**2. Alpha-synuclein toxicity**

Alpha-synuclein is a 144aa protein encoded by the gene SNCA, localized at 4q chromosome. Three different SNCA transcripts can be detected in neurons. The physiological functions of alpha-synuclein is still not fully understood. It is abundantly expressed in nervous tissues and it localizes in the cytoplasm or associated with lipid membranes. In particular, this protein is mainly localized in the pre-synaptic compartment, where it seems to have a role in regulating neurotransmitter release, vesicle turnover, membrane stability and neuronal plasticity.

The hypothesis of an active involvement of alpha-synuclein in the pathogenesis of Parkinson's disease was proposed when this protein was identified as the main component of Lewy bodies, intraneuronal aggregates constituting a constant report in the neuropathology of Parkinson's disease (Spillantini et al 1997). Beside alpha-synuclein, Lewy bodies also contain components of proteasome machinery, chaperone proteins, alphatubulin, synphilin-1, glyceraldehyde 3-phosphate dehydrogenase and several other proteins known to be involved in nigral neurodegeneration. Residues 71 to 82 of alpha-synuclein are essential for the assembly of Lewy bodies. The exact role of these inclusions in the disease physiopathology was unknown for several years.

The key function of alpha-synuclein protein in the pathogenesis of Parkinson's disease was first elucidated by genetic studies: the finding of familial cases of Parkinson's disease genetically linked to missense mutations in the SNCA gene and to genomic triplication of the wild type gene gave evidence of the potential neurotoxic effect of this protein. The first causal mutation of hereditary Parkinson's disease was identified in a large Italian family ("Contursi kindred", from the name of the town): it was the missense mutation Ala53Thr in the SNCA gene (Polymeropoulos et al 1997). Only two other pathogenetic missense mutations have been identified in SNCA: Ala30Pro and Glu46Lys. All these variants are located in the N-terminus of the protein and are linked to autosomal dominant forms of Parkinson's disease. Anyway, the missense mutation frequency in SNCA in different populations is very low, whereas SNCA multiplications are a bit more frequent. The

proteins; these last include the ubiquitin-proteasome system and the autophagic-lysosomal pathways. Both oxidative stress and impairment of protein degradation machinery exert a neurotoxic effect contributing to the pathogenesis of Parkinson's disease and partially mediated, in dopaminergic neurons, by qualitative and quantitative alterations in alphasynuclein. This protein in turn is able to boost the processes responsible for its toxic gain of function. Therefore, a physiopathologic circuit emerges constituted by a complex interaction among different pathogenetic mechanisms, everyone able to support the others; the death of dopaminergic neurons represents the ultimate and irreversible outcome of all these events. This chapter aims at clarifying this complex dynamics responsible for the neurodegenerative process, with particular regard to mechanisms both determining and mediating the deleterious effect of alpha-synuclein. Furthermore, we will report data on the development of biochemical tests helpful for diagnosis and prognosis and useful as objective criteria to determine the neuroprotective effectiveness of drugs in clinical trials: in this regard, levels and modifications of α-synuclein, as well as parameters of oxidative stress and autophagy, could represent suitable peripheral biomarkers of disease risk and progression. Finally, we will discuss about the opportunity of interfering with all the reported pathogenetic

Alpha-synuclein is a 144aa protein encoded by the gene SNCA, localized at 4q chromosome. Three different SNCA transcripts can be detected in neurons. The physiological functions of alpha-synuclein is still not fully understood. It is abundantly expressed in nervous tissues and it localizes in the cytoplasm or associated with lipid membranes. In particular, this protein is mainly localized in the pre-synaptic compartment, where it seems to have a role in regulating neurotransmitter release, vesicle turnover, membrane stability and neuronal

The hypothesis of an active involvement of alpha-synuclein in the pathogenesis of Parkinson's disease was proposed when this protein was identified as the main component of Lewy bodies, intraneuronal aggregates constituting a constant report in the neuropathology of Parkinson's disease (Spillantini et al 1997). Beside alpha-synuclein, Lewy bodies also contain components of proteasome machinery, chaperone proteins, alphatubulin, synphilin-1, glyceraldehyde 3-phosphate dehydrogenase and several other proteins known to be involved in nigral neurodegeneration. Residues 71 to 82 of alpha-synuclein are essential for the assembly of Lewy bodies. The exact role of these inclusions in the disease

The key function of alpha-synuclein protein in the pathogenesis of Parkinson's disease was first elucidated by genetic studies: the finding of familial cases of Parkinson's disease genetically linked to missense mutations in the SNCA gene and to genomic triplication of the wild type gene gave evidence of the potential neurotoxic effect of this protein. The first causal mutation of hereditary Parkinson's disease was identified in a large Italian family ("Contursi kindred", from the name of the town): it was the missense mutation Ala53Thr in the SNCA gene (Polymeropoulos et al 1997). Only two other pathogenetic missense mutations have been identified in SNCA: Ala30Pro and Glu46Lys. All these variants are located in the N-terminus of the protein and are linked to autosomal dominant forms of Parkinson's disease. Anyway, the missense mutation frequency in SNCA in different populations is very low, whereas SNCA multiplications are a bit more frequent. The

mechanisms as putative neuroprotective pharmacologic strategy.

**2. Alpha-synuclein toxicity** 

physiopathology was unknown for several years.

plasticity.

Ala53Thr alpha-synuclein mutant is responsible for the greater in vivo neurotoxicity in transgenic animal models.

The deleterious effects of these genetic alterations on dopaminergic neurons have been demonstrated in cellular and animal models, confirming the hypothesis that both qualitative and quantitative alterations of alpha-synuclein are able to trigger its toxic effect: mutant alpha-synuclein protein exerts a selective toxicity in dopaminergic neurons when expressed in rats; at the same time, the mutant protein causes aggregation and formation of Lewy bodies in dopaminergic neurons of mouse models.

The toxicity of both mutant and wild type alpha-synuclein seems to require the acquisition of a misfolded conformation which prevents alpha-synuclein degradation and favors its fibrillization, firstly into protofibrillar oligomeric species and then to fibrillar aggregates. Recently, it has become clear that insoluble aggregates probably do not have an intrinsic toxic function, as suggested by the finding that Lewy bodies–positive dopaminergic neurons are less vulnerable to degeneration. Furthermore, the increased size of Lewy bodies and the decreased levels of soluble alpha-synuclein correlate with a higher resistance to cytotoxic agents. Therefore, formation of Lewy bodies could represent a protective phenomenon favoring the removal of soluble oligomers. Different mechanisms can explain the negative effects exerted by alpha-synuclein oligomers, which seem to be directly responsible for the neurotoxic effect of the protein (Vekrellis et al 2004):


Alpha-synuclein oligomers directly interfere with proteasome subunits. Furthermore, alphasynuclein impairs microtubule stability through binding to alpha-tubulin and to the microtubule associated protein TAU. Alpha synuclein induces the aggregation of TAU, which in turn favors alpha-synuclein fibrillization; interestingly, polymorphisms in the TAU gene have proved to modify the risk of developing Parkinson's disease.

In monogenic forms of Parkinson's disease the direct neurotoxic effect of mutant alphasynuclein may depend on specific conformational modifications, modulating the propensity of the protein to aggregate, to interact with other proteins and to be processed by clearance mechanisms. Pathogenetic mutations of alpha-synuclein protein are known to confer a tendency to acquire abnormal conformation substantially easier than the wild type protein, which accounts for the development of Parkinson's disease in patients having these mutations.

The fact that over-expressed wild type alpha-synuclein is responsible for neurotoxicity suggests that even simple protein accumulation is able to trigger the misfolded conformation and to promote protein aggregation.

As well as in familial cases, even in sporadic Parkinson's disease the toxic gain of function of alpha-synuclein could derive from the intraneuronal accumulation of the protein or from biochemical modifications enhancing the propensity of the protein to aggregate. Anyway, the ubiquitous expression and the high levels of alpha-synuclein in the brain suggests that its simple presence in dopaminergic neurons is not sufficient to explain the selective degeneration of these cells, which must have specific characteristics able to induce or to accelerate alpha-synuclein misfolding, oligomerization and aggregation. Oxidative stress and production of highly reactive aldehydes, both depending on high levels of dopamine, can modify alpha-synuclein, partially explaining the high tendency of this protein to acquire

Alpha-Synuclein, Oxidative Stress and

**3.1 Mitochondrial dysfunction** 

inclusions (Betarbet et al 2000).

2000).

relevance of complex I activity (van der Walt et al 2003).

excitotoxicity.

Autophagy Failure: Dangerous Liaisons in Dopaminergic Neurodegeneration 115

synuclein toxicity. The NAD+ concentration-dependency of SIRT2 activity suggests a possible modulation of alpha-synuclein misfolding and aggregation by energy metabolism.

An impairment in multiple steps of the mitochondrial respiratory chain has been demonstrated in Parkinson's disease (Schapira et al 1998). The importance of this pathogenetic mechanism mainly depends on its role of main source of reactive oxygen species. At the same time, the loss of mitochondrial function and the consequent energetic deficit interfere with glutamate uptake and increase neuronal vulnerability to glutamate

Several epidemiologic and experimental data confirm the importance of mitochondrial derangement in the pathogenesis of Parkinson's disease. First of all, electron transport complex I represents the main molecular target of paraquat, rotenone and MPTP, the exogenous substances known to determine degeneration of human dopaminergic neurons. The selective toxicity of MPTP on dopaminergic neurons derives from its transformation to the metabolite 1-methyl-4-phenyl-pyridium (MPP+), which is concentrated within dopaminergic neurons by dopamine transporters. Even paraquat, which is structurally similar to MPP+, is carried into dopaminergic cells by the same transporter. In contrast to MPTP and paraquat, rotenone is not concentrated in nigral neurons, nevertheless it produces selective death of these cells. The development of parkinsonism after exposure to rotenone represents a further proof of the high susceptibility of dopaminergic neurons to mitochondrial impairment. Toxic derangement in complex I induces alpha-synuclein aggregation and formation of Lewy body-like

A reduced activity of the mitochondrial complexes I and IV has been observed in the substantia nigra of patients with Parkinson's disease and in animal models of disease (Palacino et al 2004). Decreased levels of coenzyme Q10 together with a higher ratio of oxidized vs. reduced Coenzyme Q10 have been observed in patients. An increase of cerebral lactate levels, suggesting an alteration in aerobic metabolism as a consequence of derangement of mitochondrial respiratory chain, has been detected by brain magnetic resonance spectroscopy in patients with Parkinson's disease (Henchcliffe et al 2008). Experiments performed on cytoplasmic hybrid cell lines from patients with Parkinson's disease suggest that the complex I deficit associated to the disease might be genetically determined by defects in mitochondrial DNA. A single nucleotide polymorphism within the gene encoding NADH dehydrogenase 3 of complex I has been associated to a decrease in the risk of sporadic Parkinson's disease, providing genetic evidence of the pathogenetic

An endogenous substance able to impair mitochondrial function is salsolinol, which is synthesized from dopamine and acetaldehyde by salsolinol synthase. This molecule has been found in various regions of the brain, including striatum and substantia nigra. Salsolinol inhibits mitochondrial complex II and this effect may explain the fact that high levels of this molecule are able to induce degeneration of dopaminergic neurons (Storch et al

**3. Oxidative stress, mitochondrial dysfunction and excitotoxicity** 

a misfolded conformation in dopaminergic neurons. Indeed, dopamine promotes alphasynuclein protofibrils formation. Oxidative stress, which is known to be one of the main pathogenetic factors leading to the death of dopaminergic neurons in Parkinson's disease, has been identified as a major responsible for alpha-synuclein post-translational modifications, consisting in oxidation and nitration of specific aminoacid residues; these alterations in alpha-synuclein have been detected in nigral neurons of brain samples from patients with Parkinson's disease (Giasson et al 2000) and from animal models of disease (Gao et al 2008).

An increase in alpha-synuclein levels, correlated with the degree of nigrostriatal dopamine depletion, has been demonstrated in the substantia nigra of patients with sporadic Parkinson's disease (Chu et al 2007). It is conceivable to suppose that this accumulation of the toxic protein might derive from an increase in transcription and translation of the SNCA gene or from a decrease in the protein degradation.

The variability in codifying and non-codifying SNCA gene sequences has demonstrated to modify the risk of developing sporadic Parkinson's disease, probably through a mild modulatory effect on transcription activity and, as a consequence, on intraneuronal levels of alpha-synuclein. SNCA duplication was found in sporadic cases of Parkinson's disease. Increasing length in the dinucleotide repeat sequence (REP1) of the SNCA gene promoter has been demonstrated to be correlated with an increased risk of developing sporadic Parkinson's disease (Maraganore et al 2006; Brighina et al 2008). Furthermore, single nucleotide polymorphisms at the promoter region and at the 3' end of SNCA gene have been found to modify the risk of disease.

Anyway, aging remains the most evident risk factor for sporadic Parkinson's disease. An interesting hypothesis suggests that senescence of dopaminergic neurons could promote the accumulation of misfolded alpha-synuclein through two main mechanisms: slowing alpha-synuclein turnover through the impairment of clearance machinery; impairing the function of mitochondria and antioxidant systems, with consequent increase in oxidative stress.

Great efforts have been done in order to identify genetic and environmental factors able to modulate the aging process. Caloric restriction represents the only environmental condition which has demonstrated to delay aging in a wide range of organisms. This evidence suggests that caloric restriction might guarantee protection from developing Parkinson's disease and could delay the disease progression. As it was supposed, caloric restriction has shown a protective effect in MPTP-induced animal models of Parkinson's disease, even if an epidemiologic correlation between diet and incidence of Parkinson's disease has not been demonstrated. The molecular basis of the protective role of caloric restriction in animal models has been elucidated in yeast, where a family of NAD+-dependent protein deacetylases, called sirtuins (SIRTs), have been identified as mediators of the changes induced by starvation in cells; many sirtuins analogs have been discovered in mammals. These proteins regulate cell homeostasis partially through an epigenetic mechanism: they modulate histones acetylation and chromatin condensation. SIRT1 is a major responsible for protective cell modifications during starvation, whereas other SIRTs seem to exert a specific deleterious effect on dopaminergic neurons. In particular, SIRT2, an alpha-tubulin deacetylase, promotes formation of alpha-tubulin oligomers, which destabilize microtubules and form toxic complexes with alpha-synuclein oligomers. The interaction of alphasynuclein oligomers with alpha-tubulin plays an important role in mediating alphasynuclein toxicity. The NAD+ concentration-dependency of SIRT2 activity suggests a possible modulation of alpha-synuclein misfolding and aggregation by energy metabolism.
