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Vitamin B6 Deficiency Induces Autism-Like Behaviors in Rats by Regulating mTOR-Mediated Autophagy in the Hippocampus

Vitamin B6 Deficiency Induces Autism-Like Behaviors in Rats by Regulating mTOR-Mediated Autophagy... Hindawi Behavioural Neurology Volume 2023, Article ID 6991826, 12 pages https://doi.org/10.1155/2023/6991826 Research Article Vitamin B6 Deficiency Induces Autism-Like Behaviors in Rats by Regulating mTOR-Mediated Autophagy in the Hippocampus 1 2 2 2 2 2 2 Lijuan Chen, Jing Li, Xinglian Liu, Zhiwei Zhao, Yan Jin, Yikun Fu, Aiqin Zhou, 2 3 Chengqun Wang, and Yan Zhou Department of Pediatric Neurology, Hubei Maternal and Child Health Hospital, Wuhan, China Children’s Health Department, Hubei Maternal and Child Health Hospital, Wuhan, China Mass Spectrometry Center, Wuhan KingMed Diagnostics Group Co., Ltd, Wuhan, China Correspondence should be addressed to Yan Zhou; zhouyan_zzhy@163.com Received 2 November 2022; Revised 14 April 2023; Accepted 15 April 2023; Published 9 May 2023 Academic Editor: Giuseppe Biagini Copyright © 2023 Lijuan Chen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Vitamin B6 (VB ) exhibits therapeutic effects towards autism spectrum disorder (ASD), but its specific mechanism is poorly understood. Rat dams were treated with VB standard, VB deficiency, or VB supplementary diet, and the same treatment was 6 6 6 provided to their offspring, with their body weights monitored. Three-chambered social test and open field test were employed to evaluate the effect of VB on autism-like behaviors. Gamma-aminobutyric acid (GABA) generation and synaptic inhibition of neurons in the hippocampus of rat were detected via immunofluorescence staining, followed by the measurement of GABA concentration through high-performance liquid chromatography (HPLC). The role of VB in the autophagy and apoptosis of cells was determined via Western blot and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). In order to conduct rescue experiments, the inhibition of mammalian target of rapamycin (mTOR) or the activation of GABA was achieved by drug administration to the offspring rats with VB deficiency. As a result, no evident difference in weight was observed in the offspring with varied VB treatments. VB deficiency impaired social interaction; aggravated self-grooming and 6 6 bowel frequency; decreased GABA concentration, VIAAT, GAD67, vGAT expressions, and LC3 II/LC3 I ratio; increased p62 level and p-mTOR/mTOR ratio; and promoted cell apoptosis. Inhibition of mTOR reversed the effect of VB deficiency on cell autophagy. GABA activation or mTOR inhibition offset the role of VB deficiency in autism-like behaviors and hippocampal GABA expression. Collectively, VB deficiency induces autism-like behaviors in rats by regulating mTOR-mediated autophagy in the hippocampus. 1. Introduction (GABA)ergic synaptic transmission, which in turn influence the balance of excitatory/inhibitory signal ratio, leading to Autism spectrum disorder (ASD) is a pervasive neurodeve- autism-like behaviors [4]. Interneuronal GABAergic disor- lopmental disease with core symptoms of impaired social ders have been observed in many animal models of ASD. interaction, repetitive stereotypical behaviors, and narrow Thus, repairing the dysfunction of the GABA system is interests [1–3]. Unfortunately, there is no effective treatment thought to be an important way to alleviate ASD [5]. for ASD, and children with this disease have poor social As an essential nutrient for the body, vitamin makes a adaptability, bringing great emotional and economic burdens profound impact upon the development and function of to society and families, which, therefore, has aroused wide- the brain and nervous system [6]. In recent years, multiple spread attention. It is believed that ASD is caused by a combi- vitamins have been proved to alleviate ASD, including vita- nation of both polygenic genetic and external environmental min A (VA), VB , and VD [7, 8]. VB , a pyridine derivative, 6 6 factors. Pathogenetically, during early embryonic develop- works as a coenzyme factor in diverse metabolic reactions of ment, patients with ASD are affected by a variety of factors the human body, including amino acid metabolism, syn- that impair neuronal function and gamma-aminobutyric acid thesis and breakdown of neurotransmitters, regulation of 2 Behavioural Neurology steroid hormone activity, and control of gene expression [9, for 4 weeks. During the pregnancy and lactation, in consis- 10]. Although the combination therapy using VB has been tent with the grouping of their mother rats, the rats were reported to be applied for ASD at present [11], knowledge continued to be given aforementioned VB treatments. After gaps on its specific mechanism remain. However, existing weaning, new pups (male) were collected and divided into research suggests that it might be associated with the ability the three groups: Control group (n =6, a basic diet with of VB to strengthen the neurotransmitter system [12, 13]. 6mg VB ), VB deficiency group (n =24, a basic diet with- 6 6 6 Autophagy is a process of transporting misfolded pro- out VB ), and VB supplement group (n =6, a basic diet 6 6 teins or damaged cell organelles, dysfunctional mitochon- with 30 mg VB ). The body weight of offspring rats was dria, for instance, through autophagic vesicles to lysosomes recorded from postnatal day 1 (PND 1) to PND 42. The off- for degradation [14]. Autophagy is a pivotal mechanism spring rats between PND 42 and PND 56 were subjected to for cells to maintain homeostasis, the imbalance of which the behavioral tests. The mother rats were sacrificed by cer- in neurons is usually linked to cerebral diseases, including vical dislocation under anesthesia (40 mg/kg, pentobarbital ASD and neurodegenerative diseases [15–18]. Previous stud- sodium, P-010, Sigma-Aldrich, USA). ies have pointed out that disruption of mammalian target of 2.3. Drug Administration. GABA receptor agonist Clonaze- rapamycin (mTOR)-regulated macroautophagy and autoph- pam (C-907, Sigma-Aldrich, USA) and mTOR inhibitor agy leads to autism-like abnormalities [19, 20]. Recently, it NVP-BEZ235 (A10133, Adooq Bioscience, China) were used has been additionally demonstrated that enhancing for drug administration in vivo. A total of 18 offspring rats mTOR-mediated autophagy contributes to the restoration were randomly selected from the abovementioned VB defi- of GABAergic signaling [21], which might alleviate autism- 6 ciency group and assigned into a new VB deficiency group like behaviors in rats [22]. 6 (a basic diet without VB ), VB deficiency + Clonazepam Three regions of the adult brain, the olfactory bulb, 6 6 group (intraperitoneal injection of 0.025 mg/kg Clonazepam hypothalamus, and hippocampal dentate gyrus, contain based on the VB deficiency diet), and VB deficiency newborn neurons that are crucial players in the natural func- 6 6 + NVP-BEZ235 group (intraperitoneal injection of 400 μg/ tional circuits [23]. Many neurodegenerative diseases and kg NVP-BEZ235 based on the VB deficiency diet), with six neurodevelopmental disorders involving cognitive impair- 6 rats in each group. All offspring rats in this experiment were ment may be associated with hippocampal dysfunction, reared for 42–56 days since PND 1, and the drug administra- which is at least partially attributable to adult neurogenetic tion was implemented 30 minutes prior to the behavioral disorders [24]. A recent review stated that hippocampus tests [20, 22]. may represent an important component within a system of changed brain regions that work in tandem to contribute 2.4. Behavioral Tests to the phenotype of ASD [25]. Moreover, VA deficiency induces autistic-like behaviors in rats through modulating 2.4.1. Three-Chambered Social Test. A three-chambered the RARβ-CD38-oxytocin axis in the hypothalamus [26]. apparatus (XR-XJ117) purchased from XinRuan Informa- Thus, the hippocampus was chosen for the study of VB in 6 tion Technology (Shanghai, China) was applied to conduct ASD. sociability test [22]. After 10-minute habituation, the off- In this study, to suss out the role of VB in ASD, we 6 spring rats (PND 42 and PND 56) with or without the indi- firstly constructed the models in the offspring rat with VB cated treatment of VB or drugs above were individually deficiency or VB supplement and then explored whether 6 placed into the center chamber of the apparatus and allowed the mechanism of VB in ASD was associated with mTOR- 6 to access three chambers for 10 minutes, with an identical mediated autophagy, with the aim to provide new insight cage in each side of the chamber, one of which contained into the pathogenesis of ASD. an unacquainted rat. The time the offspring rats spent in each chamber was automatically recorded. 2. Material and Methods 2.4.2. Open Field Test. The locomotor activity of the off- 2.1. Animals. The surgical procedures with animals in this spring rats (PND 42 and PND 56) with or without the indi- study were approved by the Committee of Zhejiang Baiyue cated treatment of VB or drugs above was evaluated via the Biotech Co., Ltd for Experimental Animals Welfare open field test with the assistance of an uncovered box (approval number: ZJBYLA-IACUC-20220701) and per- (100 × 100 × 50 cm, XR-XZ301, XinRuan Information Tech- formed in accordance with Guide for the Care and Use of nology, China). The box was cleaned thoroughly and steril- Laboratory Animals. A total of 24 virgin female Wistar rats ized with 75% alcohol (E299578, Aladdin, China) before (120–135 g) were purchased from Charles River Labs (Bei- the tests to eliminate any odors. The open field test was car- jing, China) and were group-housed in standard animal ried out according to the previous instructions [26]. Briefly, cages (25 ± 2 C) with consistent 12/12 hours light/dark cycle. the offspring rats were individually placed into the center of the open field and allowed to explore the new environment 2.2. Modeling. Autistic rat models with VB deficiency or without any disturbance for 15 minutes during the test. The SuperMaze software (vision 2.0, XR-Xmaze, XinRuan VB supplement were constructed using the offspring of female Wistar rats as previously described [12]. Based on a Information Technology, China) was applied to monitor basic diet, all female rats were randomly fed with 0, 6, or movement speed, self-grooming time, and bowel frequency 30 mg VB (P5669, Sigma-Aldrich, St. Louis, MO, USA) automatically during the test. 6 Behavioural Neurology 3 After behavioral test, all offspring rats were sacrificed by hippocampal hemispheres from six consecutive serial sec- cervical dislocation under anesthesia, and the hippocampal tions were used. tissues were harvested and stored in liquid nitrogen at −80 C for follow-up experiments. The right hemisphere 2.6. HPLC Assay. The content of GABA in the hippocampi was used for immunocytochemistry and terminal deoxynu- of rats from Control, VB deficiency, and VB supplement 6 6 cleotidyl transferase dUTP nick-end labeling (TUNEL) groups was measured by HPLC assay. In brief, the hippo- experiments, while the hippocampus from the other hemi- campal tissues from both hemispheres were homogenized sphere was utilized for Western blot. The hippocampal tis- in perchlorate solution (0.04 M, abs47048087, Absin, China) sues from both hemispheres were employed for high- on ice, subsequent to which 15-minute centrifugation was performance liquid chromatography (HPLC) assay. The carried out. Then, the supernatants were neutralized with experimental design was described in Supplementary potassium bicarbonate solution (2 M, abs42025446, Absin, Figure 1. China). After centrifugation, 2 ml supernatant of the sample or standard GABA solution was treated with triethylamine- 2.5. Immunofluorescence Analysis. The frozen hippocampal acetonitrile (1 ml, 1/10, v/v) and phenyl isothiocyanate tissues were cut into sections with a thickness of 5μmand fixed (PITC) acetonitrile (1 ml, 1/25, v/v) at room temperature with 4% paraformaldehyde (P0099, Beyotime, China) for 15 for 1 hour away from light, followed by 20 minute n-hex- minutes. After being washed with phosphate buffered saline ane treatment (2 ml, H100107, Aladdin, China). Follow- (PBS, P1022, Solarbio, China), the sections were subjected to ing the filtration, the lower layer solution (1 ml) was 15 minute treatment with Triton X-100 (T8200, Solarbio, harvestedfor analysis.HPLCwas conductedunder an China) and 1 hour blocking using 5% goat serum (abs933, Agilent 1100 HPLC system (Agilent, Santa Clara, CA, USA) to analyze the content of GABA according to the Absin, China). To determine the expressions of vesicular inhibitory amino acid transporter (VIAAT) and glutamate previous protocols [22]. Assay for each sample was decarboxylase 67 (GAD67) in hippocampal CA3 area, the sec- repeated twice. tions were incubated with primary antibodies against VIAAT (1 : 1000, ab101934, Abcam, Cambridge, UK) and GAD67 2.7. Western Blot. The levels of autophagy-related proteins (10μg/ml, ab26116, Abcam, UK) at 4 C overnight away from in the brain tissues from rats in VB deficiency group light. Then, the sections underwent 1 hour incubation with sec- with or without drug administration, VB supplement ondary antibodies Alexa Fluor 488-labeled Goat Anti-Rabbit group, and Control group were measured via Western IgG (1 : 1000, P0176, Beyotime, China) and Alexa Fluor 647- blot. After the extraction of total protein with RIPA Lysis labeled Goat Anti-Mouse IgG (1 : 1000, P0191, Beyotime, Buffer (E-BC-R327, Elabscience, China), BCA Protein China) at room temperature. After 5 minute counterstain with Assay Kit (23225, ThermoFisher, Waltham, MA, USA) was 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride used to detect protein concentration in the collected superna- (DAPI, C1005, Beyotime, China), fluorescent images were cap- tant following the instructions. Afterwards, equal amount of tured using a confocal microscope (LSM 780, ZEISS, Ger- protein (50μg/lane) was separated by 10% sodium dodecyl many) at ×600 magnification. sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, E-IR- To detect colocalization between GAD67 positivity R305, Elabscience, China) and loaded onto nitrocellulose mem- (GAD67 ) with vesicular gamma-aminobutyric acid trans- branes (2215, Millipore, Billerica, MA, USA). Next, the mem- porter (vGAT) in hippocampal CA3 area, the sections were branes were sequentially treated with blocking buffer at room pre-treated with Triton X-100 and 5% goat serum as above temperature for 1 hour and cultivated with primary antibodies and then were cultivated with primary antibodies against at 4 C overnight. The next day, the membranes were rinsed GAD67 and vGAT (1 : 500, 131006, Synaptic Systems, Ger- with tris-buffered saline with Tween-20 (TBS-T, abs952, Absin, many) at 4 C overnight in the dark. The next day, the sec- China) and incubated with secondary antibody at room tem- tions were cultured with secondary antibodies Alexa Fluor perature for 2 hours. For the visualization of immunoblots, 488-Labeled Goat anti-Mouse IgG (1 : 1000, P0188, Beyo- the protein bands were added with PLUS Chemiluminescent time, China) and Alexa Fluor 680-labeled Donkey Anti- Substrate (34577, ThermoFisher, USA) and developed in a Chicken IgG (1 : 100, 703-625-155, Jackson Immuno Tanon 5200 Imaging System (Shanghai, China). All antibodies Research, USA), followed by DAPI staining. Lastly, a confo- were obtained from Abcam (UK), and the primary antibodies cal microscope was used to observe the stained sections were those against LC3 I/LC3 II (1/1000, 14 kDa, 16 kDa, under ×400 magnification. ab192890), p62 (1/10000, 62 kDa, ab109012), mTOR (1/1000, For investigating the activity of GABA in hippocampal 250 kDa, ab32028), phosphor (p)-mTOR (1/1000, 289 kDa, CA3 area from hemispheres of VB -deficient offspring rats ab109268), and glyceraldehyde-3-phosphate dehydrogenase with or without drug administration, the pre-treated sec- (GAPDH, 0.1μg/ml, 36 kDa, ab9484). The secondary anti- tions were cultured with primary antibody targeting GABA bodies consisted of Rabbit Anti-Mouse IgG (1/2000, ab6728) (1 : 2000, ab8891, Abcam, UK) at 4 C overnight without and Goat Anti-Rabbit IgG (1/3000, ab6721). light, followed by the cultivation with Alexa Fluor 647- labeled Goat Anti-Rabbit IgG Secondary Antibody (P0180, 2.8. TUNEL Assay. Cell apoptosis in CA1 and CA3 areas of Beyotime, China) at room temperature for 1 hour. After rat hippocampus from Control, VB deficiency, and VB sup- 6 6 the staining with DAPI, fluorescent images were obtained plement groups was determined using Colorimetric TUNEL by a microscope at ×100 magnification. A total of three Apoptosis Assay Kit (C1091, Beyotime, China). The frozen 4 Behavioural Neurology hippocampal sections were treated with Immunol Staining signals was lessened in hippocampal CA3 area of the off- Fix Solution (P0098, Beyotime, China) at room temperature spring rats with VB deficiency, and no remarkable for 30 minutes, followed by the rinse using PBS twice. Then, change was observed in the offspring rats with VB sup- each section was immersed into Enhanced Immunostaining Per- plement. Additionally, the hippocampal CA3 area of off- meabilization Buffer (P0097, Beyotime, China) for 5 minutes, spring rats with VB deficiency exhibited conspicuously blocked with 0.3% H O in PBS for 20 minutes, and reacted with decreased GAD67 and vGAT, while no significant 2 2 50μl prepared biotin-labeled solution at 37 C in a wet box for 1 change was visible in the offspring rats with VB supple- hour without light. After the reaction was terminated, the sec- ment (Figure 3). Moreover, the results of HPLC assay tions were subjected to color development, including the treat- revealed that the concentration of GABA was dwindled ment of both streptavidin–horseradish peroxidase (HRP) in the hippocampus of offspring rats in VB deficiency (50μl) and 3,3-diaminobenzidine (DAB, 0.2 ml) according to group, compared to that in Control group (Figure 4(a), the manufacturer’s specification. Following dehydration with p <0:001). gradient alcohol and hyalinization with xylene, the number of positively apoptotic cells in the hippocampal CA1 and CA3 by 3.3. VB Deficiency Suppressed Autophagy and Accelerated cells/0.01 mm was counted using a light microscope (×100 mag- Apoptosis of Neurons in the Offspring Rats. Considering that nification, CX23, Olympus, Japan). Three hippocampal hemi- the imbalance of autophagy is closely associated with cerebral spheres from six consecutive serial sections were used. diseases including autism [15], Western blot was employed for measuring the protein levels of LC3 I, LC3 II, p62, mTOR, 2.9. Statistical Analysis. The results of triplicate assays were and p-mTOR in the hippocampal tissues. As compared with expressed as mean ± standard deviation. Kolmogorov– those in Control group, decreased LC3 II/LC3 I ratio and Smirnov test was used to verify normality. One-way analy- increased p-mTOR/mTOR ratio and p62 protein level were sis of variance was used for multi-group comparison, observed in the offspring rats in VB deficiency group, while followed by Tukey or Dunnett’s post-hoc test to detect increased LC3 II/LC3 I ratio was observed in the offspring rats any inter-group differences. Statistical analyses were real- in VB supplement group (Figures 4(b), 4(c), 4(d), and ized using Graphpad Prism 8.0 software (GraphPad Soft- 4(e), p <0:001). We also detected cell apoptosis in hippo- ware Inc., San Diego, CA, USA). P <0:05 indicated the campal CA1 and CA3 areas using TUNEL assay. Notably, data were statistically significant. more cell apoptosis in the hippocampal CA1 area emerged in VB deficiency group, while less apoptotic cells appeared in VB supplement group (Figures 4(f) and 4(g), p <0:001). 3. Results In addition, VB deficiency or VB supplement generated 6 6 no significant effect on cell apoptosis in the hippocampal 3.1. VB Deficiency Reduced Sociability and Aggravated Anxiety-Related Behaviors of Offspring Rats. No abnormal CA3area(Figures4(h)and 4(i)). deaths or loss were observed in all rats during this study. To explore the role of VB in rats with autism-like behaviors, 3.4. The Role of VB in the Autism-Like Behaviors of Rats 6 6 we obtained the offspring from pregnant rats to construct rat Was Mediated by mTOR Inhibition and GABA Activation. model with VB normality, VB deficiency, or VB supple- It has been suggested that the role of mTOR-mediated 6 6 6 ment. As exhibited in weight curves (Figure 1(a)), no obvi- autophagy in the development of autism might be correlated ous difference was reported concerning the weight of the with GABA [22]. Hence, we triggered mTOR inhibition or offspring rats among the control, VB deficiency, and VB GABA activation in the offspring rats with VB deficiency 6 6 6 supplement groups. Subsequently, we investigated whether by drug administration with NVP-BEZ235 or Clonazepam VB has an effect on autism-like behaviors of rats. The eval- and conducted rescue assays. The results of three- uation of social interactions demonstrated that when com- chambered social test unveiled that both NVP-BEZ235 and pared to the control group, in VB deficiency group, the Clonazepam promoted social interaction of the offspring offspring rats had fewer social interactions with unfamiliar rats with VB deficiency, compared to those without drug rats but more interactions with the object, and in VB sup- administration (Figure 5(a), p <0:001). As demonstrated in plement group, the offspring rats had more social interac- Figures 5(b), 5(c), and 5(d), neither NVP-BEZ235 nor Clo- tions with unfamiliar rats and less interactions with the nazepam signally influenced the movement speed, and VB object (Figure 1(b), p <0:05). In line with the open field deficiency-induced increments in self-grooming time and test results, we observed that VB deficiency and VB sup- bowel frequency were dwindled by both NVP-BEZ235 and 6 6 plement exerted no effect on movement speed of the off- Clonazepam (p <0:05). At molecular level, NVP-BEZ235 spring rats (Figure 1(c)), while VB deficiency increased increased LC3 II/LC3 I ratio, decreased p-mTOR/mTOR self-grooming time and bowel frequency (Figures 1(d) ratio, and down-regulated p62 protein level, which reversed and 1(e), p <0:01). the effects of VB deficiency (Figures 6(a), 6(b), 6(c), and 6(d), p <0:001). No observable effect of Clonazepam, how- 3.2. VB Deficiency Diminished Synaptic Inhibition in the ever, was detected on LC3 II/LC3 I ratio, p-mTOR/mTOR Hippocampi of Rats. The immunofluorescence analysis was ratio, and p62 protein level (Figures 6(a), 6(b), 6(c), and conducted to reveal the effect of VB on synaptic inhibition 6(d)). Interestingly, the results of immunofluorescence stain- of neurons in the hippocampus of offspring rats. As illustrated ing demonstrated that both NVP-BEZ235 and Clonazepam in Figure 2, the density of VIAAT and GAD67 fluorescence enhanced GABA in the hippocampi of the offspring rats Behavioural Neurology 5 150 500 ⁎⁎⁎ ^^^ ⁎⁎⁎ ^^^ 0 0 1 7 14 21 28 35 42 After PND time (days) Control VB lack VB supplement Rat Middle Object (a) (b) 0.04 150 ⁎⁎⁎ 0.03 0.02 0.01 ^^^ 0.00 0 (c) (d) ⁎⁎ ^^ (e) Figure 1: VB deficiency reduced sociability and aggravated anxiety-related behaviors of offspring rats. The offspring rats obtained from pregnant rats with VB normality, VB deficiency, or VB supplement were subjected to the modeling. (a) The body weight of offspring 6 6 6 rats was recorded from PND 1 to PND 42. (b) Three-chambered social test was utilized to evaluate the social interactions of offspring rats. (c, d, and e) The movement speed, self-grooming time, and bowel frequency of offspring rats were detected by open field test. Six rats/group. One-way analysis of variance with Tukey’s post-hoc test. VB : vitamin B6; PND: postnatal day; **p <0:01; ***p <0:001 vs. Control; ^^p <0:01; ^^^p <0:001 vs. VB lack. with VB deficiency, compared to those without drug 4. Discussion administration (Figure 6(e)). Finally, a scheme showing the roles of VB , GABA transmission, mTOR, and apoptosis in The treatment for ASD using VB could date back to the 6 6 the proposed mechanism of the autism etiology was pre- 1960s, and years of clinical studies have proven that oral sented in Supplementary Figure 2. administration with high-dose VB can significantly alleviate Wistar rats (g) Movement speed (m/s) Control VB lack Bowel frequency VB supplement Control Self-grooming time (s) Stay time (s) VB lack VB supplement Control Control VB lack VB lack VB supplement 6 6 VB supplement Control VB lack VB supplement Control VB lack VB supplement 6 6 Behavioural Neurology VIAAT GAD67 DAPI Merge Figure 2: VB deficiency reduced GABAergic inhibition in hippocampal CA3 area of offspring rats. The offspring rats obtained from their pregnant mothers with VB normality, VB deficiency, or VB supplement were modeled to evaluate autism-like behaviors. Hippocampal 6 6 6 tissues were collected after euthanasia of offspring rats. The expressions of VIAAT and GAD67 in the hippocampal CA3 area of rat were determined using immunofluorescence staining (×600 magnification, scale bar: 40 μm). Three rats/group. One-way analysis of variance with Tukey’s post-hoc test. VB : vitamin B6; VIAAT: vesicular inhibitory amino acid transporter; GAD67: glutamate decarboxylase 67. Merge GAD67 vGAT Figure 3: VB deficiency diminished GAD67 inhibitory synapses in hippocampal CA3 area of offspring rats. The offspring rats obtained from pregnant mothers with VB normality, VB deficiency, or VB supplement were modeled. Hippocampal tissues were collected after 6 6 6 euthanasia. Immunofluorescence analysis was used to detect synaptic inhibition of neurons in the hippocampus by staining GAD67 and vGAT (×400 magnification, scale bar: 50 μm). Three rats/group. One-way analysis of variance with Tukey’s post-hoc test. VB : vitamin B6; GAD67 : glutamate decarboxylase 67-positive; vGAT: vesicular gamma-aminobutyric acid transporter. autism-like behaviors in patients [27]. Persistent stereotypi- Through the behavioral tests in animal, we observed a nota- cal and repetitive behaviors, narrow interests, and lagging ble lack of social interest and strong repetitive stereotypic social skills constitute the main symptoms of ASD [28]. behaviors in the VB -deficient model rats, but the deficiency VB supplement VB lack Control 6 6 VB supplement VB lack Control 6 6 ×400 ×600 Behavioural Neurology 7 0.4 –16 kDa LC3 I ^^^ 0.3 –14 kDa LC3 II 0.2 ⁎⁎⁎ –62 kDa p62 0.1 –289 kDa p-mTOR 0.0 –250 kDa mTOR –36 kDa GAPDH (a) (b) 6 3 ⁎⁎⁎ ^^^ 4 2 ^^^ 2 1 ⁎⁎⁎ (c) (d) 1.5 Control VB lack VB supplement 6 6 ⁎⁎⁎ 1.0 ^^^ 0.5 0.0 CA1 area (e) (f) Figure 4: Continued. Control VB lack VB supplement GABA ( g/g) p-mTOR/mTOR LC3 II/LC3 I Control Control Control VB lack VB lack VB lack 6 6 VB supplement VB supplement VB supplement 6 6 6 Relative p62 protein expression levels Control VB lack VB supplement ×100 8 Behavioural Neurology Control VB lack VB supplement 6 6 ⁎⁎⁎ ^^^ CA3 area (g) (h) (i) Figure 4: VB deficiency suppressed GABA content and autophagy and accelerated apoptosis of neurons in hippocampus of offspring rats. The offspring rats obtained from pregnant mothers with VB normality, VB deficiency, or VB supplement were constructed to evaluate 6 6 6 autism-like behaviors. Hippocampal tissues were collected after sacrifice of offspring rats. (a) The concentration of GABA in rat hippocampus was measured by high-performance liquid chromatography. (b, c, d, and e) Western blot was applied to measure the levels of LC3 I, LC3 II, p62, mTOR, and p-mTOR proteins in the hippocampal tissues. GAPDH was used as the loading control. (f, g, h, and i) The number of apoptotic cells in rat hippocampal CA1 and CA3 areas was determined using TUNEL assay (×100 magnification, scale bar: 200 μm). Three rats/group. One-way analysis of variance with Tukey’s post-hoc test. VB : vitamin B6; GABA: gamma-aminobutyric acid; mTOR: mammalian target of rapamycin; p-mTOR: phosphor-mTOR; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; TUNEL: TdT-mediated dUTP nick-end labeling; ***p <0:001 vs. Control; ^^^p <0:001 vs. VB lack. patients with ASD [32–34]. The study of Almeida et al. indi- and supplementation of VB did not markedly affect body weight and locomotion in the model rats. It can be con- cated that the appearance of GABA neurotransmitter perturba- cluded that VB deficiency indeed participates in the devel- tions in the offspring may be attributed to maternal VB 6 6 opment of ASD, anxiety, and cognitive behaviors in deficiency [12]. The above findings enlightened us that the particular. Nevertheless, its regulatory mechanism in this repair of GABA system via VB could be a potential mecha- process has not been fully understood. nism in alleviating ASD. Excitation/inhibition synaptic imbal- Since VB can promote amino acid absorption and protein ance is considered as one of the most dominant synthesis, catalyze the conversion of glutamate into GABA, and neurophysiological features of ASD [35]. Synapses are special- inhibit the central nervous system, it is often used clinically to ized structures that connect and transmit between neurons or treat epilepsy, isoniazid accumulation-induced central nervous between neurons and effector cells, and their structural and system (CNS) excitation, and peripheral neuritis [29–31]. The quantitative integrity ensure the normal brain function. It has deficiency of behavioral flexibility frequently observed in been shown that GABA is critical in regulating feedback and patients with ASD may be, at least in part, due to the inade- feedforward inhibition of neuronal excitability [36]. Therefore, quate control of GABAergic transmission by the regulatory sys- we investigated the generation of GABA in the hippocampal tem. GABA, produced by GABAergic inhibitory interneurons, CA3 area by immunofluorescence staining, and uncovered that is an important inhibitory transmitter in the CNS, and its the fluorescence density of GAD67, VIAAT as well as + + down-regulation can cause dysfunction of central neurotrans- GAD67 ,and VIAAT cells was prominently reduced in the mitters, which results in delayed brain response, distraction, neurons of VB -deficient rat. Moreover, the results of HPLC and emotional variability, thus affecting social interaction of assay verified that VB deficiency induced the diminution of Number of positive cells/0.01 mm Control VB lack VB supplement Number of positive cells/0.01 mm Control VB lack VB supplement ×100 Behavioural Neurology 9 500 0.04 ^^^ 0.03 ^^^ 0.02 ^^^ ^^^ ^^^ 0.01 100 ^ 0 0.00 Rat Middle Object (a) (b) 150 6 100 4 ^^ ^^^ 50 2 ^^^ 0 0 (c) (d) Figure 5: VB deficiency induced autism-like behaviors by inhibiting the generation of GABAergic interneurons or activating mTOR pathway in the hippocampus of offspring rats. The modeling of autism-like behaviors was constructed in offspring rats obtained from pregnant mothers with VB deficiency, and the offspring was intraperitoneally injected with NVP-BEZ235 (400 μg/kg) or Clonazepam (0.025 mg/kg) 30 minutes before behavioral tests. (a) After drug administration, three-chambered social test was utilized to evaluate the social interactions of offspring rats. (b, c, and d) The movement speed, self-grooming time, and bowel frequency of offspring rats were detected by open field test. Six rats/group. One-way analysis of variance with Dunnett’s post-hoc test. VB : vitamin B6; ^p <0:05; ^^p <0:01; ^^^p <0:001 vs. VB deficiency. GABA concentration in rat hippocampus. GAD67 is an iso- to promote the degradation of autophagy. Numerous form of GABA synthase that can catalyze the production of researches have revealed that the abnormally initiated GABA from L-glutamate [37, 38]. VIAAT, as a marker of autophagy-related pathways result in reduced autophagic GABAergic presynaptic elements, is responsible for the uptake activity, which is associated with the development of ASD and transport of GABA from the synaptic gap to the synaptic [15, 40, 41]. Hui et al. unraveled that autophagy deficiency vesicles to regulate the concentration of GABA in the synaptic in the hippocampus contributes to the formation of gap and terminate inhibitory synaptic transmission. The SQSTM1/p62-positive aggregates, thereby compromise the expression level of VIAAT directly reflects the strength of syn- transport function of GABA receptors [21]. mTOR is a aptic transmission [39]. Our findings identified that VB defi- critical downstream signaling molecule of adenosine 5- ciency impaired the production of GABA and reduces monophosphate (AMP)-activated protein kinase (AMPK) inhibitory synapses, thus enhancing the release of excitatory and plays a negative regulatory role in the autophagy of cells. neurotransmitters from neurons, which may be an underlying The research by Zhang et al. confirmed that autophagy acts mechanism of autism-like behaviors in rats. as a link between mTOR and GABA signaling, and that LC3 is the most important marker during autophagy and mTOR inhibition promotes PI3K/AKT/mTOR-mediated is primarily involved in the formation of autophagosomes. autophagic activity, thereby enhancing social interaction in p62 acts as a link between ubiquitinated proteins and LC3 autistic rats [20]. As expected, we detected the elevated p62 Stay time (s) VB lack VB lack + NVP-BEZ235 VB lack + Clonazepam Self-grooming time (s) VB lack VB lack + NVP-BEZ235 VB lack + Clonazepam VB lack VB lack VB lack + NVP-BEZ235 6 6 VB lack + NVP-BEZ235 VB lack + Clonazepam VB lack + Clonazepam Bowel frequency Movement speed (m/s) VB lack VB lack VB lack + NVP-BEZ235 VB lack + NVP-BEZ235 VB lack + Clonazepam VB lack + Clonazepam 6 10 Behavioural Neurology 4 1.5 LC3 I –16 kDa ^^^ LC3 II –14 kDa 1.0 –62 kDa p62 0.5 1 ^^^ –289 kDa p-mTOR 0 0.0 –250 kDa mTOR –36 kDa GAPDH (a) (b) (c) 1.0 + GABA DAPI Merge 0.8 0.6 ^^^ 0.4 0.2 0.0 (d) (e) Figure 6: VB deficiency suppressed generation of GABAergic interneurons and autophagy by activating mTOR pathway in hippocampus in offspring rats. The offspring rats obtained from their pregnant mothers with VB deficiency was subjected to the modeling and were intraperitoneally injected with NVP-BEZ235 (400 μg/kg) or Clonazepam (0.025 mg/kg) 30 minutes before behavioral tests. Following drug administration, hippocampal tissues were collected after euthanasia of offspring. (a, b, c, and d) The levels of autophagy-related proteins (LC3 I, LC3 II, and p62) and mTOR pathway-related proteins (mTOR and p-mTOR) were measured by Western blot. GAPDH was used as the loading control. (e) The expression of GABA in the hippocampal CA3 area of offspring rats was determined using immunofluorescence staining (×100 magnification, scale bar: 50 μm). Three rats/group. One-way analysis of variance with Dunnett’s post-hoc test. VB : vitamin B6; GABA : gamma-aminobutyric acid-positive; mTOR: mammalian target of rapamycin; p-mTOR: phosphor-mTOR; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ^^^p <0:001 vs. VB deficiency. protein level and the reduced LC3 II/LC3 I and p-mTOR/ hippocampus. In order to clarify the interaction between mTOR ratios in the hippocampal tissues of VB -deficient mTOR-mediatedautophagy andGABA, we separately rat and demonstrated that VB deficiency can inhibit the inhibited mTOR and activated GABA in VB -deficent rat 6 6 autophagy in rat hippocampus by the activation of mTOR, models by NVP-BEZ235 (mTOR inhibitor) and Clonaze- thereby reducing GABA generation and inhibitory synap- pam (which has been validated to enhance the function of ses. Additionally, we verified that VB deficiency triggered GABA receptors) in animal models of ASD [42]. Based on cell apoptosis in the hippocampal CA1 area, which was in the analysis of rescue experiments, we found that NVP- consistent with the study of Zhang et al. [20], proving that BEZ235 acted similarly to Clonazepam to improve autism- the decrease in inhibitory synapses was accompanied by an like behaviors and enhance hippocampal GABA expression increase in the number of apoptotic interneurons in the in rats with VB deficiency, and meanwhile Clonazepam did VB lack VB lack ++ NVP-BEZ235 VB lack ++ Clonazepam p-mTOR/mTOR VB lack VB lack + NVP-BEZ235 VB lack + Clonazepam VB lack + VB lack + 6 6 Clonazepam NVP-BEZ235 VB lack LC3 II/LC3 I VB lack VB lack + NVP-BEZ235 VB lack + Clonazepam Relative p62 protein expression levels VB lack VB lack + NVP-BEZ235 VB lack + Clonazepam ×100 Behavioural Neurology 11 not affect autophagic processes. 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Vitamin B6 Deficiency Induces Autism-Like Behaviors in Rats by Regulating mTOR-Mediated Autophagy in the Hippocampus

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0953-4180
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1875-8584
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10.1155/2023/6991826
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Abstract

Hindawi Behavioural Neurology Volume 2023, Article ID 6991826, 12 pages https://doi.org/10.1155/2023/6991826 Research Article Vitamin B6 Deficiency Induces Autism-Like Behaviors in Rats by Regulating mTOR-Mediated Autophagy in the Hippocampus 1 2 2 2 2 2 2 Lijuan Chen, Jing Li, Xinglian Liu, Zhiwei Zhao, Yan Jin, Yikun Fu, Aiqin Zhou, 2 3 Chengqun Wang, and Yan Zhou Department of Pediatric Neurology, Hubei Maternal and Child Health Hospital, Wuhan, China Children’s Health Department, Hubei Maternal and Child Health Hospital, Wuhan, China Mass Spectrometry Center, Wuhan KingMed Diagnostics Group Co., Ltd, Wuhan, China Correspondence should be addressed to Yan Zhou; zhouyan_zzhy@163.com Received 2 November 2022; Revised 14 April 2023; Accepted 15 April 2023; Published 9 May 2023 Academic Editor: Giuseppe Biagini Copyright © 2023 Lijuan Chen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Vitamin B6 (VB ) exhibits therapeutic effects towards autism spectrum disorder (ASD), but its specific mechanism is poorly understood. Rat dams were treated with VB standard, VB deficiency, or VB supplementary diet, and the same treatment was 6 6 6 provided to their offspring, with their body weights monitored. Three-chambered social test and open field test were employed to evaluate the effect of VB on autism-like behaviors. Gamma-aminobutyric acid (GABA) generation and synaptic inhibition of neurons in the hippocampus of rat were detected via immunofluorescence staining, followed by the measurement of GABA concentration through high-performance liquid chromatography (HPLC). The role of VB in the autophagy and apoptosis of cells was determined via Western blot and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). In order to conduct rescue experiments, the inhibition of mammalian target of rapamycin (mTOR) or the activation of GABA was achieved by drug administration to the offspring rats with VB deficiency. As a result, no evident difference in weight was observed in the offspring with varied VB treatments. VB deficiency impaired social interaction; aggravated self-grooming and 6 6 bowel frequency; decreased GABA concentration, VIAAT, GAD67, vGAT expressions, and LC3 II/LC3 I ratio; increased p62 level and p-mTOR/mTOR ratio; and promoted cell apoptosis. Inhibition of mTOR reversed the effect of VB deficiency on cell autophagy. GABA activation or mTOR inhibition offset the role of VB deficiency in autism-like behaviors and hippocampal GABA expression. Collectively, VB deficiency induces autism-like behaviors in rats by regulating mTOR-mediated autophagy in the hippocampus. 1. Introduction (GABA)ergic synaptic transmission, which in turn influence the balance of excitatory/inhibitory signal ratio, leading to Autism spectrum disorder (ASD) is a pervasive neurodeve- autism-like behaviors [4]. Interneuronal GABAergic disor- lopmental disease with core symptoms of impaired social ders have been observed in many animal models of ASD. interaction, repetitive stereotypical behaviors, and narrow Thus, repairing the dysfunction of the GABA system is interests [1–3]. Unfortunately, there is no effective treatment thought to be an important way to alleviate ASD [5]. for ASD, and children with this disease have poor social As an essential nutrient for the body, vitamin makes a adaptability, bringing great emotional and economic burdens profound impact upon the development and function of to society and families, which, therefore, has aroused wide- the brain and nervous system [6]. In recent years, multiple spread attention. It is believed that ASD is caused by a combi- vitamins have been proved to alleviate ASD, including vita- nation of both polygenic genetic and external environmental min A (VA), VB , and VD [7, 8]. VB , a pyridine derivative, 6 6 factors. Pathogenetically, during early embryonic develop- works as a coenzyme factor in diverse metabolic reactions of ment, patients with ASD are affected by a variety of factors the human body, including amino acid metabolism, syn- that impair neuronal function and gamma-aminobutyric acid thesis and breakdown of neurotransmitters, regulation of 2 Behavioural Neurology steroid hormone activity, and control of gene expression [9, for 4 weeks. During the pregnancy and lactation, in consis- 10]. Although the combination therapy using VB has been tent with the grouping of their mother rats, the rats were reported to be applied for ASD at present [11], knowledge continued to be given aforementioned VB treatments. After gaps on its specific mechanism remain. However, existing weaning, new pups (male) were collected and divided into research suggests that it might be associated with the ability the three groups: Control group (n =6, a basic diet with of VB to strengthen the neurotransmitter system [12, 13]. 6mg VB ), VB deficiency group (n =24, a basic diet with- 6 6 6 Autophagy is a process of transporting misfolded pro- out VB ), and VB supplement group (n =6, a basic diet 6 6 teins or damaged cell organelles, dysfunctional mitochon- with 30 mg VB ). The body weight of offspring rats was dria, for instance, through autophagic vesicles to lysosomes recorded from postnatal day 1 (PND 1) to PND 42. The off- for degradation [14]. Autophagy is a pivotal mechanism spring rats between PND 42 and PND 56 were subjected to for cells to maintain homeostasis, the imbalance of which the behavioral tests. The mother rats were sacrificed by cer- in neurons is usually linked to cerebral diseases, including vical dislocation under anesthesia (40 mg/kg, pentobarbital ASD and neurodegenerative diseases [15–18]. Previous stud- sodium, P-010, Sigma-Aldrich, USA). ies have pointed out that disruption of mammalian target of 2.3. Drug Administration. GABA receptor agonist Clonaze- rapamycin (mTOR)-regulated macroautophagy and autoph- pam (C-907, Sigma-Aldrich, USA) and mTOR inhibitor agy leads to autism-like abnormalities [19, 20]. Recently, it NVP-BEZ235 (A10133, Adooq Bioscience, China) were used has been additionally demonstrated that enhancing for drug administration in vivo. A total of 18 offspring rats mTOR-mediated autophagy contributes to the restoration were randomly selected from the abovementioned VB defi- of GABAergic signaling [21], which might alleviate autism- 6 ciency group and assigned into a new VB deficiency group like behaviors in rats [22]. 6 (a basic diet without VB ), VB deficiency + Clonazepam Three regions of the adult brain, the olfactory bulb, 6 6 group (intraperitoneal injection of 0.025 mg/kg Clonazepam hypothalamus, and hippocampal dentate gyrus, contain based on the VB deficiency diet), and VB deficiency newborn neurons that are crucial players in the natural func- 6 6 + NVP-BEZ235 group (intraperitoneal injection of 400 μg/ tional circuits [23]. Many neurodegenerative diseases and kg NVP-BEZ235 based on the VB deficiency diet), with six neurodevelopmental disorders involving cognitive impair- 6 rats in each group. All offspring rats in this experiment were ment may be associated with hippocampal dysfunction, reared for 42–56 days since PND 1, and the drug administra- which is at least partially attributable to adult neurogenetic tion was implemented 30 minutes prior to the behavioral disorders [24]. A recent review stated that hippocampus tests [20, 22]. may represent an important component within a system of changed brain regions that work in tandem to contribute 2.4. Behavioral Tests to the phenotype of ASD [25]. Moreover, VA deficiency induces autistic-like behaviors in rats through modulating 2.4.1. Three-Chambered Social Test. A three-chambered the RARβ-CD38-oxytocin axis in the hypothalamus [26]. apparatus (XR-XJ117) purchased from XinRuan Informa- Thus, the hippocampus was chosen for the study of VB in 6 tion Technology (Shanghai, China) was applied to conduct ASD. sociability test [22]. After 10-minute habituation, the off- In this study, to suss out the role of VB in ASD, we 6 spring rats (PND 42 and PND 56) with or without the indi- firstly constructed the models in the offspring rat with VB cated treatment of VB or drugs above were individually deficiency or VB supplement and then explored whether 6 placed into the center chamber of the apparatus and allowed the mechanism of VB in ASD was associated with mTOR- 6 to access three chambers for 10 minutes, with an identical mediated autophagy, with the aim to provide new insight cage in each side of the chamber, one of which contained into the pathogenesis of ASD. an unacquainted rat. The time the offspring rats spent in each chamber was automatically recorded. 2. Material and Methods 2.4.2. Open Field Test. The locomotor activity of the off- 2.1. Animals. The surgical procedures with animals in this spring rats (PND 42 and PND 56) with or without the indi- study were approved by the Committee of Zhejiang Baiyue cated treatment of VB or drugs above was evaluated via the Biotech Co., Ltd for Experimental Animals Welfare open field test with the assistance of an uncovered box (approval number: ZJBYLA-IACUC-20220701) and per- (100 × 100 × 50 cm, XR-XZ301, XinRuan Information Tech- formed in accordance with Guide for the Care and Use of nology, China). The box was cleaned thoroughly and steril- Laboratory Animals. A total of 24 virgin female Wistar rats ized with 75% alcohol (E299578, Aladdin, China) before (120–135 g) were purchased from Charles River Labs (Bei- the tests to eliminate any odors. The open field test was car- jing, China) and were group-housed in standard animal ried out according to the previous instructions [26]. Briefly, cages (25 ± 2 C) with consistent 12/12 hours light/dark cycle. the offspring rats were individually placed into the center of the open field and allowed to explore the new environment 2.2. Modeling. Autistic rat models with VB deficiency or without any disturbance for 15 minutes during the test. The SuperMaze software (vision 2.0, XR-Xmaze, XinRuan VB supplement were constructed using the offspring of female Wistar rats as previously described [12]. Based on a Information Technology, China) was applied to monitor basic diet, all female rats were randomly fed with 0, 6, or movement speed, self-grooming time, and bowel frequency 30 mg VB (P5669, Sigma-Aldrich, St. Louis, MO, USA) automatically during the test. 6 Behavioural Neurology 3 After behavioral test, all offspring rats were sacrificed by hippocampal hemispheres from six consecutive serial sec- cervical dislocation under anesthesia, and the hippocampal tions were used. tissues were harvested and stored in liquid nitrogen at −80 C for follow-up experiments. The right hemisphere 2.6. HPLC Assay. The content of GABA in the hippocampi was used for immunocytochemistry and terminal deoxynu- of rats from Control, VB deficiency, and VB supplement 6 6 cleotidyl transferase dUTP nick-end labeling (TUNEL) groups was measured by HPLC assay. In brief, the hippo- experiments, while the hippocampus from the other hemi- campal tissues from both hemispheres were homogenized sphere was utilized for Western blot. The hippocampal tis- in perchlorate solution (0.04 M, abs47048087, Absin, China) sues from both hemispheres were employed for high- on ice, subsequent to which 15-minute centrifugation was performance liquid chromatography (HPLC) assay. The carried out. Then, the supernatants were neutralized with experimental design was described in Supplementary potassium bicarbonate solution (2 M, abs42025446, Absin, Figure 1. China). After centrifugation, 2 ml supernatant of the sample or standard GABA solution was treated with triethylamine- 2.5. Immunofluorescence Analysis. The frozen hippocampal acetonitrile (1 ml, 1/10, v/v) and phenyl isothiocyanate tissues were cut into sections with a thickness of 5μmand fixed (PITC) acetonitrile (1 ml, 1/25, v/v) at room temperature with 4% paraformaldehyde (P0099, Beyotime, China) for 15 for 1 hour away from light, followed by 20 minute n-hex- minutes. After being washed with phosphate buffered saline ane treatment (2 ml, H100107, Aladdin, China). Follow- (PBS, P1022, Solarbio, China), the sections were subjected to ing the filtration, the lower layer solution (1 ml) was 15 minute treatment with Triton X-100 (T8200, Solarbio, harvestedfor analysis.HPLCwas conductedunder an China) and 1 hour blocking using 5% goat serum (abs933, Agilent 1100 HPLC system (Agilent, Santa Clara, CA, USA) to analyze the content of GABA according to the Absin, China). To determine the expressions of vesicular inhibitory amino acid transporter (VIAAT) and glutamate previous protocols [22]. Assay for each sample was decarboxylase 67 (GAD67) in hippocampal CA3 area, the sec- repeated twice. tions were incubated with primary antibodies against VIAAT (1 : 1000, ab101934, Abcam, Cambridge, UK) and GAD67 2.7. Western Blot. The levels of autophagy-related proteins (10μg/ml, ab26116, Abcam, UK) at 4 C overnight away from in the brain tissues from rats in VB deficiency group light. Then, the sections underwent 1 hour incubation with sec- with or without drug administration, VB supplement ondary antibodies Alexa Fluor 488-labeled Goat Anti-Rabbit group, and Control group were measured via Western IgG (1 : 1000, P0176, Beyotime, China) and Alexa Fluor 647- blot. After the extraction of total protein with RIPA Lysis labeled Goat Anti-Mouse IgG (1 : 1000, P0191, Beyotime, Buffer (E-BC-R327, Elabscience, China), BCA Protein China) at room temperature. After 5 minute counterstain with Assay Kit (23225, ThermoFisher, Waltham, MA, USA) was 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride used to detect protein concentration in the collected superna- (DAPI, C1005, Beyotime, China), fluorescent images were cap- tant following the instructions. Afterwards, equal amount of tured using a confocal microscope (LSM 780, ZEISS, Ger- protein (50μg/lane) was separated by 10% sodium dodecyl many) at ×600 magnification. sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, E-IR- To detect colocalization between GAD67 positivity R305, Elabscience, China) and loaded onto nitrocellulose mem- (GAD67 ) with vesicular gamma-aminobutyric acid trans- branes (2215, Millipore, Billerica, MA, USA). Next, the mem- porter (vGAT) in hippocampal CA3 area, the sections were branes were sequentially treated with blocking buffer at room pre-treated with Triton X-100 and 5% goat serum as above temperature for 1 hour and cultivated with primary antibodies and then were cultivated with primary antibodies against at 4 C overnight. The next day, the membranes were rinsed GAD67 and vGAT (1 : 500, 131006, Synaptic Systems, Ger- with tris-buffered saline with Tween-20 (TBS-T, abs952, Absin, many) at 4 C overnight in the dark. The next day, the sec- China) and incubated with secondary antibody at room tem- tions were cultured with secondary antibodies Alexa Fluor perature for 2 hours. For the visualization of immunoblots, 488-Labeled Goat anti-Mouse IgG (1 : 1000, P0188, Beyo- the protein bands were added with PLUS Chemiluminescent time, China) and Alexa Fluor 680-labeled Donkey Anti- Substrate (34577, ThermoFisher, USA) and developed in a Chicken IgG (1 : 100, 703-625-155, Jackson Immuno Tanon 5200 Imaging System (Shanghai, China). All antibodies Research, USA), followed by DAPI staining. Lastly, a confo- were obtained from Abcam (UK), and the primary antibodies cal microscope was used to observe the stained sections were those against LC3 I/LC3 II (1/1000, 14 kDa, 16 kDa, under ×400 magnification. ab192890), p62 (1/10000, 62 kDa, ab109012), mTOR (1/1000, For investigating the activity of GABA in hippocampal 250 kDa, ab32028), phosphor (p)-mTOR (1/1000, 289 kDa, CA3 area from hemispheres of VB -deficient offspring rats ab109268), and glyceraldehyde-3-phosphate dehydrogenase with or without drug administration, the pre-treated sec- (GAPDH, 0.1μg/ml, 36 kDa, ab9484). The secondary anti- tions were cultured with primary antibody targeting GABA bodies consisted of Rabbit Anti-Mouse IgG (1/2000, ab6728) (1 : 2000, ab8891, Abcam, UK) at 4 C overnight without and Goat Anti-Rabbit IgG (1/3000, ab6721). light, followed by the cultivation with Alexa Fluor 647- labeled Goat Anti-Rabbit IgG Secondary Antibody (P0180, 2.8. TUNEL Assay. Cell apoptosis in CA1 and CA3 areas of Beyotime, China) at room temperature for 1 hour. After rat hippocampus from Control, VB deficiency, and VB sup- 6 6 the staining with DAPI, fluorescent images were obtained plement groups was determined using Colorimetric TUNEL by a microscope at ×100 magnification. A total of three Apoptosis Assay Kit (C1091, Beyotime, China). The frozen 4 Behavioural Neurology hippocampal sections were treated with Immunol Staining signals was lessened in hippocampal CA3 area of the off- Fix Solution (P0098, Beyotime, China) at room temperature spring rats with VB deficiency, and no remarkable for 30 minutes, followed by the rinse using PBS twice. Then, change was observed in the offspring rats with VB sup- each section was immersed into Enhanced Immunostaining Per- plement. Additionally, the hippocampal CA3 area of off- meabilization Buffer (P0097, Beyotime, China) for 5 minutes, spring rats with VB deficiency exhibited conspicuously blocked with 0.3% H O in PBS for 20 minutes, and reacted with decreased GAD67 and vGAT, while no significant 2 2 50μl prepared biotin-labeled solution at 37 C in a wet box for 1 change was visible in the offspring rats with VB supple- hour without light. After the reaction was terminated, the sec- ment (Figure 3). Moreover, the results of HPLC assay tions were subjected to color development, including the treat- revealed that the concentration of GABA was dwindled ment of both streptavidin–horseradish peroxidase (HRP) in the hippocampus of offspring rats in VB deficiency (50μl) and 3,3-diaminobenzidine (DAB, 0.2 ml) according to group, compared to that in Control group (Figure 4(a), the manufacturer’s specification. Following dehydration with p <0:001). gradient alcohol and hyalinization with xylene, the number of positively apoptotic cells in the hippocampal CA1 and CA3 by 3.3. VB Deficiency Suppressed Autophagy and Accelerated cells/0.01 mm was counted using a light microscope (×100 mag- Apoptosis of Neurons in the Offspring Rats. Considering that nification, CX23, Olympus, Japan). Three hippocampal hemi- the imbalance of autophagy is closely associated with cerebral spheres from six consecutive serial sections were used. diseases including autism [15], Western blot was employed for measuring the protein levels of LC3 I, LC3 II, p62, mTOR, 2.9. Statistical Analysis. The results of triplicate assays were and p-mTOR in the hippocampal tissues. As compared with expressed as mean ± standard deviation. Kolmogorov– those in Control group, decreased LC3 II/LC3 I ratio and Smirnov test was used to verify normality. One-way analy- increased p-mTOR/mTOR ratio and p62 protein level were sis of variance was used for multi-group comparison, observed in the offspring rats in VB deficiency group, while followed by Tukey or Dunnett’s post-hoc test to detect increased LC3 II/LC3 I ratio was observed in the offspring rats any inter-group differences. Statistical analyses were real- in VB supplement group (Figures 4(b), 4(c), 4(d), and ized using Graphpad Prism 8.0 software (GraphPad Soft- 4(e), p <0:001). We also detected cell apoptosis in hippo- ware Inc., San Diego, CA, USA). P <0:05 indicated the campal CA1 and CA3 areas using TUNEL assay. Notably, data were statistically significant. more cell apoptosis in the hippocampal CA1 area emerged in VB deficiency group, while less apoptotic cells appeared in VB supplement group (Figures 4(f) and 4(g), p <0:001). 3. Results In addition, VB deficiency or VB supplement generated 6 6 no significant effect on cell apoptosis in the hippocampal 3.1. VB Deficiency Reduced Sociability and Aggravated Anxiety-Related Behaviors of Offspring Rats. No abnormal CA3area(Figures4(h)and 4(i)). deaths or loss were observed in all rats during this study. To explore the role of VB in rats with autism-like behaviors, 3.4. The Role of VB in the Autism-Like Behaviors of Rats 6 6 we obtained the offspring from pregnant rats to construct rat Was Mediated by mTOR Inhibition and GABA Activation. model with VB normality, VB deficiency, or VB supple- It has been suggested that the role of mTOR-mediated 6 6 6 ment. As exhibited in weight curves (Figure 1(a)), no obvi- autophagy in the development of autism might be correlated ous difference was reported concerning the weight of the with GABA [22]. Hence, we triggered mTOR inhibition or offspring rats among the control, VB deficiency, and VB GABA activation in the offspring rats with VB deficiency 6 6 6 supplement groups. Subsequently, we investigated whether by drug administration with NVP-BEZ235 or Clonazepam VB has an effect on autism-like behaviors of rats. The eval- and conducted rescue assays. The results of three- uation of social interactions demonstrated that when com- chambered social test unveiled that both NVP-BEZ235 and pared to the control group, in VB deficiency group, the Clonazepam promoted social interaction of the offspring offspring rats had fewer social interactions with unfamiliar rats with VB deficiency, compared to those without drug rats but more interactions with the object, and in VB sup- administration (Figure 5(a), p <0:001). As demonstrated in plement group, the offspring rats had more social interac- Figures 5(b), 5(c), and 5(d), neither NVP-BEZ235 nor Clo- tions with unfamiliar rats and less interactions with the nazepam signally influenced the movement speed, and VB object (Figure 1(b), p <0:05). In line with the open field deficiency-induced increments in self-grooming time and test results, we observed that VB deficiency and VB sup- bowel frequency were dwindled by both NVP-BEZ235 and 6 6 plement exerted no effect on movement speed of the off- Clonazepam (p <0:05). At molecular level, NVP-BEZ235 spring rats (Figure 1(c)), while VB deficiency increased increased LC3 II/LC3 I ratio, decreased p-mTOR/mTOR self-grooming time and bowel frequency (Figures 1(d) ratio, and down-regulated p62 protein level, which reversed and 1(e), p <0:01). the effects of VB deficiency (Figures 6(a), 6(b), 6(c), and 6(d), p <0:001). No observable effect of Clonazepam, how- 3.2. VB Deficiency Diminished Synaptic Inhibition in the ever, was detected on LC3 II/LC3 I ratio, p-mTOR/mTOR Hippocampi of Rats. The immunofluorescence analysis was ratio, and p62 protein level (Figures 6(a), 6(b), 6(c), and conducted to reveal the effect of VB on synaptic inhibition 6(d)). Interestingly, the results of immunofluorescence stain- of neurons in the hippocampus of offspring rats. As illustrated ing demonstrated that both NVP-BEZ235 and Clonazepam in Figure 2, the density of VIAAT and GAD67 fluorescence enhanced GABA in the hippocampi of the offspring rats Behavioural Neurology 5 150 500 ⁎⁎⁎ ^^^ ⁎⁎⁎ ^^^ 0 0 1 7 14 21 28 35 42 After PND time (days) Control VB lack VB supplement Rat Middle Object (a) (b) 0.04 150 ⁎⁎⁎ 0.03 0.02 0.01 ^^^ 0.00 0 (c) (d) ⁎⁎ ^^ (e) Figure 1: VB deficiency reduced sociability and aggravated anxiety-related behaviors of offspring rats. The offspring rats obtained from pregnant rats with VB normality, VB deficiency, or VB supplement were subjected to the modeling. (a) The body weight of offspring 6 6 6 rats was recorded from PND 1 to PND 42. (b) Three-chambered social test was utilized to evaluate the social interactions of offspring rats. (c, d, and e) The movement speed, self-grooming time, and bowel frequency of offspring rats were detected by open field test. Six rats/group. One-way analysis of variance with Tukey’s post-hoc test. VB : vitamin B6; PND: postnatal day; **p <0:01; ***p <0:001 vs. Control; ^^p <0:01; ^^^p <0:001 vs. VB lack. with VB deficiency, compared to those without drug 4. Discussion administration (Figure 6(e)). Finally, a scheme showing the roles of VB , GABA transmission, mTOR, and apoptosis in The treatment for ASD using VB could date back to the 6 6 the proposed mechanism of the autism etiology was pre- 1960s, and years of clinical studies have proven that oral sented in Supplementary Figure 2. administration with high-dose VB can significantly alleviate Wistar rats (g) Movement speed (m/s) Control VB lack Bowel frequency VB supplement Control Self-grooming time (s) Stay time (s) VB lack VB supplement Control Control VB lack VB lack VB supplement 6 6 VB supplement Control VB lack VB supplement Control VB lack VB supplement 6 6 Behavioural Neurology VIAAT GAD67 DAPI Merge Figure 2: VB deficiency reduced GABAergic inhibition in hippocampal CA3 area of offspring rats. The offspring rats obtained from their pregnant mothers with VB normality, VB deficiency, or VB supplement were modeled to evaluate autism-like behaviors. Hippocampal 6 6 6 tissues were collected after euthanasia of offspring rats. The expressions of VIAAT and GAD67 in the hippocampal CA3 area of rat were determined using immunofluorescence staining (×600 magnification, scale bar: 40 μm). Three rats/group. One-way analysis of variance with Tukey’s post-hoc test. VB : vitamin B6; VIAAT: vesicular inhibitory amino acid transporter; GAD67: glutamate decarboxylase 67. Merge GAD67 vGAT Figure 3: VB deficiency diminished GAD67 inhibitory synapses in hippocampal CA3 area of offspring rats. The offspring rats obtained from pregnant mothers with VB normality, VB deficiency, or VB supplement were modeled. Hippocampal tissues were collected after 6 6 6 euthanasia. Immunofluorescence analysis was used to detect synaptic inhibition of neurons in the hippocampus by staining GAD67 and vGAT (×400 magnification, scale bar: 50 μm). Three rats/group. One-way analysis of variance with Tukey’s post-hoc test. VB : vitamin B6; GAD67 : glutamate decarboxylase 67-positive; vGAT: vesicular gamma-aminobutyric acid transporter. autism-like behaviors in patients [27]. Persistent stereotypi- Through the behavioral tests in animal, we observed a nota- cal and repetitive behaviors, narrow interests, and lagging ble lack of social interest and strong repetitive stereotypic social skills constitute the main symptoms of ASD [28]. behaviors in the VB -deficient model rats, but the deficiency VB supplement VB lack Control 6 6 VB supplement VB lack Control 6 6 ×400 ×600 Behavioural Neurology 7 0.4 –16 kDa LC3 I ^^^ 0.3 –14 kDa LC3 II 0.2 ⁎⁎⁎ –62 kDa p62 0.1 –289 kDa p-mTOR 0.0 –250 kDa mTOR –36 kDa GAPDH (a) (b) 6 3 ⁎⁎⁎ ^^^ 4 2 ^^^ 2 1 ⁎⁎⁎ (c) (d) 1.5 Control VB lack VB supplement 6 6 ⁎⁎⁎ 1.0 ^^^ 0.5 0.0 CA1 area (e) (f) Figure 4: Continued. Control VB lack VB supplement GABA ( g/g) p-mTOR/mTOR LC3 II/LC3 I Control Control Control VB lack VB lack VB lack 6 6 VB supplement VB supplement VB supplement 6 6 6 Relative p62 protein expression levels Control VB lack VB supplement ×100 8 Behavioural Neurology Control VB lack VB supplement 6 6 ⁎⁎⁎ ^^^ CA3 area (g) (h) (i) Figure 4: VB deficiency suppressed GABA content and autophagy and accelerated apoptosis of neurons in hippocampus of offspring rats. The offspring rats obtained from pregnant mothers with VB normality, VB deficiency, or VB supplement were constructed to evaluate 6 6 6 autism-like behaviors. Hippocampal tissues were collected after sacrifice of offspring rats. (a) The concentration of GABA in rat hippocampus was measured by high-performance liquid chromatography. (b, c, d, and e) Western blot was applied to measure the levels of LC3 I, LC3 II, p62, mTOR, and p-mTOR proteins in the hippocampal tissues. GAPDH was used as the loading control. (f, g, h, and i) The number of apoptotic cells in rat hippocampal CA1 and CA3 areas was determined using TUNEL assay (×100 magnification, scale bar: 200 μm). Three rats/group. One-way analysis of variance with Tukey’s post-hoc test. VB : vitamin B6; GABA: gamma-aminobutyric acid; mTOR: mammalian target of rapamycin; p-mTOR: phosphor-mTOR; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; TUNEL: TdT-mediated dUTP nick-end labeling; ***p <0:001 vs. Control; ^^^p <0:001 vs. VB lack. patients with ASD [32–34]. The study of Almeida et al. indi- and supplementation of VB did not markedly affect body weight and locomotion in the model rats. It can be con- cated that the appearance of GABA neurotransmitter perturba- cluded that VB deficiency indeed participates in the devel- tions in the offspring may be attributed to maternal VB 6 6 opment of ASD, anxiety, and cognitive behaviors in deficiency [12]. The above findings enlightened us that the particular. Nevertheless, its regulatory mechanism in this repair of GABA system via VB could be a potential mecha- process has not been fully understood. nism in alleviating ASD. Excitation/inhibition synaptic imbal- Since VB can promote amino acid absorption and protein ance is considered as one of the most dominant synthesis, catalyze the conversion of glutamate into GABA, and neurophysiological features of ASD [35]. Synapses are special- inhibit the central nervous system, it is often used clinically to ized structures that connect and transmit between neurons or treat epilepsy, isoniazid accumulation-induced central nervous between neurons and effector cells, and their structural and system (CNS) excitation, and peripheral neuritis [29–31]. The quantitative integrity ensure the normal brain function. It has deficiency of behavioral flexibility frequently observed in been shown that GABA is critical in regulating feedback and patients with ASD may be, at least in part, due to the inade- feedforward inhibition of neuronal excitability [36]. Therefore, quate control of GABAergic transmission by the regulatory sys- we investigated the generation of GABA in the hippocampal tem. GABA, produced by GABAergic inhibitory interneurons, CA3 area by immunofluorescence staining, and uncovered that is an important inhibitory transmitter in the CNS, and its the fluorescence density of GAD67, VIAAT as well as + + down-regulation can cause dysfunction of central neurotrans- GAD67 ,and VIAAT cells was prominently reduced in the mitters, which results in delayed brain response, distraction, neurons of VB -deficient rat. Moreover, the results of HPLC and emotional variability, thus affecting social interaction of assay verified that VB deficiency induced the diminution of Number of positive cells/0.01 mm Control VB lack VB supplement Number of positive cells/0.01 mm Control VB lack VB supplement ×100 Behavioural Neurology 9 500 0.04 ^^^ 0.03 ^^^ 0.02 ^^^ ^^^ ^^^ 0.01 100 ^ 0 0.00 Rat Middle Object (a) (b) 150 6 100 4 ^^ ^^^ 50 2 ^^^ 0 0 (c) (d) Figure 5: VB deficiency induced autism-like behaviors by inhibiting the generation of GABAergic interneurons or activating mTOR pathway in the hippocampus of offspring rats. The modeling of autism-like behaviors was constructed in offspring rats obtained from pregnant mothers with VB deficiency, and the offspring was intraperitoneally injected with NVP-BEZ235 (400 μg/kg) or Clonazepam (0.025 mg/kg) 30 minutes before behavioral tests. (a) After drug administration, three-chambered social test was utilized to evaluate the social interactions of offspring rats. (b, c, and d) The movement speed, self-grooming time, and bowel frequency of offspring rats were detected by open field test. Six rats/group. One-way analysis of variance with Dunnett’s post-hoc test. VB : vitamin B6; ^p <0:05; ^^p <0:01; ^^^p <0:001 vs. VB deficiency. GABA concentration in rat hippocampus. GAD67 is an iso- to promote the degradation of autophagy. Numerous form of GABA synthase that can catalyze the production of researches have revealed that the abnormally initiated GABA from L-glutamate [37, 38]. VIAAT, as a marker of autophagy-related pathways result in reduced autophagic GABAergic presynaptic elements, is responsible for the uptake activity, which is associated with the development of ASD and transport of GABA from the synaptic gap to the synaptic [15, 40, 41]. Hui et al. unraveled that autophagy deficiency vesicles to regulate the concentration of GABA in the synaptic in the hippocampus contributes to the formation of gap and terminate inhibitory synaptic transmission. The SQSTM1/p62-positive aggregates, thereby compromise the expression level of VIAAT directly reflects the strength of syn- transport function of GABA receptors [21]. mTOR is a aptic transmission [39]. Our findings identified that VB defi- critical downstream signaling molecule of adenosine 5- ciency impaired the production of GABA and reduces monophosphate (AMP)-activated protein kinase (AMPK) inhibitory synapses, thus enhancing the release of excitatory and plays a negative regulatory role in the autophagy of cells. neurotransmitters from neurons, which may be an underlying The research by Zhang et al. confirmed that autophagy acts mechanism of autism-like behaviors in rats. as a link between mTOR and GABA signaling, and that LC3 is the most important marker during autophagy and mTOR inhibition promotes PI3K/AKT/mTOR-mediated is primarily involved in the formation of autophagosomes. autophagic activity, thereby enhancing social interaction in p62 acts as a link between ubiquitinated proteins and LC3 autistic rats [20]. As expected, we detected the elevated p62 Stay time (s) VB lack VB lack + NVP-BEZ235 VB lack + Clonazepam Self-grooming time (s) VB lack VB lack + NVP-BEZ235 VB lack + Clonazepam VB lack VB lack VB lack + NVP-BEZ235 6 6 VB lack + NVP-BEZ235 VB lack + Clonazepam VB lack + Clonazepam Bowel frequency Movement speed (m/s) VB lack VB lack VB lack + NVP-BEZ235 VB lack + NVP-BEZ235 VB lack + Clonazepam VB lack + Clonazepam 6 10 Behavioural Neurology 4 1.5 LC3 I –16 kDa ^^^ LC3 II –14 kDa 1.0 –62 kDa p62 0.5 1 ^^^ –289 kDa p-mTOR 0 0.0 –250 kDa mTOR –36 kDa GAPDH (a) (b) (c) 1.0 + GABA DAPI Merge 0.8 0.6 ^^^ 0.4 0.2 0.0 (d) (e) Figure 6: VB deficiency suppressed generation of GABAergic interneurons and autophagy by activating mTOR pathway in hippocampus in offspring rats. The offspring rats obtained from their pregnant mothers with VB deficiency was subjected to the modeling and were intraperitoneally injected with NVP-BEZ235 (400 μg/kg) or Clonazepam (0.025 mg/kg) 30 minutes before behavioral tests. Following drug administration, hippocampal tissues were collected after euthanasia of offspring. (a, b, c, and d) The levels of autophagy-related proteins (LC3 I, LC3 II, and p62) and mTOR pathway-related proteins (mTOR and p-mTOR) were measured by Western blot. GAPDH was used as the loading control. (e) The expression of GABA in the hippocampal CA3 area of offspring rats was determined using immunofluorescence staining (×100 magnification, scale bar: 50 μm). Three rats/group. One-way analysis of variance with Dunnett’s post-hoc test. VB : vitamin B6; GABA : gamma-aminobutyric acid-positive; mTOR: mammalian target of rapamycin; p-mTOR: phosphor-mTOR; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ^^^p <0:001 vs. VB deficiency. protein level and the reduced LC3 II/LC3 I and p-mTOR/ hippocampus. In order to clarify the interaction between mTOR ratios in the hippocampal tissues of VB -deficient mTOR-mediatedautophagy andGABA, we separately rat and demonstrated that VB deficiency can inhibit the inhibited mTOR and activated GABA in VB -deficent rat 6 6 autophagy in rat hippocampus by the activation of mTOR, models by NVP-BEZ235 (mTOR inhibitor) and Clonaze- thereby reducing GABA generation and inhibitory synap- pam (which has been validated to enhance the function of ses. Additionally, we verified that VB deficiency triggered GABA receptors) in animal models of ASD [42]. Based on cell apoptosis in the hippocampal CA1 area, which was in the analysis of rescue experiments, we found that NVP- consistent with the study of Zhang et al. [20], proving that BEZ235 acted similarly to Clonazepam to improve autism- the decrease in inhibitory synapses was accompanied by an like behaviors and enhance hippocampal GABA expression increase in the number of apoptotic interneurons in the in rats with VB deficiency, and meanwhile Clonazepam did VB lack VB lack ++ NVP-BEZ235 VB lack ++ Clonazepam p-mTOR/mTOR VB lack VB lack + NVP-BEZ235 VB lack + Clonazepam VB lack + VB lack + 6 6 Clonazepam NVP-BEZ235 VB lack LC3 II/LC3 I VB lack VB lack + NVP-BEZ235 VB lack + Clonazepam Relative p62 protein expression levels VB lack VB lack + NVP-BEZ235 VB lack + Clonazepam ×100 Behavioural Neurology 11 not affect autophagic processes. 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Behavioural NeurologyHindawi Publishing Corporation

Published: May 9, 2023

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