1 in ATG5 Reduces and Leads to

2 Ataxia with Developmental Delay

3

4 Authors: Myungjin Kim1,14, Erin Sandford2,14, Damian Gatica3,4, Yu Qiu5, Xu Liu3,4, Yumei

5 Zheng5, Brenda A. Schulman5,6, Jishu Xu7, Ian Semple1, Seung-Hyun Ro1, Boyoung Kim1, R.

6 Nehir Mavioglu8, Aslıhan Tolun8, Andras Jipa9,10, Szabolcs Takats9, Manuela Karpati9, Jun Z.

7 Li7,11, Zuhal Yapici12, Gabor Juhasz9,10, Jun Hee Lee1*, Daniel J. Klionsky3,4*, Margit

8 Burmeister2,7,11, 13*

9

10 Affiliations

11 1Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI

12 48109, USA.

13 2Molecular & Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, MI 48109,

14 USA.

15 3Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann

16 Arbor, MI 48109, USA.

17 4Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA.

18 5Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105,

19 USA.

20 6Howard Hughes Medical Institute, St. Jude Children’s Research Hospital, Memphis, TN 38105,

21 USA.

22 7Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109, USA.

23 8Department of Molecular Biology and Genetics, Boğaziçi University, 34342 Istanbul, Turkey.

1

24 9Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Budapest

25 H-1117, Hungary.

26 10Institute of Genetics, Biological Research Centre, Hungarian Academy of Sciences, Szeged H-

27 6726, Hungary

28 11Department of Computational Medicine & Bioinformatics, University of Michigan, Ann Arbor,

29 MI 48109, USA.

30 12Department of Neurology, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey.

31 13Department of Psychiatry, University of Michigan, Ann Arbor, MI 48109, USA.

32 14co-first authors

33 *For correspondence: [email protected] (JHL); [email protected] (DJK); [email protected]

34 (MB).

35

2

36 Abstract

37 Autophagy is required for the homeostasis of cellular material and is proposed to be involved in

38 many aspects of health. Defects in the autophagy pathway have been observed in

39 neurodegenerative disorders; however, no genetically-inherited pathogenic in any of

40 the core autophagy-related (ATG) have been reported in human patients to date. We

41 identified a homozygous missense mutation, changing a conserved amino acid, in ATG5 in two

42 siblings with congenital ataxia, mental retardation, and developmental delay. The subjects’ cells

43 display a decrease in autophagy flux and defects in conjugation of ATG12 to ATG5. The

44 homologous mutation in yeast demonstrates a 30-50% reduction of induced autophagy. Flies in

45 which Atg5 is substituted with the mutant human ATG5 exhibit severe movement disorder, in

46 contrast to flies expressing the wild-type human . Our results demonstrate the critical role

47 of autophagy in preventing neurological diseases and maintaining neuronal health.

48

49

50

3

51 Introduction

52 Macroautophagy, referred to hereafter as autophagy, is a cellular process by which and

53 are degraded and recycled through sequestration within and delivery

54 to (Levine and Klionsky, 2004). The autophagy pathway is highly conserved and

55 required for organismal development and function. Defects in autophagy are associated with

56 diseases including cancer, metabolic disruption, and neurodegenerative disorder (Choi et al.,

57 2013; Cuervo and Wong, 2014; Frake et al., 2015). Patients with mutations in any of the non-

58 redundant core autophagy-related (ATG) genes have not previously been reported.

59

60 Ataxia is a neurodegenerative disease caused by disruption of the cerebellum and Purkinje cells,

61 which results in the lack of coordinated muscle movements. Large diversity is present

62 in individuals with ataxia, including age of onset, rate of progression, and other accompanying

63 neurological and non-neurological features (Jayadev and Bird, 2013), with corresponding

64 genotypic heterogeneity (Sandford and Burmeister, 2014). Even within the more defined

65 phenotype of childhood ataxia with developmental delay, there are a large number of associated

66 genes, such that similar phenotypic features alone are often insufficient information for an

67 accurate diagnosis (Burns et al., 2014; De Michele and Filla, 2012; Jayadev and Bird, 2013).

68 Identification of genetic causes of childhood ataxia is important for understanding disease

69 pathogenesis and for possible future treatment development.

70

71 Whole exome sequencing has been successfully utilized to identify known and novel genetic

72 mutations responsible for ataxia (Burns et al., 2014; Fogel et al., 2014). Identification of

73 candidate genes can be further verified through additional molecular analysis and utilization of

4

74 specific and general animal models. Here we identified a novel mutation in a core autophagy

75 , ATG5, in two children with ataxia, and demonstrate a reduction in autophagic response,

76 also reproducing the phenotype in yeast and fly models.

77

78 Results

79 E122D Mutation in ATG5 is Associated with Familial Ataxia

80 Two Turkish siblings presented with ataxia and developmental delay in childhood, as previously

81 described (Yapici and Eraksoy, 2005). We performed linkage analysis on both affected siblings,

82 their unaffected siblings, and their unaffected mother, using a model of remote parental

83 consanguinity and identified a single broad (>14 Mbp) peak with LOD score 3.16 on

84 6q21, between 102 and 116 Mb (Figure 1). Whole exome sequencing identified a

85 homozygous missense mutation, hg19 chr6:106,727,648 T>A, corresponding to E122D in ATG5

86 (Figure 2A) as the only damaging mutation within the genetically identified chromosomal

87 linkage interval. The mutation was Sanger verified and found absent from variant databases and

88 from Turkish controls.

89

90 Cells from ATG5E122D/E122D Patients Exhibit Reduced ATG12–ATG5 Expression

91 ATG5 plays a role in elongation of the phagophore and its subsequent maturation into the

92 complete . ATG12 is a -like protein that covalently binds ATG5

93 (Mizushima et al., 1998a), and this conjugate noncovalently binds ATG16L1. Crystal structure

94 of the resulting ATG12–ATG5-ATG16L1 complex indicated that E122 is located in the vicinity

95 of the ATG12–ATG5 interaction surface (Figure 2B); hence, we predicted that the mutation in

96 ATG5 could affect the conjugation of ATG12. Comparison of protein isolated from control

5

97 lymphoblastoid cell lines (LCL) and of affected subjects revealed a severe reduction of the

98 ATG12–ATG5 conjugate in the mutant cells under basal conditions (Figure 3A), suggesting that

99 the E122D mutation may have impaired autophagy by inhibiting conjugation between ATG12

100 and ATG5.

101

102 Cells from ATG5E122D/E122D Patients Exhibit Autophagic Flux Attenuation

103 The ATG12–ATG5-ATG16L1 complex functions in part as an E3 ligase to facilitate the

104 conjugation of LC3 to phosphatidylethanolamine, generating LC3-II (Fujita et al., 2008; Hanada

105 et al., 2007). Compared to control cells, LCLs from patients with the E122D mutation exposed to

106 bafilomycin A1 showed a substantial reduction in LC3-II accumulation under basal conditions

107 (Figure 3B), suggesting a possible decrease in E3 activity and subsequent attenuation of basal

108 autophagic flux. The patient LCLs were also unable to upregulate their autophagic flux in

109 response to Torin 1 (Figure 3C), which is a strong inducer of autophagy (Thoreen et al., 2009).

110 ATG5E122D LCLs also showed elevated levels of SQSTM1/p62, an autophagy substrate, further

111 indicating disruption of basal autophagy (Figure 3C).

112

113 E122D Mutation of ATG5 Impairs ATG12–ATG5 Conjugation

114 To examine the effect of the ATG5E122D mutation on formation of the ATG12–ATG5-ATG16L1

115 complex, we expressed the recombinant human proteins in insect Hi5 cells and analyzed the

116 complexes by affinity isolation. We could detect the ATG12–ATG5 complex when both wild-

117 type proteins were co-expressed, but we could only detect a minimal amount of the ATG12–

118 ATG5E122D complex (Figure 4A). Although overexpression of human ATG5WT in HEK293 cells

119 or Drosophila tissues results in efficient covalent conjugation with overexpressed human ATG12

6

120 (Figure 4, B and C) or endogenous Drosophila Atg12 (Figure 4D), mutant ATG5E122D was

121 dramatically impaired in this process (Figure 4, B, C, and D). Interestingly, expression levels of

122 ATG5WT and ATG5E122D monomers were comparable to each other, indicating that the mutation

123 affects the conjugation process, rather than the stability of proteins. This was consistent with the

124 structural location of ATG5 E122 adjacent to the surface that interacts with ATG12 (Figure 2B).

125 To confirm that the mutation does not overtly alter the structure of ATG5 or binding to ATG16,

126 we analyzed formation of the noncovalent ATG5-ATG16L1 complex using constructs

127 containing a TEV protease site. Both wild-type and mutant ATG5 protein were efficiently co-

128 precipitated with ATG16L1 (Figure 4E). Indeed, the co-crystal structure of a human ATG5E122D-

129 ATG16L1 complex (Figure 2 and Table 1) superimposes well with the previously determined

130 structure of the WT proteins (Figure 2D), with the major obvious difference being replacement

131 of the side-chain (Figure 2, E and F). Thus, it appears that the E122D mutation interferes with

132 the ATG12–ATG5 conjugation process, but not with ATG5 folding or binding of ATG16L1.

133

134 ATG5 Mutation in Yeast Results in Decreased Autophagy

135 ATG5 is a highly conserved protein, and sequence alignment demonstrated that E122

136 corresponds to yeast E141 (Figure 2, A and C). We extended our analysis of the effect of the

137 mutation on autophagy activity, by taking advantage of the yeast system. To test whether

138 autophagy was affected by the Atg5 mutation in yeast, we initially relied on the GFP-Atg8

139 processing assay (Shintani and Klionsky, 2004). During autophagy a population of Atg8 is

140 continuously transported to the inside of autophagosomes. Tagging the N terminus of

141 Atg8 with GFP makes it possible to monitor autophagy flux because Atg8 is rapidly degraded

142 inside the vacuole whereas GFP is relatively resistant to vacuolar hydrolases; the generation of

7

143 free GFP is an indication of autophagic activity. We observed a consistent decrease in autophagy

144 activity with the Atg5E141D mutant relative to Atg5WT following autophagy induction by

145 starvation (Figure 5A). Atg8, or GFP-Atg8, does not measure autophagic cargo per se (Klionsky,

146 2016), and the amount of GFP-Atg8 processing only corresponds to the inner surface of the

147 autophagosome. To corroborate the effects observed through the GFP-Atg8 processing assay we

148 examined autophagy using the quantitative Pho8∆60 assay (Noda and Klionsky, 2008). Pho8∆60

149 is an altered form of a phosphatase that is only delivered to the vacuole via autophagy;

150 subsequent proteolytic processing generates an active form of the hydrolase. After 4 and 6 hr of

151 starvation, yeast cells expressing the plasmid-based Atg5E141D mutant showed a significant

152 decrease in autophagy levels compared to cells expressing Atg5WT (Figure 5B), and similar

153 results were obtained when the WT and mutant ATG5 genes were integrated back into the

154 chromosomal ATG5 (Figure 5C).

155 To determine the reason for reduced autophagic activity we tested the effects of the

156 Atg5E141D mutant on Atg8 lipidation. As shown by the ratio of Atg8–PE:total Atg8, cells

157 expressing Atg5E141D displayed a decrease in Atg8–PE conjugation at 30 and 60 min of

158 starvation compared to cells expressing Atg5WT (Figure 5D). We extended this analysis using the

159 in vivo reconstitution of Atg8–PE conjugation as described previously (Cao et al., 2008). In

160 brief, we examined Atg8 lipidation in a multiple-knockout (MKO) strain in which 23 ATG genes

161 are deleted, when expressing only the E1, E2 and E3-like conjugation enzymes of the autophagy

162 machinery. Atg8∆R that lacks the C-terminal arginine was used in the assay to bypass the initial

163 activation step mediated by Atg4; due to the absence of Atg4, there is no cleavage of Atg8–PE

164 from the membrane, resulting in stabilization of this form of the protein. We found that the

165 Atg5E141D mutant was significantly defective in Atg8–PE conjugation compared to the cells with

8

166 Atg5WT when Atg16 was not present (Figure 5E). Atg16 is not required mechanistically for Atg8

167 conjugation, but its presence increases the efficiency of this process and may dictate the site of

168 conjugation (Cao et al., 2008; Hanada et al., 2007). Thus, the presence of Atg16 may partially

169 mask the Atg8 lipidation defects of the Atg5E141D mutant, and this may explain why the

170 E122D/E141D mutation induces a hypomorphic rather than a complete null phenotype.

171

172 ATG5E122D Fails to Complement the Ataxic Phenotype of Atg5-null flies

173 To further characterize the effect of the E122D mutation on the development of ataxia, we

174 generated knockouts for Atg5 (Figure 6A), and reconstituted the Atg5-

175 null mutant flies with transgenes expressing wild-type (WT) or E122D human ATG5 (Figure 6,

176 B-D). Unlike mouse models, Atg5-null flies are viable, although they exhibit severe mobility

177 defects after adult eclosion as demonstrated by a negative geotaxis assay (Figure 6, E and I, and

178 Video 1), similar to Atg7 null mutant flies (Juhasz et al., 2007). These mobility defects were

179 substantially restored by expression of ATG5WT (Figure 6, F and I, and Video 2), suggesting that

180 the molecular function of ATG5 is conserved between human and Drosophila. However, Atg5-

181 null mutant flies expressing ATG5E122D were still defective in mobility although slightly better

182 than Atg5-null controls (Figure 6, G-I, and Videos 3 and 4), demonstrating again that ATG5

183 activity is compromised but not eliminated by the E122D mutation. ATG5E122D was also inferior

184 to ATG5WT in suppressing Ref(2)P (fly SQSTM1) accumulation (Figure 6, J and K) and cell

185 death (Figure 6, L and M) in the brain of Atg5-null mutant flies.

186

187 Discussion

188 In summary, we demonstrate that the homozygous E122D mutation of ATG5, a unique mutation

9

189 found in two human subjects with ataxia, results in reduced conjugation to ATG12 and in an

190 overall decrease in autophagy activity. The homologous mutation in yeast also interferes with

191 autophagy, and the ataxia phenotype was replicated in a fly model. Based on these results we

192 propose that this ATG5 mutation, and the consequent disruption in autophagy activity, is the

193 cause of the ataxic phenotype and disturbance of the cerebellum in the affected siblings. This

194 hypothesis is in agreement with previously characterized mouse models, in which neuron-

195 specific knockout of Atg5 results in ataxia-like (Hara et al., 2006; Nishiyama et al.,

196 2007). By contrast, mice with complete knockout of Atg5 die shortly after birth, demonstrating

197 that autophagy is essential for mammalian survival (Kuma et al., 2004). Our results indicate that

198 E122D is a partial loss-of-function allele that impairs but does not completely abolish ATG5

199 activity. Although the overall structure of the E122D mutant ATG5 superimposes well with the

200 wild-type protein, the mutation causes a striking decrease in the level of ATG12–ATG5

201 conjugate that is formed when the C terminus of ATG12 is covalently linked to Lys130 of ATG5.

202 We speculate that the E122D mutation causes subtle changes in the conformational dynamics

203 that propagate to Lys130, which is less than 10 Å away, resulting in less ATG12–ATG5, which

204 in turn leads to reduced LC3/Atg8 conjugation.

205

206 In neurons, which are among the cells most dependent on autophagy for tissue homeostasis

207 (Button et al., 2015), the residual function of the E122D allele is inadequate, resulting in

208 predominantly neurological symptoms in the two patients. Since homozygous mutations with

209 complete loss-of-function have not been reported, we predict that individuals carrying such

210 mutations, similar to Atg5-null mice (Kuma et al., 2004), might not be viable.

211

10

212 Autophagy is quickly gaining importance for its roles in preventing neurodegeneration. WDR45

213 is a redundant, non-core autophagy gene, one of four mammalian homologs to Atg18, and

214 mutations in WDR45 cause SENDA, static encephalopathy of childhood with neurodegeneration

215 in adulthood (Haack et al., 2012; Saitsu et al., 2013). Autophagy appears to be critical in ataxia,

216 whether mutant proteins evade autophagy processes or normal autophagy is disrupted. Several

217 ataxias are attributed to intranuclear or cytoplasmic aggregation of mutant proteins within the

218 cell (Matilla-Duenas et al., 2014). These protein aggregates, in humans and in mouse models, not

219 only evade autophagic sequestration but may even inhibit autophagy (Alves et al., 2014), or lead

220 to reduction in autophagy available for other proteins due to saturation. Assessment of autophagy

221 in patient cells may be used to refine and identify the genetic cause of a patient’s ataxia.

222

223 Further discovery of the role of autophagy in neurodegenerative diseases should be used to

224 investigate therapies targeted at the autophagy process. Many drugs enhance autophagy and their

225 effects on a multitude of neurodegenerative diseases has been studied (Sarkar et al., 2009).

226 Recently more studies have been conducted assessing the value of autophagy enhancers in ataxia

227 models and patients. Induction in autophagy through administration of Temsirolimus, a

228 rapamycin ester, and lentiviral overexpression of BECN1 in SCA3 model mice increase

229 autophagy and the clearance of mutant protein aggregates, and reduce the ataxic phenotype

230 (Menzies et al., 2010; Nascimento-Ferreira et al., 2013). In a single patient trial, trehalose

231 treatment of patient fibroblasts increased autophagy and alleviated cellular pathogenic features

232 by improving mitochondrial morphology and reducing free radicals in the cell (Casarejos et al.,

233 2014; Sarkar et al., 2007). Trehalose also showed success in trials involving models of SCA17

234 (Chen et al., 2015). Lithium, another inducer of autophagy, improved symptoms in a SCA1

11

235 mouse model (Watase et al., 2007), but did not slow or reduce symptoms in a treatment trial in

236 SCA2 patients (Sacca et al., 2015). An autophagy enhancer may be an appropriate treatment to

237 test in the presented subjects, as autophagic flux is attenuated, but not completely abrogated, by

238 the ATG5E122D/E122D mutation.

239

240 This study’s finding of the pathogenic human E122D mutation in ATG5, a gene encoding part of

241 the autophagy-controlling core machinery, is important and novel, but consistent with reports of

242 neurodegenerative disorders in other autophagy-related genes (Frake et al., 2015). Our results

243 suggest that other mutations in this and other ATG genes, which impair but do not completely

244 abolish autophagy, may result in similar forms of ataxia, intellectual disability and

245 developmental delay. This study exemplifies the utility of exome sequencing in the identification

246 of rare disease-causing variants, and supports the role of impaired autophagy in

247 neurodegenerative disease. In addition, we demonstrate the utility of a combined genetic,

248 biochemical and cell biological analysis in multiple model systems to elucidate the underlying

249 pathogenic mechanism of rare human diseases.

250

251 Materials and Methods

252 Subjects

253 Study protocols including written informed consents have been approved by the University of

254 Michigan Institutional Review Board and the Boğaziçi University Institutional Review Board for

255 Research with Human Participants. Two Turkish brothers, ages 5 and 7 in 2004, presented with

256 ataxia and developmental delay, as previously described (Yapici and Eraksoy, 2005). Parents

257 were initially reported to be unrelated, but recently suggested they might be remotely related.

12

258 Both patients were delayed in walking, had truncal ataxia and dysmetria, nystagmus, and lower

259 IQ (68 and 70). MRI revealed cerebellar hypoplasia. Follow-up examinations showed no

260 progression of symptoms.

261

262 Genetic Analysis

263 DNA was isolated from peripheral whole blood using the Qiagen (Germantown, MD) Gentra

264 Puregene isolation kit. Linkage analysis was performed using the genotype data generated with

265 Illumina HumanOmniExpress-24 chip for the mother and the four sibs. The Allegro module

266 (Gudbjartsson et al., 2000) of easyLINKAGE software was used, assuming autosomal recessive

267 inheritance and parents as third cousins. No deletion or duplications common to just the two

268 affected brothers were detected using cnvPartition plug-in in Illumina Genome Studio v.1.02

269 software.

270

271 Exome sequencing was performed independently twice on one subject. Capture for whole exome

272 sequencing was performed with NimbleGen SeqCap EZ Exome Library v1.0 kit (Roche,

273 Indianapolis, IN). Captured regions were sequenced with Illumina HiSeq2000 instruments.

274 Variants were filtered to remove common variants based on 1000 Genomes, Exome Sequencing

275 Project, and Exome Aggregation Consortium databases, variants outside of identified linkage

276 regions, variants not expected to change protein coding, and variants not following a recessive

277 model of inheritance (Exome Aggregation Consortium (ExAC), 2015; Genomes Project et al.,

278 2012; NHLBI Go Exome Sequencing Project, 2015).

279

13

280 PCR followed by Sanger sequencing was performed to validate the variant identified through

281 exome sequencing and test for segregation within the family. The variant of interest was further

282 examined in two separate collections of a total of 500 Turkish samples, and found absent.

283

284 Lymphoblast Cell Culture

285 Lymphoblastoid cell lines (LCL) of both subjects were generated from heparinized whole blood

286 samples and cultured as described (Doyle, 1990). As they are made in house and cultured briefly,

287 mycoplasma contamination risk is minimized.

288

289 Protein Co-Expression and Affinity Purification from Insect Cells

290 We used a baculovirus/insect cell expression system to examine formation of the human

291 ATG12–ATG5 conjugate in a heterologous system described previously (Qiu et al., 2013). Hi5

292 insect cells (Invitrogen) were infected with baculoviruses expressing human ATG7, ATG10, a

293 GST-tagged version of ATG12 (residues 53-140, corresponding to the ubiquitin-like domain)

294 and a His-tagged version of either ATG5WT or ATG5E122D. 3 days post infection, lysates were

295 subjected to glutathione affinity chromatography, and the GST-ATG12–His-ATG5 conjugate

296 was detected by SDS-PAGE followed by Coomassie Blue staining. To confirm that the

297 baculoviruses produce protein, Hi5 cells were coinfected with baculoviruses expressing the His-

298 tagged WT and mutant versions of ATG5 and the N-terminal domain of ATG16L1 (residues 1-

299 69, as an MBP fusion). At 3 days post infection, lysates were subjected to nickel affinity

300 purification. The ATG5-ATG16L1 complex formation was detected by SDS-PAGE and

301 Coomassie Blue staining.

302

14

303 Crystallization and Structure Determination

304 The complex containing ATG5E122D and the N-terminal domain of ATG16L1 (residues 1-69) was

305 expressed in Hi5 insect cells, and purified by nickel affinity, ion exchange, and size exclusion

306 chromatography into a final buffer of 20 mM Tris, pH 8.5, 50 mM NaCl, 10 mM DTT. The

307 complex was concentrated to 18.5 mg/ml, aliquoted, flash-frozen and stored at -80°C until

308 further use. Crystals were grown by the hanging drop vapor diffusion method by mixing purified

309 protein 1:1 with reservoir solutions of 37.5 mM MES, pH 5.2–5.8, 0.2 M sodium tartrate, and

310 11–13% polyethylene glycol 3350. Final crystals were obtained by micro-seeding with reservoir

311 solution of 40 mM MES, pH 5.5, 0.2 M sodium tartrate, 8.5% PEG3350, 10 mM DTT. Crystals

312 were cryoprotected in reservoir solution supplemented with 25% xylitol, and flash frozen in

313 liquid nitrogen prior to data collection. Diffraction data were processed with XDS. The structure

314 was determined by molecular replacement using Phaser (McCoy et al., 2007) using the structure

315 of the WT ATG5-ATG16L1 (1-69) (PDB: 4TQ0) complex as a search model (Kim et al., 2015a).

316 Model construction and rebuilding were performed using Coot (Emsley et al., 2010). The

317 structure was refined using Phenix (Adams et al., 2010). Diffraction data and refinement

318 statistics are provided in Table 1.

319

320 Immunoblotting

321 Cells or tissues were lysed in cell lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM

322 EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM

323 Na3VO4, 1% Triton X-100) or RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1%

324 sodium deoxycholate, 1% NP-40, 0.1% SDS) containing protease inhibitor cocktail (Roche).

325 After being clarified with centrifugation, lysates were boiled in SDS sample buffer, separated by

15

326 SDS-PAGE, transferred to polyvinylidene difluoride membranes and probed with the indicated

327 antibodies. ATG5 (12994), LC3 (3868) and SQSTM1/p62 (5114) antibodies were purchased

328 from Cell Signaling Technology. Hemagglutinin (HA, 3F10) antibody was from Roche. Actin

329 (JLA20) and tubulin (T5168) antibodies were from Developmental Studies Hybridoma Bank and

330 Sigma, respectively. Ref(2)P/SQSTM1 antibody was previously described (Pircs et al., 2012).

331

332 HEK293 Cell Culture

333 Wild-type human ATG5-coding sequence was from Addgene #24922 (deposited by Dr. Toren

334 Finkel) (Lee et al., 2008). The E122D mutation was introduced into ATG5 by PCR-based site-

335 directed mutagenesis. ATG5WT and ATG5E122D were cloned into the plasmid pLU-CMV-Flag.

336 The HA-ATG12-expressing plasmid was from Addgene #22950 (deposited by Dr. Noboru

337 Mizushima) (Mizushima et al., 1998b). HEK293 cells (the 293 A substrain from Invitrogen,

338 tested negative for mycoplasma by PCR) were cultured in Dulbecco’s modified Eagle’s medium

339 (DMEM, Invitrogen) containing 10% fetal bovine serum (FBS) and penicillin/streptomycin at

340 37°C in 5% CO2. For transient expression of proteins, HEK293 cells were transfected with

341 purified plasmid constructs and polyethylenimine (PEI, Sigma) as previously described

342 (Horbinski et al., 2001). Cells were harvested 24 hr after transfection for immunoblot analyses.

343

344 Yeast Model

345 strain WLY176 was used to generate an ATG5 knockout strain

346 (∆) as previously described (Gueldener et al., 2002). The MKO strain YCY137 (SEY6210

347 atg1∆, 2∆, 4∆, 5∆, 6∆, 7∆, 8∆, 9∆, 10∆, 11∆, 12∆, 13∆, 14∆, 16∆, 17∆, 18∆, 19∆, 20∆, 21∆,

348 23∆, 24∆, 27∆, 29∆) (Cao et al., 2008), was used for in vivo reconstitution of Atg8 conjugation.

16

349 Site-directed mutagenesis was performed to generate ATG5 amplicons with the E141D mutation

350 as previously described (Liu and Naismith, 2008). A pRS406 empty plasmid was digested with

351 Spel and SalI, and then ligated with a DNA fragment encoding either wild-type or mutant Atg5-

352 PA. atg5∆ was transformed with an empty pRS406 vector, or plasmids encoding Atg5-PA WT

353 or Atg5-PA E141D. Wild-type WLY176 colonies were transformed with empty pRS406 vector

354 as a control. Colonies were grown on SMD-URA medium and starved in nitrogen-deficient

355 medium. Pho8Δ60 and western blot analyses were performed as described previously (Noda and

356 Klionsky, 2008; Shintani and Klionsky, 2004). Quantification was performed using ImageJ

357 software. The pATG8∆R-ATG7-ATG10(414), pATG5(WT)-HA-ATG12(416) and

358 pATG5(WT)-HA-ATG12-ATG16(416) plasmids were described previously (Cao et al., 2008).

359 The pATG5(E141D)-HA-ATG12(416) and pATG5(E141D)-HA-ATG12-ATG16(416) plasmids

360 were made by site-directed mutagenesis based on the wild-type constructs.

361

362 Drosophila Genetics

363 Atg5-null Drosophila flies (Atg55cc5) were generated by CRISPR-Cas9-mediated genome editing,

364 using a double gRNA approach, both targeting the same gene, as described (Kondo and Ueda,

365 2013). The Atg55cc5 mutant was recovered by screening viable candidate lines for accumulation

366 of the specific autophagy cargo p62/Ref(2)P using western blots, followed by PCR and

367 sequencing. Atg55cc5 mutants have a deletion in X:7,322,242-7,323,717 residues (Drosophila

368 melanogaster R6.06), which deletes five out of six exons of the Atg5 gene, eliminating more than

369 85% of protein-coding sequences including the translation start site (Figure 6A). The PhiC31

370 integrase-mediated site-specific transformation method was used to express human ATG5WT and

371 ATG5E122D from an identical genomic locus (Bateman et al., 2006; Bischof et al., 2007; Venken

17

372 et al., 2006). In brief, flag-tagged ATG5WT and ATG5E122D were cloned into a pUAST-attB

373 vector (Bischof et al., 2007) and fully sequenced. pUAST-attB-ATG5WT and pUAST-attB-

374 ATG5E122D were microinjected into y1 M{vas-int.Dm}ZH-2A w*; M{3xP3-RFP.attP}ZH-51D

375 flies and stable transformants were isolated by the presence of the mini-white+ marker (Figure

376 6B). The UAS-ATG5WT or E122D transgenes were crossed with a double balancer strain (Bl/CyO;

377 TM2/TM6B) and then with +/CyO; Tub-Gal4/TM2 to be constructed as stable Tub>ATG5 lines

378 (UAS-ATG5/UAS-ATG5; Tub-Gal4/TM6B). The Tub>ATG5 male flies were crossed with

379 Atg55cc5/FM7 female flies to generate Atg5-null flies expressing human ATG5 transgenes.

380 Climbing assays and TUNEL staining were performed as previously described (Kim et al.,

381 2015b).

382

383 Acknowledgments

384 This research was funded by the National Institutes of Health (NIH) grants R01-NS078560 (MB),

385 R21-OD018265 (JHL), R01-GM077053 (BAS) and R01-GM053396 (DJK), HHMI (BAS),

386 American Heart Association 14POST19890021 (YQ), the Boğaziçi University Research Fund

387 Grant 6655 (AT), ALSAC/St. Jude (BAS), the Wellcome Trust 087518/Z/08/Z and Lendulet

388 LP2014-2 (GJ). We thank Miriam H. Meisler for critical reading, Karen Majczenko, MD

389 (deceased) and Linda Gates for EBV transformation and tissue culture, Dr. Ke Wang and Kendal

390 Walker for technical assistance, the University of Michigan DNA Sequencing Core for Exome

391 sequencing, Dr. Mustafa Cengiz Yakicier (ACIBADEM University, Turkey, Department of

392 Medical Biology and Genetic Diagnostic Center) for Turkish control samples, and the

393 TUBITAK Advanced Genomics and Bioinformatics Group (IGBAM) for sharing the Turkish

394 exome sequence database.

18

395

396 Author Contributions

397 Z.Y. provided samples and clinical information on subjects, E.S., N.M., A.T. performed linkage

398 analysis; E.S., J.X., N.M., A.T., J.Z.L., M.B. performed exome sequencing and analysis; M.Kim,

399 I.S., S.-H.R., B. K., J.H.L. performed LCL/HEK293 cell culture and Drosophila experiments;

400 Y.Q., B.A.S. performed structure analysis; D.G., X.L., D.J.K. performed yeast experiments; A.J.,

401 S.T., M.Karpati, G.J. generated Atg5-null flies; E.S., M.B. drafted the manuscript; M.Kim,

402 B.A.S., A.T., J.H.L., D.J.K., Y.Q., M.B. revised the manuscript. All authors approved the final

403 manuscript.

404

19

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566

567

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568 Figure Legends:

569

570 Figure 1. Linkage analysis in consanguineous family with two siblings with ataxia, mental

571 retardation and developmental delay maps defect to chromosomal interval containing

572 ATG5. Remote consanguinity was detected between parents of two previously described siblings

573 with ataxia (Yapici and Eraksoy, 2005), illustrated here as third cousins. SNP and linkage results

574 for (B) are illustrated below the pedigree (A), with the shared homozygous region

575 between rs4334996 and rs1204817 encompassing ATG5, at 106.6 Mb. Father (501)’s alleles

576 were inferred, 0 denotes unknown alleles. Affected siblings, 601 and 602, are denoted by black

577 squares and unaffected family members by open symbols. The proximal boundary is defined by

578 a recombination event between rs1547384 and rs4334996 in affected individual 602, while the

579 distal boundary is defined as an ancestral recombination event (lack of homozygosity, dark

580 green) between rs1204817 and rs648248. Orange arrows indicate the position of ATG5.

581

28

582 Figure 2. The primary sequence of ATG5, including the mutant E122 residue, as well as the

583 protein structure is highly conserved across eukaryotic species. (A) Amino acid sequence

584 alignment between ATG5 orthologs from human (HsaATG5), mouse (MmuAtg5), Drosophila

585 melanogaster (DmeAtg5) and Saccharomyces cerevisiae (SceAtg5) was constructed at

586 GenomeNet (Kyoto University Bioinformatics Center) through CLUSTALW and rendered in

587 Genedoc v.2.7 using default settings. E122 in human ATG5 and E141 in yeast Atg5, which are

588 homologous residues, are indicated by red arrows. (B) Location of E122 residue is highlighted in

589 yellow on the crystal structure of a human ATG12–ATG5 (residues 53-140)-ATG16L1 (residues

590 11-43) complex (PDB ID: 4NAW). ATG5 is shown in cyan, ATG16L1 in magenta, and ATG12

591 in green (Otomo et al., 2013). (C) Location of the E141 residue in yeast Atg5, which corresponds

592 to the E122 in human ATG5, is indicated in yellow on the crystal structure of a yeast Atg12–

593 Atg5 (100-186)-Atg16 (1-46) complex, colored as for the human counterparts as in panel B

594 (PDB ID: 3W1S) (Noda et al., 2013). (D) Superimposition of crystal structure of ATG5E122D-

595 ATG16L1 with ATG5WT-ATG16L1 (PDB: 4TQ0) (Kim et al., 2015a). Close-up view of ATG5

596 structure around WT (E) and E122D (F) mutation.

597

29

598

599 Figure 3. Cells from ataxia patients with ATG5E122D/E122D mutation exhibit autophagy

600 defects. (A) Decreased expression of ATG12–ATG5 conjugates in cells from ataxia patients

601 with ATG5E122D/E122D mutation. ATG5 immunoblotting (IB) of ATG12-ATG5 conjugates of

602 LCLs from individuals whose ATG5 genotype is wild type (A to I) or E122D (J and K). (B)

603 Decreased autophagic flux in ATG5E122D/E122D LCL cells. A subset of LCLs from (A) were

604 treated with 0.1 μM bafilomycin A1 (Baf) for the indicated hours and analyzed by IB. LC3-II is

605 an autophagosome marker, and LC3-I is a precursor for LC3-II. Baf inhibits lysosomal

606 degradation of LC3-II. Actin is shown as a loading control. (C) Decreased autophagic flux and

607 increased expression of SQSTM1, an autophagy substrate, in ATG5E122D/E122D LCL cells. A

608 subset of LCLs from (A) were treated with 250 nM Torin 1 or 0.1 μM Baf, for 2 hr and analyzed

609 by IB. Torin 1 is an autophagic flux activator.

610

30

611

612 Figure 4. E122D mutation interferes with formation of the ATG12–ATG5 conjugate. (A)

613 Coomassie Blue-stained SDS-PAGE gel following glutathione affinity purification from lysates

614 of Hi5 cells infected with baculoviruses expressing GST-ATG12 and either WT or E122D

615 mutant ATG5. (B and C) HEK293 cells expressing the indicated proteins were analyzed by IB.

616 (D) Drosophila whole bodies expressing the indicated transgenes under the control of Tub-Gal4

617 were analyzed by IB. (E) Lysates from Hi5 cells expressing the indicated proteins were subjected

618 to His/Ni-NTA purification and subsequent TEV protease treatment. Proteins were analyzed by

619 Coomassie Blue staining.

620

31

621 Figure 5. E141D mutation of yeast Atg5 attenuates autophagy. (A-D) Yeast cells were grown

622 in SMD to mid-log phase and nitrogen starved for the indicated times. (A) WLY176 atg5∆ yeast

623 cells expressed GFP-Atg8 through its endogenous promoter and plasmid-based Atg5WT-PA,

624 Atg5E141D-PA or an empty vector. Protein extracts were analyzed for GFP-Atg8 processing by

625 western blot. The ratio of free GFP to Dpm1 (loading control) is presented below the blots, and

626 quantification is presented on the right (Student’s t test, n=4; *, p < 0.05); the value for Atg5WT

627 at 6 h was set to 1.0 and other values were normalized. (B) WLY176 atg5∆ yeast cells expressed

628 either plasmid-based Atg5WT-PA, Atg5E141D-PA or an empty vector. Protein extracts were used

629 to measure autophagy through the Pho8Δ60 assay (Student’s t test, n=6; *, p < 0.05). (C)

630 WLY176 cells with genomic integrated Atg5WT or Atg5E141D were used to generate protein

631 extracts and autophagy was monitored through the Pho8Δ60 assay (Student’s t test, n=3; *, p <

632 0.05). (D) WLY176 atg5∆ yeast cells expressing plasmid-based Atg5WT-PA, Atg5E141D-PA or an

633 empty vector were used to generate protein extracts. The ratio of Atg8–PE to total Atg8 is

634 presented below the blots based on western blot using antiserum to Atg8. Dpm1 was used as a

635 loading control. (E) MKO ATG3 (YCY137) cells were co-transformed with pATG8∆R-ATG7-

636 ATG10(414), and either pATG5WT-HA-ATG12(416), pATG5E141D-HA-ATG12(416),

637 pATG5WT-HA-ATG12-ATG16(416), or pATG5E141D-HA-ATG12-ATG16(416). Overnight

638 cultures were diluted to OD=0.02 in SMD -Ura -Trp. The cells were incubated at 30°C for 18 hr

639 to mid-log phase before they were shifted to SD-N for nitrogen starvation. Samples at the

640 corresponding time points were collected, TCA precipitated and subsequently analyzed by

641 western blot. S.E., short exposure; L.E., long exposure.

642

643

32

644 Figure 6. Ataxic phenotype of Atg5-null flies is suppressed by human ATG5WT but not by

645 ATG5E122D. (A) Genomic organization of the Atg5 locus and the Atg5-null mutant (Atg55cc5).

646 Atg55cc5 mutants have a CRISPR-Cas9-mediated deletion in approximately 1.5 kb residues that

647 eliminate more than 85% of Atg5-coding sequences including the translation start site. Open

648 boxes, untranslated exons; closed boxes, protein-coding exons. Scale bar, relative length of 1 kb

649 genomic span. (B) Schematic representation of how ATG5 transgenic flies were made. Plasmid

650 which can express wild-type or E122D-mutated human ATG5 was inserted into an identical

651 genomic location (the attP site) through phiC31-mediated recombination (Bateman et al., 2006;

652 Bischof et al., 2007; Venken et al., 2006). The scheme was adapted from a previous publication

653 (Kim and Lee, 2015). (C) Genetic scheme of how ATG5 transgenes were placed into the Atg5-

654 null mutant flies. Atg5, UAS-ATG5 and Tub-Gal4 loci are on the X-chromosome, second

655 chromosome and third chromosome, respectively. (D) Whole flies of indicated genotypes were

656 analyzed by IB. (E to H) Photographs of the vials containing 2-week-old adult male flies of

657 indicated genotypes taken at 3 sec after negative geotaxis induction: (E) Atg5-null flies exhibit

658 severely impaired mobility. (F) Ataxic phenotype of Atg5-null flies is complemented by human

659 ATG5WT expression. (G and H) Human ATG5E122D is less capable than human ATG5WT in

660 suppressing the fly ataxia phenotype. (I) Quantification of the climbing speeds of 2-week-old

661 adult male flies (n≥20) of the indicated genotype. Climbing speed is presented as mean ±

662 standard deviation (n=5). P values were calculated using the Student’s t test (*** P<0.001). (J)

663 Drosophila heads from two-weeks-old flies of the indicated genotypes were analyzed by IB. (K)

664 Ref(2)P/SQSTM1 is an autophagy substrate. Relative protein expression was measured by

665 densitometry and presented in a bar graph (mean ± standard error; n=4). (L) Terminal

666 deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) of Drosophila brain (middle

33

667 layer of the medial compartment). (M) TUNEL-positive cells per field were quantified and

668 presented in a bar graph (mean ± standard error; n≥5). K and M: P values were calculated using

669 the Student’s t test (* P<0.05, ** P<0.01, *** P<0.001).

34

670 Table 1: Crystallography Data Collection and Refinement Statistics

671 Data collection 672 673 Beam line APS 24-ID-C 674 Space group C2 675 Unit cell parameters 676 a, b, c (Å) 217.1, 84.5, 151.9677 678 α, β, γ (°) 90, 133.8, 90 679 Resolution (Å) (highest shell) 50–3.0 (3.12-3.0)680 Wavelength (Å) 0.9792 681 Number of measured reflections 179,310 682 683 Number of unique refections 39,496 684 Overall Rsym 0.057 (0.645)685 686 Completeness (%) 98.9 (99.3) 687 688 Overall I/σI 14.3 (2.3) 689 690 Multiplicity 4.5 691 692 Refinement 693 694 Resolution (Å) 50–3.0 695 696

Rwork/Rfree 0.198/0.244 697 698 rmsd bond lengths (Å) 0.008 699 700 rmsd bond angles (°) 0.994 701 702 Number of protein atoms 9,403 703 704 Ramachandran statistics 705 706 Preferred (%) 97.69 707 708 Allowed (%) 2.22 709 710 Disallowed (%) 0.09 711 712 713 714

35

715 716 Video 1. Climbing assay in 2 weeks-old wild-type flies (left) and Atg5-null flies (right). 717 718 Video 2. Climbing assay in 2 weeks-old Atg5-null flies (left) and Atg5-null flies expressing 719 ATG5WT (right). 720 721 Video 3. Climbing assay in 2 weeks-old Atg5-null flies (left) and Atg5-null flies expressing 722 ATG5E122D (right). 723 724 Video 4. Climbing assay in 2 weeks-old Atg5-null flies expressing ATG5WT (left) or ATG5E122D 725 (right). 726

36

Figure 1

A

B pLOD (MPT)

Marker coverage, genetic position from 6pter [cM] Figure 2

A

E122 in human ATG5

E141 in yeast Atg5

Human ATG12–ATG5-ATG16L1 complex PDB ID: 4NAW B ATG16L1 E ATG12–ATG5 ATG5 (WT) linkage ATG5 (E122D)-ATG16L1 E122 D ATG5 (WT)-ATG16L1 ATG12 E122

ATG5

Yeast Atg12–Atg5-Atg16 complex C PDB ID: 3W1S F Atg16 Atg12–Atg5 ATG5 (E122D) linkage

E141 Atg12 D122

Atg5 Figure3

A ATG5WT/WT ATG5E122D/E122D A BCDEFGH I JK ATG12–ATG5

Actin

ATG5WT/WT ATG5E122D/E122D B H J Baf (hr): 0 0 0.5 1 1.5 2 0 0 0.5 1 1.5 2 LC3-I LC3 LC3-II

Actin ATG5WT/WT ATG5E122D/E122D I K Baf (hr): 0 0 0.5 1 1.5 2 0 0 0.5 1 1.5 2 LC3 LC3-I LC3-II

Actin

ATG5WT/WT ATG5ED/ED ATG5WT/WT ATG5ED/ED C H J I K Baf: - -++--+ + - -++--+ + Torin1: - +-+-+- + - +-+-+- + ATG12–ATG5 SQSTM1

LC3-I LC3 LC3-II Actin Figure 4

A Hi5 cells B HEK293 cells His-TEV-ATG5WT + - Myc-ATG12- - + - + + His-TEV-ATG5E122D - + Flag-ATG5WT - + + - - - GST-TEV-ATG12 + + Flag-ATG5E122D - - - + + - GFP- + - + - +

* Non-specific ATG12–ATG5 GST-ATG12 –His-ATG5 IB: Myc (ATG12) Elution from * Non-specific GST affinity GST affinity isolation ATG12

D Drosophila (Tub-Gal4) ATG12–ATG5 Flag-ATG5WT - + - * Non-specific Flag-ATG5E122D - - + IB: ATG5

* Non-specific ATG5

IB: ATG5 Atg12–ATG5 (human- specific) ATG5 C HEK293 cells HA-ATG12- - + - + + Flag-ATG5WT - + + - - - Flag-ATG5E122D - - - + + - IB: Tubulin Tubulin GFP- + - + - +

E Hi5 cells ATG12–ATG5 His-TEV-ATG5WT + + - - IB: HA His-TEV-ATG5E122D - - + + (ATG12) MBP-TEV-ATG16L1 + + + + TEV Protease - + - + ATG12

MBP-ATG16L1 MBP His-ATG5 ATG5 ATG12–ATG5 IB: * Non-specific ATG5

ATG5 ATG16L1 and TEV protease treatment and TEV Elution from Ni affinity Elution from Ni affinity isolation A atg5∆ GFP-Atg8 SMD SD-N 3 h Atg5-PA: WT E141D — SD-N 1 h SD-N 6 h -N(h): 0 1 3 6 0 1 3 6 0 1 3 6 150 GFP-Atg8 * 100 GFP 50 Dpm1 Free GFP:Dpm1 Free 0 GFP/Dpm1: 0 .1 .7 1 0 .1 .4 .6 Atg5-PA: WT E141D atg5∆ B 150 SMD * SD-N 2 h * 100 SD-N 3 h SD-N 4 h SD-N 6 h 50 Pho8∆60 activity

0 Atg5WT-PA Atg5E141D-PA Vector atg5∆ C 150 SMD * SD-N (3 h) 100

50 Pho8∆60 activity (%) 0 Strain: WT E141D atg5∆

D atg5∆ Atg5-PA: WT E141D — -N (min): 0 15 30 60 0 15 30 60 0 15 30 60 Atg8 Atg8–PE Dpm1 Atg8–PE/total Atg8: .4 .59 .75 .7 .5 .53 .53 .54

E Atg5: WT E141D WT E141D -N (min): 0 10 20 0 10 20 0 10 20 0 10 20 Atg8 Atg8–PE S.E. Atg8 Atg8–PE L.E. Atg8–PE/total Atg8: .88 .88 .87 .61 .57 .53 .93 .92 .91 .93 .90 .91

Pgk1 HA-Atg12 HA-Atg12, Atg16 Figure 6

A Atg5 1-kb

Atg5WT Atg55cc5 (Atg5-null) BCD Atg5+/+ Atg5-/- WT WT or E122D Flag-ATG5 - - + - UAS-ATG5 ; Flag-ATG5E122D - - - + Tub-Gal4/TM6B * Non-specific

on second IB: ATG5 Atg12–ATG5 chromosome Atg55cc5/FM7GFP (human- specific) ATG5 Atg55cc5/Y; WT or E122D UAS-ATG5 /+; IB: Tubulin Tub-Gal4/+ Tubulin E F GIH -/- -/- -/- -/- *** +/+ -/- -/- Atg5 -/- Atg5 Atg5 Atg5 Atg5 Atg5 Atg5 WT Atg5 E122D WT E122D 1.2 *** ATG5 ATG5 ATG5 ATG5 *** *** 1 0.8 0.6 0.4 0.2 Climbing speed (cm/s) Climbing speed 0 Atg5+/+ - ATG5WTATG5E122D Atg5-/-

K *** L M * 30 ** Atg5+/+ Atg5-/- J -/- 8 Atg5+/+ Atg5 25 ** * WT Flag-ATG5 - - + - 6 Flag-ATG5E122D - - - + 20 IB:Ref(2)P 15 TUNEL TUNEL 4 (SQSTM1) 10 Atg5-/- Atg5-/- ATG5WT ATG5E122D 2 5 IB: Tubulin TUNEL(+) cells Ref(2)P expression 0 0 - ATG5WT ATG5E122D - ATG5WT ATG5E122D Atg5+/+ TUNEL TUNEL Atg5+/+ Atg5-/- Atg5-/-