**3. GBA: the principal genetic risk factor for Parkinson disease**

The *GBA* gene was initially described in association with a rare lysosomal storage disease (LSD) called Gaucher disease (GD). When mutated in homozygosis, depending on the mutation present, the resulting enzyme is malformed or even no enzyme is synthesized leading to enzyme glucocerebrosidase (GCase) partial or total deficiency and glucosylceramide (GlcCer) accumulation. The symptoms are multisystemic, with the brain, spleen, liver and bone marrow being the main organs affected. The presence and intensity of those symptoms differ between the three types of GD (GD1, GD2 and GD3). The heterozygous individuals do not present any clinical manifestation; however, in the last years, this perspective has changed [25, 26].

Further a number of studies have recorded the occurrence of parkinsonian manifestations in patients with GD and their relatives [27, 28]. In ref. [29] was showed that *GBA* mutations homozygous individuals have 21.4 fold increased risk to develop PD with probability of 9–12% to manifest motor symptoms before 80 years old. Despite being low, this risk is considerably higher than in the same age group in general population, 3%. The *GBA* and PD association was confirmed in the Jewish Ashkenazi, which showed a prevalence of *GBA* mutations in heterozygosis and homozygosis individuals in the PD population that by far outweighs the reported prevalence of mutations in other susceptibility genes for PD, as *Parkin* and *SNCA* [30].

Researchers worldwide have attempted to validate the same association in populations from many different genetic backgrounds [31–41]. In 2009, an international and multicenter study with a great sample of approximately 5000 PD patients and equal number of controls provided the definitive proof found for this association with an odds ratio greater than five (OR 5.3) and showed that mutations N370S and L444P are the most frequent in this gene. In other words, *GBA* mutation genes were recognized as the major genetic risk factor for PD until now [42].

In addition to alter the risk to manifest the disease, the presence of *GBA* mutations has also the potential to modify PD phenotype such as age of onset. The modulatory effects best described in the literature investigated the association with age of onset and declined cognitive. The association to other symptoms and patients survival rate has been not yet approached in more detail.

Whether in heterozygosis or in homozygosis individuals (GD patients), the age of onset of symptoms apparently occurs earlier than in PD patients without mutations, usually between the fourth and sixth decade of life [28–43]. In relation to symptoms is noticeable a cognitive decline earlier in PD patients with *GBA* mutations (PD-GBA) [26–45]. Dementia is one of the clinical manifestations that most affects the patient's quality of life and it is more frequent in GBA mutation carriers than in non-carriers. Longitudinal studies have shown that PD-GBA patients have a risk three times higher than patients without GBA mutations to present dementia [26, 44, 46]. Neuroimaging exams support this association by showing more expansive synucleinopathy in the neocortical and subcortical areas of PD-GBA patients, increasing the risk to dementia, psychosis and postural hypotension [46].

A few studies have evaluated the survival rate, if there is a greater risk of death in PD-GBA patients than in those without any mutations. A 2014 study found lower survival for the carrier group, but had a weak statistical value [47]. However, in 2016, a study with the largest sample number ever described replicated the same result with powerful statistical significance reinforcing this conclusion. It was defined in this study that there is a twice greater risk of mortality among PD-GBA patients. One explanation may be the increased presence of non-responsive levodopa motor impairments such as dysphagia and non-motor impairments such as orthostatic hypotension in the group of PD-GBA. There was no difference in disease duration compared to non-carriers, but patients were significantly younger at the time of death [46].

#### **3.1 Genotype-phenotype correlations**

Researchers have also observed different *GBA* mutations having a different impact in the clinical manifestations. Mutations generally associated with defined neuropathic forms of GD (GD type 2 and 3), such as L444P, are classified as severe mutations, while others associated with GD type 1, such as N370S, are classified as mild mutations [26–48].

A meta-analysis study included populations from North, Central and South America, Western and Eastern Europe, North Africa, Asia and Ashkenazi Jews, and its results showed a clear and significant differentiated effect comparing mild and severe mutations on the risk of developing PD and the age of onset of symptoms not only in Ashkenazi populations, but also worldwide. Severe mutations such as L444P confer a three to four times increased risk for its carriers to develop PD and are associated with the onset of symptoms 5 years earlier than mild mutations. The average age found for severe mutations was 53.1 (±11.2), whereas the average age for mild mutations was 58.1 (±10.6) [26].

The type of mutation was also relevant in modulating the cognitive impairments of PD patients. Ref. [46] observed that severe mutations conferred a higher risk of dementia for its carriers. The risk was three times greater compared to patients with mild mutations and five times greater when compared to the risk for PD without

**69**

control [49].

*Genetic Risk Factors and Lysosomal Function in Parkinson Disease*

**4. GBA-associated PD in different populations**

ment (p = 0.113, OR 2.5, 95% CI = 0.77–8.42) [34].

*GBA* mutations. Another longitudinal study with a similar sample number corroborated with the increased risk of dementia for patients with severe mutations versus non-carriers [44]. With regard to other symptoms, motor impairments appear to be similar between patients with mild mutations and non-carriers, while those with severe mutations appear to be more aggravated and seem to have a higher frequency of non-motor symptoms such as psychosis, apathy and postural hypotension [46]. Patients' survival does not seem to differ when comparing the types of mutations with each other. However, when compared separately with non-carriers, mild mutations do not differ statistically from non-carriers, while mortality was shown

to be greater for carriers of severe mutations than in non-carriers [46].

Due to the discovery and increasing number of proofs supporting the great influence of *GBA* mutations in PD, some authors consider the possibility of reclassifying them from risk factors for agents causing autosomal dominant PD [43, 49].

Given the multifactorial etiology of PD, the different environment and ethnicity of a population may impact in the different results seen among the papers that investigated the frequency of *GBA* mutations in PD patients. Other possible causes

The highest frequencies of mutations of the *GBA* gene have been found in PD patients of Ashkenazi Jewish ancestry, with rates of 13.7–31.3% in comparison with 4.5–6.2% in control groups [26–28]. The frequencies recorded in PD patients in non-Jewish populations representing other populations, such as Italians, Caucasian Americans, Greeks, Brazilians, British and Taiwanese, are invariably much lower— 3.5% to 12.0%—while controls from the same populations range from 0% to 5.3% [31–41]. Previously, in North Africa, a study found no association between PD and mutations of the *GBA* gene; however, a more recent African study data suggested a risk association between mutations in the *GBA* gene and PD [37]. The lowest rate recorded to date was 2.3% in Norwegian PD patients, compared with 1.7% in the

The genetic background can also impact the frequency even within the same country. In Brazil, four studies evaluated the association between GBA and PD, with variances in frequencies depending on the region (**Table 1**). The North region had twice as many cases of PD patients with GBA mutations (7.4%) compared to the frequencies of the South and Southeast regions (3.5%) with a similar sample number. The fact may be explained by the different genetic composition of the North region, which, despite also has a European origin, has a higher Amerindian ancestry than the Southern Brazil, which is almost exclusively from European ancestry [31–50]. Greek and Italian studies have found significant differences comparing PD patients and controls from urban and rural areas, and from the North and South regions, respectively. In the Greek study, the frequency of *GBA* mutations between PD patients and controls was statically significant. However, when the cohorts were analyzed separately, there was a difference of frequencies. The difference between PD patients and controls was statistically significant only in the case of the patients of cohort A that is originated from Thessaly, a mainly rural area (p = 0.021, OR 4.2, 95% CI = 1.14–15.54) and not in the case of cohort B patients, the majority of which were residents and/or originated from the greater area of Athens, an urban environ-

In the Italian study, there was a lower frequency of mutations in PD patients (11/395, 2.8%) and in controls (1/483, 0.2%) from the Southern region, and the most common mutation was p.L444P. Conversely, in the Northern region, the most

of this variation can be the use of different techniques and methodologies.

*DOI: http://dx.doi.org/10.5772/intechopen.91850*

*Genetic Risk Factors and Lysosomal Function in Parkinson Disease DOI: http://dx.doi.org/10.5772/intechopen.91850*

*Methods in Molecular Medicine*

and multicenter study with a great sample of approximately 5000 PD patients and equal number of controls provided the definitive proof found for this association with an odds ratio greater than five (OR 5.3) and showed that mutations N370S and L444P are the most frequent in this gene. In other words, *GBA* mutation genes were

In addition to alter the risk to manifest the disease, the presence of *GBA* mutations has also the potential to modify PD phenotype such as age of onset. The modulatory effects best described in the literature investigated the association with age of onset and declined cognitive. The association to other symptoms and patients

Whether in heterozygosis or in homozygosis individuals (GD patients), the age of onset of symptoms apparently occurs earlier than in PD patients without mutations, usually between the fourth and sixth decade of life [28–43]. In relation to symptoms is noticeable a cognitive decline earlier in PD patients with *GBA* mutations (PD-GBA) [26–45]. Dementia is one of the clinical manifestations that most affects the patient's quality of life and it is more frequent in GBA mutation carriers than in non-carriers. Longitudinal studies have shown that PD-GBA patients have a risk three times higher than patients without GBA mutations to present dementia [26, 44, 46]. Neuroimaging exams support this association by showing more expansive synucleinopathy in the neocortical and subcortical areas of PD-GBA patients,

A few studies have evaluated the survival rate, if there is a greater risk of death in PD-GBA patients than in those without any mutations. A 2014 study found lower survival for the carrier group, but had a weak statistical value [47]. However, in 2016, a study with the largest sample number ever described replicated the same result with powerful statistical significance reinforcing this conclusion. It was defined in this study that there is a twice greater risk of mortality among PD-GBA patients. One explanation may be the increased presence of non-responsive levodopa motor impairments such as dysphagia and non-motor impairments such as orthostatic hypotension in the group of PD-GBA. There was no difference in disease duration compared to non-carriers, but patients were significantly younger at the

Researchers have also observed different *GBA* mutations having a different impact in the clinical manifestations. Mutations generally associated with defined neuropathic forms of GD (GD type 2 and 3), such as L444P, are classified as severe mutations, while others associated with GD type 1, such as N370S, are classified as

A meta-analysis study included populations from North, Central and South America, Western and Eastern Europe, North Africa, Asia and Ashkenazi Jews, and its results showed a clear and significant differentiated effect comparing mild and severe mutations on the risk of developing PD and the age of onset of symptoms not only in Ashkenazi populations, but also worldwide. Severe mutations such as L444P confer a three to four times increased risk for its carriers to develop PD and are associated with the onset of symptoms 5 years earlier than mild mutations. The average age found for severe mutations was 53.1 (±11.2), whereas the average age for

The type of mutation was also relevant in modulating the cognitive impairments of PD patients. Ref. [46] observed that severe mutations conferred a higher risk of dementia for its carriers. The risk was three times greater compared to patients with mild mutations and five times greater when compared to the risk for PD without

increasing the risk to dementia, psychosis and postural hypotension [46].

recognized as the major genetic risk factor for PD until now [42].

survival rate has been not yet approached in more detail.

**68**

time of death [46].

mild mutations [26–48].

**3.1 Genotype-phenotype correlations**

mild mutations was 58.1 (±10.6) [26].

*GBA* mutations. Another longitudinal study with a similar sample number corroborated with the increased risk of dementia for patients with severe mutations versus non-carriers [44]. With regard to other symptoms, motor impairments appear to be similar between patients with mild mutations and non-carriers, while those with severe mutations appear to be more aggravated and seem to have a higher frequency of non-motor symptoms such as psychosis, apathy and postural hypotension [46].

Patients' survival does not seem to differ when comparing the types of mutations with each other. However, when compared separately with non-carriers, mild mutations do not differ statistically from non-carriers, while mortality was shown to be greater for carriers of severe mutations than in non-carriers [46].

Due to the discovery and increasing number of proofs supporting the great influence of *GBA* mutations in PD, some authors consider the possibility of reclassifying them from risk factors for agents causing autosomal dominant PD [43, 49].

#### **4. GBA-associated PD in different populations**

Given the multifactorial etiology of PD, the different environment and ethnicity of a population may impact in the different results seen among the papers that investigated the frequency of *GBA* mutations in PD patients. Other possible causes of this variation can be the use of different techniques and methodologies.

The highest frequencies of mutations of the *GBA* gene have been found in PD patients of Ashkenazi Jewish ancestry, with rates of 13.7–31.3% in comparison with 4.5–6.2% in control groups [26–28]. The frequencies recorded in PD patients in non-Jewish populations representing other populations, such as Italians, Caucasian Americans, Greeks, Brazilians, British and Taiwanese, are invariably much lower— 3.5% to 12.0%—while controls from the same populations range from 0% to 5.3% [31–41]. Previously, in North Africa, a study found no association between PD and mutations of the *GBA* gene; however, a more recent African study data suggested a risk association between mutations in the *GBA* gene and PD [37]. The lowest rate recorded to date was 2.3% in Norwegian PD patients, compared with 1.7% in the control [49].

The genetic background can also impact the frequency even within the same country. In Brazil, four studies evaluated the association between GBA and PD, with variances in frequencies depending on the region (**Table 1**). The North region had twice as many cases of PD patients with GBA mutations (7.4%) compared to the frequencies of the South and Southeast regions (3.5%) with a similar sample number. The fact may be explained by the different genetic composition of the North region, which, despite also has a European origin, has a higher Amerindian ancestry than the Southern Brazil, which is almost exclusively from European ancestry [31–50].

Greek and Italian studies have found significant differences comparing PD patients and controls from urban and rural areas, and from the North and South regions, respectively. In the Greek study, the frequency of *GBA* mutations between PD patients and controls was statically significant. However, when the cohorts were analyzed separately, there was a difference of frequencies. The difference between PD patients and controls was statistically significant only in the case of the patients of cohort A that is originated from Thessaly, a mainly rural area (p = 0.021, OR 4.2, 95% CI = 1.14–15.54) and not in the case of cohort B patients, the majority of which were residents and/or originated from the greater area of Athens, an urban environment (p = 0.113, OR 2.5, 95% CI = 0.77–8.42) [34].

In the Italian study, there was a lower frequency of mutations in PD patients (11/395, 2.8%) and in controls (1/483, 0.2%) from the Southern region, and the most common mutation was p.L444P. Conversely, in the Northern region, the most


#### **Table 1.**

**71**

**Figure 2.**

*Ref. [6].*

*Genetic Risk Factors and Lysosomal Function in Parkinson Disease*

frequent genetic defect found was p.N370S and the frequency of mutations in PD was 4.5% and 0.63% in controls. Therefore, the difference may be due to a particu

ship, that is, the risk of healthy heterozygous for *GBA* mutations in developing PD, since it is not known for sure the degree of influence that this genetic alteration has

Anheim M et al. [43] published in 2012 an estimate of the penetration of PD in healthy heterozygous people for *GBA* mutations and reached a value of 7.6%, 13.7%, 21.4% and 29.7% for 50, 60, 70 and 80 years, respectively, based on a dominance model. However, in the same year, [51] found a lower value: 5% for 60 years and 15% for 80 years of age. Such difference is perhaps due to additional genetic fac

tors or environmental factors, a fact that emphasizes the possibility of variance of the risk of developing PD according to the genetic background of the population. Families that have *GBA* mutations segregation through generations are a group at risk for developing PD and should be monitored for a possible early diagnosis to

α-Synuclein is a key protein in the neuropathogenesis of PD, involved in several

stood, but studies show that it is normally located in presynaptic terminals where it binds to lipids and plays the role of regulating in more than one step the traffic of synaptic vesicles to be released. As cited above, the insoluble forms (oligomers and fibrils) of this protein accumulate and compound the Lewy bodies found in most

The reason behind this accumulation can be due to increased synthesis or decreased degradation (**Figure 2**). Mutations, as the triplications of the *SNCA*

oligomers and fibrils formed with other mutant proteins can result in the deficiency

olysis. On the other hand, the initial deficiency of certain metabolic pathway caused by mutations in genes, advanced age or environmental factors can also be the trigger

α-synuclein resulting in the insoluble forms. This last theory has been

lar frequency of *GBA* mutations in regions of Italy or to the sample size [36]. Although the frequency of *GBA* mutations in populations of PD patients has been well characterized worldwide, few data are available on the inverse relation






α-synuclein is not well under

α-synuclein prote

*α-synuclein in neurons. Adapted from* 

α-synuclein, while the interaction between the

*DOI: http://dx.doi.org/10.5772/intechopen.91850*

have better chances in modifying the disease.

pathogenic processes. The physiological function of

PD patients and also contribute to neuronal cell death [5].

of certain metabolic pathways and contribute to slowing down

*The proposed physiological and PD-associated pathological functions of* 

**5. Pathogenic mechanisms in PD**

gene, can enhance the production of

to accumulate

on the onset of the disease.

*GBA mutation among PD patients in different Brazilian regions.*

#### *Genetic Risk Factors and Lysosomal Function in Parkinson Disease DOI: http://dx.doi.org/10.5772/intechopen.91850*

*Methods in Molecular Medicine*

**70**

**Studies** Spitz et al.

65 PD patients and

Early onset

PCR-RFLP, restriction

N370S and

2/65 **(3%)**;

0/267

Patient 1 at

46 yr old and

patient 2 at 42 yr

old

L444P 2/2

(100%);

N370S 0/2

(0%)

L444P

endonucleases and

electrophoresis

(<55 years).

267 control subjects

from Southeastern

Brazil

Socal et al.

62 PD patients from

All patients

PCR-RFLP, restriction

N370S,

2/62 **(3.5%)**;

Not

Patients with

Not informed.

mutation

37 ± 4 yr

Patients without

mutation

41.4 ± 10.8 yr

Patients with

Those with FH

and those without

FH did not

present statistical

significance.

mutation

49.9 ± 11.3 yr

Patients without

mutation

52.5 ± 13.3 yr

Patients with

From the 6 patients,

2 had FH. No

statistical test was

mutation

49.6 ± 17.4 yr

Patients without

used.

55.1 ± 11.6 yr

informed

L444P 1/2

(50%); N370S

1/2 (50%)

L444P and

IVS2þ1

endonucleases

diagnosed

were included.

Southern Brazil

(2008)

De

347 PD patients and

All patients

Direct sequencing

N370S and

13/347 **(3.7%)**;

0/341

L444P 8/13

(62%); N370S

5/13 (38%)

L444P

diagnosed

were included.

341 control subjects

from Southeastern,

Midwestern and

Northern Brazil

Carvalho

et al.

(2012)

Amaral

81 PD patients and 81

All patients

Amplification of the

N370S and

6/81 **(7.4%);**

0/81

L444P 3/6

(50%); N370S

3/6 (50%)

L444P

exon 8–exon 11, PCR-RFLP

for N370S and L444P,

restriction endonucleases

and direct sequencing of

N370S and L444P

diagnosed

were included.

control subjects from

Northern Brazil

et al.

(2018)

*Bold valor are percentage.*

**Table 1.** *GBA mutation among PD patients in different Brazilian regions.*

(2007)

**Population studied**

**PD inclusion** 

**Method**

**Mutation** 

**Patients** 

**Control** 

**Age of onset**

*GBA* **mutated** 

**PD and familiar** 

**history (FH)**

The two patients

had FH, no

statistical test was

used.

**mutation** 

**frequency**

**mutation** 

**frequency**

**analyzed**

**criteria**

frequent genetic defect found was p.N370S and the frequency of mutations in PD was 4.5% and 0.63% in controls. Therefore, the difference may be due to a particular frequency of *GBA* mutations in regions of Italy or to the sample size [36].

Although the frequency of *GBA* mutations in populations of PD patients has been well characterized worldwide, few data are available on the inverse relationship, that is, the risk of healthy heterozygous for *GBA* mutations in developing PD, since it is not known for sure the degree of influence that this genetic alteration has on the onset of the disease.

Anheim M et al. [43] published in 2012 an estimate of the penetration of PD in healthy heterozygous people for *GBA* mutations and reached a value of 7.6%, 13.7%, 21.4% and 29.7% for 50, 60, 70 and 80 years, respectively, based on a dominance model. However, in the same year, [51] found a lower value: 5% for 60 years and 15% for 80 years of age. Such difference is perhaps due to additional genetic factors or environmental factors, a fact that emphasizes the possibility of variance of the risk of developing PD according to the genetic background of the population. Families that have *GBA* mutations segregation through generations are a group at risk for developing PD and should be monitored for a possible early diagnosis to have better chances in modifying the disease.

### **5. Pathogenic mechanisms in PD**

α-Synuclein is a key protein in the neuropathogenesis of PD, involved in several pathogenic processes. The physiological function of α-synuclein is not well understood, but studies show that it is normally located in presynaptic terminals where it binds to lipids and plays the role of regulating in more than one step the traffic of synaptic vesicles to be released. As cited above, the insoluble forms (oligomers and fibrils) of this protein accumulate and compound the Lewy bodies found in most PD patients and also contribute to neuronal cell death [5].

The reason behind this accumulation can be due to increased synthesis or decreased degradation (**Figure 2**). Mutations, as the triplications of the *SNCA* gene, can enhance the production of α-synuclein, while the interaction between the oligomers and fibrils formed with other mutant proteins can result in the deficiency of certain metabolic pathways and contribute to slowing down α-synuclein proteolysis. On the other hand, the initial deficiency of certain metabolic pathway caused by mutations in genes, advanced age or environmental factors can also be the trigger to accumulate α-synuclein resulting in the insoluble forms. This last theory has been

#### **Figure 2.**

*The proposed physiological and PD-associated pathological functions of α-synuclein in neurons. Adapted from Ref. [6].*

reinforced by the confirmed association of diverse genes involved in autophagy, endocytosis and lysosomal pathways as the *GBA* and *LRRK2* gene [1–6].

#### **5.1 Lysosomal function-related genes and PD**

The endosome-lysosome traffic processes, autophagy and lysosomal degradation, are essential functions for cell homeostasis, especially for neurons. The differentiated neurons have to maintain their homeostasis during the aging through degradation pathways since they do not divide in the same way as other eukaryotic cells. Moreover, cellular and animal models have also shown that the process of lysosome-autophagy and ubiquitin-proteasome has its activity reduced with natural aging. It may cause the accumulation of proteins whose homeostasis depends on those processes, such as α-synuclein. Indeed, the stimulation of degradation by macroautophagy through drugs proved to decrease intracellular levels of α-synuclein in experimental models [1–6].

Reciprocally, the accumulation of α-synuclein in the substantia nigra in experimental models leads to a reduction in lysosomal enzymes such as GCase, cathepsin B, β-galactosidase and hexosaminidase causing the inhibition of macroautophagy and ubiquitin-proteasome processes as a consequence enzyme transport to the lysosome interruption through dysfunction of vesicles and endosomes. The result is a vicious cycle where α-synuclein degradation mechanisms are inefficient resulting in the protein accumulation and it reinforces the inhibition of degradation activity [5–52].

Both GCase deficiency and the accumulation of its substrate (GlcCer) have been described to be associated with neurodegeneration (**Figure 3**). Feany M et al. [53] suggested that the connection of the α-synuclein to lipidic membranes would protect this protein from inadequate and clumped folding. Mutations of the *GBA* gene would alter the lipid composition of the membrane, which would favor a build-up of α-synuclein in the cytosol and subsequently in the Lewy bodies. Knockdown *GBA* in neuronal cells or in mouse models impairs α-synuclein clearance, whereas increasing glucocerebrosidase activity has the opposite effect, perhaps giving support to the loss-of-function theory in which the reduced or absent lysosomal enzyme is the trigger to α-synuclein accumulation [54].

Even excluding *GBA*, there was evidence for a burden damaging alleles in association with PD. In 2017, Ref. [55] performed a large study to examine the overlap between genes responsible for LSD and PD. More than half of PD cases in their cohort harbors one or more putative damaging variants among the 54 LSD genes. Specially, risk alleles in the genes *SMPD1* (Niemann-Pick type A/B), *GALC* (Krabbe disease), *SLC17A5* (Salla disease), *ASAH1* (Farber lipogranulomatosis) and *CTSD* (neuronal ceroid lipofuscinosis) have been candidate genes well replicated in different studies. *SMPD1* and *ASAH1,* along with *GBA*, participate in ceramide metabolism, and this fact can be evidence of the ceramide-associated process being relevant in a scenery of lysosomal dysfunction in PD [10–55].

The genes appointed as risk factor for PD to date explain only a fraction of PD heritability, suggesting the involvement of additional loci. Besides, the fact that *GBA* is the major genetic risk factor for PD makes other LSD genes attractive candidate risk factors. The results of [55] suggest that many genes that encode lysosomal enzymes besides *GBA* likely contribute to susceptibility for PD in Caucasian population.

Not only the lysosomal function is important, but also the previous steps necessary for the vesicle content to reach this organelle. In [10], a GWAS meta-analysis study found that PD-associated signals were enriched for autophagy and lysosomal function. *SCARB2* encodes a membrane protein (LIMP-2) required for correct targeting of GCase enzyme to the lysosome. Independent large GWAS have replicated

**73**

accumulation.

**Figure 3.**

membranes, including the lysosome [6–18].

(also known as RAB29) [9–16].

*Genetic Risk Factors and Lysosomal Function in Parkinson Disease*

common risk alleles in this gene. Functional analysis in cellular and animal model has shown that the reduction of LIMP-2 impairs the clearance of α-synuclein [55]. Those data reinforce that both the malfunction and the absence of the GCase, through mutations or impairment in the pathway, can result in α-synuclein

*The vicious cycle between the GCase and α-synuclein. Decreased glucocerebrosidase increases the lysosomal concentrations of glucosylceramide, which increases the formation of soluble α-synuclein oligomers. These oligomers also disrupt transport of newly synthesized glucocerebrosidase between the endoplasmic reticulum* 

*and Golgi apparatus, further compounding the problem. Adapted from reference [52].*

The protein LRRK2 is complex and can work together with diverse proteins in different pathways, but for PD, the most relevant seems to be its endosome-to-lysosome trafficking function. Mutations in the kinase domain of the LRRK2 protein, such as the most common G2019S, compromise the traffic of the endosomal content to the lysosome through accentuated phosphorylation resulting in the dysregulation of proteins of the Rab family, responsible to target vesicles to the correct organelle

In support of vesicular trafficking to lysosome impairment in PD, in 2009, two GWAS collaborative studies examining Caucasian and Asian subjects revealed significant risk alleles in *PARK16* locus for PD. This locus is a large linkage disequilibrium block that includes a Rab protein member of a subfamily that is implicated in vesicular transport to lysosomes and to lysosome-like organelles, the Rab-7 L1

Mutations in the *VPS35* gene are one of the causes for autosomal dominant PD. Its protein is also involved in trafficking to lysosomes as a member of the

*DOI: http://dx.doi.org/10.5772/intechopen.91850*

*Genetic Risk Factors and Lysosomal Function in Parkinson Disease DOI: http://dx.doi.org/10.5772/intechopen.91850*

#### **Figure 3.**

*Methods in Molecular Medicine*

**5.1 Lysosomal function-related genes and PD**

α-synuclein in experimental models [1–6].

enzyme is the trigger to α-synuclein accumulation [54].

relevant in a scenery of lysosomal dysfunction in PD [10–55].

reinforced by the confirmed association of diverse genes involved in autophagy,

The endosome-lysosome traffic processes, autophagy and lysosomal degradation, are essential functions for cell homeostasis, especially for neurons. The differentiated neurons have to maintain their homeostasis during the aging through degradation pathways since they do not divide in the same way as other eukaryotic cells. Moreover, cellular and animal models have also shown that the process of lysosome-autophagy and ubiquitin-proteasome has its activity reduced with natural aging. It may cause the accumulation of proteins whose homeostasis depends on those processes, such as α-synuclein. Indeed, the stimulation of degradation by macroautophagy through drugs proved to decrease intracellular levels of

Reciprocally, the accumulation of α-synuclein in the substantia nigra in experimental models leads to a reduction in lysosomal enzymes such as GCase, cathepsin B, β-galactosidase and hexosaminidase causing the inhibition of macroautophagy and ubiquitin-proteasome processes as a consequence enzyme transport to the lysosome interruption through dysfunction of vesicles and endosomes. The result is a vicious cycle where α-synuclein degradation mechanisms are inefficient resulting in the protein accumulation and it reinforces the inhibition of degradation activity [5–52]. Both GCase deficiency and the accumulation of its substrate (GlcCer) have been described to be associated with neurodegeneration (**Figure 3**). Feany M et al. [53] suggested that the connection of the α-synuclein to lipidic membranes would protect this protein from inadequate and clumped folding. Mutations of the *GBA* gene would alter the lipid composition of the membrane, which would favor a build-up of α-synuclein in the cytosol and subsequently in the Lewy bodies. Knockdown *GBA* in neuronal cells or in mouse models impairs α-synuclein clearance, whereas increasing glucocerebrosidase activity has the opposite effect, perhaps giving support to the loss-of-function theory in which the reduced or absent lysosomal

Even excluding *GBA*, there was evidence for a burden damaging alleles in association with PD. In 2017, Ref. [55] performed a large study to examine the overlap between genes responsible for LSD and PD. More than half of PD cases in their cohort harbors one or more putative damaging variants among the 54 LSD genes. Specially, risk alleles in the genes *SMPD1* (Niemann-Pick type A/B), *GALC* (Krabbe disease), *SLC17A5* (Salla disease), *ASAH1* (Farber lipogranulomatosis) and *CTSD* (neuronal ceroid lipofuscinosis) have been candidate genes well replicated in different studies. *SMPD1* and *ASAH1,* along with *GBA*, participate in ceramide metabolism, and this fact can be evidence of the ceramide-associated process being

The genes appointed as risk factor for PD to date explain only a fraction of PD heritability, suggesting the involvement of additional loci. Besides, the fact that *GBA* is the major genetic risk factor for PD makes other LSD genes attractive candidate risk factors. The results of [55] suggest that many genes that encode lysosomal enzymes besides *GBA* likely contribute to susceptibility for PD in Caucasian

Not only the lysosomal function is important, but also the previous steps necessary for the vesicle content to reach this organelle. In [10], a GWAS meta-analysis study found that PD-associated signals were enriched for autophagy and lysosomal function. *SCARB2* encodes a membrane protein (LIMP-2) required for correct targeting of GCase enzyme to the lysosome. Independent large GWAS have replicated

endocytosis and lysosomal pathways as the *GBA* and *LRRK2* gene [1–6].

**72**

population.

*The vicious cycle between the GCase and α-synuclein. Decreased glucocerebrosidase increases the lysosomal concentrations of glucosylceramide, which increases the formation of soluble α-synuclein oligomers. These oligomers also disrupt transport of newly synthesized glucocerebrosidase between the endoplasmic reticulum and Golgi apparatus, further compounding the problem. Adapted from reference [52].*

common risk alleles in this gene. Functional analysis in cellular and animal model has shown that the reduction of LIMP-2 impairs the clearance of α-synuclein [55]. Those data reinforce that both the malfunction and the absence of the GCase, through mutations or impairment in the pathway, can result in α-synuclein accumulation.

The protein LRRK2 is complex and can work together with diverse proteins in different pathways, but for PD, the most relevant seems to be its endosome-to-lysosome trafficking function. Mutations in the kinase domain of the LRRK2 protein, such as the most common G2019S, compromise the traffic of the endosomal content to the lysosome through accentuated phosphorylation resulting in the dysregulation of proteins of the Rab family, responsible to target vesicles to the correct organelle membranes, including the lysosome [6–18].

In support of vesicular trafficking to lysosome impairment in PD, in 2009, two GWAS collaborative studies examining Caucasian and Asian subjects revealed significant risk alleles in *PARK16* locus for PD. This locus is a large linkage disequilibrium block that includes a Rab protein member of a subfamily that is implicated in vesicular transport to lysosomes and to lysosome-like organelles, the Rab-7 L1 (also known as RAB29) [9–16].

Mutations in the *VPS35* gene are one of the causes for autosomal dominant PD. Its protein is also involved in trafficking to lysosomes as a member of the

retromer complex, which has the role to regulate the delivery of the protein content within endosomes to organelles. Some of the proteins carried by this complex are cation-independent mannose-6-phosphate receptors, necessary for the transport of lysosomal enzymes to the lysosome. In the dysfunction of the retromer complex, the receptors are not returned to the Golgi complex, thus impairing the lysosomal function. In addition, mutations in *ATP13A2* are a rare cause of recessive juvenileonset Parkinsonism and dementia and are associated to lysosomal dysfunction. This gene codifies a lysosomal P-type ATPase [1, 6].

Interestingly, potentiated retromer function might suppress the altered trafficking and toxicity that are associated with mutations in *LRRK2* or the overexpression of α-synuclein85, which suggests a potential therapeutic avenue. This fact emphasizes the possibility that different genes can interact with each other influencing the lysosomal function and as a consequence modifying the PD progression.

These common and rare risk alleles in *ATP13A1, RAB7L1*, *LRRK2* and *VPS35*, which support a model of partial loss-of-function variants in genes regulating lysosomal activity by cellular trafficking, result in an increased vulnerability to α-synuclein mechanisms in PD [55]. Ref. [10], the largest GWAS meta-analysis study, concluded that PD-associated signals were enriched for autophagy and lysosomal function. It replicated the results for *GBA* and *TMEM175* genes, which encode a potassium channel involved in the regulation of lysosome and identified three novel candidate genes, *CTSB* (a lysosomal cysteine protease)*, ATP60A1* (an ATPase) and *GALC* (a lysosomal enzyme).

#### **Figure 4.**

*Some of the PD-related genes associated with trafficking to the lysosome. Genes that encode intracellular trafficking components are associated with common sporadic and familial forms of PD, as well as related syndromes that share some of the clinical features of PD. Most of these genes are known to affect trafficking to the lysosome in the context of late endosome-to-lysosome pathways, clathrin-dependent endocytosis, macroautophagy or mitophagy. Wild-type α-synuclein (blue) can also enter lysosomes through chaperonemediated autophagy. Adapted from Ref. [6].*

**75**

*Genetic Risk Factors and Lysosomal Function in Parkinson Disease*

studies and experiments in PD cellular or animal models.

Besides *GBA*, loss-of-function alleles are known as frequent PD risk factors, and

Therefore, advances in genetic and experimental model for PD have illuminated

Currently, genetic testing for PD is not a routine procedure, being restricted only to cases with a positive family history, with early onset or with the presence of specific atypical symptoms. In the future with the advance of genetic research, however, there is a possibility to use genetic variants to provide a perspective of the patient's clinical evolution. For this purpose, it is important to replicate risk variants for PD in large and genetically diverse samples due to the different results among populations. Genetic studies need to be a collaboration of the whole world to understand the genetics of a complex disease. In addition, candidate genes here appointed need further experiments in PD cellular or animal models understanding of the underlying pathology and molecular pathogenesis to provide perhaps the basis for the development of new therapies able to target mutated proteins that cause impairment in relevant pathways for PD as endosome trafficking, lysosome

This study was performed with research grants from Instituto Nacional de Genética Médica e Populacional—INAGEMP (CNPq: 573993/2008-4), Fundação de Amparo à Pesquisa do Estado do Pará (FAPESPA) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) from Brazil.

some of those genes had the functional characterization made by analysis studies that showed knockout mice manifesting tremor phenotype with cerebral and cerebellar atrophy, thus corroborating with lysosome loss-of-function hypothesis to

an important role for defects in intracellular transport pathways to lysosomes (**Figure 4**). The probability of discovering rare PD disease risk alleles at a single locus is low; however, if a set of lysosomal-related genes is investigated in conjunction, the chance of finding significant genetic variations is increased. Also, the candidate genes here appointed need further studies including even larger case–control

be involved with α-synuclein dysfunction and PD pathogenesis [10].

*DOI: http://dx.doi.org/10.5772/intechopen.91850*

**6. Conclusion**

function and autophagy.

**Acknowledgements**

*Genetic Risk Factors and Lysosomal Function in Parkinson Disease DOI: http://dx.doi.org/10.5772/intechopen.91850*

Besides *GBA*, loss-of-function alleles are known as frequent PD risk factors, and some of those genes had the functional characterization made by analysis studies that showed knockout mice manifesting tremor phenotype with cerebral and cerebellar atrophy, thus corroborating with lysosome loss-of-function hypothesis to be involved with α-synuclein dysfunction and PD pathogenesis [10].

Therefore, advances in genetic and experimental model for PD have illuminated an important role for defects in intracellular transport pathways to lysosomes (**Figure 4**). The probability of discovering rare PD disease risk alleles at a single locus is low; however, if a set of lysosomal-related genes is investigated in conjunction, the chance of finding significant genetic variations is increased. Also, the candidate genes here appointed need further studies including even larger case–control studies and experiments in PD cellular or animal models.

### **6. Conclusion**

*Methods in Molecular Medicine*

gene codifies a lysosomal P-type ATPase [1, 6].

ATPase) and *GALC* (a lysosomal enzyme).

**74**

**Figure 4.**

*mediated autophagy. Adapted from Ref. [6].*

*Some of the PD-related genes associated with trafficking to the lysosome. Genes that encode intracellular trafficking components are associated with common sporadic and familial forms of PD, as well as related syndromes that share some of the clinical features of PD. Most of these genes are known to affect trafficking to the lysosome in the context of late endosome-to-lysosome pathways, clathrin-dependent endocytosis, macroautophagy or mitophagy. Wild-type α-synuclein (blue) can also enter lysosomes through chaperone-*

retromer complex, which has the role to regulate the delivery of the protein content within endosomes to organelles. Some of the proteins carried by this complex are cation-independent mannose-6-phosphate receptors, necessary for the transport of lysosomal enzymes to the lysosome. In the dysfunction of the retromer complex, the receptors are not returned to the Golgi complex, thus impairing the lysosomal function. In addition, mutations in *ATP13A2* are a rare cause of recessive juvenileonset Parkinsonism and dementia and are associated to lysosomal dysfunction. This

Interestingly, potentiated retromer function might suppress the altered trafficking and toxicity that are associated with mutations in *LRRK2* or the overexpression of α-synuclein85, which suggests a potential therapeutic avenue. This fact emphasizes the possibility that different genes can interact with each other influencing the

These common and rare risk alleles in *ATP13A1, RAB7L1*, *LRRK2* and *VPS35*, which support a model of partial loss-of-function variants in genes regulating lysosomal activity by cellular trafficking, result in an increased vulnerability to α-synuclein mechanisms in PD [55]. Ref. [10], the largest GWAS meta-analysis study, concluded that PD-associated signals were enriched for autophagy and lysosomal function. It replicated the results for *GBA* and *TMEM175* genes, which encode a potassium channel involved in the regulation of lysosome and identified three novel candidate genes, *CTSB* (a lysosomal cysteine protease)*, ATP60A1* (an

lysosomal function and as a consequence modifying the PD progression.

Currently, genetic testing for PD is not a routine procedure, being restricted only to cases with a positive family history, with early onset or with the presence of specific atypical symptoms. In the future with the advance of genetic research, however, there is a possibility to use genetic variants to provide a perspective of the patient's clinical evolution. For this purpose, it is important to replicate risk variants for PD in large and genetically diverse samples due to the different results among populations. Genetic studies need to be a collaboration of the whole world to understand the genetics of a complex disease. In addition, candidate genes here appointed need further experiments in PD cellular or animal models understanding of the underlying pathology and molecular pathogenesis to provide perhaps the basis for the development of new therapies able to target mutated proteins that cause impairment in relevant pathways for PD as endosome trafficking, lysosome function and autophagy.

#### **Acknowledgements**

This study was performed with research grants from Instituto Nacional de Genética Médica e Populacional—INAGEMP (CNPq: 573993/2008-4), Fundação de Amparo à Pesquisa do Estado do Pará (FAPESPA) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) from Brazil.

*Methods in Molecular Medicine*
