**3. Autosomal dominant PD**

### **3.1 SNCA (PARK1 and 4, 4p21)**

It was the first gene to be inequivocally associated with familial autosomal dominant PD. It encodes for α-synuclein, a small protein, which is aboundantly expressed in the brain and localized mostly to presynaptic nerve terminals. This protein has a central role in the learning process, brain plasticity, vescicular trafficking and dopamine synthesis but many aspects of the normal function of alpha-synuclein are still unknown. There is still no good explanation for the selectivity of neural damage in PD, which is prominent in dopaminergic cells whereas α-synuclein is expressed in many areas of the brain.

Alpha synuclein plays a role in both familial and sporadic form of PD and for this reason can be an interesting target for the development of new therapies.

The protein is linked to the phospolipid membrane strate through the N-terminal edge and to a lesser extent it is free in the cytoplasm. It is hypothesized that a possible pathological role derives from a conformational change that lead to an imbalance between the protein linked to the membrane and that free in the cytoplasm, with a consequent aggregation and fibril formation.

Three missense mutations are causal factor for the familial autosomic dominant form of the disease

to cosegregate with the disease in several families and are the most common cause of mendelian PD identified so far. In studies across several populations, 5–15% of autosomal dominant PD families carried mutations in *LRRK2*. One particularly common mutation, a base pair change at position 6055,better known as the 'G2019S-mutation', is responsible for familial PD in up to 7% of cases in different Caucasian populations , but was found, somewhat surprisingly, also in 1–2% of sporadic patients. Even higher G2019S prevalence rates of up to 40%were found in genetically isolated populations,such as the Ashkenazi Jewish and the North-African Arab populations, both in sporadic and familial cases. P.G2019S is the most common mutation among Caucasian patients and it is responsible for the 0.5-2% of the cases of the sporadic disease and for the 5% of the familial cases. This mutation is particularly frequent among the Askenazi Jews and the Arabs from North Africa, where it is responsible for 18-30% of the cases. The substitution has been identified also in the Iberian Peninsula, where it is involved in the 2.5-65% of the cases of the sporadic disease. It seems that the mutation has been originated in North Africa or in the Middle East and probably later it has been spread to Europe and Northern America. It has also been hypothesized a common founder for the p.G2019S substitution probably dated back to the 13th Century. The presence of the mutation in the Middle East and in North Africa lead to guess that the mutation should be even more older. The Fenices were known to be the principal merchants of the ancient world and probably they were responsible for the

Due to its relatively high frequency, the p.G2019S mutation offers for the first time the possibility of looking at gene–gene interactions. Three Spanish patients simultaneously harbouring heterozygous mutations in *LRRK2* and in the parkin gene did not present with an earlier age at onset or a more severe disease. The G2019S mutation also seems to be fully dominant, as homozygous mutation carriers have been identified who also do not differ

Another, even more common *LRRK2* variant, G2385R, has been found in the Asian population in 6–10% of sporadic PD patients, as opposed to 3–5% of controls. Consequently, this variant confers a relative risk of about 2 to 3 of developing PD,suggesting that different alterations in one and the same gene may act as a high-penetrance disease-causing mutation or as a genetic risk variant in sporadic populations.To date, more than 20 potentially pathogenic mutations in *LRRK2* have been identified, but in only six of them pathogenicity can be considered to be highly likely (R1441C, R1441G,R1441H, Y1699C, G2019S and I2020T), because of firm evidence of cosegregation in affected families and functional data suggesting an alteration of kinase activity.The most extensive clinicogenetic study so far estimated the overall frequency of *LRRK2* mutations in the European population to be 1.5%in sporadic and 4%in familial cases, with a geographic gradient decreasing from Mediterranean countries (Spain, Portugal, Italy) to northern countries. The average age of onset was 58 years, with a wide range from the mid-20s (rarely) to over 90 years. The clinical picture was that of typical asymmetric L-dopa responsive parkinsonism that was indistinguishable by any single criterion from PD in individuals without *LRRK2* mutations. As a group, the disease appeared to be somewhat more benign in patients with mutations in *LRRK2*, with slower progression and a lower frequency of dementia and psychiatric

In contrast to the finding that α-synuclein stains Lewy bodies and tau stains neurofibrillary tangles and grains, LRRK2 immunocytochemistry has so far failed to highlight any specific,

from heterozygotes with respect to disease severity or age of onset.

diffusion of this substitution.

complications.


The pathogenic mechanism is supposed to be a conformational change due to the amminoacid substitution in the protein chain, which facilitates α-synuclein aggregation. Clinically, the phenotype is more aggressive with an higher incidence of cognitive impairment and autonomic disfunction. *SNCA* point mutations have so far been found only in large, multigenerational PD families, never in sporadic PD. The phenotype of patients with *SNCA* point mutations is that of L-dopa responsive parkinsonism with a relatively early age at onset, rapid progression, and high prevalence of dementia, psychiatric and autonomic disturbances, reminiscent of Lewy body dementia. Several family members from these kindreds have come to autopsy and they invariably showed cell loss of dopaminergic neurons of the substantia nigra, and severe and widespread Lewy pathology, particularly in the form of Lewy neuritis. The identification of *SNCA* point mutations as a cause of PD soon led to the discovery that the encoded protein, a-synuclein, is the major fibrillar component of Lewy bodies and Lewy neuritis in familial as well as sporadic cases. The currently favoured hypothesis settles that the ammoniacid changes within alpha-synuclein lead to an increased tendency of the protein to form oligomers and later on fibrillar aggregates, representing a 'toxic gain of function'. However, the precise sequence of events which lead from aggregation to cellular dysfunction and cell death is still not obvious. Some studies favour the hypothesis that the mature aggregates (Lewy bodies) are not themselves the toxic moiety, but rather an attempt to the cell to clear small toxic oligomers. A direct link between α-synuclein and PD was further supported by the discovery that multiplications of the wildtype sequence of *SNCA* (duplications and triplications) (Singleton et al., 2003) cause PD with or without dementia in some families. This finding was of major mechanicistic importance because indicates that an increase in wild-type a-synuclein protein expression appears to be toxic to neurons. A dose dependency of this effect is demonstrated by the fact that patients with *SNCA* triplications (4 copies of the gene) have an early age of onset (mean of around 35 years) and high prevalence of dementia, while patients with *SNCA* duplications (3 copies) have a more typical late-onset PD phenotype.

The presence of α-synuclein-containing aggregates in the absence of coding *SNCA* mutations in sporadic disease suggest that other α-synuclein modifications, such as alternative splicing, phosporylation,, alterations in gene expression, or additional interacting genes may contribute to sporadic PD.

Then interaction of α-synuclein with proteins aggregating in other neurodegenerative diseases is coming increasingly into focus. 'Cross-seeding' of α-synuclein and tau has been hypothesized by a recent study. Interestingly this molecular mechanism may turn out to be the biological basis of the recently confirmed and refined association of *MAPT* haplotypes with PD and of an interaction of genetic variants in the *SNCA* and *MAPT* genes.

#### **3.2 LRRK2 (PARK8, 12p21)**

The gene spans a genomic region of 144 Kb, with 51 exons encoding LRRK2 or Dardarin, a 2527 amino acids protein, with various conserved domains recognized in its primary ammino-acid sequence. More than 40 variants have been identified in the gene and at least 16 of them are recognized as pathogenic ones. Missense mutations in this gene were found

• A53T, identified within an Italian family and three Greek families (Polymeropoulous et

The pathogenic mechanism is supposed to be a conformational change due to the amminoacid substitution in the protein chain, which facilitates α-synuclein aggregation. Clinically, the phenotype is more aggressive with an higher incidence of cognitive impairment and autonomic disfunction. *SNCA* point mutations have so far been found only in large, multigenerational PD families, never in sporadic PD. The phenotype of patients with *SNCA* point mutations is that of L-dopa responsive parkinsonism with a relatively early age at onset, rapid progression, and high prevalence of dementia, psychiatric and autonomic disturbances, reminiscent of Lewy body dementia. Several family members from these kindreds have come to autopsy and they invariably showed cell loss of dopaminergic neurons of the substantia nigra, and severe and widespread Lewy pathology, particularly in the form of Lewy neuritis. The identification of *SNCA* point mutations as a cause of PD soon led to the discovery that the encoded protein, a-synuclein, is the major fibrillar component of Lewy bodies and Lewy neuritis in familial as well as sporadic cases. The currently favoured hypothesis settles that the ammoniacid changes within alpha-synuclein lead to an increased tendency of the protein to form oligomers and later on fibrillar aggregates, representing a 'toxic gain of function'. However, the precise sequence of events which lead from aggregation to cellular dysfunction and cell death is still not obvious. Some studies favour the hypothesis that the mature aggregates (Lewy bodies) are not themselves the toxic moiety, but rather an attempt to the cell to clear small toxic oligomers. A direct link between α-synuclein and PD was further supported by the discovery that multiplications of the wildtype sequence of *SNCA* (duplications and triplications) (Singleton et al., 2003) cause PD with or without dementia in some families. This finding was of major mechanicistic importance because indicates that an increase in wild-type a-synuclein protein expression appears to be toxic to neurons. A dose dependency of this effect is demonstrated by the fact that patients with *SNCA* triplications (4 copies of the gene) have an early age of onset (mean of around 35 years) and high prevalence of dementia, while patients with *SNCA* duplications (3 copies)

The presence of α-synuclein-containing aggregates in the absence of coding *SNCA* mutations in sporadic disease suggest that other α-synuclein modifications, such as alternative splicing, phosporylation,, alterations in gene expression, or additional interacting

Then interaction of α-synuclein with proteins aggregating in other neurodegenerative diseases is coming increasingly into focus. 'Cross-seeding' of α-synuclein and tau has been hypothesized by a recent study. Interestingly this molecular mechanism may turn out to be the biological basis of the recently confirmed and refined association of *MAPT* haplotypes

The gene spans a genomic region of 144 Kb, with 51 exons encoding LRRK2 or Dardarin, a 2527 amino acids protein, with various conserved domains recognized in its primary ammino-acid sequence. More than 40 variants have been identified in the gene and at least 16 of them are recognized as pathogenic ones. Missense mutations in this gene were found

with PD and of an interaction of genetic variants in the *SNCA* and *MAPT* genes.

• A30P, identified in a German family (Krüger et al.,1998) • E56K, identified within a Spanish family (Zarranz et al., 2004)

have a more typical late-onset PD phenotype.

genes may contribute to sporadic PD.

**3.2 LRRK2 (PARK8, 12p21)** 

al., 1997)

to cosegregate with the disease in several families and are the most common cause of mendelian PD identified so far. In studies across several populations, 5–15% of autosomal dominant PD families carried mutations in *LRRK2*. One particularly common mutation, a base pair change at position 6055,better known as the 'G2019S-mutation', is responsible for familial PD in up to 7% of cases in different Caucasian populations , but was found, somewhat surprisingly, also in 1–2% of sporadic patients. Even higher G2019S prevalence rates of up to 40%were found in genetically isolated populations,such as the Ashkenazi Jewish and the North-African Arab populations, both in sporadic and familial cases. P.G2019S is the most common mutation among Caucasian patients and it is responsible for the 0.5-2% of the cases of the sporadic disease and for the 5% of the familial cases. This mutation is particularly frequent among the Askenazi Jews and the Arabs from North Africa, where it is responsible for 18-30% of the cases. The substitution has been identified also in the Iberian Peninsula, where it is involved in the 2.5-65% of the cases of the sporadic disease. It seems that the mutation has been originated in North Africa or in the Middle East and probably later it has been spread to Europe and Northern America. It has also been hypothesized a common founder for the p.G2019S substitution probably dated back to the 13th Century. The presence of the mutation in the Middle East and in North Africa lead to guess that the mutation should be even more older. The Fenices were known to be the principal merchants of the ancient world and probably they were responsible for the diffusion of this substitution.

Due to its relatively high frequency, the p.G2019S mutation offers for the first time the possibility of looking at gene–gene interactions. Three Spanish patients simultaneously harbouring heterozygous mutations in *LRRK2* and in the parkin gene did not present with an earlier age at onset or a more severe disease. The G2019S mutation also seems to be fully dominant, as homozygous mutation carriers have been identified who also do not differ from heterozygotes with respect to disease severity or age of onset.

Another, even more common *LRRK2* variant, G2385R, has been found in the Asian population in 6–10% of sporadic PD patients, as opposed to 3–5% of controls. Consequently, this variant confers a relative risk of about 2 to 3 of developing PD,suggesting that different alterations in one and the same gene may act as a high-penetrance disease-causing mutation or as a genetic risk variant in sporadic populations.To date, more than 20 potentially pathogenic mutations in *LRRK2* have been identified, but in only six of them pathogenicity can be considered to be highly likely (R1441C, R1441G,R1441H, Y1699C, G2019S and I2020T), because of firm evidence of cosegregation in affected families and functional data suggesting an alteration of kinase activity.The most extensive clinicogenetic study so far estimated the overall frequency of *LRRK2* mutations in the European population to be 1.5%in sporadic and 4%in familial cases, with a geographic gradient decreasing from Mediterranean countries (Spain, Portugal, Italy) to northern countries. The average age of onset was 58 years, with a wide range from the mid-20s (rarely) to over 90 years. The clinical picture was that of typical asymmetric L-dopa responsive parkinsonism that was indistinguishable by any single criterion from PD in individuals without *LRRK2* mutations. As a group, the disease appeared to be somewhat more benign in patients with mutations in *LRRK2*, with slower progression and a lower frequency of dementia and psychiatric complications.

In contrast to the finding that α-synuclein stains Lewy bodies and tau stains neurofibrillary tangles and grains, LRRK2 immunocytochemistry has so far failed to highlight any specific,

been suggested to reduce the risk of developing sporadic PD. Interestingly, another study hypothesizes that UCHL1 may possess two enzymatic activities, hydrolase and ligase activity. Overexpression of UCH-L1 variant I93M resulted in an accumulation of αsynuclein. Furthermore, they suggest that this accumulation is due to an ubiquitination of αsynuclein by dimerized UCH-L1. However, the S18Y polymorphic variant of UCH-L1 has

which may explain the 'protective' effect of the S18Y polymorphism. With evidence of UCH-L1 and parkin being involved in certain forms of familial PD, this gives credence to the involvement of the UPS in PD. However, it is unclear how UCH-L1 can promote the specific neurodegeneration of dopaminergic neurons in familial PD and what role it plays in the

The strategies of linkage mapping and positional cloning can also be used to identify loci in genes responsible for autosomal-recessive monogenic diseases. This mode of inheritance is characterized typically by the occurrence of the disease in siblings while the parents are obligatory heterozygous mutation carriers and usually remain healthy. Autosomal-recessive PD has clinically been first recognized and characterized in Japan (Ishikawa and Tsuji, 1996). Sibling pairs with PD often have much earlier age of onset compared with patients with the sporadic disease, which is why the term 'autosomal-recessive juvenile parkinsonism'(AR-JP): has been coined. Since families with a recessive disease are usually much smaller than multigenerational dominant pedigrees, linkage analysis is only successful if several families

So far, mutations in three genes have been identified in clinically 'pure' forms of autosomal recessive PD: *PARKIN* (PRKN, or PARK2), *PINK1* (PARK6) and *DJ-1*(PARK7). *PRKN* encodes for parkin, a cytoplasmatic protein which functions in the cellular ubiquitination/protein degradation pathway as an ubiquitin ligase. *DJ-1* and *PINK1* encode for mitochondrial proteins. Genetics mutations in these genes cause PD through the loss of the wild-type protein neuroprotective function, leading to oxidative stress, iron

They cause PD with earlier onset (<45 years) and slower progression compared to idiopatic PD. Three additional recessive genes have been added to the list more recently-*ATP13A2*, *PLA2G6* and *FBXO7*- which all also cause, when mutated, an early-onset disease with parkinsonism, but also with additional features such as dystonia, which often is seen as the

Juvenile cases of parkinsonism in siblings were first recognized in Japan. The first genetic locus for autosomal- recessive juvenile parkinsonism (AR-JP), as this form of PD was called,

Mutations were then identified in a large gene in that region that was called parkin. Clinically, these patients suffer from L-dopa-responsive parkinsonism and often develop early and severe L-dopa-induced motor fluctuations and dyskinesias. Some show diurnal fluctuations, with symptoms becoming worse later in the day. Dystonia at onset of the disease is common. *Parkin* mutations turned out to be a common cause of parkinsonism

reduced ligase activity but normal hydrolase activity,

mapping to the same locus are included into a study.

accumulation and mitochondrial dysfunction.

**4.1 PARKIN (PARK2, 6q25.2-q27)** 

was mapped to chromosome 6.

first symptom, dementia, oculomotor disturbances and spasticity.

sporadic form.

**4. Autosomal recessive PD** 

neurodegenerative lesion , and it is unclear how LRRK2 substitutions result in neuropathology. *LRRK2* mutations may therefore be an upstream event in the cascade leading to neurodegeneration with different pathologies.

*LRRK2* is widely expressed in the brain, with highest levels in the striatum and the hippocampus but a relatively low abundance in the substantia nigra; it can also be detected in other organs, such as the spleen, the lung and the liver. It has been found in the cytoplasm as well as associated with membranes. Its function and the mechanism by which LRRK2 mutations cause neuronal degeneration are unknown. By sequence homology, LRRK2 can be assigned to the group of recently identified ROCO proteins and contains a protein kinase domain of the mitogen-activated kinase class, suggesting a role in intracellular signalling pathways. Although the natural substrate of LRRK2 is unknown, cell culture studies using generic substrates or LRRK2 autophosphorylation paradigms suggested that at least some pathogenic mutations seem to be associated with an increase, rather than a loss, of kinase activity, and that kinase activity appears to be necessary for neurotoxicity in vitro. This discovery raises the interesting possibility that kinase inhibition may be a potential therapeutic strategy. An interesting aspect of LRRK2-associated PD is its heterogeneous pathology. Post-mortem changes in patients with *LRRK2* mutations are those of typical Lewy-body PD in most cases, but also include diffuse Lewy-body disease, nigral degeneration without distinctive histopathology and, rarely, even aggregates of the microtubule-associated protein tau, suggestive of progressive supranuclear palsy or frontotemporal dementia. Different pathological findings were even reported in a single family with an R1441C mutation. *LRRK2* mutations may therefore be an upstream event in the cascade leading to neurodegeneration with different pathologies. However, the vast majority of patients with a G2019S mutation, in whom pathology has been reported, seem to conform to the typical α-synuclein Lewy-body type of PD. No direct link between LRRK2 and α-synuclein has so far been demonstrated. α-synuclein does not seem to be phosphorylated by LRRK2.

Despite its still poorly understood role in pathogenesis, LRRK2-associated PD is of particular interest since it is the first example of a mendelian form of PD that is common enough to provide the opportunity to study the development

of the disease in a sizeable population. Longitudinal studies in presymptomatic mutation carriers may reveal premotor changes by clinical, biochemical or imaging methods, indicating the very early phases of the neurodegenerative process. It is in this population that studies exploring neuroprotective or preventive measures are most promising to yield first results

#### **3.3 UCHL1 (Park 5, 4p14)**

The ubiquitin C-terminal hydrolase-L1 (*UCH-L1*) was first identified as an abundant (1–2% of total brain protein), neuronspecific protein encoded by a 9.5 kb gene product (Day and Thompson, 1987; Doran et al., 1983). UCH-L1 is a de-ubiquitinating enzyme that removes carboxy-terminal ubiquitin *UCH-L1* from substrates but does not break bonds within polyubiquitin complexes and results in the recycling of ubiquitin. In addition,UCHL1 protein is found in several pathological structures including Lewy bodies and some Alzheimer's neurofibrillary tangles (Lowe

et al., 1990). Furthermore, recent studies have identified a single dominant mutant (I93M) in two members of a PD-affected family. Inversely, a polymorphism in *UCH-L1* (S18Y) has

neurodegenerative lesion , and it is unclear how LRRK2 substitutions result in neuropathology. *LRRK2* mutations may therefore be an upstream event in the cascade

*LRRK2* is widely expressed in the brain, with highest levels in the striatum and the hippocampus but a relatively low abundance in the substantia nigra; it can also be detected in other organs, such as the spleen, the lung and the liver. It has been found in the cytoplasm as well as associated with membranes. Its function and the mechanism by which LRRK2 mutations cause neuronal degeneration are unknown. By sequence homology, LRRK2 can be assigned to the group of recently identified ROCO proteins and contains a protein kinase domain of the mitogen-activated kinase class, suggesting a role in intracellular signalling pathways. Although the natural substrate of LRRK2 is unknown, cell culture studies using generic substrates or LRRK2 autophosphorylation paradigms suggested that at least some pathogenic mutations seem to be associated with an increase, rather than a loss, of kinase activity, and that kinase activity appears to be necessary for neurotoxicity in vitro. This discovery raises the interesting possibility that kinase inhibition may be a potential therapeutic strategy. An interesting aspect of LRRK2-associated PD is its heterogeneous pathology. Post-mortem changes in patients with *LRRK2* mutations are those of typical Lewy-body PD in most cases, but also include diffuse Lewy-body disease, nigral degeneration without distinctive histopathology and, rarely, even aggregates of the microtubule-associated protein tau, suggestive of progressive supranuclear palsy or frontotemporal dementia. Different pathological findings were even reported in a single family with an R1441C mutation. *LRRK2* mutations may therefore be an upstream event in the cascade leading to neurodegeneration with different pathologies. However, the vast majority of patients with a G2019S mutation, in whom pathology has been reported, seem to conform to the typical α-synuclein Lewy-body type of PD. No direct link between LRRK2 and α-synuclein has so far been demonstrated. α-synuclein does not seem to be

Despite its still poorly understood role in pathogenesis, LRRK2-associated PD is of particular interest since it is the first example of a mendelian form of PD that is common

of the disease in a sizeable population. Longitudinal studies in presymptomatic mutation carriers may reveal premotor changes by clinical, biochemical or imaging methods, indicating the very early phases of the neurodegenerative process. It is in this population that studies exploring neuroprotective or preventive measures are most promising to yield

The ubiquitin C-terminal hydrolase-L1 (*UCH-L1*) was first identified as an abundant (1–2% of total brain protein), neuronspecific protein encoded by a 9.5 kb gene product (Day and Thompson, 1987; Doran et al., 1983). UCH-L1 is a de-ubiquitinating enzyme that removes carboxy-terminal ubiquitin *UCH-L1* from substrates but does not break bonds within polyubiquitin complexes and results in the recycling of ubiquitin. In addition,UCHL1 protein is found in several pathological structures including Lewy bodies and some

et al., 1990). Furthermore, recent studies have identified a single dominant mutant (I93M) in two members of a PD-affected family. Inversely, a polymorphism in *UCH-L1* (S18Y) has

enough to provide the opportunity to study the development

leading to neurodegeneration with different pathologies.

phosphorylated by LRRK2.

**3.3 UCHL1 (Park 5, 4p14)** 

Alzheimer's neurofibrillary tangles (Lowe

first results

been suggested to reduce the risk of developing sporadic PD. Interestingly, another study hypothesizes that UCHL1 may possess two enzymatic activities, hydrolase and ligase activity. Overexpression of UCH-L1 variant I93M resulted in an accumulation of αsynuclein. Furthermore, they suggest that this accumulation is due to an ubiquitination of αsynuclein by dimerized UCH-L1. However, the S18Y polymorphic variant of UCH-L1 has reduced ligase activity but normal hydrolase activity,

which may explain the 'protective' effect of the S18Y polymorphism. With evidence of UCH-L1 and parkin being involved in certain forms of familial PD, this gives credence to the involvement of the UPS in PD. However, it is unclear how UCH-L1 can promote the specific neurodegeneration of dopaminergic neurons in familial PD and what role it plays in the sporadic form.
