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1 SYNAPTIC RTP801 CONTRIBUTES TO MOTOR LEARNING DYSFUNCTION IN

2 HUNTINGTON’S DISEASE

3

4 RUNNING HEAD: Synaptic RTP801 and motor learning in HD

5

6 Núria Martín-Flores, PhD. 1,2 *, Leticia Pérez-Sisqués, BSc 1,2, Jordi Creus-Muncunill,

7 PhD1,2,3,4, Mercè Masana, PhD 1,2,3,4, Sílvia Ginés, PhD 1,2,3,4 , Jordi Alberch, MD/PhD

8 1,2,3,4, Esther Pérez-Navarro, PhD 1,2,3,4 and Cristina Malagelada PhD 1,2,4 *

9 1 Department of Biomedicine, Faculty of Medicine, University of Barcelona, 10 Barcelona, 08036 Catalonia, Spain 11 2 Institut de Neurociències, University of Barcelona, Barcelona 08036, Catalonia, 12 Spain 13 3 IDIBAPS-Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, 14 08036 Catalonia, Spain 15 4 Centro de Investigación Biomédica en Red sobre Enfermedades 16 Neurodegenerativas (CIBERNED), Barcelona, 08036 Spain 17 Authors’ Emails: [email protected]; [email protected];

18 [email protected]; [email protected]; [email protected];

19 [email protected]; [email protected]; [email protected]

20 *Correspondence should be addressed to :

21 Núria Martín-Flores, Department of Cell and Developmental Biology, University 22 College London, London, UK (current affiliation) E-mail: [email protected] 23 24 Cristina Malagelada, PhD. Unit of Biochemistry, Department de Biomedicine, Faculty 25 of Medicine. Address: Casanova 143, north wing 3rd floor; Universitat de Barcelona, 26 Barcelona 08036 Catalonia, (Spain) +34-934021919 27 E-mail: [email protected] 28 Twitter: @crismalagelada 29 30

31

32

33

34

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35 ABSTRACT

36 RTP801/REDD1 is a stress responsive protein that mediates mutant huntingtin

37 (mhtt) toxicity in cellular models and is up regulated in Huntington’s disease (HD)

38 patients’ putamen. Here, we investigated whether RTP801 is involved in motor

39 impairment in HD by affecting striatal synaptic plasticity.

40 Ectopic mhtt was over expressed in cultured rat primary neurons. The protein levels

41 of RTP801 were assessed in homogenates and crude synaptic fractions from human

42 postmortem HD brains and mouse models of HD. Striatal RTP801 expression was

43 knocked down with adeno-associated viral particles containing a shRNA in the R6/1

44 mouse model of HD and motor learning was then tested.

45 Ectopic mhtt elevated RTP801 in synapses of cultured neurons. RTP801 was also

46 up regulated in striatal synapses from HD patients and mouse models. Knocking

47 down RTP801 in the R6/1 mouse striatum prevented motor learning impairment.

48 RTP801 silencing normalized the Ser473 Akt hyperphosphorylation by

49 downregulating Rictor and it induced synaptic elevation of calcium permeable GluA1

50 subunit and TrkB receptor levels, suggesting an enhancement in synaptic plasticity.

51 These results indicate that mhtt-induced RTP801 mediates motor dysfunction in a

52 HD murine model, revealing a potential role in the human disease. These findings

53 open a new therapeutic framework focused on the RTP801/Akt/mTOR axis.

54

55

56

57 KEY WORDS: Huntington’s disease, plasticity, motor learning, mTOR, RTP801, Akt.

58

59

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60 INTRODUCTION

61 Huntington’s disease (HD) is an autosomal-dominant neurodegenerative

62 disorder caused by a CAG expansion in the exon 1 of the Huntingtin gene. This

63 expansion encodes for a mutant form of the huntingtin (htt) protein that has been

64 traditionally pointed out as responsible for the specific loss of medium-sized spiny

65 neurons in the human striatum 1–3. However, the expanded CAG RNA was identified

66 also as toxic and an active contributor to the HD pathogenesis 4. HD manifests a

67 triad of signs including severe motor dysfunction with involuntary movements

68 (chorea), cognitive impairment and neuropsychiatric symptoms. Even though mutant

69 htt severely affects striatal neurons, other areas, such as cortex, hippocampus,

70 amygdala or cerebellum, display synaptic alterations, atrophy and/or neuronal death

71 5,6.

72 Although neuronal death does not occur until late stages of HD, abnormal

73 synaptic plasticity and neuronal dysfunction are the main early pathogenic events

74 that lead to neurodegeneration 7–9. Due to the early aberrant synaptic function,

75 observed both in the human and the mouse pathology, HD is considered a

76 synaptopathy 10–12. In this regard, one of the pathways that controls synaptic

77 plasticity is the mechanistic target of rapamycin (mTOR) pathway, since it regulates

78 translation and more notably, local protein synthesis at the spines 13,14. Importantly,

79 mTOR pathway is also involved in cytoskeleton remodeling to ensure proper

80 formation and function of dendritic spines 15.

81 mTOR kinase is the central component of mTOR complex (mTORC) 1 and 2.

82 Both complexes share protein partners, but they have unique elements that define

83 substrates’ specificity and therefore, functionality. First, mTORC1 binds specifically

84 to Raptor and controls mostly protein synthesis and autophagy. Second, mTORC2

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85 specifically binds to Rictor and phosphorylates the Serine 473 residue (Ser473) in

86 Akt kinase to mediate neuronal survival. Among other functions, mTORC2 controls

87 actin polymerization and, as a consequence, it is required for LTP and LTD induction

88 to mediate synaptic strength 16,17.

89 Synaptic plasticity is impaired in HD patients 18 and mouse models 19–21.

90 These plastic alterations correlate well with mTOR/Akt signaling axis impairment. For

91 example, several HD mouse models show increased phosphorylation levels of

92 striatal mTOR and the mTORC2 substrate Akt at the Ser473 residue 22–24.

93 Interestingly, PHLPP1 (PH domain leucine-rich repeat protein phosphatase 1), the

94 phosphatase that dephosphorylates the Ser473 residue in Akt 25,26, is decreased in

95 the putamen of HD patients and in the striatum of HD mice 23. Moreover, mTORC2-

96 regulator protein Rictor, but not mTORC1-regulator protein Raptor, is increased in

97 the striatum of HD mouse models and in the putamen of HD patients 24. This

98 evidence suggested the activation of a compensatory mechanism to counteract mhtt

99 toxicity promoting cell survival. However, the exacerbation on the phosphorylation

100 status of mTOR pathway components could eventually be counterproductive for

101 synaptic plasticity 27,28.

102 In our previous work, we described RTP801/REDD1, an mTOR/Akt

103 modulator, as a mediator of mhtt toxicity in in vitro models of HD. We also showed

104 that RTP801 was elevated in the putamen and iPSC from HD patients 29. RTP801,

105 by interacting with TSC1/2 complex, regulates Rheb promoting its GDP-bound form.

106 Rheb-GDP is not able to promote mTOR kinase activity of both mTORC1 and 2

107 complexes, inactivating S6 kinase and Akt activities, as readouts of mTORC1 and

108 mTORC2, respectively. This mechanism was described to mediate neuronal death in

109 Parkinson’s disease (PD) models 30,31. However, since in HD there is an

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110 hyperphosphorylation of mTOR in the striatum of both HD mouse models and

111 patients, the mechanism by which RTP801 contributes to the specific mTOR/Akt axis

112 over activation and how this is translated to impaired plasticity has not been

113 elucidated yet. For this reason, here we investigated whether mhtt-induced RTP801

114 increase could alter mTOR/Akt signaling at a synaptic level and mediate motor

115 impairment in HD.

116

117

118 RESULTS

119

120 Ectopic mhtt increases RTP801 at soma and dendritic spines in rat cortical

121 primary neurons

122

123 We have previously shown that overexpression of the pathogenic exon-1 of mhtt

124 elevates RTP801 protein levels in rat cortical neuronal cultures 29. Here, we

125 extended these studies by analyzing whether RTP801 increase takes place in the

126 dendrites. The use of cortical primary cultures facilitated this analysis since striatal

127 mono-cultures generally produce morphologically immature MSNs with low densities

128 of dendritic spines 32–35.

129 Hence, rat cortical primary neurons (DIV13) were transfected with eGFP, Q25, Q72-

130 or Q103-htt-expressing plasmids. Twenty-four hours after transfection, neurons were

131 stained against endogenous RTP801 and dendritic F-actin puncta were visualized

132 with phalloidin (Figure 1A). As indicated (yellow arrowheads), RTP801 was found

133 within the dendritic spines. The overexpression of exon-1-mhtt (Q72 and Q103

134 pathogenic forms) induced a reduction in spine density (Figure 1B) along with an

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135 increase in somatic RTP801 (Figure 1C), as previously reported 29,36. Importantly,

136 mhtt overexpression increased around 50% the levels of RTP801 protein at the

137 remaining dendritic spines (Figure 1D).

138

139 RTP801 is increased in the synaptic compartment in the putamen of HD

140 patients

141

142 Our previous work showed that RTP801 was elevated in whole putamen lysates

143 from HD patients. To investigate further whether the increase in RTP801 also

144 extends to synaptic contacts, we isolated crude synaptosomes from the putamen of

145 non-affected (CT) and HD individuals (Table S1). We found that RTP801 was

146 enriched at the synaptosome subcellular compartment, along with synaptic markers

147 SV2A and PSD-95. However, phospho-Ser473-Akt (P-Ser473-Akt) and Phospho-

148 Ser235/236-S6 (P-Ser235/236-S6) were not significantly enriched in the crude

149 synaptosomes (Figure S1). According to our previous results, we detected higher

150 levels of RTP801 in homogenates derived from HD putamen (Figure S1A). Still, in

151 homogenates, we confirmed that mTOR activities were elevated in HD as judged by

152 increased levels of P-Ser473-Akt and P-Ser235/236-S6 (Figure S1A). Interestingly,

153 we observed that RTP801 was significantly increased in HD synaptosomes (Figure

154 2B) in comparison to controls. As expected and in line with previous data 37,

155 postsynaptic marker PSD-95 was diminished in synaptosomes from HD patients and

156 no differences were observed in pre-synaptic marker SV2A (Figure 2B). No

157 significant differences were found in P-Ser473-Akt or P-Ser235/236-S6 at the

158 synaptosomal compartment between non affected and HD individuals (Figure 2B).

159

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160 In order to correct protein levels for the remaining synapses, synaptosomal levels of

161 each protein were relativized to the levels of pre-synaptic marker SV2A (Figure 2C)

162 and post-synaptic marker PSD-95 (Figure 2D). Hence, we confirmed that versus the

163 SV2A- and PSD-95-synaptic proteins amount, RTP801 was increased in the HD

164 group in comparison to controls. Furthermore, both P-Ser473-Akt and P-Ser235/236-

165 S6 were elevated in the PSD-95-synaptic fraction of HD patients respect to

166 unaffected individuals and showed the same tendency in the SV2A-synaptic fraction.

167 These data are in line with the previously described impairment of the mTOR

168 pathway in HD 38–40.

169 Taken together, these results show that RTP801 is increased in the remaining

170 synaptic terminals of HD patients’ putamen, along with the mTOR substrates P-

171 Ser473-Akt and P-Ser235/236-S6.

172

173 To check whether these differences were region-specific, we performed the same

174 analyses in the prefrontral cortex, a less affected brain region in the disease.

175 Similarly, as in the putamen, RTP801, SV2A, PSD-95, P-Ser473-Akt and P-

176 Ser235/236-S6 were highly enriched in the synaptosomal compartment (Figure S2,

177 Enrichment), although no differences in RTP801 levels were observed neither in

178 homogenates (Figure S2, Homogenates) nor in synaptosomes (Figure S3) between

179 HD and non-affected individuals. Only synaptosomal P-Ser235/236-S6 normalized to

180 SV2A or to PSD-95 showed a significant decrease in HD samples in contrast to

181 control cases (Figure S3C&D).

182

183 RTP801 is increased in striatal synapses of HD mouse models

184

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185 To assess whether synaptic elevation of RTP801 could be reproduced in HD mouse

186 models, we investigated the synaptic levels of RTP801 in the striatum of 10-month-

187 old HdhQ7/Q111 (KI) (Figure 3 & S4) and 16-week-old R6/1 mice (Figure 4 & S5). At

188 this disease stage, these mice already display abnormal neuronal plasticity and

189 motor and cognitive dysfunctions 41–46.

190

191 Synaptosomal fractions of wild-type (WT) and HD mouse striata were enriched for

192 RTP801, SV2A, PSD-95 and P-S6-Ser235/236 (Figure S4A-C, E; Figure S5A-C, E),

193 as in human putamen. However, RTP801 protein levels did not differ between

194 genotypes in either homogenates (Figure S4A and S5A) or synaptosomes (Figure

195 3B and 4B). PSD-95 displayed decreased protein levels in comparison to the WT

196 group in KI and R6/1 mouse striatal synaptosomes (Figure 3B and 4B). After

197 correcting the synaptic loss by expressing protein levels relative to the synaptic

198 markers SV2A (Figure 3C& 4C) and PSD-95 (Figure 3D & 4D), RTP801 was

199 increased in the synaptic fraction of KI and R6/1 mice. Interestingly, only P-Ser473-

200 Akt was also significantly increased at the synaptic fraction of KI mice whereas in

201 R6/1 mice, P-Ser235/236-S6 levels were also increased.

202

203 RTP801 knockdown prevents motor learning deficits in symptomatic R6/1 mice

204

205 Since we found alterations in the striatal synaptic protein levels of RTP801 in HD

206 mouse models and human HD brains, we investigated whether RTP801 could

207 contribute to plasticity dysfunction in HD. To answer this question, 9-week-old WT

208 and R6/1 mice were bilaterally injected with AAV-shCtr or AAV-shRTP801 into the

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209 striatum and 5 weeks later motor learning, a corticostriatal dependent function, was

210 assessed by using the accelerating rotarod.

211 Comparable GFP signal was detected in the striatum of WT and R6/1 mice

212 confirming the transduction of AAV-shCtr and AAV-shRTP801 (Figure 5A).

213 Moreover, striatal injections of AAV-shRTP801 reduced RTP801 protein levels

214 between 25-30%, both in WT and R6/1 mice (Figure 5B). The accelerating rotarod

215 test showed that, as expected, shCtr-injected R6/1 animals performed poorly in this

216 motor task in comparison to WT-shCtr-injected animals. Remarkably, RTP801

217 silencing in the R6/1 mice striatum prevented motor learning deficits as shown by the

218 increased latency to fall, which was comparable to that in WT animals. Indeed, AAV-

219 shCtr or AAV-shRTP801-injected WT mice showed no differences in motor learning

220 skills (Figure 5C). These results indicate that mhtt-induced RTP801 synaptic

221 increase is contributing to motor learning dysfunction in the R6/1 mouse model of

222 HD.

223

224 RTP801 knockdown normalizes phospho-Akt Ser473 and enhances the

225 expression of synaptic proteins in the R6/1 mouse striatum

226

227 In order to understand the pathological function of synaptic RTP801 and how its

228 knockdown prevents motor learning dysfunction, we analyzed by WB downstream

229 effectors of the mTOR pathway in striatal synaptic fractions obtained from WT and

230 R6/1 mice transduced with AAV-shCtr or AAV-shRTP801.

231 Knocking down RTP801 affected neither the levels of P-Ser2448-mTOR nor the

232 levels of P-Ser235/236-S6, as mTORC1 readout (Figure 6A & 6B) but interestingly, it

233 restored P-Ser473-Akt levels to basal levels both in homogenates and

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234 synaptosomes, with no significant effect in WT animals (Figure 6C). Therefore, this

235 result indicates that RTP801 regulates the phosphorylation levels of Serine 473 Akt

236 in this pathological context.

237 Since increased levels of Rictor and reduced levels of PHLPP1 phosphatase are

238 responsible for Akt hyperactivation in the R6/1 mouse model 23,24, we investigated

239 whether RTP801 knockdown in R6/1 mice could affect these two Akt modulators.

240 Strikingly, RTP801 knockdown normalized specifically the synaptic levels of Rictor in

241 R6/1 mice (Figure 6D) without altering the levels of PHLPP1 (Figure 6E). Hence,

242 these results suggest that RTP801 knockdown prevents Akt hyperactivation by

243 decreasing Rictor protein levels and thus downregulating the activity of mTORC2.

244 We next asked whether RTP801 downregulation could be translated to

245 synaptic transmission modulation. Striatal RTP801 silencing induced an increase of

246 AMPA receptor subunit GluA1 at the homogenate fraction in shRTP801-injected

247 R6/1 mice in comparison to shCtr-injected R6/1 mice whereas non-significant

248 tendencies were observed at the synaptic compartment (Fig 7A). One of the

249 mechanisms contributing to synaptic impairment in the striatum of HD mouse models

250 is the imbalance between BDNF receptors, TrkB and p75NTR 47,48. We found that

251 RTP801 silencing increased TrkB levels in both homogenates and synaptosomes of

252 R6/1 mice striatum in comparison to their shCtr-injected R6/1 littermates (Figure 7B)

253 whereas p75NTR was nor modified in any compartment analyzed (Figure 7C). Neither

254 synaptosomal levels of PSD-95 (Figure 7D) nor NR1 (Figure 7E) were affected by

255 the knockdown of RTP801.

256 These evidences indicate that striatal RTP801 knockdown prevents R6/1 motor

257 learning deficit by decreasing Rictor levels, normalizing the bulk of P-Ser473-Akt and

258 possibly, by enhancing postsynaptic signaling.

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259

260 Altogether, the results indicate that RTP801 is increased in the synapses from the

261 striatum, the most susceptible brain area to mhtt toxicity in both humans and HD

262 mouse models, and its upregulation contributes to impair motor learning-related

263 synaptic plasticity (Figure 8).

264

265

266 DISCUSSION

267

268 HD is characterized by the appearance of aberrant synaptic plasticity and the

269 progressive and selective loss of neuronal subpopulations 10,11,19,49–51, in which

270 mTOR plays a critical role 13. In this work, we have investigated whether mhtt-

271 induced RTP801 increase contributes to the impairment of synaptic plasticity in HD.

272 Our results show that RTP801 is elevated in cultured neurons’ synapses

273 overexpressing mhtt exon-1. Moreover, putamen, but not cortex, of HD patients

274 displays increased levels of synaptic RTP801 respect to unaffected individuals. In

275 addition, in HD mouse models, RTP801 is highly enriched in the striatal synaptic

276 compartment when compared to WT animals. Notably, the knockdown of RTP801

277 expression in the striatum of R6/1 mice prevents motor learning deficits, which is

278 accompanied by a synaptic restoration of Rictor and P-Ser473-Akt levels and an

279 increase of GluA1 and TrkB receptors in the striatum.

280

281 We first demonstrated that mhtt elevates RTP801 protein in the soma and in the

282 dendritic spines of primary cortical neurons, in accordance with our previous results

283 obtained in differentiated PC12 cells 29. Interestingly, mhtt-induced RTP801 up

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284 regulation courses along with a reduction in spine density. These results are in line

285 with extensive work reporting the loss of dendritic spines in neurons overexpressing

286 mhtt 36,52 and suggest that RTP801 synaptic increase may be contributing to this

287 phenomena.

288 Indeed, in human HD putamen, RTP801 protein levels were not only increased in

289 whole lysates, as we previously described 29, but also in synaptosomes. Structural

290 and morphological synaptic alterations in striatal medium-sized spiny neurons 53,54

291 and loss of dendritic spines in HD post-mortem brains have been widely reported

292 37,53,54. In accordance with our findings, PSD-95 is also decreased in the striatum of

293 HD patients 37. Since we observed that RTP801 is enriched in the synaptic fraction

294 and is highly accumulated in HD synaptic contacts in the striatum, we speculated

295 that synaptic RTP801 in the remaining striatal dendritic spines could have a role in

296 the altered plasticity associated to this neurodegenerative process. In addition, we

297 found that RTP801 is also enriched in human cortical synapses, but no differences

298 were found between controls and HD patients in the synaptic fraction. Even though

299 morphological alterations in HD prefrontal cortical pyramidal neurons have been

300 described 18, our results indicate that RTP801 elevation is specific for the striatum. In

301 line with this, neither the postsynaptic marker PSD-95 or the presynaptic marker

302 SV2A were altered in the prefrontal cortex. Our results thus suggest that may be

303 intrinsic neuronal properties modulate the effect of mhtt depending on the subcellular

304 type.

305

306 In HD pathogenesis, synaptic impairment precedes motor symptoms 7–9. In fact, HD

307 mouse models are reported to exhibit cognitive deficits before the appearance of

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308 motor symptoms, indicating that abnormal synaptic plasticity occurs at early stages

309 of the disease also in mouse models 46,55.

310 In accordance with the lack of neuronal death in HD mouse models 41,42,56–58, striatal

311 levels of RTP801 in homogenates were not altered either in R6/1 or KI mice.

312 However, specifically analyzing protein levels in synaptosomal preparations, we

313 concluded that mhtt leads to a localized aberrant accumulation of RTP801 in

314 synapses that could contribute to plasticity impairment in the pathology. In fact,

315 structural alterations at the morphology and/or spine number in the striatum of the

316 R6/1 59 and the KI 60 have been reported. This observation is also extended to other

317 HD murine models such as N-terminal fragment R6/2 mice 61–63 and transgenic full-

318 length htt YAC128 mice 64. As a synaptic marker, PSD-95 levels are reduced in

319 those HD animal models 46,65,66, supporting our results.

320

321 To evaluate the cortico-striatal function, responsible for motor learning 67, R6/1 mice

322 transduced with AAV-shCtr or AAV-shRTP801 were subjected to the accelerating

323 rotarod test. Interestingly, silencing RTP801 in the striatum of R6/1 mice preserves

324 their performance in the accelerating rotarod, comparable to WT mice, indicating that

325 RTP801 knockdown retains the ability to learn new motor skills in the HD mice. The

326 fact that silencing RTP801 in WT animals does not affect their performance at this

327 paradigm could suggest that remaining basal levels of RTP801 are sufficient to

328 maintain proper motor learning plasticity and, therefore, cortico-striatal functionality.

329 Highlighting the function of RTP801 in synaptic plasticity, its downregulation in the

330 Substantia Nigra pars compacta has also been shown to restore motor learning skills

331 in a PD mice model subjected to chronic stress 68.

332

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333 The only known function of RTP801 is as a mTOR signaling repressor. In PD studies

334 using cellular and animal models, RTP801 upregulation upon cell stress represses

335 mTOR and finally inhibits Akt pro-survival signals, triggering neuronal death. In PD

336 cellular models and nigral neurons of PD brains, RTP801 increases along with a

337 decrease in the phosphorylation of Akt and S6 30,31. Indeed, in HD, alterations in the

338 activity of both mTORC1 40 and mTORC2 24 have been described. Here, we confirm

339 that both mTOR complexes display aberrant activity and, importantly, that alterations

340 found at total cell level are extended to the synaptic compartment.

341 Unlike in PD models, we show that mhtt increases RTP801 despite the

342 hyperphosphorylation of Akt at Ser473 residue in the synaptosomes. Interestingly, P-

343 Ser235/236-S6 displays the same trend. The fact that RTP801 levels are increased

344 in HD models may also be a consequence of mTOR pathway hyperactivity, since

345 RTP801 protein synthesis is mTORC1-dependent 69.

346

347 Supporting its synaptic role, RTP801 silencing also alters the levels of mTOR

348 pathway-associated protein components and enhances the expression of

349 postsynaptic proteins. Our data indicate that RTP801 downregulation in R6/1 mice

350 striatum prevents Akt hyperphosphorylation at Ser473 residue, both at homogenates

351 and synaptosomes. Overactivation of Akt has been proposed to be a pro-survival

352 response to counteract mhtt toxicity in the disease 22–24 However, some evidences

353 have also pointed out that synaptic plasticity alterations occur along with

354 hyperphosphorylation of Akt 70 and that sustained overactivation of Akt could be

355 detrimental for cell survival and synaptic function 71,72. Moreover, the function of

356 mTOR complexes can differ between neuronal compartments. For instance,

357 mTORC2 at the soma mediates the activation of Akt to ensure neuronal survival,

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358 whereas mTORC2 close to the synapse mediates the activation of PKC, responsible

359 for the polymerization of actin and receptor trafficking (reviewed in 73. Thus, RTP801

360 function could also be different between cell compartments. Altogether, this evidence

361 confirms the regulation of RTP801 over mTORC2-dependent Akt phosphorylation

362 but, in the presence of mhtt, the regulation is not negative.

363

364 Phosphorylation levels of Akt at Ser473 depend on a proper activity of the mTOR

365 kinase complexed with Rictor 74 and the phosphatase PHLPP1 25,26, that specifically

366 dephosphorylates this residue. In fact, in HD, increased Rictor and decreased

367 PHLPP1 levels are both responsible for Akt overactivation 23,24. In line with that, we

368 found that Rictor synaptic up regulation in the R6/1 mice is sensitive to RTP801

369 levels since RTP801 knockdown specifically reduces synaptosomal Rictor levels

370 without affecting PHLPP1. Still the regulation of RTP801 towards Rictor or whether it

371 exists any feedback mechanisms over Akt phosphorylation remains uncertain.

372 Interestingly, we observed that RTP801 knockdown in the striatum elevated the

373 levels of AMPA subunit receptor GluA1. AMPA receptors normally are

374 heterotetramers composed mostly by calcium impermeable GluA2 subunits.

375 However, GluA1 subunit confers calcium permeability to the pore, allowing signaling

376 activation and synaptic regulation 75–77. Therefore, we speculate that this GluA1 up

377 regulation could affect the composition of the AMPA receptors and promote synaptic

378 strength. In line with this, RTP801 knockdown elevated TrkB levels in R6/1

379 homogenates and the same trend was observed in the synaptosomal fraction, with

380 no changes in p75NTR. Both TrkB and p75NTR are BDNF receptors, although each

381 receptor activates different signaling pathways. BDNF mediates neuronal survival by

382 the activation of TrkB receptor 78,79 whereas p75NTR can activate apoptotic signals

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383 leading to neuronal death 47,80–83. In fact, imbalance of TrkB and p75NTR has been

384 proposed to mediate impaired plasticity in HD 47,84. Hence, increased TrkB and

385 GluA1 levels, as a result of RTP801 silencing, could promote neuroprotection and

386 enhancement of synaptic transmission 78,79,84.

387

388 CONCLUSIONS

389 Our results indicate that downregulation of striatal RTP801 prevents motor learning

390 deficits in R6/1 mouse model, in part by by preventing Akt hyperphosphorylation at

391 the Ser473 residue via Rictor downregulation and by increasing postsynaptic GluA1

392 and TrkB receptors. Hence, RTP801 emerges as a promising target for future

393 therapeutic strategies to prevent or at least halt the progression of HD.

394

395

396 METHODS

397

398 Cortical primary neurons. Primary cortical cultures were obtained from rat

399 Sprague-Dawley embryos at day 18 (E18) as previously described 29. Briefly,

400 cortices were dissected out, dissociated in 0.05% trypsin (Sigma-Aldrich) and

401 maintained in Neurobasal medium supplemented with B27 and 2mM GlutaMAXTM

402 (all from Thermo Fisher Scientific) in a 5% CO2 humified atmosphere at 37ºC. For

403 imaging experiments, neurons were plated at a density of 300 cells/mm2 on poly-L-

404 lysine-coated (Sigma-Aldrich) in seeding medium containing MEM medium (Thermo

405 Fisher Scientific) supplemented with 10% heat-inactivated horse serum (Sigma-

406 Aldrich). After 60 minutes, medium was replaced for supplemented Neurobasal.

407 Medium was replenished one third every 7 days.

16 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030080; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

408 Transfection of the constructs eGFP, Q25, Q72 and Q103 and immunocytochemistry

409 of neuronal cultures were performed as described elsewhere 85. Briefly, cultures

410 were incubated with the primary antibody mouse monoclonal against eGFP (1:1000,

411 Santa Cruz Biotechnology) and rabbit polyclonal against RTP801 (1:150, Proteintech

412 Group Inc.). AlexaFlour Phalloidin 568 (1:1000, Thermo Fisher Scientific) was diluted

413 along with the secondary antibody during incubation. The secondary antibodies used

414 were goat anti-Mouse IgG (H+L) conjugated to AlexaFluorTM 488 or goat anti-Rabbit

415 IgG (H+L) conjugated with Alexa FluorTM 568 or AlexaFluorTM 647 (all from Thermo

416 Fisher Scientific). For nuclear staining, nuclei were revealed with Hoechst 3342

417 (Thermo Fisher Scientific) diluted 1:5000 along with the secondary antibody

418 incubation. In the case of actin filamentous labelling, AlexaFluorTM 568 Phalloidin

419 diluted 1:10000 was incubated along with the secondary antibody and Hoechst 3342.

420 Spine density was calculated by scoring the number of spines for each 15 μm

421 dendrite-length of at least 10 dendrites per condition, in three independent

422 experiments. ImageJ was used to quantify RTP801 intensity as the Integrated

423 Density in the soma and in the dendrites.

424

425 HD mouse models. R6/1 transgenic mice expressing the exon-1 of mhtt with 145

426 CAG repeats were obtained from Jackson Laboratory and maintained in B6CBA

427 background. HdhQ7 WT mice, with 7 CAG repeats, and HdhQ111 knock-in mice, with

428 targeted insertion of 109 CAG repeats that extends the glutamine segment in murine

429 htt to 111 residues, were maintained on a C57Bl/6 genetic background. Male and

430 female HdhQ7/Q111 heterozygous were intercrossed to generate age-matched

431 HdhQ7/Q7 WT and HdhQ7/Q111 knock-in littermates. All mice used were males and were

432 housed together in numerical birth order in groups of mixed genotypes. Data were

17 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030080; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

433 recorded for analysis by microchip mouse number. Animals were housed with

434 access to food and water ad libitum in a colony room kept at 19-22ºC and 40-60%

435 humidity, under a 12:12 hours light/dark cycle. All procedures were carried out in

436 accordance with the National Institutes of Health Guide for the Care and Use of

437 Laboratory Animals and approved by the local animal care committee of the

438 Universitat de Barcelona, following European (2010/63/UE) and Spanish

439 (RD53/2013) regulations for the care and use of laboratory animals.

440

441 Human postmortem tissue. Postmortem striatal and frontal cortical brain tissues

442 from control and HD patients were obtained from the Neurological Tissue Bank

443 (Biobank-HC-IDIBAPS) thanks to Dr. Ellen Gelpi collaboration. (See Supplementary

444 Table 1)

445

446 Crude synaptosomal preparations. Synaptosomes were prepared from mouse

447 striata and human putamen and frontal cortex as previously described elsewhere

448 with minor modifications. Briefly, dissected tissue was homogenized in Krebs-Ringer

449 (KR) buffer (125 mM Nacl, 1.2 mM KCl, 22 mM NaHCO3, 1 mM HaH2PO4, 1.2 mM

450 MgSO4, 1.2 mM CaCl2, 10 mM glucose (pH=7.4)) supplemented with 0.32 M

451 Sucrose. Then, samples were subjected to a first centrifugation at 1,000g for 10

452 minutes (4ºC) to discard debris. A sample of the supernatant was kept as the

453 homogenate fraction. After, the supernatant was subjected to a second

454 centrifugation at 16,000g for 15 minutes (4ºC) to obtain the crude synaptosomal

455 fraction. The pellet was finally resuspended in KR 0.32M sucrose buffer and

456 subjected to WB to analyze protein content.

457

18 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030080; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

458 Western blotting. Homogenate and synaptosomal protein analysis was performed

459 by Western blotting as previously described 86. The following primary antibodies

460 were used: Akt rabbit polyclonal (1:1000, Cell Signaling), Akt-phospho Ser473 rabbit

461 polyclonal (1:1000, Cell Signaling), mTOR rabbit polyclonal (1:1000, Cell Signaling),

462 mTOR-phospho Ser2448 (1:1000, Cell Signaling), anti-NR1 mouse monoclonal

463 (1:1000, Chemicon), p75NTR rabbit polyclonal (1:1000, Thermo Fisher Scientific),

464 PHLPP1 rabbit polyclonal (1:500, Cayman Chemical), PSD-95 mouse monoclonal

465 (1:1000, Thermo Fisher Scientific), RTP801 (1:500, Proteintech Group Inc.), RPS6-

466 phospho Ser235/236 rabbit polyclonal (1:1000, Cell Signaling), SV2A mouse

467 monoclonal (1:1000, Santa Cruz Biotechnology) and TrkB mouse monoclonal

468 (1:1000, BD Bioscience). Then, membranes were incubated with the corresponding

469 secondary antibodies: goat anti-Mouse IgG (H+L) and goat anti-Rabbit IgG (H+L)

470 conjugated to horseradish peroxidase (1:10,000, Thermo Fisher Scientific). Loading

471 control was obtained incubating with anti-actin-horseradish peroxidase conjugated

472 (1:100,000, Sigma Aldrich). Chemiluminescent images were acquired using a LAS-

473 3000 imager (Fuji) or ChemiDoc (Bio-Rad) imaging systems and quantified by

474 computer-assisted densitometric analysis (Image J software).

475

476 Intrastriatal injection of adeno-associated vectors. To knockdown RTP801

477 expression, shRNA verified scrambled sequence (5’-GTGCGTTGCTAGTACCAAC-

478 3’) and the one against RTP801 (5’-AAGACTCCTCATACCTGGATG-3’) were cloned

479 into a rAAV2/8-GFP adenoviral vector at restriction sites BamHI at 5’ and Agel at the

480 3’. The rAAV2/8 plasmids and infectious AAV viral particles containing GFP

481 expression cassette with shCtr or shRTP801 were generated by the Vectors

482 Production Unit from the Center of Animal Biotechnology and Gene Therapy at the

19 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030080; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

483 Universitat Autònoma de Barcelona. Nine-week-old WT and R6/1 mice were

484 subjected to bilateral intrastriatal injections of rAVV2/8 expressing shRTP801 or

485 control shRNA. Animals were deeply anesthetized with a mixture of oxygen and

486 isoflurane (4-5 for induction and 1-2 for maintaining anesthesia) and placed in a

487 stereotaxic apparatus for bilateral intrastriatal injections (2 μl; 15×109 genomic

488 copies). Two injections were performed in the striatum at the following coordinates

489 relative to bregma: (1) anteroposterior (AP), +0.8; mediolateral (ML), +/-1.8; and

490 profundity, -2.6 mm and (2) AP, +0.3; ML, +/- 2; and profundity, -2.6 mm, below

491 the dural surface. Viral vectors were injected using a 10 μl-Hamilton microliter

492 syringe at an infusion rate of 200 nl/min. The needle was left in place for 5 min to

493 ensure complete diffusion of the viruses and then slowly retracted from the brain.

494 Both hemispheres were injected with the same shRNA.

495

496 Accelerating rotarod. Five weeks after intrastriatal injection of rAAV2/8-shRNAs,

497 animals were subjected to the accelerating rotarod test to analyze motor learning.

498 Mice were placed on a 3 cm rod (Panlab) with an increasing speed from 4 to 40 rpm

499 over 5 minutes. Latency to fall was recorded as the time mice spent in the rod before

500 falling. Accelerating rotarod test was performed for 4 days, 3 trials per day. Trials in

501 the same day were separated by 1 hour.

502

503 Immunohistofluorescence of mouse brain sections. Animals were deeply

504 anesthetized with dolethal (Vétoquinol) (60 mg/kg), and intracardially perfused with

505 4% paraformaldehyde in PBS buffer (pH=7.2-7.4). Brains were removed and post-

506 fixed for 18-24h, and cryoprotected with 30% sucrose in PBS 0.02% sodium azide

507 and frozen in dry-ice cooled 2-methylbutane. Serial cryostat 25 µm-thick sections

20 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030080; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

508 were collected in PBS 0.2% sodium azide as free-floating sections and processed for

509 immunohistofluorescence. Sections were washed with PBS and incubated with 50

510 mM NH4Cl for 30 minutes to block aldehyde free-induced fluorescence. Tissue was

511 blocked with SuperBlock-PBS containing 0.3% Triton X-100 for 2 hours at room

512 temperature. After, slices were incubated overnight at 4ºC with the primary

513 antibodies anti-GFP chicken polyclonal (Synaptic Systems) diluted 1:500 in

514 SuperBlock-PBS 0,3% Triton X-100. Later, sections were washed in PBS and

515 incubated for 2 hours at room temperature with the secondary antibody goat anti-

516 Chicken IgY (H+L) conjugated to AlexaFluorTM 488 diluted 1:500 in SuperBlock-PBS

517 0.3% Triton X-100. For nuclear staining, nuclei were revealed with Hoechst 3342

518 diluted 1:5000 along with the secondary antibody incubation. Slices were washed

519 with PBS before being mounted with ProLongTM Gold Antifade Mountant on

520 SuperFrostTM Plus Adhesion Slides (Thermo Fisher Scientific). Stained mouse brain

521 sections were observed by epifluorescent microscopy.

522

523 Statistical analysis. All experiments were performed at least in triplicate, and results

524 are reported as mean+SEM. Student’s T-test was performed as unpaired, two-tailed

525 sets of arrays and presented as probability P values. One-way ANOVA with

526 Bonferroni’s multiple comparison test and Two-way ANOVA followed by Bonferroni’s

527 post-hoc tests were performed for the comparison of multiple groups. Values of P <

528 0.05 were considered as statistically significant.

529

530 ABBREVIATIONS: Huntington’s disease (HD); knock out (KO); mechanistic target of

531 rapamycin (mTOR); mTOR complex (mTORC); mutant huntingtin (mhtt);

532 postsynaptic density (PSD); Western blot (WB); wild type (WT)

21 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030080; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

533

534 ACKNOWLEDGEMENTS: The authors thank Dr. M MacDonald (Massachusetts

535 General Hospital, Boston, Massachusetts, USA) for the HdhQ7/Q111 mice. The

536 constructs Q25 Q72 and Q103 were a kind gift of Dr. G.M. Lawless (Cure HD

537 Initiative, Reagent Resource Bank of the Hereditary Disease Foundation, New York,

538 NY). We also thank the Neurological Tissue Bank of the Biobanc-Hospital Clínic-

539 IDIBAPS (Barcelona, Spain) and Dr. Ellen Gelpi for providing human tissue samples,

540 and Ana López for her technical support. We thank Maria Calvo from the Advanced

541 Microscopy Unit, Scientific and Technological Centers, University of Barcelona, for

542 their support and advice in confocal techniques.

543

544

545 DECLARATIONS:

546 Declarations:

547 Ethical Approval and Consent to participate:

548 All procedures were performed in compliance with the NIH Guide for the Care and

549 Use of Laboratory Animals and approved by the local animal care committee of

550 Universitat de Barcelona following European (2010/63/UE) and Spanish

551 (RD53/2013) regulations for the care and use of laboratory animals.

552 Human samples were obtained following the guidelines and approval of the local

553 ethics committee (Hospital Clínic of Barcelona’s Clinical Research Ethics

554 Committee).

555 Consent for publication: Non applicable

556 Availability of supporting data: All data generated or analyzed during this study

557 are included in this published article and the supplementary information files.

22 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030080; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

558 Competing interests: The authors declare that they have no competing interests to

559 disclose.

560 Funding: This work was funded by the Spanish Ministry of Economy and

561 Competitivity with the following grants: SAF2017-8812-R and SAF2014-57160R

562 Authors' contributions: N.M-F., L.P-S., J.C-M., M.M., S.G., J.A., E.P-N. and C.M.

563 have contributed in the conception and design of the study, acquisition and analysis

564 of data and in drafting the manuscript and figures. All authors read and approved

565 the final manuscript.

566

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846

847

848 FIGURE TITLES AND LEGENDS

849

850 Figure 1. Overexpression of exon-1 mhtt increases RTP801 protein levels in

851 dendrites and in synaptic spines. (A1) Rat cortical cultures at DIV13 were

852 transfected with eGFP, Q25, Q72 or Q103 plasmids. Twenty-four hours after

853 transfection cultures were fixed and stained against GFP (in green), RTP801 (in

854 grey) and phalloidin (in red) to visualize the actin cytoskeleton. Images were

855 acquired by confocal microscopy. Yellow rectangles show digital zoom of dendrites

856 with spines and yellow arrows show RTP801 staining at the puncta. Scale bar, 10μm

857 and in higher magnification images, 1.5μm. (B) Graphs display the number of spines

34 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030080; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

858 scored for each 15 µm dendrite-length and RTP801 staining intensity quantification

859 at (C) the neuronal soma and at (D) the spines. Data are shown as percentage of

860 RTP801 intensity (mean ± SEM) of four independent experiments and were

861 analysed with One-way ANOVA followed by Dunnett’s multiple comparisons test

862 (*P0.05 and **P0.01 vs. eGFP and #P0.05 vs. Q25, n=13 dendrites for eGFP,

863 n=10 dendrites for Q25, Q72 and Q103 per experiment).

864

865 Figure 2. RTP801 is increased in the synaptic fraction derived from the

866 putamen of HD patients. (A,B) Putamen of 6 HD patients and 5 control individuals

867 lysates and synaptosomes were subjected to WB. Membranes were probed against

868 RTP801, SV2A, PSD-95, P-Akt (Ser473), P-S6 (Ser235/236), total S6 and total Akt

869 was used as a loading control. Graphs show the densitometric quantification of

870 synaptosomal levels. (C,D) Graphs indicate the levels of RTP801, P-Akt (Ser473)

871 and P-S6 (235/236) relative to synaptic markers (C) SV2A and (D) PSD-95 in the

872 synaptosomes. The results are shown as mean ± SEM. Data were analyzed by

873 Student’s T-test (*P<0.05).

874

875 Figure 3. RTP801 is increased in the synaptic fraction derived from the

876 striatum of HdhQ7/Q111 mice. (A,B) Striatal lysates and synaptosomes of 6 KI and

877 6 WT animals at 10-months of age were subjected to WB. Membranes were probed

878 against RTP801, P-Akt (Ser473), P-S6 (Ser235/236), PSD-95, SV2A and total Akt

879 was used as loading control. Graphs show the densitometric quantification of

880 synaptosomal levels. (C,D) Graphs indicate the levels of RTP801, P-Akt (Ser473)

881 and P-S6 (235/236) relative to synaptic markers (C) SV2A and (D) PSD-95 in the

35 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030080; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

882 synaptosomes. The results are shown as mean ± SEM. Data were analysed by

883 Student’s T-test (*P<0.05, **P<0.01).

884

885 Figure 4. RTP801 is increased in the synaptic fraction derived from the

886 striatum of R6/1 mice. Striatal lysates and synaptosomes of 7 R6/1 and 6 WT

887 animals at 16-weeks of age were subjected to WB. Membranes were probed against

888 RTP801, P-Akt (Ser473), P-S6 (Ser235/236), PSD-95, SV2A and total Akt as a

889 loading control. Graphs show the densitometric quantification of synaptosomal

890 levels. (C,D) Graphs indicate the levels of RTP801, P-Akt (Ser473) and P-S6

891 (235/236) relative to synaptic markers (C) SV2A and (D) PSD-95 in the

892 synaptosomes. The results are shown as mean ± SEM. Data were analyzed by

893 Student’s T-test (**P<0.01, ***P<0.001).

894

895 Figure 5. Striatal RTP801 knockdown preserves motor learning in symptomatic

896 R6/1 mouse. (A) GFP immunofluorescence confirmed the transduction of AAV-shCtr

897 and AAV-shRTP801 in WT and R6/1 mice striatal cells. A secondary control was

898 performed without the incubation of primary antibody (anti-GFP). Scale bar 450μm.

899 (B) Striatal lysates of WT and R6/1 injected with AAV-shCtr (n=6 WT and n=6 R6/1)

900 or AAV-shRTP801 (n=6 WT and n=7 R6/1) were subjected to WB. Membranes were

901 probed against RTP801, GFP and actin as a loading control. Graph show the

902 densitometric quantification of RTP801 signal. Data are shown as a mean ± SEM

903 and were analyzed with Two-way ANOVA followed by Bonferroni’s multiple

904 comparisons test for post-hoc analyses (*P<0.05 vs. WT AAV-shCtr #P<0.05 vs.

905 R6/1 AAV-shCtr). (C) Five weeks after adeno-associated transduction, motor

906 learning was assessed in the same animals by the accelerating rotarod. The graph

36 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030080; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

907 shows the latency to fall as the mean of three trials tested each day. Values are

908 expressed as mean ± SEM and were analysed with Two-way ANOVA followed by

909 Bonferroni’s multiple comparisons test for post-hoc analyses (*P<0.05, **P<0.01 and

910 ***P<0.001 R6/1 shCtr vs. WT-shCtr and #P<0.05 R6/1-shRTP801 vs. R6/1-shCtr).

911

912 Figure 6. Knockdown of striatal RTP801 in R6/1 mice prevents

913 hyperphosphorylation of Akt-(Ser473) by decreasing Rictor levels. Striatal

914 lysates and synaptosomes of WT and R6/1 injected with AAV-shCtr (n=6 WT and

915 n=6 R6/1) or AAV-shRTP801 (n=6 WT and n=7 R6/1) were subjected to WB.

916 Membranes were probed against (A) P-mTOR (Ser2448), (B) P-S6 (Ser235/236),

917 (C) P-Akt (Ser273), (D) Rictor and (E) PHLPP1. Total mTOR, S6, Akt and actin were

918 used as loading controls. Graphs show the densitometric quantification. Values are

919 shown as a mean ± SEM. Homogenates and synaptosomes data were analysed with

920 Two-way ANOVA followed by Bonferroni’s multiple comparisons test for post-hoc

921 analyses(*P<0.05, **P<0.01, **P<0.010 vs. WT AAV-shCtr; $P<0.05 vs. WT AAV-

922 shRTP801; #P<0.05, ##P<0.01 vs. R6/1 AAV-shCtr).

923

924 Figure 7. Knockdown of RTP801 in the striatum of R6/1 mice enhances the

925 expression of synaptic proteins. Striatal lysates and synaptosomes of WT and

926 R6/1 injected with AAV-shCtr (n=6 WT and n=6 R6/1) or AAV-shRTP801 (n=6 WT

927 and n=7 R6/1) were subjected to WB. Membranes were probed against (A) GluA1,

928 (B) TrkB, (C) p75NTR, (D) PSD-95 and (E) NR-1. Total actin was used as loading

929 controls. Graphs show the densitometric quantification. Values are shown as a mean

930 ± SEM. Homogenates and synaptosomes data were analysed with Two-way ANOVA

37 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030080; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

931 followed by Bonferroni’s multiple comparisons test for post-hoc analyses; #P<0.05

932 vs. R6/1 AAV-shCtr).

933 Figure 8. RTP801 knockdown prevents corticostriatal learning deficits. In

934 comparison to WT mice, R6/1 mice display increased RTP801 at the striatal synapse

935 along with increased Rictor and P-Akt (Ser473) and motor learning deficits. RTP801

936 silencing preserves corticostriatal motor learning function by decreasing the levels of

937 Rictor and P-Akt Ser473 and also enhancing postsynaptic signaling by increasing

938 GluA1 and TrkB receptors.

939

940 SUPPLEMENTAL INFORMATION TITLES AND LEGENDS:

941

942 Figure S1. Synaptosomal enrichment of proteins in the putamen of HD

943 patients. Putaminal lysates and synaptosomes of 5 HD patients and 5 control

944 individuals were subjected to WB. Membranes were probed against (A) RTP801, (B)

945 SV2A, (C) PSD-95, (D) P-Akt (Ser473) and (E) P-S6 (Ser235/236) and total Akt as a

946 loading control. Graphs show the densitometric quantification. Data is shown as a

947 mean ± SEM. Data were analyzed with Two-way ANOVA followed by Bonferroni’s

948 multiple comparisons test for post-hoc analyses (***P<0.001 vs. CT homogenate)

949 and data of homogenates and synaptosomes were analyzed by Student’s T-test

950 (*P<0.05, **P<0.01).

951

952 Figure S2. Synaptosomal enrichment of proteins in the cortex of HD patients.

953 Prefrontal cortex lysates and synaptosomes of 5 HD patients and 6 control

954 individuals were subjected to WB. Membranes were probed against (A) RTP801, (B)

955 SV2A, (C) PSD-95, (D) P-Akt (Ser473) and (E) P-S6 (Ser235/236) and total Akt as a

38 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030080; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

956 loading control. Graphs show the densitometric quantification. Data is shown as a

957 mean ± SEM. Enrichment data was analyzed with Two-way ANOVA followed by

958 Bonferroni’s multiple comparisons test for post-hoc analyses (*P<0.05, **P<0.01,

959 ***P<0.001 vs. CT homogenate; ### P<0.001 vs. HD homogenate). Data from

960 homogenates was analyzed with Student’s t-test.

961

962 Figure S3. RTP801 is not altered in the synaptic fraction derived from the

963 cortex of HD patients. (A, B) Prefrontal cortex lysates and synaptosomes of 6 HD

964 patients and 6 control individuals were subjected to WB. Membranes were probed

965 against RTP801, P-Akt (Ser473), P-S6 (Ser235/236), PSD-95, SV2A and total Akt as

966 a loading control. Graphs show the densitometric quantification of synaptosomal

967 levels. (C, D) Graphs indicate the levels of RTP801, P-Akt (Ser473) and P-S6

968 (235/236) relative to synaptic markers (C) SV2A and (D) PSD-95 in the

969 synaptosomes. The results are shown as mean ± SEM. Data were analyzed by

970 Student’s T-test (*P<0.05, **P<0.01).

971

972 Figure S4. Synaptosomal enrichment of proteins in the in the striatum of

973 HdhQ7/Q111 mice. Striatal lysates and synaptosomes of 6 KI and 6 WT animals at

974 10-months of age were subjected to WB. Membranes were probed against (A)

975 RTP801, (B) SV2A, (C) PSD-95, (D) P-Akt (Ser473) and (E) P-S6 (Ser235/236) and

976 total Akt as a loading control. Graphs show the densitometric quantification. Data is

977 shown as a mean ± SEM. Data were analyzed with Two-way ANOVA followed by

978 Bonferroni’s multiple comparisons test for post-hoc analyses (**P<0.01, ***P<0.001

979 vs. WT homogenate, ##P<0.01, ###P<0.001 vs. KI homogenate) and data of

980 homogenates were analysed by Student’s T-test (*P<0.05).

39 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030080; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

981

982 Figure S5. Synaptosomal enrichment of proteins in the striatum of R6/1 mice.

983 Striatal lysates and synaptosomes of 7 R6/1 and 6 WT animals at 16-weeks of age

984 were subjected to WB. Membranes were probed against (A) RTP801, (B) SV2A, (C)

985 PSD-95, (D) P-Akt (Ser473) and (E) P-S6 (Ser235/236) and total Akt as a loading

986 control. Graphs show the densitometric quantification. Data in shown as a mean ±

987 SEM. Data were analysed with Two-way ANOVA followed by Bonferroni’s multiple

988 comparisons test for post-hoc analyses (*P<0.05, **P<0.01, ***P<0.001 vs. WT

989 homogenate, #P<0.05, ###P<0.001 vs. R6/1 homogenate) and data of

990 homogenates were analysed by Student’s T-test (***P<0.001).

991

992 Figure S6. Enrichment of proteins in the striatum of R6/1 after RTP801

993 knockdown. Striatal lysates and synaptosomes of WT and R6/1 injected with AAV-

994 shCtr (n=6 WT and n=6 R6/1) or AAV-shRTP801 (n=6 WT and n=7 R6/1) were

995 subjected to WB. Graphs show the densitometric quantification of (A) P-mTOR

996 (Ser2448), (B) P-S6 (Ser235/236), (C) P-Akt (Ser273), (D) Rictor, (E) PHLPP1, (F)

997 GluA1, (G) TrkB and (H) p75NTR. Values are shown as a mean ± SEM. Enrichment

998 data were analyzed with Two-way ANOVA followed by Bonferroni’s multiple

999 comparisons test for post-hoc analyses (**P<0.01, ***P<0.001 vs. WT AAV-shCtr

1000 Hom; #P<0.05, ##P<0.01, ###P<0.001 vs. R6/1 AAV-shCtr Hom; ; ++P<0.01,

1001 +++P<0.001 vs. WT AAV-shRTP801; $P<0.05, $$P<0.01, $$$P<0.001 vs. R6/1-

1002 shRTP801 Hom).

1003

40 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030080; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030080; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030080; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030080; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030080; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030080; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030080; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030080; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.