the presence of the correct structure of ASN. Moreover, elevated expression of *SNCA*-mRNA levels have been found in the affected regions of PD brain (Chiba-Falek et al., 2006). Increased of ASN level has been also associated with progress and worsening of the disease symptoms (Singleton et al., 2003). However, there are only few reports investigating the level of ASN in the blood of PD patients (Bialek et al., 2011; Fuchs et al., 2008; Lee et al., 2006).

Mutations of PARK Genes and Alpha-Synuclein and Parkin Concentrations in Parkinson's Disease

http://dx.doi.org/10.5772/57107

19

The aim of the study was to estimate the concentration of ASN in plasma of patients with PD

The studies were conducted on 32 patients with PD, including 18 women and 14 men aging 35-82 years. Control group included 24 individuals, 20 women and 4 men, 40-69 years of age.

Patients with PD were diagnosed using the criteria of UK Parkinson's Disease Society Brain Bank (Litvan et al., 2003), however stage of disease according to the scale of Hoehn and Yahr (Hoehn & Yahr, 1967). None of the control subjects had verifiable symptoms of dementia or

All patients were recruited from the Neurology Clinic of Chair and Department of Neurology, University of Medical Sciences, Poznan in Poland. Only Caucasian, Polish subjects were

A Local Ethical Committee approved the study and the written consent of all patients or their

**Factor Controls Patients with PD**

**Table 13.** Demographic data of patients with PD and control subjects analyzed for ASN concentrations. SD – standard

**Individuals** 24 32 **Age** 40-69 35-82 **Mean age ±SD** 55.3±6.8 62.5±10.5 **F/M** 20/4 18/14

Immunohistochemical studies have shown that in the cells, there is essentially ASN bonded to both the nuclear membrane, and in the synaptic vesicles (Totterdel & Meredith, 2005). To a lesser extent, ASN occurs in the free form in the cytoplasm.

Functions of ASN are not fully understood, however, due to cellular location of this protein it is suggested, that function of ASN may be related with the synaptic transport (Alim et al., 2002). There are also reports indicating that ASN participate in the process of differentiation and survival of the dopaminergic neuron progenitor cells of the mouse and human (Michell et al., 2007; Schneider et al., 2007).

Under pathological conditions ASN may change the structure and take the form of beta harmonica, what may lead to aggregation of ASN and formation of soluble oligomers, and then the insoluble filaments and deposits in the nerve cells (Bodles et al., 2001). As it have been shown, ASN is one of the main components of Lewy's bodies (LB), pathology, round or polymorphonuclear cellular inclusions in the cytoplasm of nerve cells. Moreover, it is sug‐ gested, that the formation of insoluble deposits of ASN and the aggregation process may give rise to the formation of LB (Halliday et al., 2006).

It is obvious that the process of aggregation of the ASN is a negative phenomenon for neural cells not only because of the high toxicity of the resulting aggregates, but also because of the ASN physiological function disorders caused by the reduction of bioavailability of this protein (Conway et al., 2000). It has been shown, that in PD, the process of ASN aggregation may be modulated by a number factors (Fig. 2) [Haggerty et al., 2011; Li et al., 2008; Ren et al., 2009; Sherer et al., 2002].

**Figure 2.** Selected factors affect alpha-synuclein (ASN) level.

#### **3.1. Alpha-synuclein concentration in Parkinson's disease**

It has been shown that aggregation of the ASN may be caused among others by multiplication of *SNCA* gene. Furthermore, it has been shown that triplication of *SNCA* gene leads to twofold increase of ASN level, while duplication of *SNCA* gene increases the level of this protein one and a half-fold (Farrer et al., 2001; Singleton et al., 2003). Therefore it is believed that increased level of ASN may be related with PD manifestation (Farrer et al., 2001; Mata et al., 2004). It is also known that over-expression of ASN in neuron facilitates aggregation of this protein even in the presence of the correct structure of ASN. Moreover, elevated expression of *SNCA*-mRNA levels have been found in the affected regions of PD brain (Chiba-Falek et al., 2006). Increased of ASN level has been also associated with progress and worsening of the disease symptoms (Singleton et al., 2003). However, there are only few reports investigating the level of ASN in the blood of PD patients (Bialek et al., 2011; Fuchs et al., 2008; Lee et al., 2006).

The aim of the study was to estimate the concentration of ASN in plasma of patients with PD and in control group.

#### *3.1.1. Patients*

form of tetramers, and may take the helical form without connection to the lipid membrane

Immunohistochemical studies have shown that in the cells, there is essentially ASN bonded to both the nuclear membrane, and in the synaptic vesicles (Totterdel & Meredith, 2005). To a

Functions of ASN are not fully understood, however, due to cellular location of this protein it is suggested, that function of ASN may be related with the synaptic transport (Alim et al., 2002). There are also reports indicating that ASN participate in the process of differentiation and survival of the dopaminergic neuron progenitor cells of the mouse and human (Michell

Under pathological conditions ASN may change the structure and take the form of beta harmonica, what may lead to aggregation of ASN and formation of soluble oligomers, and then the insoluble filaments and deposits in the nerve cells (Bodles et al., 2001). As it have been shown, ASN is one of the main components of Lewy's bodies (LB), pathology, round or polymorphonuclear cellular inclusions in the cytoplasm of nerve cells. Moreover, it is sug‐ gested, that the formation of insoluble deposits of ASN and the aggregation process may give

It is obvious that the process of aggregation of the ASN is a negative phenomenon for neural cells not only because of the high toxicity of the resulting aggregates, but also because of the ASN physiological function disorders caused by the reduction of bioavailability of this protein (Conway et al., 2000). It has been shown, that in PD, the process of ASN aggregation may be modulated by a number factors (Fig. 2) [Haggerty et al., 2011; Li et al., 2008; Ren et al., 2009;

lesser extent, ASN occurs in the free form in the cytoplasm.

(Bartels et al., 2011).

18 A Synopsis of Parkinson's Disease

Sherer et al., 2002].

et al., 2007; Schneider et al., 2007).

rise to the formation of LB (Halliday et al., 2006).

**Figure 2.** Selected factors affect alpha-synuclein (ASN) level.

The studies were conducted on 32 patients with PD, including 18 women and 14 men aging 35-82 years. Control group included 24 individuals, 20 women and 4 men, 40-69 years of age. Demographic data of all groups summarized in Table 13.

Patients with PD were diagnosed using the criteria of UK Parkinson's Disease Society Brain Bank (Litvan et al., 2003), however stage of disease according to the scale of Hoehn and Yahr (Hoehn & Yahr, 1967). None of the control subjects had verifiable symptoms of dementia or any other neurological disorders.

All patients were recruited from the Neurology Clinic of Chair and Department of Neurology, University of Medical Sciences, Poznan in Poland. Only Caucasian, Polish subjects were included in the study.

A Local Ethical Committee approved the study and the written consent of all patients or their caregivers was obtained.


**Table 13.** Demographic data of patients with PD and control subjects analyzed for ASN concentrations. SD – standard deviation, F – female, M – male.

#### *3.1.2. Analysis of ASN concentrations*

*Preparation of samples.* Blood samples from these subjects were drawn using EDTA as an anticoagulant in the morning after an overnight fast and the samples were centrifuge for 15 min at 1000xg at 4° C within 30 min and plasma was frozen at −80° C for later use.

**Duration of disease ASN concentrations in PD patients [pg/ml]**

Mutations of PARK Genes and Alpha-Synuclein and Parkin Concentrations in Parkinson's Disease

http://dx.doi.org/10.5772/57107

21

< 5 years [55.2-293.67] 5-10 years [1294.9] >10 years -

**Table 16.** Detectable (above 0.058 pg/ml in ELISA test) concentrations of ASN in PD patients depending on duration of the disease. Results are expressed as an interval between the minimum and maximum measurement/single result.

In this study and Bialek et al. (2011) have been shown higher concentration of plasma ASN level in PD patients as compared to controls. However, it seems that aggregation of the ASN in the nerve cells may reduce ASN ability to pass through the blood-brain barrier, which in turn may result in significantly reduced levels of this protein in the peripheral blood. More‐ over, a high concentration of ASN has been detected only in the initial period of PD (in two first stages of PD progress in Hoehn and Yahr scale, and in the first ten years of the disease), probably even before the accumulation of deposits in the form of LB in the brain of PD patients. However, in the study by Pchelina et al. (2011) the level of ASN was significantly lower in patients with *LRRK2*-associated PD compared with SPD and controls what may be caused also

Parkin is a cytoplasmic protein which plays a vital role in the proper functioning of the mitochondria and functions as an E3 ligase ubiquitin stimulating protein binding (directed to degradation in the proteasome) with ubiquitin, consequently preventing the cell apoptosis (Zhang et al., 2000). Ubiquitination is a vital cellular quality control mechanism that prevents accumulation of misfolded and damaged proteins in the cell. It is thought that substrates of Parkin include among others synphilin-1, ASN, CDC-rel1, cyclin E, p38 tRNA synthase, Pael-R and synaptotagmin XI. It has been shown in the study by Zhang et al, [2000] Parkin is also

Recent studies have shown that Parkin may play a role in decision-making, choosing between two systems of degradation: the proteasome activity (through its ability to promote ubiquiti‐ nation K48 associated with the proteasome) and macroautophagy (through K63 ubiquitination

The aim of the study was to estimate the concentration of Parkin in plasma of patients with

related to cell signaling and the formation of LB) [Henn et al., 2007; Lim et al., 2006].

by severed ASN aggregation in this group.

**4.1. Parkin concentration in Parkinson's disease**

PD and in control group.

Patients (see point 3.1.1.)

Analysis of Parkin concentrations

**4. Role of Parkin in pathogenesis of Parkinson's disease**

responsible for their own ubiquitination and degradation in the proteasome.

*Determination of ASN concentration.* ASN ELISA was performed using the Enzyme-linked Immunosorbent Assay Kit for Human Synuclein Alpha (Uscn Life Science Inc., China) according to the manufacturer's protocol. The minimal detection limits in this assay is typically less than 4.8 pg/ml. The standard curve concentrations used were 1000; 5000; 250; 125; 62.5; 31.2 and 15.6 pg/ml. The intra- and interassay precision of coefficiences of variation were <10% and <12% respectively. After completion of each assay the plate(s) were read at 450 nm on an EPOCH Multi-Volume Spectrophotometer (BioTek, USA) and the results were analyzed using Gen5 2.1 Software (BioTek, USA).

#### *3.1.3. Results*

Detectable concentrations of ASN have been detected in higher percentage of controls than in PD patients. However, in patients with PD has been shown higher concentration of ASN (Table 14).


**Table 14.** ASN concentrations in the patients with PD and in control group. Results are expressed as an interval between the minimum and maximum measurement, n – number, % - of subjects; detectable concentrations of ASN – above 0.058 pg/ml in ELISA test.

In PD patients, the highest concentrations of ASN were present in two first stages of disease progress (Hoehn and Yahr scale) [Table 15] and in the first ten years of the disease (Table 16).


**Table 15.** Detectable (above 0.058 pg/ml in ELISA test) concentrations of ASN in PD patients depending on stage of disease in Hoehn and Yafr scale. Results are expressed as an interval between the minimum and maximum measurement/single result.


**Table 16.** Detectable (above 0.058 pg/ml in ELISA test) concentrations of ASN in PD patients depending on duration of the disease. Results are expressed as an interval between the minimum and maximum measurement/single result.

In this study and Bialek et al. (2011) have been shown higher concentration of plasma ASN level in PD patients as compared to controls. However, it seems that aggregation of the ASN in the nerve cells may reduce ASN ability to pass through the blood-brain barrier, which in turn may result in significantly reduced levels of this protein in the peripheral blood. More‐ over, a high concentration of ASN has been detected only in the initial period of PD (in two first stages of PD progress in Hoehn and Yahr scale, and in the first ten years of the disease), probably even before the accumulation of deposits in the form of LB in the brain of PD patients. However, in the study by Pchelina et al. (2011) the level of ASN was significantly lower in patients with *LRRK2*-associated PD compared with SPD and controls what may be caused also by severed ASN aggregation in this group.

## **4. Role of Parkin in pathogenesis of Parkinson's disease**

Parkin is a cytoplasmic protein which plays a vital role in the proper functioning of the mitochondria and functions as an E3 ligase ubiquitin stimulating protein binding (directed to degradation in the proteasome) with ubiquitin, consequently preventing the cell apoptosis (Zhang et al., 2000). Ubiquitination is a vital cellular quality control mechanism that prevents accumulation of misfolded and damaged proteins in the cell. It is thought that substrates of Parkin include among others synphilin-1, ASN, CDC-rel1, cyclin E, p38 tRNA synthase, Pael-R and synaptotagmin XI. It has been shown in the study by Zhang et al, [2000] Parkin is also responsible for their own ubiquitination and degradation in the proteasome.

Recent studies have shown that Parkin may play a role in decision-making, choosing between two systems of degradation: the proteasome activity (through its ability to promote ubiquiti‐ nation K48 associated with the proteasome) and macroautophagy (through K63 ubiquitination related to cell signaling and the formation of LB) [Henn et al., 2007; Lim et al., 2006].

#### **4.1. Parkin concentration in Parkinson's disease**

The aim of the study was to estimate the concentration of Parkin in plasma of patients with PD and in control group.

Patients (see point 3.1.1.)

*3.1.2. Analysis of ASN concentrations*

20 A Synopsis of Parkinson's Disease

Gen5 2.1 Software (BioTek, USA).

above 0.058 pg/ml in ELISA test.

measurement/single result.

**Stage of disease according to**

*3.1.3. Results*

14).

*Preparation of samples.* Blood samples from these subjects were drawn using EDTA as an anticoagulant in the morning after an overnight fast and the samples were centrifuge for 15

*Determination of ASN concentration.* ASN ELISA was performed using the Enzyme-linked Immunosorbent Assay Kit for Human Synuclein Alpha (Uscn Life Science Inc., China) according to the manufacturer's protocol. The minimal detection limits in this assay is typically less than 4.8 pg/ml. The standard curve concentrations used were 1000; 5000; 250; 125; 62.5; 31.2 and 15.6 pg/ml. The intra- and interassay precision of coefficiences of variation were <10% and <12% respectively. After completion of each assay the plate(s) were read at 450 nm on an EPOCH Multi-Volume Spectrophotometer (BioTek, USA) and the results were analyzed using

Detectable concentrations of ASN have been detected in higher percentage of controls than in PD patients. However, in patients with PD has been shown higher concentration of ASN (Table

Detectable concentrations of ASN, n [%] 4 [17%] 4 [12%]

In PD patients, the highest concentrations of ASN were present in two first stages of disease progress (Hoehn and Yahr scale) [Table 15] and in the first ten years of the disease (Table 16).

**the scale of Hoehn and Yahr ASN concentrations in PD patients [pg/ml]**

1 [55.2] 2 [60.94-1294.9]

3 -

**Table 15.** Detectable (above 0.058 pg/ml in ELISA test) concentrations of ASN in PD patients depending on stage of disease in Hoehn and Yafr scale. Results are expressed as an interval between the minimum and maximum

**Table 14.** ASN concentrations in the patients with PD and in control group. Results are expressed as an interval between the minimum and maximum measurement, n – number, % - of subjects; detectable concentrations of ASN –

ASN concentrations [pg/ml] [68.19-645.57] [55.2-1294.9]

**Parameter Controls Patients with PD**

min at 1000xg at 4° C within 30 min and plasma was frozen at −80° C for later use.

Analysis of Parkin concentrations

*Preparation of samples.* Blood samples from these subjects were drawn using EDTA as an anticoagulant in the morning after an overnight fast and the samples were centrifuge for 20 min at 1000xg at 4° C within 30 min and plasma was frozen at −80° C for later use.

**Duration of disease Parkin concentrations in PD patients [ng/ml]**

Mutations of PARK Genes and Alpha-Synuclein and Parkin Concentrations in Parkinson's Disease

[0.076] mutation in 11 exon of *PRKN*

http://dx.doi.org/10.5772/57107

23

< 5 years [0.549-2.123 ] 5-10 years [0.158-2.054]

**Table 19.** Detectable (above 0.058 ng/ml in ELISA test) concentrations of Parkin in PD patients depending on duration of the disease. Results are expressed as an interval between the minimum and maximum measurement/single result.

It is known that dysfunction of Parkin may lead to manifestation of PD in several mechanism including mitochondrial and ubiquitination disturbances. It also seems that expression and cellular level of Parkin may be essential factor for proper function of this protein. However, the presence of Parkin protein has been demonstrated in human serum using Western blotting, there is few analysis of the level of this protein in the blood of PD patients (Dorszewska et al., 2012; Kasap et al., 2009). In the study by Dorszewska et al. (2012) has been detected lower plasma Parkin concentration in PD patients than in controls. Moreover, increased levels of the Parkin have been detected in PD patients in the early stages of PD (Hoehn and Yahr scale) and decreasing with the progress and duration of this disease. It seems that in the early stages of PD development may occur to increase of Parkin expression through the ongoing degenerative process and to the accumulation of pathological proteins-Parkin substrates. However, as the disease progresses, probably, resources of the Parkin running out and occurs weaken its

**5. Relationship between alpha-synuclein and Parkin levels in Parkinson's**

In 2001, Shimura et al. first described the presence in the human brain complex containing Parkin with the glycosylated form of the ASN (alpha-SP22), thus indicating the involvement of Parkin in ASN degradation in ubiquitin-proteasome system [Shimura et al., 2001; Chung et al., 2004]. It has been also shown that dysfunction of the Parkin can lead to ineffective elimi‐ nation of ASN and the aggregation of this protein [Haass & Kahle, 2001]. In addition, according to the reports, the Parkin may also interact with the dopamine and indirectly influence the aggregation of the ASN in the nerve cell (Oyama et al., 2010). Therefore, it seems that the levels

In patients with PD detectable levels of Parkin occurred in a nearly two-fold higher incidence

of these two proteins may be related and dependent on each other.

Analysis of ASN (see point 3.1.2.), and Parkin (see point 4.1.2.) concentrations

>10 years

neuroprotective function.

Patients (see point 3.1.1.)

than the ASN (Tables 14, 17).

**5.1. Results**

**disease**

*Determination of Parkin concentration.* Parkin ELISA was performed using the Enzyme-linked Immunosorbent Assay Kit for Human Parkinson Disease Protein 2 (Uscn Life Science Inc., China) according to the manufacturer's protocol. The minimal detection limits in this assay is typically less than 0.058 ng/ml. The standard curve concentrations used were 10; 5; 2.5; 1.25; 0.625; 0.312; and 0.156 ng/ml. The intra- and interassay precision of coefficiences of variation were <10% and <12% respectively. After completion of each assay the plate(s) were read at 450 nm on an EPOCH Multi-Volume Spectrophotometer (BioTek, USA) and the results were analyzed using Gen5 2.1 Software (BioTek, USA).

#### *4.1.1. Results*

Detectable concentrations of Parkin have been detected in similar percentage of controls and PD patients. However, in patients with PD has been shown lower concentration of Parkin (Table 17).


**Table 17.** Parkin concentrations in the patients with PD and control group. Results are expressed as an interval between the minimum and maximum measurement. n – number, % - of subjects; detectable concentrations of Parkin – above 0.058 ng/ml in ELISA test.

In PD patients, the highest concentration of Parkin occurred in 2 stage of disease progress with tendency to reduce the concentration in the 3 stage of the disease (Hoehn and Yahr scale) [Table 18] and in the first ten years of the disease (Table 19).


**Table 18.** Detectable (above 0.058 ng/ml in ELISA test) concentrations of Parkin in PD patients depending on stage of disease in Hoehn and Yahr scale. Results are expressed as an interval between the minimum and maximum measurement/single result.


**Table 19.** Detectable (above 0.058 ng/ml in ELISA test) concentrations of Parkin in PD patients depending on duration of the disease. Results are expressed as an interval between the minimum and maximum measurement/single result.

It is known that dysfunction of Parkin may lead to manifestation of PD in several mechanism including mitochondrial and ubiquitination disturbances. It also seems that expression and cellular level of Parkin may be essential factor for proper function of this protein. However, the presence of Parkin protein has been demonstrated in human serum using Western blotting, there is few analysis of the level of this protein in the blood of PD patients (Dorszewska et al., 2012; Kasap et al., 2009). In the study by Dorszewska et al. (2012) has been detected lower plasma Parkin concentration in PD patients than in controls. Moreover, increased levels of the Parkin have been detected in PD patients in the early stages of PD (Hoehn and Yahr scale) and decreasing with the progress and duration of this disease. It seems that in the early stages of PD development may occur to increase of Parkin expression through the ongoing degenerative process and to the accumulation of pathological proteins-Parkin substrates. However, as the disease progresses, probably, resources of the Parkin running out and occurs weaken its neuroprotective function.

## **5. Relationship between alpha-synuclein and Parkin levels in Parkinson's disease**

In 2001, Shimura et al. first described the presence in the human brain complex containing Parkin with the glycosylated form of the ASN (alpha-SP22), thus indicating the involvement of Parkin in ASN degradation in ubiquitin-proteasome system [Shimura et al., 2001; Chung et al., 2004]. It has been also shown that dysfunction of the Parkin can lead to ineffective elimi‐ nation of ASN and the aggregation of this protein [Haass & Kahle, 2001]. In addition, according to the reports, the Parkin may also interact with the dopamine and indirectly influence the aggregation of the ASN in the nerve cell (Oyama et al., 2010). Therefore, it seems that the levels of these two proteins may be related and dependent on each other.

Patients (see point 3.1.1.)

Analysis of ASN (see point 3.1.2.), and Parkin (see point 4.1.2.) concentrations

#### **5.1. Results**

*Preparation of samples.* Blood samples from these subjects were drawn using EDTA as an anticoagulant in the morning after an overnight fast and the samples were centrifuge for 20

*Determination of Parkin concentration.* Parkin ELISA was performed using the Enzyme-linked Immunosorbent Assay Kit for Human Parkinson Disease Protein 2 (Uscn Life Science Inc., China) according to the manufacturer's protocol. The minimal detection limits in this assay is typically less than 0.058 ng/ml. The standard curve concentrations used were 10; 5; 2.5; 1.25; 0.625; 0.312; and 0.156 ng/ml. The intra- and interassay precision of coefficiences of variation were <10% and <12% respectively. After completion of each assay the plate(s) were read at 450 nm on an EPOCH Multi-Volume Spectrophotometer (BioTek, USA) and the results were

Detectable concentrations of Parkin have been detected in similar percentage of controls and PD patients. However, in patients with PD has been shown lower concentration of Parkin

Detectable concentrations of Parkin, n [%] 5 [21%] 7 [22%]

In PD patients, the highest concentration of Parkin occurred in 2 stage of disease progress with tendency to reduce the concentration in the 3 stage of the disease (Hoehn and Yahr scale) [Table

**the scale of Hoehn and Yahr Parkin concentrations in PD patients [ng/ml]**

1 - 2 [0.158-2.123] 3 [0.076-0.409]

**Table 18.** Detectable (above 0.058 ng/ml in ELISA test) concentrations of Parkin in PD patients depending on stage of

disease in Hoehn and Yahr scale. Results are expressed as an interval between the minimum and maximum

**Table 17.** Parkin concentrations in the patients with PD and control group. Results are expressed as an interval between the minimum and maximum measurement. n – number, % - of subjects; detectable concentrations of Parkin

Parkin concentrations [ng/ml] [0.036-4.436] [0.076-2.123]

**Parameter Controls Patients with PD**

min at 1000xg at 4° C within 30 min and plasma was frozen at −80° C for later use.

analyzed using Gen5 2.1 Software (BioTek, USA).

18] and in the first ten years of the disease (Table 19).

**Stage of disease according to**

*4.1.1. Results*

22 A Synopsis of Parkinson's Disease

(Table 17).

– above 0.058 ng/ml in ELISA test.

measurement/single result.

In patients with PD detectable levels of Parkin occurred in a nearly two-fold higher incidence than the ASN (Tables 14, 17).


**6. Mutations in PARK (***PRKN, SPR, HTRA2, SNCA***) genes and ASN and**

Mutations of PARK Genes and Alpha-Synuclein and Parkin Concentrations in Parkinson's Disease

http://dx.doi.org/10.5772/57107

25

Our study on Polish population shown that in PD patients *PRKN* (exons 4, 8, 11) mutations were more than four times frequency as compared to controls. Moreover, in PD patients more frequently occurred genotypes +2/+2 and +2/+3 of the promoter region *SNCA* gene than in controls. In patients with PD shown higher concentration of ASN while higher Parkin level in controls. In PD patients without mutations in PARK, highest concentration of ASN and Parkin was present in two first stages of disease progress (Hoehn and Yahr scale) and in the first ten years of the disease. However, only in PD patient with mutation in 11 exon of *PRKN* gene shown presence of Parkin without ASN after ten years of disease duration (Table 19, 22).

It seems that analysis of these pathological proteins with PARK gene mutations may be useful

In Polish population, in *PRKN* gene point mutations occur few times more often in PD patients than controls. Control subjects tend to show higher level of plasma Parkin whereas patients suffering from PD tend to generate higher level of plasma ASN. In PD patients without point mutations in PRKN gene Parkin and ASN plasma levels increase until 2nd stage of disease in

Analysis of the variations of PARK gene as well as plasma levels of ASN and Parkin may

, Wojciech Kozubski2

1 Poznan University of Medical Sciences, Laboratory of Neurobiology Department of Neu‐

2 Poznan University of Medical Sciences, Chair and Department of Neurology, Poznan, Po‐

[1] Alim, M.A., Hossain, M.S., Arima, K., Takeda, K., Izumiyama, Y., Nakamura, M., Ka‐ ji, H., Shinoda, T., Hisanaga, S., & Ueda, K. (2002). Tubulin seeds alpha-synuclein fi‐

and Jolanta Dorszewska1

**Parkin concentrations in Parkinson's disease**

in the diagnostic and monitoring of the PD progress in the future.

Hoehn and Yahr scale and during first 10 years of disease.

, Margarita Lianeri1

consist an additional diagnostic factor for PD.

**7. Conclusion**

**Author details**

Anna Oczkowska1

land

**References**

rology, Poznan, Poland

**Table 20.** ASN and Parkin concentrations in the patients with PD and control group. Detectable concentrations of ASN and Parkin – above 0.058 pg/ml in ELISA test. Results are expressed as an interval between the minimum and maximum measurement.


**Table 21.** Detectable (above 0.058 pg/ml in ELISA test) concentrations of ASN and Parkin in PD patients depending on stage of disease in Hoehn and Yahr scale. Results are expressed as an interval between the minimum and maximum measurement/single result.


**Table 22.** Detectable (above 0.058 pg/ml in ELISA test) concentrations of ASN and Parkin in PD patients depending on duration of the disease. Results are expressed as an interval between the minimum and maximum measurement/ single result.

In this study and studies by Bialek et al. (2011) and Dorszewska et al. (2012) have been shown, that in PD patients increased level of ASN was associated with the decreased level of Parkin in contrast to control group (Tables 20-22). Independently for the analyzed group, the highest levels of ASN have been observed in the subjects who had very low Parkin levels. It suggested that low concentration of Parkin may contribute to increased ASN level in the nerve cells and combined with over-expression of ASN intensify or accelerate neurodegenerative process. Moreover, it has been also shown that configuration: increased plasma level of ASN and decreased of Parkin was associated with earlier onset of this disease.

## **6. Mutations in PARK (***PRKN, SPR, HTRA2, SNCA***) genes and ASN and Parkin concentrations in Parkinson's disease**

Our study on Polish population shown that in PD patients *PRKN* (exons 4, 8, 11) mutations were more than four times frequency as compared to controls. Moreover, in PD patients more frequently occurred genotypes +2/+2 and +2/+3 of the promoter region *SNCA* gene than in controls. In patients with PD shown higher concentration of ASN while higher Parkin level in controls. In PD patients without mutations in PARK, highest concentration of ASN and Parkin was present in two first stages of disease progress (Hoehn and Yahr scale) and in the first ten years of the disease. However, only in PD patient with mutation in 11 exon of *PRKN* gene shown presence of Parkin without ASN after ten years of disease duration (Table 19, 22).

It seems that analysis of these pathological proteins with PARK gene mutations may be useful in the diagnostic and monitoring of the PD progress in the future.

## **7. Conclusion**

**Parameter Controls Patients with PD**

ASN concentration [pg/ml] [68.19-645.57] [55.2-1294.9] Parkin concentration [pg/ml] [36.0-4436.0] [76.0-2123.0]

**Table 20.** ASN and Parkin concentrations in the patients with PD and control group. Detectable concentrations of ASN and Parkin – above 0.058 pg/ml in ELISA test. Results are expressed as an interval between the minimum and

> **ASN concentrations in PD patients [pg/ml]**

1 [55.2] -

**Table 21.** Detectable (above 0.058 pg/ml in ELISA test) concentrations of ASN and Parkin in PD patients depending on stage of disease in Hoehn and Yahr scale. Results are expressed as an interval between the minimum and maximum

**Table 22.** Detectable (above 0.058 pg/ml in ELISA test) concentrations of ASN and Parkin in PD patients depending on duration of the disease. Results are expressed as an interval between the minimum and maximum measurement/

In this study and studies by Bialek et al. (2011) and Dorszewska et al. (2012) have been shown, that in PD patients increased level of ASN was associated with the decreased level of Parkin in contrast to control group (Tables 20-22). Independently for the analyzed group, the highest levels of ASN have been observed in the subjects who had very low Parkin levels. It suggested that low concentration of Parkin may contribute to increased ASN level in the nerve cells and combined with over-expression of ASN intensify or accelerate neurodegenerative process. Moreover, it has been also shown that configuration: increased plasma level of ASN and

**Duration of disease ASN concentrations in PD patients**

**[pg/ml]**

>10 years - [76.0]

decreased of Parkin was associated with earlier onset of this disease.

< 5 years [55.2-293.67] [549.0-2123.0 ] 5-10 years [1294.9] [158.0-2054.0]

2 [60.94-1294.9] [158.0-2123.0] 3 - [76.0-409.0]

**Parkin concentrations in PD patients [pg/ml]**

**Parkin concentrations in PD**

mutation in 11 exon of *PRKN*

**patients [pg/ml]**

maximum measurement.

24 A Synopsis of Parkinson's Disease

measurement/single result.

single result.

**Stage of disease according to the scale of Hoehn and Yahr**

> In Polish population, in *PRKN* gene point mutations occur few times more often in PD patients than controls. Control subjects tend to show higher level of plasma Parkin whereas patients suffering from PD tend to generate higher level of plasma ASN. In PD patients without point mutations in PRKN gene Parkin and ASN plasma levels increase until 2nd stage of disease in Hoehn and Yahr scale and during first 10 years of disease.

> Analysis of the variations of PARK gene as well as plasma levels of ASN and Parkin may consist an additional diagnostic factor for PD.

## **Author details**

Anna Oczkowska1 , Margarita Lianeri1 , Wojciech Kozubski2 and Jolanta Dorszewska1

1 Poznan University of Medical Sciences, Laboratory of Neurobiology Department of Neu‐ rology, Poznan, Poland

2 Poznan University of Medical Sciences, Chair and Department of Neurology, Poznan, Po‐ land

## **References**

[1] Alim, M.A., Hossain, M.S., Arima, K., Takeda, K., Izumiyama, Y., Nakamura, M., Ka‐ ji, H., Shinoda, T., Hisanaga, S., & Ueda, K. (2002). Tubulin seeds alpha-synuclein fi‐ bril formation. *Journal of Biological Chemistry*, Vol.277, No.3, (January 2002), pp. 2112-2117, ISSN 0021-9258

its aggregation and toxicity. *Journal of Neurochemistry*, Vol.78, No.2, (July 2001), pp.

Mutations of PARK Genes and Alpha-Synuclein and Parkin Concentrations in Parkinson's Disease

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27

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**Chapter 2**

**Sleep Disturbances in Patients with Parkinson's Disease**

Parkinson's disease (PD) is clinically characterized by motor symptoms such as rigidity, bradykinesia, resting tremor, and postural instability, which are caused by the degenera‐ tion of striatonigral dopaminergic neurons [1]. Reflecting recent advances in therapeutic options and management in motor disabilities in PD, non-motor symptoms have received considerable attention due to substantial evidence that shows their significant impact on quality of life. Although some non-motor symptoms can be effectively treated by dopami‐ nergic agents, their management and treatment remain clinically challenging [2]. Nonmotor symptoms include sleep disturbances, cognitive impairment, mood disorders, hyposmia, pain, and dysautonomia, among which sleep disturbances are the central issue when considering their impact on disease course and clinical correlation. The presence of sleep disorders in PD was first described by James Parkinson in his "An Essay on the

*– "In this stage, the sleep becomes much disturbed. The tremulous motion of the limbs occur during sleep, and augment until they*

Sleep disturbances in PD are common and multifactorial problems with a reported incidence ranging from approximately 40% to 90% [4-7]. Disease-related intrinsic causes include impairment in thalamocortical arousal and degeneration of the brainstem regulatory centers for sleep/wakefulness maintenance and REM sleep [8]. Other causes include nocturnal motor

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Keisuke Suzuki, Tomoyuki Miyamoto,

Masaoki Iwanami and Koichi Hirata

Additional information is available at the end of the chapter

Hiroaki Fujita, Yuji Watanabe,

http://dx.doi.org/10.5772/57320

Shaking Palsy" published in 1817 [3].

*awaken the patient, and frequently with much agitation and alarm" –*

**1. Introduction**

Masayuki Miyamoto, Ayaka Numao, Hideki Sakuta,


**Chapter 2**

## **Sleep Disturbances in Patients with Parkinson's Disease**

Keisuke Suzuki, Tomoyuki Miyamoto, Masayuki Miyamoto, Ayaka Numao, Hideki Sakuta, Hiroaki Fujita, Yuji Watanabe, Masaoki Iwanami and Koichi Hirata

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/57320

## **1. Introduction**

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[117] Zhang, Y., Gao, J., Chung, K.K., Huang, H., Dawson, V.L., & Dawson, T.M. (2000). Parkin functions as an E2-dependent ubiquitin- protein ligase and promotes the deg‐ radation of the synaptic vesicle-associated protein, CDCrel-1. *Proceedings of the Na‐ tional Academy of Sciences USA*, Vol.97, No.24, (November 2000), pp. 13354-13359,

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tober 2001), pp. 485-494, ISSN 1387-2877

1672-0733

38 A Synopsis of Parkinson's Disease

ISSN 0027-8424

Parkinson's disease (PD) is clinically characterized by motor symptoms such as rigidity, bradykinesia, resting tremor, and postural instability, which are caused by the degenera‐ tion of striatonigral dopaminergic neurons [1]. Reflecting recent advances in therapeutic options and management in motor disabilities in PD, non-motor symptoms have received considerable attention due to substantial evidence that shows their significant impact on quality of life. Although some non-motor symptoms can be effectively treated by dopami‐ nergic agents, their management and treatment remain clinically challenging [2]. Nonmotor symptoms include sleep disturbances, cognitive impairment, mood disorders, hyposmia, pain, and dysautonomia, among which sleep disturbances are the central issue when considering their impact on disease course and clinical correlation. The presence of sleep disorders in PD was first described by James Parkinson in his "An Essay on the Shaking Palsy" published in 1817 [3].

*– "In this stage, the sleep becomes much disturbed. The tremulous motion of the limbs occur during sleep, and augment until they awaken the patient, and frequently with much agitation and alarm" –*

Sleep disturbances in PD are common and multifactorial problems with a reported incidence ranging from approximately 40% to 90% [4-7]. Disease-related intrinsic causes include impairment in thalamocortical arousal and degeneration of the brainstem regulatory centers for sleep/wakefulness maintenance and REM sleep [8]. Other causes include nocturnal motor

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

symptoms, psychiatric symptoms, dementia, medication use, circadian rhythm sleep disor‐ ders, and comorbidities involving sleep apnea syndrome (SAS), restless legs syndrome (RLS), and rapid eye movement sleep behavior disorder (RBD). Table 1 shows the various causes of sleep problems in PD; when patients complain of or are likely to suffer from sleep problems such as difficulty initiating or maintaining sleep, early awakening, or daytime sleepiness, all of the possibilities should be considered in clinical practice, but they are often underestimated. In this chapter, we review the current understanding of sleep problems in PD and the methods used to evaluate these problems, focusing on the PD sleep scale (PDSS) and PDSS-2.

## **2. Evaluation of sleep disturbance**

#### **2.1. Polysomnography**

Polysomnography is the gold standard in the assessment of sleep disorders because it provides sleep status information, including sleep efficiency, sleep latency, and sleep architecture. However, the cost, special equipment required, and limited availability may hamper its routine application. Sleep structure may be altered, reflecting disease-related changes in the brainstem in patients with PD: the degeneration of cholinergic neurons in the basal forebrain and brainstem, including the pedunculopontine nucleus and noradrenergic neurons in the locus coeruleus, results in disorders of REM sleep. A loss of serotoninergic neurons in the raphe nucleus is associated with a decreased percentage of slow-wave sleep [9] (Table 2). However, reported PSG findings on sleep architecture vary [10]. Reduced total sleep time and sleep efficiency were observed in PD patients compared with controls [11-13], although other studies did not find a difference. Reduced total sleep time was associated with increased age and increased levodopa dose [13], whereas the other study showed a weak positive correlation between the mean sleep latency and the daily dose of levodopa [14]. Sleep stages 1 and 2 appear to be unchanged in PD patients [15, 16]. Slow-wave sleep was reported to be unchanged, decreased, or increased [11, 17, 18]. REM sleep was also shorter or unchanged [11, 13, 19, 20].

These discrepancies may reflect individual night-to-night variation, medication effects, and differences in patient selection in the studies. PSG may detect abnormal REM sleep in PD, namely, REM sleep without atonia, excessive sustained or intermittent elevation of submental tone, or excessive phasic submental or limb twitching, which are required for a diagnosis of RBD. A recent review of case-control PSG studies provides a higher prevalence of RBD in PD than in controls (0-47% vs. 0-1.8%), but no significant increase of obstructive sleep apnea (27-60% vs. 13-65%) or periodic limb movements of sleep compared with controls [10]. In contrast, slightly but significantly increased periodic limb movements during sleep were described in patients with PD [21]. The clinical significance of sleep apnea in PD will be discussed later.

and slow-wave sleep and an increased amount of time spent awake were found in PD patients whose usual dopaminergic treatments were discontinued after noon and that the administra‐ tion of levodopa had no impact on any of these PSG parameters. In a study evaluating sleep status in de novo patients with PD, the sleep continuity, sleep architecture, and periodic limb movements index were similar between patients and controls, but an increased alpha activity in REM sleep was observed in de novo PD patients [16]. In another case control study including 15 de novo PD patients and 15 controls, compared with controls, the total sleep time and sleep efficiency in the de novo PD group decreased, the stage 1 sleep and the time spent awake increased, and REM sleep was reduced. A higher percentage of REM sleep without atonia in

Raphe nuclei Serotonin Regulation of slow-wave sleep Reduction of slow-wave sleep

**Nucleus/area Main transmitter Function Consequence of dysfunction**

nucleus Acetylcholine Regulation of REM sleep Disorders of REM sleep Locus coeruleus Noradrenaline Regulation of REM sleep Reduction/absence of REM sleep

motoneurons

Loss of muscle atonia during REM sleep

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**1. PD-related pathological changes**

**2. Nocturnal motor symptoms**

**4. Medication use**

Sleep apnea syndrome REM sleep behavior disorder

Pedunculopontine

Area peri-locus

**3. Nocturnal non-motor symptoms**

Dopaminergic drugs, anti-psychotics **5. Comorbid primary sleep disorders**

Impairment of the sleep-wake cycle, circadian rhythm sleep disorder, sundown syndrome

Wearing-off phenomenon, rigidity, akinesia, tremor, medication-related dyskinesia, dystonia

Impairment of the arousal system (orexin, serotonin, noradrenaline, acetylcholine)

Neuropsychiatric symptoms (depression, psychosis, cognitive impairment) Sensory symptoms (pain, dysesthesia, restlessness of the arms or legs)

Impairment of sleep architecture (REM and non-REM sleep)

Hallucinations, nightmares and vivid dreams, nocturia

Restless legs syndrome, periodic limb movement disorder

coeruleus GABA, glutamate? Inhibition of spinal

**Table 1.** The cause of sleep-related problems in PD

**Table 2.** Impaired sleep architecture in PD [9].

Regarding the effect of dopaminergic medication on the sleep architecture in PD, relative to controls, Wailke et al [18] reported that a significantly decreased total sleep time, REM sleep,


**Table 1.** The cause of sleep-related problems in PD

symptoms, psychiatric symptoms, dementia, medication use, circadian rhythm sleep disor‐ ders, and comorbidities involving sleep apnea syndrome (SAS), restless legs syndrome (RLS), and rapid eye movement sleep behavior disorder (RBD). Table 1 shows the various causes of sleep problems in PD; when patients complain of or are likely to suffer from sleep problems such as difficulty initiating or maintaining sleep, early awakening, or daytime sleepiness, all of the possibilities should be considered in clinical practice, but they are often underestimated. In this chapter, we review the current understanding of sleep problems in PD and the methods

Polysomnography is the gold standard in the assessment of sleep disorders because it provides sleep status information, including sleep efficiency, sleep latency, and sleep architecture. However, the cost, special equipment required, and limited availability may hamper its routine application. Sleep structure may be altered, reflecting disease-related changes in the brainstem in patients with PD: the degeneration of cholinergic neurons in the basal forebrain and brainstem, including the pedunculopontine nucleus and noradrenergic neurons in the locus coeruleus, results in disorders of REM sleep. A loss of serotoninergic neurons in the raphe nucleus is associated with a decreased percentage of slow-wave sleep [9] (Table 2). However, reported PSG findings on sleep architecture vary [10]. Reduced total sleep time and sleep efficiency were observed in PD patients compared with controls [11-13], although other studies did not find a difference. Reduced total sleep time was associated with increased age and increased levodopa dose [13], whereas the other study showed a weak positive correlation between the mean sleep latency and the daily dose of levodopa [14]. Sleep stages 1 and 2 appear to be unchanged in PD patients [15, 16]. Slow-wave sleep was reported to be unchanged, decreased, or increased [11, 17, 18]. REM sleep was also shorter or unchanged [11, 13, 19, 20].

These discrepancies may reflect individual night-to-night variation, medication effects, and differences in patient selection in the studies. PSG may detect abnormal REM sleep in PD, namely, REM sleep without atonia, excessive sustained or intermittent elevation of submental tone, or excessive phasic submental or limb twitching, which are required for a diagnosis of RBD. A recent review of case-control PSG studies provides a higher prevalence of RBD in PD than in controls (0-47% vs. 0-1.8%), but no significant increase of obstructive sleep apnea (27-60% vs. 13-65%) or periodic limb movements of sleep compared with controls [10]. In contrast, slightly but significantly increased periodic limb movements during sleep were described in patients with PD [21]. The clinical significance of sleep apnea in PD will be

Regarding the effect of dopaminergic medication on the sleep architecture in PD, relative to controls, Wailke et al [18] reported that a significantly decreased total sleep time, REM sleep,

used to evaluate these problems, focusing on the PD sleep scale (PDSS) and PDSS-2.

**2. Evaluation of sleep disturbance**

**2.1. Polysomnography**

40 A Synopsis of Parkinson's Disease

discussed later.


**Table 2.** Impaired sleep architecture in PD [9].

and slow-wave sleep and an increased amount of time spent awake were found in PD patients whose usual dopaminergic treatments were discontinued after noon and that the administra‐ tion of levodopa had no impact on any of these PSG parameters. In a study evaluating sleep status in de novo patients with PD, the sleep continuity, sleep architecture, and periodic limb movements index were similar between patients and controls, but an increased alpha activity in REM sleep was observed in de novo PD patients [16]. In another case control study including 15 de novo PD patients and 15 controls, compared with controls, the total sleep time and sleep efficiency in the de novo PD group decreased, the stage 1 sleep and the time spent awake increased, and REM sleep was reduced. A higher percentage of REM sleep without atonia in patients compared with controls was observed, whereas only one patient clinically manifested RBD [22]. Because RBD precedes the onset of PD and can manifest during the early phase of PD, abnormalities in REM sleep in the early phase of PD are supported by PSG findings.

an impaired total PDSS score in patients with PD compared with controls (112.8±25.4 vs. 126.6±17.8) and revealed more severe nocturnal disturbances measured by PDSS in advancedstage PD patients (HY stage 4) compared with those with early/moderate stages (HY stage 1-3) (Figure 1). With regard to the subitems in PDSS, compared with controls, almost all the subitems were significantly impaired in PD patients except for item 2 (sleep onset insomnia), item 11 (painful muscle cramp), and item 14 (refreshment after sleep), and both groups had the most severe ratings in item 3 (sleep maintenance insomnia) and item 8 (nocturia) (Figure 2). Patients with HY 4 had significantly worse scores on item 1 (quality of sleep), item 3 (sleep maintenance insomnia), item 6 (distressing dream), item 11 (painful muscle cramps), and item 15 (daytime dozing) than those with HY 1-3 (Figure 3). In addition, sleep disturbances as measured by the total PDSS score were associated with a longer disease duration, depressive symptoms, and complications in the dopaminergic treatment (dyskinesia and wearing off) [29]. When dividing the PD patients into patients with and without depressive symptoms, we found that patients with depressive symptoms (Zung Self-Rating Depression Scale, SDS score ≥ 40) had significantly impaired scores in almost all PDSS items except item 2 (difficulty in initiating sleep) and item 11 (painful muscle cramps) compared with patients without depressive symptoms and controls [31]. The lack of significant differences between controls and nondepressed patients in PDSS subitems suggests that depressive symptoms play an important role in nocturnal disturbances in PD (Figure 4). From a detailed evaluation of nocturnal symptoms, it was determined that early morning tremor (item 13) and nocturnal

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dystonia (item 12) were closely associated with depressive symptoms.

In contrast, untreated PD patients can manifest various nocturnal symptoms as assessed by PDSS, such as nocturia, nighttime cramps, dystonia, and tremor [32], emphasizing that nocturnal disturbances should be recognized and managed early even in the early stage of PD.

*\*p*<0.05, \*\* *p*<0.01. Bonferroni test after adjustment for age. PD patients with HY stage 4 had a lower score compared

with those with HY stages 1 to 3.

**Figure 1.** Total PDSS score classified by H&Y stage in patients with PD [29].

#### **2.2. Multiple sleep latency test**

The multiple sleep latency test (MSLT) records the initial sleep latency of four or five sequential naps to evaluate objective daytime sleepiness. In 54 levodopa-treated PD patients who were referred for sleepiness, pathological sleepiness (mean SL < 5 min) was observed in 50% of the 54 patients [14], and a narcolepsy-like phenotype (2≥ sleep-onset REM periods) was found in 39% of the patients. Additionally, Rye et al [23] found that although the mean SL was similar between patients with PD and controls, abnormal sleepiness (mean SL≤5 min) was common (40 of 134 nap opportunities), and sleep-onset REM periods were also observed (13 of 134 nap opportunities) in PD patients. In contrast, Yong et al [13] reported that mean SL did not differ between PD and controls. No differences in mean SL between the levodopa-alone group and the levodopa and dopamine agonist group were reported [20]. In untreated PD patients, the mean SL on MSLT was not different compared with controls (11.7±4 vs. 12.5±2 min) [22]. The mean SL was in the pathological range (<8 min) in three PD patients and in none of the controls, and one patient with PD had a single sleep onset REM on MSLT. This observation suggests that some but not all patients exhibit pathological daytime sleepiness as measured by MSLT, irrespective of the quality and amount of nighttime sleep. However, one should note that sleepiness measured by MSLT does not always reflect subjective sleepiness.

#### **2.3. Questionnaire-based assessment**

#### *2.3.1. Parkinson's disease sleep scale*

To comprehensively and clinically address PD's common, disease-specific sleep problems, Chaudhuri et al [24] have developed the Parkinson's disease sleep scale (PDSS), a visual analogue scale, including 15 PD-related nocturnal symptoms for assessing nocturnal disability in PD. The subitems of the PDSS address the follows: item 1, overall quality of the night's sleep; items 2/3, sleep onset and maintenance insomnia; items 4/5, nocturnal restlessness; items 6/7, nocturnal psychosis; items 8/9, nocturia; items 10-13, nocturnal motor symptoms; item 14, sleep refreshment; and item 15, daytime dozing. This scale measures the patient's subjective evaluation of sleep and does not address the frequency of sleep problems. Scores for a given individual item range from 0 to 10; 10 represents the best and 0 represents the worst score. The maximum total score for PDSS is 150 (patient is free of symptoms associated with sleep disorders).

In the original study [24], PD patients showed a significantly impaired (lower) total PDSS score compared with controls (101.1±21.7 vs. 120.7±21.0), and advanced PD patients (Hoehn and Yahr (HY) stage 4-5) had a lower total PDSS score than early/moderate PD patients (HY stage 1-3) and controls. This scale is now regarded as a recommended and reliable scale for screening and assessing sleep disturbance in PD [25]. The PDSS has been validated and used extensively in a number of countries, with a high reliability [26-30]. Our multicenter study also showed an impaired total PDSS score in patients with PD compared with controls (112.8±25.4 vs. 126.6±17.8) and revealed more severe nocturnal disturbances measured by PDSS in advancedstage PD patients (HY stage 4) compared with those with early/moderate stages (HY stage 1-3) (Figure 1). With regard to the subitems in PDSS, compared with controls, almost all the subitems were significantly impaired in PD patients except for item 2 (sleep onset insomnia), item 11 (painful muscle cramp), and item 14 (refreshment after sleep), and both groups had the most severe ratings in item 3 (sleep maintenance insomnia) and item 8 (nocturia) (Figure 2). Patients with HY 4 had significantly worse scores on item 1 (quality of sleep), item 3 (sleep maintenance insomnia), item 6 (distressing dream), item 11 (painful muscle cramps), and item 15 (daytime dozing) than those with HY 1-3 (Figure 3). In addition, sleep disturbances as measured by the total PDSS score were associated with a longer disease duration, depressive symptoms, and complications in the dopaminergic treatment (dyskinesia and wearing off) [29]. When dividing the PD patients into patients with and without depressive symptoms, we found that patients with depressive symptoms (Zung Self-Rating Depression Scale, SDS score ≥ 40) had significantly impaired scores in almost all PDSS items except item 2 (difficulty in initiating sleep) and item 11 (painful muscle cramps) compared with patients without depressive symptoms and controls [31]. The lack of significant differences between controls and nondepressed patients in PDSS subitems suggests that depressive symptoms play an important role in nocturnal disturbances in PD (Figure 4). From a detailed evaluation of nocturnal symptoms, it was determined that early morning tremor (item 13) and nocturnal dystonia (item 12) were closely associated with depressive symptoms.

patients compared with controls was observed, whereas only one patient clinically manifested RBD [22]. Because RBD precedes the onset of PD and can manifest during the early phase of PD, abnormalities in REM sleep in the early phase of PD are supported by PSG findings.

The multiple sleep latency test (MSLT) records the initial sleep latency of four or five sequential naps to evaluate objective daytime sleepiness. In 54 levodopa-treated PD patients who were referred for sleepiness, pathological sleepiness (mean SL < 5 min) was observed in 50% of the 54 patients [14], and a narcolepsy-like phenotype (2≥ sleep-onset REM periods) was found in 39% of the patients. Additionally, Rye et al [23] found that although the mean SL was similar between patients with PD and controls, abnormal sleepiness (mean SL≤5 min) was common (40 of 134 nap opportunities), and sleep-onset REM periods were also observed (13 of 134 nap opportunities) in PD patients. In contrast, Yong et al [13] reported that mean SL did not differ between PD and controls. No differences in mean SL between the levodopa-alone group and the levodopa and dopamine agonist group were reported [20]. In untreated PD patients, the mean SL on MSLT was not different compared with controls (11.7±4 vs. 12.5±2 min) [22]. The mean SL was in the pathological range (<8 min) in three PD patients and in none of the controls, and one patient with PD had a single sleep onset REM on MSLT. This observation suggests that some but not all patients exhibit pathological daytime sleepiness as measured by MSLT, irrespective of the quality and amount of nighttime sleep. However, one should note that

sleepiness measured by MSLT does not always reflect subjective sleepiness.

To comprehensively and clinically address PD's common, disease-specific sleep problems, Chaudhuri et al [24] have developed the Parkinson's disease sleep scale (PDSS), a visual analogue scale, including 15 PD-related nocturnal symptoms for assessing nocturnal disability in PD. The subitems of the PDSS address the follows: item 1, overall quality of the night's sleep; items 2/3, sleep onset and maintenance insomnia; items 4/5, nocturnal restlessness; items 6/7, nocturnal psychosis; items 8/9, nocturia; items 10-13, nocturnal motor symptoms; item 14, sleep refreshment; and item 15, daytime dozing. This scale measures the patient's subjective evaluation of sleep and does not address the frequency of sleep problems. Scores for a given individual item range from 0 to 10; 10 represents the best and 0 represents the worst score. The maximum total score for PDSS is 150 (patient is free of symptoms associated with sleep

In the original study [24], PD patients showed a significantly impaired (lower) total PDSS score compared with controls (101.1±21.7 vs. 120.7±21.0), and advanced PD patients (Hoehn and Yahr (HY) stage 4-5) had a lower total PDSS score than early/moderate PD patients (HY stage 1-3) and controls. This scale is now regarded as a recommended and reliable scale for screening and assessing sleep disturbance in PD [25]. The PDSS has been validated and used extensively in a number of countries, with a high reliability [26-30]. Our multicenter study also showed

**2.2. Multiple sleep latency test**

42 A Synopsis of Parkinson's Disease

**2.3. Questionnaire-based assessment**

*2.3.1. Parkinson's disease sleep scale*

disorders).

In contrast, untreated PD patients can manifest various nocturnal symptoms as assessed by PDSS, such as nocturia, nighttime cramps, dystonia, and tremor [32], emphasizing that nocturnal disturbances should be recognized and managed early even in the early stage of PD.

*<sup>\*</sup>p*<0.05, \*\* *p*<0.01. Bonferroni test after adjustment for age. PD patients with HY stage 4 had a lower score compared with those with HY stages 1 to 3.

**Figure 1.** Total PDSS score classified by H&Y stage in patients with PD [29].

PD patients had a lower score on almost all PDSS scores compared with controls except for items 2 (sleep onset insom‐ nia), 11 (painful muscle cramps), and 14 (tired and sleepy after waking).

Significant differences in PDSS subitems, except items 2 (sleep onset insomnia) and 11 (painful muscle cramps), are

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Trenkwalder et al have recently published the PDSS-2, a revised version of the PDSS, to address the frequency of nocturnal symptoms observed in PD patients [33]. A visual analog scale used in the original version of PDSS was transformed into a frequency measure in the PDSS-2. A question regarding daytime sleepiness (item 15 in PDSS) was removed because it reflects complex regulation in PD, and a screening question for SAS was added instead. The PDSS-2 consists of 15 questions about various sleep and nocturnal disturbances rated by the patients using one of five categories, from 0 (never) to 4 (very frequent). Unlike the PDSS, in which a lower score represents worse conditions in sleep, the PDSS-2 total score ranges from 0 (no disturbance) to 60 (maximum nocturnal disturbance) (Figure 5). The study showed satisfactory internal consistency, stability, construct validity, and precision for the PDSS-2 [33]. Nocturnal problems assessed by the PDSS-2 total score were correlated with impaired quality of life and motor impairment. The factor analysis of PDSS resulted in three domain scales: 1) motor problems at night; 2) PD symptoms at night, representing disease-specific symptoms; and 3) disturbed sleep, representing sleep-specific disturbances (Table 3). We have performed a validation study and confirmed the usefulness of the Japanese version of the PDSS-2 [34]. PD patients had significantly impaired scores for the PDSS-2 total score compared with control subjects (15.0±9.7 vs. 9.1±6.6, p<0.001) (Figure 6). Significant differences were found between PD patients and controls in three PDSS-2 domain scores and subscores (Figure 7). The PDSS-2 total score was correlated with the Pittsburgh Sleep Quality Index (PSQI), Epworth Sleepiness Scale (ESS), Beck Depression Inventory-II (BDI-II), Parkinson Fatigue Scale (PFS), Parkinson's Disease Questionnaire (PDQ-39) summary index, all of the PDQ-39 domains, and the Unified Parkinson's Disease Rating Scale part III (motor function). The PDSS-2 is simple and easy to use at the outpatient clinic or bedside and is suitable for assessing not only the current status

observed among depressed patients, nondepressed patients, and controls.

*2.3.2. Parkinson's disease sleep scale-2*

**Figure 4.** PDSS subitems in depressed PD, nondepressed PD, and controls [31].

**Figure 2.** PDSS subitems in patients with PD and controls [29].

<sup>\*</sup> *p*<0.05, \*\* *p*<0.01. One-way ANOVA followed by a Bonferroni test. Compared with patients with HY stages 1-3, PDSS subitems in patients with HY stage 4 were lower for difficulty staying asleep (item 3), nightmares (item 6), painful muscle cramps (item 11), and daytime sleepiness (item 15).

**Figure 3.** PDSS subitems classified by HY stage in patients with PD [29].

Significant differences in PDSS subitems, except items 2 (sleep onset insomnia) and 11 (painful muscle cramps), are observed among depressed patients, nondepressed patients, and controls.

**Figure 4.** PDSS subitems in depressed PD, nondepressed PD, and controls [31].

#### *2.3.2. Parkinson's disease sleep scale-2*

PD patients had a lower score on almost all PDSS scores compared with controls except for items 2 (sleep onset insom‐

\* *p*<0.05, \*\* *p*<0.01. One-way ANOVA followed by a Bonferroni test. Compared with patients with HY stages 1-3, PDSS subitems in patients with HY stage 4 were lower for difficulty staying asleep (item 3), nightmares (item 6), painful

nia), 11 (painful muscle cramps), and 14 (tired and sleepy after waking).

**Figure 2.** PDSS subitems in patients with PD and controls [29].

44 A Synopsis of Parkinson's Disease

muscle cramps (item 11), and daytime sleepiness (item 15).

**Figure 3.** PDSS subitems classified by HY stage in patients with PD [29].

Trenkwalder et al have recently published the PDSS-2, a revised version of the PDSS, to address the frequency of nocturnal symptoms observed in PD patients [33]. A visual analog scale used in the original version of PDSS was transformed into a frequency measure in the PDSS-2. A question regarding daytime sleepiness (item 15 in PDSS) was removed because it reflects complex regulation in PD, and a screening question for SAS was added instead. The PDSS-2 consists of 15 questions about various sleep and nocturnal disturbances rated by the patients using one of five categories, from 0 (never) to 4 (very frequent). Unlike the PDSS, in which a lower score represents worse conditions in sleep, the PDSS-2 total score ranges from 0 (no disturbance) to 60 (maximum nocturnal disturbance) (Figure 5). The study showed satisfactory internal consistency, stability, construct validity, and precision for the PDSS-2 [33]. Nocturnal problems assessed by the PDSS-2 total score were correlated with impaired quality of life and motor impairment. The factor analysis of PDSS resulted in three domain scales: 1) motor problems at night; 2) PD symptoms at night, representing disease-specific symptoms; and 3) disturbed sleep, representing sleep-specific disturbances (Table 3). We have performed a validation study and confirmed the usefulness of the Japanese version of the PDSS-2 [34]. PD patients had significantly impaired scores for the PDSS-2 total score compared with control subjects (15.0±9.7 vs. 9.1±6.6, p<0.001) (Figure 6). Significant differences were found between PD patients and controls in three PDSS-2 domain scores and subscores (Figure 7). The PDSS-2 total score was correlated with the Pittsburgh Sleep Quality Index (PSQI), Epworth Sleepiness Scale (ESS), Beck Depression Inventory-II (BDI-II), Parkinson Fatigue Scale (PFS), Parkinson's Disease Questionnaire (PDQ-39) summary index, all of the PDQ-39 domains, and the Unified Parkinson's Disease Rating Scale part III (motor function). The PDSS-2 is simple and easy to use at the outpatient clinic or bedside and is suitable for assessing not only the current status on sleep but also evaluating treatment response [35]. For further improvement in the PDSS-2, subitems for the screening for RLS (items 4 and 5) and RBD (item 6) may not be sufficient to differentiate those conditions from RLS mimics, psychosis, and delirium. Adding a subjective evaluation of severity for each item may be useful.


\**p*<0.001, Mann–Whitney U-test

**Figure 6.** PDSS-2 total score in PD patients and controls

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**Figure 7.** The mean PDSS-2 scores in the three domains between PD patients and controls


Total score ranges from 0 (no disturbance) to 60 (maximum nocturnal disturbance).

**Figure 5.** PDSS-2 (reproduced with permission from [33]).

\**p*<0.001, Mann–Whitney U-test

on sleep but also evaluating treatment response [35]. For further improvement in the PDSS-2, subitems for the screening for RLS (items 4 and 5) and RBD (item 6) may not be sufficient to differentiate those conditions from RLS mimics, psychosis, and delirium. Adding a subjective

evaluation of severity for each item may be useful.

46 A Synopsis of Parkinson's Disease

Total score ranges from 0 (no disturbance) to 60 (maximum nocturnal disturbance).

**Figure 5.** PDSS-2 (reproduced with permission from [33]).

**Figure 6.** PDSS-2 total score in PD patients and controls

**Figure 7.** The mean PDSS-2 scores in the three domains between PD patients and controls


than during the day [32]. Therefore, nocturnal motor symptoms can interfere with sleep and are not always parallel with daytime motor symptoms, which is supported by several studies showing a weak or nonexistent correlation between sleep disturbances and daytime motor symptoms (UPDRS motor score) [7, 38] and no correlation between nocturnal motor symptoms obtained by PDSS-2 and UPDRS motor scores [33]. In patients with an early to moderate stage of PD, a substantial number of patients may suffer from nocturnal problems; however, this

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An international cross-sectional study comprising 242 patients with PD (HY 2 (n=121) being most frequent) revealed that a significant number of sleep-related symptoms were undeclared by patients before the administration of the non-motor questionnaire: nocturia, 43.9%; daytime sleepiness, 52.4%; insomnia, 43.9%; vivid dreams, 52.4%; acting out during dreams, 44.1%; and restless legs, 36.4% [39]. Patients in the advanced stages of the disease have motor dysfunction throughout the day; therefore, its impact on the nighttime period should always be considered.

**Symptoms Experienced by (%) Most troublesome (%)**

Although nocturia is associated with normal aging, 80% of PD patients show two or more episodes of nocturia per night resulting from overflow incontinence and a spastic bladder [4].Urinary bladder–related symptoms, such as frequency, urgency, and urge incontinence, are common in PD, resulting in frequent nocturnal awakenings. In animal studies, the stimulation of D1 receptors inhibits the micturition reflex, whereas the stimulation of D2 receptors facilitates the micturition reflex. D2 depletion of dopaminergic neurons induces an overactive bladder, and D1 receptor agonists produce a dose-dependent inhibition of the micturition reflex [40]. For the treatment of nocturia, first, a urologic examination is recom‐ mended to rule out underlying urologic diseases. Switching from bromocriptine to pergolide improved nocturia, thereby improving sleep status in patients with PD [41]. Anticholinergic drugs, such as oxybutynin and tolterodine, are commonly used for detrusor hyperreflexia. Subthalamic deep brain stimulation improved detrusor hyperreflexia [42]. When nocturia is related to wearing-off symptoms, adding a long-acting dopamine agonist before bedtime

Need to visit lavatory 79 29 Inability to turn over in bed 65 39 Painful leg cramps 55 15 Vivid dreams / nightmares 48 9 Inability to get out of bed unaided 35 15 Limb or facial dystonia 34 10 Back pain 34 9 Jerks of legs 33 5 Visual hallucinations 16 3

may be missed in clinical practice unless physicians screen for it.

None 4

should be considered.

**Table 4.** Nocturnal problems in PD patients (adapted from Lees et al [4])

**Table 3.** PDSS-2 domain scale

## **3. Nocturnal problems in PD**

The significant impact of nocturnal problems in PD patients has been emphasized by the study by Lees et al [4], which found that among 220 patients with PD, 215 (98%) reported sleep problems, and 29% took hypnotics or sedatives, but only 6% took any anti-parkinsonian drug during the night (Table 4). In the study using continuous activity monitoring, compared with the healthy elderly subjects, patients with PD showed an elevated nocturnal activity level and an increased proportion of time with movement [36]. Nocturnal motor symptoms such as worsened rigidity, tremor, dystonia, and akinesia can lead to nocturnal awakening and sleep maintenance insomnia, a common form of insomnia in PD [4, 7, 24, 29, 37]. Sleep-onset insomnia does not seem to account for the majority of insomnia cases in PD when compared with age-matched controls. In a community-based sleep study by Tandberg et al, sleep-onset insomnia, sleep-maintenance insomnia, and early awakening were observed in 31.8%, 38.9%, and 23.4% of PD patients compared with 22%, 12%, and 11% of healthy controls, respectively [7]. The frequency of sleep onset insomnia did not significantly differ between the groups. Selfreported sleep problems occurred significantly more often in patients with PD (60%) than in healthy controls (33%) and patients with diabetes mellitus (45%). Our cross-sectional studies have also found sleep-maintenance insomnia, but not sleep-onset insomnia, to be significantly more prevalent in patients with PD than in controls [29, 34]. Sleep disturbances correlate with motor impairment in PD patients [6]. However, even in untreated patients with PD, in addition to the changes in sleep architecture, motor symptoms may predominately occur at night rather than during the day [32]. Therefore, nocturnal motor symptoms can interfere with sleep and are not always parallel with daytime motor symptoms, which is supported by several studies showing a weak or nonexistent correlation between sleep disturbances and daytime motor symptoms (UPDRS motor score) [7, 38] and no correlation between nocturnal motor symptoms obtained by PDSS-2 and UPDRS motor scores [33]. In patients with an early to moderate stage of PD, a substantial number of patients may suffer from nocturnal problems; however, this may be missed in clinical practice unless physicians screen for it.

An international cross-sectional study comprising 242 patients with PD (HY 2 (n=121) being most frequent) revealed that a significant number of sleep-related symptoms were undeclared by patients before the administration of the non-motor questionnaire: nocturia, 43.9%; daytime sleepiness, 52.4%; insomnia, 43.9%; vivid dreams, 52.4%; acting out during dreams, 44.1%; and restless legs, 36.4% [39]. Patients in the advanced stages of the disease have motor dysfunction throughout the day; therefore, its impact on the nighttime period should always be considered.


**Table 4.** Nocturnal problems in PD patients (adapted from Lees et al [4])

**PDSS-2 Item**

Poor sleep quality 1 Difficulty falling asleep 2 Difficulty staying asleep 3 Tired/sleepy in the morning 14 Get up to pass urine 8

Restlessness of arms or legs 4 Urge to move arms or legs 5 Distressing dreams 6 Painful posturing in morning 12 Tremor on waking 13

Distressing hallucinations 7 Uncomfortable and immobile 9 Pain in arms or legs 10 Muscle cramps in arms or legs 11 Breathing problems/snoring 15

The significant impact of nocturnal problems in PD patients has been emphasized by the study by Lees et al [4], which found that among 220 patients with PD, 215 (98%) reported sleep problems, and 29% took hypnotics or sedatives, but only 6% took any anti-parkinsonian drug during the night (Table 4). In the study using continuous activity monitoring, compared with the healthy elderly subjects, patients with PD showed an elevated nocturnal activity level and an increased proportion of time with movement [36]. Nocturnal motor symptoms such as worsened rigidity, tremor, dystonia, and akinesia can lead to nocturnal awakening and sleep maintenance insomnia, a common form of insomnia in PD [4, 7, 24, 29, 37]. Sleep-onset insomnia does not seem to account for the majority of insomnia cases in PD when compared with age-matched controls. In a community-based sleep study by Tandberg et al, sleep-onset insomnia, sleep-maintenance insomnia, and early awakening were observed in 31.8%, 38.9%, and 23.4% of PD patients compared with 22%, 12%, and 11% of healthy controls, respectively [7]. The frequency of sleep onset insomnia did not significantly differ between the groups. Selfreported sleep problems occurred significantly more often in patients with PD (60%) than in healthy controls (33%) and patients with diabetes mellitus (45%). Our cross-sectional studies have also found sleep-maintenance insomnia, but not sleep-onset insomnia, to be significantly more prevalent in patients with PD than in controls [29, 34]. Sleep disturbances correlate with motor impairment in PD patients [6]. However, even in untreated patients with PD, in addition to the changes in sleep architecture, motor symptoms may predominately occur at night rather

**Disturbed sleep**

**Motor symptoms at night**

48 A Synopsis of Parkinson's Disease

**PD symptoms at night**

**Table 3.** PDSS-2 domain scale

**3. Nocturnal problems in PD**

Although nocturia is associated with normal aging, 80% of PD patients show two or more episodes of nocturia per night resulting from overflow incontinence and a spastic bladder [4].Urinary bladder–related symptoms, such as frequency, urgency, and urge incontinence, are common in PD, resulting in frequent nocturnal awakenings. In animal studies, the stimulation of D1 receptors inhibits the micturition reflex, whereas the stimulation of D2 receptors facilitates the micturition reflex. D2 depletion of dopaminergic neurons induces an overactive bladder, and D1 receptor agonists produce a dose-dependent inhibition of the micturition reflex [40]. For the treatment of nocturia, first, a urologic examination is recom‐ mended to rule out underlying urologic diseases. Switching from bromocriptine to pergolide improved nocturia, thereby improving sleep status in patients with PD [41]. Anticholinergic drugs, such as oxybutynin and tolterodine, are commonly used for detrusor hyperreflexia. Subthalamic deep brain stimulation improved detrusor hyperreflexia [42]. When nocturia is related to wearing-off symptoms, adding a long-acting dopamine agonist before bedtime should be considered.

Pain has been reported in approximately 60% of PD patients [43] in association with sleep disturbances and depressive symptoms [44, 45], in addition to tremor, rigidity, akinesia, dystonia, and akathisia. Pain is classified into the following categories: musculoskeletal pain, radicular or neuropathic pain, dystonia-related pain, akathitic discomfort, and primary central parkinsonian pain [46]. Nocturnal pain is related to nocturnal awakening. To evaluate whether pain is related to wearing off is important because it can worsen during wearing-off periods. Primary central parkinsonian, akathitic, and dystonia-related pain may respond to dopami‐ nergic treatment.

REM sleep and complex motor behavior in association with dream content [60]. Table 5

Difficulty initiating sleep Unknown cause Hypnotics (short-acting type; zolpidem,

Wearing off, resting tremor,

rigidity, akinesia,

Excessive daytime sleepiness Unknown cause Daytime rehabilitation

zopiclone, eszopiclone, brotizolam)

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dose of levodopa administration, add

Increase frequency of levodopa administration, add dopamine agonist, or switch to a different

Drug related (alerting effect) Remove or reduce dose of causative drug

Unknown cause Hypnotics (intermediate type; flunitrazepam)

Drug-induced dyskinesia Increase frequency of levodopa and reduce

Depression, anxiety Antidepressant (SSRI, SNRI, tricyclic

Nocturia Oxybutynin, flavoxate

Refractory Modafinil, caffeine

Hallucination, delusion, delirium Reduce dopaminergic drugs, consider Yi-Gan

Sleep apnea syndrome Continuous positive airway pressure therapy

Restless legs syndrome, periodic limb movement disorder Adjustment of dopaminergic treatment, use

REM sleep behavior disorder Hazard avoidance (remove potentially

dopamine agonist

antidepressant) Anti-anxiety drug Dopamine agonist (D3 R)

Drug related (sedative effect) Remove or reduce dose of causative drug

Dopamine agonist (D1 R)

(including dopamine agonist)

San and atypical antipsychotics

clonazepam and Yi-Gan San

µg/L) and clonazepam

(severe case)

dangerous objects from the bedroom and place a mattress on the floor), consider

dopamine agonist before bedtime, consider iron supplement (if serum ferritin are below 50

type of dopamine agonist

summarizes the management of sleep problems in PD.

Difficulty maintaining sleep / early

**Table 5.** Management of sleep problems in PD

morning awakening

**Type of insomnia Cause Treatment**

Increased severity in nocturnal motor symptoms such as rigidity, bradykinesia, and resting tremor may benefit from increasing the bedtime dose of dopaminergic treatment. A doubleblinded, placebo-controlled trial demonstrated the efficacy of 24-h rotigotine on daytime motor function (UPDRS part III) and nocturnal disabilities, as evaluated by the PDSS-2 [35]. Subcu‐ taneous overnight apomorphine infusion markedly reduced nocturnal awakenings, nocturnal off periods, pain, dystonia, and nocturia [47]. High-frequency subthalamic nucleus stimulation in 10 PD patients with insomnia reduced nighttime akinesia by 60% and completely sup‐ pressed axial and early-morning dystonia [48]. Furthermore, a 24-week, double-blind study showed that once-daily ropinirole prolonged release improved nocturnal symptoms (as assessed by PDSS) in patients with advanced PD who were not optimally controlled with levodopa and suffered troublesome nocturnal disturbances [49]. In contrast, some studies indicate that dopaminergic drugs can have alerting effects, possibly interfering with sleep continuity in patients with PD [14, 36, 50].

Hallucinations and psychosis affect 30 to 45% of PD patients who have been treated with levodopa for a long period [51]. Among a wide spectrum of hallucinations, visual hallucina‐ tions are commonly observed. Sleep disturbances, daily levodopa doses, older age, depression, and cognitive impairment are associated with an increased risk for hallucinations in PD patients [52, 53]. In contrast to nocturnal motor symptoms, nocturnal psychiatric symptoms including hallucination and psychosis can be effectively treated by reducing the bedtime dose of dopaminergic treatment or adding antipsychotics.

The prevalence of depression in PD patients varies, ranging from 2.7% to 89% [54]. Depression is associated with sleep disturbances and impaired quality of life in patients with PD [55, 56]. Nortripryline, desipramine, and selective serotonin reuptake inhibitors (venlafaxine and paroxetine) are more effective than placebo in treating depression in PD [57]. Additionally, it should be noted that depressive symptoms worsen during wearing-off periods and also contribute to worsened motor symptoms [58]. In this regard, antiparkinsonian drugs show beneficial effects not only on motor symptoms but also on a patient's mood. Pramipexole improved depressive symptoms in patients with PD mainly through a direct antidepressant effect rather than through improved motor symptoms [59].

Untreated nocturnal disturbances contribute not only to sleep fragmentation but also to daytime sleepiness and thus daytime motor dysfunction. The primary sleep disorders include RBD, RLS, and SAS, all of which should be properly managed because of their clinical significance in disease. RBD is a REM parasomnia characterized by loss of muscle atonia during REM sleep and complex motor behavior in association with dream content [60]. Table 5 summarizes the management of sleep problems in PD.


**Table 5.** Management of sleep problems in PD

Pain has been reported in approximately 60% of PD patients [43] in association with sleep disturbances and depressive symptoms [44, 45], in addition to tremor, rigidity, akinesia, dystonia, and akathisia. Pain is classified into the following categories: musculoskeletal pain, radicular or neuropathic pain, dystonia-related pain, akathitic discomfort, and primary central parkinsonian pain [46]. Nocturnal pain is related to nocturnal awakening. To evaluate whether pain is related to wearing off is important because it can worsen during wearing-off periods. Primary central parkinsonian, akathitic, and dystonia-related pain may respond to dopami‐

Increased severity in nocturnal motor symptoms such as rigidity, bradykinesia, and resting tremor may benefit from increasing the bedtime dose of dopaminergic treatment. A doubleblinded, placebo-controlled trial demonstrated the efficacy of 24-h rotigotine on daytime motor function (UPDRS part III) and nocturnal disabilities, as evaluated by the PDSS-2 [35]. Subcu‐ taneous overnight apomorphine infusion markedly reduced nocturnal awakenings, nocturnal off periods, pain, dystonia, and nocturia [47]. High-frequency subthalamic nucleus stimulation in 10 PD patients with insomnia reduced nighttime akinesia by 60% and completely sup‐ pressed axial and early-morning dystonia [48]. Furthermore, a 24-week, double-blind study showed that once-daily ropinirole prolonged release improved nocturnal symptoms (as assessed by PDSS) in patients with advanced PD who were not optimally controlled with levodopa and suffered troublesome nocturnal disturbances [49]. In contrast, some studies indicate that dopaminergic drugs can have alerting effects, possibly interfering with sleep

Hallucinations and psychosis affect 30 to 45% of PD patients who have been treated with levodopa for a long period [51]. Among a wide spectrum of hallucinations, visual hallucina‐ tions are commonly observed. Sleep disturbances, daily levodopa doses, older age, depression, and cognitive impairment are associated with an increased risk for hallucinations in PD patients [52, 53]. In contrast to nocturnal motor symptoms, nocturnal psychiatric symptoms including hallucination and psychosis can be effectively treated by reducing the bedtime dose

The prevalence of depression in PD patients varies, ranging from 2.7% to 89% [54]. Depression is associated with sleep disturbances and impaired quality of life in patients with PD [55, 56]. Nortripryline, desipramine, and selective serotonin reuptake inhibitors (venlafaxine and paroxetine) are more effective than placebo in treating depression in PD [57]. Additionally, it should be noted that depressive symptoms worsen during wearing-off periods and also contribute to worsened motor symptoms [58]. In this regard, antiparkinsonian drugs show beneficial effects not only on motor symptoms but also on a patient's mood. Pramipexole improved depressive symptoms in patients with PD mainly through a direct antidepressant

Untreated nocturnal disturbances contribute not only to sleep fragmentation but also to daytime sleepiness and thus daytime motor dysfunction. The primary sleep disorders include RBD, RLS, and SAS, all of which should be properly managed because of their clinical significance in disease. RBD is a REM parasomnia characterized by loss of muscle atonia during

nergic treatment.

50 A Synopsis of Parkinson's Disease

continuity in patients with PD [14, 36, 50].

of dopaminergic treatment or adding antipsychotics.

effect rather than through improved motor symptoms [59].

## **4. Excessive daytime sleepiness and sudden-onset sleep episodes**

Sudden-onset sleep episodes while driving have been reported in 3.8%-22.8% of PD patients and are associated with a high score on the ESS [61, 64, 73], although sleepiness is sometimes unrecognized by patients. However, similar to the findings by Tan et al, which showed that ESS scores ≥10 had a 71.4% sensitivity and 88.4% specificity for predicting a sleep attack [64], our study showed that an ESS score of ≥10 had a 75% sensitivity and 82.4% specificity for predicting sleep episodes [62]. This evidence suggests that PDS patients with EDS have a

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**Nucleus Main neurotransmitter Neuronal loss in Parkinsonian brain (%)**

Tuberomammillary nucleus Histamine Unchanged enzymatic activity

5-HT, 5-hydroxytryptamine (serotonin);ACh, acetylcholine; BF, basal forebrain; DA, dopamine; GABA, γ-aminobutyric acid; His, histamine; LC, locus ceruleus; LH, lateral hypothalamus; NE, norepinephrine; ORX, orexin; Raphe, median ra‐

phe nucleus; TMN, tuberomamillary nucleus; vPAG, ventral periaqueductal gray matter.

**Figure 8.** The ascending arousal systems in the human brain (reproduced with permission from [80]).

Locus coeruleus Noradrenaline 40-50 Median raphe Serotonin 20-40 Ventral periaqueductal gray matter Dopamine 9 Pedunculopontine nucleus Acetylcholine 57

Lateral hypothalamus Orexin (hypocretin) 23-62 Basal forebrain Acetylcholine 32-93

**Table 6.** Neuronal loss in arousal systems in the brains of patients with PD (adapted from [79]).

significant increased risk for sudden-onset sleep episodes.

Excessive daytime sleepiness (EDS) occurs in approximately 15%-50% of PD patients [61-63]. A high Epworth sleepiness scale (ESS) score, male gender status, longer disease duration, and high disease severity and dopaminergic medication have been associated with EDS [61, 62, 64]. Multifactorial nocturnal problems (Table 1) are the causes of sleep disturbances in PD and also lead to EDS, reflecting poor sleep quality and duration. In addition to nocturnal problems, PD-related pathological changes play a role in sleepiness: an impaired arousal system has been suggested in PD (Table 6; Figure 8). A subset of patients with PD exhibit EDS and a sudden onset of sleep episodes with a short sleep latency and a short sleep-onset REM period, independent of the nighttime sleep conditions. This finding suggests a narcolepsy phenotype in PD patients. Narcolepsy is a sleep disorder characterized by severe daytime sleepiness, cataplexy, hypnagogic hallucination, and sleep paralysis caused by loss of orexin neurons. However, cataplexy is lacking in patients with PD [65], and orexin levels in the cerebrospinal fluid in PD patients with EDS remains controversial [66-68]. Decreased orexin levels in the hypothalamus and a loss of orexin neurons have been observed in PD patients in correlation with clinical disease progression; however, no description was provided for EDS [69, 70].

Dopaminergic medication, i.e., taking dopamine agonists or levodopa, is associated with increased daytime sleepiness in patients with PD [61, 62, 64, 71, 72]. Although a higher levodopa equivalent dose is correlated with increased daytime sleepiness in PD [62, 64, 71], the association between the specific type of dopamine agonist and EDS is unclear [61, 62, 72, 73] (Figure 9). Untreated PD patients do not seem to have EDS compared with controls [22, 74]. However, the study consisting of 3078 men aged 71 to 93 years showed that there was more than a threefold excess (odds ratio 3.3) in the risk of PD in men with EDS versus men without EDS [75]. In a prospective study, of the 232 patients included at baseline, 138 were available for reevaluation after 4 years, and 89 patients were available after 8 years. The EDS frequency increased from 5.6% at the baseline to 22.5% at the 4-year follow up and 40.8% at the 8-year follow up. EDS was related to age, gender, and use of dopamine agonists in the logistic regression model, whereas in patients never having used dopamine agonists, hyper‐ somnia was associated with the HY stage only, suggesting that age- and disease-related disturbances of the sleep-wake regulation contribute to hypersomnia in PD and that treatment with dopamine agonists also contribute to EDS [76]. Regarding the interaction between dopamine and sleep [77], the D1 receptor agonist promotes wakefulness and decreases slowwave sleep and REM sleep. In contrast, the D2 receptor agonist has a biphasic action: a lower dose reduces wakefulness and increases slow-wave sleep and REM sleep via the pre-synaptic auto D2 receptor, whereas a higher dose stimulates wakefulness via the post-synaptic D2 receptor. D3 receptor stimulation increases slow-wave sleep and promotes sleep. This result for D2 receptor stimulation differs from that observed in patients with PD: a higher dose of dopaminergic drugs is associated with EDS. Bliwise et al reported that increasing dosages of dopamine agonists were associated with less daytime alertness, whereas higher levels of levodopa were associated with higher levels of alertness [78].

Sudden-onset sleep episodes while driving have been reported in 3.8%-22.8% of PD patients and are associated with a high score on the ESS [61, 64, 73], although sleepiness is sometimes unrecognized by patients. However, similar to the findings by Tan et al, which showed that ESS scores ≥10 had a 71.4% sensitivity and 88.4% specificity for predicting a sleep attack [64], our study showed that an ESS score of ≥10 had a 75% sensitivity and 82.4% specificity for predicting sleep episodes [62]. This evidence suggests that PDS patients with EDS have a significant increased risk for sudden-onset sleep episodes.


**Table 6.** Neuronal loss in arousal systems in the brains of patients with PD (adapted from [79]).

**4. Excessive daytime sleepiness and sudden-onset sleep episodes**

52 A Synopsis of Parkinson's Disease

Excessive daytime sleepiness (EDS) occurs in approximately 15%-50% of PD patients [61-63]. A high Epworth sleepiness scale (ESS) score, male gender status, longer disease duration, and high disease severity and dopaminergic medication have been associated with EDS [61, 62, 64]. Multifactorial nocturnal problems (Table 1) are the causes of sleep disturbances in PD and also lead to EDS, reflecting poor sleep quality and duration. In addition to nocturnal problems, PD-related pathological changes play a role in sleepiness: an impaired arousal system has been suggested in PD (Table 6; Figure 8). A subset of patients with PD exhibit EDS and a sudden onset of sleep episodes with a short sleep latency and a short sleep-onset REM period, independent of the nighttime sleep conditions. This finding suggests a narcolepsy phenotype in PD patients. Narcolepsy is a sleep disorder characterized by severe daytime sleepiness, cataplexy, hypnagogic hallucination, and sleep paralysis caused by loss of orexin neurons. However, cataplexy is lacking in patients with PD [65], and orexin levels in the cerebrospinal fluid in PD patients with EDS remains controversial [66-68]. Decreased orexin levels in the hypothalamus and a loss of orexin neurons have been observed in PD patients in correlation with clinical disease progression; however, no description was provided for EDS [69, 70].

Dopaminergic medication, i.e., taking dopamine agonists or levodopa, is associated with increased daytime sleepiness in patients with PD [61, 62, 64, 71, 72]. Although a higher levodopa equivalent dose is correlated with increased daytime sleepiness in PD [62, 64, 71], the association between the specific type of dopamine agonist and EDS is unclear [61, 62, 72, 73] (Figure 9). Untreated PD patients do not seem to have EDS compared with controls [22, 74]. However, the study consisting of 3078 men aged 71 to 93 years showed that there was more than a threefold excess (odds ratio 3.3) in the risk of PD in men with EDS versus men without EDS [75]. In a prospective study, of the 232 patients included at baseline, 138 were available for reevaluation after 4 years, and 89 patients were available after 8 years. The EDS frequency increased from 5.6% at the baseline to 22.5% at the 4-year follow up and 40.8% at the 8-year follow up. EDS was related to age, gender, and use of dopamine agonists in the logistic regression model, whereas in patients never having used dopamine agonists, hyper‐ somnia was associated with the HY stage only, suggesting that age- and disease-related disturbances of the sleep-wake regulation contribute to hypersomnia in PD and that treatment with dopamine agonists also contribute to EDS [76]. Regarding the interaction between dopamine and sleep [77], the D1 receptor agonist promotes wakefulness and decreases slowwave sleep and REM sleep. In contrast, the D2 receptor agonist has a biphasic action: a lower dose reduces wakefulness and increases slow-wave sleep and REM sleep via the pre-synaptic auto D2 receptor, whereas a higher dose stimulates wakefulness via the post-synaptic D2 receptor. D3 receptor stimulation increases slow-wave sleep and promotes sleep. This result for D2 receptor stimulation differs from that observed in patients with PD: a higher dose of dopaminergic drugs is associated with EDS. Bliwise et al reported that increasing dosages of dopamine agonists were associated with less daytime alertness, whereas higher levels of

levodopa were associated with higher levels of alertness [78].

<sup>5-</sup>HT, 5-hydroxytryptamine (serotonin);ACh, acetylcholine; BF, basal forebrain; DA, dopamine; GABA, γ-aminobutyric acid; His, histamine; LC, locus ceruleus; LH, lateral hypothalamus; NE, norepinephrine; ORX, orexin; Raphe, median ra‐ phe nucleus; TMN, tuberomamillary nucleus; vPAG, ventral periaqueductal gray matter.

**Figure 8.** The ascending arousal systems in the human brain (reproduced with permission from [80]).

Lesions of the locus coeruleus perialpha in cats and of the sublaterodorsal nucleus in rats have been shown to cause REM sleep without atonia with complex movements [93, 94]. The equivalent of these nuclei in humans is the subcoeruleus nucleus in the pons, which is first affected during the early stages of PD. In addition, other brainstem nuclei such as the choli‐ nergic nuclei, pedunculopontine nucleus, and laterodorsal tegmental nucleus also play a role in regulating REM sleep [95]. In the study using neuromelanin-sensitive imaging, reduced signal intensity is found in the locus coeruleus/subcoeruleus area in patients with PD relative to controls, and that difference is more marked in patients with PSG-confirmed RBD than in those without RBD. A reduced signal intensity in those areas is found to be correlated with the percentage of abnormally increased muscle tone during REM sleep. This study first shows the involvement of the coeruleus/subcoeruleus complex in PD patients and, more markedly,

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The frequency of RBD is reported to be 15- 60% of PD patients (Table 7) [79]. Co-occurrence of RBD in PD may represent distinct characteristics of PD: akinetic rigid phenotype, an increased frequency of falls, and a poor response to dopaminergic medications, orthostatic hypotension, and impaired color vision have been described compared with PD patients without RBD [97, 98]. A decreased uptake of cardiac MIBG is also reported in PD patients with RBD compared with those without RBD [99, 100]. In a prospective study with 42 patients with PD (27 patients with PSG-confirmed RBD and 15 patients without RBD) for 4 years, 48% with RBD developed dementia, compared with 0% of those without RBD, suggesting that RBD was associated with an increased risk of dementia in patients with PD [101]. Similarly, Nomura et al found coexistence of clinical RBD, but not subclinical RBD, was associated with the development of dementia in PD. In their study, RBD, but not subclinical RBD, was associated with orthostatic

hypotension and levodopa equivalent dose equivalents in patients with PD [102].

With respect to the clinical motor subtype, not all the studies support the akinetic rigid phenotype as the clinical phenotype associated with PD-RBD. A study of 457 PD patients with sleep disturbances did not find a characteristic clinical subtype for PD with RBD but did report a higher disease severity and longer disease duration in PD patients with RBD than in those without [103]. In early PD (disease duration ≤5 years and HY stage 1- 2.5), the RBD comorbidity (confirmed by clinical history during the preceding 6 months and a cut-off score > 4 on the RBD screening questionnaire) was significantly higher (55%) in PD patients, but clinical subtype and disease severity did not differ between patients with and without RBD [104]. It has been reported that a tremor-dominant type may transition into a non-tremor dominant type over time and with increased age [105]. In our study including 93 patients with PD and controls using the Japanese version of the RBD screening questionnaire (RBDSQ-J) [106], 29% of PD patients had probable RBD (5≥RBDSQ-J) compared with 8.6% of controls [107]. Patients with probable RBD had a higher score of PDQ-39 cognition and emotional wellbeing and more frequent sleep onset insomnia, distressing dreams, and hallucinations. However, there were no differences between these two groups with respect to the clinical subtype, disease severity, or motor function. In the study consisting of 57 newly diagnosed drug-naïve patients with PD, 17 PD patients (30%) were diagnosed with RBD by overnight PSG. Non-RBD patients and RBD patients did not differ with respect to age, gender, disease duration, motor symptom subtype and severity, and cognitive performance. PSG parameters such as total sleep time, REM sleep percentage, apnea-hypopnea index, and mean oxygen

in those with concomitant RBD [96].

The ESS score is significantly higher in the group with multiple dopamine agonists plus levodopa/DCI; however, no differences in EDS among different types of dopamine agonist groups are shown.

**Figure 9.** Excessive daytime sleepiness and dopamine agonists in patients with PD (created based on the data from [62]).

## **5. Primary sleep disorders**

#### **5.1. Rapid eye movement sleep behavior disorder**

REM sleep behavior disorder (RBD) is characterized by a loss of muscle atonia during REM sleep, resulting in dream-enacting behavior, leading to injury to the individual or bed partner [81]. The important implication is that idiopathic RBD patients are found to be at high risk of later developing neurodegenerative diseases in the synucleinopathies, such as PD, multiple system atrophy, and dementia with Lewy bodies [82]. Schenck and colleagues first reported that 3.7 ± 1.4 years after an initial diagnosis of idiopathic RBD, 38% of patients developed a parkinsonian syndrome [83]. In a prospective study of idiopathic RBD patients, the estimated 5-year risk of neurodegenerative disease was 17.7%, the 10-year risk was 40.6%, and the 12 year risk was 52.4% [84]. A recent clinicopathological study of 172 cases of RBD with or without coexisting neurological diseases revealed that among the neurodegenerative disorders associated with RBD (n=170), 160 (94%) were synucleinopathies [85]. The association between RBD and synucleinopathy was particularly high when RBD preceded the onset of other neurodegenerative syndrome features. Before the onset of motor symptoms, idiopathic RBD subjects already possess characteristics, such as olfactory impairment, impaired color vision, autonomic dysfunction, mild cognitive impairment, a decreased uptake of cardiac (123) Imetaiodobenzylguanidine (MIBG), and hyperechogenicity in the substantia nigra [86-90], that are similar to PD. These may be potential predictive markers for future development of PD. In the subjects with idiopathic RBD who were initially free of neurodegenerative disease, the severity of the REM atonia loss on the baseline PSG findings was associated with the devel‐ opment of PD [91]. Employing noninvasive imaging techniques such as transcranial sonogra‐ phy, single-photon emission computed tomography, and positron emission tomography may be helpful in identifying patients with iRBD potentially at future risk for PD [92].

Lesions of the locus coeruleus perialpha in cats and of the sublaterodorsal nucleus in rats have been shown to cause REM sleep without atonia with complex movements [93, 94]. The equivalent of these nuclei in humans is the subcoeruleus nucleus in the pons, which is first affected during the early stages of PD. In addition, other brainstem nuclei such as the choli‐ nergic nuclei, pedunculopontine nucleus, and laterodorsal tegmental nucleus also play a role in regulating REM sleep [95]. In the study using neuromelanin-sensitive imaging, reduced signal intensity is found in the locus coeruleus/subcoeruleus area in patients with PD relative to controls, and that difference is more marked in patients with PSG-confirmed RBD than in those without RBD. A reduced signal intensity in those areas is found to be correlated with the percentage of abnormally increased muscle tone during REM sleep. This study first shows the involvement of the coeruleus/subcoeruleus complex in PD patients and, more markedly, in those with concomitant RBD [96].

The frequency of RBD is reported to be 15- 60% of PD patients (Table 7) [79]. Co-occurrence of RBD in PD may represent distinct characteristics of PD: akinetic rigid phenotype, an increased frequency of falls, and a poor response to dopaminergic medications, orthostatic hypotension, and impaired color vision have been described compared with PD patients without RBD [97, 98]. A decreased uptake of cardiac MIBG is also reported in PD patients with RBD compared with those without RBD [99, 100]. In a prospective study with 42 patients with PD (27 patients with PSG-confirmed RBD and 15 patients without RBD) for 4 years, 48% with RBD developed dementia, compared with 0% of those without RBD, suggesting that RBD was associated with an increased risk of dementia in patients with PD [101]. Similarly, Nomura et al found coexistence of clinical RBD, but not subclinical RBD, was associated with the development of dementia in PD. In their study, RBD, but not subclinical RBD, was associated with orthostatic hypotension and levodopa equivalent dose equivalents in patients with PD [102].

The ESS score is significantly higher in the group with multiple dopamine agonists plus levodopa/DCI; however, no

**Figure 9.** Excessive daytime sleepiness and dopamine agonists in patients with PD (created based on the data from

REM sleep behavior disorder (RBD) is characterized by a loss of muscle atonia during REM sleep, resulting in dream-enacting behavior, leading to injury to the individual or bed partner [81]. The important implication is that idiopathic RBD patients are found to be at high risk of later developing neurodegenerative diseases in the synucleinopathies, such as PD, multiple system atrophy, and dementia with Lewy bodies [82]. Schenck and colleagues first reported that 3.7 ± 1.4 years after an initial diagnosis of idiopathic RBD, 38% of patients developed a parkinsonian syndrome [83]. In a prospective study of idiopathic RBD patients, the estimated 5-year risk of neurodegenerative disease was 17.7%, the 10-year risk was 40.6%, and the 12 year risk was 52.4% [84]. A recent clinicopathological study of 172 cases of RBD with or without coexisting neurological diseases revealed that among the neurodegenerative disorders associated with RBD (n=170), 160 (94%) were synucleinopathies [85]. The association between RBD and synucleinopathy was particularly high when RBD preceded the onset of other neurodegenerative syndrome features. Before the onset of motor symptoms, idiopathic RBD subjects already possess characteristics, such as olfactory impairment, impaired color vision, autonomic dysfunction, mild cognitive impairment, a decreased uptake of cardiac (123) Imetaiodobenzylguanidine (MIBG), and hyperechogenicity in the substantia nigra [86-90], that are similar to PD. These may be potential predictive markers for future development of PD. In the subjects with idiopathic RBD who were initially free of neurodegenerative disease, the severity of the REM atonia loss on the baseline PSG findings was associated with the devel‐ opment of PD [91]. Employing noninvasive imaging techniques such as transcranial sonogra‐ phy, single-photon emission computed tomography, and positron emission tomography may

be helpful in identifying patients with iRBD potentially at future risk for PD [92].

differences in EDS among different types of dopamine agonist groups are shown.

[62]).

**5. Primary sleep disorders**

54 A Synopsis of Parkinson's Disease

**5.1. Rapid eye movement sleep behavior disorder**

With respect to the clinical motor subtype, not all the studies support the akinetic rigid phenotype as the clinical phenotype associated with PD-RBD. A study of 457 PD patients with sleep disturbances did not find a characteristic clinical subtype for PD with RBD but did report a higher disease severity and longer disease duration in PD patients with RBD than in those without [103]. In early PD (disease duration ≤5 years and HY stage 1- 2.5), the RBD comorbidity (confirmed by clinical history during the preceding 6 months and a cut-off score > 4 on the RBD screening questionnaire) was significantly higher (55%) in PD patients, but clinical subtype and disease severity did not differ between patients with and without RBD [104]. It has been reported that a tremor-dominant type may transition into a non-tremor dominant type over time and with increased age [105]. In our study including 93 patients with PD and controls using the Japanese version of the RBD screening questionnaire (RBDSQ-J) [106], 29% of PD patients had probable RBD (5≥RBDSQ-J) compared with 8.6% of controls [107]. Patients with probable RBD had a higher score of PDQ-39 cognition and emotional wellbeing and more frequent sleep onset insomnia, distressing dreams, and hallucinations. However, there were no differences between these two groups with respect to the clinical subtype, disease severity, or motor function. In the study consisting of 57 newly diagnosed drug-naïve patients with PD, 17 PD patients (30%) were diagnosed with RBD by overnight PSG. Non-RBD patients and RBD patients did not differ with respect to age, gender, disease duration, motor symptom subtype and severity, and cognitive performance. PSG parameters such as total sleep time, REM sleep percentage, apnea-hypopnea index, and mean oxygen saturation also did not differ [108]. Differences in clinical background factors between PD patients with and without RBD await confirmation in longitudinal studies. Interestingly, restored motor control (movements, speech, and facial expressions) during REM sleep with enacted dreams has been reported in PD patients with RBD [109].

of a marked beneficial response to dopaminergic treatment observed in both PD and RLS, a shared dopaminergic dysfunction has been suggested in PD and RLS [118]. However, unlike RBD, there is no evidence suggesting RLS as a risk factor of PD, and the co-morbidity of RLS in PD patients varies widely from 0 to 50% [119]. In contrast to PD, which shows degeneration of the striatonigral dopaminergic neurons, confirmed by the reduced striatal uptake observed in neuroimaging studies, in idiopathic RLS, PET/SPECT studies have not produced consistent findings of striatonigral dopaminergic dysfunction [120, 121]. Postmortem studies of RLS patients have found an increased total and phosphorylated tyrosine hydroxylase in the putamen and substantia nigra [122]. Cerebrospinal fluid iron insufficiency has been demon‐ strated in idiopathic RLS independent of serum iron levels [123, 124]. The transcranial sonography findings of substantia nigra hypoechogenicity and brain imaging studies also suggest brain iron insufficiency in RLS patients [125, 126]. In contrast, the hyperechogenicity in the substantia nigra commonly observed in patients with PD may reflect increased levels of iron in the substantia nigra. No difference in the substantia nigra echogenicity has been reported between the PD with RLS and PD without RLS groups [127]. Thus, there are some similarities between PD and idiopathic RLS, including a marked response to dopamine agonist treatment, although the two disorders may have different pathogenic mechanisms [118, 128].

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57

In PD, there are several conditions that mimic RLS, including sensory symptoms, pain, and the wearing-off phenomenon [118]. Moreover, the clinical overlap between RLS, wearing-offrelated lower limb discomfort, and restlessness and akathisia has been suggested [129, 130]. In the study evaluating RLS in PD, 20.8% of PD patients had RLS, and patients with PD with RLS had an older age at onset and were much less likely to report a family history of RLS compared with patients with isolated RLS [131]. Complicatedly, dopaminergic treatment may either mask or augment coexisting RLS symptoms in PD [129], and there is an association between long-term dopaminergic treatment and RLS development in PD patients [132].

In addition, in recent studies, an increased frequency of leg motor restlessness (LMR) without fulfilling the criteria for RLS has been described in patients with PD [128]. A total of 200 early, drug-naive patients with PD derived from a population-based incident cohort and 173 ageand gender-matched control subjects were examined, and 31 (15.5%) of PD patients and 16 (9.2%) of control subjects met RLS criteria (p=0.07). However, LMR (OR 2.84, 95% CI 1.43–5.61, p=0.001) but not RLS (OR 1.76, 95% CI 0.90–3.43, p=0.089) occurs with a near 3-fold higher risk in early PD compared with controls [128]. Shimohata and Nishizawa reported that among 158 patients with PD, 11% had RLS and 19% had LMR without fulfilling the criteria for RLS (total LMR, 30%). The frequencies of insomnia and EDS in patients with LMR were lower than those of patients with RLS but higher than in patients without LMR or RLS, highlighting the impact of LMR and RLS on sleep disturbance in PD [133]. Likewise, our study showed that LMR was more frequent in patients with PD than in controls (32.3% vs. 14.0%; scores of PDSS-2 subitem 4 (restlessness of arms or legs) or subitem 5 (urge to move arms or legs) ≥2), although RLS frequency was similar between the patients with PD and controls (5.5% vs. 2.2%) [34].

For the treatment of RLS, iron replacement therapy should be considered when the serum levels of ferritin are lower than 50 µg / L. Using long-acting dopamine agonists at bedtime is

effective.

Before starting treatment of RBD, it should be noted that RBD can be triggered or worsened by antidepressants [110]. Clonazepam (0.5 to 1.5 mg) at bedtime is the most effective treatment for RBD patients. Melatonin (3-12 mg) at bedtime has been shown to ameliorate RBD [111, 112]. Administration of Yi-Gan San, an herbal medication, at 2.5 g three times a day, alone or in conjunction with 0.25 mg clonazepam, may also be effective [113]. Some patients may respond to 1 evening or bedtime dose of 2.5g.


**Table 7.** RBD in neurodegenerative diseases (adapted from [79])

#### **5.2. Restless leg syndrome**

RLS is characterized by an urge to move the legs, uncomfortable leg sensations, and motor restlessness, typically occurring during the evening and night. The pathogenesis of RLS remains unclear; however, dysfunction of the dopaminergic A11 nucleus of the hypothalamus has been implicated [114]. The hypothalamic A11 nucleus has projections to the suprachias‐ matic nuclei and dorsal raphe, and it provides descending projections to the preganglionic sympathetic neurons, dorsal horn region, interneurons, and somatic motor neurons [114]. Neurophysiological studies have revealed disinhibition of inhibitory cortical controls, decreased intracortical inhibition, and hyperexcitability of spinal pathways [115-117]. In view of a marked beneficial response to dopaminergic treatment observed in both PD and RLS, a shared dopaminergic dysfunction has been suggested in PD and RLS [118]. However, unlike RBD, there is no evidence suggesting RLS as a risk factor of PD, and the co-morbidity of RLS in PD patients varies widely from 0 to 50% [119]. In contrast to PD, which shows degeneration of the striatonigral dopaminergic neurons, confirmed by the reduced striatal uptake observed in neuroimaging studies, in idiopathic RLS, PET/SPECT studies have not produced consistent findings of striatonigral dopaminergic dysfunction [120, 121]. Postmortem studies of RLS patients have found an increased total and phosphorylated tyrosine hydroxylase in the putamen and substantia nigra [122]. Cerebrospinal fluid iron insufficiency has been demon‐ strated in idiopathic RLS independent of serum iron levels [123, 124]. The transcranial sonography findings of substantia nigra hypoechogenicity and brain imaging studies also suggest brain iron insufficiency in RLS patients [125, 126]. In contrast, the hyperechogenicity in the substantia nigra commonly observed in patients with PD may reflect increased levels of iron in the substantia nigra. No difference in the substantia nigra echogenicity has been reported between the PD with RLS and PD without RLS groups [127]. Thus, there are some similarities between PD and idiopathic RLS, including a marked response to dopamine agonist treatment, although the two disorders may have different pathogenic mechanisms [118, 128].

saturation also did not differ [108]. Differences in clinical background factors between PD patients with and without RBD await confirmation in longitudinal studies. Interestingly, restored motor control (movements, speech, and facial expressions) during REM sleep with

Before starting treatment of RBD, it should be noted that RBD can be triggered or worsened by antidepressants [110]. Clonazepam (0.5 to 1.5 mg) at bedtime is the most effective treatment for RBD patients. Melatonin (3-12 mg) at bedtime has been shown to ameliorate RBD [111, 112]. Administration of Yi-Gan San, an herbal medication, at 2.5 g three times a day, alone or in conjunction with 0.25 mg clonazepam, may also be effective [113]. Some patients may

RBD RWA

enacted dreams has been reported in PD patients with RBD [109].

**DISEASE PREVALENCE (%)**

Parkinson's disease 15-60 Multiple system atrophy 90 Dementia with Lewy bodies 86

Corticobasal degeneration Case reports Frontotemporal dementia None

Guadeloupean parkinsonism 78

Huntington's disease 12 Spinocerebellar ataxia type 3 56 Parkin mutation 60

RBD, REM sleep behavior disorder; RWA, REM sleep without atonia

**Table 7.** RBD in neurodegenerative diseases (adapted from [79])

Progressive supranuclear palsy 10-11 0-33 Alzheimer's disease 7 29

Pallidopontonigral degeneration 0 0

RLS is characterized by an urge to move the legs, uncomfortable leg sensations, and motor restlessness, typically occurring during the evening and night. The pathogenesis of RLS remains unclear; however, dysfunction of the dopaminergic A11 nucleus of the hypothalamus has been implicated [114]. The hypothalamic A11 nucleus has projections to the suprachias‐ matic nuclei and dorsal raphe, and it provides descending projections to the preganglionic sympathetic neurons, dorsal horn region, interneurons, and somatic motor neurons [114]. Neurophysiological studies have revealed disinhibition of inhibitory cortical controls, decreased intracortical inhibition, and hyperexcitability of spinal pathways [115-117]. In view

respond to 1 evening or bedtime dose of 2.5g.

**Synucleinopathies**

56 A Synopsis of Parkinson's Disease

**Tauopathies**

**Genetic Diseases**

**5.2. Restless leg syndrome**

In PD, there are several conditions that mimic RLS, including sensory symptoms, pain, and the wearing-off phenomenon [118]. Moreover, the clinical overlap between RLS, wearing-offrelated lower limb discomfort, and restlessness and akathisia has been suggested [129, 130]. In the study evaluating RLS in PD, 20.8% of PD patients had RLS, and patients with PD with RLS had an older age at onset and were much less likely to report a family history of RLS compared with patients with isolated RLS [131]. Complicatedly, dopaminergic treatment may either mask or augment coexisting RLS symptoms in PD [129], and there is an association between long-term dopaminergic treatment and RLS development in PD patients [132].

In addition, in recent studies, an increased frequency of leg motor restlessness (LMR) without fulfilling the criteria for RLS has been described in patients with PD [128]. A total of 200 early, drug-naive patients with PD derived from a population-based incident cohort and 173 ageand gender-matched control subjects were examined, and 31 (15.5%) of PD patients and 16 (9.2%) of control subjects met RLS criteria (p=0.07). However, LMR (OR 2.84, 95% CI 1.43–5.61, p=0.001) but not RLS (OR 1.76, 95% CI 0.90–3.43, p=0.089) occurs with a near 3-fold higher risk in early PD compared with controls [128]. Shimohata and Nishizawa reported that among 158 patients with PD, 11% had RLS and 19% had LMR without fulfilling the criteria for RLS (total LMR, 30%). The frequencies of insomnia and EDS in patients with LMR were lower than those of patients with RLS but higher than in patients without LMR or RLS, highlighting the impact of LMR and RLS on sleep disturbance in PD [133]. Likewise, our study showed that LMR was more frequent in patients with PD than in controls (32.3% vs. 14.0%; scores of PDSS-2 subitem 4 (restlessness of arms or legs) or subitem 5 (urge to move arms or legs) ≥2), although RLS frequency was similar between the patients with PD and controls (5.5% vs. 2.2%) [34].

For the treatment of RLS, iron replacement therapy should be considered when the serum levels of ferritin are lower than 50 µg / L. Using long-acting dopamine agonists at bedtime is effective.

#### **5.3. Sleep apnea syndrome**

Upper airway dysfunction associated with parkinsonism, such as bradykinesia and rigidity, and fluctuations in the respiratory muscles that occur with motor complications can contribute to obstructive sleep apnea in patients with PD [17]. A significant correlation between the apnea hypopnea index (AHI) and the severity of PD was reported [21]. Earlier studies suggested SAS is more frequent in patients with PD than in control subjects [17, 21]. However, in PD patients, lower levels of decline in the minimal or mean nocturnal oxygen saturation levels than in AHImatched controls were observed [17, 21]. In contrast, recent studies suggest that the comor‐ bidity of SAS is not more frequent in PD patients than in the general population, and thus, it may not a relevant issue in PD [10, 11, 134]. Moreover, it has been argued that SAS may not play a major role in EDS in PD. De Cock et al. [11] reported that sleep apnea (defined as an AHI > 5) was less frequent in the PD group than in the in-hospital control group (27% vs. 40%); however, PD patients with sleep apnea had greater motor disability than patients without sleep apnea. EDS was not correlated with sleep apnea.

**6. Conclusion**

**Author details**

Keisuke Suzuki1

Hideki Sakuta1

**References**

, Tomoyuki Miyamoto2

1 Department of Neurology, Dokkyo Medical University, Japan

, Yuji Watanabe1

2 Department of Neurology, Dokkyo Medical University Koshigaya Hospital, Japan

, Hiroaki Fujita1

Psychiatry 2008;79(4):368-376.

2002;14(2):223-236; discussion 222.

2002;17(4):775-781.

ease. Clin Neuropharmacol 1988;11(6):512-519.

Sleep disorders can occur in the early stages of PD and worsen as the disease progresses. The cause of sleep disorders in PD is multifactorial, reflecting PD-related pathology, various aspects of PD-related motor and non-motor symptoms, and comorbidity of primary sleep disorders. Early recognition of and active intervention for sleep disturbance is of great importance, as sleep disturbances significantly impair the quality of life of patients with PD.

, Masayuki Miyamoto1

[1] Jankovic J. Parkinson's disease: clinical features and diagnosis. J Neurol Neurosurg

[2] Chaudhuri KR, Schapira AH. Non-motor symptoms of Parkinson's disease: dopami‐

[3] Parkinson J. An essay on the shaking palsy. 1817. J Neuropsychiatry Clin Neurosci

[4] Lees AJ, Blackburn NA, Campbell VL. The nighttime problems of Parkinson's dis‐

[5] Suzuki K, Miyamoto M, Miyamoto T, Iwanami M, Hirata K. Sleep disturbances asso‐

[6] Kumar S, Bhatia M, Behari M. Sleep disorders in Parkinson's disease. Mov Disord

[7] Tandberg E, Larsen JP, Karlsen K. A community-based study of sleep disorders in

[8] Diederich NJ, McIntyre DJ. Sleep disorders in Parkinson's disease: many causes, few

nergic pathophysiology and treatment. Lancet Neurol 2009;8(5):464-474.

ciated with Parkinson's disease. Parkinsons Dis 2011;2011:219056.

patients with Parkinson's disease. Mov Disord 1998;13(6):895-899.

therapeutic options. J Neurol Sci 2012;314(1-2):12-19.

, Masaoki Iwanami2

, Ayaka Numao1

Sleep Disturbances in Patients with Parkinson's Disease

http://dx.doi.org/10.5772/57320

59

,

and Koichi Hirata1

In our questionnaire-based study, snoring was more frequent in PD patients than in controls (14.0% vs. 1.1%), and snoring in PD patients was associated with disease severity, impaired motor function, and a decreased quality of life, but it was not associated with EDS [135]. Table 8 shows the previous PSG studies that evaluated sleep-related breathing disorders in PD.


When a patient has severe obstructive SAS, continuous positive airway pressure therapy should be considered.

\*Referred for sleepiness, \*\*Controls were obtained from a population-based study, #drug-naïve PD patients SL, sleep latency; SRBD, sleep-related breathing disorders

**Table 8.** Polysomnographic studies evaluating sleep-related breathing disorders in PD

## **6. Conclusion**

**5.3. Sleep apnea syndrome**

58 A Synopsis of Parkinson's Disease

should be considered.

Arnulf et al [14] <sup>2002</sup>

Maria et al

De Cock et

Trotti et al [134] <sup>2010</sup>

Buskova et al [22] <sup>2011</sup>

Yong et al

**Authors Year Sample No.**

[21] <sup>2003</sup> 15 (12 M)/

al [11] <sup>2010</sup> 50 (35 M)/

[13] <sup>2011</sup> 56 (34 M)/

**PD/Controls**

\*54 (44 M)/NA

50 (35 M)

\*\*55 (37 M)/6132

> #15 (14 M)/ 15(14 M)

68 (38 M)

SL, sleep latency; SRBD, sleep-related breathing disorders

sleep apnea. EDS was not correlated with sleep apnea.

Upper airway dysfunction associated with parkinsonism, such as bradykinesia and rigidity, and fluctuations in the respiratory muscles that occur with motor complications can contribute to obstructive sleep apnea in patients with PD [17]. A significant correlation between the apnea hypopnea index (AHI) and the severity of PD was reported [21]. Earlier studies suggested SAS is more frequent in patients with PD than in control subjects [17, 21]. However, in PD patients, lower levels of decline in the minimal or mean nocturnal oxygen saturation levels than in AHImatched controls were observed [17, 21]. In contrast, recent studies suggest that the comor‐ bidity of SAS is not more frequent in PD patients than in the general population, and thus, it may not a relevant issue in PD [10, 11, 134]. Moreover, it has been argued that SAS may not play a major role in EDS in PD. De Cock et al. [11] reported that sleep apnea (defined as an AHI > 5) was less frequent in the PD group than in the in-hospital control group (27% vs. 40%); however, PD patients with sleep apnea had greater motor disability than patients without

In our questionnaire-based study, snoring was more frequent in PD patients than in controls (14.0% vs. 1.1%), and snoring in PD patients was associated with disease severity, impaired motor function, and a decreased quality of life, but it was not associated with EDS [135]. Table 8 shows the previous PSG studies that evaluated sleep-related breathing disorders in PD.

When a patient has severe obstructive SAS, continuous positive airway pressure therapy

**ESS score and EDS PD/Control**

ESS; 14.3±4.1/NA EDS; 50%/NA (mean SL<5 min)

15 (12 M) 63±4 / 60±4 ESS; 12.3 / 6.1 66.7%/NA 11.7/5.7

ESS; 9.2±4.7/5.8±4.0 EDS; 24%/72% (ESS>10)

NA/NA

EDS; 66.1%/2.9% (ESS≥10) EDS; 23.6%/39.4% (Mean SL<8 min.)

60.2±10.0 ESS; 5.6±3.0/6,1±2.0 26.7%/20.0% 9.5/4.6

**Frequency of SRBD PD/Controls**

48.1%/NA (AHI>5)

20%/40% (AHI>5)

43.6%/46.4% (AHI>5)

49.1%/65.1% (AHI≥5)

20%/NA (AHI>15) NA/NA

**AHI (/h) PD/Controls**

> 6±11/ 23±23

6.3-8.0(9.2-10.6)/ NA

> 12.5±15.6/ 12.2±13.1

**Age (years) PD/ Controls**

> 68±7.0/ NA

62.1 ± 9.8/ 62.4 ± 13.8

63.9±9.1/ 62.9±11.0

59.8±10.0/

65.4±9.1/ 59.3±9.1

**Table 8.** Polysomnographic studies evaluating sleep-related breathing disorders in PD

\*Referred for sleepiness, \*\*Controls were obtained from a population-based study, #drug-naïve PD patients

Sleep disorders can occur in the early stages of PD and worsen as the disease progresses. The cause of sleep disorders in PD is multifactorial, reflecting PD-related pathology, various aspects of PD-related motor and non-motor symptoms, and comorbidity of primary sleep disorders. Early recognition of and active intervention for sleep disturbance is of great importance, as sleep disturbances significantly impair the quality of life of patients with PD.

## **Author details**

Keisuke Suzuki1 , Tomoyuki Miyamoto2 , Masayuki Miyamoto1 , Ayaka Numao1 , Hideki Sakuta1 , Hiroaki Fujita1 , Yuji Watanabe1 , Masaoki Iwanami2 and Koichi Hirata1

1 Department of Neurology, Dokkyo Medical University, Japan

2 Department of Neurology, Dokkyo Medical University Koshigaya Hospital, Japan

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68 A Synopsis of Parkinson's Disease


**Chapter 3**

**Melatonin in Parkinson's Disease**

Alessia Carocci, Maria Stefania Sinicropi, Alessia Catalano, Graziantonio Lauria and

Additional information is available at the end of the chapter

and cognitive decline in the later stages of PD [2].

Parkinson's disease (PD) is characterized by the progressive depletion of pigmented neurons containing dopamine (DA) in the region known as substantia nigra pars compacta (SNpc) and by the presence of intraneuronal aggregates called Lewy bodies, which are enriched in filamentous α-synuclein and other proteins, that are often ubiquitinated before being de‐ stroyed [1]. The locus coeruleus, the dorsal motor nucleus, the autonomic nervous system and the cerebral cortex are additional neuronal fields and neurotransmitter systems involved in PD with consequent loss of noradrenergic, serotonergic and cholinergic neurons. These neuronal changes led to progressive non-motor symptoms like sleep abnormalities, depression

Currently, levodopa is widely prescribed for the treatment of PD. Although it is highly effective as a symptomatic treatment, levodopa is incapable of providing the long-term protection that is needed to impair the onset or progress of the disease [3]. In fact, in addition to a few specific mutations, oxidative stress and generation of free radicals from both mitochondrial impair‐ ment and DA metabolism play critical and important roles in PD etiology. Deficits in mito‐ chondrial functions, oxidative and nitrosative stress, accumulation of aberrant and misfolded proteins, and ubiquitin-proteasome system dysfunction can represent the main molecular

It is known that about 15% of PD patients has a family background of the disease and few specific mutations have been identified to be responsible for rare familial forms of the path‐ ology: α-synuclein, parkin, UCH-L1, DJ-1, and PINK1 are genes found to be related to PD [5]. These genetic defects seem to affect a common molecular pathway related to the ubiquitinproteasome system with exception of PINK1, which is related to mitochondrial metabolism [6].

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

pathways that trigger the pathogenesis of sporadic and familiar forms of PD [4].

Giuseppe Genchi

**1. Introduction**

http://dx.doi.org/10.5772/57352

**Chapter 3**

## **Melatonin in Parkinson's Disease**

Alessia Carocci, Maria Stefania Sinicropi, Alessia Catalano, Graziantonio Lauria and Giuseppe Genchi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/57352

## **1. Introduction**

Parkinson's disease (PD) is characterized by the progressive depletion of pigmented neurons containing dopamine (DA) in the region known as substantia nigra pars compacta (SNpc) and by the presence of intraneuronal aggregates called Lewy bodies, which are enriched in filamentous α-synuclein and other proteins, that are often ubiquitinated before being de‐ stroyed [1]. The locus coeruleus, the dorsal motor nucleus, the autonomic nervous system and the cerebral cortex are additional neuronal fields and neurotransmitter systems involved in PD with consequent loss of noradrenergic, serotonergic and cholinergic neurons. These neuronal changes led to progressive non-motor symptoms like sleep abnormalities, depression and cognitive decline in the later stages of PD [2].

Currently, levodopa is widely prescribed for the treatment of PD. Although it is highly effective as a symptomatic treatment, levodopa is incapable of providing the long-term protection that is needed to impair the onset or progress of the disease [3]. In fact, in addition to a few specific mutations, oxidative stress and generation of free radicals from both mitochondrial impair‐ ment and DA metabolism play critical and important roles in PD etiology. Deficits in mito‐ chondrial functions, oxidative and nitrosative stress, accumulation of aberrant and misfolded proteins, and ubiquitin-proteasome system dysfunction can represent the main molecular pathways that trigger the pathogenesis of sporadic and familiar forms of PD [4].

It is known that about 15% of PD patients has a family background of the disease and few specific mutations have been identified to be responsible for rare familial forms of the path‐ ology: α-synuclein, parkin, UCH-L1, DJ-1, and PINK1 are genes found to be related to PD [5]. These genetic defects seem to affect a common molecular pathway related to the ubiquitinproteasome system with exception of PINK1, which is related to mitochondrial metabolism [6].

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Some, if not all, of these mutations are partially related to free-radical generation. High levels of free-radical, reactive oxygen species (ROS) and reactive nitrogen species (RNS) damage not only phospholipids and polyunsaturated fatty acids of mitochondrial bilayers but also mitochondrial DNA (mtDNA) and mitochondrial proteins [7]. Uncontrolled increase in these metabolites lead to free radical-mediated chain reactions which indiscriminately target proteins, lipids and DNA resulting in cell death [8], producing neurodegeneration, at least in part, through the mitochondrial apoptotic pathway [9]. Several experimentally PD models are used to study the pathogenesis of the disease. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin able to produce experimentally Parkinson's disease in humans and monkeys (Figure 1). When administered to animals, MPTP readily crosses the blood-brain barrier (BBB), where it selectively destroys DA neurons in the substantia nigra (SN). Once MPTP crosses the BBB, it enters astrocytes, where it is converted into the active metabolite 1 methyl-4-phenylpyridinium (MPP+ ) by the action of the enzyme monoamine oxidase B (MAO B) [10]. MPP+ leaves the astrocytes and via the DA transporter enters the dopaminergic neurons. First of all, MPP+ accumulates into mitochondrial matrix, where it inhibits the Krebs cycle enzyme α-ketoglutarate dehydrogenase [11]. In addition, this metabolite inhibits complex I of the electron transport chain (ETC), causing increased generation of ROS, de‐ creased adenosine triphosphate (ATP) production and nigral cell death [12,13]. MPP+ , by inducing nitric oxide synthase (NOS) expression in SNpc, has been shown to produce large amounts of nitric oxide (NO) that, reacting with O2 – generates the highly toxic peroxynitrite (ONOO– ), a molecule that impairs mitochondrial functions causing irreversible inhibition of all ETC complexes [14] and neuronal cell death [15].

There are several agents that are currently under investigation for their potential neuropro‐ tective effects based on their capacity to modify mitochondrial dysfunction. These include creatine, melatonin (MLT), nicotine, nicotinamide, lipoic acid, acetyl-L-carnitine, resveratrol etc. (Table 1) [21]. Among these compounds, melatonin has shown to be effective in preventing neuronal cell death and ameliorating PD symptoms in several *in vivo* and *in vitro* PD models.

MLT is a natural hormone secreted by the pineal gland that easily crosses BBB. This hormone regulates and modulates a wide variety of physiological functions. Besides the well-known chronobiotic and sleep inducing properties [22], many other physiological effects have been ascribed to MLT, such as the modulation of cardiovascular [23] and immune [24] systems and the influence on hormone secretion and metabolism [25]. Other effects of MLT described in the literature include antitumor [26,27], anti-inflammatory [28], pain modulator [29], neuro‐

<sup>N</sup> N+

O

H3CO

S - OCH3

N H

<sup>H</sup> <sup>S</sup>

O O

Melatonin in Parkinson's Disease http://dx.doi.org/10.5772/57352 73

O

**Rotenone**

N

**Maneb**

S

S - Mn2+

H

H

MAO B

**MPTP MPP+**

protective [30,31], and antioxidant [32] activities.

OH NH2

**6-OHDA**

**Paraquat**

**Figure 1.** Toxins in experimental PD models.

N+ Cl-

OH OH

N+

Cl-

Together with MPTP, other toxin-based models frequently used to induce dopaminergic neurodegeneration include the neurotoxin 6-hydroxydopamine (6-OHDA), the herbicides paraquat (*N,N′-*dimethyl-4,4′-bipyridinium dichloride) and rotenone, and the fungicide maneb (Figure 1). They are capable of inducing the pathological hallmark of PD, the neuronal cell loss in the SN. The main contributing factor to this cell loss is mitochondrial dysfunction by inhibiting complex I, resulting in oxidative stress and eventually cell death [16]. In partic‐ ular, neurotoxin 6-OHDA induces reduction of the antioxidant glutathione (GSH) and antioxidant enzyme superoxide dismutase (SOD) [17], increase of iron levels in SN [18] and inhibition of complexes I and IV in mitochondria [19] which lead to further oxidative stress. The herbicide paraquat having a structural similarity to MPP+ directly inhibits complex I [20] and produces oxidative stress through redox cycling. The herbicide rotenone, extracted from tropical plants, easily crosses the BBB and accumulates inside the mitochondrial dopaminergic neuron, where it inhibits complex I. Maneb, on the other hand, induces the nigrostriatal dopaminergic neurodegeneration by inhibiting complex III [16].

Actually, considering that the existence of mitochondrial damage, due to oxidative stress, is the base of the disease which may lead to a decrease in the activities of mitochondrial com‐ plexes and ATP production, and as a consequence, a further increase in free radical generation, with the final consequence being cell death by necrosis or apoptosis, the use of antioxidants as an important co-treatment with traditional therapies has been suggested.

There are several agents that are currently under investigation for their potential neuropro‐ tective effects based on their capacity to modify mitochondrial dysfunction. These include creatine, melatonin (MLT), nicotine, nicotinamide, lipoic acid, acetyl-L-carnitine, resveratrol etc. (Table 1) [21]. Among these compounds, melatonin has shown to be effective in preventing neuronal cell death and ameliorating PD symptoms in several *in vivo* and *in vitro* PD models.

MLT is a natural hormone secreted by the pineal gland that easily crosses BBB. This hormone regulates and modulates a wide variety of physiological functions. Besides the well-known chronobiotic and sleep inducing properties [22], many other physiological effects have been ascribed to MLT, such as the modulation of cardiovascular [23] and immune [24] systems and the influence on hormone secretion and metabolism [25]. Other effects of MLT described in the literature include antitumor [26,27], anti-inflammatory [28], pain modulator [29], neuro‐ protective [30,31], and antioxidant [32] activities.

Some, if not all, of these mutations are partially related to free-radical generation. High levels of free-radical, reactive oxygen species (ROS) and reactive nitrogen species (RNS) damage not only phospholipids and polyunsaturated fatty acids of mitochondrial bilayers but also mitochondrial DNA (mtDNA) and mitochondrial proteins [7]. Uncontrolled increase in these metabolites lead to free radical-mediated chain reactions which indiscriminately target proteins, lipids and DNA resulting in cell death [8], producing neurodegeneration, at least in part, through the mitochondrial apoptotic pathway [9]. Several experimentally PD models are used to study the pathogenesis of the disease. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin able to produce experimentally Parkinson's disease in humans and monkeys (Figure 1). When administered to animals, MPTP readily crosses the blood-brain barrier (BBB), where it selectively destroys DA neurons in the substantia nigra (SN). Once MPTP crosses the BBB, it enters astrocytes, where it is converted into the active metabolite 1-

) by the action of the enzyme monoamine oxidase B (MAO

generates the highly toxic peroxynitrite

directly inhibits complex I [20]

, by

leaves the astrocytes and via the DA transporter enters the dopaminergic

–

), a molecule that impairs mitochondrial functions causing irreversible inhibition of

neurons. First of all, MPP+ accumulates into mitochondrial matrix, where it inhibits the Krebs cycle enzyme α-ketoglutarate dehydrogenase [11]. In addition, this metabolite inhibits complex I of the electron transport chain (ETC), causing increased generation of ROS, de‐ creased adenosine triphosphate (ATP) production and nigral cell death [12,13]. MPP+

inducing nitric oxide synthase (NOS) expression in SNpc, has been shown to produce large

Together with MPTP, other toxin-based models frequently used to induce dopaminergic neurodegeneration include the neurotoxin 6-hydroxydopamine (6-OHDA), the herbicides paraquat (*N,N′-*dimethyl-4,4′-bipyridinium dichloride) and rotenone, and the fungicide maneb (Figure 1). They are capable of inducing the pathological hallmark of PD, the neuronal cell loss in the SN. The main contributing factor to this cell loss is mitochondrial dysfunction by inhibiting complex I, resulting in oxidative stress and eventually cell death [16]. In partic‐ ular, neurotoxin 6-OHDA induces reduction of the antioxidant glutathione (GSH) and antioxidant enzyme superoxide dismutase (SOD) [17], increase of iron levels in SN [18] and inhibition of complexes I and IV in mitochondria [19] which lead to further oxidative stress.

and produces oxidative stress through redox cycling. The herbicide rotenone, extracted from tropical plants, easily crosses the BBB and accumulates inside the mitochondrial dopaminergic neuron, where it inhibits complex I. Maneb, on the other hand, induces the nigrostriatal

Actually, considering that the existence of mitochondrial damage, due to oxidative stress, is the base of the disease which may lead to a decrease in the activities of mitochondrial com‐ plexes and ATP production, and as a consequence, a further increase in free radical generation, with the final consequence being cell death by necrosis or apoptosis, the use of antioxidants

methyl-4-phenylpyridinium (MPP+

amounts of nitric oxide (NO) that, reacting with O2

all ETC complexes [14] and neuronal cell death [15].

The herbicide paraquat having a structural similarity to MPP+

dopaminergic neurodegeneration by inhibiting complex III [16].

as an important co-treatment with traditional therapies has been suggested.

B) [10]. MPP+

72 A Synopsis of Parkinson's Disease

(ONOO–

**6-OHDA**

**Rotenone**

**Maneb**

**Figure 1.** Toxins in experimental PD models.

Many *in vitro* and *in vivo* experimental models have contributed to demonstrate the role of MLT as an efficient radical scavenger against several reactive oxygen species (ROS), for example, the hydroxyl radical, the peroxynitrite anion, the superoxide anion, and singlet oxygen [33]. MLT has also been shown to enhance the production and the activity of several antioxidant enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GRd), catalase, and glucose-6-phosphate dehydrogenase [34,35]. Furthermore, *in vivo* observations on the protective role of MLT in ischemic brain injury [36] or in animal models of PD [37] emphasize the therapeutic potential of this compound as a neuroprotective agent [38]. Moreover, MLT increases the efficiency of the electron transport chain thereby limiting electron integrity of the mitochondria and helps to maintain cell functions and survival [39]. Treatment with MLT counteracts the effects of MPTP in brain nuclei, increasing complex I activity, and the effects of MPTP on lipid peroxidation and nitrite levels in the cytosol and in the mitochondria of mice brain [40]. There is growing evidence that MLT antiapoptotic effects play an important role in neurodegeneration as well [41].

**Agent Structure**

OH

S S

H3CO

OH O

OH

O O

> N H

> > N

NH2

N S

NH2

OH

O

N

N

OH

F3CO

OH

OH

Melatonin in Parkinson's Disease http://dx.doi.org/10.5772/57352 75

OH

OH

OH

OH

COOH

NHCOCH3

(–)-Epigallocatechin gallate

(EGCG)

(R)-Lipoic acid

Melatonin (MLT)

(–)-Nicotine

Nicotinamide

Resveratrol

Riluzole

**Table 1.** Neuroprotective agents in PD models.

**Table 1.** Neuroprotective agents in PD models.

Many *in vitro* and *in vivo* experimental models have contributed to demonstrate the role of MLT as an efficient radical scavenger against several reactive oxygen species (ROS), for example, the hydroxyl radical, the peroxynitrite anion, the superoxide anion, and singlet oxygen [33]. MLT has also been shown to enhance the production and the activity of several antioxidant enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GRd), catalase, and glucose-6-phosphate dehydrogenase [34,35]. Furthermore, *in vivo* observations on the protective role of MLT in ischemic brain injury [36] or in animal models of PD [37] emphasize the therapeutic potential of this compound as a neuroprotective agent [38]. Moreover, MLT increases the efficiency of the electron transport chain thereby limiting electron integrity of the mitochondria and helps to maintain cell functions and survival [39]. Treatment with MLT counteracts the effects of MPTP in brain nuclei, increasing complex I activity, and the effects of MPTP on lipid peroxidation and nitrite levels in the cytosol and in the mitochondria of mice brain [40]. There is growing evidence that

MLT antiapoptotic effects play an important role in neurodegeneration as well [41].

**Agent Structure**

Creatine <sup>2</sup> NNH

N+

O

O OH

O

O

OH N+ COO-

N <sup>N</sup> <sup>N</sup> O N

COOH

OCH3

OH

NH

O O

H3CO

OH

O

O

COO-

Acetyl-L-carnitine

74 A Synopsis of Parkinson's Disease

(acetylsalicylic acid)

Aspirin

Carnitine

Caffeine

Curcumin

## **2. Neuroprotective agents for Parkinson's disease**

Relevant preclinical studies have identified several compounds such as MLT, estrogen, nicotine, caffeine, riluzole, curcumin, aspirin, epigallocatechin-3-gallate (EGCG) and resvera‐ trol, as neuroprotective agents in PD [42] (Table 1). Various prospective studies have suggested a strong association between tobacco smoking and a decreased risk of PD. Nicotine is one of the main constituents of tobacco and is known for its pharmacological effects, exerted by interaction with cholinergic nicotinic receptors in both central and peripheral nervous systems [43]. A recent clinical trial among six male PD patients demonstrated that chronic high doses of nicotine improved motor scores, reduced dopaminergic treatment and had a potential beneficial effect on striatal dopamine transporter density [44]. Chronic nicotine treatment partly protects against the MPTP-induced degeneration of nigrostriatal dopamine neurons in the black mouse, counteracts the disappearance of tyrosine-hydroxylase-immunoreactive nerve cell bodies, dendrites and terminals in the mesostriatal dopamine system and prevent striatal dopamine loss provoked by 6-OHDA administration in the substantia nigra [45-47].

Many evidences reported that an important risk factor for the disease is aging [60]. It contrib‐ utes to PD progression because of accumulative oxidative damage and decrease of antioxidant capacity. Genetic studies have also revealed that aging can be controlled by changes in intracellular NAD/NADH ratio regulating sirtuins, a group of proteins linked to aging, metabolism and stress tolerance in several organisms. Consistently, the neuroprotective roles of dietary antioxidants including for example, acetyl-L-carnitine, curcumin, epigallocate‐ chin-3-gallate (EGCG), carnosine, resveratrol, etc. have been demonstrated through the

Melatonin in Parkinson's Disease http://dx.doi.org/10.5772/57352 77

In particular, acetyl-L-carnitine has been proposed to have beneficial effects in preventing the loss of brain function which typically occurs during aging and neurodegenerative disorders [21]. In fact, acetyl-L-carnitine treatment has been shown to prevent age-related changes in mitochondrial respiration and decrease oxidative stress biomarkers through the up-regulation of HO-1 (heme oxygenase-1), Hsp70 (heat shock protein 70) and superoxide dismutase-2 in senescent rats [61]. Acetyl-L-carnitine has shown to be neuroprotective through a variety of other effects such as the increase in protein kinase C (PKC) activity [62]. Moreover acetyl-Lcarnitine has also been reported to attenuate the occurrence of parkinsonian symptoms associated with MPTP *in vivo*, and protects *in vitro* against the toxicity of neurotoxic MPP+ [63]. Curcumin is an active polyphenolic compound of Turmeric (*Curcuma longa*), which is exten‐ sively used as dietary spice in Indian food. Curcumin is used as a food additive because of its yellow colouring properties and presents anti-inflammatory and antioxidant properties. Recent studies demonstrated the neuroprotective effects of pretreatment with curcumin in the 6-OHDA model in rats. Both motor deficits and neuronal damage were prevented by curcumin and by one of its main metabolites, tetrahydrocurcumin, which also had beneficial effects on the antioxidant status, with increasing GSH levels and activity of antioxidant enzymes. Curcumin inhibited, in fact, MAO-B activity which prevents the conversion of MPTP to its

EGCG is a catechin ubiquitously found in plants and is an important substance in green tea. Interestingly, there are several epidemiological studies that investigated an association between tea and PD. Among tea drinkers, the risk of developing PD was lower than in nontea drinkers [65]. This effect was thought to be especially influenced by EGCG, to which has been ascribed a wide range of therapeutic properties, including neuroprotection. In fact, green tea and EGCG prevented MPTP-induced neuron loss and inhibited the upregulation of striatal

Inflammation is believed to be one of the important factors in the pathogenesis of PD. More‐ over, it had been demonstrated that the enzyme cyclooxygenase (COX) and other inflamma‐ tory proteins are elevated in PD. Therefore, there is a significant interest in non-steroidal antiinflammatory drugs (NSAIDs), especially aspirin [42]. The aspirin has an additional free radical scavenging property in addition to COX2 inhibition. In a study reported by Marahaj and co-authors, aspirin (100 mg/kg) and paracetamol (100 mg/kg) prevented KCN-induced superoxide generation and lipid peroxidation. While paracetamol was a more effective antioxidant, aspirin completely blocked the debilitating effects of MPP+ on striatal DA in rats,

whereas paracetamol was only able to partially block this effect [67].

activation of these redox-sensitive intracellular pathways.

toxic metabolite MPP+ [64].

SOD and catalase enzymes [66].

17β-estradiol (E2) is a predominant sex hormone that acts on the whole body. Since several epidemiological studies have shown a greater incidence of PD in men than women, extensive research have investigated the possible neuroprotective effects of E2 in MPTP mice models and in 6-OHDA-injury model [48,49]. Estrogens alters MPTP-induced neurotoxicity in female mice with effects on striatal DA concentrations and release [50]. E2 prevents loss of dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) in substantia nigra, induces regulation of striatal preproenkephalin mPRNA levels in MPTP-lesioned mice, protects the SNpc of female rats from lesion induced by 6-OHDA and interacts with the insulin-like growth factor-1 (IGF-1) system to protect nigrostriatal dopamine and maintain motoric behavior after 6-OHDA lesions [51-53].

Caffeine is the most widely used psychoactive substance in the world due to its presence in coffee and other beverages. Several epidemiological studies have linked coffee intake with a lower incidence of PD, suggesting neuroprotective properties for caffeine and demonstrating its strong neuroprotective role in rodents for various injury models [54,55]. In particular, Chen and co-authors found that caffeine (10 mg/kg) was neuroprotective when administered 10 min prior to four injections of MPTP [56], attenuating the depletions in striatal DA, 3,4-dihydrox‐ yphenylacetic acid (DOPAC) and DAT-binding sites. The same effects were also established in a 6-OHDA model [57]. Several epidemiological studies suggested an interaction between estrogen and caffeine. It has been reported that caffeine attenuated the toxic effects of MPTP in male mice in a dose-dependent manner. In contrast, this results was not found in female mice and estrogen treatment also prevented this effect in young male mice [58].

Riluzole is a selective Na+ -channel blocker and some researchers have demonstrated its neuroprotective effects in rodents and in a primate model. Boireau and co-authors reported that riluzole neuroprotection in combination with MPTP was due to interference with MPP+ production by MAO-B inhibition. The protective effect was confirmed in MPTP- treated mice, partially due to astrocyte activation [59].

Many evidences reported that an important risk factor for the disease is aging [60]. It contrib‐ utes to PD progression because of accumulative oxidative damage and decrease of antioxidant capacity. Genetic studies have also revealed that aging can be controlled by changes in intracellular NAD/NADH ratio regulating sirtuins, a group of proteins linked to aging, metabolism and stress tolerance in several organisms. Consistently, the neuroprotective roles of dietary antioxidants including for example, acetyl-L-carnitine, curcumin, epigallocate‐ chin-3-gallate (EGCG), carnosine, resveratrol, etc. have been demonstrated through the activation of these redox-sensitive intracellular pathways.

**2. Neuroprotective agents for Parkinson's disease**

6-OHDA lesions [51-53].

76 A Synopsis of Parkinson's Disease

Riluzole is a selective Na+

partially due to astrocyte activation [59].

Relevant preclinical studies have identified several compounds such as MLT, estrogen, nicotine, caffeine, riluzole, curcumin, aspirin, epigallocatechin-3-gallate (EGCG) and resvera‐ trol, as neuroprotective agents in PD [42] (Table 1). Various prospective studies have suggested a strong association between tobacco smoking and a decreased risk of PD. Nicotine is one of the main constituents of tobacco and is known for its pharmacological effects, exerted by interaction with cholinergic nicotinic receptors in both central and peripheral nervous systems [43]. A recent clinical trial among six male PD patients demonstrated that chronic high doses of nicotine improved motor scores, reduced dopaminergic treatment and had a potential beneficial effect on striatal dopamine transporter density [44]. Chronic nicotine treatment partly protects against the MPTP-induced degeneration of nigrostriatal dopamine neurons in the black mouse, counteracts the disappearance of tyrosine-hydroxylase-immunoreactive nerve cell bodies, dendrites and terminals in the mesostriatal dopamine system and prevent striatal dopamine loss provoked by 6-OHDA administration in the substantia nigra [45-47].

17β-estradiol (E2) is a predominant sex hormone that acts on the whole body. Since several epidemiological studies have shown a greater incidence of PD in men than women, extensive research have investigated the possible neuroprotective effects of E2 in MPTP mice models and in 6-OHDA-injury model [48,49]. Estrogens alters MPTP-induced neurotoxicity in female mice with effects on striatal DA concentrations and release [50]. E2 prevents loss of dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) in substantia nigra, induces regulation of striatal preproenkephalin mPRNA levels in MPTP-lesioned mice, protects the SNpc of female rats from lesion induced by 6-OHDA and interacts with the insulin-like growth factor-1 (IGF-1) system to protect nigrostriatal dopamine and maintain motoric behavior after

Caffeine is the most widely used psychoactive substance in the world due to its presence in coffee and other beverages. Several epidemiological studies have linked coffee intake with a lower incidence of PD, suggesting neuroprotective properties for caffeine and demonstrating its strong neuroprotective role in rodents for various injury models [54,55]. In particular, Chen and co-authors found that caffeine (10 mg/kg) was neuroprotective when administered 10 min prior to four injections of MPTP [56], attenuating the depletions in striatal DA, 3,4-dihydrox‐ yphenylacetic acid (DOPAC) and DAT-binding sites. The same effects were also established in a 6-OHDA model [57]. Several epidemiological studies suggested an interaction between estrogen and caffeine. It has been reported that caffeine attenuated the toxic effects of MPTP in male mice in a dose-dependent manner. In contrast, this results was not found in female

neuroprotective effects in rodents and in a primate model. Boireau and co-authors reported that riluzole neuroprotection in combination with MPTP was due to interference with MPP+ production by MAO-B inhibition. The protective effect was confirmed in MPTP- treated mice,


mice and estrogen treatment also prevented this effect in young male mice [58].

In particular, acetyl-L-carnitine has been proposed to have beneficial effects in preventing the loss of brain function which typically occurs during aging and neurodegenerative disorders [21]. In fact, acetyl-L-carnitine treatment has been shown to prevent age-related changes in mitochondrial respiration and decrease oxidative stress biomarkers through the up-regulation of HO-1 (heme oxygenase-1), Hsp70 (heat shock protein 70) and superoxide dismutase-2 in senescent rats [61]. Acetyl-L-carnitine has shown to be neuroprotective through a variety of other effects such as the increase in protein kinase C (PKC) activity [62]. Moreover acetyl-Lcarnitine has also been reported to attenuate the occurrence of parkinsonian symptoms associated with MPTP *in vivo*, and protects *in vitro* against the toxicity of neurotoxic MPP+ [63].

Curcumin is an active polyphenolic compound of Turmeric (*Curcuma longa*), which is exten‐ sively used as dietary spice in Indian food. Curcumin is used as a food additive because of its yellow colouring properties and presents anti-inflammatory and antioxidant properties. Recent studies demonstrated the neuroprotective effects of pretreatment with curcumin in the 6-OHDA model in rats. Both motor deficits and neuronal damage were prevented by curcumin and by one of its main metabolites, tetrahydrocurcumin, which also had beneficial effects on the antioxidant status, with increasing GSH levels and activity of antioxidant enzymes. Curcumin inhibited, in fact, MAO-B activity which prevents the conversion of MPTP to its toxic metabolite MPP+ [64].

EGCG is a catechin ubiquitously found in plants and is an important substance in green tea. Interestingly, there are several epidemiological studies that investigated an association between tea and PD. Among tea drinkers, the risk of developing PD was lower than in nontea drinkers [65]. This effect was thought to be especially influenced by EGCG, to which has been ascribed a wide range of therapeutic properties, including neuroprotection. In fact, green tea and EGCG prevented MPTP-induced neuron loss and inhibited the upregulation of striatal SOD and catalase enzymes [66].

Inflammation is believed to be one of the important factors in the pathogenesis of PD. More‐ over, it had been demonstrated that the enzyme cyclooxygenase (COX) and other inflamma‐ tory proteins are elevated in PD. Therefore, there is a significant interest in non-steroidal antiinflammatory drugs (NSAIDs), especially aspirin [42]. The aspirin has an additional free radical scavenging property in addition to COX2 inhibition. In a study reported by Marahaj and co-authors, aspirin (100 mg/kg) and paracetamol (100 mg/kg) prevented KCN-induced superoxide generation and lipid peroxidation. While paracetamol was a more effective antioxidant, aspirin completely blocked the debilitating effects of MPP+ on striatal DA in rats, whereas paracetamol was only able to partially block this effect [67].

Also resveratrol, a polyphenol compound, found in grapes and in red wine, has shown antiinflammatory, anti-oxidant, and neuroprotective properties. The effects of resveratrol on the 6-OHDA injury in rats were studied by Khan and colleagues [68]. They have demonstrated that resveratrol was not only capable to protect neurons, but also to increase the activity of antioxidant enzymes and decrease the levels of thiobarbituric acid reactive substances (TBARS), protein carbonyl (PC), and phospholipase A2 (PA2), providing evidence for a possible antioxidant property. Then, pretreatment with resveratrol (50 and 100 mg/kg) prevented neuronal cell loss in the SN and striatal DA depletion, in 6-OHDA-injury model in rats it was neuroprotective and it has been shown to decrease mRNA and protein levels of TNF-α in COX2, suggesting that an anti-inflammatory mechanism underlies the protective effects of this polyphenol [69,70].

sensitive form of the human enzyme quinine reductase 2 [81]. MLT is also a ligand for retinoid orphan nuclear hormone receptors referred to as RZRα and RZRβ at concentrations in the low nanomolar range. Both receptors are present in the central and peripheral nervous system and have been associated with cell differentiation and immune response regulation [82,83]. The melatonin MT1 receptor is coupled to different G proteins that mediate the inhibition of adenylyl cyclase and the activation of phospholipase C [84], while the MT2 receptor is coupled to a number of signal transduction mechanisms, among them phosphoinositide production,

Melatonin in Parkinson's Disease http://dx.doi.org/10.5772/57352 79

Tryptophan serves as the precursor for the biosynthesis of MLT (Figure 2). It is converted into serotonin via 5-hydroxytryptophan. Serotonin is then acetylated to form *N*-acetylserotonin by arylalkylamine *N*-acetyltransferase (AANAT or NAT), one of the key enzyme in MLT synthesis. *N*-acetylserotonin is then converted to MLT by hydroxyindole-*O*-methyltransferase (HIOMT) which has been identified as the rate-limiting enzyme in the biosynthesis of pineal MLT [85]. In all mammals pineal MLT biosynthesis is synchronized to light/dark cycle by the SCN, which receives its input from the retinohypothalamic tract. Special photoreceptive retinal ganglion cells containing melanopsin as a photopigment are involved in the projection from retina [86]. Fibers from the SCN pass through a circuitous route involving the paraventricular nucleus of the hypothalamus and then proceed to innervate pineal gland as postganglionic sympathetic fibers. Norepinephrine released from these fibers binds to postsynaptic adreno‐ ceptors whose activation induces an increase in cyclic adenosine-3′,5′-monophosphate (cyclic

MLT has two important functional groups which determine its specificity and amphiphilicity: the 5-methoxy group and the *N*-acetyl side chain. Due to its lipophilic nature and p*K*a, MLT readily crosses the BBB. Once formed within the pineal gland, the majority of MLT diffuses directly towards the cerebrospinal fluid of the brain's third ventricle, while another fraction is released into the blood stream where it is distributed to all tissues. The brain has much higher

Circulating MLT is partially bound to albumin and can also binds to hemoglobin [89,90]. MLT is mainly metabolized in the liver via hydroxylation reaction by cytochrome P450 monooxygenases. This reaction is followed by conjugation with sulfuric or glucuronic acid, to produce the principal urinary metabolite, 6-sulfatoxymelatonin. Conjugated MLT and minute quantities of unmetabolized MLT are eliminated through the kidney. In addition to hepatic metabolism, oxidative pyrrole-ring cleavage appears to be the major metabolic pathway in

MLT seems to function *via* a number of means to reduce oxidative stress. It can develop its action at two levels: as a direct antioxidant, due its ability to act as a free radical scavenger, and as an indirect antioxidant, since it is able to induce the expression and/or the activity of

MLT is a powerful free radical scavenger since it is able to remove H2O2, •OH, peroxinitrite

rich molecule, is able to interact with free radicals through consecutive reactions giving rise to

•– and peroxyl radical (LOO•). MLT, as an electron-

O2), O2

inhibition of adenylyl cyclase and guanylyl cyclase [80].

AMP) accumulation and a subsequent activation of NAT [87].

concentrations of MLT than any other tissue in the body [88].

other tissues, including CNS [91].

the main antioxidant enzymes.

), singlet oxygen (1

anion (ONOO–

## **3. Melatonin**

Melatonin (*N*-acetyl-5-methoxy triptamine, MLT), a triptophan derivative, is a highly conser‐ vative naturally occurring molecule present in a wide spectrum of organisms, including bacteria, fungi, plants, protozoa, invertebrates [71] and vertebrates. In vertebrates, MLT is primarily produced by the pineal gland with a marked circadian rhythm that is governed by the central circadian pacemaker in the suprachiasmatic nuclei (SCN) of the hypothalamus, the highest levels occurring during the period of darkness [72]. Extrapineal sites of MLT produc‐ tion include retina, Harderian gland, gut, bone marrow [74], platelets, and skin [75]. However, with the exception of retina, the physiological significance of these extrapineal sites is still a matter of debate. MLT was first isolated and identified in the bovine pineal gland by Lerner and coworkers in 1958 [76].

MLT acts as time-giver (*Zeitgeber*) in the regulation of circadian rhythms [77,78] and in synchronizing the reproductive cycle with the appropriate season of the year in photoperiodic species [8]. In non-photoperiodic species such as humans, MLT actions consist in consolidation of sleep and regulation of the circadian rhythm [9]. MLT actions, however, are not restricted to its role in the neuroendocrine physiology. Many other physiological effects have been ascribed to MLT, such as the modulation of cardiovascular [23] and immune [24] systems and the influence on hormone secretion and metabolism [25]. Other effects of MLT described in the literature include antitumor [26, 27], anti-inflammatory [28], pain modulator [29], neuro‐ protective [30, 31] and antioxidant [32] properties. MLT have also been associated with the cellular antioxidant defence since it is a powerful free radical scavenger, and it is able to induce the expression and/or the activity of the main antioxidant enzymes [79].

MLT exerts its actions by multiple mechanisms. Many of its physiological actions are mediated through activation of distinct MLT receptors expressed in a wide variety of tissues. Cloning studies have revealed at least three MLT receptor subtypes, two of which (MT1 and MT2) have been found in mammals and are localized in different areas of the central nervous system (CNS) as well as in peripheral tissues [80]. Moreover, a non-mammalian MLT binding site with a lower affinity profile (MT3) has been found in hamster brain and characterized as a MLT- sensitive form of the human enzyme quinine reductase 2 [81]. MLT is also a ligand for retinoid orphan nuclear hormone receptors referred to as RZRα and RZRβ at concentrations in the low nanomolar range. Both receptors are present in the central and peripheral nervous system and have been associated with cell differentiation and immune response regulation [82,83]. The melatonin MT1 receptor is coupled to different G proteins that mediate the inhibition of adenylyl cyclase and the activation of phospholipase C [84], while the MT2 receptor is coupled to a number of signal transduction mechanisms, among them phosphoinositide production, inhibition of adenylyl cyclase and guanylyl cyclase [80].

Also resveratrol, a polyphenol compound, found in grapes and in red wine, has shown antiinflammatory, anti-oxidant, and neuroprotective properties. The effects of resveratrol on the 6-OHDA injury in rats were studied by Khan and colleagues [68]. They have demonstrated that resveratrol was not only capable to protect neurons, but also to increase the activity of antioxidant enzymes and decrease the levels of thiobarbituric acid reactive substances (TBARS), protein carbonyl (PC), and phospholipase A2 (PA2), providing evidence for a possible antioxidant property. Then, pretreatment with resveratrol (50 and 100 mg/kg) prevented neuronal cell loss in the SN and striatal DA depletion, in 6-OHDA-injury model in rats it was neuroprotective and it has been shown to decrease mRNA and protein levels of TNF-α in COX2, suggesting that an anti-inflammatory mechanism underlies the protective

Melatonin (*N*-acetyl-5-methoxy triptamine, MLT), a triptophan derivative, is a highly conser‐ vative naturally occurring molecule present in a wide spectrum of organisms, including bacteria, fungi, plants, protozoa, invertebrates [71] and vertebrates. In vertebrates, MLT is primarily produced by the pineal gland with a marked circadian rhythm that is governed by the central circadian pacemaker in the suprachiasmatic nuclei (SCN) of the hypothalamus, the highest levels occurring during the period of darkness [72]. Extrapineal sites of MLT produc‐ tion include retina, Harderian gland, gut, bone marrow [74], platelets, and skin [75]. However, with the exception of retina, the physiological significance of these extrapineal sites is still a matter of debate. MLT was first isolated and identified in the bovine pineal gland by Lerner

MLT acts as time-giver (*Zeitgeber*) in the regulation of circadian rhythms [77,78] and in synchronizing the reproductive cycle with the appropriate season of the year in photoperiodic species [8]. In non-photoperiodic species such as humans, MLT actions consist in consolidation of sleep and regulation of the circadian rhythm [9]. MLT actions, however, are not restricted to its role in the neuroendocrine physiology. Many other physiological effects have been ascribed to MLT, such as the modulation of cardiovascular [23] and immune [24] systems and the influence on hormone secretion and metabolism [25]. Other effects of MLT described in the literature include antitumor [26, 27], anti-inflammatory [28], pain modulator [29], neuro‐ protective [30, 31] and antioxidant [32] properties. MLT have also been associated with the cellular antioxidant defence since it is a powerful free radical scavenger, and it is able to induce

MLT exerts its actions by multiple mechanisms. Many of its physiological actions are mediated through activation of distinct MLT receptors expressed in a wide variety of tissues. Cloning studies have revealed at least three MLT receptor subtypes, two of which (MT1 and MT2) have been found in mammals and are localized in different areas of the central nervous system (CNS) as well as in peripheral tissues [80]. Moreover, a non-mammalian MLT binding site with a lower affinity profile (MT3) has been found in hamster brain and characterized as a MLT-

the expression and/or the activity of the main antioxidant enzymes [79].

effects of this polyphenol [69,70].

and coworkers in 1958 [76].

**3. Melatonin**

78 A Synopsis of Parkinson's Disease

Tryptophan serves as the precursor for the biosynthesis of MLT (Figure 2). It is converted into serotonin via 5-hydroxytryptophan. Serotonin is then acetylated to form *N*-acetylserotonin by arylalkylamine *N*-acetyltransferase (AANAT or NAT), one of the key enzyme in MLT synthesis. *N*-acetylserotonin is then converted to MLT by hydroxyindole-*O*-methyltransferase (HIOMT) which has been identified as the rate-limiting enzyme in the biosynthesis of pineal MLT [85]. In all mammals pineal MLT biosynthesis is synchronized to light/dark cycle by the SCN, which receives its input from the retinohypothalamic tract. Special photoreceptive retinal ganglion cells containing melanopsin as a photopigment are involved in the projection from retina [86]. Fibers from the SCN pass through a circuitous route involving the paraventricular nucleus of the hypothalamus and then proceed to innervate pineal gland as postganglionic sympathetic fibers. Norepinephrine released from these fibers binds to postsynaptic adreno‐ ceptors whose activation induces an increase in cyclic adenosine-3′,5′-monophosphate (cyclic AMP) accumulation and a subsequent activation of NAT [87].

MLT has two important functional groups which determine its specificity and amphiphilicity: the 5-methoxy group and the *N*-acetyl side chain. Due to its lipophilic nature and p*K*a, MLT readily crosses the BBB. Once formed within the pineal gland, the majority of MLT diffuses directly towards the cerebrospinal fluid of the brain's third ventricle, while another fraction is released into the blood stream where it is distributed to all tissues. The brain has much higher concentrations of MLT than any other tissue in the body [88].

Circulating MLT is partially bound to albumin and can also binds to hemoglobin [89,90]. MLT is mainly metabolized in the liver via hydroxylation reaction by cytochrome P450 monooxygenases. This reaction is followed by conjugation with sulfuric or glucuronic acid, to produce the principal urinary metabolite, 6-sulfatoxymelatonin. Conjugated MLT and minute quantities of unmetabolized MLT are eliminated through the kidney. In addition to hepatic metabolism, oxidative pyrrole-ring cleavage appears to be the major metabolic pathway in other tissues, including CNS [91].

MLT seems to function *via* a number of means to reduce oxidative stress. It can develop its action at two levels: as a direct antioxidant, due its ability to act as a free radical scavenger, and as an indirect antioxidant, since it is able to induce the expression and/or the activity of the main antioxidant enzymes.

MLT is a powerful free radical scavenger since it is able to remove H2O2, •OH, peroxinitrite anion (ONOO– ), singlet oxygen (1 O2), O2 •– and peroxyl radical (LOO•). MLT, as an electronrich molecule, is able to interact with free radicals through consecutive reactions giving rise to

The pineal production of MLT exhibits an unambiguous circadian rhythm with its peak near the middle of scotophase and basal levels during the photophase. The amount of MLT produced by the pineal gland of mammals changes as animals age. The tendency is that pineal MLT production wanes with advanced age. In humans, MLT production not only decreases in the aged but also is significantly lower in many age-related diseases as Alzheimer's,

H3CO

*N1*

H3CO

HCOOH

H2O2 CO2

N

**Cyclic-3-hydroxymelatonin (3-OHM)**

H

HO

N

**-acetyl-5-methoxykynuramine (AMK)**

NH2

N CH3 O

H

O

CH3 O

Melatonin in Parkinson's Disease http://dx.doi.org/10.5772/57352 81

Parkinson's and Huntington's disease [104,105] and cardiovascular disease [106,107].

OH .

Arylamine formaminidase

or Hemoperoxidase

*-N***<sup>2</sup>***- <sup>N</sup>***<sup>1</sup>***-*

Mitochondria are organelles found almost ubiquitously in eukaryotes, that play a central role in the cell physiology; in fact, besides their classic function of energy metabolism, these organelles perform many other functions including the distribution of energy through the cells, energy/heat modulation, ROS regulation, calcium homeostasis, and apoptosis control. In mitochondria important metabolic pathways take place including fatty acids β-oxidation, pyruvate oxidation, Krebs cycle, lipids and cholesterol biosynthesis. Many of these processes are functions required for the wellbeing of the cells and of the human beings. The inner mitochondrial membrane is rich in proteins, half of which are involved in oxidation-reduction reactions with transport of electrons and in oxidative phosphorylation (OXPHOS). The oxidative phosphorylation, coupled to electron transport chain (ETC), allows the synthesis of

N H

**Melatonin**

H3CO

H3CO N CH3

*N1* **-acetyl-N2 -formyl-5-methoxykynuramine (AFMK)**

**Figure 3.** Melatonin oxidation.

N

H

O

**4. Mitochondria and melatonin**

H

O

H2O2 Catalase O

N CH3 O

H

**Figure 2.** Biosynthetic pathway of melatonin.

many stable compounds that can be excreted by urine. In fact, the MLT antioxidant mechanism implied a free radical scavengers cascade, since secondary, and even tertiary metabolites are also efficient free radicals scavengers, like *N*-acetyl-*N*-formyl-5-methoxykynuramine (AFMK) and *N*-acetyl-methoxykynuramine (AMK) (Figure 3) [92,93]. The formation of such metabo‐ lites from MLT implies that, unlike classic antioxidants, melatonin does not produce prooxi‐ dant reactions and, even more, AMK and AFMK, in all the mitochondrial studies where comparisons were made, were more potent than MLT itself [94].

The large subcellular distribution of MLT allows its interaction with almost any kind of molecule, diminishing oxidative damage in both lipid and aqueous environments. This is supported experimentally by numerous data that show that MLT is able to protect lipids in the cellular membranes, proteins in the cytosol and DNA in the nucleus from free radical damage [95]. MLT gets free access to all cell components especially in the nucleus [96] and mitochondria [97], where it seems to accumulate in high concentration. In addition, MLT interacts with lipid bilayers of mitochondria, stabilizing its inner membrane [98], an effect that improves ETC activity [99].

Apart from its direct scavenging activity, MLT confers indirect protection against oxygen species through its capability to increase the gene expression and/or activities of antioxidant enzymes. This regulatory role is also mediated by the metabolites of MLT [34,35]. The expression of enzymes, such as GPx, GRd and SOD, related to the endogenous antioxidant system of the cells and the mitochondria, are under genomic regulation of MLT [100,101]. Some antioxidant properties of MLT are attributable to a genomic effect in the regulation of the activities of other antioxidant enzymes such as inducible (iNOS) and mitochondrial (mtNOS) isoforms of nitric oxide synthase [102]. MLT also inhibits neuronal nitric oxide synthase (nNOS) activity because of its binding to the calcium-calmodulin complex [103].

The pineal production of MLT exhibits an unambiguous circadian rhythm with its peak near the middle of scotophase and basal levels during the photophase. The amount of MLT produced by the pineal gland of mammals changes as animals age. The tendency is that pineal MLT production wanes with advanced age. In humans, MLT production not only decreases in the aged but also is significantly lower in many age-related diseases as Alzheimer's, Parkinson's and Huntington's disease [104,105] and cardiovascular disease [106,107].

**Figure 3.** Melatonin oxidation.

many stable compounds that can be excreted by urine. In fact, the MLT antioxidant mechanism implied a free radical scavengers cascade, since secondary, and even tertiary metabolites are also efficient free radicals scavengers, like *N*-acetyl-*N*-formyl-5-methoxykynuramine (AFMK) and *N*-acetyl-methoxykynuramine (AMK) (Figure 3) [92,93]. The formation of such metabo‐ lites from MLT implies that, unlike classic antioxidants, melatonin does not produce prooxi‐ dant reactions and, even more, AMK and AFMK, in all the mitochondrial studies where

The large subcellular distribution of MLT allows its interaction with almost any kind of molecule, diminishing oxidative damage in both lipid and aqueous environments. This is supported experimentally by numerous data that show that MLT is able to protect lipids in the cellular membranes, proteins in the cytosol and DNA in the nucleus from free radical damage [95]. MLT gets free access to all cell components especially in the nucleus [96] and mitochondria [97], where it seems to accumulate in high concentration. In addition, MLT interacts with lipid bilayers of mitochondria, stabilizing its inner membrane [98], an effect that

Apart from its direct scavenging activity, MLT confers indirect protection against oxygen species through its capability to increase the gene expression and/or activities of antioxidant enzymes. This regulatory role is also mediated by the metabolites of MLT [34,35]. The expression of enzymes, such as GPx, GRd and SOD, related to the endogenous antioxidant system of the cells and the mitochondria, are under genomic regulation of MLT [100,101]. Some antioxidant properties of MLT are attributable to a genomic effect in the regulation of the activities of other antioxidant enzymes such as inducible (iNOS) and mitochondrial (mtNOS) isoforms of nitric oxide synthase [102]. MLT also inhibits neuronal nitric oxide synthase

(nNOS) activity because of its binding to the calcium-calmodulin complex [103].

comparisons were made, were more potent than MLT itself [94].

N H

3CO NH

**Figure 2.** Biosynthetic pathway of melatonin.

N H

O

H

OH NH2

COOH

5-Hydroxytryptophan decarboxylase

**5-Hydroxytryptophan Serotonin**

+ *S*-Adenosylmethionine

Hydroxyindole-*O*-methyltransferase

**Melatonin** *N***-Acetylserotonin**

N H

N H

H NO CH3

O

*N*-Acetyltranferase + Acetyl-CoA

H

OH NH2

improves ETC activity [99].

N H

**Tryptophan**

NH2 COOH

80 A Synopsis of Parkinson's Disease

Tryptophan hydroxylase

## **4. Mitochondria and melatonin**

Mitochondria are organelles found almost ubiquitously in eukaryotes, that play a central role in the cell physiology; in fact, besides their classic function of energy metabolism, these organelles perform many other functions including the distribution of energy through the cells, energy/heat modulation, ROS regulation, calcium homeostasis, and apoptosis control. In mitochondria important metabolic pathways take place including fatty acids β-oxidation, pyruvate oxidation, Krebs cycle, lipids and cholesterol biosynthesis. Many of these processes are functions required for the wellbeing of the cells and of the human beings. The inner mitochondrial membrane is rich in proteins, half of which are involved in oxidation-reduction reactions with transport of electrons and in oxidative phosphorylation (OXPHOS). The oxidative phosphorylation, coupled to electron transport chain (ETC), allows the synthesis of adenosine triphosphate (ATP), a molecule rich in energy, via the enzyme complex ATP synthase.

and in the mitochondria. Under normal conditions, MLT reduces mitochondrial hydroperox‐ ide levels and stimulates the activity of GPx and GRd, enzymes involved in the GSH-GSSG balance [116]. The indoleamine MLT is also able to neutralize the oxidative stress induced by high doses of *t*-butyl hydroperoxide, restoring GSH levels and GPx and GRd activities.

Other antioxidants such as ascorbate, ubiquinone and α-tocopherol can participate in the

However, uncontrolled increase in these metabolites leads to a series of reactions which target proteins, lipids and DNA resulting in cell death by necrosis or apoptosis. In recent years, several findings support the antioxidant effect of MLT in mitochondrial homeostasis

Apoptosis and necrosis are two types of cell death occurring in neurodegeneration. Apoptosis (programmed cell death) occurs naturally under normal physiological conditions; on the contrary, necrosis is caused by external factors such as toxins, infections and trauma. Apoptosis is characterized by cell shrinkage, cytoplasm contraction, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation, plasma membrane bleb formation and apoptotic body formation [119]. Many of these changes are activated by a family of caspases, i.e. proteases that in their active site possess a cysteine and cleaves the substrates after aspartate residues. Apoptotic cells are rapidly sequestered by phagocytosis before they can lyse and cause an inflammatory process [120]. Necrosis does not involve any DNA or protein degra‐ dation and is accompanied by swelling of the cytoplasm and of the mitochondria with membrane ruptures. Both apoptosis and necrosis involve a change in mitochondrial mem‐

MMP causes the opening of a nonspecific pore in the mitochondrial membranes, known as the mitochondrial transition pore (MTP), that allows the passage of any molecules of >1500 Da across this membrane. This pore can be rapidly closed by chelation of calcium ion. Because

chondria and uncoupling of oxidative phosphorylation without synthesis of ATP. If the MTP remains open, ATP levels can be totally depleted; on the contrary, transient opening of the MTP can be involved in the mitochondrial-mediated apoptosis through the proteins released from mitochondria. Among these apoptogenic proteins we know cytochrome c [122], the serine

Permeabilization events, which occur at points where outer and inner mitochondrial mem‐ branes are in contact, involve association of several proteins from different districts of the cell and the mitochondria [125]: cytosol (hexokinase), outer mitochondrial membrane (peripheral benzodiazepine receptor and voltage dependent anion channel or VDAC), mitochondrial inner membrane space (creatine kinase), inner mitochondrial membrane (adenine nucleotide

Two main considerations suggest a role for MLT in mitochondrial homeostasis. As it is known, mitochondria produce high amounts of ROS and RNS. Besides, mitochondria depend on the GSH uptake from the cytosol, even if they have GPx and GRd to maintain redox cycling. Thus,

), its opening causes depolarization of mito‐

•– to O2.

83

Melatonin in Parkinson's Disease http://dx.doi.org/10.5772/57352

However, vitamins C and E have no such effect under the same conditions [116].

[99,117,118].

brane permeabilization (MMP) [121].

MTP allows also rapid passage of protons (H+

protease HtrA2/Omi [123], and endonuclease G [124].

translocator or ANT) and mitochondrial matrix (cyclophilin D).

mitochondrial antioxidative defense system, but without to be able to convert O2

Human mitochondria contain their own genome (mitochondrial DNA, mtDNA), a circular double stranded-molecule. The human mitochondrial chromosome contains 37 genes (16,569 base pairs), including 13 that encode subunits of respiratory chain/oxidative phosphorylation proteins; the remaining genes code for rRNA and tRNA molecules necessary to the proteinsynthesizing complex of mitochondria. About 99% of the mitochondrial proteins are encoded by nuclear DNA (nDNA); so these proteins have to be imported into mitochondria. Mito‐ chondrial proteins synthesized in the cytosol possess mitochondrial targeting signals that direct them to the appropriate compartment (outer or inner membranes, intermembranes space and matrix) within the organelle. Transport across outer and inner membranes needs a complex machinery including the presence of ATP, docking proteins, chaperonins and proteases, and it involves unfolding and refolding of the proteins to be translocated.

NADH produced in the cytosol by glycolysis and in the mitochondria by oxidation of pyruvate, fatty acids β-oxidation, and Krebs cycle, are oxidized by respiratory chain transferring electrons to O2, that is converted to water. The primary function of mitochondria is to generate ATP (from ADP and phosphate by adenin nucleotide and phosphate translocators and FoF1 ATP synthase) through the ETC resulting in OXPHOS. The ETC, located in the inner mito‐ chondrial membrane, comprises a series of electron carriers grouped into four enzyme complexes: complex I or NADH ubiquinone reductase, complex II or succinate ubiquinone reductase, complex III or ubiquinol cytochrome c reductase, and complex IV or cytochrome c oxidase. The end product of the respiratory chain is water generated after reduction of O2 by mitochondrial complex IV; this process needs the addition of four electrons to each oxygen molecule. However, about 5-10% of the oxygen is involved in production of hydrogen peroxide (H2O2), superoxide anion radical (O2 •–), and the extremely reactive hydroxyl radical (•OH) [108]. These three molecules are ROS and represent endogenous oxidotoxins. The mitochon‐ dria for action of the enzyme nitric oxide synthase (mtNOS) can also produce nitric oxide (NO•) from L-arginine [109], which can be converted into various reactive nitrogen species (RNS), such as nitrosonium cation (NO+ ), nitroxyl anion (NO– ) and peroxynitrite (ONOO– ) [110]. These free radicals are detoxified or their peroxidation products are decomposed by the natural antioxidant defense system as SOD, glutathione redox cycle, catalase and coenzyme Q. Mitochondria not only generate ROS/RNS, but are also the main target of their actions [111]. Small fluctuations in the steady-state concentration of ROS/RNS may play a role in intracellular signaling [112]. Several mechanisms take part in the control of ROS/RNS production. Among these the enzyme SOD, localized in the inner side of the inner mitochondrial membrane, remove O2 •– [113]. When formed, O2 •– is immediately dismutated to H2O2 by cytosolic or mitochondrial superoxide dismutase. As H2O2 is the precursor of the highly damaging •OH, it is imperative that H2O2 is removed very quickly.

The enzyme GPx metabolizes H2O2 to water and O2; GPx in this reaction also converts reduced GSH to its oxidized form (GSSG). In turn, GSSG is reduced to GSH by the action of the enzyme glutathione reductase (GRd) in the presence of NADPH [114,115]. These enzymes form part of the endogenous antioxidant defense system suppressing ROS/RNS levels both in the cells and in the mitochondria. Under normal conditions, MLT reduces mitochondrial hydroperox‐ ide levels and stimulates the activity of GPx and GRd, enzymes involved in the GSH-GSSG balance [116]. The indoleamine MLT is also able to neutralize the oxidative stress induced by high doses of *t*-butyl hydroperoxide, restoring GSH levels and GPx and GRd activities. However, vitamins C and E have no such effect under the same conditions [116].

adenosine triphosphate (ATP), a molecule rich in energy, via the enzyme complex ATP

Human mitochondria contain their own genome (mitochondrial DNA, mtDNA), a circular double stranded-molecule. The human mitochondrial chromosome contains 37 genes (16,569 base pairs), including 13 that encode subunits of respiratory chain/oxidative phosphorylation proteins; the remaining genes code for rRNA and tRNA molecules necessary to the proteinsynthesizing complex of mitochondria. About 99% of the mitochondrial proteins are encoded by nuclear DNA (nDNA); so these proteins have to be imported into mitochondria. Mito‐ chondrial proteins synthesized in the cytosol possess mitochondrial targeting signals that direct them to the appropriate compartment (outer or inner membranes, intermembranes space and matrix) within the organelle. Transport across outer and inner membranes needs a complex machinery including the presence of ATP, docking proteins, chaperonins and

proteases, and it involves unfolding and refolding of the proteins to be translocated.

NADH produced in the cytosol by glycolysis and in the mitochondria by oxidation of pyruvate, fatty acids β-oxidation, and Krebs cycle, are oxidized by respiratory chain transferring electrons to O2, that is converted to water. The primary function of mitochondria is to generate ATP (from ADP and phosphate by adenin nucleotide and phosphate translocators and FoF1 ATP synthase) through the ETC resulting in OXPHOS. The ETC, located in the inner mito‐ chondrial membrane, comprises a series of electron carriers grouped into four enzyme complexes: complex I or NADH ubiquinone reductase, complex II or succinate ubiquinone reductase, complex III or ubiquinol cytochrome c reductase, and complex IV or cytochrome c oxidase. The end product of the respiratory chain is water generated after reduction of O2 by mitochondrial complex IV; this process needs the addition of four electrons to each oxygen molecule. However, about 5-10% of the oxygen is involved in production of hydrogen peroxide

[108]. These three molecules are ROS and represent endogenous oxidotoxins. The mitochon‐ dria for action of the enzyme nitric oxide synthase (mtNOS) can also produce nitric oxide (NO•) from L-arginine [109], which can be converted into various reactive nitrogen species

[110]. These free radicals are detoxified or their peroxidation products are decomposed by the natural antioxidant defense system as SOD, glutathione redox cycle, catalase and coenzyme Q. Mitochondria not only generate ROS/RNS, but are also the main target of their actions [111]. Small fluctuations in the steady-state concentration of ROS/RNS may play a role in intracellular signaling [112]. Several mechanisms take part in the control of ROS/RNS production. Among these the enzyme SOD, localized in the inner side of the inner mitochondrial membrane,

mitochondrial superoxide dismutase. As H2O2 is the precursor of the highly damaging •OH,

The enzyme GPx metabolizes H2O2 to water and O2; GPx in this reaction also converts reduced GSH to its oxidized form (GSSG). In turn, GSSG is reduced to GSH by the action of the enzyme glutathione reductase (GRd) in the presence of NADPH [114,115]. These enzymes form part of the endogenous antioxidant defense system suppressing ROS/RNS levels both in the cells

), nitroxyl anion (NO–

•–), and the extremely reactive hydroxyl radical (•OH)

•– is immediately dismutated to H2O2 by cytosolic or

) and peroxynitrite (ONOO–

)

synthase.

82 A Synopsis of Parkinson's Disease

(H2O2), superoxide anion radical (O2

(RNS), such as nitrosonium cation (NO+

•– [113]. When formed, O2

it is imperative that H2O2 is removed very quickly.

remove O2

Other antioxidants such as ascorbate, ubiquinone and α-tocopherol can participate in the mitochondrial antioxidative defense system, but without to be able to convert O2 •– to O2. However, uncontrolled increase in these metabolites leads to a series of reactions which target proteins, lipids and DNA resulting in cell death by necrosis or apoptosis. In recent years, several findings support the antioxidant effect of MLT in mitochondrial homeostasis [99,117,118].

Apoptosis and necrosis are two types of cell death occurring in neurodegeneration. Apoptosis (programmed cell death) occurs naturally under normal physiological conditions; on the contrary, necrosis is caused by external factors such as toxins, infections and trauma. Apoptosis is characterized by cell shrinkage, cytoplasm contraction, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation, plasma membrane bleb formation and apoptotic body formation [119]. Many of these changes are activated by a family of caspases, i.e. proteases that in their active site possess a cysteine and cleaves the substrates after aspartate residues. Apoptotic cells are rapidly sequestered by phagocytosis before they can lyse and cause an inflammatory process [120]. Necrosis does not involve any DNA or protein degra‐ dation and is accompanied by swelling of the cytoplasm and of the mitochondria with membrane ruptures. Both apoptosis and necrosis involve a change in mitochondrial mem‐ brane permeabilization (MMP) [121].

MMP causes the opening of a nonspecific pore in the mitochondrial membranes, known as the mitochondrial transition pore (MTP), that allows the passage of any molecules of >1500 Da across this membrane. This pore can be rapidly closed by chelation of calcium ion. Because MTP allows also rapid passage of protons (H+ ), its opening causes depolarization of mito‐ chondria and uncoupling of oxidative phosphorylation without synthesis of ATP. If the MTP remains open, ATP levels can be totally depleted; on the contrary, transient opening of the MTP can be involved in the mitochondrial-mediated apoptosis through the proteins released from mitochondria. Among these apoptogenic proteins we know cytochrome c [122], the serine protease HtrA2/Omi [123], and endonuclease G [124].

Permeabilization events, which occur at points where outer and inner mitochondrial mem‐ branes are in contact, involve association of several proteins from different districts of the cell and the mitochondria [125]: cytosol (hexokinase), outer mitochondrial membrane (peripheral benzodiazepine receptor and voltage dependent anion channel or VDAC), mitochondrial inner membrane space (creatine kinase), inner mitochondrial membrane (adenine nucleotide translocator or ANT) and mitochondrial matrix (cyclophilin D).

Two main considerations suggest a role for MLT in mitochondrial homeostasis. As it is known, mitochondria produce high amounts of ROS and RNS. Besides, mitochondria depend on the GSH uptake from the cytosol, even if they have GPx and GRd to maintain redox cycling. Thus, the anti-oxidant effect of melatonin and its ability to increase the levels of GSH may be of great importance for mitochondrial physiology [126]. The fact that the inhibition of CN– on complex IV of the mitochondrial ETC is removed by MLT, also supports its intramitochondrial role [127]. A protective effect of MLT against MPP+ -induced inhibition of complex I of ETC has been also shown [128].

When the 6-OHDA model was used instead of MPTP ones to induce dopaminergic degener‐ ation, MLT administration restored the motor deficits elicited by apomorphine co-treatment with 6-OHDA [137] and also completely prevented the rise in neural lipid peroxidation products and partially rescued striatal dopaminergic levels after lesioning with 6-OHDA [138]. The protective action of MLT against dopaminergic neuronal degeneration was also expressed by reduction of the DNA fragmentation induced by MTPT [139] and of mitochondrial complex I deficiency observed after 6-OHDA administration [140]. MLT also counters MPTP-induced c-Jun-N-terminal kinase and caspase-dependent signaling leading to the dopaminergic neurodegeneration [141]. It has been reported that MLT partially preserves the GSH concen‐ trations in SN of MPTP-treated rats [142,143]. The antioxidant activity of MLT was supposed to be the major mechanism underlying MLT's protection in these PD models. The protective function of MLT also include its antiapoptotic effects. MLT has been reported to rescue

Melatonin in Parkinson's Disease http://dx.doi.org/10.5772/57352 85

dopamine neurons from spontaneous cell death in low-density seeding culture [144].

may be beneficial in the treatment of parkinsonism [145].

to preserve mitochondrial metabolism.

PD epidemiological studies have suggested an association with the environmental toxin rotenone, a mitochondrial complex I inhibitor. In recent years, *Drosophila melanogaster* has been used as a model for several neurodegenerative diseases, including PD. Coulom and Birman studied for several days the neurodegenerative effects of a chronic exposure to rotenone in *Drosophila melanogaster*. After several days of treatment, flies presented characteristic locomo‐ tor impairments that increased with the dose of herbicide. Immunocytochemistry analysis demonstrated a dramatic and selective loss of dopaminergic neurons in the brain of all treated flies. The addition of L-dopa into the feeding medium rescued the behavioral deficits but not neuronal death, as is the case in human PD patients. On the contrary, the antioxidant MLT alleviated both symptomatic impairment and neuronal loss, supporting the idea that this agent

MLT has been shown to protects PC12 cells from both apoptosis and necrosis induced by high doses of 6-OHDA [146,147]. Since 6-OHDA induced cellular toxicity is mediated by increased free-radical generation, the antioxidant properties of MLT presumably account for its ability to suppress both necrosis and apoptosis. Numerous data suggest a role for MLT in mitochon‐ drial homeostasis [148]. It has been reported that MLT increases the activities of respiratory complexes I and IV in a time-dependent manner after *in vivo* administration to rats [129] and maintains GSH homeostasis in the mitochondrial matrix under increased oxidative stress; these actions are not shared by either vitamin C or vitamin E [117]. Mitochondria in the cell are the major source of ROS, owing to the leakage of electrons through the electron transport chain. Due the critical role of mitochondria in programmed cell death and PD, it is conceivable that actions at the mitochondrial level mediate at least some of MLT apoptotic effects. It has been reported that MLT induces ATP production, increasing the activity of the mitochondrial oxidative phosphorylation (OXPHOS) enzymes [129]. The indoleamine also protects mito‐ chondrial DNA, which is particularly vulnerable to oxidative damage, thus indirectly helping

Since mitochondria play a critical role in the pathogenesis of PD, it is conceivably that actions at mitochondria level mediate some of MLT antiapoptotic effects. The beneficial actions of MLT in PD has been widely investigated not only on the basis of its neuroprotective efficacy

The effects of MLT on mitochondrial ETC have been also studied on submitochondrial particles from rat liver and brain mitochondria [129]. MLT at 1 nM concentration significantly increased the activity of the complexes I and IV of ETC in rat liver submitochondrial particles, whereas 10-100 nM MLT stimulated the activity of the same complexes but in brain submitochondrial particles. The indoleamine counteracted CN– -induced inhibition of complex IV, restoring the levels of Cyt aa3. This effect was of physiological significance, since the MLT increased the ETC and OXPHOS activities with a consequent increase of ATP synthesis [129]. In addition, due the high redox potential of MLT (-0.98 V), this molecule can donate directly electrons to complex I of the ETC [130].

The effect of MLT (10 mg/kg) on ETC complexes from rat liver and brain mitochondria has been also studied in vivo. Martin et al. [116] have found that MLT increases the activity of the respiratory chain complexes I and IV and ATP synthesis in a time-dependent manner after mitochondrial damage induced by ruthenium red [116].

Recently, the role of MLT on cardiolipin and mitochondrial biogenesis was studied [131]. Cardiolipin, a phospholipid located in inner mitochondrial membrane, is required for several mitochondrial bioenergetic processes as well as for the activity of transport proteins. Altera‐ tions in cardiolipin structure and acyl chain composition have been associated with mito‐ chondrial dysfunction under a variety of pathological dysfunctions. The authors [131] reported that MLT protects the mitochondrial membranes from oxidation-reduction damage by preventing cardiolipin oxidation.

## **5. Melatonin and Parkinson's**

In the last decade, many research findings provide scientific evidence for the protective role of MLT in a number of oxidative stress related diseases, especially Alzheimer's [132] and Parkinson's diseases [133], being the protective actions of the indoleamine attributable to its direct and indirect antioxidative properties. The first evidence of a significant relationship between Parkinson's disease and MLT derived from the evidence of a reduction in the concentration of circulating MLT in PD patients as a consequence of a decreased activity of the pineal gland [134]. After its antioxidant properties were uncovered, melatonin has been successfully tested in several *in vivo* and *in vitro* PD models.

MLT was found to inhibit *in vitro* the prooxidant effects of dopamine and L-dopa [135] and to be more effective than the vitamin E analog, trolox, in preventing dopamine autooxidation [136]. Melatonin was also reported to prevent in the MPTP model the rise in lipid peroxidation products in the substantia nigra (SN) of MPP+ -treated rats and, additionally, to preserve tyrosine hydroxylase (TH) activity, which is normally decreased after toxin treatment [69].

When the 6-OHDA model was used instead of MPTP ones to induce dopaminergic degener‐ ation, MLT administration restored the motor deficits elicited by apomorphine co-treatment with 6-OHDA [137] and also completely prevented the rise in neural lipid peroxidation products and partially rescued striatal dopaminergic levels after lesioning with 6-OHDA [138]. The protective action of MLT against dopaminergic neuronal degeneration was also expressed by reduction of the DNA fragmentation induced by MTPT [139] and of mitochondrial complex I deficiency observed after 6-OHDA administration [140]. MLT also counters MPTP-induced c-Jun-N-terminal kinase and caspase-dependent signaling leading to the dopaminergic neurodegeneration [141]. It has been reported that MLT partially preserves the GSH concen‐ trations in SN of MPTP-treated rats [142,143]. The antioxidant activity of MLT was supposed to be the major mechanism underlying MLT's protection in these PD models. The protective function of MLT also include its antiapoptotic effects. MLT has been reported to rescue dopamine neurons from spontaneous cell death in low-density seeding culture [144].

the anti-oxidant effect of melatonin and its ability to increase the levels of GSH may be of great

IV of the mitochondrial ETC is removed by MLT, also supports its intramitochondrial role

The effects of MLT on mitochondrial ETC have been also studied on submitochondrial particles from rat liver and brain mitochondria [129]. MLT at 1 nM concentration significantly increased the activity of the complexes I and IV of ETC in rat liver submitochondrial particles, whereas 10-100 nM MLT stimulated the activity of the same complexes but in brain submitochondrial

levels of Cyt aa3. This effect was of physiological significance, since the MLT increased the ETC and OXPHOS activities with a consequent increase of ATP synthesis [129]. In addition, due the high redox potential of MLT (-0.98 V), this molecule can donate directly electrons to

The effect of MLT (10 mg/kg) on ETC complexes from rat liver and brain mitochondria has been also studied in vivo. Martin et al. [116] have found that MLT increases the activity of the respiratory chain complexes I and IV and ATP synthesis in a time-dependent manner after

Recently, the role of MLT on cardiolipin and mitochondrial biogenesis was studied [131]. Cardiolipin, a phospholipid located in inner mitochondrial membrane, is required for several mitochondrial bioenergetic processes as well as for the activity of transport proteins. Altera‐ tions in cardiolipin structure and acyl chain composition have been associated with mito‐ chondrial dysfunction under a variety of pathological dysfunctions. The authors [131] reported that MLT protects the mitochondrial membranes from oxidation-reduction damage by

In the last decade, many research findings provide scientific evidence for the protective role of MLT in a number of oxidative stress related diseases, especially Alzheimer's [132] and Parkinson's diseases [133], being the protective actions of the indoleamine attributable to its direct and indirect antioxidative properties. The first evidence of a significant relationship between Parkinson's disease and MLT derived from the evidence of a reduction in the concentration of circulating MLT in PD patients as a consequence of a decreased activity of the pineal gland [134]. After its antioxidant properties were uncovered, melatonin has been

MLT was found to inhibit *in vitro* the prooxidant effects of dopamine and L-dopa [135] and to be more effective than the vitamin E analog, trolox, in preventing dopamine autooxidation [136]. Melatonin was also reported to prevent in the MPTP model the rise in lipid peroxidation

tyrosine hydroxylase (TH) activity, which is normally decreased after toxin treatment [69].

on complex




importance for mitochondrial physiology [126]. The fact that the inhibition of CN–

[127]. A protective effect of MLT against MPP+

particles. The indoleamine counteracted CN–

mitochondrial damage induced by ruthenium red [116].

successfully tested in several *in vivo* and *in vitro* PD models.

products in the substantia nigra (SN) of MPP+

been also shown [128].

84 A Synopsis of Parkinson's Disease

complex I of the ETC [130].

preventing cardiolipin oxidation.

**5. Melatonin and Parkinson's**

PD epidemiological studies have suggested an association with the environmental toxin rotenone, a mitochondrial complex I inhibitor. In recent years, *Drosophila melanogaster* has been used as a model for several neurodegenerative diseases, including PD. Coulom and Birman studied for several days the neurodegenerative effects of a chronic exposure to rotenone in *Drosophila melanogaster*. After several days of treatment, flies presented characteristic locomo‐ tor impairments that increased with the dose of herbicide. Immunocytochemistry analysis demonstrated a dramatic and selective loss of dopaminergic neurons in the brain of all treated flies. The addition of L-dopa into the feeding medium rescued the behavioral deficits but not neuronal death, as is the case in human PD patients. On the contrary, the antioxidant MLT alleviated both symptomatic impairment and neuronal loss, supporting the idea that this agent may be beneficial in the treatment of parkinsonism [145].

MLT has been shown to protects PC12 cells from both apoptosis and necrosis induced by high doses of 6-OHDA [146,147]. Since 6-OHDA induced cellular toxicity is mediated by increased free-radical generation, the antioxidant properties of MLT presumably account for its ability to suppress both necrosis and apoptosis. Numerous data suggest a role for MLT in mitochon‐ drial homeostasis [148]. It has been reported that MLT increases the activities of respiratory complexes I and IV in a time-dependent manner after *in vivo* administration to rats [129] and maintains GSH homeostasis in the mitochondrial matrix under increased oxidative stress; these actions are not shared by either vitamin C or vitamin E [117]. Mitochondria in the cell are the major source of ROS, owing to the leakage of electrons through the electron transport chain. Due the critical role of mitochondria in programmed cell death and PD, it is conceivable that actions at the mitochondrial level mediate at least some of MLT apoptotic effects. It has been reported that MLT induces ATP production, increasing the activity of the mitochondrial oxidative phosphorylation (OXPHOS) enzymes [129]. The indoleamine also protects mito‐ chondrial DNA, which is particularly vulnerable to oxidative damage, thus indirectly helping to preserve mitochondrial metabolism.

Since mitochondria play a critical role in the pathogenesis of PD, it is conceivably that actions at mitochondria level mediate some of MLT antiapoptotic effects. The beneficial actions of MLT in PD has been widely investigated not only on the basis of its neuroprotective efficacy assessment but also because of the down regulation of MLT receptors in the nigrostriatal region of PD brain [149]. There is growing evidence of sleep–wake boundary dysfunction in PD. REM sleep behavior disorder (RBD) which is characterized by loss of normal skeletal muscle tone with prominent motor activity and dreaming, has been associated with PD and/or other forms of dementia, with a tendency for RBD to precede the onset of parkinsonism. There is some clinical evidence that MLT can be a useful add-on therapy for RBD in PD [150].

synthesis, reducing at the same time the oxygen consumption; then, it avoids an excess of ROS/

Considering that this hormone is an endogenous, nontoxic, antioxidant molecule without known side-effects, it should be considered as a useful agent in PD patients as a treatment with other conventional therapies. Although MLT is an important molecule and possibly has a great future in PD research, it should be extensively tested across multiple populations for efficacy and real effects along with the side effects at the efficacious doses. Future therapeutic strategies could be directed at identifying and developing MLT analogues as drugs with more powerful inhibitory effects on the mitochondrial cell death pathway, slowing the progression of

, Graziantonio Lauria2

and

Melatonin in Parkinson's Disease http://dx.doi.org/10.5772/57352 87

, Maria Stefania Sinicropi2\*, Alessia Catalano1

1 Department of Pharmacy-Drug Sciences, University of Bari "Aldo Moro", Bari, Italy

2 Department of of Pharmacy, Health and Nutritional Sciences, University of Calabria, Co‐

[1] Lee VM, Trojanowsky JQ. Mechanisms of Parkinson's disease linked to pathological alpha-synuclein: new targets for drug discovery. Neuron 2006;52(1): 33-38.

[2] Braak H, Del Tredici K, Rub U, de Vos RA, Jansen Steur EN, Braak E. Staging of brain pathology related to sporadic Parkinson's disease. Neurobiology of Aging

[3] Yacoubian TA, Standaert DG. Targets for neuroprotection in Parkinson's disease

[4] Schapira AHV. Mitochondria in the aetiology and pathogenesis of Parkinson's dis‐

[5] Huang Y, Cheung L, Rowe D, Halliday G. Genetic contributions to Parkinson's dis‐

[6] Vila M, Przedborski S. Genetic clues to the pathogenesis of Parkinson's disease. Na‐

ease. Brain Research Brain Research Reviews 2004;46(1): 44-70.

\*Address all correspondence to: s.sinicropi@unical.it; genchi@unical.it

RNS, preventing PTP opening and apoptosis.

neurodegenerative diseases.

**Author details**

Alessia Carocci1

senza, Italy

**References**

2003;24(2): 197-211.

2009;1792(7): 676-687.

ease. The Lancet Neurology 2008;7(1): 97-109.

ture Medicine 2004;10(7s): S58-S62.

Giuseppe Genchi2

## **6. Conclusions**

PD is a highly debilitating condition that concerns thousands of family in the world and annually cost millions of euro for treatment. This disease has occasionally a genetic basis, but the signs of PD develop after free-radical damage to the substantia nigra pars compacta. Moreover, neuroinflammation and mitochondrial dysfunction participate in the ethiology of this neurodegenerative disorder and contribute to the increase of oxidative damage to the dopaminergic neurons.

The mitochondria in cells play a myriad of different and important functions, so any alteration in these organelles could have a considerable impact on the functionality of the cells and also the entire body. Mitochondria are also the site of generation of reactive oxygen and nitrogen species (ROS/RNS) and the subsequent widespread deleterious effects (oxidation and/or nitrosylation of mtDNA, oxidation of phospholipids and proteins) of these intermediates. These effects lead also to the opening of the mitochondrial transition pore, release of Cyt c and the activation of the events that culminate in apoptosis.

Abnormal mitochondrial functions (decreased respiratory complexes activities, increased electron leakage, opening of the mitochondrial transition pore) have all been shown to play a role in the pathophysiology of neurodegenerative disorders such as PD, AD and HD. Mito‐ chondrial involvement in PD is revealed by deficiency of mitochondrial complexes I and IV, decreased ATP production with a parallel reduction in GSH levels.

Among the substances involved in maintaining mitochondrial biogenetics, a number of *in vivo* and *in vitro* studies indicate that MLT may emerge as a major therapeutic candidate to preserve bioenergetic function of mitochondria.

MLT is a molecule present in all creatures from prokaryotes to human beings. It is an antiox‐ idant that protected organisms from oxidative stresses and apoptosis and mediates seasonal physiological functions, is a signal of dark/light promoting also sleep, modulates the immune system, and inhibits the growth of several cancer. Indoleamine is an antioxidant that directly scavenges ROS/RNS produced during the normal metabolism of mitochondria and it indi‐ rectly promotes the activity of the antioxidant enzymes including SOD, catalase, GPx and GRd.

It has also been documented that the ability of MLT to quell the oxidation-reduction processes, with the formation of free radicals, is due to its conversion to metabolites, such as cyclic 3- OHM, AFMK and AMK. Considering the cascade of reactions that include AFMK and AMK, a MLT can scavenge about ten ROS/RNS. MLT increases the activity of ETC and the ATP synthesis, reducing at the same time the oxygen consumption; then, it avoids an excess of ROS/ RNS, preventing PTP opening and apoptosis.

Considering that this hormone is an endogenous, nontoxic, antioxidant molecule without known side-effects, it should be considered as a useful agent in PD patients as a treatment with other conventional therapies. Although MLT is an important molecule and possibly has a great future in PD research, it should be extensively tested across multiple populations for efficacy and real effects along with the side effects at the efficacious doses. Future therapeutic strategies could be directed at identifying and developing MLT analogues as drugs with more powerful inhibitory effects on the mitochondrial cell death pathway, slowing the progression of neurodegenerative diseases.

## **Author details**

assessment but also because of the down regulation of MLT receptors in the nigrostriatal region of PD brain [149]. There is growing evidence of sleep–wake boundary dysfunction in PD. REM sleep behavior disorder (RBD) which is characterized by loss of normal skeletal muscle tone with prominent motor activity and dreaming, has been associated with PD and/or other forms of dementia, with a tendency for RBD to precede the onset of parkinsonism. There is some

PD is a highly debilitating condition that concerns thousands of family in the world and annually cost millions of euro for treatment. This disease has occasionally a genetic basis, but the signs of PD develop after free-radical damage to the substantia nigra pars compacta. Moreover, neuroinflammation and mitochondrial dysfunction participate in the ethiology of this neurodegenerative disorder and contribute to the increase of oxidative damage to the

The mitochondria in cells play a myriad of different and important functions, so any alteration in these organelles could have a considerable impact on the functionality of the cells and also the entire body. Mitochondria are also the site of generation of reactive oxygen and nitrogen species (ROS/RNS) and the subsequent widespread deleterious effects (oxidation and/or nitrosylation of mtDNA, oxidation of phospholipids and proteins) of these intermediates. These effects lead also to the opening of the mitochondrial transition pore, release of Cyt c and

Abnormal mitochondrial functions (decreased respiratory complexes activities, increased electron leakage, opening of the mitochondrial transition pore) have all been shown to play a role in the pathophysiology of neurodegenerative disorders such as PD, AD and HD. Mito‐ chondrial involvement in PD is revealed by deficiency of mitochondrial complexes I and IV,

Among the substances involved in maintaining mitochondrial biogenetics, a number of *in vivo* and *in vitro* studies indicate that MLT may emerge as a major therapeutic candidate to

MLT is a molecule present in all creatures from prokaryotes to human beings. It is an antiox‐ idant that protected organisms from oxidative stresses and apoptosis and mediates seasonal physiological functions, is a signal of dark/light promoting also sleep, modulates the immune system, and inhibits the growth of several cancer. Indoleamine is an antioxidant that directly scavenges ROS/RNS produced during the normal metabolism of mitochondria and it indi‐ rectly promotes the activity of the antioxidant enzymes including SOD, catalase, GPx and GRd. It has also been documented that the ability of MLT to quell the oxidation-reduction processes, with the formation of free radicals, is due to its conversion to metabolites, such as cyclic 3- OHM, AFMK and AMK. Considering the cascade of reactions that include AFMK and AMK, a MLT can scavenge about ten ROS/RNS. MLT increases the activity of ETC and the ATP

clinical evidence that MLT can be a useful add-on therapy for RBD in PD [150].

**6. Conclusions**

86 A Synopsis of Parkinson's Disease

dopaminergic neurons.

the activation of the events that culminate in apoptosis.

preserve bioenergetic function of mitochondria.

decreased ATP production with a parallel reduction in GSH levels.

Alessia Carocci1 , Maria Stefania Sinicropi2\*, Alessia Catalano1 , Graziantonio Lauria2 and Giuseppe Genchi2

\*Address all correspondence to: s.sinicropi@unical.it; genchi@unical.it

1 Department of Pharmacy-Drug Sciences, University of Bari "Aldo Moro", Bari, Italy

2 Department of of Pharmacy, Health and Nutritional Sciences, University of Calabria, Co‐ senza, Italy

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**Chapter 4**

**Parkinson's Disease and Peripheral Neuropathy**

Idiopathic Parkinson's disease (IPD), an age-dependent neurodegenerative disorder without known cause, is well known to manifest with rest tremor, rigidity, bradykinesia and gait instability on examination [1, 2]. However, other ancillary manifestations have become accepted over the years, including cognitive decline, depression, and autonomic dysfunction [3]. All of these clinically presenting features have long been known to result from disease within the central nervous system, where IPD is pathologically associated with degeneration

The possibility of peripheral nervous system functional or pathological involvement in IPD has only recently been considered. One form of peripheral nervous system disease is a peripheral neuropathy, a distal-predominant process affecting the feet and legs, and in more severe cases, the hands and torso [5]. Peripheral neuropathy can manifest as a disease of the axons or the myelin, or both, within nerve fibers. In addition, sensory nerve fibers carrying information for touch, pain and temperature sensations (small nerve fibers termed Aδ and C fibers) as well as for vibration detection and proprioception (large nerve fibers termed Aα and Aβ fibers) can be selectively affected. In most cases, sensory dysfunction appears first, followed by further disease of large nerve fibers leading to loss of reflexes and the possible development of weakness. Symptoms of a peripheral neuropathy may include the early onset of numbness, tingling or prickling sensations, followed by the later onset of incoordination, weakness and pain [6]. The presence of such symptoms in patients should lead to a detailed neurological examination of the peripheral nervous system. Physical examination findings in a patient with peripheral neuropathy will often include abnormal responses to touch, pinprick, temperature, vibration and proprioception, as well as abnormal reflexes, weakness and ataxia. These features will typically display a stocking-glove pattern of distribution due to the distal

predominance of peripheral neuropathy with the feet being implicated first.

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Peter Podgorny and Cory Toth

http://dx.doi.org/10.5772/57106

**1. Introduction**

of the substantia nigra [4].

Additional information is available at the end of the chapter
