Astrocytes Protect Human Dopaminergic Neurons from Α-Synuclein Accumulation and Propagation

Research Articles: Cellular/Molecular Astrocytes Protect Human Dopaminergic Neurons from α-Synuclein Accumulation and Propagation https://doi.org/10.1523/JNEUROSCI.0954-20.2020

Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.0954-20.2020 Received: 23 April 2020 Revised: 18 August 2020 Accepted: 1 September 2020

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1 Astrocytes protect human dopaminergic neurons from D-synuclein accumulation and 2 propagation 3 4 5 Authors: Taiji Tsunemi1,2*, Yuta Ishiguro2, Asako Yoroisaka2, Clarissa Valdez3, Kengo Miyamoto2, 6 Keiichi Ishikawa3, Shinji Saiki2, Wado Akamatsu3, Nobutaka Hattori2 and Dimitri Krainc1* 7 8 Affiliations: 9 1. Ken & Ruth Davee Department of Neurology, Northwestern University Feinberg School of 10 Medicine, 303 East Chicago Avenue, Ward 12-2, Chicago, Illinois 60611, USA. 11 2. Department of Neurology, Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo- 12 ku, Tokyo 113-8421, Japan. 13 3. Center for Genomic and Regenerative Medicine, Juntendo University School of Medicine, 2- 14 1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. 15 16 *To whom correspondence should be addressed: 17 18 Dr. Dimitri Krainc 19 Department of Neurology 20 Northwestern University Feinberg School of Medicine 21 303 East Chicago Avenue 22 Ward 12-140 (office) 23 312-503-3936 (phone) 24 312-503-3951 (fax) 25 Email: [email protected] 26 27 Or 28 29 Dr. Taiji Tsunemi 30 Department of Neurology 31 Juntendo University School of Medicine 32 2-1-1, Hongo, Bunkyo-ku 33 Tokyo 113-8421 34 Japan 35 Email: [email protected] 36 37 38 Key Words: Parkinson’s disease (PD), Kufor-Rakeb syndrome (KRS), ATP13A2/PARK9, alpha 39 synuclein, induced pluripotent stem cells (iPSCs), astrocytes, dopaminergic neurons 40

41x Abstract: The pathological hallmark of Parkinson’s disease is the accumulation of D-synuclein - 42 containing Lewy bodies/neurites almost exclusively in neurons, and rarely in glial cells. 43 However, emerging evidence suggests that glia such as astrocytes play an important role in 44 development of D-synuclein pathology. Using iPS derived dopaminergic neurons and astrocytes 45 from healthy subjects and patients carrying mutations in lysosomal ATP13A2, a monogenic 46 form of synucleinopathy, we found that astrocytes rapidly internalized D-synuclein, and 47 exhibited higher lysosomal degradation rates compared to neurons. Moreover, co-culturing 48 astrocytes and neurons led to decreased accumulation of D-synuclein in neurons and 49 consequently diminished inter-neuronal transfer of D-synuclein. These protective functions of 50 astrocytes were attenuated by ATP13A2 deficiency, suggesting that loss of ATP13A2 function in 51 astrocytes, at least partially contributes to neuronal D-synuclein pathology. Together, our 52 results highlight the importance of lysosomal function in astrocytes in the pathogenesis of 53 synucleinopathies.

54 55 Significance Statement: While most neurodegenerative disorders are characterized by the 56 accumulation of aggregated mutant proteins exclusively in neurons, the contribution of glial 57 cells in this process remains poorly explored. Here, we demonstrate that astrocytes contribute 58 to removal of extracellular D-synuclein and that disruption of this pathway caused by mutations 59 in the Parkinson’s disease-linked gene ATP13A2 result in D-synuclein accumulation in human 60 dopaminergic neurons. We found astrocytes also protect neurons from D-synuclein 61 propagation, whereas ATP13A2 deficiency in astrocytes compromises this protective function. 62 These results highlight astrocyte-mediated D-synuclein clearance as a potential therapeutic 63 target in disorders characterized by the accumulation of D-synuclein including Parkinson’s 64 disease. 65 66

67 Main Text: 68 Introduction: Parkinson’s disease (PD), the most common neurodegenerative disease next to 69 Alzheimer’s disease, is characterized by the accumulation of protein aggregates, Lewy bodies 70 and neurites, which are immunoreactive for alpha-synuclein (D-syn), a presynaptic protein 71 associated with the pathogenesis of sporadic and familial PD (Wong and Krainc, 2017). 72 Abnormally elevated D-syn levels are toxic to neurons, as D-syn locus duplication causes late- 73 onset PD, while triplication leads to early-onset PD, suggesting dose-dependent α-syn-mediated 74 neurotoxicity (Singleton et al., 2003; Chartier-Harlin et al., 2004; Ibanez et al., 2004). Therefore, 75 it is critical for neurons to maintain D-syn within a certain level, which is conducted by a 76 complex cellular machinery, including exocytosis, which is mainly conducted by two pathways – 77 exosomes and lysosomal exocytosis. Both of these pathways are impaired in Kufor-Rakeb 78 syndrome (KRS) (Tsunemi et al., 2014 and 2019), which was originally described as a rare 79 hereditary neurodegenerative disorder caused by loss of function mutations in ATP13A2 80 (Ramirez et al., 2006). 81 82 ATP13A2 encodes a lysosomal Type 5 P-type ATPase that has been extensively studied, but the 83 precise mechanism of ATP13A2-mediated neurodegeneration remains to be explored (Dehay et 84 al., 2012; Usenovic et al., 2012; Kett et al., 2015; Bento et al., 2016; Lopes da Fonseca et al., 85 2016; van Veen et al., 2020). We and others have reported that mutant ATP13A2 results in 86 insufficient generation of intraluminal vesicles (ILVs) and consequently decreased release of 87 exosomes and D-syn into extracellular space (Kong et al., 2014; Tsunemi et al., 2014). ATP13A2 88 also regulates another exocytotic pathway, lysosomal exocytosis, in which lysosomes directly 89 fuse with plasma membrane and release their contents (Tsunemi et al., 2019). Importantly, 90 intracellular levels of D-syn are inversely correlated with the amount of D-syn released from 91 neurons by either pathway, suggesting that secretory pathways, at least in part, regulate 92 intracellular D-syn levels (Tsunemi et al., 2014; Tsunemi et al., 2019). While D-syn secretion 93 would be beneficial for host neurons, it may enhance the progression of PD pathology as 94 extracellularly released D-syn may be subsequently taken up by neighboring neurons, 95 mediating D-syn propagation. 96 97 Here, we showed that iPSC-derived astrocytes as well as rat primary astrocytes exhibit highly 98 active phagocytosis, endocytosis, and proteolysis, and play a critical role in removing 99 extracellular D-syn. We further found that co-cultured astrocytes prevent not only neuronal D- 100 syn accumulation in iPSC-derived dopaminergic neurons, but also D-syn transfer between 101 neurons, suggesting a protective role against the progression of PD pathology. Importantly, 102 these astrocytic protective functions are partially impaired by ATP13A2 mutations, resulting in 103 the increased accumulation and propagation of D-syn. Taken together, these results suggest 104 that astrocytic protective functions against D-syn toxicity may be potential targets for 105 developing therapeutics for KRS and other related synucleinopathies such as PD. 106 Materials and Methods 107

108 Cell culture: Human induced pluripotent stem (iPS) cells were taken from four normal of either 109 sex and two male KRS patients (1550 C>T; MUT1 and 3176 T>G, 3253 delC; MUT2) were 110 cultured and reprogrammed as described previously (Tsunemi et al., 2019). Differentiation 111 towards dopaminergic neurons was conducted following the protocol described previously 112 (Mazzulli et al., 2016; Tsunemi et al., 2019). At 40 days after the initiation of differentiation, we 113 infected lentiviruses depending on the experiments. Differentiation into astrocytes and 114 maturation were performed following the manufacturer’s instructions (STEMCELL Tech, 115 Cambridge MA). At 12 days after generation of embryonic bodies, astrocyte differentiation was 116 induced. At 21 days after differentiation, the medium was replaced to the Astrocyte Maturation 117 Medium. The astrocytes at 35 days after differentiation were used for the experiments (Fig. 118 1AB). For neuron-astrocyte co-culture experiments, the coverslips for DA neurons were 119 prepared following a published protocol with a few modifications (Kaech and Banker, 2006). 120 The paraffin wax (Sigma) was applied to the 18 mm coverslips on Parafilm®. After being coated 121 with 5 mg/ml poly-D-Lysine overnight, the coverslips were washed three times in sterile water 122 every 2 hours and coated with 50 ug/ml laminin overnight. DA neurons at 21-30 days after the 123 start of differentiation were plated on the coated coverslips at the density of 5 X 105 124 cells/coverslip. At day 40, neurons on a coverslip were placed in each well of 6 well dishes on 125 the bottom of which differentiated astrocytes were plated at the density of 2 X 106 cells/well 126 (Fig. 2). For D-syn transfer experiment, Cont and ATP13A2 Mut DA neurons that were 127 separately plated at the density of 5 X 105 cells/coverslip were co-cultured in a 10 cm dish (Fig. 128 5AB), on the bottom of which astrocytes were plated at the density of 1 X 107 cells/dish (Fig. 129 5C-F) for 5 to 7 days. Then, each DA neurons on the coverslips and astrocytes on the bottom of 130 the plates were separately harvested for immunoblotting. Rat primary neurons and astrocytes 131 were cultured as described previously (Meberg and Miller, 2003; Al-Bader et al., 2011). 132 Astrocyte-conditioned medium was generated by culturing astrocytes for 24 hours. Rat primary 133 cortical neurons were cultured in the medium containing Neurobasal medium with B27 134 Supplement (Gibco) and 10% astrocyte-conditioned medium for 24 hours (Fig. 2E-H). 135 Fluorescent-tagged D-syn (D-syn Alexa555) was generated as described previously (Tsunemi et 136 al., 2019). 137 138 Immunocytochemistry 139 Immunocytochemical analysis was conducted as described previously (Tsunemi et al., 2014; 140 Tsunemi and Krainc, 2014; Tsunemi et al., 2019). Briefly, after fixation in 4% paraformaldehyde, 141 the cells were permeabilized/blocked in PBS containing 0.1% saponin, 1% BSA and 5% normal 142 goat serum for 20 min. Specimens were then incubated with primary antibodies overnight, 143 washed in PBS, and then incubated with Alexa conjugated anti-rabbit or anti-mouse antibodies 144 at 1:400 dilution for one hour. Confocal imaging was conducted on the Zeiss LSM 880 confocal 145 system with the Zeiss AX10 inverted microscope equipped with D Plan-APOCHROMAT 63× (1.4 146 numerical aperture) oil-immersion objective. 147 148 Electron microscopy 149 Electron microscopic analysis was conducted as described previously with modifications 150 (Tsunemi et al., 2014). Briefly, the cells were fixed with 2.5% glutaraldehyde in 0.1 M phosphate 151 buffer, pH7.4, for overnight. After post-fixed with 1% osmium tetroxide in phosphate buffer,

152 the cells were dehydrated through a series of graded ethanol and embedded in Epon812 (Oken 153 Shoji). The resin blocks were thin sectioned with ultramicrotome UC6 (Leica). The sections 154 were stained with uranyl acetate and lead citrate, and then analyzed with a transmission EM 155 HT7700 (Hitachi). 156 157 Western blotting 158 Immunoblotting was conducted as described previously (Tsunemi et al., 2014; Tsunemi and 159 Krainc, 2014; Tsunemi et al., 2019). The antibodies used were anti-human S100 beta (Abcam), 160 anti-human GFAP (Cell Signaling), anti-human alpha synuclein C-20 (Santa Cruz), anti-human 161 alpha synuclein 211 (Santa Cruz Biotechnology), anti-human GAPDH (Millipore), human 162 Vimentin (BD Biosciences), anti-human LAMP 1 (Santa Cruz Biotechnology), anti-human EEA1 163 (Cell Signaling), anti-human EGFR (Cell Signaling), anti-human CD63 (Developmental Studies 164 Hybridoma Bank), anti-human Flotillin-1 (BD Biosciences), anti-human β-III tubulin (Covance) 165 and anti-human TH (Millipore). 166 167 Exosome isolation and nanoparticle tracking analysis 168 Exosomes were purified as described previously (Tsunemi et al., 2014). Briefly, exosomes were 169 collected from cell-conditioned media using a basic differential centrifugation method (200 × g 170 for 5 min, 1200 × g for 10 min, and 16,500 × g for 30 min), followed by ultracentrifugation at 171 110,000 × g for 60 min. After washing in PBS, exosomes were collected by a centrifugation at 172 110,000 × g for 60 min. Analysis of extracellular vesicles was conducted by NanoSight LM10 173 system (NanoSight), configured with a 405 nm laser and a high-sensitivity digital camera system 174 (OrcaFlash2.8, Hamamatsu C11440, NanoSight). Samples were administered and recorded for 1 175 min under sustained flow controlled by script control system equipped with the NanoSight 176 syringe pump. Videos were analyzed by the NTA-software (v2.3). 177 178 Lysosomal proteolysis in live neurons and lysosomal enzyme activity assays 179 EGFR degradation assay was conducted as described previously (Usenovic et al., 2012). Briefly, 180 DA neurons or astrocytes were treated with 150 ng/ml of human epidermal growth factor (EGF; 181 Preprotech) to stimulate endocytosis of epidermal growth factor receptor (EGFR). The levels of 182 EGFR were monitored with anti-EGFR antibody. Long-lived protein degradation assays were 183 performed by radioactive pulse-chase using tritium-labeled leucine (Perkin-Elmer, 184 #NET460A001MC) as previously described (Kaushik and Cuervo, 2009; Tsunemi et al., 2019). 185 Glucocerebrosidase activity assays in lysosome-enriched P2 fractions isolated from DA neurons 186 and astrocytes were performed using the artificial enzyme substrates 4MU-glucopyranoside 187 (for GCase) and 4MU-sulfate potassium salt (for a-i-2-sulf) as described previously (Mazzulli et 188 al., 2011; Tsunemi et al., 2019). 189 190 Alpha synuclein detection 191 Alpha synuclein oligomers/fibrils were formed as described previously (Mazzulli et al., 2011). 192 Briefly, after D-syn monomers were incubated at 37°C for 10 days under continuous agitation of 193 1000 rpm, D-syn oligomers/fibrils were centrifuged at 10,000 x g for 30 min. The pellets were 194 re-suspended in PBS and fibril formation was assessed by Thioflavin T spectroscopic assay and 195 electron microscopic analysis. For analyzing alpha synuclein internalization, 100 ng/ml alpha

196 synuclein oligomers/fibrils were added in the media for indicated time, DA neurons or 197 astrocytes were washed 4 times in PBS and fixed in 4% paraformaldehyde. 198 199 Statistical analysis 200 All data were prepared for analysis with standard spreadsheet software (Microsoft Excel). 201 Statistical analysis was performed by one-way ANOVA post hoc Tukey test or Student t test. All 202 error bars represent SEM in figures. 203 204 RESULTS 205 206 Astrocytes can uptake D-synuclein secreted from DA neurons 207 We have reported that increased neuronal secretion of D-syn, via either exosomal secretion or 208 lysosomal exocytosis, results in lower intracellular D-syn (Tsunemi et al., 2014; Tsunemi et al., 209 2019). Secreted D-syn is taken up by glia cells such as astrocytes and causes inflammatory 210 responses (Lee et al., 2010), ((Loria et al., 2017). Using astrocytes differentiated from control 211 iPSCs (Fig. 1AB), we found that under normal conditions, D-syn level in astrocytes was very low 212 (Fig. 1C top, D left). However, after culturing astrocytes in the media containing α-syn 213 monomers, astrocytic D-syn levels increased (Fig. 1C middle, D center), suggesting that 214 astrocytes took up D-syn from the extracellular space, consistent with previous reports (Lee et 215 al., 2010; Loria et al., 2017). Astrocytes were also able to take up D-syn oligomer/fibrils (Fig. 1C 216 bottom, D right). This increase in α-syn levels in astrocytes was further confirmed by sensitive 217 ELISA assay (Tsunemi et al., 2014; Tsunemi et al., 2019) (Fig. 1E), and the uptake of D-syn was 218 confirmed by the trypan blue assay to exclude the possibility that D-syn attached to the outside 219 of plasma membranes (Fig. 1F) (Tsunemi et al., 2019). 220 221 We next examined the relationship between the amounts of D-syn in the media and D-syn 222 levels in astrocytes. While α-syn was not detectable in control astrocytes prior to co-culturing 223 with neurons (Fig. 1GH leftmost lane), it became detectable after 24 hours of co-culturing with 224 DA neurons. (Fig. 1GH lane 2-5). Interestingly, the increase in astrocytic D-syn was more 225 pronounced when astrocytes were co-cultured with control neurons compared to ATP13A2- 226 mutant DA neurons, consistent with our prior observations that ATP13A2-mutant DA neurons 227 secreted less D-syn than control neurons (Cont1 and 2 vs ATP13A2 Mut; 0.73 r 0.02, p = 0.042) 228 (Fig. 1GH lane 4) (Tsunemi et al., 2014; Tsunemi et al., 2019). In contrast, co-culturing of 229 astrocytes with neurons carrying D-syn triplication, which express higher levels of α-syn, 230 resulted in dramatically increased uptake of D-syn into astrocytes (Cont1 and 2 vs ATP13A2 231 Mut; 2.21 r 0.03, p = 0.001) (Fig. 1GH lane 5). 232 233 To exclude the possibility that D-syn directly transferred from neurons to astrocytes, we 234 cultured astrocytes in cell media used for neuronal cultures, and found that D-syn levels in 235 astrocytes were comparable to those co-cultured with neurons, further suggesting that 236 astrocytic D-syn originated from neuronally secreted D-syn (Fig. 1GH lane 6-9). To examine 237 whether astrocytes can uptake D-syn oligomers/fibrils secreted from neurons, we cultured 238 astrocytes either in the media containingD-syn oligomers/fibrils, or co-cultured with DA

239 neurons. These experiments revealed that D-syn oligomers/fibrils were detected in astrocytes 240 in the presence of control or mutant ATP13A2 neurons (Cont 1, Cont 2, Mut 1 and Mut 2) or DA 241 neurons carrying SNCA triplications (Fig. 1IJ leftmost lane) (Fig. 1IJ lane 2-5). Similarly, 242 astrocytic D-syn oligomers/fibrils were detected after culturing astrocytes with neuronal media 243 (Fig. 1IJ lane 6-9). Taken together, these data demonstrate that astrocytes can take up 244 different D-syn species secreted from DA neurons into the media. 245 246 Co-culturing ATP13A2 neurons with astrocytes lowers levels of neuronal D-syn levels 247 Next, we examined how D-syn uptake by astrocytes affects D-syn levels in neurons in a steady- 248 state condition. Using rat primary neurons and astrocytes (Meberg and Miller, 2003; Al-Bader 249 et al., 2011), we found that both Tx soluble (Fig. 2A left two, B) and SDS soluble fractions of 250 neuronal D-syn (Fig. 2A right two, C) were decreased after co-culturing neurons with 251 astrocytes, indicating that astrocyte-mediated D-syn uptake was able to lower intracellular α- 252 syn levels in neurons. To further confirm that astrocytes uptake neuronal D-syn, we labeled 253 synthetic D-syn fibrils with Alexa-555 (D-syn 555) (Tsunemi et al., 2019). After DA neurons were 254 exposed to D-syn 555 overnight and then co-cultured with astrocytes, we were able to detect 255 α-syn 555 in astrocytes, further demonstrating that astrocytic D-syn was of neuronal origin (Fig. 256 2D). To examine whether astrocytes generate and release some factors which could reduce D- 257 syn expression in neurons, we analyzed expression and protein levels of neuronal D-syn before 258 and after culturing neurons with astrocyte-derived conditioned media. We did not find any 259 differences in either D-syn expression (Fig. 2E), or D-syn protein levels (Fig. 2F-H) before and 260 after culturing neurons with astrocyte-derived conditioned media. These results suggest that it 261 is unlikely that astrocytes send some sort of a signal that would lower neuronal D-syn. To 262 examine the effect of ATP13A2 on neuronal D-syn levels, iPSC-derived DA neurons and 263 astrocytes were used. We found that D-syn levels in control DA neurons did not change when 264 these neurons were co-cultured with control or mutant astrocytes (Fig. 2I left four, JK left 265 four). In contrast, levels of D-syn in ATP13A2-mutant DA neurons were significantly reduced 266 when the neurons were co-cultured with control or mutant astrocytes (Fig. 2I right four, JK 267 right four). Importantly, the reduction of D-syn levels in ATP13A2-mutant neurons was more 268 profound when the neurons were co-cultured with control compared to mutant ATP13A2 269 astrocytes (Fig. 2JK right four). Thus, astrocytes offer an important mechanism for regulating 270 neuronal D-syn levels by taking up secreted D-syn, but this regulation is less efficient in mutant 271 ATP13A2 astrocytes. 272 273 Proteolysis and degradation of α-syn is more efficient in astrocytes compared to neurons 274 Our data so far indicate that astrocytes can uptake secreted D-syn and therefore protect 275 neurons from accumulation of D-syn. Based on these results, we hypothesized that D-syn gets 276 efficiently degraded in astrocytes. To test this hypothesis, we first measured degradation of 277 epidermal growth factor receptor (EGFR), which is expressed on the plasma membrane, but 278 endocytosed and degraded in lysosomes upon binding to EGF (Usenovic et al., 2012). These 279 experiments revealed significantly increased average EGFR degradation rates in both control 280 and PARK 9 mutant astrocytes compared with DA neurons (Fig. 3AB). However, ATP13A2 281 deficiency due to mutations, decreased EGFR degradation rates in either DA neurons or

282 astrocytes (Fig. 3AB), suggesting that ATP13A2 mutations impair the endocytic pathway in both 283 cell types. 284 285 We then measured D-syn internalization (Fig. 3CD), by exposing the cells to oligomeric/fibrillar 286 human D-syn (Tremblay et al., 2019). We found that oligomeric/fibrillar D-syn internalization 287 was faster in astrocytes than neurons, demonstrating a higher rate of endocytic activity (Fig. 288 3D), whereas ATP13A2 deficiency decreased the D-syn internalization rates in astrocytes. Next, 289 we compared the proteolytic capacity in control (Fig. 3E) and ATP13A2-mutated DA neurons 290 (Fig. 3F) and control (Fig. 3G) and ATP13A2-mutated astrocytes (Fig. 3H) by pulse-chase analysis 291 (Tsunemi and Krainc, 2014; Tsunemi et al., 2019). The results showed that lysosomal 292 proteolysis was significantly higher in astrocytes than in neurons (Fig. 3I), whereas ATP13A2- 293 deficiency leads to decreased lysosomal proteolysis in both DA neurons and astrocytes (Fig. 294 3I). The activity of glucocerebrosidase in lysosome-enriched fractions in astrocytes was higher 295 than those in DA neurons (Fig. 3J, p = 0.037), further supporting the notion of higher proteolytic 296 capacity in astrocytes. 297 298 To begin exploring the reasons for increased proteolytic capacity of astrocytes, we compared 299 endo-lysosomal protein levels and found that astrocytes contain increased levels of both EEA1, 300 an early endosomal protein, and LAMP-1, a lysosomal protein, compared to neurons (Fig. 4A-C). 301 ATP13A2 deficiency resulted in increased EEA1 and LAMP-1 levels both in astrocytes and 302 neurons (Fig. 4BC). Using TEM to quantify electron dense vesicles (EDVs), we found that EDVs 303 were abundant in astrocytes (Fig. 4DE, Cont astrocytes vs Cont DA neurons, p = 0.017), whereas 304 ATP13A2 deficiency resulted in further increase of EDVs both in astrocytes and neurons. These 305 results were confirmed by immunocytochemistry, showing that astrocytes contained more 306 LAMP-1-positive vesicles than neurons (Cont astrocytes vs Cont DA neurons, p = 0.009), while 307 ATP13A2 deficiency resulted in increased LAMP-1-positive vesicles and EDVs both in astrocytes 308 and neurons (Fig. 4FG). Together, these results suggest that both endocytic activities and 309 lysosomal proteolytic activities were higher in astrocytes compared to neurons, possibly due to 310 higher density of endo-lysosomes in astrocytes. 311 312 Co-culturing astrocytes and neurons decreases transfer of D-syn between neurons 313 Based on these results, we hypothesized that secretion of D-syn from neurons, followed by 314 uptake and degradation of D-syn by astrocytes leads to less D-syn available for neuron-to- 315 neuron propagation. To this end, we used a triple co-culture system in which mutant and 316 control DA neurons were cultured on the cover slips and astrocytes were cultured on the 317 bottom of a dish, allowing us to analyze the changes in D-syn levels in each type of cells 318 separately. We tested whether the presence of astrocytes affected neuron-to-neuron D-syn 319 transmission (Fig. 5). In the absence of astrocytes, lentivirus-mediated expression of wild-type 320 ATP13A2 in mutant DA neurons led to an decrease in D-syn levels in mutant DA neurons due to 321 increased exocytosis (Tsunemi et al., 2014; Tsunemi et al., 2019) (Fig. 5AB first and third from 322 left), and an increase in D-syn levels in control DA neurons (Fig. 5AB second and forth from 323 left), showing that D-syn secreted from mutant DA neurons was taken up by control DA 324 neurons. Then we examined the effect of co-culturing astrocytes on this D-syn transfer

325 between neurons (Fig. 5C-F). Similarly, lentivirus-mediated expression of wild-type ATP13A2 in 326 mutant DA neurons increased D-syn secretion and resulted in reduced intracellular D-syn levels 327 (Fig. 5CD first and third from left). While D-syn was expressed in control astrocytes at very low 328 levels, the secreted D-syn was taken up by astrocytes (Fig. 5CD right two), therefore D-syn 329 levels remained unchanged in co-cultured control DA neurons (Fig. 5CD second and 4th from 330 left). These results suggested that the presence of astrocytes protects healthy neurons from 331 extracellular D-syn. 332 333 We next examined whether ATP13A2 deficiency in astrocytes affects this neuronal D-syn 334 transmission. Indeed, D-syn levels increased in control DA neurons, suggesting that reduced 335 uptake and degradation of D-syn in ATP13A2-mutated astrocytes resulted in increased D-syn 336 transmission between DA neurons (Fig. 5EF first and third from left). Taken together, our data 337 demonstrated that astrocytes have the capacity to clear extracellular D-syn more efficiently 338 than neurons, whereas ATP13A2 deficiency partially disrupts this protective role of astrocytes, 339 potentially contributing to PD pathology. 340 341 DISCUSSION 342 343 Recent studies using ATP13A2-mutant DA neurons demonstrated that decreased exosomal or 344 lysosomal secretion of D-syn results in neuronal accumulation of D-syn, whereas enhancing 345 these pathways leads to decreased levels of neuronal D-syn (Tsunemi et al., 2014; Filippini et 346 al., 2019; Tsunemi et al., 2019). However, the fate of secreted D-syn has not been explored in 347 detail. In this study, we focused on the role of astrocytes and ATP13A2 in the regulation of D- 348 syn secreted from neurons. 349 350 Astrocytes are known to provide metabolic and trophic support to neurons, and more recent 351 studies also suggest that astrocytes play important roles in modulating neurotransmission, cell 352 signaling, inflammation, synapse modulation and metabolite and electrolyte homeostasis 353 (Belanger et al., 2011). For example, glutamate transporters (EAAT1 and EAAT2) and Kir4.1 are 354 able to clear excess extracellular glutamate and potassium released from neurons, respectively 355 (Ben Haim and Rowitch, 2017), whereas their malfunction results in aberrant neuronal 356 excitability leading to epilepsy (Barker-Haliski and White, 2015). In addition, astrocytes can 357 uptake misfolded toxic protein aggregates including huntingtin (Shin et al., 2005), amyloid-β 358 (Pihlaja et al., 2008; Xiao et al., 2014), and D-syn (Lee et al., 2010; Bliederhaeuser et al., 2016; 359 Loria et al., 2017). In this study, we observed astrocyte-mediated clearance of neuronal D-syn 360 (Fig. 1, 2), revealing an important role of astrocytes in regulation of neuronal D-syn. 361 Interestingly, the astrocytic uptake was decreased by the presence of ATP13A2 deficiency (Fig. 362 3 CD), leading to increased D-syn levels in DA neurons (Fig. 2 D, F). Astrocyte-mediated 363 neuroprotection was also recently observed in another PD-associated mutation, LRRK2 G2019S 364 (di Domenico et al., 2019). In addition, the astrocyte-specific overexpression of the redox- 365 sensitive transcriptional factor erythroid 1 related factor 2 (Nrf2) lead to decreased D-syn levels 366 in neurons from mice expressing mutant D-synA53T (Gan et al., 2012). These results suggest a 367 significant involvement of astrocytes in in regulation of D-syn in PD. Of note, astrocytes which

368 uptake mutant proteins may also become reactive astrocytes which exhibit neurotoxicity (Shin 369 et al., 2005) or induce inflammation (Lee et al., 2010). Indeed, a lack of ATP13A2 induces 370 activation of nod-like receptor protein 3 (NLRP3) inflammasome to produce excess IL-1β from 371 astrocytes, whereas overexpression of ATP13A2 can reverse it (Qiao et al., 2016). However, our 372 results favor a neuroprotective function for astrocytes through the removal of D-syn, as 373 astrocyte-mediated D-syn clearance mitigates its accumulation in neurons, suggesting an 374 important astrocytic regulation of neuronal protein homeostasis (Fig. 2, 3). Importantly, this 375 neuroprotective D-syn clearance is reduced in ATP13A2-mutant astrocytes, contributing to D- 376 syn accumulation in DA neurons (Fig.2 DF). 377 378 We found that D-syn was abundantly expressed in neurons, but rarely in astrocytes (Fig. 1), 379 which is consistent with previous reports (Zhang et al., 2014). In line with previous studies 380 (Loria et al., 2017), we found that phagocytosis and the endosomal pathway were more active 381 in astrocytes compared to neurons. Indeed, internalization and recycling of D-syn were more 382 active in astrocytes compared to neurons (Fig. 3CD), but were reduced in both cell types in the 383 presence of ATP13A2 deficiency (Fig 3CD). However, D-syn internalization was still more 384 efficient in mutant astrocytes compared to neurons, indicating that extracellular D-syn is more 385 likely taken up by astrocytes even in the presence of ATP13A2 mutations. While D-syn is 386 degraded through multiple pathways (Wong and Krainc, 2017), recent evidence emphasized the 387 importance of lysosomal degradation of D-syn in neurons as defects in lysosomal hydrolases 388 have been linked to several genetic forms of Parkinson’s disease (Ramirez et al., 2006; Mazzulli 389 et al., 2011). We found that lysosomal degradation was more active in astrocytes compared to 390 neurons (Fig. 3E-I), suggesting that astrocytes have better capacity to digest lipids, amino acids, 391 complex sugars and proteins including D-syn (Loria et al., 2017). Interestingly, D-syn 392 accumulation was observed in astrocytes in sporadic PD patients, but with lower frequency 393 compared to neurons, possibly due to higher proteolysis in astrocytes (Wakabayashi et al., 394 2000). Moreover, PD-associated mutations in GBA1 also resulted in decreased lysosomal 395 function in iPSC-derived astrocytes, raising the possibility that dysfunctional astrocytic 396 proteolysis contributes to neuronal D-syn accumulation in PD (Aflaki et al., 2020). 397 398 Recent studies showed that D-syn can be transmitted from neuron to neuron, and D-syn 399 pathology may spread in a ‘prion-like’ manner (Guo and Lee, 2014), but the role of astrocytic 400 ATP13A2 in modulating this transmission was previously not known. While the precise 401 propagation mechanisms remain to be determined, initial studies in cultured cells 402 demonstrated that D-syn can be transmitted via exosome secretion (Danzer et al., 2012), 403 whereas later studies using microfluidic chambers revealed that D-syn can be released from 404 axon terminals, suggesting synaptic propagation (Brahic et al., 2016; Mao et al., 2016). Studies 405 in mouse brains have further documented that D-syn first spreads to anatomically connected 406 lesions, and subsequently to lesions without direct connections, suggesting that both forms of 407 transmissions may occur but at different rates (Luk et al., 2012). While some studies proposed 408 direct transfer via tunnel nanotubes (Rostami et al., 2017), most of previous results suggest that 409 D-syn can be released and transmitted by multiple pathways including exosomal secretion and 410 lysosomal exocytosis (Tsunemi et al., 2014; Tsunemi et al., 2019). Our co-culturing experiments

411 demonstrated that astrocytes play a protective role in D-syn propagation by preventing indirect 412 D-syn transfer, consistent with the previous report (Loria et al., 2017) (Fig. 5A-D). We found 413 that ATP13A2-deficiency impaired this protective role of astrocytes, leading to increased D-syn 414 transfer between neurons (Fig. 5EF). 415 416 In conclusion, using iPSC-derived astrocytes and neurons, we showed that intraneuronal D-syn 417 levels were, at least in part, regulated by astrocytic uptake and lysosomal degradation. 418 Impairment of these pathways by familial PD-associated ATP13A2-mutations contributed to 419 increased accumulation and propagation of D-syn in DA neurons. Although patients 420 with ATP13A2 mutations present with parkinsonism, supranuclear gaze palsy and dementia, 421 they also respond well to treatment with levodopa, suggesting degeneration of nigral 422 dopaminergic (DA) neurons that is typically seen in PD. In addition, Atp13a2 mouse models and 423 DA neurons differentiated from patient-derived iPSCs all demonstrated increased levels of D- 424 syn, as commonly seen in PD (Schultheis et al., 2013; Tsunemi et al., 2019). Importantly, most 425 sporadic PD patients and patients with genetic forms of PD also develop cognitive decline and 426 dementia (Kalia and Lang, 2015), and patients with sporadic PD have been also shown to have 427 altered ATP13A2 in the nigra (Ramirez et al., 2006; Dehay et al., 2012; Ramonet et al., 2012; 428 Murphy et al., 2013), suggesting that our studies of ATP13A2 may be more broadly informative 429 for PD and other synucleinopathies. Therefore, targeting astrocytic clearance of D-syn may be 430 an important therapeutic strategy for ameliorating D-syn-mediated pathology not only for KRS 431 but also other synucleinopathies. 432 433 Acknowledgments: This work is supported by National Institute of Health (R37 NS096241) to 434 D.K., JSPS KAKENHI Grants (18K07510), Juntendo University Research Institute for 435 Environmental & Gender-specific Medicine to T.T.; Strategic Research Foundation Grant-in-Aid 436 for Private Universities to T.T., Y.I., K.I., S.S., W.A., and N.H. and the Rare/Intractable Disease 437 Project of the Japan from AMED (JP17ek0109244 and JP20ek0109429) to T.T., K.I., S.S., 438 W.A., and N.H. 439 440 Author contributions: D.K. and T.T. organized the project, interpreted the results and wrote the 441 manuscript. T.T., Y.I., A.Y. and C.V. performed experiments. W.A. and N.H. provided helpful 442 suggestions for data interpretation. Competing interests: D.K. is the Founder and Scientific 443 Advisory Board Chair of Lysosomal Therapeutics Inc. and Vanqua Bio; D.K. serves on the 444 scientific advisory boards of The Silverstein Foundation, Intellia Therapeutics, and Prevail 445 Therapeutics and is a Venture Partner at OrbiMed. 446

447 Figures: 448 Figure 1. Astrocytes absorb different D-syn species from culture media

449

450 A. Astrocytes differentiated from induced pluripotent stem cells (iPSCs) that were taken from 451 two normal individuals (Cont 1 and Cont 2) and two patients with ATP13A2 mutations (Mut 1 452 and Mut 2). The cells were stained with astrocyte markers, Glial fibrillary acidic protein (GFAP) 453 (upper) or E-S100 (bottom). 454 B. Immunoblot characterization of astrocytes with astrocyte markers including Vimentin (top), 455 GFAP (second from top), Glutamine synthetase (second from bottom) and E-S100 (bottom). 456 C-E. iPSC-derived astrocytes can absorb D-syn from the media 457 C. Representative images of D-syn immunofluorescence in Cont 1 astrocytes before (top) and 458 after culturing in the media containing monomeric D-syn (middle) and oligomeric/fibrillar D-syn 459 for 24 hours (bottom). GFAP is used for astrocytic marker. 460 D. Quantification of D-syn fluorescence intensities normalized by a nuclear marker, 4',6- 461 diamidino-2-phenylindole (DAPI) intensities in astrocytes (n = 3, *p = 0.001, **p = 0.015, one- 462 way ANOVA Tukey post hoc test). 463 E. The quantification of D-syn levels in astrocytes that were cultured in the normal media or in 464 the media containing D-syn (conditioned media) (n = 3, *p = 0.001, **p = 0.0011, one-way 465 ANOVA Tukey post hoc test). 466 F. Trypan blue assay with Cont 1 and Mut 1 astrocytes that were cultured in the media 467 containing oligomeric/fibrillar D-syn for 24 hours. 468 G. Representative immunoblot for alpha synuclein (D-syn) of lysates from astrocytes before 469 (leftmost lane), after co-cultured with Cont1, 2 and Mut 1 dopaminergic (DA) neurons and DA 470 neurons harboring D-syn triplication (lanes 2-5) and after cultured in the leftover media of Cont 471 1, 2 and Mut 1 DA neurons and DA neurons harboring D-syn triplication. (lanes 6-9). 472 H. Densitometric quantification is shown as the relative D-syn levels against GFAP in astrocytes 473 (n = 3, *p = 0.003, **p = 0.042, ***p = 0.001, one-way ANOVA Tukey post hoc test). 474 I. Representative immunoblot for D-syn of lysates from astrocytes before (leftmost lane), after 475 co-cultured with Cont 1, 2 and Mut 1 DA neurons and DA neurons harboring D-syn triplication 476 that were cultured in the media containing oligomeric/fibrillar D-syn for 24 hours (lanes 2-5) or 477 cultured in the leftover media of Cont 1, 2 and Mut 1 DA neurons and DA neurons harboring D- 478 syn triplication after they were cultured in the media containing oligomeric/fibrillar D-syn for 24 479 hours (lanes 6-9). 480 J. Densitometric quantification is shown as the relative D-syn levels against GFAP in astrocytes 481 (n = 3, *p = 0.001, **p = 0.0013, ***p = 0.003, one-way ANOVA Tukey post hoc test). 482 Scale bars indicate 50 Pm for Figure A, C and F. In all graphs, error bars indicate SEM. 483 484

485 Figure 2. Co-culturing of astrocytes and neurons lowers neuronal levels of D-syn

486 487 A. Representative immunoblot for D-syn, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) 488 and Vimentin in triton-soluble (right two columns) and SDS-soluble fractions (left two columns) 489 from rat primary cortical neurons that were cultured without (each left column) or with rat 490 primary astrocytes (each right column). 491 B. Densitometric quantification is shown as the relative D-syn levels against GAPDH in Triton- 492 soluble fractions in cortical neurons (n = 3, *p = 0.021, Student t test). 493 C. Densitometric quantification is shown as the relative D-syn levels against Vimentin in SDS- 494 soluble fractions in cortical neurons (n = 3, *p = 0.035, Student t test). 495 D. A representative image of rat primary neuron that was co-cultured with D-syn Alexa555 496 containing astrocytes. 497 E. Expression of D-syn mRNA in rat primary neurons before and after cultured in astrocyte- 498 conditioned media (n = 3, p = 0.80, Student t test). 499 F. Representative immunoblot for triton-soluble (right four columns) and SDS-soluble fractions 500 (left four columns) from rat primary cortical neurons before and after cultured in astrocyte- 501 conditioned media. 502 G. Densitometric quantification is shown as the relative D-syn levels against GAPDH in Triton- 503 soluble fractions in rat primary cortical neurons (n = 3, p = 0.75, Student t test). 504 H. Densitometric quantification is shown as the relative D-syn levels against Vimentin in SDS- 505 soluble fractions in rat primary cortical neurons (n = 3, p = 0.71, Student t test).

506 I. Representative immunoblot for triton-soluble (upper two lanes) and SDS-soluble fractions 507 (bottom two lanes) from Cont (Cont 1) (left four columns) and Mut (ATP13A2 Mut 1) (right 508 four columns) DA neurons that were cultured without (left) or with Cont astrocytes (right). 509 J. Densitometric quantification is shown as the relative D-syn levels against GAPDH in Triton- 510 soluble fractions in DA neurons (n = 3, *p = 0.018, **p = 0.033, Student t test). 511 K. Densitometric quantification is shown as the relative D-syn levels against Vimentin in SDS- 512 soluble fractions in DA neurons (n = 3, *p = 0.005, **p = 0.009, Student t test). 513 In all graphs, error bars indicate SEM. 514 515

15 516 Figure 3. Astrocytes exhibit higher rates of lysosomal proteolysis compared to neurons A B Astrocytes Cont Astrocytes Mut Astrocytes DA neurons 120 Neurons Cont Neurons Mut 0 1 3 5 0 1 3 5 (hr) 100 175 EGFR Cont 80 GAPDH * 37 60 *** ** ** 40 DA neurons 175 EGFR ****

Mut EGFR levels (%) 20 *** ** 37 GAPDH Astrocytes (kDa) 0 *** 0135 (hr) C Astrocytes DA neurons

01624(hr)24 (hr) D Astrocytes Cont 300 Astrocytes Mut 250 DA neurons Cont * Cont 200 DA neurons Mut 150 ** 100 a-syn (pixels/cell) ** 50 ** Mut ** 0 0 0.5 1 6 24 (hr) E F 60 60 I lysosomal non lysosomal 40 40 * 120 20 20

0 protein) 0 100 proteolysis (% /initial protein) 082028 0 8 20 28 80 time (hours) proteolysis (% /initial time (hours) 60

G H 40 *** *** 60 60 20 ** 40 ** 40 0 proteolysis (% of total protein) Cont1 Cont2 Cont3 Mut1 Mut2 Cont1 Cont3 Mut1 Mut2 20 20 DA neurons Astrocytes

protein) 0 protein) 0 082028 082028

proteolysis (% /initial time (hours) proteolysis (% /initial time (hours)

** **** J 150 * 120 ***

0 Cont1Cont3 Mut1 Mut2 Cont1Cont2 Mut1 Mut2 GCase activity (% to Cont1) activity (% to Cont1) GCase DA neurons Astrocytes 517 518 A. Representative immunoblot for epidermal growth factor receptor (EGFR) degradation in iPSC 519 derived Cont (Cont 1) and Mut (Mut 1) astrocytes and Cont and Mut DA neurons that was 520 followed for 5 h. B. Levels of EGFR were normalized to GAPDH levels and expressed as 521 percentage levels of initial time point (T0; n = 6, *p = 0.037, **p = 0.01, ***p = 0.015, ****p = 522 0.041, one-way ANOVA Tukey post hoc test). C. Representative images of D-syn internalization 523 assay in Cont (Cont 1) and Mut (ATP13A2 Mut1) astrocytes and DA neurons. At 0, 1, 6 and 24 524 hours after incubation with D-syn oligomers/fibrils, the astrocytes and DA neurons were fixed

525 and stained with GFAP with DAPI and b-iii-T with DAPI, respectively. D. Analysis of D-syn 526 internalization assay in astrocytes and DA neurons (n = 3, *p = 0.037, **p = 0.0014, one-way 527 ANOVA Tukey post hoc test). E-I. Lysosomal proteolysis of Cont (E) and Mut DA neurons (F) and 528 Cont (G) and Mut astrocytes (H) is calculated by subtracting lysosomal inhibitors (2.5 mM NH4Cl 529 and 50 PM leupeptin) sensitive proteolysis from total proteolysis at 8, 20 and 28 h after chase. 530 I. Lysosomal (red) and non-lysosomal proteolysis (light blue) in Cont 1 to 3, Mut 1 and 2 DA 531 neurons (left four columns); Cont 1 and 3 and Mut 1 and 2 astrocytes (right four columns) (n = 532 3, *p = 0.009, **p = 0.022, ***p = 0.039, one-way ANOVA Tukey post hoc test). 533 J. Glucocerebrosidase (GCase) activities in lysosome-enriched fractions extracted from Cont 1, 534 3, Mut 1 and 2 DA neurons (left four columns) and Cont 1, 2, Mut 1 and 2 astrocytes (right four 535 columns) (n = 3, *p = 0.009, **p = 0.037, ***p = 0.027, ****p = 0.019, one-way ANOVA Tukey 536 post hoc test). 537

538 Figure 4. Higher endocytic activity in astrocytes compared to neurons

539 540 A-C. Immunoblot analysis for endosomal protein levels in astrocytes and neurons 541 A. Representative immunoblot for Early Endosome Antigen 1 (EEA1) (top), Lysosome-associated 542 membrane protein 1 (LAMP-1) (second from top), GAPDH (middle), Vimentin (second from 543 bottom) and GFAP (bottom) of lysates from Cont 1, 2, Mut 1 and 2 DA neurons (left four 544 columns) and Cont 1, 2, Mut 1 and 2 astrocytes (right four columns). B. Densitometric 545 quantification is shown as the relative EEA1 levels against GAPDH in Cont 1, 2, Mut 1 and 2 546 astrocytes (left four columns) and Cont 1, 2, Mut 1 and 2 DA neurons (right four columns) (n = 547 3, *p = 0.031, **p = 0.027, one-way ANOVA Tukey post hoc test). C. Densitometric 548 quantification is shown as the relative LAMP-1 levels against GAPDH in Cont 1, 2, Mut 1 and 2 549 astrocytes (left four columns) and Cont 1, 2, Mut 1 and 2 DA neurons (right four columns) (n = 550 3, *p = 0.039, **p = 0.027, one-way ANOVA Tukey post hoc test). D. Representative EM 551 images of electron dense vesicles in Cont (Cont 1) (left upper), Mut (ATP13A2 Mut 1) (left 552 bottom) astrocytes and Cont (right upper), Mut (right bottom) DA neurons. Scale bar, 5 Pm. 553 E. Quantification analysis of the number of electron dense vesicles from Cont and Mut 554 astrocytes (left two columns) and Cont and Mut DA neurons (right two columns) (n = 10-20, *p 555 = 0.033, **p = 0.027, ***p = 0.017, ****p = 0.021, one-way ANOVA Tukey post hoc test). 556 F. Representative images of LAMP-1 positive vesicles in Cont and Mut astrocytes and Cont and 557 Mut DA neurons. Scale bars indicate 50 Pm in astrocytes and 10 Pm in neurons. 558 G. Quantification analysis of the number of LAMP-1 positive vesicles from Cont and Mut 559 astrocytes (left two columns) and Cont and Mut DA neurons (right two columns) (n = 10-20, *p 560 = 0.039, **p = 0.033, ***p = 0.009, ****p = 0.013, one-way ANOVA Tukey post hoc test). 561 Scale bars indicate 20 Pm for Figure C, 5 Pm for Figure N and 50 Pm for astrocytes and 20 Pm 562 for neurons in Figure P. In all graphs, error bars indicate SEM.

563 Figure 5. Co-culturing neurons with astrocytes prevents D-syn transfer between neurons

564

565 A-D The effect of co-culturing astrocytes on the D-syn transmission between neurons. 566 A. The D-syn transmission from Mut (ATP13A2 Mut 1) to Cont (Cont 1) DA neurons in the 567 absence of co-culturing astrocytes. Immunoblot analysis of D-syn levels in Mut and Cont DA 568 neurons (left two columns) and D-syn levels in ATP13A2 overexpressing Mut and Cont DA 569 neurons (right two columns). B. Densitometric quantification of D-syn levels normalized to 570 EIII-tubulin in neurons that were not co-cultured with astrocytes (n = 3, *p = 0.029, **p = 571 0.017, ***p = 0.008, one-way ANOVA Tukey post hoc test). C. The D-syn transmission from Mut 572 to Cont DA neurons in the presence of co-culturing Cont astrocytes. Immunoblot analysis of D- 573 syn levels in Mut and Cont neurons (first two columns) and D-syn levels in ATP13A2 574 overexpressing Mut and Cont DA neurons (next two columns) after co-cultured with Cont 575 astrocytes. Immunoblot analysis of D-syn levels in Cont astrocytes co-cultured with Mut and 576 ATP13A2 overexpressing Mut DA neurons (right two columns). D. Densitometric 577 quantification of D-syn levels normalized to EIII-tubulin in neurons in the presence of co- 578 culturing astrocytes (left graph) (n = 3, *p = 0.027, **p = 0.025, one-way ANOVA Tukey post hoc 579 test). Densitometric quantification of D-syn levels normalized to GFAP in Cont astrocytes (right 580 graph) (n = 3, *p = 0.002, Student t test) 581 E, F The D-syn transmission from Mut to Cont DA neurons in the presence of co-culturing Mut 582 astrocytes. E. Immunoblot analysis of D-syn levels in Mut and Cont neurons (left two columns) 583 and D-syn levels in ATP13A2 overexpressing Mut and Cont DA neurons (next two columns) 584 after co-cultured with Mut 1 astrocytes. Immunoblot analysis of D-syn levels in Mut astrocytes 585 that were co-cultured with Mut and ATP13A2 overexpressing Mut DA neurons (right two 586 columns). 587 F. Densitometric quantification of D-syn levels normalized to EIII-tubulin in neurons in the 588 presence of co-culturing Mut astrocytes (left graph) (n = 3, *p = 0.017, **p = 0.022, ***p = 589 0.030, one-way ANOVA Tukey post hoc test). Densitometric quantification of D-syn levels 590 normalized to GFAP in Mut astrocytes (right graph) (n = 3, *p = 0.003, Student t test). 591 Error bars indicate SEM. 592 593 594

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