Methamphetamine and HIV-1 Tat protein synergistically induce endoplasmic reticulum stress to promote TRIM13-mediated neuronal autophagy

Methamphetamine and HIV-1 Tat protein synergistically induce endoplasmic reticulum stress to promote TRIM13-mediated neuronal autophagy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Methamphetamine and HIV-1 Tat protein synergistically induce endoplasmic reticulum stress to promote TRIM13-mediated neuronal autophagy Chan Wang, Genmeng Yang, Jian Huang, Yunqing Tian, Chi-Kwan Leung, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4788696/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Dec, 2024 Read the published version in Molecular Neurobiology → Version 1 posted 12 You are reading this latest preprint version Abstract Co-exposure to methamphetamine (METH) abuse and HIV infection exacerbates central nervous system damage. However, the underlying mechanisms of this process remain poorly understood. This study aims to explore the roles of neuronal autophagy in the synergistic damage to the central nervous system caused by METH and HIV proteins. Models of METH and HIV-1 Tat protein co-exposure were established using tree shrews, primary neurons, and SH-SY5Y cells. Co-exposure to METH and HIV-1 Tat protein significantly increased the distance traveled, mean velocity, and stereotyped behaviors of tree shrews in the open field test. Western blot analysis revealed that Co-exposure to METH and HIV-1 Tat protein markedly increased the expression of endoplasmic reticulum stress (ERS)-associated proteins (p-ERK, IRE1, ATF6, and Bip) and autophagy markers (ATG7, ATG5, Beclin1, and LC3II). Conversely, Co-exposure to METH and HIV-1 Tat protein significantly downregulated the expressions of p62 and TRIM13. Immunofluorescence staining demonstrated that Pre-treatment with the ERS inhibitor 4-PBA or TRIM13-siRNA rescued the abnormal behaviors induced by METH and HIV-1 Tat protein co-exposure in tree shrews and restored the expression of ERS-related and autophagy-related proteins. Additionally, TRIM13 was found to interact with autophagy-related proteins, including p62, Beclin1, and LC3II by immunoprecipitation assays. Our findings suggest for the first time that METH and HIV-1 Tat protein synergistically induce neuronal autophagy through ERS pathways, with TRIM13 playing a pivotal regulatory role in this process. methamphetamine HIV-1 Tat endoplasmic reticulum stress autophagy TRIM13 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction According to the World Drug Report 2023, the global incidence of drug use is on the rise, with approximately 36 million individuals using amphetamine-type stimulants [ 1 ]. The relationship between drug use and HIV infection is particularly alarming, drug users are seven times more likely to contract HIV compared to the general population [ 2 ]. Moreover, substance abuse markedly elevates the risk of neurological symptoms and HIV-associated neurocognitive disorders (HAND) among those with HIV—conditions that mutually exacerbate neurological harm [ 3 ]. HAND and related neurological symptoms often manifest early in HIV infection, precipitated by the invasion of neurons by HIV-1-associated proteins. These proteins are secreted following the HIV-1 virus infecting T-cells, monocytes, and macrophages [ 4 , 5 ]. Among these, the HIV-1 Tat protein, a potent transcriptional activator released by infected cells, is critical for viral replication and is implicated as a significant pathogenetic agent in HAND development [ 6 – 10 ]. In recent years, the mechanisms underlying cellular autophagy have received considerable attention. The accumulation of the HIV-1 Tat protein in brain tissues has been demonstrated to trigger autophagy in neuronal cells, which may lead to cell damage and subsequent neurotoxicity [ 11 – 13 ]. Our previous research has shown that METH-induced significant autophagy in SH-SY5Y cells, dopaminergic neurons, and microglia, with the presence of HIV-1 Tat protein intensifying this effect [ 14 , 15 ]. While strides have been made in understanding autophagy induced by METH and HIV-1 Tat, the underlying mechanisms of neuronal autophagy caused by METH and HIV-1 Tat co-exposure remain unclear. Disruptions of endoplasmic reticulum (ER) function induce abnormal accumulation of unfolded proteins within the ER lumen. This accumulation triggers endoplasmic reticulum stress (ERS), a cellular stress response associated with the ER [ 16 – 20 ]. In the central nervous system (CNS) of patients with HAND, persistent ERS activates autophagy, leading to cell death through excessive autophagy, exacerbating neurological damage [ 21 , 22 ]. Its E3 ubiquitin ligase activity characterizes the Tripartite Motif (TRIM) protein family and plays a pivotal role in various physiological processes, including autophagy, apoptosis, and inflammation [ 23 , 24 ]. Growing evidence suggests that TRIM13 may act as a molecular link between ERS and autophagy [ 25 , 26 ]. However, the effect of TRIM13 on neuronal autophagy caused by METH and HIV-1 Tat protein co-exposure remains unclear. Given their evolutionary proximity to non-human primates and similarities to humans in the nervous and immune systems, tree shrews have increasingly been utilized as a novel model for investigating the neurotoxic effects of METH and HIV-1 Tat protein [ 27 – 29 ]. In this study, we employ tree shrews, primary cortical neurons, and the SH-SY5Y cell line to examine the synergistic effects of METH and HIV-1 Tat protein co-exposure on neuronal autophagy and to elucidate the roles of ERS and TRIM13 in this process. This study aims to uncover the mechanisms underlying neurological damage caused by novel psychoactive substances and to explore potential strategies for preventing neurocognitive impairment in HIV-infected individuals with substance use disorders. 2. Materials and methods 2.1 Reagents METH was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). HIV-1 TAT Clade-B (#HIV-129-c) was purchased from Prospecbio (Rehovot, Israel). 4-PBA (HY-15654) was purchased from MCE (Shanghai, China). 2.2 Cell culture The neonatal tree shrews were supplied by the Tree Shrew Germplasm Resource Center at the Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming, China. The cerebral cortex of neonatal tree shrews was aseptically dissected after the removal of the meninges, olfactory bulb, cerebellum, and brainstem. The tissues were subsequently enzymatically digested with 0.25% trypsin-EDTA (Gibco, China) for 10 min. Digestion was stopped using DMEM medium (Biological Industries, Israel) containing 10% fetal bovine serum (Gibco, China), followed by filtration through a 70 µm cell strainer (Biosharp, China) and centrifugation for 8 min at 4°C. Cells were resuspended in a DMEM medium (Biological Industries, Israel) containing 10% FBS and 1% penicillin-streptomycin (Gibco, China). After 24 hours, the medium was replaced with Neurobasal™-A Medium (Gibco, China) containing 15% FBS (Gibco, China), 2% penicillin-streptomycin (Gibco, China), 1% glutamine (Gibco, China) and 2% B-27™ Plus Supplement (Gibco, China). SH-SY5Y cells were cultured in DMEM F12 medium (Biological Industries, Israel) containing 15% FBS (Gibco, China) and 1% penicillin/streptomycin (Gibco, China). 2.3 Animals Male tree shrews, weighing 120–190 g and aged 1 year old, were provided by the Tree Shrew Germplasm Resource Center at the Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College in Kunming, China. The tree shrews were housed in a temperature-controlled room at 23°C ± 1°C with a humidity level of 45–55%. They were exposed to a 12-h light/dark cycle and provided ad libitum access to food and water. The tree shrews were randomly divided into various experimental groups. All procedures followed the guidelines established by the National Institutes of Health for the care and use of laboratory animals and received approval from the Experimental Animal Ethics Committee of Kunming Medical University. 2.4 Animal models The tree shrews were randomly assigned to five experimental groups: saline, HIV-1 Tat protein, METH, METH + HIV-1 Tat protein, and 4-PBA pretreatment followed by METH and HIV-1 Tat protein. The saline group received intraperitoneal (i.p.) injections of an equal volume of saline for 10 days. The HIV-1 Tat protein group was administered 10 µg of HIV-1 Tat protein directly into the left lateral ventricle (coordinates relative to the fontanelle: anterior-posterior − 0.6 mm, medial-lateral + 1.8 mm, dorsal-ventral − 4.0 mm). The METH group received METH at a dose of 2 mg/kg, i.p., for 10 days. The METH + HIV-1 Tat protein group was treated with both METH (2 mg/kg, i.p., for 10 days) and a single dose of 10 µg HIV-1 Tat protein into the left ventricle. Lastly, the 4-PBA + HIV-1 Tat protein + METH group underwent treatment with 136 mg/kg 4-PBA, i.p., 1 hour before the METH and HIV-1 Tat protein injections, with METH administered at 2 mg/kg, i.p., for 10 days, and the HIV-1 Tat protein administered as a single dose into the left ventricle. Once the tree shrews received the METH, they were immediately placed in the open-field apparatus. The tracks, total distance traveled, and average speed of tree shrews in the open-field apparatus were recorded using the VisuTrack system (Xinruan Information Technology Co., Ltd., China). 2.5 Stereotypical behavior score After watching the video recordings made during the open-field tests and referring to the GHF-Dodd scoring method[ 30 , 31 ], stereotypic behavior scores were made as follows (Table 1 ). Table 1 The scoring system of the stereotyped behavior Score Behavior description 0 Stationary, little or no movement 1 Normal movement accompanied by repeated exploration in situ or no fixed direction of repeated exploration 2 Ran fast around the open field, circled, climbed, and jumped repeatedly 3 Repetitive movements of the head or tail, either up or down, directed toward one wall or corner of the chamber, experienced repeated convulsions and hiccups or sustained an arched position 2.6 siRNA Transfection The primary cortical neurons from tree shrews and SH-SY5Y cells were subjected to siRNA transfection to knock down TRIM13 expression. The siRNAs targeting TRIM13 (LV3-TRIM13-1 sequence: 5'-TGCAGCTGATTTGTGGGATCT-3', LV3-TRIM13-2 sequence: 5'-ATGAAGAACTTTGATACCAGT-3', LV3-TRIM133 sequence: 5'-GCTCTTTCTCGCTTGGATACC-3') and a non-targeting control siRNA (NC-siRNA sequence: 5'-TTCTCCGAACGTGTCACGT-3') were acquired from Gima Genetics. For the transfection procedure, primary cortical neurons and SH-SY5Y cells were seeded at a density of 1 × 10 ^ 5 cells/well in six-well plates and allowed to attach overnight at 37°C with 5% CO 2 . The following day, the cells were transfected with TRIM13-siRNAs and NC-siRNA at a concentration of 120 pmol/ml using Lipofectamine® 2000 reagent (5 µl/ml) in Opti-MEM® I Reduced Serum Medium. This mixture was incubated with the cells for 24 hours. Post-transfection, the medium containing the viral particles was removed, and cells were replenished with complete growth medium before being returned to the incubator set at 37°C and 5% CO 2 . Subsequently, the transfected cells were harvested for the subsequent experiments. 2.7 Western Blot The total proteins of the primary neurons from tree shrews, SH-SY5Y cells, and the tree shrew's prefrontal cortex were treated with RIPA buffer containing protease inhibitors. The protein concentration of the samples was measured using the BCA Protein Kit (Beyotime, China). Subsequently, the sample proteins were separated by SDS-PAGE and transferred to a PVDF membrane using a transfer device (BioRed, USA). After blocking the membranes with 5% skim milk at room temperature for 2 hours, the membranes were subsequently incubated overnight at 4℃ with the following primary antibodies: mouse anti-β-actin (1:2000, Proteintech, China), LC3Ⅱ, ATG7, ATG5, Beclin1, p62, p-PERK, ATF6, IRE1, Bip (1:1000, Cell Signaling Technology, USA), PERK and TRIM13 (1:100, Santa Cruz Biotechnology, USA),. 2.8 Immunofluorescence The cells and brain slices were fixed using 4% paraformaldehyde for 1 hour at room temperature. After fixation, permeabilization was performed with a 0.3% Triton X-100 solution for 20 minutes at room temperature. To block non-specific binding, the wells were incubated with 10% goat serum for cells and 20% goat serum for brain slices for 2 hours at room temperature. The antibodies (LC3 II, TRIM13, and MAP2) were incubated overnight at 4℃ with each sample. The next day, fluorescent secondary antibodies were used and incubated at 37℃ for 2 hours. Leica software was utilized for image capture. The digitized images were then quantitatively analyzed and processed using ImageJ software. 2.9 Immunoprecipitation The cellular proteins were treated in lysis buffer containing protease and phosphatase inhibitors for 30 min, the supernatant was collected and 50 µl of the supernatant was taken as input group. The specific antibody and IgG were incubated with the protein A/G beads for 2 hours at room temperature. Antibody-encapsulated beads were washed to remove unbound antibodies. These prepared beads are then added to the lysate (excluding the reserved input sample) and incubated overnight at 4℃. The next day the beads were thoroughly washed to remove non-specifically bound proteins. Bound protein complexes were then eluted by adding 1× SDS-PAGE sample loading buffer and heating at 95℃ for 5 minutes. Finally, the beads are removed by centrifugation and the supernatant is collected for subsequent Western blot analysis. 2.10 Statistical analysis All the statistical analyses were performed using GraphPad Prism 6.02 (GraphPad Software, USA). One-way or two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests was performed for comparison among multiple groups and P < 0.05 was considered statistically significant. To assess the potential synergistic interaction between the HIV-1 Tat protein and methamphetamine (METH), the Bliss Independence model was applied. Prior to applying the Bliss Independence calculation, Max-Min Normalization was performed to standardize the data, utilizing the formula: \(\:\text{z}\text{i}=\frac{\text{x}\text{i}-\text{M}\text{i}\text{n}\left(\text{X}\right)}{\text{M}\text{a}\text{x}\left(\text{X}\right)-\text{M}\text{i}\text{n}\left(\text{X}\right)}\) . The individual effects of METH and the HIV-1 Tat protein are denoted as E A and E B , respectively, while the combined effect of both is represented by E AB . The interaction between the two is quantified using the formula: \(\:\frac{{E}_{A}+{E}_{B}-\left({E}_{A}\times\:{E}_{B}\right)}{{E}_{AB}}\) . An outcome greater than 1 suggests antagonism, equal to 1 indicates additivity, and less than 1 is indicative of synergy. 3. Results 3.1 METH and HIV-1 Tat protein synergistically induced autophagy in Neurons The primary cortical neurons from tree shrews and SH-SY5Y cells were exposed to METH (0.5mM or 2mM) or/and HIV-1 Tat protein (50nM or 100nM) for 24 hours [ 15 , 27 ]. Both METH and HIV-1 Tat protein treatments upregulated the expression of autophagy-related proteins (ATG5, ATG7, Beclin1, and LC3II) while downregulating the expression of p62 compared to the control group. Notably, these changes were further amplified when cells were co-exposed to METH and HIV-1 Tat protein, indicating a synergistic effect in provoking neuronal autophagy (Fig. 1 A, B, and Supplementary Fig. 1A, C). This was further confirmed using the Bliss independence model. The combination index (CI) for the expression of autophagy-related proteins was all less than 1, suggesting a synergistic interaction (Supplementary Tables 1 and 2). Immunofluorescence (IF) staining revealed that co-exposure to METH and HIV-1 Tat significantly enhanced the fluorescence intensity of LC3II in both cell types compared to the groups exposed solely METH or HIV-1 Tat (Fig. 1 I and Supplementary Fig. 1E). 3.2 METH and HIV-1 Tat Protein Synergistically Induced ERS in Neurons To further understand the effects of METH on the expression of ERS-related proteins, both types of cells were treated with varying concentrations of METH for different durations. The expression of ERS markers (p-PERK, ATF6, IRE1, and Bip) was assessed using the western blot. The data revealed that METH upregulated the expression of these ERS-related proteins in a dose and time-dependent manner (Supplementary Fig. 2A-H). Compared to the control group, the ERS-related proteins in primary cortical neurons and SH-SY5Y cells were upregulated after solely METH or HIV-1 Tat protein exposure. Notably, this upregulation was further accentuated following co-exposure to METH and HIV-1 Tat protein (Fig. 1 C, D and Supplementary Fig. 1B, D). The synergistic effect of METH and HIV-1 Tat protein on ERS was quantitatively confirmed by the CI values (Supplementary Table 1 and Table 2). 3.3 4-PBA pre-treatment reduced ERS and Autophagy Induced by METH and HIV-1 Tat Protein in Neurons In neurons that co-exposure to METH and HIV-1 Tat protein, 4-PBA pre-treatment significantly downregulated the expression of ERS-associated proteins (Fig. 1 E, F and Supplementary Fig. 3A, C). In addition, co-exposure to METH and HIV-1 Tat protein upregulated the expression of autophagy-related proteins and downregulated the p62 expression, which was partially reversed by 4-PBA pre-treatment (Fig. 1 G, H and Supplementary Fig. 3B, D). IF analysis further confirmed decreased expression of LC3II following 4-PBA pre-treatment (Fig. 1 J and Supplementary Fig. 3E). 3.4 METH and HIV-1 Tat protein Synergistically aggravated excitotoxicity and stereotypic behaviors in tree shrews via the modulation of ERS The protocol for the open field test was shown in Fig. 2 A. The tracks of the tree shrews in the open field were depicted in Fig. 2 B. Compared to the Saline, HIV-1 Tat, or METH groups, tree shrews co-exposed to METH and HIV-1 Tat protein showed a significant increase in total distance traveled and average speed in the open field (Day1-Day10). However, pre-treatment of tree shrews with 4-PBA effectively reduced the total distance traveled (Day 5-Day 10) and average speed (Day 3, Day 7-Day 10) in the open field (Fig. 2 C, D). In addition, the stereotypic behaviors of tree shrews were also analyzed following METH injection. Compared to the saline group, tree shrews exposed to METH displayed obvious spontaneous stereotypic activities, including irritability, exploring forward, repeatedly shaking their heads, curling their tails into a semicircle, hiccups, and decreased defecation. Importantly, tree shrews co-exposed to METH and HIV-1 Tat protein presented more severe spontaneous stereotypic activities compared to the solely METH-exposed group. In contrast, pretreatment of tree shrews with 4-PBA decreased spontaneous stereotypic activities (Fig. 2 E), suggesting that METH and HIV-1 Tat protein Synergistically aggravated excitotoxicity and stereotypic behaviors in tree shrews via the modulation of ERS. 3.5 METH and HIV-1 Tat Protein Synergistically Induced Autophagy and ERS in the Prefrontal Cortex of Tree Shrew Compared to the saline group, tree shrews exposed to solely METH or HIV-1 Tat protein showed the upregulated expression of autophagy-related proteins in the prefrontal cortex, along with a notable reduction in p62 expression. As expected, these effects were exacerbated in the group co-exposed to METH and HIV-1 Tat treatment (Fig. 3 A, B and Supplementary Table 3). Likewise, exposure to METH or HIV-1 Tat protein also upregulated the expression of ERS-related proteins in the prefrontal cortex of tree shrews. This upregulation was further augmented in tree shrews co-exposed to METH and HIV-1 Tat protein (Fig. 3 C, D and Supplementary Table 3). To investigate whether ERS contributes to autophagy in this context, tree shrews were pretreated with 4-PBA before co-exposure to METH and HIV-1 Tat protein. The results demonstrated that 4-PBA pretreatment rescued the upregulation of ERS-related and autophagy-related proteins caused by METH and HIV-1 Tat protein co-exposure (Fig. 3 E-H). Moreover, co-exposure to METH and HIV-1 Tat protein significantly increased the fluorescence intensity of LC3 II in the prefrontal cortex of tree shrews, while pre-treatment of 4-PBA reversed this effect (Fig. 3 I). 3.6 METH and HIV-1 Tat Protein Synergistically Decreased the Expression of TRIM13 by Modulating ERS Co-exposure to METH and HIV-1 Tat protein further downregulated the protein and fluorescence intensity of TRIM13 in the prefrontal cortex compared to solely METH or HIV-1 Tat protein exposure (Fig. 4 A, C). Pretreatment of 4-PBA rescued the downregulation of TRIM13 in the prefrontal cortex of tree shrews (Fig. 4 . B, C). In primary cortical neurons and SH-SY5Y cell lines, a dose- and time-dependent downregulation in TRIM13 expression was observed with METH exposure (Fig. 4 D, E and Supplementary Fig. 4A, B). Notably, co-exposure to METH and HIV-1 Tat protein resulted in a more significant reduction in expression compared to either agent alone (Fig. 4 F and Supplementary Fig. 4C). IF staining further confirmed that the fluorescence intensity of TRIM13 significantly diminished in both types of cells following METH and/or HIV-1 Tat exposure (Fig. 4 G, H and Supplementary Fig. 4E, G). Importantly, pretreatment of 4-PBA reversed the downregulation of TRIM13 protein and fluorescence intensity caused by co-exposure to METH and HIV-1 Tat protein (Fig. 4 I, J, and K and Supplementary Fig. 4D, F and H). 3.7 METH and HIV-1 Tat Protein Synergistically Induced Autophagy Via ERS-Mediated TRIM13 Pretreatment with the ERS inhibitor 4-PBA reduced METH and HIV-1 Tat protein synergistically induced neuronal autophagy in both in vivo and in vitro models. TRIM13, a protein involved in maintaining endoplasmic reticulum homeostasis, may play a crucial role in neuronal autophagy. Co-immunoprecipitation (IP) analyses revealed that co-exposure to METH and HIV-1 Tat protein intensified the interaction between TRIM13 and key autophagy-related proteins, including p62, LC3II, and Beclin1 (Fig. 5 A and Supplementary Fig. 4I). Pre-treatment with siRNA-TRIM13 significantly downregulated the expression of autophagy markers (ATG5, ATG7, Beclin1, and LC3II), while upregulating the p62 expression in the primary cortical neurons and SH-SY5Y cells (Fig. 5 B, C and Supplementary Fig. 4J). Additionally, IF staining confirmed that the pretreatment of TRIM13 significantly attenuated the METH and HIV-1 Tat co-exposure induced increase in LC3II protein fluorescence intensity (Fig. 5 D, E and Supplementary Fig. 4.K). 4. Discussion In this study, we found that co-exposure to METH and HIV-1 Tat proteins markedly upregulated the expression of proteins related to ERS and autophagy, as well as increased the excitotoxicity and stereotypic behaviors in tree shrews. Pretreatment with the ERS-inhibitor 4-PBA reduced excitotoxicity, stereotypic behaviors, and autophagy induced by co-exposure to METH and HIV-1 Tat proteins. Furthermore, METH and HIV-1 Tat proteins synergistically downregulated TRIM13 expression, which could be rescued by 4-PBA pretreatment. Notably, the interaction between TRIM13 and autophagy-related proteins was intensified after co-exposure to METH and HIV-1 Tat proteins. Silencing of the TRIM13 gene effectively attenuated METH and HIV-1 Tat synergistically induced neuronal autophagy. Our study demonstrated that METH and HIV-1 Tat protein synergistically induced neuronal autophagy through ERS-mediated alteration of TRIM13. ER is crucial in protein folding and translation, lipid synthesis, and Ca 2+ homeostasis, supported by its extensive membrane network and myriad associated enzymes [ 32 ]. Cells exposed to METH initiate ER stress by disrupting these normal ER functions [ 19 , 33 ]. Specifically, ER stress disturbs protein synthesis and folding within the ER, leading to the degradation of misfolded ER proteins and the activation of various transcription factors. Moreover, Rong et al. found that the HIV-1 Tat protein induces apoptosis in human brain microvascular endothelial cells (HBMECs) by inducing ER stress [ 34 ]. These findings are consistent with our observations, suggesting that ER stress contributes to the neurotoxicity induced by either HIV or METH. We also noted that co-exposure to METH and HIV-1 Tat proteins significantly increased the expression of ER stress-related proteins, indicating that ER stress may play a vital role in the synergistic neurotoxicity of METH and HIV-1 Tat proteins. ERS and autophagy are cellular adaptive responses to various stimuli, but severe ERS may induce excessive autophagy, leading to apoptosis [ 35 ]. Research involving drugs and HIV has highlighted the pivotal role of ERS pathways in autophagy regulation [ 36 – 38 ]. In this study, we found that co-exposure to METH and HIV-1 Tat protein-induced neuronal autophagy, which is consistent with the increased ERS. However, the relationship between neuronal ERS and autophagy caused by the co-exposure to METH and HIV-1 Tat protein remains poorly understood. 4-PBA is commonly employed as an ER stress inhibitor due to its aids in protein post-translational modifications and folding, thereby attenuating ERS-mediated neuronal death [ 18 ]. Pretreatment with 4-PBA alleviated METH and HIV-1 Tat protein synergistically induced neuronal autophagy, suggesting that ERS may act as a precursor in the METH and HIV-1 Tat protein-induced neuronal autophagy. Glutamate is the primary excitatory neurotransmitter in the mammalian central nervous system, and overactivation of its receptors constitutes a significant pathway for neuronal excitotoxicity, whereas calcium inward flow is critical for glutamate excitotoxicity[ 39 ]. The endoplasmic reticulum acts as an extensive network regulating Ca 2+ , thus its stability is critical for maintaining Ca 2+ homeostasis[ 40 ]. In our study, METH and HIV-1 Tat protein synergistically induced excitotoxicity and stereotypic behaviors in tree shrews. Pretreatment with 4-PBA alleviated these effects, suggesting that METH HIV-1 Tat protein synergistically induced excitotoxicity and stereotypic behaviors in tree shrews via modulation of ERS. The ERS-mediated calcium dysregulation and glutamatergic excitotoxicity may be involved in this process, although additional evidence is needed to confirm this potential mechanism. Pretreatment with 4-PBA rescued downregulation of TRIM13 induced by METH and HIV-1 Tat protein co-exposure, indicating potential links between TRIM13 and ER homeostasis [ 41 , 42 ]. TRIM13 may function as an essential initiator of autophagy, serving as an ER transmembrane receptor. TRIM13 facilitates autophagy through k63-linked ubiquitination, recruiting p62 and interacting with LC3 on the autophagic membrane to catalyze the formation of autophagic vesicles [ 41 , 43 ]. In our study, immunoprecipitation analysis also revealed the interactions between TRIM13 and autophagy makers, including Beclin1, p62, and LC3II. Furthermore, silencing of the TRIM13 gene reduced neuronal autophagy induced by METH and HIV-1 Tat protein co-exposure. These results suggest that TRIM13 may play a role in assembling an autophagic complex on the ER surface, facilitating the recruitment of autophagy-related proteins such as Beclin1, LC3, and p62. Silencing the TRIM13 gene impairs cells’ ability to properly synthesize proteins and perform subsequent ubiquitination processes, ultimately resulting in a deficiency of necessary components for establishing an autophagic platform. This deficiency may result in improper synthesis of intracellular autophagic vesicles [ 44 – 46 ]. In our experiments, we observed that TRIM13 did not interact with ATG7 and ATG5, but both proteins experienced a significant reduction in expression following TRIM13 silencing. This observation suggests that while ATG7 and ATG5 do not directly bind to TRIM13, they are nonetheless crucial for the formation of autophagic vesicles, particularly in the lipidation of LC3I to LC3II, a critical step in autophagosome maturation [ 47 ]. Although this hypothesis is plausible, further experimental verification is needed to confirm this interaction. Together, TRIM13 provides new insights into understanding the synergistic mechanisms of METH and HIV-induced damage to the central nervous system. 5. Conclusion Our study demonstrates that METH and HIV-1 Tat protein synergistically induce neuronal autophagy through the modulation of ERS. Additionally, we have uncovered that the interaction between TRIM13 and autophagy is critical for this process (Fig. 6 ). In conclusion, our study provides novel perspectives on the mechanisms underlying the synergistic effects of METH and HIV-1 Tat protein on neuronal autophagy and identifies potential targets for the prevention of METH abuse in HIV-infected individuals. Declarations Author Contributions Wang C. contributed to the conceptualization, methodology, data analysis, and writing the original manuscript; Yang G.M., Huang J., and Tian Y.Q. designed and performed the experiments. Chi-Kwan L. and Miao L. contributed to writing—review, and editing; Wang H.W., Li Y., Huang Y.Z., and Teng H.X. contributed to data analysis and validation; Liu L., Li J., and Zeng X.F. supervised data analysis, and revised manuscript. All authors reviewed the manuscript. Funding This work was supported by the National Natural Science Foundation of China (81960340, 82060382, and 82160325) and the Yunnan Applied Basic Research Projects Joint Special Project (202201AY070001-020). Data Availability The data used to support the findings of this study are available from the corresponding author upon request. Ethics Statement All procedures involving animals were carried out in strict accordance with the guidelines set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Institutional Animal Care and Use Committee of Kunming Medical University. Consent to Participate Not applicable. Consent for Publication Not applicable. Competing Interests The authors declare no competing interests. References World Drug Report 2023. In: U. N. Off. Drugs Crime. //www.unodc.org/unodc/en/data-and-analysis/world-drug-report-2023.html. Accessed 16 Nov 2023 UNAIDS leads the world’s most extensive data collection on HIV epidemiology, programme coverage and finance | UNAIDS. https://www.unaids.org/en/topic/data. Accessed 25 Sep 2023 Blackard JT, Sherman KE (2021) Drugs of Abuse and Their Impact on Viral Pathogenesis. Viruses 13:2387. https://doi.org/10.3390/v13122387 Williams ME, Zulu SS, Stein DJ, et al (2020) Signatures of HIV-1 subtype B and C Tat proteins and their effects in the neuropathogenesis of HIV-associated neurocognitive impairments. Neurobiol Dis 136:104701. https://doi.org/10.1016/j.nbd.2019.104701 Rudd H, Toborek M (2022) Pitfalls of Antiretroviral Therapy: Current Status and Long-Term CNS Toxicity. Biomolecules 12:894. https://doi.org/10.3390/biom12070894 Spector C, Mele AR, Wigdahl B, Nonnemacher MR (2019) Genetic variation and function of the HIV-1 Tat protein. Med Microbiol Immunol (Berl) 208:131–169. https://doi.org/10.1007/s00430-019-00583-z Kipp AM, Desruisseau AJ, Qian H-Z (2011) Non-injection Drug Use and HIV Disease Progression in the Era of Combination Antiretroviral Therapy. J Subst Abuse Treat 40:386–396. https://doi.org/10.1016/j.jsat.2011.01.001 Friedman H, Pross S, Klein TW (2006) Addictive drugs and their relationship with infectious diseases. FEMS Immunol Med Microbiol 47:330–342. https://doi.org/10.1111/j.1574-695X.2006.00097.x Link RW, Mele AR, Antell GC, et al (2019) Investigating the distribution of HIV-1 Tat lengths present in the Drexel Medicine CARES cohort. Virus Res 272:197727. https://doi.org/10.1016/j.virusres.2019.197727 Ye X, Zhang Y, Xu Q, et al (2017) HIV-1 Tat inhibits EAAT-2 through AEG-1 upregulation in models of HIV-associated neurocognitive disorder. Oncotarget 8:39922–39934. https://doi.org/10.18632/oncotarget.16485 Lapierre J, Karuppan MKM, Perry M, et al (2022) Different Roles of Beclin1 in the Interaction Between Glia and Neurons after Exposure to Morphine and the HIV- Trans-Activator of Transcription (Tat) Protein. J Neuroimmune Pharmacol Off J Soc NeuroImmune Pharmacol 17:470–486. https://doi.org/10.1007/s11481-021-10017-4 Halcrow PW, Kumar N, Quansah DNK, et al (2022) Endolysosome Iron Chelation Inhibits HIV-1 Protein-Induced Endolysosome De-Acidification-Induced Increases in Mitochondrial Fragmentation, Mitophagy, and Cell Death. Cells 11:1811. https://doi.org/10.3390/cells11111811 Teodorof-Diedrich C, Spector SA (2020) Human Immunodeficiency Virus Type 1 and Methamphetamine-Mediated Mitochondrial Damage and Neuronal Degeneration in Human Neurons. J Virol 94:e00924-20. https://doi.org/10.1128/JVI.00924-20 Yang G, Li J, Leung C-K, et al (2022) Methamphetamine and HIV-1 Tat proteins synergistically induce microglial autophagy via activation of the Nrf2/NQO1/HO-1 signal pathway. Neuropharmacology 220:109256. https://doi.org/10.1016/j.neuropharm.2022.109256 Zeng X-F, Li Q, Li J, et al (2018) HIV-1 Tat and methamphetamine co-induced oxidative cellular injury is mitigated by N-acetylcysteine amide (NACA) through rectifying mTOR signaling. Toxicol Lett 299:159–171. https://doi.org/10.1016/j.toxlet.2018.09.009 Nakka VP, Prakash-Babu P, Vemuganti R (2016) Crosstalk Between Endoplasmic Reticulum Stress, Oxidative Stress, and Autophagy: Potential Therapeutic Targets for Acute CNS Injuries. Mol Neurobiol 53:532–544. https://doi.org/10.1007/s12035-014-9029-6 Shah A, Kumar A (2016) Methamphetamine-mediated endoplasmic reticulum (ER) stress induces type-1 programmed cell death in astrocytes via ATF6, IRE1α and PERK pathways. Oncotarget 7:46100–46119. https://doi.org/10.18632/oncotarget.10025 Qie X, Wen D, Guo H, et al (2017) Endoplasmic Reticulum Stress Mediates Methamphetamine-Induced Blood–Brain Barrier Damage. Front Pharmacol 8:639. https://doi.org/10.3389/fphar.2017.00639 Yang L, Guo N, Fan W, et al (2020) Thioredoxin-1 blocks methamphetamine-induced injury in brain through inhibiting endoplasmic reticulum and mitochondria-mediated apoptosis in mice. Neurotoxicology 78:163–169. https://doi.org/10.1016/j.neuro.2020.03.006 Wongprayoon P, Govitrapong P (2017) Melatonin Protects SH-SY5Y Neuronal Cells Against Methamphetamine-Induced Endoplasmic Reticulum Stress and Apoptotic Cell Death. Neurotox Res 31:1–10. https://doi.org/10.1007/s12640-016-9647-z Andhavarapu S, Katuri A, Bryant J, et al (2020) Intersecting roles of ER stress, mitochondrial dysfunction, autophagy, and calcium homeostasis in HIV-associated neurocognitive disorder. J Neurovirol 26:664–675. https://doi.org/10.1007/s13365-020-00861-0 Chen X, Zhang T, Zhang Y (2020) Endoplasmic reticulum stress and autophagy in HIV-1-associated neurocognitive disorders. J Neurovirol 26:824–833. https://doi.org/10.1007/s13365-020-00906-4 Hatakeyama S (2017) TRIM Family Proteins: Roles in Autophagy, Immunity, and Carcinogenesis. Trends Biochem Sci 42:297–311. https://doi.org/10.1016/j.tibs.2017.01.002 Watanabe M, Hatakeyama S (2017) TRIM proteins and diseases. J Biochem (Tokyo) 161:135–144. https://doi.org/10.1093/jb/mvw087 Tomar D, Prajapati P, Sripada L, et al (2013) TRIM13 regulates caspase-8 ubiquitination, translocation to autophagosomes and activation during ER stress induced cell death. Biochim Biophys Acta BBA - Mol Cell Res 1833:3134–3144. https://doi.org/10.1016/j.bbamcr.2013.08.021 Fan Y, Peng X, Li B, Zhao G (2020) Development of Autophagy Signature-Based Prognostic Nomogram for Refined Glioma Survival Prognostication. BioMed Res Int 2020:1872962. https://doi.org/10.1155/2020/1872962 Li J, Wang W, Tong P, et al (2018) Autophagy Induction by HIV-Tat and Methamphetamine in Primary Midbrain Neuronal Cells of Tree Shrews via the mTOR Signaling and ATG5/ATG7 Pathway. Front Neurosci 12:921. https://doi.org/10.3389/fnins.2018.00921 Huang J, Yang G, Li Z, et al (2020) Involvement of dopamine D3 receptor and dopamine transporter in methamphetamine-induced behavioral sensitization in tree shrews. Brain Behav 10:e01533. https://doi.org/10.1002/brb3.1533 Huang J, Zhang R, Wang S, et al (2021) Methamphetamine and HIV-Tat Protein Synergistically Induce Oxidative Stress and Blood-Brain Barrier Damage via Transient Receptor Potential Melastatin 2 Channel. Front Pharmacol 12:619436. https://doi.org/10.3389/fphar.2021.619436 Huang J, Yang G, Li Z, et al (2020) Involvement of dopamine D3 receptor and dopamine transporter in methamphetamine-induced behavioral sensitization in tree shrews. Brain Behav 10:e01533. https://doi.org/10.1002/brb3.1533 Xie X-L, Zhou W-T, Zhang K-K, et al (2018) METH-Induced Neurotoxicity Is Alleviated by Lactulose Pretreatment Through Suppressing Oxidative Stress and Neuroinflammation in Rat Striatum. Front Neurosci 12:802. https://doi.org/10.3389/fnins.2018.00802 Schwarz DS, Blower MD (2016) The endoplasmic reticulum: structure, function and response to cellular signaling. Cell Mol Life Sci CMLS 73:79–94. https://doi.org/10.1007/s00018-015-2052-6 Lee H-S, Kim E-N, Jeong G-S (2021) Aromadendrin Protects Neuronal Cells from Methamphetamine-Induced Neurotoxicity by Regulating Endoplasmic Reticulum Stress and PI3K/Akt/mTOR Signaling Pathway. Int J Mol Sci 22:2274. https://doi.org/10.3390/ijms22052274 Ma R, Yang L, Niu F, Buch S (2016) HIV Tat-Mediated Induction of Human Brain Microvascular Endothelial Cell Apoptosis Involves Endoplasmic Reticulum Stress and Mitochondrial Dysfunction. Mol Neurobiol 53:132–142. https://doi.org/10.1007/s12035-014-8991-3 Mehrbod P, Ande SR, Alizadeh J, et al (2019) The roles of apoptosis, autophagy and unfolded protein response in arbovirus, influenza virus, and HIV infections. Virulence 10:376–413. https://doi.org/10.1080/21505594.2019.1605803 Huang R, Zhang Y, Han B, et al (2017) Circular RNA HIPK2 regulates astrocyte activation via cooperation of autophagy and ER stress by targeting MIR124–2HG. Autophagy 13:1722–1741. https://doi.org/10.1080/15548627.2017.1356975 Guo M-L, Liao K, Periyasamy P, et al (2015) Cocaine-mediated microglial activation involves the ER stress-autophagy axis. Autophagy 11:995–1009. https://doi.org/10.1080/15548627.2015.1052205 Sil S, Niu F, Tom E, et al (2019) Cocaine mediated neuroinflammation: Role of dysregulated autophagy in pericytes. Mol Neurobiol 56:3576–3590. https://doi.org/10.1007/s12035-018-1325-0 Dossat AM, Trychta KA, Glotfelty EJ, et al (2024) Excitotoxic glutamate levels cause the secretion of resident endoplasmic reticulum proteins. J Neurochem. https://doi.org/10.1111/jnc.16093 Fitting S, Knapp PE, Zou S, et al (2014) Interactive HIV-1 Tat and morphine-induced synaptodendritic injury is triggered through focal disruptions in Na + influx, mitochondrial instability, and Ca 2+ overload. J Neurosci Off J Soc Neurosci 34:12850–12864. https://doi.org/10.1523/JNEUROSCI.5351-13.2014 D T, R S, Ak S, et al (2012) TRIM13 regulates ER stress induced autophagy and clonogenic ability of the cells. Biochim Biophys Acta 1823:. https://doi.org/10.1016/j.bbamcr.2011.11.015 Tomar D, Singh R (2014) TRIM13 regulates ubiquitination and turnover of NEMO to suppress TNF induced NF-κB activation. Cell Signal 26:2606–2613. https://doi.org/10.1016/j.cellsig.2014.08.008 Yu B, Zhou Y, He J (2023) TRIM13 inhibits cell proliferation and induces autophagy in lung adenocarcinoma by regulating KEAP1/NRF2 pathway. Cell Cycle Georget Tex 22:1496–1513. https://doi.org/10.1080/15384101.2023.2216504 Ji CH, Kim HY, Heo AJ, et al (2019) The N-Degron Pathway Mediates ER-phagy. Mol Cell 75:1058-1072.e9. https://doi.org/10.1016/j.molcel.2019.06.028 Ji CH, Kim HY, Heo AJ, et al (2020) Regulation of reticulophagy by the N-degron pathway. Autophagy 16:373–375. https://doi.org/10.1080/15548627.2019.1695402 Yu B, Zhou Y, He J (2023) TRIM13 inhibits cell proliferation and induces autophagy in lung adenocarcinoma by regulating KEAP1/NRF2 pathway. Cell Cycle Georget Tex 22:1496–1513. https://doi.org/10.1080/15384101.2023.2216504 Urbańska K, Orzechowski A (2021) The Secrets of Alternative Autophagy. Cells 10:3241. https://doi.org/10.3390/cells10113241 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterialtable.docx Supplementary Material: Table Table 1. Synergistic Effect Quantification of HIV-1 Tat Protein and METH in the Primary Cortical Neurons from Tree Shrews. Table 2. Quantitative Analysis of Synergistic Interactions between HIV-1 Tat Protein and METH in SH-SY5Y Cells. Table 3. Evaluation of Synergistic Effects of HIV-1 Tat Protein and METH in the Prefrontal Cortex of Tree Shrews. SupplementaryFigure.1.png Supplementary Fig.1 METH and HIV-1 Tat protein synergistically induced autophagy and ERS in SH-SY5Y cells. (A, C) METH and HIV-1 Tat protein synergistically upregulated the expression of autophagy-related proteins (ATG7, Beclin1, ATG5, and LC3II), and downregulated the expression of p62 in SH-SY5Y cells. (B, D) METH and HIV-1 Tat protein synergistically upregulated the expression of ERS-related proteins (PERK, p-PERK, IRE1, ATF6, and Bip) in SH-SY5Y cells. (E) METH and HIV-1 Tat protein synergistically upregulated the fluorescent expression of LC3II, with blue fluorescence indicating DAPI and red fluorescence indicating LC3II. All data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test; all values are presented as the mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, n≥3. SupplementaryFigure.2.png Supplementary Fig. 2 METH-induced endoplasmic reticulum stress in primary cortical neurons of tree shrews and SH-SY5Y Cells (A, C) METH elevated the expression of ERS-related proteins (PERK, p-PERK, IRE1, ATF6, and Bip) in the primary cortical neurons of tree shrews in a dose-dependent manner. (B, D) METH elevated the expression of ERS-related proteins (PERK, p-PERK, IRE1, ATF6, and Bip) in the primary cortical neurons of tree shrews in a time-dependent manner. (E, G) METH elevated the expression of ERS-related proteins (PERK, p-PERK, IRE1, ATF6, and Bip) in SH-SY5Y cells in a dose-dependent manner. (F, H) METH elevated the expression of ERS-related proteins (PERK, p-PERK, IRE1, ATF6, and Bip) in SH-SY5Y cells in a time-dependent manner. All data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test; all values are presented as the mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, n≥3. SupplementaryFigure.3.png Supplementary Fig.3 Pre-treatment of 4-PBA alleviated ERS and autophagy induced by METH and HIV-1 Tat protein in SH-SY5Y cells. (A-C) Pre-treatment of 4-PBA downregulated the expression of ERS-related proteins (PERK, p-PERK, IRE1, ATF6, and Bip) in SH-SY5Y cells. (B, D) Pre-treatment of 4-PBA downregulated the expression of autophagy-related proteins (ATG5, ATG7, Beclin1, and LC3II), and upregulated the expression of p62 in SH-SY5Y cells. (E) Pre-treatment of 4-PBA downregulated the fluorescent expression of LC3II in SH-SY5Y cells, with blue fluorescence indicating DAPI and red fluorescence indicating LC3II. All data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test; all values are presented as the mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, n≥3. SupplementaryFigure.4.png Supplementary Fig.4 METH and HIV-1 Tat protein synergistically induced autophagy via ERS-Mediated TRIM13 in SH-SY5Y cell (A) METH downregulated the expression of TRIM13 in SH-SY5Y cells in a dose-dependent manner. (B) METH downregulated the expression of TRIM13 in SH-SY5Y cells in a time-dependent manner. (C) METH and HIV-1 Tat protein synergistically downregulated the expression of TRIM13 in SH-SY5Y cells. (D) Pre-treatment of 4-PBA upregulated the expression of TRIM13 in SH-SY5Y cells. (E, G) METH and HIV-1 Tat protein synergistically downregulated the fluorescent expression of TRIM13 in SH-SY5Y cells, with blue fluorescence indicating DAPI and green fluorescence indicating TRIM13. (F, H) Pre-treatment of 4-PBA upregulated the fluorescent expression of TRIM13 in SH-SY5Y cells, with blue fluorescence indicating DAPI and green fluorescence indicating TRIM13. (I) METH and HIV-1 Tat synergistically induced TRIM13 interacting with autophagy-related proteins (p62, Beclin1, and LC3II) in SH-SY5Y cells. (J) Pre-treatment of siRNA-TRIM13 downregulated the expression of autophagy-related proteins (LC3II, Beclin1, ATG5, and ATG7), and upregulated the expression of p62 induced by METH and HIV-1 Tat protein in SH-SY5Y cells. (K) Pre-treatment of siRNA-TRIM13 downregulated the fluorescent expression of LC3II in SH-SY5Y cells, with blue fluorescence indicating DAPI and red fluorescence indicating LC3II. All data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test; all values are presented as the mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, n≥3. Cite Share Download PDF Status: Published Journal Publication published 23 Dec, 2024 Read the published version in Molecular Neurobiology → Version 1 posted Editorial decision: Revision requested 01 Nov, 2024 Reviews received at journal 31 Oct, 2024 Reviews received at journal 17 Oct, 2024 Reviewers agreed at journal 17 Oct, 2024 Reviewers agreed at journal 14 Oct, 2024 Reviewers agreed at journal 11 Oct, 2024 Reviewers agreed at journal 04 Aug, 2024 Reviewers agreed at journal 02 Aug, 2024 Reviewers invited by journal 30 Jul, 2024 Editor assigned by journal 27 Jul, 2024 Submission checks completed at journal 26 Jul, 2024 First submitted to journal 23 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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HIV-1 Tat protein synergistically upregulated the expression of autophagy-related proteins (ATG7, Beclin1, ATG5, and LC3II), and downregulated the expression of p62 in the primary cortical neurons of tree shrews. (C-D) METH and HIV-1 Tat protein synergistically upregulated the expression of ERS-related proteins (PERK, p-PERK, IRE1, ATF6, and Bip) in the primary cortical neurons of tree shrews. (E-F) Pre-treatment of 4-PBA downregulated the expression of ERS-related proteins (PERK, p-PERK, IRE1, ATF6, and Bip) in the primary cortical neurons of tree shrews. (G-H) Pre-treatment of 4-PBA downregulated the expression of autophagy-related proteins (ATG5, ATG7, Beclin1, and LC3II), and upregulated the expression of p62 in the primary cortical neurons of tree shrews. (I-J) Pre-treatment of 4-PBA downregulated the fluorescent expression of LC3II in the primary cortical neurons of tree shrews, with blue fluorescence indicating DAPI and red fluorescence indicating LC3II. All data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test; all values are presented as the mean ± SD, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, n≥3.\u003c/p\u003e","description":"","filename":"Figure.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4788696/v1/f2ff79d08ec8380aa3581033.png"},{"id":63018055,"identity":"96ff9136-3999-4ce4-b020-3268dd6216cb","added_by":"auto","created_at":"2024-08-22 07:03:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1081532,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMETH and HIV-1 Tat protein synergistically aggravated excitotoxicity and stereotypic behaviors in tree shrews via the modulation of ERS.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The schedule of animal administration protocols and behavioral tests. (B) The tracks of tree shrews in the open-field apparatus. (C) The total distance traveled by the tree shrew in the open field. (D) The average speed of the tree shrew in the open field. (E) The stereotyped behavioral scores of tree shrews. The data were analyzed by two-way ANOVA or one-way ANOVA followed by Tukey's multiple comparison test; all values are presented as the mean ± SEM, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, n≥3.\u003c/p\u003e","description":"","filename":"Figure.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4788696/v1/fed8d0464ba948c26178155f.png"},{"id":63018054,"identity":"2f8129a2-069c-416f-92da-058098ad0be4","added_by":"auto","created_at":"2024-08-22 07:03:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1267630,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePre-treatment of 4-PBA alleviated ERS and autophagy induced by METH and HIV-1 Tat protein in the Prefrontal Cortex of Tree Shrew.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) METH and HIV-1 Tat protein synergistically upregulated the expression of autophagy-related proteins (ATG7, Beclin1, ATG5, and LC3II), and downregulated the expression of p62 in the prefrontal cortex of tree shrews. (C-D) METH and HIV-1 Tat protein synergistically upregulated the expression of ERS-related proteins (PERK, p-PERK, IRE1, ATF6, and Bip) in the prefrontal cortex of tree shrews. (E-F) Pre-treatment of 4-PBA downregulated the expression of ERS-related proteins (PERK, p-PERK, IRE1, ATF6, and Bip) in the prefrontal cortex of tree shrews. (G-H) Pre-treatment of 4-PBA downregulated the expression of autophagy-related proteins (ATG5, ATG7, Beclin1, and LC3II), and upregulated the expression of p62 in the prefrontal cortex of tree shrews. (I) Pre-treatment of 4-PBA downregulated the fluorescent expression of LC3II in the prefrontal cortex of tree shrews, with blue fluorescence indicating DAPI, red fluorescence indicating LC3II, and green fluorescence indicating MAP2. All data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test; all values are presented as the mean ± SD, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, n≥3.\u003c/p\u003e","description":"","filename":"Figure.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4788696/v1/082ea4b9640d3b82b4f9629b.png"},{"id":63017012,"identity":"f7614c9a-2d3e-4631-b21b-4323350a2f37","added_by":"auto","created_at":"2024-08-22 06:55:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1309476,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePre-treatment with 4-PBA improves TRIM13 expression synergistically reduced by METH and HIV-1 Tat proteins in tree shrew neurons.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) METH and HIV-1 Tat protein synergistically downregulated the expression of TRIM13 in the prefrontal cortex of tree shrews. (B) Pre-treatment of 4-PBA upregulated the expression of TRIM13 in the prefrontal cortex of tree shrews. (C) Pre-treatment of 4-PBA upregulated the fluorescent expression of TRIM13 in the prefrontal cortex of tree shrews, with blue fluorescence indicating DAPI, red fluorescence indicating TRIM13, and green fluorescence indicating MAP2. (D) METH downregulated the expression of TRIM13 in the primary cortical neurons of tree shrews in a dose-dependent manner. (E) METH downregulated the expression of TRIM13 in the primary cortical neurons of tree shrews in a time-dependent manner. (F) METH and HIV-1 Tat protein synergistically downregulated the expression of TRIM13 in the primary cortical neurons of tree shrews. (G-J) Pre-treatment of 4-PBA upregulated the fluorescent expression of TRIM13 in the primary cortical neurons of tree shrews, with blue fluorescence indicating DAPI and green fluorescence indicating TRIM13. (K) Pre-treatment of 4-PBA upregulated the expression of TRIM13 in the primary cortical neurons of tree shrews. All data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test; all values are presented as the mean ± SD, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, n≥3.\u003c/p\u003e","description":"","filename":"Figure.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4788696/v1/6c1449e0eb3647351ce26eef.png"},{"id":63018051,"identity":"60acd215-8955-40d9-8d32-b495dc22dad9","added_by":"auto","created_at":"2024-08-22 07:03:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":854733,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003esiRNA-TRIM13 alleviated the effect of METH and HIV-1 Tat protein synergistically induced autophagy\u003c/strong\u003e \u003cstrong\u003ein the primary cortical neurons of tree shrews.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) METH and HIV-1 Tat synergistically induced TRIM13 interacting with autophagy-related proteins (p62, Beclin1, and LC3II) in the primary cortical neurons of tree shrews. (B-C) Pre-treatment of siRNA-TRIM13 downregulated the expression of autophagy-related proteins (LC3II, Beclin1, ATG5, and ATG7), and upregulated the expression of p62 induced by METH and HIV-1 Tat protein in the primary cortical neurons of tree shrews. (D-E) Pre-treatment of siRNA-TRIM13 downregulated the fluorescent expression of LC3II, with blue fluorescence indicating DAPI and red fluorescence indicating LC3II in the primary cortical neurons of tree shrews.All data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test; all values are presented as the mean ± SD (n = 5), *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, n≥3.\u003c/p\u003e","description":"","filename":"Figure.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4788696/v1/a8b2bce5a575f8085396f3a7.png"},{"id":63017008,"identity":"697fbce8-7de0-4c14-b9a0-b493f3def9c1","added_by":"auto","created_at":"2024-08-22 06:55:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":562938,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of METH and HIV-1 Tat protein synergy in neuronal autophagy induction through TRIM13 modulation during endoplasmic reticulum stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMETH and HIV-1 Tat protein synergistically induce neuronal autophagy through the modulation of ERS. Additionally, the interaction between TRIM13 and autophagy is critical for this process.\u003c/p\u003e","description":"","filename":"Figure.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4788696/v1/50640ea45fe2f3d28b1bc978.png"},{"id":72641010,"identity":"4c8f0f4d-6959-44ae-89af-9f15575596c0","added_by":"auto","created_at":"2024-12-30 16:10:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6930062,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4788696/v1/2f1f31ea-156f-45f8-8322-b0b08ee57eed.pdf"},{"id":63017011,"identity":"08bd8357-ba54-4053-8000-9c30e2a58d4b","added_by":"auto","created_at":"2024-08-22 06:55:20","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16150,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Material: Table\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 1. Synergistic Effect Quantification of HIV-1 Tat Protein and METH in the Primary Cortical Neurons from Tree Shrews.\u003c/p\u003e\n\u003cp\u003eTable 2. Quantitative Analysis of Synergistic Interactions between HIV-1 Tat Protein and METH in SH-SY5Y Cells.\u003c/p\u003e\n\u003cp\u003eTable 3. Evaluation of Synergistic Effects of HIV-1 Tat Protein and METH in the Prefrontal Cortex of Tree Shrews.\u003c/p\u003e","description":"","filename":"Supplementarymaterialtable.docx","url":"https://assets-eu.researchsquare.com/files/rs-4788696/v1/e71fe98d227fe8503b7d1b65.docx"},{"id":63017022,"identity":"2f832a0f-6fd7-4374-a302-2308e1b73f00","added_by":"auto","created_at":"2024-08-22 06:55:22","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":806472,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig.1 METH and HIV-1 Tat protein synergistically induced autophagy and ERS in SH-SY5Y cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, C) METH and HIV-1 Tat protein synergistically upregulated the expression of autophagy-related proteins (ATG7, Beclin1, ATG5, and LC3II), and downregulated the expression of p62 in SH-SY5Y cells. (B, D) METH and HIV-1 Tat protein synergistically upregulated the expression of ERS-related proteins (PERK, p-PERK, IRE1, ATF6, and Bip) in SH-SY5Y cells. (E) METH and HIV-1 Tat protein synergistically upregulated the fluorescent expression of LC3II, with blue fluorescence indicating DAPI and red fluorescence indicating LC3II. All data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test; all values are presented as the mean ± SD, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, n≥3.\u003c/p\u003e","description":"","filename":"SupplementaryFigure.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4788696/v1/48fc8f0dfd2249e755b3a2fb.png"},{"id":63018565,"identity":"d03549dd-830b-40e5-b731-673e035c9bbf","added_by":"auto","created_at":"2024-08-22 07:11:20","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":726729,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 2 METH-induced endoplasmic reticulum stress in primary cortical neurons of tree shrews and SH-SY5Y Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, C) METH elevated the expression of ERS-related proteins (PERK, p-PERK, IRE1, ATF6, and Bip) in the primary cortical neurons of tree shrews in a dose-dependent manner. (B, D) METH elevated the expression of ERS-related proteins (PERK, p-PERK, IRE1, ATF6, and Bip) in the primary cortical neurons of tree shrews in a time-dependent manner. (E, G) METH elevated the expression of ERS-related proteins (PERK, p-PERK, IRE1, ATF6, and Bip) in SH-SY5Y cells in a dose-dependent manner. (F, H) METH elevated the expression of ERS-related proteins (PERK, p-PERK, IRE1, ATF6, and Bip) in SH-SY5Y cells in a time-dependent manner. All data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test; all values are presented as the mean ± SD, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, n≥3.\u003c/p\u003e","description":"","filename":"SupplementaryFigure.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4788696/v1/4f15a9152bb42d3e8b50b4ef.png"},{"id":63018052,"identity":"8d80b5f9-4ba5-4174-83b1-e978e9497c7e","added_by":"auto","created_at":"2024-08-22 07:03:20","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":780096,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig.3 Pre-treatment of 4-PBA alleviated ERS and autophagy induced by METH and HIV-1 Tat protein in SH-SY5Y cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-C) Pre-treatment of 4-PBA downregulated the expression of ERS-related proteins (PERK, p-PERK, IRE1, ATF6, and Bip) in SH-SY5Y cells. (B, D) Pre-treatment of 4-PBA downregulated the expression of autophagy-related proteins (ATG5, ATG7, Beclin1, and LC3II), and upregulated the expression of p62 in SH-SY5Y cells. (E) Pre-treatment of 4-PBA downregulated the fluorescent expression of LC3II in SH-SY5Y cells, with blue fluorescence indicating DAPI and red fluorescence indicating LC3II. All data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test; all values are presented as the mean ± SD, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, n≥3.\u003c/p\u003e","description":"","filename":"SupplementaryFigure.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4788696/v1/30e2986703085431793654a9.png"},{"id":63017024,"identity":"997c9ad4-9bb8-4212-8539-48a0becb3c6e","added_by":"auto","created_at":"2024-08-22 06:55:22","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":924707,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig.4\u003c/strong\u003e \u003cstrong\u003eMETH and HIV-1 Tat protein synergistically induced autophagy via ERS-Mediated TRIM13 in SH-SY5Y cell\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) METH downregulated the expression of TRIM13 in SH-SY5Y cells in a dose-dependent manner. (B) METH downregulated the expression of TRIM13 in SH-SY5Y cells in a time-dependent manner. (C) METH and HIV-1 Tat protein synergistically downregulated the expression of TRIM13 in SH-SY5Y cells. (D) Pre-treatment of 4-PBA upregulated the expression of TRIM13 in SH-SY5Y cells. (E, G) METH and HIV-1 Tat protein synergistically downregulated the fluorescent expression of TRIM13 in SH-SY5Y cells, with blue fluorescence indicating DAPI and green fluorescence indicating TRIM13. (F, H) Pre-treatment of 4-PBA upregulated the fluorescent expression of TRIM13 in SH-SY5Y cells, with blue fluorescence indicating DAPI and green fluorescence indicating TRIM13. (I) METH and HIV-1 Tat synergistically induced TRIM13 interacting with autophagy-related proteins (p62, Beclin1, and LC3II) in SH-SY5Y cells. (J) Pre-treatment of siRNA-TRIM13 downregulated the expression of autophagy-related proteins (LC3II, Beclin1, ATG5, and ATG7), and upregulated the expression of p62 induced by METH and HIV-1 Tat protein in SH-SY5Y cells. (K) Pre-treatment of siRNA-TRIM13 downregulated the fluorescent expression of LC3II in SH-SY5Y cells, with blue fluorescence indicating DAPI and red fluorescence indicating LC3II. All data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test; all values are presented as the mean ± SD, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, n≥3.\u003c/p\u003e","description":"","filename":"SupplementaryFigure.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4788696/v1/e3decda355a6acbfdd6d3e36.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Methamphetamine and HIV-1 Tat protein synergistically induce endoplasmic reticulum stress to promote TRIM13-mediated neuronal autophagy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAccording to the World Drug Report 2023, the global incidence of drug use is on the rise, with approximately 36\u0026nbsp;million individuals using amphetamine-type stimulants [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The relationship between drug use and HIV infection is particularly alarming, drug users are seven times more likely to contract HIV compared to the general population [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Moreover, substance abuse markedly elevates the risk of neurological symptoms and HIV-associated neurocognitive disorders (HAND) among those with HIV\u0026mdash;conditions that mutually exacerbate neurological harm [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. HAND and related neurological symptoms often manifest early in HIV infection, precipitated by the invasion of neurons by HIV-1-associated proteins. These proteins are secreted following the HIV-1 virus infecting T-cells, monocytes, and macrophages [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Among these, the HIV-1 Tat protein, a potent transcriptional activator released by infected cells, is critical for viral replication and is implicated as a significant pathogenetic agent in HAND development [\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn recent years, the mechanisms underlying cellular autophagy have received considerable attention. The accumulation of the HIV-1 Tat protein in brain tissues has been demonstrated to trigger autophagy in neuronal cells, which may lead to cell damage and subsequent neurotoxicity [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Our previous research has shown that METH-induced significant autophagy in SH-SY5Y cells, dopaminergic neurons, and microglia, with the presence of HIV-1 Tat protein intensifying this effect [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. While strides have been made in understanding autophagy induced by METH and HIV-1 Tat, the underlying mechanisms of neuronal autophagy caused by METH and HIV-1 Tat co-exposure remain unclear.\u003c/p\u003e \u003cp\u003eDisruptions of endoplasmic reticulum (ER) function induce abnormal accumulation of unfolded proteins within the ER lumen. This accumulation triggers endoplasmic reticulum stress (ERS), a cellular stress response associated with the ER [\u003cspan additionalcitationids=\"CR17 CR18 CR19\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In the central nervous system (CNS) of patients with HAND, persistent ERS activates autophagy, leading to cell death through excessive autophagy, exacerbating neurological damage [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Its E3 ubiquitin ligase activity characterizes the Tripartite Motif (TRIM) protein family and plays a pivotal role in various physiological processes, including autophagy, apoptosis, and inflammation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Growing evidence suggests that TRIM13 may act as a molecular link between ERS and autophagy [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, the effect of TRIM13 on neuronal autophagy caused by METH and HIV-1 Tat protein co-exposure remains unclear.\u003c/p\u003e \u003cp\u003eGiven their evolutionary proximity to non-human primates and similarities to humans in the nervous and immune systems, tree shrews have increasingly been utilized as a novel model for investigating the neurotoxic effects of METH and HIV-1 Tat protein [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In this study, we employ tree shrews, primary cortical neurons, and the SH-SY5Y cell line to examine the synergistic effects of METH and HIV-1 Tat protein co-exposure on neuronal autophagy and to elucidate the roles of ERS and TRIM13 in this process. This study aims to uncover the mechanisms underlying neurological damage caused by novel psychoactive substances and to explore potential strategies for preventing neurocognitive impairment in HIV-infected individuals with substance use disorders.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Reagents\u003c/h2\u003e \u003cp\u003eMETH was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). HIV-1 TAT Clade-B (#HIV-129-c) was purchased from Prospecbio (Rehovot, Israel). 4-PBA (HY-15654) was purchased from MCE (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cell culture\u003c/h2\u003e \u003cp\u003eThe neonatal tree shrews were supplied by the Tree Shrew Germplasm Resource Center at the Institute of Medical Biology, Chinese Academy of Medical Sciences \u0026amp; Peking Union Medical College, Kunming, China. The cerebral cortex of neonatal tree shrews was aseptically dissected after the removal of the meninges, olfactory bulb, cerebellum, and brainstem. The tissues were subsequently enzymatically digested with 0.25% trypsin-EDTA (Gibco, China) for 10 min. Digestion was stopped using DMEM medium (Biological Industries, Israel) containing 10% fetal bovine serum (Gibco, China), followed by filtration through a 70 \u0026micro;m cell strainer (Biosharp, China) and centrifugation for 8 min at 4\u0026deg;C. Cells were resuspended in a DMEM medium (Biological Industries, Israel) containing 10% FBS and 1% penicillin-streptomycin (Gibco, China). After 24 hours, the medium was replaced with Neurobasal\u0026trade;-A Medium (Gibco, China) containing 15% FBS (Gibco, China), 2% penicillin-streptomycin (Gibco, China), 1% glutamine (Gibco, China) and 2% B-27\u0026trade; Plus Supplement (Gibco, China). SH-SY5Y cells were cultured in DMEM F12 medium (Biological Industries, Israel) containing 15% FBS (Gibco, China) and 1% penicillin/streptomycin (Gibco, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Animals\u003c/h2\u003e \u003cp\u003eMale tree shrews, weighing 120\u0026ndash;190 g and aged 1 year old, were provided by the Tree Shrew Germplasm Resource Center at the Institute of Medical Biology, Chinese Academy of Medical Sciences \u0026amp; Peking Union Medical College in Kunming, China. The tree shrews were housed in a temperature-controlled room at 23\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C with a humidity level of 45\u0026ndash;55%. They were exposed to a 12-h light/dark cycle and provided ad libitum access to food and water. The tree shrews were randomly divided into various experimental groups. All procedures followed the guidelines established by the National Institutes of Health for the care and use of laboratory animals and received approval from the Experimental Animal Ethics Committee of Kunming Medical University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Animal models\u003c/h2\u003e \u003cp\u003e The tree shrews were randomly assigned to five experimental groups: saline, HIV-1 Tat protein, METH, METH\u0026thinsp;+\u0026thinsp;HIV-1 Tat protein, and 4-PBA pretreatment followed by METH and HIV-1 Tat protein. The saline group received intraperitoneal (i.p.) injections of an equal volume of saline for 10 days. The HIV-1 Tat protein group was administered 10 \u0026micro;g of HIV-1 Tat protein directly into the left lateral ventricle (coordinates relative to the fontanelle: anterior-posterior \u0026minus;\u0026thinsp;0.6 mm, medial-lateral\u0026thinsp;+\u0026thinsp;1.8 mm, dorsal-ventral \u0026minus;\u0026thinsp;4.0 mm). The METH group received METH at a dose of 2 mg/kg, i.p., for 10 days. The METH\u0026thinsp;+\u0026thinsp;HIV-1 Tat protein group was treated with both METH (2 mg/kg, i.p., for 10 days) and a single dose of 10 \u0026micro;g HIV-1 Tat protein into the left ventricle. Lastly, the 4-PBA\u0026thinsp;+\u0026thinsp;HIV-1 Tat protein\u0026thinsp;+\u0026thinsp;METH group underwent treatment with 136 mg/kg 4-PBA, i.p., 1 hour before the METH and HIV-1 Tat protein injections, with METH administered at 2 mg/kg, i.p., for 10 days, and the HIV-1 Tat protein administered as a single dose into the left ventricle. Once the tree shrews received the METH, they were immediately placed in the open-field apparatus. The tracks, total distance traveled, and average speed of tree shrews in the open-field apparatus were recorded using the VisuTrack system (Xinruan Information Technology Co., Ltd., China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Stereotypical behavior score\u003c/h2\u003e \u003cp\u003eAfter watching the video recordings made during the open-field tests and referring to the GHF-Dodd scoring method[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], stereotypic behavior scores were made as follows (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe scoring system of the stereotyped behavior\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eScore\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBehavior description\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStationary, little or no movement\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNormal movement accompanied by repeated exploration in situ or no fixed direction of repeated exploration\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRan fast around the open field, circled, climbed, and jumped repeatedly\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRepetitive movements of the head or tail, either up or down, directed toward one wall or corner of the chamber, experienced repeated convulsions and hiccups or sustained an arched position\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 siRNA Transfection\u003c/h2\u003e \u003cp\u003eThe primary cortical neurons from tree shrews and SH-SY5Y cells were subjected to siRNA transfection to knock down TRIM13 expression. The siRNAs targeting TRIM13 (LV3-TRIM13-1 sequence: 5'-TGCAGCTGATTTGTGGGATCT-3', LV3-TRIM13-2 sequence: 5'-ATGAAGAACTTTGATACCAGT-3', LV3-TRIM133 sequence: 5'-GCTCTTTCTCGCTTGGATACC-3') and a non-targeting control siRNA (NC-siRNA sequence: 5'-TTCTCCGAACGTGTCACGT-3') were acquired from Gima Genetics. For the transfection procedure, primary cortical neurons and SH-SY5Y cells were seeded at a density of 1 \u0026times; 10\u003csup\u003e^\u003c/sup\u003e5 cells/well in six-well plates and allowed to attach overnight at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThe following day, the cells were transfected with TRIM13-siRNAs and NC-siRNA at a concentration of 120 pmol/ml using Lipofectamine\u0026reg; 2000 reagent (5 \u0026micro;l/ml) in Opti-MEM\u0026reg; I Reduced Serum Medium. This mixture was incubated with the cells for 24 hours. Post-transfection, the medium containing the viral particles was removed, and cells were replenished with complete growth medium before being returned to the incubator set at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Subsequently, the transfected cells were harvested for the subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Western Blot\u003c/h2\u003e \u003cp\u003eThe total proteins of the primary neurons from tree shrews, SH-SY5Y cells, and the tree shrew's prefrontal cortex were treated with RIPA buffer containing protease inhibitors. The protein concentration of the samples was measured using the BCA Protein Kit (Beyotime, China). Subsequently, the sample proteins were separated by SDS-PAGE and transferred to a PVDF membrane using a transfer device (BioRed, USA). After blocking the membranes with 5% skim milk at room temperature for 2 hours, the membranes were subsequently incubated overnight at 4℃ with the following primary antibodies: mouse anti-β-actin (1:2000, Proteintech, China), LC3Ⅱ, ATG7, ATG5, Beclin1, p62, p-PERK, ATF6, IRE1, Bip (1:1000, Cell Signaling Technology, USA), PERK and TRIM13 (1:100, Santa Cruz Biotechnology, USA),.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Immunofluorescence\u003c/h2\u003e \u003cp\u003eThe cells and brain slices were fixed using 4% paraformaldehyde for 1 hour at room temperature. After fixation, permeabilization was performed with a 0.3% Triton X-100 solution for 20 minutes at room temperature. To block non-specific binding, the wells were incubated with 10% goat serum for cells and 20% goat serum for brain slices for 2 hours at room temperature. The antibodies (LC3 II, TRIM13, and MAP2) were incubated overnight at 4℃ with each sample. The next day, fluorescent secondary antibodies were used and incubated at 37℃ for 2 hours. Leica software was utilized for image capture. The digitized images were then quantitatively analyzed and processed using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Immunoprecipitation\u003c/h2\u003e \u003cp\u003eThe cellular proteins were treated in lysis buffer containing protease and phosphatase inhibitors for 30 min, the supernatant was collected and 50 \u0026micro;l of the supernatant was taken as input group. The specific antibody and IgG were incubated with the protein A/G beads for 2 hours at room temperature. Antibody-encapsulated beads were washed to remove unbound antibodies. These prepared beads are then added to the lysate (excluding the reserved input sample) and incubated overnight at 4℃. The next day the beads were thoroughly washed to remove non-specifically bound proteins. Bound protein complexes were then eluted by adding 1\u0026times; SDS-PAGE sample loading buffer and heating at 95℃ for 5 minutes. Finally, the beads are removed by centrifugation and the supernatant is collected for subsequent Western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll the statistical analyses were performed using GraphPad Prism 6.02 (GraphPad Software, USA). One-way or two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests was performed for comparison among multiple groups and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. To assess the potential synergistic interaction between the HIV-1 Tat protein and methamphetamine (METH), the Bliss Independence model was applied. Prior to applying the Bliss Independence calculation, Max-Min Normalization was performed to standardize the data, utilizing the formula:\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{z}\\text{i}=\\frac{\\text{x}\\text{i}-\\text{M}\\text{i}\\text{n}\\left(\\text{X}\\right)}{\\text{M}\\text{a}\\text{x}\\left(\\text{X}\\right)-\\text{M}\\text{i}\\text{n}\\left(\\text{X}\\right)}\\)\u003c/span\u003e\u003c/span\u003e. The individual effects of METH and the HIV-1 Tat protein are denoted as E\u003csub\u003eA\u003c/sub\u003e and E\u003csub\u003eB\u003c/sub\u003e, respectively, while the combined effect of both is represented by E\u003csub\u003eAB\u003c/sub\u003e. The interaction between the two is quantified using the formula: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{{E}_{A}+{E}_{B}-\\left({E}_{A}\\times\\:{E}_{B}\\right)}{{E}_{AB}}\\)\u003c/span\u003e\u003c/span\u003e. An outcome greater than 1 suggests antagonism, equal to 1 indicates additivity, and less than 1 is indicative of synergy.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 METH and HIV-1 Tat protein synergistically induced autophagy in Neurons\u003c/h2\u003e \u003cp\u003eThe primary cortical neurons from tree shrews and SH-SY5Y cells were exposed to METH (0.5mM or 2mM) or/and HIV-1 Tat protein (50nM or 100nM) for 24 hours [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Both METH and HIV-1 Tat protein treatments upregulated the expression of autophagy-related proteins (ATG5, ATG7, Beclin1, and LC3II) while downregulating the expression of p62 compared to the control group. Notably, these changes were further amplified when cells were co-exposed to METH and HIV-1 Tat protein, indicating a synergistic effect in provoking neuronal autophagy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B, and Supplementary Fig.\u0026nbsp;1A, C). This was further confirmed using the Bliss independence model. The combination index (CI) for the expression of autophagy-related proteins was all less than 1, suggesting a synergistic interaction (Supplementary Tables\u0026nbsp;1 and 2). Immunofluorescence (IF) staining revealed that co-exposure to METH and HIV-1 Tat significantly enhanced the fluorescence intensity of LC3II in both cell types compared to the groups exposed solely METH or HIV-1 Tat (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI and Supplementary Fig.\u0026nbsp;1E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 METH and HIV-1 Tat Protein Synergistically Induced ERS in Neurons\u003c/h2\u003e \u003cp\u003eTo further understand the effects of METH on the expression of ERS-related proteins, both types of cells were treated with varying concentrations of METH for different durations. The expression of ERS markers (p-PERK, ATF6, IRE1, and Bip) was assessed using the western blot. The data revealed that METH upregulated the expression of these ERS-related proteins in a dose and time-dependent manner (Supplementary Fig.\u0026nbsp;2A-H).\u003c/p\u003e \u003cp\u003eCompared to the control group, the ERS-related proteins in primary cortical neurons and SH-SY5Y cells were upregulated after solely METH or HIV-1 Tat protein exposure. Notably, this upregulation was further accentuated following co-exposure to METH and HIV-1 Tat protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D and Supplementary Fig.\u0026nbsp;1B, D). The synergistic effect of METH and HIV-1 Tat protein on ERS was quantitatively confirmed by the CI values (Supplementary Table\u0026nbsp;1 and Table\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 4-PBA pre-treatment reduced ERS and Autophagy Induced by METH and HIV-1 Tat Protein in Neurons\u003c/h2\u003e \u003cp\u003eIn neurons that co-exposure to METH and HIV-1 Tat protein, 4-PBA pre-treatment significantly downregulated the expression of ERS-associated proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, F and Supplementary Fig.\u0026nbsp;3A, C). In addition, co-exposure to METH and HIV-1 Tat protein upregulated the expression of autophagy-related proteins and downregulated the p62 expression, which was partially reversed by 4-PBA pre-treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, H and Supplementary Fig.\u0026nbsp;3B, D). IF analysis further confirmed decreased expression of LC3II following 4-PBA pre-treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ and Supplementary Fig.\u0026nbsp;3E).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4 METH and HIV-1 Tat protein Synergistically aggravated excitotoxicity and stereotypic behaviors in tree shrews via the modulation of ERS\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe protocol for the open field test was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. The tracks of the tree shrews in the open field were depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. Compared to the Saline, HIV-1 Tat, or METH groups, tree shrews co-exposed to METH and HIV-1 Tat protein showed a significant increase in total distance traveled and average speed in the open field (Day1-Day10). However, pre-treatment of tree shrews with 4-PBA effectively reduced the total distance traveled (Day 5-Day 10) and average speed (Day 3, Day 7-Day 10) in the open field (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). In addition, the stereotypic behaviors of tree shrews were also analyzed following METH injection. Compared to the saline group, tree shrews exposed to METH displayed obvious spontaneous stereotypic activities, including irritability, exploring forward, repeatedly shaking their heads, curling their tails into a semicircle, hiccups, and decreased defecation. Importantly, tree shrews co-exposed to METH and HIV-1 Tat protein presented more severe spontaneous stereotypic activities compared to the solely METH-exposed group. In contrast, pretreatment of tree shrews with 4-PBA decreased spontaneous stereotypic activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), suggesting that METH and HIV-1 Tat protein Synergistically aggravated excitotoxicity and stereotypic behaviors in tree shrews via the modulation of ERS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.5 METH and HIV-1 Tat Protein Synergistically Induced Autophagy and ERS in the Prefrontal Cortex of Tree Shrew\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCompared to the saline group, tree shrews exposed to solely METH or HIV-1 Tat protein showed the upregulated expression of autophagy-related proteins in the prefrontal cortex, along with a notable reduction in p62 expression. As expected, these effects were exacerbated in the group co-exposed to METH and HIV-1 Tat treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B and Supplementary Table\u0026nbsp;3). Likewise, exposure to METH or HIV-1 Tat protein also upregulated the expression of ERS-related proteins in the prefrontal cortex of tree shrews. This upregulation was further augmented in tree shrews co-exposed to METH and HIV-1 Tat protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D and Supplementary Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate whether ERS contributes to autophagy in this context, tree shrews were pretreated with 4-PBA before co-exposure to METH and HIV-1 Tat protein. The results demonstrated that 4-PBA pretreatment rescued the upregulation of ERS-related and autophagy-related proteins caused by METH and HIV-1 Tat protein co-exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-H). Moreover, co-exposure to METH and HIV-1 Tat protein significantly increased the fluorescence intensity of LC3 II in the prefrontal cortex of tree shrews, while pre-treatment of 4-PBA reversed this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.6 METH and HIV-1 Tat Protein Synergistically Decreased the Expression of TRIM13 by Modulating ERS\u003c/h2\u003e \u003cp\u003eCo-exposure to METH and HIV-1 Tat protein further downregulated the protein and fluorescence intensity of TRIM13 in the prefrontal cortex compared to solely METH or HIV-1 Tat protein exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, C). Pretreatment of 4-PBA rescued the downregulation of TRIM13 in the prefrontal cortex of tree shrews (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. B, C). In primary cortical neurons and SH-SY5Y cell lines, a dose- and time-dependent downregulation in TRIM13 expression was observed with METH exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E and Supplementary Fig.\u0026nbsp;4A, B). Notably, co-exposure to METH and HIV-1 Tat protein resulted in a more significant reduction in expression compared to either agent alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF and Supplementary Fig.\u0026nbsp;4C). IF staining further confirmed that the fluorescence intensity of TRIM13 significantly diminished in both types of cells following METH and/or HIV-1 Tat exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, H and Supplementary Fig.\u0026nbsp;4E, G). Importantly, pretreatment of 4-PBA reversed the downregulation of TRIM13 protein and fluorescence intensity caused by co-exposure to METH and HIV-1 Tat protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI, J, and K and Supplementary Fig.\u0026nbsp;4D, F and H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.7 METH and HIV-1 Tat Protein Synergistically Induced Autophagy Via ERS-Mediated TRIM13\u003c/h2\u003e \u003cp\u003ePretreatment with the ERS inhibitor 4-PBA reduced METH and HIV-1 Tat protein synergistically induced neuronal autophagy in both in vivo and in vitro models. TRIM13, a protein involved in maintaining endoplasmic reticulum homeostasis, may play a crucial role in neuronal autophagy. Co-immunoprecipitation (IP) analyses revealed that co-exposure to METH and HIV-1 Tat protein intensified the interaction between TRIM13 and key autophagy-related proteins, including p62, LC3II, and Beclin1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and Supplementary Fig.\u0026nbsp;4I). Pre-treatment with siRNA-TRIM13 significantly downregulated the expression of autophagy markers (ATG5, ATG7, Beclin1, and LC3II), while upregulating the p62 expression in the primary cortical neurons and SH-SY5Y cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C and Supplementary Fig.\u0026nbsp;4J). Additionally, IF staining confirmed that the pretreatment of TRIM13 significantly attenuated the METH and HIV-1 Tat co-exposure induced increase in LC3II protein fluorescence intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E and Supplementary Fig.\u0026nbsp;4.K).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we found that co-exposure to METH and HIV-1 Tat proteins markedly upregulated the expression of proteins related to ERS and autophagy, as well as increased the excitotoxicity and stereotypic behaviors in tree shrews. Pretreatment with the ERS-inhibitor 4-PBA reduced excitotoxicity, stereotypic behaviors, and autophagy induced by co-exposure to METH and HIV-1 Tat proteins. Furthermore, METH and HIV-1 Tat proteins synergistically downregulated TRIM13 expression, which could be rescued by 4-PBA pretreatment. Notably, the interaction between TRIM13 and autophagy-related proteins was intensified after co-exposure to METH and HIV-1 Tat proteins. Silencing of the \u003cem\u003eTRIM13\u003c/em\u003e gene effectively attenuated METH and HIV-1 Tat synergistically induced neuronal autophagy. Our study demonstrated that METH and HIV-1 Tat protein synergistically induced neuronal autophagy through ERS-mediated alteration of TRIM13.\u003c/p\u003e \u003cp\u003eER is crucial in protein folding and translation, lipid synthesis, and Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis, supported by its extensive membrane network and myriad associated enzymes [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Cells exposed to METH initiate ER stress by disrupting these normal ER functions [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Specifically, ER stress disturbs protein synthesis and folding within the ER, leading to the degradation of misfolded ER proteins and the activation of various transcription factors. Moreover, Rong et al. found that the HIV-1 Tat protein induces apoptosis in human brain microvascular endothelial cells (HBMECs) by inducing ER stress [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These findings are consistent with our observations, suggesting that ER stress contributes to the neurotoxicity induced by either HIV or METH. We also noted that co-exposure to METH and HIV-1 Tat proteins significantly increased the expression of ER stress-related proteins, indicating that ER stress may play a vital role in the synergistic neurotoxicity of METH and HIV-1 Tat proteins.\u003c/p\u003e \u003cp\u003eERS and autophagy are cellular adaptive responses to various stimuli, but severe ERS may induce excessive autophagy, leading to apoptosis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Research involving drugs and HIV has highlighted the pivotal role of ERS pathways in autophagy regulation [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In this study, we found that co-exposure to METH and HIV-1 Tat protein-induced neuronal autophagy, which is consistent with the increased ERS. However, the relationship between neuronal ERS and autophagy caused by the co-exposure to METH and HIV-1 Tat protein remains poorly understood. 4-PBA is commonly employed as an ER stress inhibitor due to its aids in protein post-translational modifications and folding, thereby attenuating ERS-mediated neuronal death [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Pretreatment with 4-PBA alleviated METH and HIV-1 Tat protein synergistically induced neuronal autophagy, suggesting that ERS may act as a precursor in the METH and HIV-1 Tat protein-induced neuronal autophagy.\u003c/p\u003e \u003cp\u003eGlutamate is the primary excitatory neurotransmitter in the mammalian central nervous system, and overactivation of its receptors constitutes a significant pathway for neuronal excitotoxicity, whereas calcium inward flow is critical for glutamate excitotoxicity[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The endoplasmic reticulum acts as an extensive network regulating Ca\u003csup\u003e2+\u003c/sup\u003e, thus its stability is critical for maintaining Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In our study, METH and HIV-1 Tat protein synergistically induced excitotoxicity and stereotypic behaviors in tree shrews. Pretreatment with 4-PBA alleviated these effects, suggesting that METH HIV-1 Tat protein synergistically induced excitotoxicity and stereotypic behaviors in tree shrews via modulation of ERS. The ERS-mediated calcium dysregulation and glutamatergic excitotoxicity may be involved in this process, although additional evidence is needed to confirm this potential mechanism.\u003c/p\u003e \u003cp\u003ePretreatment with 4-PBA rescued downregulation of TRIM13 induced by METH and HIV-1 Tat protein co-exposure, indicating potential links between TRIM13 and ER homeostasis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. TRIM13 may function as an essential initiator of autophagy, serving as an ER transmembrane receptor. TRIM13 facilitates autophagy through k63-linked ubiquitination, recruiting p62 and interacting with LC3 on the autophagic membrane to catalyze the formation of autophagic vesicles [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In our study, immunoprecipitation analysis also revealed the interactions between TRIM13 and autophagy makers, including Beclin1, p62, and LC3II. Furthermore, silencing of the \u003cem\u003eTRIM13\u003c/em\u003e gene reduced neuronal autophagy induced by METH and HIV-1 Tat protein co-exposure. These results suggest that TRIM13 may play a role in assembling an autophagic complex on the ER surface, facilitating the recruitment of autophagy-related proteins such as Beclin1, LC3, and p62.\u003c/p\u003e \u003cp\u003eSilencing the \u003cem\u003eTRIM13\u003c/em\u003e gene impairs cells\u0026rsquo; ability to properly synthesize proteins and perform subsequent ubiquitination processes, ultimately resulting in a deficiency of necessary components for establishing an autophagic platform. This deficiency may result in improper synthesis of intracellular autophagic vesicles [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In our experiments, we observed that TRIM13 did not interact with ATG7 and ATG5, but both proteins experienced a significant reduction in expression following TRIM13 silencing. This observation suggests that while ATG7 and ATG5 do not directly bind to TRIM13, they are nonetheless crucial for the formation of autophagic vesicles, particularly in the lipidation of LC3I to LC3II, a critical step in autophagosome maturation [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Although this hypothesis is plausible, further experimental verification is needed to confirm this interaction. Together, TRIM13 provides new insights into understanding the synergistic mechanisms of METH and HIV-induced damage to the central nervous system.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eOur study demonstrates that METH and HIV-1 Tat protein synergistically induce neuronal autophagy through the modulation of ERS. Additionally, we have uncovered that the interaction between TRIM13 and autophagy is critical for this process (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In conclusion, our study provides novel perspectives on the mechanisms underlying the synergistic effects of METH and HIV-1 Tat protein on neuronal autophagy and identifies potential targets for the prevention of METH abuse in HIV-infected individuals.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWang C. contributed to the conceptualization, methodology, data analysis, and writing the original manuscript; Yang G.M., Huang J., and Tian Y.Q. designed and performed the experiments. Chi-Kwan L. and Miao L. contributed to writing\u0026mdash;review, and editing; Wang H.W., Li Y., Huang Y.Z., and Teng H.X. contributed to data analysis and validation; Liu L., Li J., and Zeng X.F. supervised data analysis, and revised manuscript. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (81960340, 82060382, and 82160325) and the Yunnan Applied Basic Research Projects Joint Special Project (202201AY070001-020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used to support the findings of this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures involving animals were carried out in strict accordance with the guidelines set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Institutional Animal Care and Use Committee of Kunming Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWorld Drug Report 2023. In: U. N. Off. Drugs Crime. //www.unodc.org/unodc/en/data-and-analysis/world-drug-report-2023.html. Accessed 16 Nov 2023\u003c/li\u003e\n\u003cli\u003eUNAIDS leads the world\u0026rsquo;s most extensive data collection on HIV epidemiology, programme coverage and finance | UNAIDS. https://www.unaids.org/en/topic/data. Accessed 25 Sep 2023\u003c/li\u003e\n\u003cli\u003eBlackard JT, Sherman KE (2021) Drugs of Abuse and Their Impact on Viral Pathogenesis. Viruses 13:2387. https://doi.org/10.3390/v13122387\u003c/li\u003e\n\u003cli\u003eWilliams ME, Zulu SS, Stein DJ, et al (2020) Signatures of HIV-1 subtype B and C Tat proteins and their effects in the neuropathogenesis of HIV-associated neurocognitive impairments. Neurobiol Dis 136:104701. https://doi.org/10.1016/j.nbd.2019.104701\u003c/li\u003e\n\u003cli\u003eRudd H, Toborek M (2022) Pitfalls of Antiretroviral Therapy: Current Status and Long-Term CNS Toxicity. Biomolecules 12:894. https://doi.org/10.3390/biom12070894\u003c/li\u003e\n\u003cli\u003eSpector C, Mele AR, Wigdahl B, Nonnemacher MR (2019) Genetic variation and function of the HIV-1 Tat protein. Med Microbiol Immunol (Berl) 208:131\u0026ndash;169. https://doi.org/10.1007/s00430-019-00583-z\u003c/li\u003e\n\u003cli\u003eKipp AM, Desruisseau AJ, Qian H-Z (2011) Non-injection Drug Use and HIV Disease Progression in the Era of Combination Antiretroviral Therapy. J Subst Abuse Treat 40:386\u0026ndash;396. https://doi.org/10.1016/j.jsat.2011.01.001\u003c/li\u003e\n\u003cli\u003eFriedman H, Pross S, Klein TW (2006) Addictive drugs and their relationship with infectious diseases. FEMS Immunol Med Microbiol 47:330\u0026ndash;342. https://doi.org/10.1111/j.1574-695X.2006.00097.x\u003c/li\u003e\n\u003cli\u003eLink RW, Mele AR, Antell GC, et al (2019) Investigating the distribution of HIV-1 Tat lengths present in the Drexel Medicine CARES cohort. Virus Res 272:197727. https://doi.org/10.1016/j.virusres.2019.197727\u003c/li\u003e\n\u003cli\u003eYe X, Zhang Y, Xu Q, et al (2017) HIV-1 Tat inhibits EAAT-2 through AEG-1 upregulation in models of HIV-associated neurocognitive disorder. Oncotarget 8:39922\u0026ndash;39934. https://doi.org/10.18632/oncotarget.16485\u003c/li\u003e\n\u003cli\u003eLapierre J, Karuppan MKM, Perry M, et al (2022) Different Roles of Beclin1 in the Interaction Between Glia and Neurons after Exposure to Morphine and the HIV- Trans-Activator of Transcription (Tat) Protein. J Neuroimmune Pharmacol Off J Soc NeuroImmune Pharmacol 17:470\u0026ndash;486. https://doi.org/10.1007/s11481-021-10017-4\u003c/li\u003e\n\u003cli\u003eHalcrow PW, Kumar N, Quansah DNK, et al (2022) Endolysosome Iron Chelation Inhibits HIV-1 Protein-Induced Endolysosome De-Acidification-Induced Increases in Mitochondrial Fragmentation, Mitophagy, and Cell Death. Cells 11:1811. https://doi.org/10.3390/cells11111811\u003c/li\u003e\n\u003cli\u003eTeodorof-Diedrich C, Spector SA (2020) Human Immunodeficiency Virus Type 1 and Methamphetamine-Mediated Mitochondrial Damage and Neuronal Degeneration in Human Neurons. J Virol 94:e00924-20. https://doi.org/10.1128/JVI.00924-20\u003c/li\u003e\n\u003cli\u003eYang G, Li J, Leung C-K, et al (2022) Methamphetamine and HIV-1 Tat proteins synergistically induce microglial autophagy via activation of the Nrf2/NQO1/HO-1 signal pathway. Neuropharmacology 220:109256. https://doi.org/10.1016/j.neuropharm.2022.109256\u003c/li\u003e\n\u003cli\u003eZeng X-F, Li Q, Li J, et al (2018) HIV-1 Tat and methamphetamine co-induced oxidative cellular injury is mitigated by N-acetylcysteine amide (NACA) through rectifying mTOR signaling. Toxicol Lett 299:159\u0026ndash;171. https://doi.org/10.1016/j.toxlet.2018.09.009\u003c/li\u003e\n\u003cli\u003eNakka VP, Prakash-Babu P, Vemuganti R (2016) Crosstalk Between Endoplasmic Reticulum Stress, Oxidative Stress, and Autophagy: Potential Therapeutic Targets for Acute CNS Injuries. Mol Neurobiol 53:532\u0026ndash;544. https://doi.org/10.1007/s12035-014-9029-6\u003c/li\u003e\n\u003cli\u003eShah A, Kumar A (2016) Methamphetamine-mediated endoplasmic reticulum (ER) stress induces type-1 programmed cell death in astrocytes via ATF6, IRE1\u0026alpha; and PERK pathways. Oncotarget 7:46100\u0026ndash;46119. https://doi.org/10.18632/oncotarget.10025\u003c/li\u003e\n\u003cli\u003eQie X, Wen D, Guo H, et al (2017) Endoplasmic Reticulum Stress Mediates Methamphetamine-Induced Blood\u0026ndash;Brain Barrier Damage. Front Pharmacol 8:639. https://doi.org/10.3389/fphar.2017.00639\u003c/li\u003e\n\u003cli\u003eYang L, Guo N, Fan W, et al (2020) Thioredoxin-1 blocks methamphetamine-induced injury in brain through inhibiting endoplasmic reticulum and mitochondria-mediated apoptosis in mice. Neurotoxicology 78:163\u0026ndash;169. https://doi.org/10.1016/j.neuro.2020.03.006\u003c/li\u003e\n\u003cli\u003eWongprayoon P, Govitrapong P (2017) Melatonin Protects SH-SY5Y Neuronal Cells Against Methamphetamine-Induced Endoplasmic Reticulum Stress and Apoptotic Cell Death. Neurotox Res 31:1\u0026ndash;10. https://doi.org/10.1007/s12640-016-9647-z\u003c/li\u003e\n\u003cli\u003eAndhavarapu S, Katuri A, Bryant J, et al (2020) Intersecting roles of ER stress, mitochondrial dysfunction, autophagy, and calcium homeostasis in HIV-associated neurocognitive disorder. J Neurovirol 26:664\u0026ndash;675. https://doi.org/10.1007/s13365-020-00861-0\u003c/li\u003e\n\u003cli\u003eChen X, Zhang T, Zhang Y (2020) Endoplasmic reticulum stress and autophagy in HIV-1-associated neurocognitive disorders. J Neurovirol 26:824\u0026ndash;833. https://doi.org/10.1007/s13365-020-00906-4\u003c/li\u003e\n\u003cli\u003eHatakeyama S (2017) TRIM Family Proteins: Roles in Autophagy, Immunity, and Carcinogenesis. Trends Biochem Sci 42:297\u0026ndash;311. https://doi.org/10.1016/j.tibs.2017.01.002\u003c/li\u003e\n\u003cli\u003eWatanabe M, Hatakeyama S (2017) TRIM proteins and diseases. J Biochem (Tokyo) 161:135\u0026ndash;144. https://doi.org/10.1093/jb/mvw087\u003c/li\u003e\n\u003cli\u003eTomar D, Prajapati P, Sripada L, et al (2013) TRIM13 regulates caspase-8 ubiquitination, translocation to autophagosomes and activation during ER stress induced cell death. Biochim Biophys Acta BBA - Mol Cell Res 1833:3134\u0026ndash;3144. https://doi.org/10.1016/j.bbamcr.2013.08.021\u003c/li\u003e\n\u003cli\u003eFan Y, Peng X, Li B, Zhao G (2020) Development of Autophagy Signature-Based Prognostic Nomogram for Refined Glioma Survival Prognostication. BioMed Res Int 2020:1872962. https://doi.org/10.1155/2020/1872962\u003c/li\u003e\n\u003cli\u003eLi J, Wang W, Tong P, et al (2018) Autophagy Induction by HIV-Tat and Methamphetamine in Primary Midbrain Neuronal Cells of Tree Shrews via the mTOR Signaling and ATG5/ATG7 Pathway. Front Neurosci 12:921. https://doi.org/10.3389/fnins.2018.00921\u003c/li\u003e\n\u003cli\u003eHuang J, Yang G, Li Z, et al (2020) Involvement of dopamine D3 receptor and dopamine transporter in methamphetamine-induced behavioral sensitization in tree shrews. Brain Behav 10:e01533. https://doi.org/10.1002/brb3.1533\u003c/li\u003e\n\u003cli\u003eHuang J, Zhang R, Wang S, et al (2021) Methamphetamine and HIV-Tat Protein Synergistically Induce Oxidative Stress and Blood-Brain Barrier Damage via Transient Receptor Potential Melastatin 2 Channel. Front Pharmacol 12:619436. https://doi.org/10.3389/fphar.2021.619436\u003c/li\u003e\n\u003cli\u003eHuang J, Yang G, Li Z, et al (2020) Involvement of dopamine D3 receptor and dopamine transporter in methamphetamine-induced behavioral sensitization in tree shrews. Brain Behav 10:e01533. https://doi.org/10.1002/brb3.1533\u003c/li\u003e\n\u003cli\u003eXie X-L, Zhou W-T, Zhang K-K, et al (2018) METH-Induced Neurotoxicity Is Alleviated by Lactulose Pretreatment Through Suppressing Oxidative Stress and Neuroinflammation in Rat Striatum. Front Neurosci 12:802. https://doi.org/10.3389/fnins.2018.00802\u003c/li\u003e\n\u003cli\u003eSchwarz DS, Blower MD (2016) The endoplasmic reticulum: structure, function and response to cellular signaling. Cell Mol Life Sci CMLS 73:79\u0026ndash;94. https://doi.org/10.1007/s00018-015-2052-6\u003c/li\u003e\n\u003cli\u003eLee H-S, Kim E-N, Jeong G-S (2021) Aromadendrin Protects Neuronal Cells from Methamphetamine-Induced Neurotoxicity by Regulating Endoplasmic Reticulum Stress and PI3K/Akt/mTOR Signaling Pathway. Int J Mol Sci 22:2274. https://doi.org/10.3390/ijms22052274\u003c/li\u003e\n\u003cli\u003eMa R, Yang L, Niu F, Buch S (2016) HIV Tat-Mediated Induction of Human Brain Microvascular Endothelial Cell Apoptosis Involves Endoplasmic Reticulum Stress and Mitochondrial Dysfunction. Mol Neurobiol 53:132\u0026ndash;142. https://doi.org/10.1007/s12035-014-8991-3\u003c/li\u003e\n\u003cli\u003eMehrbod P, Ande SR, Alizadeh J, et al (2019) The roles of apoptosis, autophagy and unfolded protein response in arbovirus, influenza virus, and HIV infections. Virulence 10:376\u0026ndash;413. https://doi.org/10.1080/21505594.2019.1605803\u003c/li\u003e\n\u003cli\u003eHuang R, Zhang Y, Han B, et al (2017) Circular RNA HIPK2 regulates astrocyte activation via cooperation of autophagy and ER stress by targeting MIR124\u0026ndash;2HG. Autophagy 13:1722\u0026ndash;1741. https://doi.org/10.1080/15548627.2017.1356975\u003c/li\u003e\n\u003cli\u003eGuo M-L, Liao K, Periyasamy P, et al (2015) Cocaine-mediated microglial activation involves the ER stress-autophagy axis. Autophagy 11:995\u0026ndash;1009. https://doi.org/10.1080/15548627.2015.1052205\u003c/li\u003e\n\u003cli\u003eSil S, Niu F, Tom E, et al (2019) Cocaine mediated neuroinflammation: Role of dysregulated autophagy in pericytes. Mol Neurobiol 56:3576\u0026ndash;3590. https://doi.org/10.1007/s12035-018-1325-0\u003c/li\u003e\n\u003cli\u003eDossat AM, Trychta KA, Glotfelty EJ, et al (2024) Excitotoxic glutamate levels cause the secretion of resident endoplasmic reticulum proteins. J Neurochem. https://doi.org/10.1111/jnc.16093\u003c/li\u003e\n\u003cli\u003eFitting S, Knapp PE, Zou S, et al (2014) Interactive HIV-1 Tat and morphine-induced synaptodendritic injury is triggered through focal disruptions in Na\u003csup\u003e+\u003c/sup\u003e influx, mitochondrial instability, and Ca\u003csup\u003e2+\u003c/sup\u003e overload. J Neurosci Off J Soc Neurosci 34:12850\u0026ndash;12864. https://doi.org/10.1523/JNEUROSCI.5351-13.2014\u003c/li\u003e\n\u003cli\u003eD T, R S, Ak S, et al (2012) TRIM13 regulates ER stress induced autophagy and clonogenic ability of the cells. Biochim Biophys Acta 1823:. https://doi.org/10.1016/j.bbamcr.2011.11.015\u003c/li\u003e\n\u003cli\u003eTomar D, Singh R (2014) TRIM13 regulates ubiquitination and turnover of NEMO to suppress TNF induced NF-\u0026kappa;B activation. Cell Signal 26:2606\u0026ndash;2613. https://doi.org/10.1016/j.cellsig.2014.08.008\u003c/li\u003e\n\u003cli\u003eYu B, Zhou Y, He J (2023) TRIM13 inhibits cell proliferation and induces autophagy in lung adenocarcinoma by regulating KEAP1/NRF2 pathway. Cell Cycle Georget Tex 22:1496\u0026ndash;1513. https://doi.org/10.1080/15384101.2023.2216504\u003c/li\u003e\n\u003cli\u003eJi CH, Kim HY, Heo AJ, et al (2019) The N-Degron Pathway Mediates ER-phagy. Mol Cell 75:1058-1072.e9. https://doi.org/10.1016/j.molcel.2019.06.028\u003c/li\u003e\n\u003cli\u003eJi CH, Kim HY, Heo AJ, et al (2020) Regulation of reticulophagy by the N-degron pathway. Autophagy 16:373\u0026ndash;375. https://doi.org/10.1080/15548627.2019.1695402\u003c/li\u003e\n\u003cli\u003eYu B, Zhou Y, He J (2023) TRIM13 inhibits cell proliferation and induces autophagy in lung adenocarcinoma by regulating KEAP1/NRF2 pathway. Cell Cycle Georget Tex 22:1496\u0026ndash;1513. https://doi.org/10.1080/15384101.2023.2216504\u003c/li\u003e\n\u003cli\u003eUrbańska K, Orzechowski A (2021) The Secrets of Alternative Autophagy. Cells 10:3241. https://doi.org/10.3390/cells10113241\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"methamphetamine, HIV-1 Tat, endoplasmic reticulum stress, autophagy, TRIM13","lastPublishedDoi":"10.21203/rs.3.rs-4788696/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4788696/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCo-exposure to methamphetamine (METH) abuse and HIV infection exacerbates central nervous system damage. However, the underlying mechanisms of this process remain poorly understood. This study aims to explore the roles of neuronal autophagy in the synergistic damage to the central nervous system caused by METH and HIV proteins. Models of METH and HIV-1 Tat protein co-exposure were established using tree shrews, primary neurons, and SH-SY5Y cells. Co-exposure to METH and HIV-1 Tat protein significantly increased the distance traveled, mean velocity, and stereotyped behaviors of tree shrews in the open field test. Western blot analysis revealed that Co-exposure to METH and HIV-1 Tat protein markedly increased the expression of endoplasmic reticulum stress (ERS)-associated proteins (p-ERK, IRE1, ATF6, and Bip) and autophagy markers (ATG7, ATG5, Beclin1, and LC3II). Conversely, Co-exposure to METH and HIV-1 Tat protein significantly downregulated the expressions of p62 and TRIM13. Immunofluorescence staining demonstrated that Pre-treatment with the ERS inhibitor 4-PBA or TRIM13-siRNA rescued the abnormal behaviors induced by METH and HIV-1 Tat protein co-exposure in tree shrews and restored the expression of ERS-related and autophagy-related proteins. Additionally, TRIM13 was found to interact with autophagy-related proteins, including p62, Beclin1, and LC3II by immunoprecipitation assays. Our findings suggest for the first time that METH and HIV-1 Tat protein synergistically induce neuronal autophagy through ERS pathways, with TRIM13 playing a pivotal regulatory role in this process.\u003c/p\u003e","manuscriptTitle":"Methamphetamine and HIV-1 Tat protein synergistically induce endoplasmic reticulum stress to promote TRIM13-mediated neuronal autophagy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-22 06:55:15","doi":"10.21203/rs.3.rs-4788696/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-02T02:09:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-31T05:59:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-17T13:54:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"152391383055940370009533754288776323284","date":"2024-10-17T11:36:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"162605815863551064553494603587215969233","date":"2024-10-14T04:25:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"83794703571496036345394210845105613463","date":"2024-10-11T16:35:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"174170009209933256842085696469786256909","date":"2024-08-04T13:23:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"47477276705423540334122912120533017632","date":"2024-08-02T08:20:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-30T07:57:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-27T12:20:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-26T13:28:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurobiology","date":"2024-07-23T12:16:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"152ff02f-a37a-4bd7-b6e6-d863ad84d88a","owner":[],"postedDate":"August 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-30T16:05:59+00:00","versionOfRecord":{"articleIdentity":"rs-4788696","link":"https://doi.org/10.1007/s12035-024-04667-7","journal":{"identity":"molecular-neurobiology","isVorOnly":false,"title":"Molecular Neurobiology"},"publishedOn":"2024-12-23 15:57:10","publishedOnDateReadable":"December 23rd, 2024"},"versionCreatedAt":"2024-08-22 06:55:15","video":"","vorDoi":"10.1007/s12035-024-04667-7","vorDoiUrl":"https://doi.org/10.1007/s12035-024-04667-7","workflowStages":[]},"version":"v1","identity":"rs-4788696","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4788696","identity":"rs-4788696","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}