Evidence That R-Synuclein Does Not Inhibit Phospholipase D

R-Synuclein (RSyn) is a small cytosolic protein of unknown function, which is highly enriched in the brain. It is genetically linked to Parkinson’s disease (PD) in that missense mutations or multiplication of the gene encoding RSyn causes early onset familial PD. Furthermore, the neuropathological hallmarks of both sporadic and familial PD, Lewy bodies and Lewy neurites, contain insoluble aggregates of RSyn. Several studies have reported evidence that RSyn can inhibit phospholipase D (PLD), which hydrolyzes phosphatidylcholine to form phosphatidic acid and choline. Although various hypotheses exist regarding the roles of RSyn in health and disease, no other specific biochemical function for this protein has been reported to date. Because PLD inhibition could represent an important function of RSyn, we sought to extend existing reports on this interaction. Using purified proteins, we tested the ability of RSyn to inhibit PLD activity in cell-free assays. We also examined several cell lines and transfection conditions to assess whether RSyn inhibits endogenous or overexpressed PLD in cultured mammalian cells. In yeast, we extended our previous report of an interaction between RSyn and PLD-dependent phenotypes, for which PLD activity is absolutely necessary. Despite testing a range of experimental conditions, including those previously published, we observed no significant inhibition of PLD by RSyn in any of these systems. We propose that the previously reported effects of RSyn on PLD activity could be due to increased endoplasmic reticulum-related stress associated with RSyn overexpression in cells, but are not likely due to a specific and direct interaction between RSyn and PLD. Parkinson’s disease (PD) is a debilitating neurodegenerative disorder that affects over one million people in the United States (for review, see ref 1). Although more than 90% of PD cases are believed to occur sporadically, several genes are known to cause familial forms of PD. Among these, point mutations or increased dosage of the R-synuclein (RSyn) gene are known to cause rare cases of early onset familial PD (2-7). The RSyn protein is also the major component of Lewy bodies and Lewy neurites, the insoluble aggregates that are neuropathological hallmarks of both familial and sporadic PD (8). Thus, dysregulation of RSyn at either the genetic or protein level can contribute to PDtype neurodegeneration. RSyn is a small, highly conserved cytosolic protein of unknown function. It is enriched in the brain, particularly at presynaptic terminals. Proposed functions of RSyn include lipid binding, regulation of membrane composition, and regulation of neurotransmitter release and/or of the reserve pool of synaptic vesicles (9-15). Given the hypothesis that loss of axonal terminals in the striatum may precede the death of nigral neurons in PD (1, 16), these observations suggest that the dysfunction of RSyn at the synapse could be an early event in the pathogenesis of PD. Phospholipase D (PLD) is a membrane-associated enzyme that hydrolyzes phosphatidylcholine to form phosphatidic acid (PA) and choline. PA is an essential metabolic intermediate and an intracellular signaling molecule that can be further hydrolyzed into diacylglycerol, another important signaling molecule (for review, see ref 17). Mammalian cells express two PLD isoforms, PLD1 and PLD2. They are regulated by distinct cellular mechanisms but perform a similar hydrolytic reaction. In cell-free systems, PLD1 requires an activator such as Rho or ARF GTPases, while PLD2 is highly active in these systems (18, 19). The precise function of PLD in the cell has not yet been determined, though proposed functions include roles in exocytosis, endocytosis, and intracellular signaling (16, 20, 21). † This work was supported by the Brigham and Women’s Hospital Udall Center for Excellence in Parkinson’s Disease, NIH/NINDS Grant NS038375 (D.J.S.). * To whom correspondence should be addressed. D.J.S.: Center for Neurologic Diseases, Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, HIM 730, Boston, MA 02115. Phone: 617-525-5200. Fax: 617525-5252. E-mail: [email protected]. S.L.: Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA02142.Phone:617-258-5184.Fax:617-258-7226.E-mail: lindquist@ wi.mit.edu. ‡ Department of Neurology, Harvard Medical School and Brigham & Women’s Hospital. § Whitehead Institute and Massachusetts Institute of Technology. | Current address: Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA. ⊥ Vanderbilt University Medical Center. # Department of Internal Medicine, Harvard Medical School and Brigham & Women’s Hospital. 1 Abbreviations: RSyn, R-synuclein; Syn, -synuclein; BSA, bovine serum albumin; ER, endoplasmic reticulum; hPLD1b, human phospholipase D 1b; hPLD2a, human phospholipase D 2a; PD, Parkinson’s disease; PA, phosphatidic acid; PBut, phosphatidylbutanol; PLD, phospholipase D; PMA, phorbol-12-myristate-13-acetate. Biochemistry 2009, 48, 1077–1083 1077 10.1021/bi801871h CCC: $40.75  2009 American Chemical Society Published on Web 01/15/2009 In 1998, in Vitro studies suggested that RSyn and its close family member, -synuclein ( Syn), could act as inhibitors of PLD2 (22). Three other laboratories extended these findings over the next six years, suggesting that RSyn can inhibit PLD activity in mammalian cells (23), in yeast (24), and in cell-free assays (25). Although several hypotheses exist regarding the physiological and pathological roles of RSyn in the brain, no other specific biochemical function for RSyn has been proposed to date. Furthermore, if PLD regulation is a physiological function of RSyn, then PLD dysregulation due to gain or loss of RSyn function could be an early event in the pathogenesis of PD and other synucleinopathies. Here, we sought to further explore the mechanism of the interaction between RSyn and PLD. We used cell-free and cell-based systems to directly assay PLD activity in the presence or absence of RSyn. In yeast, we extended our previously published genetic findings on RSyn inhibition of PLD. These various approaches did not yield evidence that RSyn significantly inhibits PLD. Based on our data, as well as previous work showing that RSyn expression can induce cytotoxicity associated with endoplasmic reticulum (ER) stress, we suggest that the previously published effects of RSyn on PLD activity may be nonspecific or attributable to general ER stress, rather than a direct and physiological inhibition of PLD by the RSyn protein. EXPERIMENTAL PROCEDURES Materials. Chromatographically enriched human PLD1b (hPLD1b) and 6-his-tagged human PLD2a (hPLD2a) were generated as previously described (26, 27) and tested for activity using an exogenous substrate assay (17, 28). Recombinant RSyn protein was generously provided by P. T. Lansbury (29) and by J. M. George (25). Following resuspension in phosphate buffered saline, the protein was filtered through a 0.22 μm syringe filter followed by a 100,000 MWCO centrifugal filter. The filtered solution was probed by SDS-PAGE and Coomassie stain to confirm the monomeric nature of the recombinant protein preparation. Lipids, including phosphatidylbutanol, were purchased from Avanti Polar Lipids (Alabaster, AL). 3H-Oleic acid was purchased from PerkinElmer Life Science Products (Newtown, CT). The RSyn expression plasmid was provided by R. Sharon. Unless otherwise stated, all other reagents were purchased from Sigma-Aldrich (St. Louis, MO). Cell-Based PLD ActiVity Assay. Endogenous PLD activity was measured as described previously (30). Briefly, 24 h before the assay, HEK 293, HeLa, or undifferentiated PC12 cells were transfected as indicated using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA). Cells were labeled with 3H-oleic acid (10 μCi/mL) overnight in serum-free medium supplemented with 0.25 mg/mL fatty acid free bovine serum albumin (BSA). On the day of the assay, some cells were preincubated for 5 min in serum-free medium containing the PLD inhibitor VU0155056 (2 μM) (27). Cells were then stimulated for 30 min with serum-free medium containing 1 μM phorbol-12-myristate-13-acetate (PMA) with 0.3% butanol and/or VU0155056, as indicated. Lipids were extracted using chloroform:methanol:HCl 0.1 N (1:1:1), then spotted onto silica-coated TLC plates. The plates were developed in chloroform:methanol:acetic acid:acetone:water (50:10:10:20:5), and the radioactivity was visualized on autoradiographic film with a Kodak Transcreen LE enhancing screen, scanned, and quantified using Quantity One software (BioRad Laboratories, Hercules, CA). A phosphatidylbutanol standard was run on each plate and visualized with sublimed iodine (17). Cell-Free PLD ActiVity Assay. PLD activity was assayed as described previously (28). Briefly, purified hPLD2a (15 nM final concentration) was added to reaction buffer (50 mM HEPES pH 7.5, 80 mM KCl, 3 mM EGTA, 0.1 mM DTT, 4.5 mM MgCl2, 4.5 mM CaCl2, 10 μM GTPγS) containing lipid vesicles and 3H-PC (total DPPC:POPE:PI(4,5)P2:cholesterol at a molar ratio of 10:100:6.2:1.4) in a total volume of 60 μL per assay tube. Various concentrations of RSyn were added to some reactions as indicated, and the PLD inhibitor VU0155056 (20 μM) was added to other reactions as a control. Reactions were incubated at 37 °C for 30 min, then terminated with 200 μL trichloroacetic acid (10% v/v) and 100 μL BSA (10% w/v). Free 3H-choline released by hPLD2a activity was measured by scintillation counting. Similar assays were performed using purified hPLD1b. Yeast Plasmids and Strains. The sec14-1, cki1∆, and sec14-1 cki1∆ strains were described previously (24). Here, we generated the sec14-1 cki1∆ spo14∆ strain by replacing SPO14 with a KanMX cassette by homologous recombination in the sec14-1 cki1∆ strain. Colony PCR was used to verify the gene disruption. A SPO14 entry clone (pDONR221) and the Advanced Gateway destination vectors pAG413GPDccdB or pAG416GPD-ccdB (31) were used in a Gateway LR reaction (Invitrogen) to construct pAG413GPD-SPO14 and pAG416GPD-SPO14, respectively. To construct the pAG413GPD-RSyn plasmid, an RSyn entry clone was used in a Gateway LR reaction with the pAG413GPD-ccdB destination vector. Yeast Transformation and Spotting Assays. Yeast cultures were maintained according to standard protocols (32). We used the PEG/lithium acetate method to transform yeast with DNA (33). Yeast cells carrying the RSyn and/or SPO14 plasmids were grown overnight at 30 °C in liquid media containing glucose until they reached log or midlog phase. Cultures were then normalized for OD600, serially diluted and spotted onto synthetic solid media containing glucose and lacking either uracil (for pAG416GPD plasmids) or histidine (for pAG413GPD plasmids) and were grown at 23 °C or 37 °C for 2-3 days. Statistical Analyses. For each assay, at least three independent experiments were conducted, each in triplicate. Results were analyzed by one-way ANOVA with Tukey’s post-hoc tests, as indicated in the figure legends.