α-Synuclein facilitates endocytosis by elevating the steady-state levels of phosphatidylinositol 4,5-bisphosphate

α-Synuclein (α-Syn) is a protein implicated in the pathogenesis of Parkinson’s disease (PD). It is an intrinsically disordered protein that binds acidic phospholipids. Growing evidence supports a role for α-Syn in membrane trafficking, including, mechanisms of endocytosis and exocytosis, although the exact role of α-Syn in these mechanisms is currently unclear. Here we have investigated the role of α-Syn in membrane trafficking through its association with acidic phosphoinositides (PIPs), such as phosphatidylinositol 4,5-bisphosphate (PI4,5P2) and phosphatidylinositol 3,4-bisphosphate (PI3,4P2). Our results show that α-Syn colocalizes with PIP2 and the phosphorylated active form of the clathrin adaptor AP2 at clathrin-coated pits. Using endocytosis of transferrin, an indicator of clathrin mediated endocytosis (CME), we find that α-Syn involvement in endocytosis is specifically mediated through PI4,5P2 levels. We further show that the rate of synaptic vesicle (SV) endocytosis is differentially affected by α-Syn mutations. In accord with their effects on PI4,5P2 levels at the plasma membrane, the PD associated E46K and A53T mutations further enhance SV endocytosis. However, neither A30P mutation, nor Lysine to Glutamic acid substitutions at the KTKEGV repeat domain of α-Syn, that interfere with phospholipid binding, affect SV endocytosis. This study provides evidence for a critical involvement of PIPs in α-Syn-mediated membrane trafficking. Significance Statement α-Synuclein (α-Syn) protein is known for its causative role in Parkinson’s disease. α-Syn is normally involved in mechanisms of membrane trafficking, including endocytosis, exocytosis and synaptic vesicles cycling. However, a certain degree of controversy regarding the exact role of α-Syn in these mechanisms persists. Here we show that α-Syn acts to increase plasma membrane levels PI4,5P2 and PI3,4P2 to facilitate clathrin mediated and synaptic vesicles endocytosis. Based on the results, we suggest that α-Syn interactions with the acidic phosphoinositides facilitate a shift in their homeostasis to support endocytosis.


Introduction 59
α-Synuclein (α-Syn) protein is critically implicated in the pathogenesis of Parkinson's disease (PD). α-60 Syn reversibly interacts with membrane lipids. It preferentially binds acidic phospholipids (Davidson 61 et al., 1998;Stockl et al., 2008;Bodner et al., 2009) and fatty acids (Sharon et al., 2001(Sharon et al., , 2003a(Sharon et al., , 62 2003b. Upon lipid binding, the intrinsically disordered α-Syn protein acquires a a-helical structure 63 (Davidson et al., 1998). AAV1/2 particles were produced as previously described (Orenbuch et al., 2012). Briefly, HEK293T 171 cells were co-transfected with the pD1 and pD2 helper plasmids and a plasmid containing the cDNA 172 of interest located between AAV2 ITRs, preceded by the hSyn promotor. After 3 days of incubation at 173 37 °C in a humidified 5% CO2 incubator, cells were lysed in lysis solution (150 mM NaCl, 50 mM 174 Tris-HCl pH 8.5) using 3 rapid freeze-thaw cycles (in an ethanol bath chilled to -80 °C and a heated 37 175 °C water bath). The supernatant was treated with 10 units/ml benzonaze (Sigma-Aldrich, Rehovot, 176 Israel), cleared by centrifugation and filtrated through a 0.45 μm membrane. The viral particles were 177 maintained at 4°C until use. Viral titer was determined by infecting neuronal cultures, aiming for 80-178 90% infection efficiency, verified by immunofluorescence or direct fluorescence imaging, as 179 applicable. Viral titer was determined by adding 0.2-2 μl of the viral prep directly to the growth 180 medium at 5 DIV. 181 182 Lentiviral particles were produced as described ) by co-transfecting HEK 293T 183 cells with a set of three plasmids: pCMVΔR8.91; pMD2.G; and a transfer plasmid pLKO-1-puro. 184 Virus titer was determined for each preparation following transduction of cells, by quantitative PCR 185 using specific primers for Puromycin resistance gene: forward, 5'-TCACCGAGCTGCAAGAACTCT-cells were acid washed at pH 5.3 (0.2 M sodium acetate, 0.2 M sodium chloride) on ice for 1.5 min, to 218 remove surface-bound transferrin. Cells were then washed 2 additional times with ice-cold PBS, fixed 219 in 2% paraformaldehyde (PFA) for 20 min on ice and processed for ICC. 220 221 PI4,5P2 detection by the PH-PLCδ1-GFP biosensor. Cells were grown in 12-well plates, on cover 222 slides that were pre-coated with poly-D-Lysine (100 µg/ml, for 1 hour). Cells were co-transfected to 223 express PH-PLCδ1-GFP together with WT α-Syn or one of the specified α-Syn mutations ( The detection of PSer129 α-Syn levels was performed as described previously (Davidi et al., 2020). plasma membrane and colocalized with α-Syn (Fig. 1A). To assess the specificity of pAP2 signal we 303 utilized a specific inhibitor (LP-935509) for Numb-associated kinases (NAKs), that phosphorylate the was confirmed in cells that their α-Syn expression was silenced with shSNCA and treated with the LP-308 935509 inhibitor. The results show a substantial loss of both signals, α-Syn and pAP2 (Fig. 1C). 309 To confirm that α-Syn colocalizes with pAP2 on CCP, the slides were immunoreacted also with 310 antibodies against PI4,5P2 ( Fig. 1)  These results suggest that endogenous α-Syn localizes, at least in part, to PI4,5P2 / PI3,4P2-positive 328 endocytic clathrin-coated pits, consistent with a possible function in CME. 329 330 α-Syn involvement in endocytosis of transferrin associates with alterations in PIP2 levels 331 Endocytosis of fluorescently labelled transferrin was utilized as a functional readout for CME. 332 These data suggest that α-Syn regulates the levels of PI3,4P2 and PI4,5P2 phosphoinositides that 359 control CME of transferrin. We therefore decided to test the hypothesis that α-Syn increases PIP2 360 levels to enhance CME. 361 362 α-Syn-mediated endocytosis of transferrin is PI4,5P2-dependent 363 To experimentally regulate the levels of PI4,5P2, we utilized an inducible enzymatic system to acutely 364 deplete PI4,5P2 from the plasma membrane (Suh et al., 2006). This system enables rapamycin-induced 365 targeting of Inp45p, a PI4P-5-phosphatase, to the plasma membrane. HEK293T cells were transfected 366 to co-express the inducible phosphatase together either with WT α-Syn or pcDNA mock plasmid. 48 hours post DNA-transfection, cells were processed simultaneously for 568-Tf endocytosis together 368 with activation of the phosphatase with rapamycin (see methods). 369

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The Inp45p phosphatase is recruited to the plasma membrane in cells treated with rapamycin but 371 remains in the cytoplasm in DMSO-treated cells (Fig. 3A). In accord, PI4,5P2 levels were lower in 372 rapamycin (43%) compared with the DMSO treated cells (set at 100%), demonstrating phosphatase 373 activity (ICC; Fig. 3B; P<0.01; t-test). To find out whether plasma membrane PI4,5P2 levels play a 374 role in α-Syn' effect to enhance endocytosis, we quantified 568-Tf internalization in cells that co-375 express Inp45p together with WT α-Syn and treated with rapamycin or DMSO (Fig. 3A, C). The 376 results show that rapamycin-induced depletion of PI4,5P2 completely abolished the ability of 377 overexpressed α-Syn to stimulate CME (Fig. 3C), whereas CME was stimulated by α-Syn expression 378 in the DMSO-treated cells, measuring a higher degree of 568-Tf internalization (163%) compared with 379 the control cells that express a mock pcDNA (set at 100%). In control cultures, in which cells were 380 transfected and treated in parallel but without Inp45p expression, we found no effect for rapamycin on 381 α-Syn-induced CME (Fig. 3C). Mean ± SE of n=4 experiments. *, P< 0. We tested three PD-associated mutations in α-Syn, A30P, E46K and A53T, and two synthetic 406 K10,12E and K21,23E α-Syn mutation. The synthetic mutations were generated by replacing two 407 positively charged Lysine residues within the KTKEGV repeat domain, with negatively charged 408 Glutamic acid residues. In a previous study, these K to E mutations were shown to interfere with α-409 Syn binding to membrane phospholipids (Zarbiv et al., 2014). We thus concluded that α-Syn increases PIP2 levels to facilitate CME and decided to test the 427 hypothesis that it similarly acts to enhance SV endocytosis. 428 429 α-Syn accelerates the rate of SV endocytosis alongside with reducing the fraction of released SVs. 430 The involvement of α-Syn in SVs cycling was tested using Synaptophysin-pHluorin (sypHy) 431 The PD-associated mutations in α-Syn, E46K and A53T, further inhibited SV recycling (Fig. 5E) and 458 in accord, further accelerated the rate of endocytosis (Fig. 5F). However, the A30P mutation and both 459 K to E mutations were not different from control cells in their effects on SV recycling (Fig. 5 E,F). 460 Together, measurements of α-Syn effects of SVs cycling, as determined by sypHy, reveal its complex 461 effects on SV pools and architecture. However, considering the actual segment of SVs trafficking, α-462 Syn appears to accelerate the rate of endocytosis. 463

464
We next assessed PI4,5P2 levels in primary neurons, infected to express WT α-Syn or the specified 465 mutations as above. At 13 DIV, neurons were fixed and processed for ICC with anti α-Syn and anti 466 PI4,5P2 antibodies. Similar to the results in cell lines (Fig. 4) we found that expression of α-Syn 467 mutations in hippocampal neurons differentially affected PI4,5P2 levels (Fig. 5G). That is, WT α-Syn 468 increased PI4,5P2 levels (139%) over the levels detected in control cells (set at 100%); the PD-469 associated A30P, E46K and A53T mutations further increased PI4,5P2 levels (152-200%), however, 470 PI4,5P2 levels in primary neurons expressing the K to E mutations did not differ from control cells. 471

474
The results demonstrate a correlation between α-Syn -dependent increases in PI4,5P2 levels and its 475 capacity to enhance the rate of endocytosis. That is, an inverse correlation of r= -0.75 was calculated 476 between the decay-constant of sypHy signal and PI4,5P2 levels with the different α-Syn mutations. 477 Excluding A30P mutation, which appears ineffective in endocytosis, yet increases PI4,5P2 levels, 478 results in a stronger correlation (r= -0.87). 479

481
Increases in cellular PI4,5P2 levels are associated with α-Syn toxicity. 482 methods). PSer129 to total α-Syn ratio was calculated and found to be significantly higher in cells 487 harboring high PI4,5P2 levels upon the expression of PIPKIγ (Fig. 6). 488 489

Discussion 490
We address the controversy over α-Syn's role in membrane trafficking and SV cycling by 491 investigating α-Syn associations with PIP2 and specifically with PI4,5P2. We show that α-Syn 492 colocalizes with components of clathrin coated pits/vesicles, e.g., pAP2, PI4,5P2 and PI3,4P2. We 493 further show that α-Syn-mediated CME requires PI4,5P2 at the plasma membrane. Utilizing a 494 recruitable 5-phosphatase, that hydrolyses PI4,5P2 at the plasma membrane, we demonstrate that α-495 Syn mediated CME is PI4,5P2-dependent. In addition, lowering PI4,5P2 levels by means of silencing 496 the PI-transfer protein, Nir2, abolished the enhancing effect of α-Syn on 568-Tf endocytosis. Finally, 497 utilizing specific α-Syn mutations, with differential effects on PI4,5P2 levels, we demonstrate a 498 correlation between neuronal PI4,5P2 levels and the rate of SVs endocytosis, assessed by acidification 499 of sypHy. Based on these results and the established role of PI4,5P2 in CME (Cremona et al., 1999; with their effects on PI4,5P2 levels at the synapse. However, the A30P mutation in α-Syn is There is a general agreement in the field concerning two features of α-Syn protein, its preference for 517 binding acidic phospholipids (Davidson et al., 1998) and its preference for curved membranes, akin the 518 curvature that typifies synaptic vesicles (Antonny, 2011). Thus, the findings herein, indicating that α-519 Syn interacts with and regulates PI4,5P2 levels, fit well with these two features. PI4,5P2 is an acidic 520 phospholipid enriched on presynaptic membranes and due to its enrichment with PUFAs, it helps to 521 form membrane curvature (Balla, 2013). PI4,5P2 is critical for both mechanisms, endocytosis and 522 exocytosis. Thus, recruiting PIP2 to one mechanism will inevitable affect the other. Together with our 523 previous findings, indicating a role for α-Syn to enrich membrane phospholipids with PUFA and 524 increase membrane fluidity (Sharon et al., 2003b), it appears that α-Syn plays major roles in shaping 525 membrane content and in accord, membrane function. 526 527 An emerging question in α-Syn's involvement in mechanisms of membrane trafficking is Why do 528 different studies report a different outcome and how can we get better consistency? To be able to solve 529 this problem we may need to consider the following: 1. The type of endocytic mechanism; 2. 530 Neuronal activity; 3. α-Syn expression; and 4. The lipid content at the plasma membrane. While CME 531 is a key mechanism of SV endocytosis, additional routes of SV endocytosis, including, kiss-and-run, 532 ultrafast and bulk endocytosis take place at the synapse (Gan and Watanabe, 2018; Milosevic, 2018). 533 The degree of involvement and relative importance of each of these mechanisms during physiological 534 neuronal function and in different neuronal types is not fully clear. It is possible that different neurons 535 rely on different mechanisms of endocytosis, depending on their electrophysiological activity and the 536 accompanied need in vesicle recycling (Gan and Watanabe, 2018;Milosevic, 2018). In relevance to 537 neuronal activity, it was suggested that α-Syn role in endocytosis may differ between basal and 538 intense neurotransmission (Lautenschläger et al., 2017). Moreover, neural activity has been shown to 539 control the synaptic accumulation of α-Syn (Fortin, 2005). Thus, the type of α-Syn expression model 540 may affect the outcome, whether α-Syn -/-; a,b,g Syn -/-; stable (long term) or transient α-Syn over-541 expression; exogenously added or endogenously expressed α-Syn. α-Syn is a highly dynamic protein 542 which responds to changes in its environment with structural changes that may affects its activity. Due 543 to its multifaceted nature, it is not possible to consider an opposite outcome when comparing the 544 results obtained in α-Syn silencing vs α-Syn over-expressing models. The results herein indicate that and the additional cellular mechanisms that rely on PI4,5P2 levels, it is important to also take PIPs 547 homeostasis into consideration when analyzing α-Syn effects in membrane trafficking.