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α-Syn has a natively unfolded monomeric state with no significant secondary structure (Weinreb, Zhen, Poon, Conway, & Lansbury, 1996). However, it is thought to gain structure upon binding to substrates. With numerous phosphorylation sites possessed, it can undergo several post-translational modifications that regulate substrate affinity and stabilise intermediate conformations (Esposito, Dohm, Kermer, Bahr, & Woutersa, 2007). Taken together, α-Syn is believed to undergo major structural change from its natively unfolded state to α-helical conformation upon interaction with membrane lipids or to the characteristic crossed β-conformation in highly organised amyloid- like fibrils under conditions that trigger aggregation (Moussa, Mahmoodian, Tomita, & Sidhu, 2007).
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long-term potentiation in hippocampus of young and aged rats (Stephan et al., 2002).
A study done by Cabin et al. (2002) demonstrated that following prolonged repetitive stimulation, mice lacking α-Syn exhibited attenuated synaptic response. Moreover, hippocampal synapses of α-Syn knock-out mice showed fewer synaptic vesicles, particularly in the reserve pool, and the replenishment of docked vesicles by reserve pool vesicles after depletion was also slower, compared to that of their control littermates, thus, suggesting that α-Syn may be required for the genesis and/or maintenance of a reserve pool of presynaptic vesicles and regulation of synaptic vesicle mobilisation at nerve terminals (Cabin et al., 2002).
α-Syn-depleted neurons also showed reduced expression levels of synapsin, a protein vital for synaptic vesicle recycling, suggesting that a putative key function of α-Syn may be to regulate synaptic vesicle recycling through modulation of phospholipase D2 (PLD2) activity and its FABP properties (Cabin et al., 2002; Sidhu et al, 2004c). α-Syn may also regulate synaptic vesicle recycling by assisting in the folding and refolding of SNAREs (Bonini
& Giasson, 2005). SNAREs are protein complex consisting of synaptobrevin, syntaxin, and SNAP25, which play a role in vesicle priming, transferring of docked vesicles into an exocytosis-competent state, and vesicle fusion to the membrane (Goda, 1997). A study involving analysis of cysteine-string protein α (CSPα)-deficient mice demonstrated that up-regulation of α-syn compensates for the loss of CSPα activity by restoring SNARE complexes to their correct
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levels and suppressing presynaptic degeneration, motor dysfunction, and death of mice lacking CSPα (Chandra et al., 2005). Furthermore, α-Syn has been suggested to interfere with axonal transport of synaptic vesicles by its interaction with proteins that either bind to or are part of the cytoskeleton, such as tau, heterodimeric but not microtubule tubulin, MAP1B, MAP2, synphilin-1, and torsin A (Payton, Perrin, Clayton, & George, 2001).
Specifically, α-Syn may be instrumental in the physiological maintenance of dopamine homeostasis and dopamine synaptic tone in dopaminergic neurons of the substantia nigra pars compacta through regulation of the functional activity of tyrosine hydroxylase (TH) and dopamine transporter (DAT), as extensively reviewed by a few groups (Lotharius & Brundin, 2002; Perez & Hastings, 2004; Sidhu, Wersinger, & Vernier, 2004a).
Dopamine biosynthesis begins with a rate-limiting step: the hydroxylation of tyrosine into dihydroxyphenylalanine (L-DOPA) catalysed by phosphorylated TH followed by conversion of L-DOPA into dopamine by aromatic amino acid decarboxylase. Since TH is only active in phosphorylated form, the regulation phosphorylation/dephosphorylation of TH is essential in dopamine biosynthesis (Perez & Hastings, 2004; Sidhu Wersinger, & Vernier, 2004b). In particular, there may be a reciprocal interplay between α-Syn and 14-3-3 proteins for the regulation of TH activity. TH has been reported to require binding of chaperone protein, 14-3-3 for optimal activation through maximal phosphorylation (Xu et al., 2002). Conversely, α-Syn appears to colocalise with TH and directly bind to the dephosphorylated form of TH. It tends to
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maintain TH in an inactive form in an opposite manner to 14-3-3 proteins, causing an overall net decrease in enzymatic activity and dopamine synthesis (Perez et al., 2002).
DAT is expressed in the presynaptic terminals of dopaminergic neurons where it mediates the re-uptake of dopamine back into the dopaminergic nerve terminals (Chen & Reith, 2000). Enhanced DAT activity would increase intracellular levels of dopamine with a resulting increased oxidative stress within the dopaminergic neuron and neuronal death (Sidhu et al, 2004a). The regulation of DAT which involve the rapid and transient shuttling of the transporter molecule to and from the plasma membrane may thus be a key component in the maintenance of dopaminergic neurotransmission and the integrity of dopaminergic neurons (Sidhu et al., 2004a). In agreement with these theories, a study demonstrated that in the presence of wild type α-Syn, the transporter was dynamically trafficked away from the plasma membrane into the cytoplasm, as indexed by reduced DAT presence at the plasma membrane by biotinylation experiments (Wersinger, Prou, Vernier, & Sidhu, 2003). Moron et al. (2003) reported that activation of mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1/2 was shown to increase the amount of DAT at the cell surface and dopamine re-uptake. Since α-Syn can bind to the MAP kinase ERK and inhibit its activity, it is very likely that the presence of α-Syn at the nerve terminals tends to blur the MAP kinase activity and decrease the amount of DAT at the cell surface of dopaminergic nerve terminals (Iwata, Miura, Kanazawa, Sawada, & Nukina, 2001; Sidhu et
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al., 2004c). Figure 2.4 illustrates the collective functions of α-Syn in dopaminergic neurons.
Figure 2.4: Schematic illustration of the function of α-Syn in dopamine metabolic pathway in neurons. (1) In normal situations or when synaptic plasticity needs to be increased, α-Syn contributes to the formation of synaptic vesicles, both by enhancing PLD2 activity and by its lipid binding properties.
At the synaptic terminals, vesicles bearing the vesicular transporter quickly and efficiently accumulate dopamine. (2) α-Syn modulates the activity of TH, the limiting enzyme of catecholamine biosynthesis, by preventing its interactions with kinases. In contrast, 14-3-3 proteins favour TH activity by enhancing TH phosphorylation by calmodulin kinases or ERKs. (3) The shuttling of DAT to and away from the plasma membrane is also modified by the action of α-Syn, thereby changing the efficiency of dopamine uptake at the nerve terminal (Figure taken from Sidhu et al., 2004c).