Muscarinic Receptor Stimulation Induces Translocation of an a -Synuclein Oligomer from Plasma Membrane to a Light Vesicle Fraction in Cytoplasm*

The close correspondence between the distribution of brain a -synuclein and that of muscarinic M 1 and M 3 receptors suggests a role for this protein in cholinergic transmission. We thus examined the effect of muscarinic stimulation on a -synuclein in SH-SY5Y, a human dopaminergic cell line that expresses this protein. Under basal conditions, a -synuclein was detected in all subcellular compartments isolated as follows: plasma membrane, cytoplasm, nucleus, and two vesicle fractions. The lipid fractions contained only a 45-kDa a -synuclein oligomer, whereas the cytoplasmic and nuclear fractions contained both the oligomer and the monomer. This finding suggests a -synuclein exists physiologically as a lipid-bound oligomer and a soluble monomer. Muscarinic stimulation by carbachol reduced the a -synuclein oligomer in plasma membrane over a 30-min period, with a concomitant increase of both the oligomer and the monomer in the cytoplasmic fraction. The oligomer was associated with a light vesicle fraction in cytoplasm that contains uncoated endocytotic vesicles. The carbachol-induced alteration of a -synuclein was blocked by atropine. Translocation of the a -synuclein oligomer in response to carbachol stimulation corresponds closely with the time course of ligand-stimulated muscarinic receptor endocytosis. The data suggest that the muscarine receptor stimulated release of pH 6.8). Cell Culture and Lysis— Human dopaminergic neuroblastoma SH-SY5Y cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum and kept at 37 °C in humidified 5% CO 2 , 95% air. Cells grown on 100-mm plates to 80–90% confluence were rinsed with 1 ml of ice-cold wash buffer. Cells were fractionated according to a modification of the procedures of Fleischer and Kervina (10) as follows. Cells from each plate were and samples for each condition were run in duplicate. Verification of Lipid Fractions by Detection of Annexin II— Annexin II is a membrane protein localized exclusively in plasma membrane and early endosomes (13). Annexin II was assessed in subcellular fractions under both basal and carbachol-stimulated conditions. In order to esti-mate the relative purity of the subcellular fractions, fraction samples from unstimulated cells containing 5 m g of protein each were eluted by gel electrophoresis, detected by Western blot using the monoclonal antibody against annexin II, and visualized by chemiluminescence. Annexin II antigenicity was used also to examine the effect of carbachol stimulation on the early endosomes in the cytoplasmic (S 1 ) fraction from samples containing 20 m g of protein each from unstimulated and carbachol-stimulated cells. was used to detect a -synuclein as described under “Ex- perimental Procedures.” In a , two a -synuclein reactive bands with apparent molecular masses of ; 19 and 45 kDa were detected from samples under the mild extraction conditions; whereas in b , only the 19-kDa band was detected from samples extracted under the heated denaturing/reducing conditions. Disappearance of the 45-kDa band could be accounted for quantitatively by the increase in the signal of the a -synuclein monomer.

The neuronal protein ␣-synuclein, recently implicated in the pathogenesis of Parkinson's disease (1)(2)(3), has a brain distribution closely matching that of muscarinic M 1 and M 3 receptors and the muscarine receptor-linked enzymes phospholipase C and protein kinase C (4). The proximity of ␣-synuclein to brain muscarinic receptors and related enzymes suggests that this protein could contribute to cholinergic function. To evaluate this possibility, we investigated the effect of muscarinic receptor stimulation by the cholinergic agonist carbachol on the subcellular distribution of endogenous ␣-synuclein in the human neuroblastoma cell line SH-SY5Y. This dopaminergic cell line shares many properties with dopamine neurons of substantia nigra pars compacta, the major locus of neurodegeneration in Parkinson's disease, including the expression of M 1 and M 3 receptors (5). We now report that carbachol induces a translocation of a 45-kDa ␣-synuclein oligomer from plasma membrane to a light vesicle fraction in cytoplasm, with a time course and subcellular localization matching that of muscarinic receptors during carbachol-stimulated endocytosis. Previously, synucleins have been demonstrated to be endogenous inhibitors of phospholipase D 2 (PLD 2 ) 1 (6). Furthermore, activation of PLD isoforms, including PLD 2 , stimulates endocytosis (7)(8)(9). Thus, the present data are consistent with a model of muscarinic receptor endocytosis in which the release of ␣-synuclein from plasma membrane transiently disinhibits membranebound PLD 2 , freeing this lipase to function in ligand-stimulated muscarinic receptor endocytosis.

EXPERIMENTAL PROCEDURES
Materials-Anti-synuclein-1 (Syn-1) monoclonal antibody, annexin II monoclonal antibody, and goat anti-mouse IgG polyclonal horseradish peroxidase conjugate were from BD Transduction Laboratories; ECL detection reagents were from Amersham Pharmacia Biotech; 10ϫ SDS/Tris glycine electrophoresis running buffer and non-fat dry milk were from Bio-Rad; 4 -20% Tris glycine gels, Xcell II Mini-Cell electrophoresis system and Blot Module, PVDF membrane/filter paper, Mul-tiMark Multicolored and See-Blue standards were from NOVEX; 10ϫ Tris glycine Transfer Buffer was from Quality Biological Inc.; phosphate-buffered saline (PBS) and Dulbecco's modified Eagle's medium were from Cellgro-Mediatech; fetal bovine serum was from Life Technologies, Inc.; and protease inhibitor mixture was from Roche Molecular Biochemicals.
Cell Culture and Lysis-Human dopaminergic neuroblastoma SH-SY5Y cells (ATCC) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum and kept at 37°C in humidified 5% CO 2 , 95% air. Cells grown on 100-mm plates to 80 -90% confluence were rinsed with 1 ml of ice-cold wash buffer. Cells were fractionated according to a modification of the procedures of Fleischer and Kervina (10) as follows. Cells from each plate were scraped with 0.5 ml of ice-cold sucrose homogenization buffer into a 5-ml glass homogenization vessel and lysed by 10 strokes of a glass pestle with a Dounce ball tip. Homogenates from each plate were centrifuged at 1450 ϫ g for 10 min (3500 rpm, Beckman J-21, JA-20 rotor). The post-nuclear supernatants were transferred to other tubes and kept ice-cold for later separation of vesicles and cytoplasm. Nuclei and plasma membranes were isolated from the resulting pellets.
Isolation of Plasma Membrane and Nuclear Fractions-The pellet from the initial centrifugation was resuspended in 1 ml of ice-cold sucrose homogenization buffer. A volume of 1.62 ml of ice-cold high density sucrose buffer was added to the solution containing the resuspended pellet, producing a final concentration of 1.6 M sucrose. A two-layer step gradient was set up by layering a sufficient volume of 0.25 M sucrose buffer over the 1.6 M sucrose suspension to fill the tube and then centrifuged at 70,900 ϫ g R av (24,000 rpm, SW 25.2 rotor) for 70 min. The band at the gradient interface (0.25 and 1.6 M sucrose) was enriched in plasma membrane. The plasma membrane-containing band was carefully transferred to other tubes after removal by aspiration of the top layer of 0.25 M sucrose buffer. The pellet produced after the gradient centrifugation contained the nuclear fraction.
Isolation of Vesicle and Cytoplasmic Fractions-Two vesicle and cytoplasmic fractions were isolated by different centrifugation procedures used with matched samples of post-nuclear supernatant. A vesicle fraction termed V 1 was isolated by ultracentrifugation of the postnuclear supernatant at 100,000 ϫ g for 1 h. The pellet of this centrifugation contains vesicles in the density range of synaptic vesicles (11). The resulting pellet was resuspended in freezing buffer, which stabilizes the sample for freezing at Ϫ80°C or could be analyzed immediately. The supernatant of this centrifugation is the cytoplasmic fraction we termed S 1 . Matched culture plates were used to sediment a larger vesicle fraction, the V 2 , by ultracentrifugation of the post-nuclear supernatant at 200,000 ϫ g for 90 min (12). The supernatant from this centrifugation is the cytoplasmic fraction we termed S 2 .
Determination of Protein Concentration-Protein concentration was determined by the BCA Protein Assay kit using bovine serum albumin as a standard. Concentrations of total protein were determined from the absorption spectrum measured at A 562.
Detection of ␣-Synuclein by Western Blot-Aliquots containing equal amounts of protein from each sample (20 g each for cytoplasmic, nuclear, and vesicle fractions; 7 g each for the plasma membrane fractions) were dissolved in an equal volume aliquot of 2ϫ SDS sample buffer, loaded onto 4 -20% linear gradient polyacrylamide mini gels, and then subjected to electrophoresis (applied constant voltage, 125 V). After protein separation, each gel was placed on a PVDF membrane in transfer buffer, and the proteins were transferred to the membrane by the application of 25 V for 2 h. After the transfer was completed, the PVDF membrane was soaked in blocking buffer for 1 h, incubated for 1 h with the primary antibody (1:500 dilution in 0.1% Tween 20/PBS), washed three times for 20 min each time in 0.1% Tween 20/PBS, incubated for 1 h in secondary antibody (1:2000 dilution in 0.1% Tween 20/PBS), and then washed again three times as before. The reactive bands were visualized by chemiluminescence.
Carbachol Stimulation-Aliquots of 500 mM carbachol solution were added to cell cultures to a final concentration of 1 mM. Carbacholstimulated cells were incubated for intervals of up to 30 min and compared with non-treated control cells. The specificity of the carbachol stimulation effect was assessed by preincubation of cells with 200 M atropine 1 h prior to the addition of carbachol. All experiments were conducted in triplicate, and samples for each condition were run in duplicate.
Verification of Lipid Fractions by Detection of Annexin II-Annexin II is a membrane protein localized exclusively in plasma membrane and early endosomes (13). Annexin II was assessed in subcellular fractions under both basal and carbachol-stimulated conditions. In order to estimate the relative purity of the subcellular fractions, fraction samples from unstimulated cells containing 5 g of protein each were eluted by gel electrophoresis, detected by Western blot using the monoclonal antibody against annexin II, and visualized by chemiluminescence. Annexin II antigenicity was used also to examine the effect of carbachol stimulation on the early endosomes in the cytoplasmic (S 1 ) fraction from samples containing 20 g of protein each from unstimulated and carbachol-stimulated cells.

RESULTS AND DISCUSSION
The ␣-synuclein monoclonal antibody consistently detected two molecular species of this protein in the SH-SY5Y whole cell lysate. The ␣-synuclein monomer migrated in gel electrophore-sis at its expected position corresponding to an apparent molecular mass of ϳ19 kDa (14) and a second ␣-synuclein signal was detected at ϳ45 kDa. The 45-kDa band appears to be an oligomer containing at least one ␣-synuclein molecule, as it can be non-enzymatically cleaved under stringent reducing conditions, resulting in a quantitative increase in the monomer (15) (Fig. 1). The vigorous reducing conditions required for cleavage of this oligomer suggest that the bond between molecules may be an azo bond (16). This linkage may be similar to the dityrosine bonds recently characterized as being formed after tyrosine nitration (17).
The distribution of the two forms of ␣-synuclein in the various cellular compartments was determined by subcellular fractionation of whole cell lysate both under basal conditions and in response to carbachol stimulation for incubation intervals of up to 30 min. The post-nuclear supernatant was used to isolate two vesicle fractions V 1 and V 2 from matched sets of samples as described under "Experimental Procedures." The ␣-synuclein in the vesicle fraction we termed V 1 was shown in previous work to be associated exclusively with vesicles of densities 1.0995-1.259 g/ml and to distribute identically with synaptic vesicle-associated protein, SNAP-25 (11), thus providing strong evidence that all of the ␣-synuclein in this fraction is bound to synaptic-like vesicles. The V 2 fraction contains, in addition to the vesicles of the V 1 fraction, a lighter vesicle fraction that includes endocytotic vesicles that have lost their clathrin coating (12). The supernatants from each centrifugation condition were the cytoplasmic fractions termed S 1 and S 2 , respectively.
The efficacy of the separation of the cellular fractions was demonstrated by the relative concentrations of annexin II, a were added to an equal volume of 2ϫ SDS sample buffer with the addition (reducing) or omission (non-reducing) of 12% v/v 2-mercaptoethanol (2-ME). The samples containing reducing buffer were heated to 100°C for 5 min and then incubated for 2 days before elution, whereas the samples dissolved in non-reducing buffer were immediately frozen and then thawed after 2 days to room temperature (RT) before elution. Matched samples from each treatment were eluted in the same gel. Western blot was used to detect ␣-synuclein as described under "Experimental Procedures." In a, two ␣-synuclein reactive bands with apparent molecular masses of ϳ19 and 45 kDa were detected from samples under the mild extraction conditions; whereas in b, only the 19-kDa band was detected from samples extracted under the heated denaturing/reducing conditions. Disappearance of the 45-kDa band could be accounted for quantitatively by the increase in the signal of the ␣-synuclein monomer. marker of plasma membrane and early endosomes (13). Annexin II was highly concentrated in plasma membrane compared with whole cell lysate and was weakly present in the cytosolic S 1 fraction from unstimulated cells (Fig. 2). With a 4-fold increase in sample protein, however, the annexin II signal was clearly detected in the S 1 fraction of unstimulated cells and increased with carbachol stimulation (Fig. 3). This increase suggests that the annexin II antibody detected the increase in early endosomes containing muscarinic receptors, which have been demonstrated to increase in response to carbachol stimulation (12). Annexin II was absent in the V 1 fraction, consistent with its localization in lipids of early endosomes but not synaptic vesicles (Fig. 2). Thus, the detection of annexin II exclusively in the plasma membrane and the S 1 cytoplasmic fractions validates the methods of subcellular fractionation used in this experiment.
All membrane fractions, i.e. the plasma membrane fraction and vesicle fractions, contained only the 45-kDa form of ␣-synuclein (Fig. 4, a and b). In contrast, the cytoplasmic and nuclear fractions often contained both the monomer and the heavier species (Fig. 4, c and d). The lack of the ␣-synuclein monomer in all of the membrane fractions suggests that the membrane-associated ␣-synuclein is exclusively oligomeric in SY5Y cells. The 45-kDa species was evident in the S 1 supernatant but weak or absent in the S 2 supernatant, indicating that a subcellular component containing this molecule is not sedimented by the conditions yielding the V 1 fraction but can be sedimented by the higher gravitational force centrifugation that yields the V 2 pellet (Fig. 5). Thus, by comparing the results of the different centrifugation conditions, we conclude that the soluble ␣-synuclein monomer is found in the aqueous cytosol, and the 45-kDa oligomer is associated with two distinct vesicle populations differentiated by density.
A previous investigation (18) identified an oligomer of purified recombinant ␣-synuclein with an apparent molecular mass of ϳ45 kDa, similar to the membrane-associated oligomer of the present study. The ␣-synuclein oligomer found in this study was formed only when ␣-synuclein was incubated with artificial vesicles containing phosphatidylinositol (PI). The ␣-synuclein monomer bound only to small unilaminar vesicles containing at least 20% acidic phospholipids, whereas the polymerization of vesicle-bound ␣-synuclein occurred only if the phospholipid was PI. These results suggest that the oligomeric ␣-synuclein detected in the SY5Y cellular environment is likely to be a homo-oligomer that binds solely to membranes enriched in phosphatidylinositols. The oligomer of purified ␣-synuclein detected in vitro was described as a homodimer (18); however, it seems more plausible that both the recombinant oligomer and the molecule presently detected in SY5Y cell lysate are homotrimers of ␣-synuclein. The molecular mass of endogenous ␣-synuclein monomer as determined by mass spectrometry is 14,681 (19), but it migrates in gel electrophoresis with an apparent mass of ϳ19 kDa, due to the highly acidic terminal sequence of this natively unfolded molecule (20). Upon vesicle binding, ␣-synuclein adopts a stable ␣-helical structure (18). This conformational change from an elongated molecule to one with considerable secondary structure can be predicted to be accompanied by a change to more conventional electrophoretic migration characteristics. A molecule of ϳ45 kDa would be more accurately 3 times the molecular weight of the ␣-synuclein monomer, thus being more consistent with a homotrimer. Alternatively, the higher molecular weight species FIG. 2. Annexin II in subcellular fractions of SY5Y cells under basal conditions. Annexin II, a marker of plasma membrane and early endosomes, was detected in subcellular fractions of unstimulated SY5Y cells containing 5 g of protein per sample. Annexin II was greatly enriched in plasma membrane compared with whole cell lysate, weakly detected in the cytosolic S 1 fraction, and absent in the synaptic vesicle fraction (V 1 ).

FIG. 3. Carbachol stimulation increases annexin II in the cytosolic S 1 fraction.
Annexin II was readily detected in samples of the cytoplasm S 1 fraction of unstimulated SY5Y cells containing 20 g of protein, in contrast with those containing only 5 g of protein (Fig. 2). The increase in the annexin II signal in response to carbachol stimulation is consistent with an increase in early endosomes containing muscarinic receptors.

FIG. 4. Basal subcellular distribution of ␣-synuclein in SY5Y
cells. ␣-Synuclein antigenicity was detected by Western blot after separation of proteins by Tris glycine gel electrophoresis from the following subcellular fractions of SY5Y cells: a, plasma membrane; b, vesicle fraction (V 1 ); c, cytoplasm (S 1 ); d, nuclear fraction. The 45-kDa ␣-synuclein oligomer was present in all cellular fractions, whereas the monomer appeared only in the nuclear and cytoplasmic fractions. may be a heteromer of ␣-synuclein bound to an unidentified binding partner.
Nuclear binding of ␣-synuclein has been controversial (21,22). We detected both the ␣-synuclein monomer and the 45-kDa species in the nuclear fraction (Fig. 4d). In addition to subcellular fractionation, we also visualized the distribution of ␣-synuclein by immunocytochemistry (Fig. 6). By this method also, ␣-synuclein was detected in the nucleus and cytoplasm of the SY5Y cells. Typically, the Western blot nuclear signal from the 45-kDa species was much stronger than that of the monomer. In the original characterization of ␣-synuclein in vivo, Maratoeax et al. (21) reported ␣-synuclein antigenicity over discrete portions of the nuclear envelope of neurons of the Torpedo electric organ, with the antigen signal diminishing over a 10-m gradient toward the interior of the nucleus. The present findings are consistent with the 45-kDa ␣-synuclein oligomer being associated with the nuclear membrane and the soluble monomer existing in the nucleoplasm.
Carbachol stimulation induced a consistent decrease in the 45-kDa signal detected in the plasma membrane fraction (Fig.  7a) and a concomitant increase in both the oligomer and the monomer in the cytoplasmic fraction S 1 (Fig. 7b). There was little or no alteration in the amount of ␣-synuclein in the vesicle V 1 fraction in response to carbachol stimulation (Fig. 7c), although there was abundant ␣-synuclein in this fraction, which includes the synaptic-like vesicles. In contrast to the V 1 fraction, a clear and marked increase in the ␣-synuclein signal was detected in the V 2 vesicle fraction (Fig. 8). A comparison of results from the two centrifugation conditions suggests that carbachol stimulation triggers a translocation of the ␣-synuclein oligomer from the plasma membrane to a light vesicle fraction in cytoplasm that is sedimented in the V 2 but not the V 1 fraction. Typically, however, the changes in the ␣-synuclein oligomer associated with the light vesicle fraction were more evident when this lipid fraction remained in the S 1 cytoplasm (Fig. 9). The overall protein concentrations were measured and found to be unal- Representative examples of plasma membrane (a), cytoplasmic (S 1 ) fraction (b), and vesicle (V 1 ) fraction (c). There was a consistent decrease in the 45-kDa ␣-synuclein oligomer in plasma membrane over the 30 min of carbachol stimulation. Concomitant with the decrease in the ␣-synuclein oligomer in plasma membrane, there was also a consistent increase in this species in the S 1 cytoplasmic fraction. The ␣-synuclein monomer also increased over the same period in the cytoplasmic fraction. Although there is abundant oligomeric ␣-synuclein associated with the synapticlike vesicles in V 1 , the response to carbachol stimulation ranged from no effect to a slight increase. tered by carbachol stimulation, thus the compartmental alterations represent translocation of protein rather than carbacholinduced synthesis or degradation.
Muscarinic receptors in many cell types, including SY5Y cells, undergo desensitization induced by carbachol stimulation that is mediated by ligand-stimulated, clathrin-dependent endocytosis (12,23). Endocytotic vesicles containing muscarinic receptors translocate from the plasma membrane to the cytoplasm and rapidly lose their clathrin coats (23)(24)(25). The uncoated endocytotic vesicles are contained in the light membrane fraction that is included in the V 2 pellet (12). We determined also that the ␣-synuclein signal increase in the cytoplasmic S 1 fraction was matched by an increase in the annexin II signal, a marker for early endosomes (13) (Fig. 3). The carbachol-induced ␣-synuclein translocation from plasma membrane to the light vesicle compartment matches the time course and subcellular translocation of muscarinic receptors during ligand-stimulated muscarinic receptor endocytosis (12). The internalization of ␣-synuclein was blocked completely by atropine (Fig. 10), indicating the specificity of this response to muscarinic receptor stimulation. Collectively, these results provide strong evidence that ␣-synuclein participates in ligandstimulated muscarinic receptor endocytosis.
A role for ␣-synuclein in muscarinic receptor endocytosis is consistent with its co-localization with muscarinic receptors and related enzymes in brain and fits well with the first reported physiological function of synucleins as potent endogenous inhibitors of phospholipase D 2 (PLD 2 ) (6). Muscarinic stimulation is well documented to activate PLD in various cell types, including SY5Y cells, although distinctions have not been characterized between the PLD 1 and PLD 2 isoforms (26,27). PLD 2 is a constitutively active isoform of PLD that is localized primarily along the plasma membrane (9). The potent inhibition of PLD 2 by synucleins in vitro (6) suggests that its low basal activity in vivo may be a result of tonic inhibition by synucleins. Overexpression of PLD 2 in cell culture induces endocytosis coincident with a redistribution of PLD 2 from plasma membrane to submembrane vesicles and simultaneously induces actin polymerization, which is characteristic of the endocytotic phase of synaptic transmission (9). Transient disinhibition of post-synaptic PLD 2 after cholinergic stimulation would accomplish several requirements of clathrinmediated receptor endocytosis. Phospholipase D has been implicated in several steps of endocytosis, including recruitment of coat assembly proteins (28,29). The observation that the recruitment of adaptin protein-2 to plasma membrane prior to endocytosis does not require the GTPase ADP-ribosylation factor, unlike the PLD 1 -controlled recruitment of adaptin protein-1 to the Golgi network, led to the speculation that adaptin protein-2 attachment to plasma membrane may be under the control of a "constitutively active phospholipase D" (30), despite the fact that one had not been discovered at the time.
Membrane-bound disinhibited PLD 2 is also well positioned to hydrolyze plasma membrane phosphatidylcholine (PC) at the endocytotic vesicle point of attachment to plasma membrane, thus releasing the vesicle into cytosol after it is closed at its base by the GTPase dynamin (31). Dynamin does not pinch off the vesicle, and at present, the mechanism for vesicle detachment is not known. Membrane lysis by PLD 2 -mediated PC hydrolysis could provide an additional advantage of liberating free choline, approximately half of which is lost during cholinergic neurotransmission. Cholinergic stimulation of membrane FIG. 8. Effect of carbachol stimulation on the cytoplasmic S 2 and vesicle V 2 compartments. a, as shown in 7b also, both oligomers and monomers increase in the S 1 fraction in response to carbachol stimulation. c, in contrast, the ␣-synuclein oligomer signal from the cytoplasmic S 2 fraction is almost undetectable in the control lane (C) and increases only slightly with carbachol stimulation (probably reflecting the presence of unsedimented light vesicles). The monomer, however, increases as it does in the S 1 fraction. The ␣-synuclein oligomer in the V 2 fraction (d), in contrast with that in the V 1 fraction (b), shows a clear increase in response to carbachol stimulation. The V 2 fraction includes the light vesicle fraction that remains in the cytoplasmic S 1 fraction.
FIG. 9. Carbachol-induced translocation of ␣-synuclein from plasma membrane to a light vesicle fraction in cytoplasm. The translocation of the ␣-synuclein oligomer from plasma membrane (a) to the light vesicle fraction was typically more distinct when this fraction remained in the supernatant S 1 (b), where its appearance emerged in contrast to a typically low basal signal, than when it was sedimented into the V 2 fraction (compare with Fig. 8d) and the carbacholinduced increase appeared against a higher basal signal of the ␣-synuclein oligomer. The vesicle fraction V 1 (c) remains unaltered.
PC hydrolysis by PLD has been proposed by several groups (27,32,33) as a source of free choline for acetylcholine synthesis. The proposed modulation of cholinergic receptor endocytosis by the transient release of PLD 2 from ␣-synuclein inhibition suggests a compact and parsimonious sequence of linked events for processes occurring during cholinergic neurotransmission.
The present data do not resolve the issue of whether the ␣-synuclein oligomer remains associated with membrane throughout the entire process of endocytotic vesicle formation or is released from plasma membrane before reattaching to the endocytotic vesicle. Several lines of evidence from our laboratory and others favor a model in which muscarinic stimulation induces the release of ␣-synuclein from plasma membrane, with a concomitant conversion of the oligomer to soluble monomers prior to a reattachment and oligomerization of ␣-synuclein at the endocytotic vesicle. We observed a carbachol-induced increase in the soluble monomer in the cytoplasmic fraction that is consistent with a muscarinic receptor-stimulated cleavage of lipid-bound ␣-synuclein oligomer and release of the soluble monomer into cytosol (Figs. 7-9). The recent findings that G-protein receptor kinases phosphorylate synucleins and reduce their binding affinity for phospholipid (34) also support the hypothesis that ␣-synuclein is first released from plasma membrane before rebinding to the endocytotic vesicle membrane in response to muscarinic stimulation. Furthermore, Marateaux and Scheller (4) noted that a 45-kDa form of ␣-synuclein was converted to the 19-kDa form by stimulation of phospholipase C, the lipase coupled to muscarinic receptors. Taken together these data suggest that the carbachol-stimulated increase in the cytoplasmic monomer may represent a transition state for ␣-synuclein in its translocation from plasma membrane to uncoated endocytotic vesicles.
Carbachol stimulation induced changes in ␣-synuclein in the nucleus, as well as in the cytoplasmic and membrane fractions. The most consistent change was a decrease in the ␣-synuclein monomer over the 30 min of stimulation (Fig. 11a). It is unclear whether this decrease contributes to the increase in the cytoplasmic monomer. Although there were no consistent changes in the 45-kDa species, two experiments showed a biphasic effect of carbachol stimulation, with a transient decrease at 10 min and a return to a level greater than the basal value by 30 min (Fig. 11b). This effect, which was particularly pronounced in Fig. 11b, was accompanied by the appearance of a strong signal between 30 and 36 kDa at the 10-min time point. A signal at this molecular weight was detected frequently in the nuclear and cytoplasmic (e.g. Fig. 4, c and d) fractions, although the intensity of the signal was typically weak compared with the other two ␣-synuclein signals. The apparent molecular weight of this novel species corresponds roughly to an ␣-synuclein dimer and gives support to the hypothesis that ␣-synuclein can be interconverted between polymeric and monomeric states in response to carbachol stimulation. Although the present data do not suggest what the function of ␣-synuclein in the nucleus is, there are numerous reports of nuclear phosphatidylinositol, phospholipases, and related proteins, including phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol triphosphate, PI 3-kinase, and phosphatidylinositol triphosphate-binding protein (35)(36)(37)(38)(39)(40)(41)(42). The presence of nuclear phospholipids and related proteins demonstrates that phosphatidylinositol-linked activities occur in nucleus. ␣-Synuclein may play a role in regulating processes in the PI-cycle in the nucleus as it appears to in cytoplasm and plasma membrane.
In conclusion, we have determined that endogenous ␣-synuclein in the human SY5Y cell exists in at least two normal states, a soluble monomer and an oligomer of ϳ45 kDa that is exclusively associated with lipid membranes. This distinctive subcellular distribution of ␣-synuclein suggests that it is the oligomer that mediates all membrane-linked functions of ␣-synuclein. The present data support a model in which the ␣-synuclein oligomer functions in vivo to inhibit PLD 2 at the plasma membrane, consistent with the synuclein inhibition of this PLD isoform in vitro. In response to carbachol stimulation, the ␣-synuclein oligomer translocates from plasma membrane to a light vesicle fraction in cytoplasm with a time course and subcellular localization corresponding to that of muscarinic receptors during ligand-stimulated endocytosis. The data suggest that muscarinic stimulation triggers the release of the ␣-synuclein oligomer from plasma membrane and transiently disinhibits PLD 2 , freeing this lipase to mediate several processes of muscarinic receptor endocytosis.