**4. The** α**-synuclein synaptic proteome**

Several lines of evidence indicate that α-synuclein is critically involved in the regulation of synaptic functions (Fortin et al., 2010;Bellani et al., 2010;Sousa et al., 2009;Tofaris and Spillantini, 2007). In particular, it has been shown that α-synuclein is loosely associated with the distal pool of synaptic vesicles (Kahle et al., 2000;Lee et al., 2008;Zhang et al., 2008) and with the lipid rafts of the plasma membrane (Fortin et al., 2004), thus indicating that the protein is able to associate with membranes, as confirmed by other studies (Chandra et al., 2003;Davidson et al., 1998;Eliezer et al., 2001;Jo et al., 2000). Nonetheless, it seems that the distribution of α-synuclein at synapses is very dynamic and dependent upon neuronal stimulation, with rapid exchanges taking place among neighbouring synapses (Fortin et al., 2005). The critical importance of α-synuclein in the modulation of synaptic functions is confirmed by numerous studies shading light upon the α-synuclein synaptic proteome. Numerous observations indicate that α-synuclein is crucially involved in the regulation of synaptic functions as it interacts with- and modulates key synaptic components including lipidic and protein molecules.

Yet, in 2003 Sharon and colleagues (2003) elegantly showed that α-synuclein accumulation can influence cellular and brain phospholipids levels, including certain PUFA, thus affecting membrane fluidity and synaptic membrane trafficking. Later on, Jo and coauthors (2004) found that wild type α-synuclein does not penetrate to the fatty acyl chains and in isolated synaptosomes, while the A53T mutated form of the protein binds to synaptosomal membranes, increases lipid headgroup packing, induces subtle changes in the lipid interfacial space and decreases the fluidity of the fatty acyl chains. However, a recent

Targeting α-Synuclein-Related

Castelao and Castano, 2010).

and B and syntaxin-binding protein 1.

content, may critically be involved in the onset of PD.

**characteristics and functional consequences** 

functional consequences of α-synuclein accumulation at synaptic sites.

Synaptic Pathology: Novel Clues for Parkinson's Disease Therapy 147

likely unable to interact with synaptobrevin-2, and to regulate SNARE complex assembly. Furthermore, α-synuclein can indirectly affect SNAREs activation by sequestering arachidonic acid (Darios et al., 2010). Another study showed that transgenic overexpression of α-synuclein resulted in the selective reduction of synaptobrevin-2 and synapsin 1 as well as of other synaptic proteins playing a role in exocytosis and endocytosis such as piccolo or

Synphilin, an adaptor molecule which anchors α-synuclein to intracellular proteins involved in vesicle transport and cytoskeletal functions (Lee et al., 2004), is another protein member of the synaptic proteome (Engelender et al., 1999;Neystat et al., 2002;Alvarez-Castelao and Castano, 2010). In particular, it has been shown that synphilin-1 binds to α-synuclein thus promoting the formation of cytosolic inclusions (Engelender et al., 1999). This effect may be due to the fact that the interaction of the central region of synphilin-1 with the N-terminal region of α-synuclein prevents α-synuclein degradation by the proteasome (Alvarez-

More recently, a broad proteomic analysis investigated the serine 129 and serine 125 phosphorylation-dependent α-synuclein interactions (McFarland et al., 2008). In this paper, authors singled out several other synaptic protein partners such as synaptophysin, SNAP-25-interacting protein, vesicle-associated membrane protein-associated protein (VAPA) A

Finally, it has been shown that α-synuclein interacts with and modulate the membrane trafficking of several neuron-specific transporters implicated in neurotransmitter re-uptake from synaptic clefts, among them the DAT (Sidhu et al., 2004;Lee et al., 2001a;Bellucci et al., 2008) as well as the noradrenalin transporter (NET) (Wersinger et al., 2006a) and serotonine transporter (SERT) (Wersinger et al., 2006b). Remarkably, besides dopaminergic neurons of the substantia nigra, the noradrenergic locus ceruleus (oral parts) and motor vagal nucleus as well as serotonergic neurons of the Raphe nuclei are among the most vulnerable neurons in the PD brain (Jellinger, 1991), thus, a loss of DAT, NET or SERT synaptic membrane

**5. Alpha-synuclein-related synaptic pathology at the synapse: Biochemical** 

Since many recent reports have highlighted that α-synuclein plays a crucial role in the regulation of synaptic functions by modulating lipid synaptic membrane composition and fluidity as well as the trafficking and function of several key synaptic proteins, it has been hypothesized that α-synuclein loss of function at the synapse may be one of the pathophysiological mechanisms of PD. Thus, many post-mortem studies on the brain of PD patients as well as investigations on "in vitro" and "in vivo" models of PD showing αsynuclein accumulation have been focused on the evaluation of the biochemical and

Numerous findings indicate that transgenic overexpression of wild type, A53T, A30P and truncated α-synuclein results in the degeneration of TH-positive terminals with a reduction of striatal dopamine levels (Rockenstein et al., 2002;Fernagut et al., 2007;Ono et al., 2009;Gao et al., 2008;Gomez-Isla et al., 2003;Nieto et al., 2006;Chen et al., 2006). Interestingly, a couple of these reports described specific alterations of crucial synaptic proteins for dopaminergic neuronal functions such as the DAT (Chen et al., 2006;Magen and Chesselet, 2010). However, results of these studies had different conclusions, with the first one showing a

endocytosis-regulating proteins such as amphiphysin (Scott et al., 2010).

investigation concerning the lipidomic profiling of different mouse strains showed age and gender related differences that were poorly associated with α-synuclein genotype thus weakening the idea that α-synuclein-synaptic membrane interactions may underlie the onset of PD-related alterations (Rappley et al., 2009). However, these observations may be justified by the fact that α-synuclein is engaged in a foudamentally different mode of membrane interaction than the charge-dependent artificial membrane binding, and the mode of interaction is determined by the intrinsic properties of α-synuclein itself and by cytoplasmic context (Kim et al., 2006). Furthermore, it has been shown that nutrient starvation, a condition that mimics the impaired energy metabolism occurring in the aging brain (Bowling and Beal, 1995) induces α-synuclein pathological changes, that may lately affect the correct trafficking of synaptic proteins such as the DAT (Bellucci et al., 2008). Thus, we can't exclude that this effect can be related an α-synuclein accumulation-mediated change in synaptic membrane fluidity and composition.

This notwithstandings, the modulation of synaptic activity by α-synuclein is not solely mediated by the fact that the protein regulates some of the lipidic components of synaptic membrane. Hence, α-synuclein has been found to interact with numerous synaptic proteins, and it seems that this critical interaction may affect their specific localization and function. Indeed, α-synuclein binds to the actin cytoskeleton (Sousa et al., 2009) and modulates its dynamics, thus participating in the tuning of vesicle release process (Bellani et al., 2010). In 2007, Woods et al. described several new protein partners of α-synuclein by phage display and NMR spectroscopy, and among them a key protein implicated in the regulation synaptic vesicle release was identified: synapsin 1a. As a further indication of the occurrence of α-synuclein-synapsin 1 interaction, it has been shown that these two proteins are cotrasportated from the cell body to the axon by the slow component-b (Roy et al., 2007;Roy et al., 2008). These observations were lately confirmed by other findings indicating that αsynuclein transgenic overexpression results in a significant reduction of synapsin 1, thus impairing vesicle reclustering after exocytosis (Nemani et al., 2010). The authors elegantly discuss that since 1) synapsin 1 and 2 knock out mice show a reduction of the total number of synaptic vesicles, distinct from the primary defect in recycling pool size observed with αsynuclein overexpression, and 2) the overexpression of α-synuclein causes a more severe defect in recycling pool than the complete loss of synapsins and what's more, the transgenic mice only show a partial loss of synapsins, the effect of α-synuclein is unlikely to reflect the sole loss of synapsin. Rather, the inhibitory effect exerted by α-synuclein, may be related also to the modulation of other proteins, such as complexins, proteins involved in a step at or close to fusion with the plasma membrane (Sudhof and Rothman 2009), which have also been found to be reduced by α-synuclein overexpression. However, complexins levels are moderately changed in α- and β-synuclein knockout mice (Chandra et al., 2004), implying that α-synuclein is not strikingly involved in their physiological regulation at synaptic sytes. Thus, besides synapsin 1, the expression and subcellular localization of other proteins seem to be affected by α-synuclein accumulation. Remarkably, it has been shown that α-synuclein is able to restore N-ethylmaleimide sensitive fusion attachment protein receptor (SNARE) proteins (SNAREs) assembly induced by the cysteine-string protein-α (CSP-α) knockout by acting through a downstream mechanism that requires phospholipids binding, thus protecting nerve terminals against CSP-α deletion-induced injury (Chandra et al., 2005). The modulation of SNARE complex formation by α-synuclein likely occur through a direct interaction with synaptobrevin-2, which involves the C-terminal portion of α-synuclein as recently postulated by Burrè et al. (2010). Therefore, C-terminally truncated α-synuclein is

investigation concerning the lipidomic profiling of different mouse strains showed age and gender related differences that were poorly associated with α-synuclein genotype thus weakening the idea that α-synuclein-synaptic membrane interactions may underlie the onset of PD-related alterations (Rappley et al., 2009). However, these observations may be justified by the fact that α-synuclein is engaged in a foudamentally different mode of membrane interaction than the charge-dependent artificial membrane binding, and the mode of interaction is determined by the intrinsic properties of α-synuclein itself and by cytoplasmic context (Kim et al., 2006). Furthermore, it has been shown that nutrient starvation, a condition that mimics the impaired energy metabolism occurring in the aging brain (Bowling and Beal, 1995) induces α-synuclein pathological changes, that may lately affect the correct trafficking of synaptic proteins such as the DAT (Bellucci et al., 2008). Thus, we can't exclude that this effect can be related an α-synuclein accumulation-mediated

This notwithstandings, the modulation of synaptic activity by α-synuclein is not solely mediated by the fact that the protein regulates some of the lipidic components of synaptic membrane. Hence, α-synuclein has been found to interact with numerous synaptic proteins, and it seems that this critical interaction may affect their specific localization and function. Indeed, α-synuclein binds to the actin cytoskeleton (Sousa et al., 2009) and modulates its dynamics, thus participating in the tuning of vesicle release process (Bellani et al., 2010). In 2007, Woods et al. described several new protein partners of α-synuclein by phage display and NMR spectroscopy, and among them a key protein implicated in the regulation synaptic vesicle release was identified: synapsin 1a. As a further indication of the occurrence of α-synuclein-synapsin 1 interaction, it has been shown that these two proteins are cotrasportated from the cell body to the axon by the slow component-b (Roy et al., 2007;Roy et al., 2008). These observations were lately confirmed by other findings indicating that αsynuclein transgenic overexpression results in a significant reduction of synapsin 1, thus impairing vesicle reclustering after exocytosis (Nemani et al., 2010). The authors elegantly discuss that since 1) synapsin 1 and 2 knock out mice show a reduction of the total number of synaptic vesicles, distinct from the primary defect in recycling pool size observed with αsynuclein overexpression, and 2) the overexpression of α-synuclein causes a more severe defect in recycling pool than the complete loss of synapsins and what's more, the transgenic mice only show a partial loss of synapsins, the effect of α-synuclein is unlikely to reflect the sole loss of synapsin. Rather, the inhibitory effect exerted by α-synuclein, may be related also to the modulation of other proteins, such as complexins, proteins involved in a step at or close to fusion with the plasma membrane (Sudhof and Rothman 2009), which have also been found to be reduced by α-synuclein overexpression. However, complexins levels are moderately changed in α- and β-synuclein knockout mice (Chandra et al., 2004), implying that α-synuclein is not strikingly involved in their physiological regulation at synaptic sytes. Thus, besides synapsin 1, the expression and subcellular localization of other proteins seem to be affected by α-synuclein accumulation. Remarkably, it has been shown that α-synuclein is able to restore N-ethylmaleimide sensitive fusion attachment protein receptor (SNARE) proteins (SNAREs) assembly induced by the cysteine-string protein-α (CSP-α) knockout by acting through a downstream mechanism that requires phospholipids binding, thus protecting nerve terminals against CSP-α deletion-induced injury (Chandra et al., 2005). The modulation of SNARE complex formation by α-synuclein likely occur through a direct interaction with synaptobrevin-2, which involves the C-terminal portion of α-synuclein as recently postulated by Burrè et al. (2010). Therefore, C-terminally truncated α-synuclein is

change in synaptic membrane fluidity and composition.

likely unable to interact with synaptobrevin-2, and to regulate SNARE complex assembly. Furthermore, α-synuclein can indirectly affect SNAREs activation by sequestering arachidonic acid (Darios et al., 2010). Another study showed that transgenic overexpression of α-synuclein resulted in the selective reduction of synaptobrevin-2 and synapsin 1 as well as of other synaptic proteins playing a role in exocytosis and endocytosis such as piccolo or endocytosis-regulating proteins such as amphiphysin (Scott et al., 2010).

Synphilin, an adaptor molecule which anchors α-synuclein to intracellular proteins involved in vesicle transport and cytoskeletal functions (Lee et al., 2004), is another protein member of the synaptic proteome (Engelender et al., 1999;Neystat et al., 2002;Alvarez-Castelao and Castano, 2010). In particular, it has been shown that synphilin-1 binds to α-synuclein thus promoting the formation of cytosolic inclusions (Engelender et al., 1999). This effect may be due to the fact that the interaction of the central region of synphilin-1 with the N-terminal region of α-synuclein prevents α-synuclein degradation by the proteasome (Alvarez-Castelao and Castano, 2010).

More recently, a broad proteomic analysis investigated the serine 129 and serine 125 phosphorylation-dependent α-synuclein interactions (McFarland et al., 2008). In this paper, authors singled out several other synaptic protein partners such as synaptophysin, SNAP-25-interacting protein, vesicle-associated membrane protein-associated protein (VAPA) A and B and syntaxin-binding protein 1.

Finally, it has been shown that α-synuclein interacts with and modulate the membrane trafficking of several neuron-specific transporters implicated in neurotransmitter re-uptake from synaptic clefts, among them the DAT (Sidhu et al., 2004;Lee et al., 2001a;Bellucci et al., 2008) as well as the noradrenalin transporter (NET) (Wersinger et al., 2006a) and serotonine transporter (SERT) (Wersinger et al., 2006b). Remarkably, besides dopaminergic neurons of the substantia nigra, the noradrenergic locus ceruleus (oral parts) and motor vagal nucleus as well as serotonergic neurons of the Raphe nuclei are among the most vulnerable neurons in the PD brain (Jellinger, 1991), thus, a loss of DAT, NET or SERT synaptic membrane content, may critically be involved in the onset of PD.
