Putative functions of aSyn

Assessing the normal function of aSyn has been challenging, not only because it is an intrinsically disordered protein that shifts between conformations, but also because it is a promiscuous protein that interacts and interferes with a lot of biological/cellular processes. In addition, the model systems available only partially recapitulate the symptoms and molecular pathologies associated with the disease.

Furthermore, these models generally rely on aSyn overexpression, which adds another layer of complexity (for example, compensatory mechanisms) to the interpretation of its pathogenic and physiological roles.

The cellular localization of aSyn is thought to be regulated throughout brain development, during neuronal migration, maturation and synaptogenesis (Hsu et al.

1998; Murphy et al. 2000). In animal models, like rodents, aSyn levels are low in early embryogenesis (prior to E15), but increases in later stages of neuronal development (E18) extending into the postnatal period (P7) (Hsu et al. 1998). Also, increase levels of aSyn in pre-synapses are involved in critical stages of development, such as learning and synaptic plasticity (Maroteaux et al. 1988; George et al. 1995;

Clayton et al. 1998; Hsu et al. 1998; Murphy et al. 2000). A recent study performed with professional musicians reveled up-regulation of some genes after music performance. In the list, dopaminergic neurotransmission-related genes were consistently identified. Around 26% of the SNCA co-expression network, was found to be up-regulated along with SNCA, suggesting that music performance may modulate the biological pathways of aSyn (Kanduri et al. 2015).

aSyn exists in a dynamic equilibrium between a soluble and a membrane-bound state (Roy 2009). The interaction between aSyn and lipid surfaces is believed to be a key features both in physiological and pathological condition. The N-terminal region of aSyn can adopt α-helical secondary structure upon binding to detergent micelles, liposomes (Davidson et al. 1998) or to negatively charged lipids or

the 11-mer sequences, as truncation of this domain drastically reduces lipid binding (Bisaglia et al. 2006; Burre 2015). N-terminal acetylation of aSyn can also increase its helical folding propensity, membrane binding affinity, and resistance to aggregation (Fauvet et al. 2012; Kang et al. 2012; Maltsev et al. 2012). aSyn not only binds to membranes, but it can induce membrane curvature and membrane tubulation, similar to amphiphysin (curvature-inducing protein involved in endocytosis) (Figure 1.12) (Varkey et al. 2010). These membrane-curvature changes can have a significant impact on the fusogenic properties of synaptic vesicles. For example, vesicles that have a high curvature, favors fusion with flat target membranes (Auluck et al. 2010).

Interestingly, aSyn can aggregate faster in the presence of brain membranes, than in the presence of cytosolic fractions (Lee et al. 2002).

Figure 1.12 Model for aSyn-mediated membrane remodeling and curvature induction. (I) When aSyn binds to a single vesicle (II) the curvature strain causes initiation of a membrane tubule. The shape of the tube (V) is dependent of the concentration and orientation of the protein bound molecules on the membrane (III, IV) with higher concentrations favoring more curved structures. (V) Vesicular structures could originate from smaller membrane tubes or directly from large vesicles (II). B.

Insertion of aSyn helical structure on intact vesicles occurs at the phosphate level. From (Varkey et al.

2010)

The mechanism by which membranes can be disrupted by aSyn is still intensely debated. Nevertheless, the formation of pore-like structures within the lipid bilayer (Lashuel et al. 2002), and the observation of donut-shaped complexes by atomic force and electron microcopy (Quist et al. 2005) have been reported.

Mutations in aSyn also alter its phospholipid binding affinities. A30P and G51D decrease lipid affinity (Ysselstein et al. 2015).

Another role attributed to aSyn is its association with lipid metabolism. aSyn has been reported to bind to fatty acids (Sharon et al. 2001), to organize membrane components (Sharon et al. 2001), to regulate phospholipid composition (Adamczyk et al. 2007), and to inhibit phospholipase D1 and D2 in vitro and in vivo (Ahn et al.

2002; Outeiro et al. 2003). This implies that aSyn may be implicated in cleavage of membrane lipids and membrane biogenesis.

aSyn can interfere with neuronal membrane trafficking, affecting both Ras analog in brain (Rab) GTPases and certain N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNAREs) (Amaya et al. 2015). SNAREs are a family of proteins that act in membrane fusion (Bonifacino et al. 2004; Jahn et al.

2006). aSyn directly promotes presynaptic SNARE-complex assembly via a nonclassical chaperone activity (Burre et al. 2010). aSyn is able to act as a SNARE chaperone when bound to the membrane, which can adopt α-helical conformation, and associates into multimers on the membrane surface. The multimers are the active forms that will promote SNARE complex assembly (Figure 1.13) (Burre et al. 2014).

Figure 1.13 Schemating of a physiological folding pathway for aSyn. Native unstructured monomeric aSyn binds to synaptic vesicles during docking and priming of the vesicles. As a result of membrane binding, aSyn promotes SNARE complex assembly during docking and priming of synaptic vesicles. From (Burre et al. 2014)

The loss of dopaminergic neurons in the SN in PD results in a deficiency of DA signaling. In this context, it was hypothesized that aSyn could be related with DA

hydroxylase (TH) (Gao et al. 2007), limiting the conversion of tyrosine conversion to L-3,4-dihydroxypheny-lalanine (L-DOPA) (Baptista et al. 2003). This likely occurs via reducing TH phosphorylation and activation, which would lead to the activation of protein phosphatase 2A (Peng et al. 2005; Liu et al. 2008). Furthermore, aSyn affects the vesicular DA transporter VMAT2. aSyn knockout mice showed increased density of VMAT2 molecules per vesicle, and altered DA release (Abeliovich et al. 2000).

Expression of aSyn decreases the rate of DA release without changing DA levels or clearance/uptake mediated by dopamine transporter (Figure 1.14) (Yavich et al. 2005; Larsen et al. 2006). This shows that aSyn can acts as a negative regulator of DA release, by modulating vesicle function upon synaptic stimulation.

Figure 1.14 Schematic model of the role of aSyn in regulating presynaptic vesicle cycling. (a) When the levels of aSyn are low, the availability of vesicles in the reserve pool decreases and more vesicles are become available to be released. This increases dopamine release. (b) Under physiological conditions aSyn is regulates the vesicle docking and fusion. (c) Elevated levels of aSyn (or E46K and A53T), aSyn reduces dopamine release. From (Venda et al. 2010)