- Top of page
- Materials and Methods
α-Synuclein (αS) is an abundant neuronal cytoplasmic protein implicated in Parkinson’s disease (PD), but its physiological function remains unknown. Consistent with its having structural motifs shared with class A1 apolipoproteins, αS can reversibly associate with membranes and help regulate membrane fatty acid composition. We previously observed that variations in αS expression level in dopaminergic cultured cells or brains are associated with changes in polyunsaturated fatty acid (PUFA) levels and altered membrane fluidity. We now report that αS acts with PUFAs to enhance the internalization of the membrane-binding dye, FM 1-43. Specifically, αS expression coupled with exposure to physiological levels of certain PUFAs enhanced clathrin-mediated endocytosis in neuronal and non-neuronal cultured cells. Moreover, αS expression and PUFA-enhanced basal and -evoked synaptic vesicle (SV) endocytosis in primary hippocampal cultures of wild type (wt) and genetically depleted αS mouse brains. We suggest that αS and PUFAs normally function in endocytic mechanisms and are specifically involved in SV recycling upon neuronal stimulation.
The neuronal protein, α-synuclein (αS), has been implicated in the pathogenesis of Parkinson’s disease (PD) at both the genetic and the cytopathological levels (1–7). Despite the involvement of this abundant neuronal protein in sporadic and familial forms of PD and related α-synucleinopathies, both its normal function and the mechanism by which it gradually accumulates in dopaminergic and other neurons in disease remain unclear.
A portion of αS associates with membranes in vitro (8–16) and in vivo (17–22). These observations are consistent with its primary structure, which contains six imperfect apolipoprotein A1-like repeats in its N-terminal region that may mediate lipid binding (23,24). We obtained evidence that αS can associate with polyunsaturated fatty acids (PUFAs) in vitro and in neuronal cells and brain tissue (18). Importantly, we found that changes in αS expression can affect membrane and cytosolic PUFA composition and alter membrane fluidity. Specifically, we observed higher levels of certain long-chain PUFAs and higher fluidity in membranes of MES 23.5 dopaminergic cells overexpressing αS than in those of parental cells and lower levels of such PUFAs and lower fluidity in membranes of αS−/− than normal mouse brains (25). More recently, it was reported that αS can affect brain lipid metabolism and specifically PUFA metabolism (26–30). In agreement with our initial observation that αS expression affects membrane fatty acid (FA) composition (25), these studies in αS null mouse brains documented reduced incorporation of certain FAs into membrane phospholipids as well as decreases in FA uptake and turnover (26,27,29,30).
During endocytosis, a small region of the plasma membrane invaginates to form a new intracellular vesicle containing various cargo molecules. Clathrin-mediated endocytosis (CME) is the major entry route for extracellular molecules such as nutrients, hormones and signaling factors and serves to regulate the internalization of transmembrane receptors, including the recycling of pre- and postsynaptic neuronal membrane proteins (31–33). Although clathrin-coated vesicles are found in all eukaryotic cells, their components are particularly enriched in brain, where clathrin and its partner proteins are implicated in the biogenesis of presynaptic vesicles, the major secretory organelles within the nervous system (34,35). The cargo for endocytosis is usually recognized by a specific receptor on the cell surface. Most receptor-mediated endocytosis (RME) is mediated by clathrin-coated pits.
PUFAs have been found to play a role in the formation and/or maintenance of synaptic vesicles (SVs) (36,37), including dopaminergic vesicles (38–43). It has been hypothesized that because of their ‘cone shape’, PUFAs affect membrane curvature in a way that promotes vesicle budding and membrane trafficking (44,45). In this context, it is of interest that αS is localized in part to presynaptic neuronal terminals and has been found to be involved in the genesis and/or maintenance of the reserve, or resting, presynaptic vesicle pools (17,46,47).
In this study, we assess the effects of αS and PUFAs on plasma membrane trafficking. We provide evidence that αS and PUFAs affect endocytosis and vesicle recycling in both neuronal and non-neuronal cells and specifically activate SV recycling after neuronal stimulation by enhancing CME.
- Top of page
- Materials and Methods
Taken together, these experiments provide new evidence that αS and PUFAs can act in at least an additive fashion to modulate membrane trafficking, with a specific focus on their individual and combined effects on endocytosis mediated by clathrin. Overall, we found that αS expression or PUFA treatment induced endocytosis, with further induction upon their combination. In general, at the same carbon chain length and concentration, PUFAs induced and SFAs inhibited endocytosis. MUFAs effect on induced endocytosis was milder and was observed only with the longer carbon-chained FAs. Using the canonical example of ligand internalization through RME, we show that αS and PUFA induce the endocytosis and recycling of Tf by the TfR. αS- and PUFA-induced Tf endocytosis is molecularly specific as it is inhibited by clathrin siRNA. Relevant to the physiological function of αS in the brain, our findings indicate that αS and PUFAs can act together to stimulate SV formation in primary hippocampal neurons. Specifically, αS is implicated in the endocytic recycling of SV following neuronal stimulation. Importantly, primary hippocampal neurons genetically deleted of αS showed reduced number and size of synaptophysin-positive boutons and reduced FM1-43 endocytosis in SV, suggesting a reduced recycling pool. Based on these invariant results in primary neuronal cultures, we suggest that αS and PUFA are normally involved in the endocytic machinery leading to SV formation and replenishment and thus synaptic strengthening.
Early reports that αS is localized to nerve terminals (17,58,59) and associates with SV (60) suggested the possible involvement of αS in the formation/maintenance of SV and/or neurotransmitter release. Several subsequent studies have shown that αS expression has a role in the formation or maintenance of distinct synaptic pools. In particular, reduced sizes of distinct synaptic pools in primary hippocampal neurons were observed upon partially silencing αS expression (47) and also in hippocampal neurons from αS−/− brains (46). In accord, increased accumulation of docked vesicles was observed in PC12 cells upon αS overexpression (61). Furthermore, αS has been postulated to be involved in the replenishment of synaptic pools after a depleting stimulation (46). Nevertheless, these various observations were not consistent with certain findings in other αS−/− mouse models. No alterations in synaptic pool size or replenishment of recycling SV were observed in αS−/− mice or in the double knockout αS−/− and αS−/− mice (62), and αS was suggested to negatively regulate the readily releasable pool of dopamine-containing vesicles (63). These variations in observations may result from the different mouse models and experimental designs employed and highlight the complexity of synuclein’s physiological role. The new results presented herein strongly support a role for αS in the formation/maintenance of SV and/or neurotransmitter release. Furthermore, the results indicate that αS is involved in mechanisms leading to synaptic strengthening. That is, the reduced number and size of synaptophysin-positive boutons observed in αS−/− neurons indicate lower synaptic activity. This, in turn, could affect the plasticity of neuronal networks that is known to underlie cognitive functions such as learning and memory.
Recent studies have suggested that αS is involved in other aspects of membrane trafficking, possibly in exocytosis or in the secretory pathway. It has been reported that αS expression specifically inhibits ER-to-Golgi trafficking, resulting in cytotoxicity that was prevented by Rab1 expression (64). It was further shown that αS expression affected vesicle docking or fusion to the Golgi apparatus after an efficient budding from the ER (65). In PC12 cells, αS overexpression inhibited evoked catecholamine release and increased the ‘docked’ vesicle pool (61). Other studies have suggested an indirect role for αS in promoting the assembly of the SNARE complex (62,66). SNAREs catalyze the fusion of vesicles with their target membranes to enable the release of cargo from the vesicle (reviewed in 67,68). Collectively, growing evidence implicate αS in membrane trafficking, including endocytosis and exocytosis.
In addition to our observed induction of FM1-43 uptake into SV, αS plus PUFA additively induced dye uptake into neuronal cell bodies. Despite extensive investigation, it remains unclear whether the endocytic pathways at nerve terminals are specializations of the pathways that exist in all cells or rather represent a neuron-specific mechanism for rapid membrane recycling (69). In this regard, whether neurons have classical endosomes is also unresolved. Therefore, the nature of the induced vesicles we observed in the neuronal cell bodies is yet to be determined. Nevertheless, because we found that αS can activate endocytosis at both neuronal and non-neuronal vesicles, these pathways most likely share a high degree of similarity.
We previously reported that αS expression in cells and brain is associated with enriched levels of certain PUFAs in both the cytosol and the membranes, and this was associated with apparent changes in membrane biophysical properties reflected by increased membrane fluidity (25). Our initial working hypothesis after we obtained these results was that the αS effects on membrane fluidity may relate directly to its effects on membrane trafficking. Our rationale was that αS may act to enrich the cytoplasmic membrane leaflet with PUFAs. PUFAs in the membranes act as cone-shaped lipids that induce a positive curvature of the membrane leaflet (44,70), thus inducing invagination and fission of the membrane and thereby enhancing membrane trafficking. The results presented herein support this initial working hypothesis. It is supported by the specific inducing effect of PUFAs (but never MUFAs or SFAs of identical carbon chain length and concentration) – either with or without αS overexpression – on endocytosis and SV formation. Therefore, we speculate that αS and PUFA mechanically alter membrane curvature as a result of enrichment of the plasma membrane with PUFAs, thereby facilitating endocytosis, including, among other mechanisms, CME.
In addition to the general role of membrane curvature in endocytosis discussed above, the findings herein that αS+ PUFAs effects can alter actual protein levels of specific key factors in CME and RME, that is, TfR and clathrin, and also redirect TfR to the cell surface membrane indicate activation of regulatory mechanisms that may secondarily lead to the overexpression or stabilization of these specific endocytic participants. A hypothetical biological target for αS/PUFA activation is certain classes of membrane phospholipids, and specifically phosphoinositides, that are known to regulate CME (reviewed in 71). Further investigation will be needed to elucidate the specific molecular mechanisms by which αS and PUFAs activate endocytosis.
We view the data we have obtained to date as addressing a normal physiological role of αS. However, it is likely that there are pathophysiological implications of our findings. For example, we observed that the accumulation of αS into high MW assemblies, including soluble cytosolic dimers and low-n oligomers that could well serve as the nidus for αS aggregation into Lewy-type fibrillar deposits, is associated with alterations in neuronal PUFA composition. That is, we examined FA profiles in mesencephalic neuronal cells that stably express αS and found accumulation of PUFAs in cytosols as well as membrane fractions in such cells compared with parental cells that express low levels of endogenous αS. These changes could be relevant to the overexpression of wt αS in PD families with duplication or triplication of the αS locus and also to idiopathic PD cases that invariably accumulate wt αS in the neuronal cytoplasm. In this regard, it is interesting to consider the correlation between PUFA-induced αS oligomerization (48) and PUFA-induced FM1-43 uptake (herein). In both cases, the longer and more unsaturated the FA, the more αS oligomerization and the more FM1-43 internalization one observes. This correlation may suggest that the soluble, cytosolic oligomers are the active forms.
Collectively, our earlier and current results suggest that αS normally interacts with PUFAs to carry out its physiological functions, but under certain potentially pathogenic conditions, this interaction may lead to neuronal membrane dysfunction and ultimately αS aggregates and cell death. We propose that this shift on a continuum between normal and pathogenic αS–lipid interactions may be driven by transient increases in either the levels of certain PUFAs or the levels of αS monomers in the cytoplasm.