1 Mutation in ATG5 Reduces Autophagy 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 mutations in any of
40 the core autophagy-related (ATG) genes 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 protein. 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 proteins and
53 organelles are degraded and recycled through sequestration within autophagosomes and delivery
54 to lysosomes (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 phenotype 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 gene, 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 chromosome 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 autophagosome. ATG12 is a ubiquitin-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 vacuole 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 locus (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 Drosophila melanogaster 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 phenotypes (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 Saccharomyces cerevisiae strain WLY176 was used to generate an ATG5 knockout strain
346 (atg5∆) 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 chromosome 6 (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-/-