ALG13 Deficiency Associated with Increased Seizure Susceptibility

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67 8Q1 ALG13 Deﬁciency Associated with Increased Seizure Susceptibility 9 and Severity

Q310Q2 Peng Gao,a,b,† Feng Wang,a,b,† Junming Huo,a Ding Wan,a,b Jing Zhang,c Jianguo Niu,a Ji Wu,c,d Baoli Yud and Tao Suna,b,* a 11 Ningxia Key Laboratory of Cerebrocranial Diseases, Ningxia Medical University, 1160 Shengli Street, Yinchuan, Ningxia, China, 750001. b 12 Department of Neurosurgery, General Hospital of Ningxia Medical University, 804 Shengli Street, Yinchuan, Ningxia, China, 750001. c 13 Ningxia Key Laboratory of Reproduction and Genetics, Ningxia Medical University, 1160 Shengli Street, Yinchuan, Ningxia, China, 750001. d 14 Renji Hospital Shanghai Jiaotong University School of Medicine, Key Laboratory for the Genetics of Developmental & Neuropsychiatric Disorders (Ministry of 15 Education), Bio-X Institutes, Shanghai Jiao Tong University, Shanghai, China, 200240.

16 Abstract—ALG13 (asparagine-linked glycosylation 13 homolog) encodes a crucial proteinOOF involved in the process of 17 N-linked glycosylation, and abnormal N-linked glycosylation is considered an important risk factor that leads to neurolo- 18 gical deficits and disorders. However, the causal relationship between ALG13 and epilepsy remains unknown. This study 19 applied a kainic acid (KA)-induced epileptic mouse model to determine whether ALG13 deficiency resulted in increased 20 susceptibility to and severity of epileptic seizures. This report found that the expression of ALG13 in the central nervous 21 system (CNS) had histologically and cellular specificity, mainly in the neurons in the cortex and hippocampus, epilepsy 22 commonly occurs. In addition, KA-induced seizures significantly affected the expression levels of ALG13 mRNA and 23 protein in the forebrain of wild-type (WT) mice. KA-induced epileptic progressions were dramatically increased in 24 Alg13 knockout (KO) mice, including prolonged electrographic seizures, strikingly increased mortality rates, and the 25 severity of responses to epileptic seizures. Furthermore, KA-induced epilepsy-related pathological changes of the brain 26 were predominantly exacerbated in Alg13 KO mice. This study also preliminarily explored the possible mechanisms of 27 ALG13-involved epilepsy by showing hyperactive mTOR signaling pathways in the cortex and hippocampus of Alg13 28 KO mice. To the best of our knowledge, this report is the first evidence of the association between ALG13 and epilepsy 29 in experimental animals. © 2019 IBRO. Published by Elsevier Ltd. All rights reserved. 30 31 Key words: ALG13, kainic acid, epileptic seizure, epilepsy.

32 INTRODUCTION and epileptogenesis are diverse and while some have been 39 elucidated, many mechanisms have not yet been explained. 40 33 Epilepsy, characterized by the onset of recurrent sponta- ALG13 (Alg13) is an X-linked gene encoding ALG13 pro- 41 34 neous seizures, is a chronic disease of the cerebral neurologi- tein in both human and mice, together with ALG14, that forms 42 35 cal system. It accounts for 1% of global diseases and affects a functional UDP-GlcNAc glycosyltransferase that catalyzes 43 36 nearly 50 million people worldwide, of which approximately the second sugar addition of the highly conserved oligosac- 44 37 80% live in developing countries (Pitkanen and Lukasiuk, charide precursor in endoplasmic reticulum N-linked glycosy- 45 38 2011). The cellular and molecular mechanisms of seizures lation (Averbeck et al., 2007). At present, ALG13 is thought to 46 play a crucial role in the process of N-linked glycosylation and 47 *Corresponding author at: Ningxia Key Laboratory of Cerebrocranial the formation of ALG13/ALG14 complex, which is a target 48 Diseases, Ningxia Medical University, 1160 Shengli Street, Yinchuan, 49 Ningxia, China, 750001.UNCORRECTEDfor the regulation PR of N-linked glycosylation (Weerapana and Q4 E-mail address: [email protected] (Ji Wu). [email protected] (Tao Sun). Imperiali, 2006). Protein asparagine N-glycosylation, one of 50 Abbreviations: ALG13, Asparagine-linked glycosylation 13 homolog; the most frequent and common protein modifications, is 51 ANOVA, Analysis of variance; CDG, Congenital disorders of glycosylation; 52 CNS, Central nervous system; IML, Inner molecular layer.; KA, Kainic acid; known to be essential for the structure and function of glyco- KO, Knock-out; MFS, Mossy fiber sprouting; PCR, Polymerase chain proteins (Brasil et al., 2018). Almost any protein passing 53 reaction; PSD95, Postsynaptic density protein 95; RT-qPCR, Reverse through the ER-Golgi network is involved in the process of 54 transcription quantitative real time polymerase chain reaction; SE, Status N-glycosylation by adding N-acetylglucosamine (GlcNAc) to 55 epilepticus; SL, Stratum lucidum; SO, Stratum oriens; TLE, Temporal lobe 56 epilepsy; WT, Wild type; ZNT3, Zinc transporter 3. selected asparagines (Asn) of nascent proteins. The function † Peng Gao and Feng Wang contributed to the work equally and of N-glycosylation is to promote protein folding, stability, traf- 57 should be regarded as co-first authors. ficking, localization, and oligomerization, eventually building 58

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59 cell–cell interactions and intracellular signaling networks Shanghai, China). Gene knockout mice were generated by 115 60 (Dennis et al., 2009; Stanley et al., 2009). deleting 5 nucleotides of the fourth exon of the Alg13 gene 116 61 Generally, epilepsy is a presenting symptom commonly using CRISPR-Cas9 systems (Yu Baoli, et al., unpublished). 117Q5 62 observed in congenital disorders of glycosylation (CDG) Alg13 KO mice and their WT littermates were identified and 118 63 patients (Freeze et al., 2015). CDGs are rare genetic dis- only male mice were used. All of the animals were main- 119 64 eases characterized by defective glycosylation pathways tained in a 12 h/12 h light/dark cycle with a constant room 120 65 and low catalytic ability of UDP-GlcNAc glycosyltransferase temperature and housed in group (4 to 5 mice per cage) with 121 66 and affect most organ systems including the nervous system food and water ad libitum. The mice were handled according 122 67 (Freeze and Schachter, 2009). Typical symptoms include to the guidelines prescribed by the Institutional Animal Care 123 68 structural abnormalities (for example, rapidly progressing and Use Committee of Ningxia Medical University (IACUC 124 69 cerebellar atrophy), developmental delays, myopathies Animal Use Certificate No.: SCXK (Ning) 2015–0001). All 125 70 (including congenital muscular dystrophies and limb-girdle efforts were made to minimize the number of animals used 126 71 dystrophies), strokes and stroke-like episodes, epileptic sei- and their suffering. 127 72 zures, and demyelinating neuropathy (Barone et al., 2014). 73 In research by Barba et al., 4 of 17 infants with CDG pre- Genotyping the Alg13 KO mice 128 74 sented focal epilepsy (Barba et al., 2016). Timal et al. indi- The mice genomic DNA was extracted using a TIANamp 129 75 cated that ALG13 gene mutation was found in a child with Genomic DNA Kit (Tiangen Biotech, Beijing, China) accord- 130 76 microcephaly, epilepsy, and early death caused by CDG ing to the manufacturer's protocols. The DNA concentration 131 77 (280A > G) (Timal, 2012). Neuropathological studies of the was determined using a spectrophotometer (Nanodrop 132 78 brains of CDG patients with focal epileptic seizures showed 2000; Thermo Fisher Scientific, Waltham, MA, USA). The 133 79 decreases in the Purkinje cells, granule cells, and white presence of the DNA deletion was identified by the standard 134 80 matter in the cerebellum together with a reduced number of polymerase chain reaction (PCR) technique using the 135 81 neurons in the pons nucleus, inferior olivary nucleus, and cer- enzyme Hot Start High-Fidelity DNA Polymerase (New 136 82 ebral cortex (Strømme et al., 1991; Aronica et al., 2005). England Biolabs, Ipswich, MA, USA) and specific primers. 137 83 The Alg13 gene mutation in the epileptic mouse model is The sequences of the primers used for mouse genotyping 138 84 always a deletion mutation, while some patients with epileptic were as follows: sense primer 5′-CAAATGGGTAACAGGC- 139 85 encephalopathies are reported as point mutations (such as 3′ and anti-sense primer 5′-GGGTAGAAAAGGATGG-3′. 140 86 c.320 A > G) (Allen et al., 2013). This difference in gene For each sample, the reaction was performed in a mix con- 141 87 mutation types may cause varying degrees of impact on the taining 10 μl sense primer and anti-sense primer, 12 μl 142Q6 88 function of ALG13 protein. However, the study on the role nuclease-free water, 25 μl Q5 polymerase, and 60 ng/μl 143 89 of ALG13 in seizure thresholds and the severity in mice will DNA. After maintaining for 30 s at 98 °C, 35 PCR cycles 144 90 provide an experimental basis for understanding the effect were performed (98 °C for 10 s, 56 °C for 30 s, and 72 °C 145 91 of ALG13 on the mechanisms of human epilepsy. Thus, for 2 min), followed by 2 min at 72 °C. The PCR products 146 92 based on the function of ALG13 in glycosylation and pheno- were isolated by electrophoresis in a 2.0% agarose gel 147 93 types caused by ALG13 mutations, ALG13 may be poten- stained with GelRed (Biotium, Fremont, CA, USA) using a 148 94 tially linked with epilepsy. However, no report has yet 100 bp DNA ladder (Biomed, Beijing, China) as a DNA 149 95 demonstrated the effect of ALG13 on the pathogenesis of marker. The genotype results were obtained from the result- 150 96 epileptic seizures and epilepsy. ing PCR product sequencing (Sangon Biotech Co., Ltd., 151 97 Therefore, we hypothesize that ALG13 might play an Shanghai, China). 152 98 important role in epilepsy and further affect epileptic seizures. 99 Accordingly, three goals are expected to be accomplished in Immunohistochemical staining and 153 100 the present study: (1) to determine whether ALG13 defi- immunofluorescent labeling of the brain sections 154 101 ciency can increase epileptic susceptibility and severity by 102 comparing Alg13 KO with WT mice, and to verify whether epi- The mice were anesthetized under 1% pentobarbital and 155 103 leptic seizure can alter ALG13 expression; (2) to assess fixed by transcardial perfusion with saline followed by 4% 156 104 whether the lack of ALG13 exacerbates classical pathologi- paraformaldehyde. Upon decapitation, the brains were 157 105 cal manifestations of epilepsy, such as gliosis, neuronal loss, quickly removed, shaped, and fixed in 4% paraformaldehyde 158 106 and synaptic plasticity;UNCORRECTED and (3) to explore whether the knock- at room temperature PROOF for 15 h. Then the brains were soaked in 159 107 out of ALG13 affects the intracellular signaling pathway, alcohol of gradient concentrations (50%, 70%, 95%, 100%I, 160 108 which might be involved in the underlying mechanism of epi- and 100%II), xylene, and paraffin for dehydration, clearing, 161 109 lepsy. This study provides a basic understanding of ALG13 infiltration, and embedding, respectively. Paraffin-embedded 162 110 deficiency in epilepsy. brain tissues were cut using a paraffin-slicing machine (Leica, 163 Wetzlar, Germany) and 5 μm sections were collected. To 164 eliminate the endogenous peroxidase activity, the sections 165

111 MATERIALS AND METHODS were treated with 0.3% H2O2 for 10 min. Then the sections 166 underwent antigen retrieval using 0.01 M pH 6.0 citric acid 167 112 Experimental animals at 100 °C for 15 min and phosphate-buffered saline (PBS) 168 113 Alg13 KO mice created with a C57BL/6J background were at room temperature for 12 min. After washing in PBS for 169 114 provided by Dr. Yu Baoli (Shanghai Jiaotong University, 3 times, the sections were incubated in a blocking buffer 170 NSC 18934 No of Pages 18 15 March 2019

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171 previously combined with 5% normal goat serum for 1 h at antibodies were added. Subsequently, the chemilumines- 228 172 37 °C and then incubated overnight at 4 °C with rabbit anti- cence method was employed and the relative target protein 229 173 ALG13 antibody (1:300, Proteintech, Wuhan, China). After levels were evaluated after X-ray film exposure. Western 230 174 washing with PBS 3 times, the sections were incubated with blotting was performed using Image Studio Lite Software 231 175 biotinylated goat anti-rabbit IgG (1:500, ZSGB-bio, Beijing, (LI-COR). Representative results of immunoblots from 232 176 China) for 1 h at room temperature. Followed by visualization at least three independent experiments were used for 233 177 with DAB, counter-staining with hematoxylin, and dehydra- analysis. 234 178 tion with gradient alcohol, the sections were dried and 179 mounted. Image-Pro Plus 6.0 software (Media Cybernetics, Electrode implantation and intra-amygdala kainic 235 180 Bethesda, MD, USA) was employed for immunohistochemis- acid-induced epileptic model 236 181 try image analysis, and the system was calibrated first to 182 eliminate background influences. The mice (12 weeks old and 23–28 g) were anesthetized 237 183 For immunofluorescent labeling, the brains were removed using 1% pentobarbital and maintained normal body tem- 238 184 and fixed in 4% paraformaldehyde for 6 h, then cryoprotected perature via a feedback-controlled heat blanket (TR-200, 239 185 in 30% sucrose in 0.1 M PBS. Coronal sections (20 μmthick) Safebio, Shanghai, China). The mice were then transferred 240 186 were cut on a freezing microtome (Leica, Wetzlar, Germany). in a stereotaxic frame and a midline scalp incision was intro- 241 187 Non-specific binding was blocked using 3% normal goat duced, followed by the blunt separation of the subcutaneous 242 188 serum and 0.3% Triton-X-100 in PBS for 1 h at room tem- fascia to expose the skull. According to the Mice Brain Atlas, 243 189 perature. Rabbit anti-ALG13 (1:200, Proteintech 20,810–1- the animals were affixed with a guide cannula (coordinates 244 190 AP), NeuN (1:500, Abcam, ab177487), PSD95 (1:500, from the bregma: 2.9 mm lateral and 1.2 mm posterior 245 191 Abcam, ab18258), ZnT3 (1:500, Synaptic Systems, to the bregma and 4.6 mm below the dura) (Paxinos and 246 192 197,002) antibody and chicken anti-GFAP (1:500, Abcam, Franklin, 2001). EEG electrodes were skull-mounted over 247 193 ab4674), MAP2 (1:500, Abcam, ab5392), and MBP (1:300, the left amygdala (coordinates from the bregma: AP = 248 194 Abcam, ab134018) antibody were used as the primary anti- −1.2mmandL=−9 mm) and frontal cortex (coordinates 249 195 bodies based on the validation results by the manufacturers. from the bregma: AP = 2 mm and L = −2 mm). The guide 250 196 After washing in PBS 3 times, the sections were incubated canula and electrodes were anchored to the skull using stain- 251 197 with secondary antibodies Alexa Fluor 488 conjugated goat less steel screws and acrylic cement. The mice were 252 198 anti-rabbit IgG (1:500, Abcam ab150077) and Alexa Fluor removed from the stereotaxic frame and administered 253 199 647 conjugated goat anti-chicken IgG (1:500, Abcam 300 μL of 10% glucose subcutaneously. After resuming 254 200 ab150171) for 1 h at room temperature. The sections were locomotor activity on a heated blanket, the mice were then 255 201 then washed, counterstained with DAPI, and mounted with transferred in a clear Perspex observation chamber allowing 256 202 coverslips. For double labelling, primary antibodies were free movement. The animals were left for 10 days to fully 257 203 simultaneously incubated and further processed for each recover. An acquiring and processing system of biomedical 258 204 secondary antibody. Image-Pro Plus 6.0 software (Media signals (BL-420 N, Techman Software, Chengdu, China) 259 205 Cybernetics, Bethesda, MD, USA) was used for quantifica- was employed for EEG recording, and the baseline EEG 260 206 tion if applicable. was established. 261 Status epilepticus (SE) was produced per published meth- 262 ods via the intra-amygdala microinjection of KA (Mouri et al., 263 207 Western blotting 2008).Themicewereanesthetizedwithisoflurane (3–5%) to 264 208 The cortex and hippocampus were dissected from the freshly permit the lowering of an injection cannula through a guide 265 209 isolated whole mice brain and subsequently homogenized on cannula to 4.6 mm below the dura for the microinjection of 266 210 ice using a Bullet Blender (Next Advance, Inc., Troy, NY, 0.4 μg KA (Sigma-Aldrich Shanghai Ltd., Shanghai, China) 267 211 USA) in lysis buffer consisting of phosphatase inhibitors, in 0.2 μl phosphate-buffered saline into the basolateral/ 268 212 protease inhibitor, and phenylmethanesulphonyl fluoride central amygdaloid nucleus. The control mice received a 269 213 (PMSF). After centrifuging for 10 min at 14,000g (4 °C), the comparable volume of intra-amygdala vehicle. Each injection 270 214 protein concentrations were then determined using a bicinch- was administered for 5 min. The cannula was maintained 271 215 oninic acid kit (BCA kit; KeyGen, Nanjing, China). Before in position for 2 min after the injection and then slowly with- 272 216 boiling at 100 °CUNCORRECTED for 6 min to denature, 80 μgofprotein drawn to avoid PROOF liquid reflux. EEGs and behavioral seizures 273 217 sample was added into loading buffer. SDS-polyacrylamide were observed and recorded. One h after SE following the 274 218 gel electrophoresis (SDS-PAGE) was then performed, and microinjection of KA or vehicle, the mice were given intraper- 275 219 the proteins were later transferred to a polyvinylidene fluoride itoneal diazepam (6 mg/kg, Sigma-Aldrich Shanghai Ltd., 276 220 (PVDF) membrane (Millipore Co., Millipore, Billerica, MA, Shanghai, China) to weaken SE and reduce mortality. The 277 221 USA)andblockedfor1hwith5%skimmilkpowderin mice were then killed at different time points of 1 h, 4 h, 278 222 TBST at room temperature. The membrane was probed with 8 h, 24 h, and 7 d,14 d, and 21 d (n = 4 for each time point) 279 223 primary antibodies rabbit against ALG13 (1:500, Proteintech following SE. All of the operations and procedures were 280 224 20,810–1-AP), p-mTOR (1:1000, Abcam, ab84400), mTOR carried out blind to the genotype. The correct guide cannula 281 225 (1:1000, Abcam, ab2732), and β-actin (1:1000, ZSGB-bio, placement was confirmed by Nissl staining. Data derived 282 226 ZM-0001), followed by incubation at 4 °C overnight. After from the mice with the correct electrode placement were 283 227 washing 3 times with TBST, the corresponding secondary analyzed. 284 NSC 18934 No of Pages 18 15 March 2019

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285 Pilocarpine-induced epileptic model at 72 °C for 30 s. After amplification, Bio-Rad IQ5 software 339 was employed for the data analysis and GAPDH was used 340 286 The pilocarpine-induced epilepsy model was established as an internal control. The comparative threshold (△△Ct) 341 287Q7 according to the published method (Curia et al., 2008). The method was used and the results were converted to fold 342 288 mice were intraperitoneally administered 1 mg/kg methylsco- expression relative to the control group. 343 289 polamine (Sigma-Aldrich Shanghai Ltd., Shanghai, China) 290 in sterile saline to reduce the peripheral cholinergic effects, 291 followed by a single dose of pilocarpine (either 225 mg/kg Timm staining 344 292 or 250 mg/kg) (Sigma-Aldrich Shanghai Ltd., Shanghai, The mice were perfused transcardially with 150 ml sterile 345 293 China). The control mice were only dosed with an equal saline, 100 ml 1% sodium sulfide, 100 ml 4% paraformalde- 346 294 volume of saline after injection with methylscopolamine. hyde, and 50 ml 1% sodium sulfide. The brains were then 347 295 Upon pilocarpine injection, continuous seizure activities were removed and post-fixed in 4% paraformaldehyde for 18 h, 348 296 observed, recorded, and evaluated. followed by immersion in 30% sucrose at 4 °C for 48 h. After- 349 ward, the brains were dissected on a freezing microtome 350 297 Evaluation of behavioral seizures (Leica, Wetzlar, Germany) and sections of 30 μm were col- 351 298 The severity of the behavioral manifestations of the seizures lected and then dried in a fume hood overnight. The frozen 352 299 was evaluated and classified according to the Racine stages brain sections were stained with Timm Staining Kit (Shanghai 353 300 (Racine, 1972): stage 0: normal behavior; stage 1: mouth and Gefan Biotechnology Co., Ltd., Shanghai, China) per the 354 301 facial movement; stage 2: head nodding; stage 3: forelimb manufacturer's protocol. After washing with running water, 355 302 clonus; stage 4: rearing with forelimb clonus; and stage 5: the sections were dehydrated in gradient ethanol, cleared in 356 303 rearing and falling with forelimb clonus. xylene, and covered with coverslips. CA3 and DG of the hip- 357 304 Latency was the time span measured from the time of KA pocampus were selected as the regions of interest. 358 305 injection or the first abnormal behavior appearing upon KA 306 administration. The “first abnormal behavior” referred to facial Nissl staining 359 307 automatism and hypersalivation. Moreover, stage 5 seizures The mice cortex and hippocampus sections of 5 μm were 360 308 had a definite termination occurring prior to status, whereas collected per the handling procedures previously described 361 309 status epilepticus was defined as the time when a stage 5 in the section “Immunohistochemical staining and immuno- 362 310 seizure continued unless it ceased intentionally. Mortality fluorescent labeling of the brain sections.” The rehydrated 363 311 was defined as death within 24 h after the onset of status epi- sections were stained in cresyl violet solution for 1 h at 364 312 lepticus, including a sudden respiration cessation during the 56 °C and then washed with deionized water. Afterward, 365 313 state or within the next 24 h. the sections were maintained in Nissl differentiation solution 366 314 Seizure severity was also assessed using the sum scores for at least 2 min until a colorless background was observed 367 315 per mouse and the duration of the experiment per the follow- under a microscope, followed by dehydrating (3 min each in 368 316 ing formula (Giménez-Cassina et al., 2012): 50%, 60%, 70%, 80%, 95%, and 100% ethanol), clearing in 369 Seizure severity ¼ ΣðÞtotal scores of one specified mouse xylene, and mounting with neutral balsam. The cells in the 370 =time of experiment field of interest from the Nissl staining images were counted 371 using Image-Pro Plus 6.0 software (Media Cybernetics, 372 319317318 For each mouse, the total Racine scores were added and Bethesda, MD, USA). 373 320 then divided by the duration of the experiment (120 min after 321 drug administration). Statistical analysis 374 The data were presented as mean ± SEM. Histograms were 375 322 RT-qPCR plotted using GraphPad Prism software (version 5). Statisti- 376 323 The mice were killed after status epilepticus. The total RNA of cal analyses were performed using IBM SPSS Statistics soft- 377 324 the cortex and hippocampus were extracted using TRIzol ware (version 22). Normal distributions and equal variances 378 325 solution (100 mg tissue/ml; Invitrogen, Carlsbad, CA, USA) were determined using the Kolmogorov–Smirnov one- 379 326 according to the manufacturer's protocol. First-strand cDNA sample test and Levene's test. Two-group comparisons 380 327 was generated usingUNCORRECTED M-MLV reverse transcriptase (DBI between the WTPROOF and KO mice for the ALG13 expression, sei- 381 328 Bioscience, Shanghai, China). 1 μl first-strand cDNA pre- zure severity (Racine scale), seizure severity (%), latencies, 382 329 paration was used as the PCR template with the following percentage of reactive GFAP-positive cells, and p-mTOR 383 330 Alg13 sense and antisense primers: FW (5′-CGGGACCAC- expression were evaluated with two-tailed Student's t-test. 384 331 CAGTTTCGAC-3′) and RV (5′-CAGTACGGAATGGTTT Group differences in the ALG13 mRNA/protein levels and 385 332 GGGCA-3′), and GAPDH sense and antisense primers FW Nissl-positive neurons post-KA were evaluated via one-way 386 333 (5′-CTGCCCAGAACATCATCCCT-3′) and RV (5′-CCAC- ANOVA and different time point comparisons were per- 387 334 CACCCTGTTGC TGTAG-3′). The cDNA was amplified using formed using least significant difference (LSD) post hoc ana- 388 335 a one-step qPCR kit (SYBR Green, Bestar, Shanghai, lysis. The χ2-test (Fisher's exact test) was performed to 389 336 China). The PCR parameters were set as follows: denatura- analyze the difference in the percentage of mice that had a 390 337 tion at 95 °C for 2 min, followed by 40 cycles of denaturation first stage 5 seizure as the onset of status between the WT 391 338 at 95 °C for 10 s, annealing at 58 °C for 30 s, and extension and KO groups. Survival was assessed using the Kaplan– 392 NSC 18934 No of Pages 18 15 March 2019

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UNCORRECTED PROOF

Fig. 1 (Caption overleaf) NSC 18934 No of Pages 18 15 March 2019

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393 Meier analysis, and the statistically significant difference ALG13 protein production, as the band corresponding to 436 394 between two groups was determined with the log-rank test; ALG13 was absent (Fig. 1D). 437 395 P < .05 was considered statistically significant. One exception was that the Alg13 KO animals had 438 occasional spontaneous seizures, usually following routine 439 handling during cage changes. Normally, the behavioral 440 396 RESULTS manifestations of seizure started with hypertonia of the neck 441 and tail (Fig. 1Ea), followed by repeated clonic forelimbs, 442 397 Identification of Alg13-deficient mice sometimes bilaterally (Fig. 1Eb) and accompanied by a loss 443 398 Based on the fourth exon sequence of Alg13, a forward of posture. Brief immobility was observed during the postictal 444 399 primer and a reverse primer were designed to amplify DNA period (Fig. 1Ec). 445 400 fragments containing TTCAT by PCR. Due to the deletion 401 of the TTCAT sequence, the expected PCR products were − Characterization of ALG13 expression in mouse 446 402 545 bp for the X+ Y, X+ X+ mice and 540 bp for the X Y, − − brain 447 403 X X mice, as well as both 540 bp and 545 bp for the hetero- − 404 zygous X+ X mice (Fig. 1A).However,sinceitwasdifficult to To understand the function of ALG13 in the brain, we investi- 448 405 distinguish these two bands generated by agarose gel elec- gated the specific distribution of ALG13. To visually demon- 449 406 trophoresis, DNA sequencing was performed. The results strate the expression of ALG13 protein in different areas of 450 407 showed that there was a TTCAT deletion in the Alg13- the brain, the mouse brains were sliced sagittally and the sec- 451 408 deficient mice, but TTCAT was retained in the wild-type mice, tions were analyzed via immunohistochemistry (Fig. 2A). 452 409 and the heterozygous mice showed two color overlapping Overall, a wide distribution of ALG13 was observed in the 453 410 peaks in the TTCAT deleted position (Fig. 1B). In light of brain, mainly in the olfactory bulb, cortex, hippocampus, 454 411 these verification methods, the correct breeding of the hypothalamus, and pons (Fig. 2B). The data indicated that 455 412 Alg13 KO animals was guaranteed. the expression level of ALG13 varies in the functional region 456 413 Immunohistochemistry and Western blotting were required of the brain, and the expression of ALG13 in the central ner- 457 414 to further evaluate the effect of the 5 nucleotide deletion on vous system of the mice had histological specificity. To 458 415 the ALG13 expression in the mice brains. Immunohistochem- further explore whether ALG13 expression is cell-specific, 459 416 ical analysis was performed on the cortex, cerebellum, and we performed immunofluorescent labeling of ALG13 with 460 417 hippocampus. As shown in Fig. 1C, the WT and Alg13 KO the neuron-specific cytoskeletal protein MAP2, astrocyte- 461 418 littermates exhibited obviously different ALG13 protein specific marker GFAP, and oligodendrocyte-specific marker 462 419 expression in the brain. At 8 weeks after birth, robust cyto- MBP. The relevant results demonstrated that ALG13 coloca- 463 420 plasmic immunoreactivity of the neurons upon incubating lized only with MAP2 but not with GFAP and MBP in the hip- 464 421 with anti-ALG13 antibody was observed in all six cortical pocampus and cortex of the mouse brain, suggesting that 465 422 layers of the WT mice; however, no evidently positive neu- ALG13 was expressed in the neurons rather than in the 466 423 rons were observed in the same location in the Alg13 KO astrocytes and oligodendrocytes. Hence, the expression of 467 424 mice. A multitude of neurons in the hippocampus CA1 of ALG13 was proven to have cell type selectivity (Fig. 3A, B, 468 425 the WT mice demonstrated strong cytoplasmic immunoreac- and C). 469 426 tivity to ALG13 whereas a marked absence of ALG13- 427 positive neurons was observed in the Alg13 KO mice. Simi- Significant effects of seizure activity on the 470 428 larly, in the WT mice, the neurons in the cerebellar granule expression levels of ALG13 mRNA and protein 471 429 layer of the cytoplasm were labeled with ALG13, and most 430 of the Purkinje cells displayed evidence of cytoplasmic immu- To examine whether epilepsy conditions affect ALG13 472 431 noreactivity, while the Alg13 KO mice were ALG13-negative. expression, we measured ALG13 mRNA and protein in 473 432 Additionally, the ALG13 protein expression in the age- epileptic mice injected with KA at different time points. RT- 474 433 matched littermates was determined by Western blotting ana- qPCR was performed on extracted mRNA from the cortex 475 434 lysis with whole-cell protein extracts from the brain. As and hippocampus of the mice. As shown in Fig. 4E, the 476 435 expected, the Alg13 KO mice demonstrated no evidence of level of ALG13 mRNA in the cortex increased from 4 h after 477 UNCORRECTED PROOF

Fig. 1. Genotypic analysis and the absence of ALG13 protein in the Alg13 KO mice. (A) Agarose gel electrophoresis results of the PCR products. (M: DNA marker). (B) DNA sequencing of the PCR products amplified with forward primer and reverse primer. The retained and deleted TTCAT nucleotides are highlighted inside the red solid line rectangle and blue dashed line rectangle, respectively. (C) Immunohistochemical analysis was performed on the cortical brain, cerebellar granular cells, all of the Purkinje cells, and the hippocampal neurons in the CA1 sections from the WT and Alg13 KO mice. The presence of cytoplasmic immunoreactivity was noticeable in the WT mouse brains. Similar sections of the brain tissue from the Alg13 KO mice were not ALG13-positive. Scale bar: 100 μm. ML, molecular layer; EGL, external granular layer; EPL, external pyramidal layer; IGL, internal granular layer; IPL, internal pyramidal layer; PL, polymorphic layer. The histograms show the quantified ALG13 protein expression of the WT mice (n = 6) and the Alg13 KO mice (n = 6). (D) Western blotting analysis of whole-cell brain extract showed a weaker band representing the ALG13 protein in the Alg13 KO mice compared with the WT littermates. Equal amounts of protein were loaded in each lane. The histograms show the quantified ALG13 protein expression levels of the WT mice (n = 6) and the Alg13 KO mice (n = 6). The data are presented as mean ± SEM. (Student's t-test; P <.05, P <.01,and P < .001) (E) The spontaneous seizure manifestations of an Alg13-deficient mouse: (a) hypertonia of the neck and tail, (b) generalized tonic–clonic seizures, and (c) immobility. NSC 18934 No of Pages 18 15 March 2019

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KA injection (n = 24, P < .001, and 478 P < .01). In striking contrast, the expres- 479 sion of ALG13 mRNA in the hippocam- 480 pus significantly decreased from 1 h 481 after KA injection (n = 24, P < .05, and 482 P <.01)(Fig. 4F). Western blotting 483 was applied to detect ALG13 protein 484 extracted from the cortex and hippocam- 485 pus at different time points. In the 486 cortex, obvious growth in the corre- 487 sponding bands of ALG13 was 488 observed from 4 h after KA injection 489 (Fig. 4G). Statistical analysis showed a 490 significant increasing tendency of the 491 ALG13 expression in the model com- 492 pared with the control (n = 24; P < .01, 493 and P <.001)(Fig. 4H). As demon- 494 strated in Fig. 4I and J, an evident 495 decrease in the ALG13 expression can 496 be observed from 1 h post-KA in the hip- 497 pocampus (n = 24; P <.05, P < .01, 498 and P < .001). Interestingly, both the 499 ALG13 mRNA and protein in the hippo- 500 campus gradually rose after a dramatic 501 decline. Taken together, these data sug- 502 gest that both the mRNA and protein 503 expression of the Alg13 gene were 504 significantly affected by KA-induced 505 seizure activity in vivo. 506

ALG13 deﬁciency increased 507 seizure susceptibility induced by 508 kainic acid or pilocarpine 509 To explore the functional signiﬁcance of 510 seizure-regulated ALG13 expression, 511 the seizure behaviors of KA-induced 512 epileptic models were evaluated and 513 compared. Over a 2 h observation dura- 514 tion after KA injection, the WT and Alg13 515 KO mice demonstrated seizure beha- 516 vior, and both groups gradually reached 517 stage 5 seizure. Before status epilepti- 518 cus, the Alg13 KO mice experienced 519

UNCORRECTED PROOF Fig. 2. Different ALG13 distribution observed in the whole brain. Immunohistochemistry analysis of ALG13 in the WT mice brain was performed. (A) Representative immunohistochemistry images showing the distribution of ALG13 proteins in the sagittal section of the brain in the upper panel. The corresponding scope of the magnified images below is marked by black squares with the letters (a-l). Scale bar: 1 mm for the full-scale images and 100 μmforthemagnified images. (B) Histo- grams of the ALG13 protein expression quantified from immunohistochemistry analysis. Data are presented as mean ± SEM. NSC 18934 No of Pages 18 15 March 2019

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together to cause the first stage 5 sei- 554 zure and maintain the continuous 555 state (onset of status epilepticus). 556 To investigate the later phases of this 557 progression, measurements of the 558 latencies from KA injection or the 559 first abnormal behavior to the first 560 stage 5 seizure and from the first 561 stage 5 seizure to the onset of status 562 epilepticus were performed, and evi- 563 dently shorter time durations for 564 the Alg13 KO mice were observed 565 (Fig. 5G, H, and I). The results sug- 566 gested that the increased effect of 567 ALG13 deficiency on seizure sus- 568 ceptibility was sustained throughout 569 the entire process, including the early 570 and later phases after KA administra- 571 tion. We also summarized the situa- 572 tion in which the first stage 5 seizure 573 did not terminate and directly devel- 574 oped to status epilepticus. Intrigu- 575 ingly, this occurred only in the Alg13 576 KO mice but not in the WT group 577 (Fig. 5J). Furthermore, to verify 578 whether the behavioral seizures were 579 consistent with the electroencephalo- 580 graphic seizure activity, cortical elec- 581 trodes were implanted in the WT and 582 Alg13 KO mice to record the sei- 583 zures. The baseline EEG of the WT 584 Fig. 3. Cell type specific pattern of ALG13 distribution. (A) Immunofluorescent triple-staining of DAPI and Alg13 KO mice was nondistinc- 585 (blue), ALG13 (green), and astrocytic marker GFAP (red) in the cortex and hippocampus. (B) Immunofluor- tive. After KA injection, the electroen- 586 escent triple-staining of DAPI (blue), ALG13 (green), and neuronal marker MAP2 (red) in the cortex and hip- cephalographic seizure activities 587 pocampus. (C) Immunofluorescent triple-staining of DAPI (blue), ALG13 (green), and oligodendrocyte 588 marker MBP (red) in the cortex and hippocampus. The red rectangle marks the corresponding magnified were visualized synchronously with 589 image range (a: Cortex. b: Hippocampus). Scale bar: 1 mm for the full-scale images and 30 μm for the mag- the behavioral seizure manifestations nified images. in the two genotypes. As shown in 590 Fig. 5K, the differences in seizure 591 latencies as previously described 592 were apparent. As expected, the 593 520521522523524525526527528529530531532533534535 severe tonic–clonic seizures while the seizures suffered by Alg13 KO mice had higher frequency and amplitude of elec- 594 536 the WT mice appeared relatively mild (Fig. 5B). To better trographic seizure activities compared with the WT mice. 595 537 and directly demonstrate the overall difference between the In addition, to determine whether the knockout of Alg13 596 538 two groups during the experiment, we integrated the indivi- affecting susceptibility to epilepsy is a general phenomenon, 597 539 dual scoring of each animal and found that the seizure sever- a pilocarpine-induced seizure model was also used. The 598 540 ity in the Alg13 KO group was markedly higher than in the WT Alg13 KO and WT mice were injected intraperitoneally with 599 541 group (Fig. 5C). Additionally, different kinds of latencies dur- different doses of pilocarpine and compared for mortality 600 542 ing the whole progressionUNCORRECTED to final status epilepticus were and seizure severity.PROOF At the lower dose of pilocarpine 601 543 assessed. First, the latency to the initial abnormal behavior (225 mg/kg), the mice in both groups survived, but the 602 544 that occurred after KA administration reflected the drug sensi- Alg13 KO mice developed obviously more continuous and 603 545 tivity of the two groups, and the Alg13 KO mice had shorter severe seizures than the WT mice within 2 h after the admin- 604 546 latencies than the WT mice (Fig. 5D). Then, to confirm istration of pilocarpine (Fig. 6B). In addition to the seizure 605 547 whether the events leading to final status epilepticus might score, the seizure severity calculated by integrating the indivi- 606 548 also have been affected by the absence or presence of dual scores of each mouse during the experiment was also 607 549 Alg13, the latencies measured from KA injection or the first compared, and the severity of seizures in the Alg13 KO mice 608 550 abnormal behavior to status epilepticus were evaluated and was significantly higher than in the WT mice (Fig. 6C). In 609 551 the Alg13 KO mice had shorter time duration to developing striking contrast, the seizure severity of both the WT and 610 552 SE (Fig. 5E and F). Progressing to SE did not consist of a sin- Alg13 KO mice was drastically increased at the higher dose 611 553 gle event; there were many steps and control points working of pilocarpine (250 mg/kg). Kaplan–Meier survival curves 612 NSC 18934 No of Pages 18 15 March 2019

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613 showed that Alg13 deficiency caused a marked increase in ALG13 deficiency exacerbated the neuronal 618 614 mortality compared with the WT group (Fig. 6D). Collectively, damage following intra-amygdala KA-induced 619 615 these results indicate that Alg13 deficiency significantly status epilepticus 620 616 increases the susceptibility to seizures and aggravates the To investigate the function of ALG13 in KA-induced neuron 621 617 severity of seizures. damage and whether this damage was exacerbated in the 622 case of ALG13 deficiency, 623 the lesion conditions of the 624 hippocampus and cortex after 625 1 and 2 months following sta- 626 tus epileptic were analyzed 627 by Nissl staining. The results 628 demonstrated that, under the 629 same non-intervention cir- 630 cumstances (control), no gen- 631 eral structural change was 632 observed in the hippocampus 633 of the Alg13 KO mice com- 634 pared with the WT control 635 mice. However, in the Alg13 636 KO mice, neuron loss was 637 evident in the CA3 region of 638

Fig. 4. Seizure activities inﬂuenced the mRNA and protein expression levels of ALG13. (A) Schematic of the experimental protocol. (B) Two epidural screw electrodes implanted to record the EEG. One epidural screw acted as a fastener, and a guide cannula was implanted into the intra-amygdala. LA, left electrodes; RA, guide cannula; RF, right electrode; RO, right occipital screw. (C) Schematic showing the intra- amygdala injection cannula assem- bly. (D) Comparison of the position and mapping of the left lobes. On the right, a representative Nissl image from three independent experiments of the mice brain (coronal cut, +1.2 mm relative to the bregma) shows a track for microinjection into the amygdala (red oval frame). (E and F) RT = qPCR analysis of the ALG13 mRNA expression in the cortex and hippocampus of the KA-induced epileptic mice and the controls. The ALG13 mRNA was normalized to GAPDH UNCORRECTED PROOFmRNA levels and mean ± SEM values presented as a multiple of controls (one-way ANOVA). (G and I) Representative Western blotting showing ALG13 in the cortex and hippocampus of the KA-induced epileptic mice and the controls. (H and J) Quantiﬁcation of the Western blotting band intensities. ALG13 immunoreactivity was normalized to β-actin immunoreactivity (one-way ANOVA). Data are presented as mean ± SEM values; P <.05, P <.01,and P <.001. NSC 18934 No of Pages 18 15 March 2019

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UNCORRECTED PROOF

Fig. 5. NSC 18934 No of Pages 18 15 March 2019

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639 the hippocampus after 1 month (KA 1 M) and 2 months (KA invading the cavities caused by neuron loss. These phenom- 681 640 2 M) following KA injection, and the CA1 region also dis- ena were more obvious in the Alg13 KOgroupthanintheWT 682 641 played severe neuron loss after KA 2 M. In the similar brain group. For the non-intervention treatment, astrocytes were 683 642 regions, comparatively less neuron loss was observed in evenly and regularly distributed in all of the hippocampal 684 643 the WT mice. The Alg13 KO + KA 1 M group exhibited more regions of the WT and Alg13 KO mice. However, after KA 685 644 conspicuous changes in the DG region compared with the 1 M and KA 2 M, the results clearly exhibited increased 686 645 WT + KA 1 M group, such as loose, widened, and diffuse hypertrophy the GFAP-positive cells, enlarged, thickened, 687 646 granule cell layers. A more severe situation with granule cell and irregularly shaped protrusions, and glial scars formed 688 647 loss was observed in the DG region in the Alg13 KO mice by the GFAP-positive cells invading the cavities caused by 689 648 after KA 2 M. These results suggested that ALG13 deficiency neuron loss (Fig. 8). Compared with the WT mice, the mani- 690 649 aggravated neuron lesions in the hippocampus. Upon the festation was more marked in the Alg13 KO mice. Again, 691 650 development of status epilepticus by intra-amygdala KA these results verify that the absence of ALG13 aggravated 692 651 administration, the spread of epileptic activities can cause the loss of neurons in the hippocampal regions after the 693 652 histopathological changes in other ipsilateral regions outside KA-induced damage, as well as the hyperplasia of the hippo- 694 653 the hippocampus. Therefore, we measured the loss of ipsilat- campal reactive astrocytes. 695 654 eral cortical neurons. The results indicated that the number of 655 neurons in the WT and Alg13 KO groups after KA 2 M was ALG13 deficiency increased synaptic plasticity 696 656 significantly lower than in the respective control and KA 1 M following intra-amygdala KA-induced status 697 657 groups, but there was no significant difference between the epilepticus 698 658 WT and Alg13 KO groups (Fig. 7A, B, and C). Consequently, 659 the lack of Alg13 did not obviously aggravate the loss of neu- To identify the effect of ALG 13 deficiency on synaptic plasti- 699 660 rons occurring away from the injection site (for example, in city, we first used Timm's staining methods to visualize 700 661 the ipsilateral cortex). In summary, after KA administration, mossy fiber sprouting (MFS) including axonal sprouting, 701 662 ALG13 deficiency exacerbated the loss of neurons in the growth, and extension. In the WT control mice, Zn-positive 702 663 CA1 and CA3 regions of the hippocampus, resulting in the axons of DG cells were observed in the hilus and stratum 703 664 loss and abnormal distribution of dentate gyrus granule cells. lucidum (SL) but not in the stratum oriens (SO) or inner mole- 704 665 To further characterize the damage in the hippocampus cular layer (IML). However, in the Alg13 KO control mice, the 705 666 caused by KA-induced epileptic status, we stained the sec- Zn-positive axons were abnormally elongated in the SO (the 706 667 tions with neuronal marker NeuN and astrocyte marker yellow arrowheads). Two months after KA-induced status 707 668 GFAP. In normal adult mice, the labeled neurons distribute epilepticus, MFS appeared in the SO and IML of both the 708 669 in the CA1, CA3, and DG regions of the hippocampus, mainly Alg13 KO and WT mice (the yellow arrowheads). Moreover, 709 670 in the pyramidal cells and granule cells. Under the non- compared with the WT mice, the Zn-positive axons were 710 671 intervention condition (control), the results demonstrated no more aberrantly elongated in the SO and IML of the Alg13 711 672 abnormality of neuronal distribution, morphology, and num- KO mice (Fig. 9A). 712 673 ber in the hippocampal regions of the WT and Alg13 KO Zinc transporter 3 (ZNT3), a zinc transporter with synaptic 713 674 mice. On the contrary, some neuronal death, especially in vesicle specificity primarily in the zinc-enriched axon term- 714 675 the CA1 and CA3 regions, together with a looser and wider inals, is necessary for the accumulation of zinc ions within 715 676 distribution of the granule cells in the DG area were observed the synaptic vesicles (Wenzel et al., 1997; Kaneko et al., 716 677 in both groups after KA 1 M and KA 2 M. Moreover, certain 2002). The distribution of ZNT3 and Zn2+ are consistent in 717 678 neurons in the pyramidal cell layer of the hippocampus MFS of the hippocampus, so immunofluorescent labeling of 718 679 shrank with an irregular shape. The CA1 and CA3 regions ZNT3 can be used to visualize hippocampal MFS. Except 719 680 also presented neuron malalignment and labeled astrocytes in the WT control group, the Alg13 KO control, WT, and 720

Fig. 5. Alg13 deficiency increased seizure susceptibility in the KA-induced epileptic model. (A) Schematic of the experimental protocol. (B) Raw scores of the seizure severity evaluation in the WT (n = 13) and Alg13 KO (n = 13) mice using the Racine scale at each 5 min interval after KA injection. Compared with the WT mice, seizuresUNCORRECTED were more severe and lasted longer in the Alg13 KO mice (Student's t-test; PROOFP <.05,P <.01,andP < .001). (C) The total seizure severity evaluation of the WT and Alg13 KO mice. A 100% value was assigned to the average seizure severity value of the WT mice and then used to normalize the severity of the KO mice within the same scale. The Alg13 KO mice demonstrated higher seizure severity (%) than the WT mice (Student's t-test; P < .01). (D) The latency to the first abnormal behavior observed after KA administration. The latency of the Alg13 KO mice was significantly shorter than the WT mice (Student's t-test; P < .001). (E) The latency to status measured from the time of KA administration. Compared with the WT mice, the latency of the Alg13 KO mice was significantly shorter (Student's t-test; P < .001). (F) The latency to status measured from the time of the first abnormal behavior. The Alg13 KO mice had a significantly shorter latency to status than the WT mice (Student's t-test; P < .001). (G) The latency to the first stage 5 seizure measured from the time of KA injection. The WT mice had longer latency, and the difference between the two groups was also significant (Student's t-test; P < .001). (H) The latency to the first stage 5 seizure measured from the time of the first abnormal behavior. The Alg13 KO mice had a shorter latency relative to the WT group (Student's t-test; P < .01). (I) The latency from the first stage 5 seizure to the onset of status. There was significant difference between these two groups (Student's t-test; P < .001). (J) The percentage of mice that had a first stage 5 seizure as the onset of status (that is, the first stage 5 seizure did not terminate). The detailed ratio numbers are marked above the columns. None were observed in the WT group. The KO group was higher than the WT group, with statistical significance (χ2 test and Fisher's exact test; P < .001). (K) A representative EEG recording of approximately 2 h of KA-induced seizure activity in the WT and Alg13 KO mice. Data are presented as mean ± SEM. P <.05, P <.01,and P <.001. NSC 18934 No of Pages 18 15 March 2019

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post-KA, but there was a denser dis- 731 tribution of PSD95 in the Alg13 KO + 732 KA 2 M group. Nevertheless, no 733 aberrant PSD95 expression was 734 observed in both WT and Alg13 KO 735 control mice (Fig. 9C). 736

The more severe neurological 737 phenotype of the Alg13 KO 738 mice was associated with 739 mTOR hyperactivation 740 The mTOR signaling pathway regu- 741 lating neuronal development and 742 synaptic plasticity is often aberrantly 743 activated in epilepsy-related dis- 744 eases. mTOR is a key factor in this 745 signaling pathway and is the acti- 746 vated form of mTOR, which is phos- 747 phorylated at Ser2448 (Navé et al., 748 1999). Therefore, we used Western 749 blotting to examine the relative levels 750 of p-mTOR/mTOR in the hippocam- 751 pus and cortex of the mice to 752 evaluate whether mTOR activation 753 wasaffectedbyALG13deficiency. 754 The expression level of p-mTOR 755 increased while mTOR decreased in 756 the hippocampus and cortex in the 757 Alg13 KO mice compared with the 758 WT mice (Fig. 10A). The relative 759 levels of p-mTOR/mTOR in the hip- 760 pocampus and cortex markedly 761 increased both in hippocampus and 762 cortex of the Alg13 KO mice, indicat- 763 ing that the balance of mTOR and 764 p-mTOR was perturbed and the 765 Fig. 6. Alg13 deficiency increased seizure severity and mortality in the pilocarpine-induced epileptic mTOR signaling pathway was abnor- 766 model. (A) Schematic of the experimental protocol. (B) Raw scores of the seizure severity evaluation in the mally activated due to a lack of 767 WT (n = 8) and Alg13 KO (n = 8) mice using the Racine scale at each 5 min interval after pilocarpine injec- ALG13 (Fig. 10). 768 tion (225 mg/kg). Compared with the WT mice, the seizures were more severe and lasted longer in the Alg13 KO mice (Student's t-test; P <.05,P <.01,andP < .001). (C) The total seizure severity evaluation of the WT and Alg13 KO mice. The Alg13 KO mice demonstrated higher seizure severity (%) than the WT DISCUSSION 769 mice (Student's t-test; P < .001). (D) Kaplan–Meier survival curves showing a statistically significant difference (P < .05) in survival between the WT and Alg13 KO mice over 120 min after pilocarpine administration To the best of our knowledge, this is 770 (250 mg/kg). Survival was assessed using the Kaplan–Meier analysis, and the statistically significant differ- the first study reporting the associa- 771 ence between two groups was determined with the log-rank test. Data are presented as mean ± SEM. tion of ALG13 with epilepsy in animal 772 P <.05,and P <.01,and P <.001. experiments, demonstrating that 773 UNCORRECTED PROOFALG13 deficiency has an epilepsy- 774 promoting function. First, epileptic 775 721 Alg13 KO + KA 2 M had ZNT3 expression in the SO. Com- conditions had a significant effect on the expression of 776 722 pared with the WT + KA 2 M mice, the Alg13 KO + KA 2 M ALG13 in the forebrain. Second, behavioral seizures in the 777 723 group displayed a greater ZNT3 labeling area and a closer KA-induced and pilocarpine-induced epileptic models 778 724 distribution to the more caudal regions of the SO (Fig. 9B). demonstrated that ALG13 deficiency increased the suscept- 779 725 Immunofluorescence was performed by highlighting post- ibility to epileptic seizures. Third, ALG13 deficiency exacer- 780 726 synaptic density protein 95 (PSD95), an epilepsy-related pro- bated the classical pathological manifestations of epilepsy 781 727 tein implicated in synapse maturation, stability, strength, and in the KA-induced epileptic mice. 782 728 plasticity (Meyer et al., 2014). The results demonstrated that The present study began with mapping the distribution of 783 729 PSD95 was widely distributed in the hippocampal ML of both ALG13 in mouse brain. The results of histological analysis 784 730 the KA-induced epileptic WT and Alg13 KO mice after 2 M suggested that the distribution of ALG13 in the brain varies 785 NSC 18934 No of Pages 18 15 March 2019

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Fig. 7. Nissl staining showed neuron damage in the hippocampus and cortex. The neuronal cell morphology in the hippocampus and cortex was detected using Nissl staining. (A) Representative images of Nissl staining of the hippocampus and cortex. Scale bar: 250 μm. (B) Histograms of the Nissl- positive neurons obtained from Nissl staining in the hippocampus. A 100% value was assigned to the average Nissl-positive neuron count in the WT control mice in respective areasUNCORRECTED of the hippocampus and then was used to normalize the value of the other micePROOF groups within the same scale. For the hippocampus of the WT mice, there was no significant difference between the control and KA 1 M, but the number of neurons in the KA 2 M group was significantly lower than in the control; one-way ANOVA; P < .05 (CA1 and DG) and P < .01 (CA3). However, the positive neurons of the Alg13 KO mice had gradually reducing tendencies with statistical significance: in the CA1 area, the positive neurons after KA 2 M were significantly lower than in the controls (one-way ANOVA; P < .05). The positive neurons in the DG and CA3 areas of the hippocampus after KA 2 M were less than in the controls and after KA 1 M; one-way ANOVA; P <.001andP < .001 (DG); and P <.001andP < .05 (CA3). The quantified values between the WT and KO groups were also compared and the controls of the three areas in the hippocampus had no statistical difference (Student's t-test). Obviously, as demonstrated in CA1, the number of neurons after KA 2 M was less in the Alg13 KO group compared with the WT group (Student's t-test; P < .05). For the areas of DG and CA3, the Alg13 KO mice had fewer positive neurons than the WT mice after KA 1 M and KA 2 M; Student's t-test; P < .05 and P < .05 (DG); and P <.001andP < .001 (CA3). (C) Histograms of the Nissl-positive neurons obtained via Nissl staining in the cortex (Student's t-test). The same normalization procedures were executed. The number of neurons in the WT and Alg13 KO group after KA 2 M was significantly lower than in their respective control groups and KA 1 M (one-way ANOVA; P < .01 and P < .05), but there was no significant difference between the WT and Alg13 KO groups (Student's t-test). Data are presented as mean ± SEM. P < .05, P <.01,and P <.001. NSC 18934 No of Pages 18 15 March 2019

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Fig. 8. ALG13 deficiency exacerbated neuron loss and reactive astrogliosis following intra-amygdala KA-induced status epilepticus. (A) Represen- tative images of immunofluorescent triple-staining with NeuN (green), GFAP (red), and DAPI (blue) in the ipsilateral side of the hippocampus including the CA1, CA3, and DG areas of the WT and Alg13 KO mice. The yellow arrow shows neuron loss and reactive astrocyte infiltration in CA3. Scale bar: 1 mm for the full-scale images and 100 μmforthemagnified images. (B) The percentages of reactive astrocytes in the different regions of the whole hippocampus (n = 6 per group). The comparisons between the WT and Alg13 KO groups were carried out using Student's t-test. Data are presented as mean ± SEM. P < .05 and P < .01; ns, non-significant. UNCORRECTED PROOF 786 in a regional-specific manner. Moreover, the level of ALG13 (Chang and Lowenstein, 2003). Consequently, the regional- 797 787 expression was high in the neurons but hardly detected specific and cellular-specific distribution of ALG13 indicates 798 788 in the astrocytes or oligodendrocytes, indicating a cell type that Alg13 KO mice are likely prone to epileptic susceptibility. 799 789 specific expression pattern. Temporal lobe epilepsy (TLE) is This study used a model producing status epilepticus via 800 790 the most common form of epilepsy, because the temporal intra-amygdala KA microinjection (Shinoda et al., 2004; Li et 801 791 lobe structures, especially the hippocampus, amygdala, al., 2008). The animals showed recurrent seizure behaviors 802 792 and piriform cortex, are most susceptible to seizurogenic and progressive neuron death, providing a good model 803 793 and epileptogenesis-triggering brain lesions (Engel, 1989). of neurodegenerative diseases (Curia et al., 2014). We 804 794 Additionally, the main clinical manifestation of epilepsy is observed a significant change in the total mRNA and protein 805 795 recurrent seizure, which is normally caused by abnormally of ALG13 extracted from the ipsilateral hippocampal and 806 796 excessive electrical discharges of the cerebral neurons cortex tissue after KA-induced status epilepticus at different 807 NSC 18934 No of Pages 18 15 March 2019

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control group from 4 h post- 812 KA and demonstrated an 813 increasing trend. Neverthe- 814 less, the expression of 815 ALG13 mRNA and protein in 816 the hippocampus dramatically 817 decreased 1 h post-KA and 818 then recovered gradually but 819 never reached the control 820 level. In the same brain 821 region, the ALG13 expression 822 level of mRNA synchronized 823 with that of protein. Mouri 824 found that neuronal loss can 825 be observed at the injection 826 site and in the ipsilateral dor- 827 sal CA3 region at an early 828 stage after administration 829 (Mouri et al., 2008). That is, 830 epileptiform activity starts 831 from the amygdala (the site 832 of injection) and then succes- 833 sively propagates to the ipsi- 834 lateral hippocampus, cortex, 835 contralateral amygdala, and 836 contralateral hippocampus 837 (Mouri et al., 2008). That 838 might explain why ALG13 839 protein markedly decreases 840 in the ipsilateral hippocampus 841 while remaining unchanged 842 in the cortex after initial KA 843 injection. Furthermore, the 844 pathophysiological and struc- 845 tural alterations caused by 846 epilepsy exacerbate epileptic 847 seizure activity while trigger- 848 ing protective factors that 849 impede seizure activity; 850 thus, seizure activity can be 851 restrained (Dalby and Mody, 852 2001; Pitkänen and Lukasiuk, 853 2009; Pitkanen and Lukasiuk, 854 Fig. 9. Zn-positive axon terminals of DGC, the pattern of ZnT3 immunoreactivity, and the expression of synap- 855 tic marker PSD95 were aberrantly altered following intra-amygdala KA-induced status epilepticus in the 2011). In brief, the dynamic Alg13 KO mice. (A) Representative images of Timm histochemistry in the coronal cryosections in the hippocampus. expression of ALG13 sug- 856 More abnormally dark brown density of the reaction products was observed in the SO (the yellow arrowheads) and gests a probable compensa- 857 IML (the yellow arrowheads) of the Alg13 KO mice after KA-induced status epilepticus. (B) Representative images tory and protective effect of 858 of immunoﬂuorescent stainingUNCORRECTED with ZnT3. The staining distributed from the hilus to the SL and SO inPROOF CA3 (the yellow ALG13 in epilepsy. 859 arrowheads), in which the immunoreactive staining of ZnT3 more markedly increased in the SO of the Alg13 KO mice Considering the role of 860 after KA-induced status epilepticus compared with the WT mice. (C) Representative images of immunoﬂuorescent ALG13 in epilepsy following 861 staining with PSD95. The intensity of PSD95 was greater in the entire molecular layer of the Alg13 KO mice than 862 in the WT mice after KA-induced status epilepticus (the yellow lines indicate the extent of the molecular layer). SO, induced status epilepticus stratum oriens; SBI, subiculum; DGC, dentate granule cells; IML/OML, inner/outer molecular layer; GCL, granule cell as previously described, we 863 layer. Scale bar: 150 μm. hypothesize that ALG13 may 864 protect the neurons from 865 injury or promote neuronal 866 808 time points. However, the expression trends of both the repair through certain mechanisms after brain insult. To verify 867 809 ALG13 mRNA and protein in the hippocampus were this hypothesis, we used the chemical convulsant agent KA 868 810 obviously different from those in the cortex. The levels of to induce acute seizures via activating the excitatory gluta- 869 811 ALG13 mRNA and protein in the cortex increased in the mate receptors and assessed the severity of induced 870 NSC 18934 No of Pages 18 15 March 2019

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in the cortex but later than in the 898 hippocampus, likely due to distant 899 lesions that were caused by the pro- 900 pagation of seizure activities of SE 901 previously terminated by diazepam 902 after 1 h rather than KA itself. Thus, 903 neuronal damage at the same time 904 point after 2 months was detected in 905 both the Alg13 KO and WT mice, 906 and the surviving neurons in the 907 Alg13 KO mice were less than in the 908 WT mice but without statistical 909 difference. Therefore, the potential 910 neuroprotective role of ALG13 in 911 KA-induced seizure mice models is 912 increasingly evident. 913 In particular, the astrocyte 914 response to CNS damage via reac- 915 tive astrogliosis has been considered 916 a salient feature of human TLE and 917 most animal models with recurrent 918 seizures (Sofroniew, 2015; Pekny 919 et al., 2016). In this study, GFAP 920 staining revealed the abnormal struc- 921 ture of the insulted astrocytic area 922 Fig. 10. Altered expression levels of ALG13 and p-mTOR were observed in the hippocampus and caused by KA administration in the 923 cortex after Alg13 knockout. (A) Representative Western blotting showing immunoreactivity of ALG13 mice. The ratio of reactive astrocytes 924 fi and p-mTOR in the hippocampus and cortex of the WT and Alg13 KO mice. (B and C) Quanti cation of with hypertrophic cell bodies and 925 the Western blotting band intensities. ALG13 and p-mTOR immunoreactivity was normalized to β-actin thick processes in the hippocampus 926 and mTOR immunoreactivity, respectively (Student's t-test). Data are presented as mean ± SEM. 927 P <.05, P <.01,and P <.001. was higher in the Alg13 KO mice accompanied by more serious neuro- 928 degeneration in the hippocampal 929 871 seizures between the WT and Alg13 KO mice. As a result, CA3 regions. Although reactive astrocytes have pro- and 930 872 ALG13 deficiency markedly exacerbated the severity of KA- antiepileptic effects, an ever-growing body of evidence sup- 931 873 induced seizure, which was confirmed via various quantifica- ports the former (Falcón-Moya et al., 2018). Together, 932 874 tion methods. Again, to further test this hypothesis, we used the data indicate that ALG13 deficiency may influence the 933 875 the chemical convulsant agent pilocarpine to induce acute number of reactive astrocytes that play vital roles in regulat- 934 876 seizures via activating the M1 muscarinic receptors of the ing seizure susceptibility. 935 877 excitatory cholinergic system. Similar results were observed The formation of new synaptic connections is an imperative 936 878 under a lower dosage of pilocarpine (225 mg/kg) (to avoid process in epileptogenesis, and MFS is a main marker of 937 879 ceiling and floor effects). Significantly, the Alg13 KO mice abnormal synapse formation in the epileptic brain (Sutula 938 880 had greater mortality with the administration of a higher and Dudek, 2007). Under normal conditions, the Alg13 939 881 dosage of pilocarpine (250 mg/kg), reinforcing the possible KO brain exhibited obvious abnormalities in mossy fiber 940 882 role of ALG13 in the process of epilepsy. In conclusion, our innervation. Two months after KA, MFS was obviously 941 883 findings support that ALG13 may play a protective role in this increased in the SO of the Alg13 KO mice compared with 942 884 seizure model. the WT mice. Meanwhile, Timm-positive mossy fibers gradu- 943 885 Recurrent seizures are often concomitant with neuronal ally sprouted in the dentate granule cell layer and extended 944 886 loss in human patientsUNCORRECTED and animal models (Curia et al., toward the dentate PROOF IML. However, no visible Timm-positive 945 887 2014). Thus, we used Nissl staining and the neuronal marker mossy fiber staining appeared in the IML, probably due to a 946 888 NeuN to trace neuron death and degeneration and investi- lower quantity of vesicular zinc in the mossy fiber terminals. 947 889 gate the effects of ALG13 deficiency on KA-induced neuro- Fundamentally, regular spontaneous epileptic discharges 948 890 degeneration in the hippocampus and cortex. Our results occur after KA treatment in TLE mouse models, which 949 891 provided convincing evidence that the ipsilateral hippocam- depletes the zinc vesicles along with regular massive gluta- 950 892 pus and cortex of the Alg13 KO mice demonstrated distinct mate release (Riban et al., 2002; Mitsuya et al., 2009). To 951 893 vulnerability profiles to KA-induced brain damage compared exclude the possibility that the absence of MFS was caused 952 894 with WT. The lack of Alg13 dramatically aggravated KA- by a loss of sprouted mossy fibers, immunofluorescence 953 895 induced neurodegeneration in the hippocampal CA3 region, staining with ZNT3 was performed. The results also revealed 954 896 the brain region most affected by the activities of the gluta- no visible positive expression in the IML. Overall, the 955 897 mate receptors. However, neuronal damage also presented increased synaptic plasticity in the Alg13 KO mice suggested 956 NSC 18934 No of Pages 18 15 March 2019

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957 that ALG13 may hamper induced neuronal network reorgani- experiments and performed the statistical analyses. BL.Y. 1009 958 zation in the brain. and J.W. provided the experimental animals and assisted 1010 959 The activation of the mTOR pathway was reported to be with the animal breeding. P.G. wrote the manuscript. P.G. 1011 960 related to the cytopathology and epileptogenicity of epilepsy and F.W. contributed to the work equally and were the co first 1012 961 (Liu et al., 2014). Moreover, mTOR hyperactivation was authors. T.S. was the corresponding author. All of the authors 1013 962 observed in genetic and acquired epilepsy syndromes read and approved the final manuscript. 1014Q9 963 (Liu et al., 2014, Mirzaa and P, 2014, Sharma et al., 2010, 964Q8 Sosunov et al., 2012). 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