**7. Pathogenic pathways**

Familial and sporadic PD present similar clinical features, which reinforce the hypothesis that common pathways might be at the basis of a so analogous phenotype. Generally dystonia appears as the first symptom in the familial cases, while gait impairment and postural instability are one of the first manifestation for the sporadic ones. Evidence is emerging that some of the pathways covered in the rare monogenic forms of PD: dysfunction or impairment of the ubiquitin-proteosome system and/or mytochondria, may play a direct role in the etiology of the common sporadic disorder and genetic variation contribute to the risk of developing PD.

So far, the proteins that have been linked to parkinsonism by genetic studies have roles in:


These disparate functions might overlap as they all lead to the age-associated dysfunction and death of dopaminergic neurons that characterize PD. However, the relationships between these functions are not direct and the connections between them are not immediately evident.

#### **7.1 Mitochondrial dysfunction (α-synuclein,** *PARKIN***,** *PINK1***,** *DJ-1***,** *FBXO7***,** *POLG1, Omi/HtrA2***)**

The pathway that has come most clearly out of the analysis of the Mendelian genes is a mitochondrial damage repair pathway. α*-*synuclein has long been known to modulate mitochondrial function, but the mechanism remains unknown. A possible role in mitochondrial signaling has been hypothesized. Parkin enhances transcription and replication of mitochondrial DNA in proliferating cells. Furthermore, Parkin, an E3 ubiquitin ligase, and PINK1, a mitochondrial kinase, are involved in the elimination of damaged mitochondria. DJ-1, and possibly FBX07, another ubiquitin ligase, are also likely to play a role in the mitochondrial pathway. DJ-1 can protect the cell against oxidative stress and can also translocate to the mitochondria. (Cookson 2010). The similar phenotype can confirm a common role of these genes in the mitochondrial pathway (Paisan-Ruiz et al., 2010; Valente et al., 2004;Yamamura 2010).

POLG1 is a mitochondrial DNA polymerase (Polymerase gamma 1) of the inner membrane that synthesizes, replicates and repairs mitochondrial DNA. Several mutations within POLG1 have been associated with parkinsonism in addition to other clinical phenotypes.

HtrA2 also known as Omi (HtrA2/Omi) is serine protease localized to the inner membrane space of mitochondria. A variation within Omi (G399S) was found in four patients with late-

Parkin expression is correlated with cell maturation and implicates a physiological role of parkin in various types of neurons. *UCHL1* is highly expressed in cultured NPCs (neural progenitor cells) as well as in embryonic brain in general. UCHL1 has been shown to be involved in regulating morphology of NPCs and in mediating neurogenesis. Involvement of *LRRK2* in neurite outgrowth might explain high expression levels in the first two or three weeks after birth. *Omi/HtrA2* is found in various fetal tissues. Loss of Omi/HtrA2 (mouse mutant mnd2, motor neuron degeneration2) leads to muscle wasting, neurodegeneration, involution of the spleen and thymus, and death by 40 days of age. Since dopaminergic neurons have long been central to PD research and genes involved in development of these cells deserve special attention. Dominant mutations in *Nurr1* have been reported in families with late onset PD. Nurr1, a member of the nuclear receptor superfamily of transcription factors is critically involved in the development of ventral midbrain dopaminergic neurons. Mutations within *Nurr1* have not been found again, association studies turned out to be negative in most studies. In addition (mice deficient for *PITX3*), a homeobox transcription factor, which is expressed from E11 to adulthood, fail to develop dopaminergic neurons of the substantia nigra . Taken together several PD associated genes are expressed during development. The potential involvement of these genes in early stages of the disease remains to be determined. Additionally PD is by no means restricted to dopaminergic neurons. It will be of great interest to identify genes with involvement in developmental

Numerous working models have been proposed to integrate the complexities of environmental, biochemical, genetic and neuropathological evidence. However, a more simplicistic model of PD pathogenesis depicts a progressive imbalance between the forces that promote degeneration of at risk neurons by increasing mitochondrial dysfunction and oxidative stress during the aging process, and those that encompass individual or integrated

Genetic therapy should move forward, aiming to restabilize the balance between these antagonist forces: enhancing the neuroprotective ones (*PRKN, DJ-1, PINK1*) and/or

Parkinson's disease is a progressive neurodegenerative disease which shares genetic influences and pathways with other neurodegenerative diseases like Alzheimer's disease (AD). Specifically, common pathways involve protein aggregation and neuroinflammatory process. Several lines of investigation have now converged to show that the etiologies of AD and PD share common mechanisms (Bossy-Wetzel et al., 2004). One of the commonalities between AD and PD is the extra and intracellular accumulation of protein aggregates rich in β-pleated sheet conformation, representing the hallmarks of these two slowly progressive neurodegenerative disorders. Such protein aggregates might arise, in part, as a consequence of impaired proteasomal and/or autophagic removal of the damaged proteins (Taylor et al., 2002; Bence et al., 2001). The conformational change that results in the accumulation of misfolded proteins — amyloid-β (Aβ) and tau in AD and α-synuclein in PD — is associated with progressive dysfunction and death of cells in selected brain areas, determining clinical presentation. The intermediate forms of pathogenic proteins such as oligomers and protofibrils are thought to have cytotoxic effects on neurons. There

stopping and silencing potentiallty harmful effects ones (*SNCA*, *LRRK2*)

**8. Pathological mechanisms in neurodegenerative diseases** 

stages of several cell types affected in PD.

cellular-defense mechanism.

onset PD and a polymorphism (A141S) has been suggested to be a risk factor in Germans. Furthermore, a possible implication has been demonstrated by animal models: knocking out Omi in mice leads to neurodegeneration with features of motor neuron dysfunction, ataxia and parkinsonism with striatal damage.

In addition MPTP can damage in a selective way dopamine neurons and mitochondria causing parkinsonism. (Langston 1989).

### **7.2 Lysosome pathway (***GBA***,** *ATP13A2***)**

A second pathway that is likely to be involved in Parkinson disease clearly involves the lysosomes. Proteins with short half-lives are mostly degraded by the proteasome whereas most cytosolic proteins with half lives longer than 10 hours are degraded by the autophagy- lysosome pathway. Glucosecerebrosidase, GBA, and ATP13A2 are lysosomal enzymes. A role of GBA protein in PD is suggested by the clinical observation of association of PD with Gaucher's disease. Patients with this well characterized recessive neurometabolic disease, caused by mutation in the glucocerebrosidase gene (*GBA*) have a high prevalence of PD. Screening of PD patients for *GBA* mutations found a higher number of heterozygous mutations carriers as compared to healthy controls. *GBA* encodes a lysosomal enzyme that cleaves glucocerebroside. As α-synuclein is in part degraded by chaperone-mediated lysosomal pathway, it is possible that *GBA* mutations may increase the risk for PD by altering cellular α -synuclein homeostasis. Within the chaperone-mediated lysosomal uptake pathway α-synuclein binds lysosomal membrane receptors before being selectively translocated into the lysosome. Mutant α-synuclein also binds to receptors, but instead of being translocated sufficiently blocks not only its own uptake but also uptake of other substrates. Mutations in ATP13A2, a lysosomal ATPase, cause autosomal-recessive early onset PD further linking lysosomes to neurodegeneration

#### **7.3 Ubiquitin proteosome pathway (***PARKIN***,** *UCHL1***)**

The ubiquitin proteasome pathway has been strongly implicated in PD pathogenesis. *Parkin*  functions as an E3 ubiquitin ligase. Some disease causing mutations within parkin impair its ligase activity leading to intracellular accumulation of parkin substrates. Accumulation of potentially toxic proteins might be especially detrimental for vulnerable neurons like dopaminergic neurons. Furthermore a missense mutation has been described in *UCHL1*, a deubiquitylating enzyme. The I93M substitution decreases UCHL1 enzymatic activity in vitro. Deubiquitylation is an important process to recycle ubiquitin monomers from proteins that have been targeted to the proteasome. In addition overexpressed wildtype or mutant αsynuclein has been shown to inhibit proteasome function in vitro and in vivo, even if not directly involved in the proteosomal pathway.

#### **7.4 Embryonic development (α-synuclein,** *PARKIN***,** *UCHL1***,** *LRRK2***,** *Omi/HtrA2***,**  *NURR1***,** *PITX3***)**

α*-*synuclein is important not only in brain but also in peripheral tissues during normal human prenatal development. From 15 to 23 gestational weeks α*-*synuclein is expressed in almost all fetal human organs while in adult human tissues an high alpha-synuclein expression can be observed only in the brain.

onset PD and a polymorphism (A141S) has been suggested to be a risk factor in Germans. Furthermore, a possible implication has been demonstrated by animal models: knocking out Omi in mice leads to neurodegeneration with features of motor neuron dysfunction, ataxia

In addition MPTP can damage in a selective way dopamine neurons and mitochondria

A second pathway that is likely to be involved in Parkinson disease clearly involves the lysosomes. Proteins with short half-lives are mostly degraded by the proteasome whereas most cytosolic proteins with half lives longer than 10 hours are degraded by the autophagy- lysosome pathway. Glucosecerebrosidase, GBA, and ATP13A2 are lysosomal enzymes. A role of GBA protein in PD is suggested by the clinical observation of association of PD with Gaucher's disease. Patients with this well characterized recessive neurometabolic disease, caused by mutation in the glucocerebrosidase gene (*GBA*) have a high prevalence of PD. Screening of PD patients for *GBA* mutations found a higher number of heterozygous mutations carriers as compared to healthy controls. *GBA* encodes a lysosomal enzyme that cleaves glucocerebroside. As α-synuclein is in part degraded by chaperone-mediated lysosomal pathway, it is possible that *GBA* mutations may increase the risk for PD by altering cellular α -synuclein homeostasis. Within the chaperone-mediated lysosomal uptake pathway α-synuclein binds lysosomal membrane receptors before being selectively translocated into the lysosome. Mutant α-synuclein also binds to receptors, but instead of being translocated sufficiently blocks not only its own uptake but also uptake of other substrates. Mutations in ATP13A2, a lysosomal ATPase, cause autosomal-recessive early onset PD further linking lysosomes to

The ubiquitin proteasome pathway has been strongly implicated in PD pathogenesis. *Parkin*  functions as an E3 ubiquitin ligase. Some disease causing mutations within parkin impair its ligase activity leading to intracellular accumulation of parkin substrates. Accumulation of potentially toxic proteins might be especially detrimental for vulnerable neurons like dopaminergic neurons. Furthermore a missense mutation has been described in *UCHL1*, a deubiquitylating enzyme. The I93M substitution decreases UCHL1 enzymatic activity in vitro. Deubiquitylation is an important process to recycle ubiquitin monomers from proteins that have been targeted to the proteasome. In addition overexpressed wildtype or mutant αsynuclein has been shown to inhibit proteasome function in vitro and in vivo, even if not

**7.4 Embryonic development (α-synuclein,** *PARKIN***,** *UCHL1***,** *LRRK2***,** *Omi/HtrA2***,** 

α*-*synuclein is important not only in brain but also in peripheral tissues during normal human prenatal development. From 15 to 23 gestational weeks α*-*synuclein is expressed in almost all fetal human organs while in adult human tissues an high alpha-synuclein

and parkinsonism with striatal damage.

causing parkinsonism. (Langston 1989).

neurodegeneration

*NURR1***,** *PITX3***)** 

**7.2 Lysosome pathway (***GBA***,** *ATP13A2***)** 

**7.3 Ubiquitin proteosome pathway (***PARKIN***,** *UCHL1***)** 

directly involved in the proteosomal pathway.

expression can be observed only in the brain.

Parkin expression is correlated with cell maturation and implicates a physiological role of parkin in various types of neurons. *UCHL1* is highly expressed in cultured NPCs (neural progenitor cells) as well as in embryonic brain in general. UCHL1 has been shown to be involved in regulating morphology of NPCs and in mediating neurogenesis. Involvement of *LRRK2* in neurite outgrowth might explain high expression levels in the first two or three weeks after birth. *Omi/HtrA2* is found in various fetal tissues. Loss of Omi/HtrA2 (mouse mutant mnd2, motor neuron degeneration2) leads to muscle wasting, neurodegeneration, involution of the spleen and thymus, and death by 40 days of age. Since dopaminergic neurons have long been central to PD research and genes involved in development of these cells deserve special attention. Dominant mutations in *Nurr1* have been reported in families with late onset PD. Nurr1, a member of the nuclear receptor superfamily of transcription factors is critically involved in the development of ventral midbrain dopaminergic neurons. Mutations within *Nurr1* have not been found again, association studies turned out to be negative in most studies. In addition (mice deficient for *PITX3*), a homeobox transcription factor, which is expressed from E11 to adulthood, fail to develop dopaminergic neurons of the substantia nigra . Taken together several PD associated genes are expressed during development. The potential involvement of these genes in early stages of the disease remains to be determined. Additionally PD is by no means restricted to dopaminergic neurons. It will be of great interest to identify genes with involvement in developmental stages of several cell types affected in PD.

Numerous working models have been proposed to integrate the complexities of environmental, biochemical, genetic and neuropathological evidence. However, a more simplicistic model of PD pathogenesis depicts a progressive imbalance between the forces that promote degeneration of at risk neurons by increasing mitochondrial dysfunction and oxidative stress during the aging process, and those that encompass individual or integrated cellular-defense mechanism.

Genetic therapy should move forward, aiming to restabilize the balance between these antagonist forces: enhancing the neuroprotective ones (*PRKN, DJ-1, PINK1*) and/or stopping and silencing potentiallty harmful effects ones (*SNCA*, *LRRK2*)

#### **8. Pathological mechanisms in neurodegenerative diseases**

Parkinson's disease is a progressive neurodegenerative disease which shares genetic influences and pathways with other neurodegenerative diseases like Alzheimer's disease (AD). Specifically, common pathways involve protein aggregation and neuroinflammatory process.

Several lines of investigation have now converged to show that the etiologies of AD and PD share common mechanisms (Bossy-Wetzel et al., 2004). One of the commonalities between AD and PD is the extra and intracellular accumulation of protein aggregates rich in β-pleated sheet conformation, representing the hallmarks of these two slowly progressive neurodegenerative disorders. Such protein aggregates might arise, in part, as a consequence of impaired proteasomal and/or autophagic removal of the damaged proteins (Taylor et al., 2002; Bence et al., 2001). The conformational change that results in the accumulation of misfolded proteins — amyloid-β (Aβ) and tau in AD and α-synuclein in PD — is associated with progressive dysfunction and death of cells in selected brain areas, determining clinical presentation. The intermediate forms of pathogenic proteins such as oligomers and protofibrils are thought to have cytotoxic effects on neurons. There

MSA

PD DLB SNCA

Fig. 3. Mutations and variations in the *SNCA* gene, encoding for α-synuclein, are involved in several neurodegenerative diseases, synucleinopathies: Parkinson's disease, PD, Multi

MS

PD HLA AD

ALS

Fig. 4. Variations in the *HLA*, the major histocompatibility complex gene, are involved in several neurodegenerative diseases: Parkinson's disease, PD, Alzheimer disease, AD,

system atrophy, MSA, Dementia with Lewy Bodies, DLB

Multiple sclerosis, MS, Amiotrofic Lateral Sclerosis, ALS.

is considerable overlap in the mechanisms by which oligomeric Aβ and α-synuclein damage and kill neurons that may include: oxidative stress and free radical formation, impaired bioenergetics and mitochondrial dysfunction, disruption of neuronal Golgi apparatus and transport, molecular chaperones, neurotrophins and "neuroinflammatory" processes.

While variability in several genes may influence the risk for developing one disease, single genes often affect the risk for more than one trait. This diversity is best exemplified by variability in the tau and alpha-synuclein (*SNCA*) proteins, which alter risk of PD (*MAPT* and *SNCA*), progressive sopranuclear palsy corticobasal degeneration (*MAPT*), and multiple system atrophy (*SNCA*).

Fig. 2. Mutations and variants in the *MAPT* gene, encoding for tau protein, are involved in several neurodegenerative diseases, tauopathies: progressive sopranuclear palsy, PSP, corticobasal degeneration, CBD, Alzheimer disease, AD, Als-PD-dementia complex of Guam and Parkinson's disease.

is considerable overlap in the mechanisms by which oligomeric Aβ and α-synuclein damage and kill neurons that may include: oxidative stress and free radical formation, impaired bioenergetics and mitochondrial dysfunction, disruption of neuronal Golgi apparatus and transport, molecular chaperones, neurotrophins and "neuroinflammatory"

While variability in several genes may influence the risk for developing one disease, single genes often affect the risk for more than one trait. This diversity is best exemplified by variability in the tau and alpha-synuclein (*SNCA*) proteins, which alter risk of PD (*MAPT* and *SNCA*), progressive sopranuclear palsy corticobasal degeneration (*MAPT*), and multiple

CBD

PSP PD **MAPT**

AD ALS-PD-

DEMENTIA

Fig. 2. Mutations and variants in the *MAPT* gene, encoding for tau protein, are involved in several neurodegenerative diseases, tauopathies: progressive sopranuclear palsy, PSP, corticobasal degeneration, CBD, Alzheimer disease, AD, Als-PD-dementia complex of Guam

processes.

system atrophy (*SNCA*).

and Parkinson's disease.

Fig. 3. Mutations and variations in the *SNCA* gene, encoding for α-synuclein, are involved in several neurodegenerative diseases, synucleinopathies: Parkinson's disease, PD, Multi system atrophy, MSA, Dementia with Lewy Bodies, DLB

Fig. 4. Variations in the *HLA*, the major histocompatibility complex gene, are involved in several neurodegenerative diseases: Parkinson's disease, PD, Alzheimer disease, AD, Multiple sclerosis, MS, Amiotrofic Lateral Sclerosis, ALS.

Ultimately, a joint GWA study of a pooled population of individuals with Alzheimer's disease and Parkinson's disease might be a powerful approach to identify common genetic susceptibility factors for these diseases. One interesting model is that tau acts as a downstream factor involved in both β-amyloid and α-synuclein toxicity, with neurofibrillary tangles only forming in the presence of amyloid. Therefore, β-amyloid and αsynuclein might reinforce each other's effect on neurodegeneration in the aging population, and the relative proportions of each pathology could correlate with the extent of dementia or parkinsonism, respectively. Recent studies suggest that environmental factors may contribute to neurodegeneration through the induction of epigenetic modifications, such as DNA methylation, and chromatin remodeling, which may induce alterations in gene expression programs. Epigenetics, which refers to any process that modifies gene activity without changing the actual DNA sequence, and leads to modifications that can be transmitted to daughter cells, is a relatively novel area of research that is currently attracting a high level of interest. Epigenetic modulation is present since the prenatal stages, and the aging process is now accepted to be associated with a loss of phenotypic plasticity to epigenetic modifications. Since aging is the most important risk factor for idiopathic AD and PD, it is expected that epigenetic alterations on DNA and/or chromatin structure may also accumulate during neurodegeneration, explaining to some extent the etiology of these

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**9. References** 

286.

Fig. 5. Variations in *LINGO1* and *LINGO2* are risk factors for Parkinson's disease, PD, and essential tremor, ET.

Fig. 6. Mutations in *DJ-1* can be involved in several neurodegenerative diseases: Parkinson's disease, PD, dementia and Amyotrophic Lateral Sclerosis, ALS.

Fig. 5. Variations in *LINGO1* and *LINGO2* are risk factors for Parkinson's disease, PD, and

PD ET **LINGO1 LINGO2** 

Fig. 6. Mutations in *DJ-1* can be involved in several neurodegenerative diseases: Parkinson's

**DJ-1** 

DEMENTIA

ALS

disease, PD, dementia and Amyotrophic Lateral Sclerosis, ALS.

PD

essential tremor, ET.

Ultimately, a joint GWA study of a pooled population of individuals with Alzheimer's disease and Parkinson's disease might be a powerful approach to identify common genetic susceptibility factors for these diseases. One interesting model is that tau acts as a downstream factor involved in both β-amyloid and α-synuclein toxicity, with neurofibrillary tangles only forming in the presence of amyloid. Therefore, β-amyloid and αsynuclein might reinforce each other's effect on neurodegeneration in the aging population, and the relative proportions of each pathology could correlate with the extent of dementia or parkinsonism, respectively. Recent studies suggest that environmental factors may contribute to neurodegeneration through the induction of epigenetic modifications, such as DNA methylation, and chromatin remodeling, which may induce alterations in gene expression programs. Epigenetics, which refers to any process that modifies gene activity without changing the actual DNA sequence, and leads to modifications that can be transmitted to daughter cells, is a relatively novel area of research that is currently attracting a high level of interest. Epigenetic modulation is present since the prenatal stages, and the aging process is now accepted to be associated with a loss of phenotypic plasticity to epigenetic modifications. Since aging is the most important risk factor for idiopathic AD and PD, it is expected that epigenetic alterations on DNA and/or chromatin structure may also accumulate during neurodegeneration, explaining to some extent the etiology of these chronic and progressive disorders.
