**2. Alpha-synuclein: Biophysical characteristics**

Alpha-synuclein was first described in Torpedo californica (Maroteaux et al., 1988). It belongs to the synuclein family, which includes β- and γ-synucleins. These proteins have a common amino-terminal sequence containing a different number of repeat regions while they differ in the carboxy-terminal part (Tofaris and Spillantini, 2005). Alpha-synuclein from different organisms possesses an high degree of sequence conservation (Clayton and George, 1998). Three α-synuclein isoforms are produced by alternative splicing (Bayer et al., 1999) in humans. The best known isoform is 140 amino acids in length and constitutes the whole and major transcript of the protein. Two other isoforms, of 126 and 112 amino acids in length, derive from the selective deletion of exon 3 and 5, respectively.

The full length α-synuclein transcript (Figure 1) can be divided into three regions: 1) The Nterminal region, residues 1-60, includes the sites of three familial PD mutations and contains four 11-amino acid imperfect repeats with a highly conserved hexameric motif (KTKEGV). This region is involved in the formation of amphipatic α-helices, similar to the lipid-binding domain of apolipoproteins (George et al., 1995;Clayton and George, 1998); 2) The central region, containing residues 61-95, comprises the NAC highly aggregation-prone sequence (Ueda et al., 1993;Han et al., 1995); 3) The C-terminal region, from residue 96 to 140, is highly enriched in proline acidic residues, and contains three highly conserved tyrosine residues, which are considered a signature of the α- and β-synuclein family.

Fig. 1. Schematic representation of the full length 140 amino acid α-synuclein transcript. Pathogenic mutations as well as phosphorylation and nitration sites are indicated.

therapeutical targets among the α-synuclein synaptic partners. Modulation of these proteins may open new ways toward the development of disease modifying strategies to cure PD-

By performing a critical review of the PD-related α-synuclein proteome, this article will outline the most relevant findings defining the specific modulatory effects exerted by αsynuclein in the control of synaptic functions in physiological and pathological conditions. The overall conclusions of these studies will spot novel potential therapeutic targets for the development of pharmacological and gene-based strategies aimed at straightening α-

Alpha-synuclein was first described in Torpedo californica (Maroteaux et al., 1988). It belongs to the synuclein family, which includes β- and γ-synucleins. These proteins have a common amino-terminal sequence containing a different number of repeat regions while they differ in the carboxy-terminal part (Tofaris and Spillantini, 2005). Alpha-synuclein from different organisms possesses an high degree of sequence conservation (Clayton and George, 1998). Three α-synuclein isoforms are produced by alternative splicing (Bayer et al., 1999) in humans. The best known isoform is 140 amino acids in length and constitutes the whole and major transcript of the protein. Two other isoforms, of 126 and 112 amino acids in

The full length α-synuclein transcript (Figure 1) can be divided into three regions: 1) The Nterminal region, residues 1-60, includes the sites of three familial PD mutations and contains four 11-amino acid imperfect repeats with a highly conserved hexameric motif (KTKEGV). This region is involved in the formation of amphipatic α-helices, similar to the lipid-binding domain of apolipoproteins (George et al., 1995;Clayton and George, 1998); 2) The central region, containing residues 61-95, comprises the NAC highly aggregation-prone sequence (Ueda et al., 1993;Han et al., 1995); 3) The C-terminal region, from residue 96 to 140, is highly enriched in proline acidic residues, and contains three highly conserved tyrosine residues,

Fig. 1. Schematic representation of the full length 140 amino acid α-synuclein transcript. Pathogenic mutations as well as phosphorylation and nitration sites are indicated.

synuclein-related synaptic dysfunctions as new clues to cure PD.

length, derive from the selective deletion of exon 3 and 5, respectively.

which are considered a signature of the α- and β-synuclein family.

**2. Alpha-synuclein: Biophysical characteristics** 

related synaptic dysfunctions.

The amino acid sequence and subcellular localization of α-synuclein indicate that it can interact with lipid membranes. Indeed, the repeat region, mediates reversible binding to acidic phospholipids by making up a conserved apolipoprotein-like class-A2 helix which is associated with a large shift in protein secondary structure from around 3% to about 80% α-helical (Davidson et al., 1998). Consistently, it has been observed that α-synuclein can inhibit a protein which is localized to plasma membrane and submembraneous vesicles: phospholipase D2, whose activity produces phosphatidic acid, which is implicated in vesicle budding (Jenco et al., 1998). Membrane-bound α-synuclein has an high aggregation propensity and seeds the aggregation of the cytosolic form (Lee and Lee, 2002). Lipids can facilitate the incorporation of α-synuclein into membranes and influence α-synuclein fibril elongation (Gai et al., 2000) suggesting that α-synuclein may be strongly implicated in synaptic membrane biogenesis. Furthermore, the key role of α-synuclein in membrane-associated processes is supported by findings indicating that α-synuclein knock-out mice have enhanced dopamine release at nigrostriatal terminals in response to electrical stimulation, indicating that α-synuclein is an activity-dependent negative regulator of dopaminergic transmission (Abeliovich et al., 2000). On this line, it has been shown that depletion of α-synuclein results in a decrease in the distal pool of presynaptic vesicles in cultured hippocampal neurons (Murphy et al., 2000). Furthermore, its overexpression reduces neurotransmitter release by inhibiting synaptic vesicle reclustering after endocytosis (Nemani et al., 2010). Cytosolic proteins regulate αsynuclein membrane interactions, thus suggesting that cytosolic cofactors may be implicated in disease pathogenesis (Wislet-Gendebien et al., 2006;Wislet-Gendebien et al., 2008). In particular, protein-protein interactions may affect α-synuclein ability to bind to synaptic membranes, thus affecting its biochemical properties and modulating its tendency toward aggregation, suggesting that, in the α-synuclein synaptic proteome, it could be possible to modulate α-synuclein aggregation by selectively regulating the expression of some o its protein partners.

The relevance of the formation of protein complexes in the modulation of α-synuclein function is supported by several findings which suggest that the protein likely behave like a molecular chaperone. Indeed, some studies have shown that α-synuclein shares functional and physical homologies with a family of ubiquitous cytoplasmic chaperones: the 14-3-3 proteins (Ostrerova et al., 1999). Alpha-synuclein can bind 14-3-3 proteins as well as other protein members of its family proteome such as protein kinase C. Thus, it was hypothesized that an increase of α-synuclein levels could be harmful for the cells as it could affect signal transduction pathways involved in cell differentiation and survival. On this line, in 2001, Iwata and colleagues showed that α-synuclein inhibited mitogen-activated protein (MAP) kinase signaling and accelerated cell death following serum reduction in neuro2a cells. However, results from later studies indicated that overexpression of wild type α-synuclein can protect neuronal cells from apoptotic stimuli and delay cell death induced by serum withdrawal (Alves da Costa et al., 2000;Lee et al., 2001b), thus leading to controversial conclusions. Indeed, it has also been reported that α-synuclein protects against oxidative stress by inactivating c-jun-N-terminal kinase, which is strongly implicated in stress responses (Hashimoto et al., 2002). However, it is likely that the neuroprotective/toxic effects of α-synuclein may be ascribed to a different sensitivity of cells to α-synuclein levels. Indeed, it has been shown that at a nanomolar scale TAT-α-synuclein is neuroprotective against oxidative stress, while at a micromolar scale it is able to aggregate and trigger neurotoxic mechanisms (Albani et al., 2004;Batelli et al., 2008).

Targeting α-Synuclein-Related

Bender, 2000).

Synaptic Pathology: Novel Clues for Parkinson's Disease Therapy 143

neuritic pathology and formation of some argyrophilic and thioflavin S-positive α-synuclein inclusions. Remarkably, although mPrP but not Thy1 promoter drives high expression of the transgene in the substantia nigra neurons, the tyrosine hydroxylase (TH)–positive neurons of this area in the mPrP-driven transgenic mice were devoid of α-synclein aggregates, and didn't show loss of striatal dopamine or DAT (Giasson et al., 2002;Lee et al., 2002). In line with these findings another study showed that TH-driven expression of wt or mutant α-synuclein didn't result in the formation of intracellular α-synuclein aggregates. From these studies, it became evident that adeno- or lentiviral-mediated expression of αsynuclein may represent a powerful tool to induce the selective expression of the protein in dopaminergic neurons of the substantia nigra of adult rodents (Kirik et al., 2002;Klein et al., 2002;Lo et al., 2002). Indeed, overexpression of either wild type or mutant form of the protein led to cellular and axonal pathology associated with loss of nigral neurons, reduction in striatal dopamine levels and motor deficits without the formation of fibrillary inclusions (Lo et al., 2002), thus contradicting evidences from studies on transgenic animals. These observations suggest that dopaminergic neurons are vulnerable to high levels of human α-synuclein and that wild type or mutant form of the protein are both toxic. Nonetheless, it may be taken into account that these discrepancies may be related to the high rate of transgene expression that can be achieved by either technology or by the high toxicity which is induced by gene transfection in viral models. To date, neither transgenic nor viral-mediated rodent models expressing wild type or mutated α-synuclein show fibrillary inclusions. Thus the mechanisms by which wild type human α-synuclein assembles in LB in the substantia nigra of PD patients hasn't been clarified by using these models. Nonetheless, filamentous α-synuclein inclusions have been observed in a *Drosophila* model in association with loss of dopaminergic cells and locomotor defects (Feany and

Although studies of genetic mutations in α-synuclein helped in the understanding some of the function and pathogenic properties of α-synuclein, they only account for a very small proportion of PD cases. Indeed, more than 90% of PD cases are sporadic, thus characterized by the accumulation of insoluble fibrils of WT α-synuclein (Spillantini et al., 1997;Spillantini et al., 1998). Therefore, much effort has been made in order to understand what are the alterations which convert wild type α-synuclein to a toxic species. It has been shown that wild type α-synuclein aggregates form fibrils identical to those isolated from disease brains, even though the rate of fibril formation is slower than that of the mutant form (Serpell et al., 2000;Conway et al., 1998). To date, several post-translational modifications of α-synuclein can alter its biophysical properties. Thus, it has been hypothesized that these modifications are implicated in the induction of the fibrillation process (Oueslati et al., 2010). These studies indicate that α-synuclein has numerous potential sites for post-translational modifications such as phosphorylation, tyrosine nitration or protein cleavage. In transfected cells, it is constitutively phosphorylated at serine residues 87 and 129, with the latter being the predominant site (Okochi et al., 2000). Residue 129 in α-synuclein lies a consensus sequence for casein kinase 1, which is also present in β- and γ-synuclein, and casein kinase 1 and 2 have been found to phosphorylate this site. Alpha-synuclein can be phosphorylated also by several G-protein-coupled receptor kinases, events which reduce the ability of the protein to interact with phospholipids and PLD2 (Pronin et al., 2000). Tyrosine kinase 72syk can phosphorylate the tyrosine residues in the carboxy-terminus of the protein both *in vitro* and in CHO cells (Negro et al., 2002). The specific phosphorylation of serine 129 in the Cterminal region can decrease the ability of this portion to prevent fibril formation (Fujiwara

Finally, recent evidence showed that cells from transgenic mice expressing the truncated form of α-synuclein are more susceptible to environmental conditions and that overexpression of the wild type from of the protein in neuronal progenitor cells affect their fate of differentiation, thus supporting the notion that full length α-synuclein is involved in dopaminergic cell differentiation and survival (Michell et al., 2007;Schneider et al., 2007).
