The Protein Complex of Neurodegeneration-related Phosphoinositide Phosphatase Sac3 and ArPIKfyve Binds the Lewy Body-associated Synphilin-1, Preventing Its Aggregation

Abstract

The 5-phosphoinositide phosphatase Sac3, in which loss-of-function mutations are linked to neurodegenerative disorders, forms a stable cytosolic complex with the scaffolding protein ArPIKfyve. The ArPIKfyve-Sac3 heterodimer interacts with the phosphoinositide 5-kinase PIKfyve in a ubiquitous ternary complex that couples PtdIns(3,5)P2 synthesis with turnover at endosomal membranes, thereby regulating the housekeeping endocytic transport in eukaryotes. Neuron-specific associations of the ArPIKfyve-Sac3 heterodimer, which may shed light on the neuropathological mechanisms triggered by Sac3 dysfunction, are unknown. Here we conducted mass spectrometry analysis for brain-derived interactors of ArPIKfyve-Sac3 and unraveled the α-synuclein-interacting protein Synphilin-1 (Sph1) as a new component of the ArPIKfyve-Sac3 complex. Sph1, a predominantly neuronal protein that facilitates aggregation of α-synuclein, is a major component of Lewy body inclusions in neurodegenerative α-synucleinopathies. Modulations in ArPIKfyve/Sac3 protein levels by RNA silencing or overexpression in several mammalian cell lines, including human neuronal SH-SY5Y or primary mouse cortical neurons, revealed that the ArPIKfyve-Sac3 complex specifically altered the aggregation properties of Sph1-GFP. This effect required an active Sac3 phosphatase and proceeded through mechanisms that involved increased Sph1-GFP partitioning into the cytosol and removal of Sph1-GFP aggregates by basal autophagy but not by the proteasomal system. If uncoupled from ArPIKfyve elevation, overexpressed Sac3 readily aggregated, markedly enhancing the aggregation potential of Sph1-GFP. These data identify a novel role of the ArPIKfyve-Sac3 complex in the mechanisms controlling aggregate formation of Sph1 and suggest that Sac3 protein deficiency or overproduction may facilitate aggregation of aggregation-prone proteins, thereby precipitating the onset of multiple neuronal disorders.

Keywords: ArPIKfyve-Sac3 complex; Parkinson disease; Synphilin-1; aggregation; aggresome; neurodegenerative disease; neuron; phosphatidylinositol phosphatase; phospholipid; proteasome.

FIGURE 1.

ArPIKfyve-Sac3 complex interacts with Sph1. A, ArPIKfyve-HEK cells were transfected with Sph1-GFP alone or together with Myc-Sac3 when expression of HA-ArPIKfyve was induced (i). Twenty-four h post-transfection/induction, the cells were scraped in RIPA⁺ buffer. Cleared lysates were immunoprecipitated (IP) with purified anti-HA IgG and processed by SDS-PAGE and Western blotting (WB) as specified under “Experimental Procedures.” Shown are chemiluminescence detections of representative blots of three with similar results depicting the three proteins in input lysates and immunoprecipitations. Note the similar amounts of IP ArPIKfyve and that the band of Sph1-GFP (detected by anti-GFP and anti-Sph1 antibodies) is coimmunoprecipitated only when Sac3 is coexpressed/coimmunoprecipitated. B, RIPA⁺ lysate collected from SH-SY5Y neuroblastoma cells were immunoprecipitated with anti-ArPIKfyve or control anti-GDI2 antibodies, affinity-purified on an ArPIKfyve C-terminal peptide and His₆-GDI2 protein, respectively. Immunoprecipitates were processed for SDS-PAGE and Western blotting with a stripping step between the indicated antibodies. Shown are chemiluminescence detections of representative blots of two with similar results, demonstrating specific bands corresponding to Sac3 and Sph1 electrophoretic mobilities (arrowheads), coimmunoprecipitated with anti-ArPIKfyve only but not with GDI2 antibodies. C, in vitro associations of purified recombinant His₆-ArPIKfyve-Sac3 with immunopurified Sph1-Myc. Sph1-Myc and a control, Myc-GDI2, immunopurified on the anti-Myc antibody from RIPA⁺ lysates of transfected COS7 cells and immobilized on protein A-Sepharose CL-4B (PrA) beads were incubated with the His₆-ArPIKfyve/Sac3 complex, purified from infected Sf21 cells on nickel-nitrilotriacetic acid-agarose. PrA beads alone were incubated in parallel. After washing, beads were subjected to SDS-PAGE and Western blotting. Nitrocellulose membranes were probed with anti-Myc and, after a horizontal cut through the middle, with anti-ArPIKfyve and anti-Sac3 antibodies (lower panels) with a stripping step in between. Shown are chemiluminescence detections of representative blots of three separate experiments, illustrating efficient pulldown of the purified ArPIKfyve-Sac3 recombinant complex by Sph1-Myc but not by Myc-GDI2 or protein A-Sepharose CL-4B.

FIGURE 2.

Deficiency of ArPIKfyve and/or Sac3, but not PIKfyve, facilitates aggregation of Sph1-GFP in non-neuronal and neuronal cells. A and B, HEK293 cells were transfected with the indicated siRNA duplexes. Forty-eight h post-siRNAs, cells were transfected with cDNA of Sph1-GFP and 24 h later were fixed and processed for fluorescence microscopy as described under “Experimental Procedures.” Presented are typical fluorescence microscopy images of Sph1-GFP-positive cells treated with the indicated siRNAs (A); the data quantitation is expressed as a percentage of transfected cells with Sph1-GFP aggregates (B). The percentage of cells with multiple cytoplasmic aggregates was determined by viewing >100 GFP-positive cells from at least three random fields/condition in three separate experiments. C–E, mouse cortical neurons, prepared from E18 mice and cultured on poly-d-lysine-coated glass coverslips for 3 days, were transfected with the indicated siRNA duplexes by RNAiMax as described under “Experimental Procedures.” Forty-eight h later, neuronal cultures were transfected with cDNA of Sph1-GFP. Twenty-four to 48 h post-cDNA transfection, the neurons were fixed and processed for immunofluorescence with anti-MAP2 monoclonal, as a primary antibody, and anti-mouse Alexa Fluor 568 as a secondary antibody. Shown are representative fluorescence/immunofluorescence microscopy images of Sph1-GFP-expressing cells, for which the dendrites and soma are visualized by anti-MAP2 (C); and the quantitation of the Sph1-GFP aggregation under different conditions as a percentage of the transfected neurons shows depicting multiple small aggregates in soma and dendrites, determined by viewing >25 GFP-positive cells/condition in three separate experiments (D). Note that Sph1-GFP is predominantly diffuse in control neurons but forms multiple small aggregates under Sac3 depletion. Total RNA was harvested and analyzed by quantitative RT-PCR for Sac3 mRNA expression as described under “Experimental Procedures” (n = 3, p < 0.001) (E). F–H, SH-SY5Y neuroblastoma were transfected with the indicated siRNA duplexes. Forty-eight h later, cells were transfected with cDNA of Sph1-GFP. Twenty-four h post-cDNA transfection, cells were either fixed for fluorescence microscopy (F) or lysed for Western blotting analysis (H) as described under “Experimental Procedures.” Shown are representative fluorescence microscopy images of Sph1-GFP-positive cells treated with the indicated siRNAs, and the corresponding phase image, captured by Hoffman modulation contrast (F). Quantitation of the Sph1-GFP aggregation demonstrates a significant increase under Sac3 depletion (G). Chemiluminescence detection of a representative blot (in duplicates) of two separate experiments with similar results indicates marked decreases in endogenous Sac3 levels by Sac3-siRNA; the equal intensities of the unspecific bands (Unsp.) above Sac3 attest to equal loading (H). The percentage of cells with multiple small cytoplasmic aggregates was determined by viewing >50 GFP-positive cells from at least three random fields/condition in three separate experiments. *, significant increase in the proportion of cells with multiple small Sph1-GFP aggregates versus control (p < 0.05). Bar, 10 μm.

FIGURE 3.

Co-expression of Sac3 with ArPIKfyve^WT, but not with ArPIKfyve mutant unable to bind Sac3, reduces aggregation of Sph1-GFP. COS7 cells were transfected with Sph1-GFP cDNA alone or together with cDNAs of Myc-Sac3^WT and either HA-ArPIKfyve^WT or HA-ArPIKfyve-(1–511) as detailed under “Experimental Procedures.” On the next day, cells were fixed and immunostained consecutively with anti-Myc and anti-HA antibodies to visualize Myc-Sac3 and HA-ArPIKfyve^WT or HA-ArPIKfyve-(1–511) proteins, respectively. A, representative images of singly Sph1-GFP-expressing cells with multiple small aggregates (a) or a triply Sph1-GFP/Myc-Sac3/HA-ArPIKfyve^WT (b–d)-expressing cell showing diffuse Sph1-GFP staining. Bar, 10 μm. B, quantitation of cells with multiple small aggregates of Sph1-GFP by viewing more than 100 GFP-positive cells from at least three random fields from three independent experiments, indicating reduced aggregation only in the presence of coexpressed Myc-Sac3-HA-ArPIKfyve^WT. *, p < 0.05.

FIGURE 4.

Elevation of ArPIKfyve-Sac3 complex decreases aggregation of Sph1-GFP in ArPIKfyve-HEK cells. ArPIKfyve-HEK cells were transfected with cDNA of Sph1-GFP alone or together with that of Myc-Sac3 when HA-ArPIKfyve expression was induced (i) or not induced. On the next day cells were either processed for immunofluorescence microscopy (A and B) or underwent protein fractionation to obtain total protein, Triton X-100-soluble, and Triton X-100-insoluble fractions (C and D) as detailed under “Experimental Procedures.” A, following fixation and permeabilization, cells were stained with anti-Myc and observed by immunofluorescence microscopy. Shown are typical images of cells with multiple small aggregates by single expression of Sph1-GFP (a) or with diffuse distribution of Sph1-GFP if Myc-Sac3 and HA-ArPIKfyve were coexpressed (b and c). Bar, 10 μm. B, quantitation of Sph1-GFP aggregates under these conditions, presented as a percentage of cells with multiple cytoplasmic aggregates as determined by viewing more than 100 GFP-positive cells from at least three random fields/condition. *, different versus singly Sph1-GFP-transfected cells, p < 0.05. Note that expression of HA-ArPIKfyve alone did not significantly alter the percentage of cells with multiple aggregates of Sph1-GFP. C, transfected ArPIKfyve-HEK cells were treated with Triton X-100 in PBS to extract the soluble proteins and then scraped in RIPA⁺ buffer + 0.1% SDS to collect the insoluble proteins. Cells from a duplicate dish were scraped in RIPA⁺/0.1% SDS to recover total proteins. Equal volumes of the respective fractions were analyzed by Western blotting. Shown are chemiluminescence detections of representative blots illustrating that the partitioning of Sph1-GFP in the insoluble fraction was diminished when coexpressed with Myc-Sac3 and HA-ArPIKfyve. Note that levels of total Sph1-GFP were identical between the singly and triply expressing cells. D, relative quantitation by densitometry from three separate Western blots with similar results. *, p < 0.05.

FIGURE 5.

Elevation of ArPIKfyve-Sac3 complex fails to alter aggregation of GFP-GFAP. A and B, COS7 cells were transfected with cDNA of GFP-GFAP alone (a), in a double combination with cDNA of HA-ArPIKfyve or in a triple combination with cDNAs of HA-ArPIKfyve and Myc-Sac3 (b–d). Twenty-four h post-transfection, cells were fixed and processed for immunofluorescence microscopy as detailed under “Experimental Procedures.” Shown are representative cells with multiple small aggregates of GFP-GFAP in singly transfected cells (a) which persisted in triply transfected cells (b–d), whereas Myc-Sac3 (c) and HA-ArPIKfyve (d) remained diffuse, and quantitative analysis (B) of fluorescence microscopy images by monitoring >100 cells/condition in random fields in two separate experiments. C and D, ArPIKfyve-HEK cells were transfected with GFP-GFAP cDNA alone (a) or together with Myc-Sac3 cDNA in the presence of HA-ArPIKfyve induction (i) (b and d). Note the presence of GFP-GFAP aggregates (b), despite overexpression of Myc-Sac3 and HA-ArPIKfyve in the iArPIKfyve-HEK cells (c). Quantitative analysis (D) of fluorescence microscopy images by monitoring >100 cells/condition on random fields in two separate experiments. Bar, 10 μm.

FIGURE 6.

Aggresome formation of Sph1-GFP under proteasome inhibition is reduced by elevation of ArPIKfyve-Sac3. A and B, 24 h post-transfection with the indicated cDNAs, COS7 cells were treated with MG132 (5 μm) for 5 h, fixed, and processed for immunofluorescence microscopy. A, representative images illustrating the appearance of large single perinuclear aggresomes independent of the expression level of singly transfected Sph1-GFP (a) and the typical diffuse appearance of Sph1-GFP (b) in the presence of Myc-Sac3 (c) and HA-ArPIKfyve (d) in triply transfected cells. Arrowheads in b and c point to the aggresome formed when Myc-Sac3 is expressed without HA-ArPIKfyve. B, percentage of cells with Sph1-GFP aggresomes from three separate experiments. Note that HA-ArPIKfyve alone does not affect Sph1-GFP aggresome formation. *, p < 0.05. C, HA-ArPIKfyve-HEK cells were transfected with cDNA of Sph1-GFP alone or with cDNA of Myc-Sac3 in the presence or absence of induced HA-ArPIKfyve expression. Twenty-four h later, cells were treated with MG132 (5 μm) for 5 h and then processed for immunofluorescence microscopy. Shown is quantitation of cells with a single aggresome presented as the percentage of transfected Sph1-GFP. *, p < 0.05. D and E, aggresome formation of HA-Sac3 under proteasome inhibition is reduced by elevation of ArPIKfyve. D, HEK293 cells were transfected with cDNA of HA-Sac3 alone (a and b) or together with Myc-ArPIKfyve cDNA (c and d). Twenty-four h post-transfection cells were treated with MG132 (5 μm) for 5 h in the presence or absence of nocodazole (NOC, 5 μm) as indicated. Note the reduced aggresome formation and the diffuse distribution of HA-Sac3 by co-expression of Myc-ArPIKfyve (c and d). Bar, 10 μm. E, quantitation of the proportion of transfected cells with a single aggresome or multiple aggregates of HA-Sac3 and the fold decrease by Myc-ArPIKfyve. *, different versus single HA-Sac3 transfection (p < 0.05).

FIGURE 7.

Elevation of ArPIKfyve-Sac3 complex fails to reduce aggregation of Sph1-GFP under inhibition of basal autophagy. ArPIKfyve-HEK cells were transfected with cDNA of Sph1-GFP alone or together with that of Myc-Sac3 and then induced (i) or not induced to express HA-ArPIKfyve as indicated. Twenty-four h later, cells were treated without or with 3-MA (10 mm) in a complete 10% FBS-containing medium. Twenty-four h post-treatment, cells were fixed and processed for fluorescence/immunofluorescence microscopy with the anti-Myc/Alexa Fluor 568 anti-mouse as primary/secondary antibody as described under “Experimental Procedures.” Representative fluorescence images of singly expressed Sph1-GFP with and without 3-MA treatment show the significantly increased proportion of cells with multiple aggregates by 3-MA (A), and the relative fold change in the number of Sph1-GFP-transfected cells containing multiple small aggregates upon treatment with 3-MA under different conditions is quantitated (B). Quantitation is based on counting >100 cells/condition in three separate experiments. *, different versus single Sph1-GFP transfection without 3-MA (p < 0.05). Bar, 10 μm.

FIGURE 8.

Reduced aggregation of Sph1-GFP by ArPIKfyve-Sac3 requires active Sac3 phosphatase. ArPIKfyve-HEK cells were co-transfected with cDNAs of Sph1-GFP and either HA-Sac3^WT or the phosphatase inactive mutant HA-Sac3^D488A when ArPIKfyve expression was induced (i). On the next day, iArPIKfyve-HEK cells were fixed, permeabilized, and stained with affinity-purified anti-Sac3 antibodies and CY3-conjugated anti-rabbit secondary antibody as described under “Experimental Procedures.” A, shown are representative images of iArPIKfyve-HEK cells co-transfected with HA-Sac3^WT and Sph1-GFP (a and b) or co-transfected with HA-Sac3^D488A and Sph1-GFP (c and d). Note the diffuse Sph1-GFP distribution with coexpressed HA-Sac3^WT but multiple small aggregates with coexpressed HA-Sac3^D488A (arrowheads). Bar, 10 μm. B, the proportion of Sac3^WT/Sac3^D488A-positive cells with multiple small Sph1^WT-GFP aggregates was determined by counting >100 doubly transfected cells from random fields per condition from three separate experiments, and presented as the relative fold difference. *, p < 0.05.

FIGURE 9.

Coexpressed Sph1 and Sac3 potentiate each other's aggregation in a manner reversible by ArPIKfyve. ArPIKfyve-HEK cells were co-transfected with cDNAs of Sph1-GFP and Myc-Sac3. Twenty-four h post-transfection, HA-ArPIKfyve expression was induced where indicated. On the next day cells were processed for fluorescence microscopy (A–C) or underwent protein fractionation to obtain Triton X-100-soluble and -insoluble fractions (D) as detailed under “Experimental Procedures.” A and B, quantitation of the fluorescence microscopy indicating the percentage of cells with multiple aggregates of Sph1-GFP and/or Myc-Sac3 determined by viewing >100 doubly transfected cells/condition in three separate experiments. Note the marked increase of cells with multiple aggregates of Sph1-GFP and Myc-Sac3 in the co-transfected cells (#, different versus singly transfected cells, p < 0.05) and the significant diminution of aggregates by both proteins upon ArPIKfyve expression (*, different versus singly and doubly transfected cells, p < 0.05). C, confocal microscopy of co-transfected Sph1-GFP/Myc-Sac3 non-induced cells performed as detailed under “Experimental Procedures.” Representative images illustrating three typical observations: a–c, individual Sph1-GFP-positive small aggregates with no colocalization with Myc-Sac3 as apparent by the green aggregates in the merged images and inset in c. d–f, coalescence of some aggregates formed by Sph1-GFP (d, small arrowheads) and Myc-Sac3 (e, arrowheads) as apparent by the yellow aggregates in the merged image and inset in f. g–i, Myc-Sac3 inclusions forming rings surrounding aggregates by Sph1-GFP as evident by the presence of yellow as well as separate green and red in the merged image and inset in i. Bar, 10 μm. D, cells were treated with Triton X-100 to extract the soluble proteins and then scraped in RIPA⁺/0.1% SDS to collect the insoluble proteins. An equal volume of the respective fraction was analyzed by Western blotting. Shown are chemiluminescence detections of representative blots illustrating that HA-ArPIKfyve expression diminished Sph1-GFP and Myc-Sac3 levels in the Triton X-100-insoluble fraction but increased these levels in the Triton X-100-soluble fraction.

FIGURE 10.

Functional significance of the ArPIKfyve-Sac3 complex interactions. A, Sac3 is stabilized in the cytosol by binding ArPIKfyve and forming the ArPIKfyve-Sac3 (ArS) complex (6). A subfraction of the cytosolic ArPIKfyve-Sac3 complex is recruited to endosomal membranes where it interacts with PIKfyve (PAS) to facilitate the synthesis and turnover of PtdIns(3,5)P₂ (1, 2) or forms a triple complex with Sph1 in the cytosol (SAS, Sac3-ArPIKfyve-Sph1; this study). B, proposed mechanisms by which the ArS complex reduces aggregate formation by Sph1. Through direct physical interaction, ArPIKfyve stabilizes and solubilizes Sac3, which prevents Sac3 proteasome degradation and aggregation. The ArS complex interacts with Sph1 in the cytosol preventing misfolding, aggregation, and aggresome formation upon proteolytic stress of Sph1 (a). Formation of multiple small aggregates of Sph1 is attenuated upon interaction with the ArS complex and the formation of a soluble SAS complex (b) or by clearance through constitutive autophagy facilitated by the ArS complex (c). A single large aggresome of Sph1 formed by proteasome inhibition can be removed by induced autophagy (42). The effect of the ArS complex in this step has not been examined.