Hippocampal TRAP1 Overexpression Mitigates Hypoxia-Evoked Learning and Memory Impairments via MERCS * major project of the National Natural Science Foundation of China （82130054）

Hippocampal TRAP1 Overexpression Mitigates Hypoxia-Evoked Learning and Memory Impairments via MERCS * major project of the National Natural Science Foundation of China （82130054） | 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 Article Hippocampal TRAP1 Overexpression Mitigates Hypoxia-Evoked Learning and Memory Impairments via MERCS * major project of the National Natural Science Foundation of China （82130054） Guan Lu, Hao Chengxiao, Cao Rui, Guo Yanrong, Ma Shuang, Ge Rili This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9067186/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Chronic exposure to high-altitude hypoxia impairs hippocampus-dependent learning and memory. However, the upstream mitochondrial mechanisms by which oxygen deficiency triggers synaptic dysfunction remain incompletely understood. Therefore, the present study aimed to investigate the role of tumor necrosis factor receptor-associated protein 1 (TRAP1), a mitochondria-restricted chaperone, in this pathological process. In vivo, adult male Sprague-Dawley rats were exposed to a hypobaric chamber simulating an altitude of 5,000 m for 28 days. In vitro, HT22 hippocampal neurons were exposed to 1% oxygen (O₂) for 48 h to establish hypoxic cell models. TRAP1 was overexpressed using lentivirus or pharmacologically activated by a selective WNT5a agonist. Hippocampal structure and cognitive function were evaluated using the Morris Water Maze test for cognitive function assessment, high-resolution magnetic resonance imaging (MRI) for hippocampal volumetric and structural analysis, and whole-cell patch-clamp for neuronal recording. Moreover, the following series of experiments was conducted: transmission electron microscopy (TEM) for ultrastructural characterization of mitochondria–endoplasmic reticulum (ER) contact sites (MERCS), fluorometric assays for intracellular calcium (Ca²⁺) and reactive oxygen species (ROS) detection, reverse transcription quantitative PCR for mitochondrial DNA (mtDNA) integrity and gene expression quantification, and western blotting and co-immunoprecipitation analysis to assess the expression of synaptic-related proteins and their interactions. Hypoxia exposure led to a significant downregulation of hippocampal TRAP1 and WNT5a expression. It also caused a reduction in the MERCS gap width, a three-fold increase in mitochondrial ROS production, a two-fold elevation in mitochondrial matrix Ca²⁺ concentration, a 50% decrease in mtDNA copy number, and significant reductions in the expression of brain-derived neurotrophic factor, doublecortin, and postsynaptic density protein 95. These pathological changes were accompanied by decreased platform crossings in the Morris water maze test and loss of hippocampal definition on MRI. Notably, Trap1 overexpression reversed these hypoxia-induced deficits: it restored MERCS gap width, normalized redox balance and intracellular Ca²⁺levels, promoted transcription factor A mitochondrial-dependent mtDNA synthesis, recovered synaptic protein expression levels, and ultimately ameliorated neurodevelopmental impairment. In contrast, Trap1 knockdown recapitulated the hypoxic injury phenotypes. Additionally, treatment with a WNT5a agonist significantly upregulated TRAP1 expression, inhibited dynamin-related protein 1-mediated mitochondrial fission, and exerted a neuroprotective effect against hypoxia-induced neuronal damage. TRAP1 downregulation is an early and reversible event that links hypoxic stress to mitochondria ER dysfunction and cognitive impairment. Direct Trap1 overexpression or indirect upregulation via WNT5a agonism represents a promising dual therapeutic strategy for hypoxia-induced neurodegeneration and memory deficits. Biological sciences/Cell biology Biological sciences/Neuroscience TRAP1 hypoxia Learning and Memory Mitochondria ROS ER Wnt Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Background High-altitude environments impose persistent hypobaric hypoxia, triggering a series of systemic pathophysiological adaptations. Acute hypoxia initially induces compensatory vasodilation, leading to a transient increase in cerebral oxygen delivery. However, prolonged or severe hypoxic exposure leads to a pathological cascade that is strongly implicated in the development of neurological diseases(Midha et al., 2023 ).The brain, particularly the hippocampus, exhibits high sensitivity to changes in oxygen availability(Chattopadhyaya et al. 2025). CA1 pyramidal neurons within the hippocampus integrate multi - modal spatial and contextual information to establish activity - dependent synaptic plasticityWithin the hippocampus, CA1 pyramidal neurons integrate multi-modal spatial and contextual information to establish activity-dependent synaptic plasticity (Jain et al., 2024 , Zhao et al. , 2022).This synaptic plasticity is fundamentally reliant on normal hippocampal neurodevelopmental programming, which requires preservation of dendritic architecture and efficient dendritic signal transduction. These signal transduction processes are essential for acquiring new spatial memories and enabling long-term retention of episodic information. These functions are collectively referred to as the “cognitive map” (O'Hare et al., 2025 , Ben-Simon et al., 2022 , Cone and Clopath, 2024 ) —and constitute the neurobiological substrate for learning and memory. Consequently, hypoxia-induced synaptic loss and ultrastructural remodeling within the CA1 region are strongly correlated with measurable cognitive impairments(Vanderlinden et al., 2025 , Han et al., 2024 , Chen et al., 2024a ). In addition to generating ATP to meet the energy demands of neuronal activity, mitochondria act as the primary cellular oxygen sensors. The dynamic homeostasis of mitochondria is highly sensitive to hypoxia. Mild hypoxia initially promotes mitochondrial fusion, forming elongated mitochondrial networks as an adaptive response (Hao et al., 2023 ). However, prolonged hypoxic stress reverses this protective response and results in mitochondrial dysfunction (Wu et al., 2023 ). Hypoxia-induced bursts of reactive oxygen species (ROS) inhibit oxidative phosphorylation and suppress mitochondrial biogenesis, compromising cellular energy metabolism (Guo et al., 2024 , Wang et al., 2024 , Zhong et al., 2022 ). Dynamin-related protein 1 (DRP1) is recruited to mitochondria–endoplasmic reticulum (ER) contact sites (MERCS), where it mediates mitochondrial fission and amplifies organellar fragmentation. This excessive fission disrupts inter-organellar communication, accelerates energy failure, and promotes neuronal death(Janbandhu et al., 2022 , Duan et al., 2023a )Notably, MERCS further facilitate pathological crosstalk: mitochondria-derived ROS, including superoxide and hydroxyl radicals, traverse MERCS to oxidize ER membranes and induce ER stress (Shiiba et al., 2025a ). The resultant accumulation of misfolded or excessive proteins in the ER triggers ER stress. Suppression of the proapoptotic transcription factor C/EBP homologous protein (CHOP) can confer cytoprotection (Chen et al., 2022 ). To restore ER luminal homeostasis, the stressed ER redistributes Ca²⁺ to mitochondria and other cellular compartments via MERCS. However, this compensatory Ca²⁺ transfer imposes an additional burden on mitochondria, leading to mitochondrial Ca²⁺ overload. When the mitochondrial Ca²⁺ buffering capacity is exceeded, the mitochondrial permeability transition pore (mPTP) opens, releasing Ca²⁺ into the cytoplasm. Although this process temporarily alleviates mitochondrial matrix Ca²⁺ overload, it ultimately amplifies cellular Ca²⁺ dysregulation and activates cell death signaling pathways(Xian et al., 2022 ). Mitochondria contain an organelle-specific chaperone tumor necrosis factor receptor-associated protein 1 (TRAP1). This ATP-dependent heat-shock protein 90 (HSP90) paralog plays a crucial role in regulating client protein folding and facilitating the proteolysis of hypoxia-inducible factor-1α (HIF-1α)(Li et al., 2024 a, Yoon et al., 2021 ). Despite extensive studies on TRAP1 in oncology (Kim et al., 2024a ), its role in hypoxic brain injury, particularly in the associated learning and memory impairments, remains largely unknown. In this study, we provide the first evidence that TRAP1 is a critical determinant of hypoxia-induced cognitive dysfunction. Our results indicate that TRAP1 regulates hippocampal neurodevelopment and synaptic plasticity by fine-tuning the structure and function of MERCS and synaptic plasticity to preserve hippocampus-dependent memory processes under hypoxic stress. Materials and methods Rat and cell line The rats in this experiment were purchased from Beijing Weitong Lihua Company and were healthy male SD rats aged 6 weeks, weighing between 200 and 240g. Qualified certificate No. : SCXK (Beijing) 2021-0006. The rats in the hypoxia group were exposed to a hypobaric chamber simulating an altitude of 5,000 m, while the control group was maintained in the natural environment of Xining, Qinghai Province. The rats were sacrificed by intraperitoneal injection of 20% urethane solution at a dose of 1g/kg. The HT22 hippocampal neuron cell line(Cat NO.: CL-0697) was obtained from Procell Life Science & Technology Co., Ltd. and authenticated by short tandem repeat (STR) profiling. The cells were cultured in DMEM/F12 medium (supplemented with AML12) containing 10% fetal bovine serum. The hypoxic cell model was established by exposing the cells to an atmosphere of 1% O₂. Plasmid and transfection Lentiviral vectors encoding Trap1 (Trap1 overexpression, Trap1 OE) and small interfering RNAs (siRNAs) targeting Trap1 were purchased from Genechem Co., Ltd. (Shanghai, China). The sequences of the Trap1-specific siRNAs used for Trap1 silencing in HT22 cells are provided in the Supplementary Materials. Morris Water Maze Test A black circular pool (150 cm in diameter, 60 cm in height) was filled with water to a depth of 40 cm and maintained at a temperature of 23 ± 2°C. Rats were subjected to a 5-day acquisition training period, followed by a probe trial. All behavioral data were recorded and analyzed using the same video-tracking software. Magnetic resonance imaging(MRI) Inhalation anesthesia with 5% isofluranewas used for small animal MRI, and 1% isoflurane was used for maintenance anesthesia during scanning. Their respiratory status was continuously monitored using a pneumatic pillow sensor (SA Instruments, Stony Brook, NY, USA). High-resolution T2-weighted images were acquired in the axial, coronal, and sagittal planes with the following parameters: repetition time (TR) = 3,000 ms, echo time (TE) = 33 ms, echo-train length = 8, field of view (FOV) = 30 × 30 mm2, matrix size = 256 × 256, 30 contiguous slices, and slice thickness = 1.0 mm. Hematoxylin - Eosin (HE) Staining Paraffin-embedded hippocampal tissue sections were deparaffinized by sequential immersion in xylene followed by graded ethanol solutions (100%, 90%, 80%, and 70%), and then air-dried. Sections were subsequently stained with H&E according to standard protocols, examined under a light microscope at 40× magnification, and images were captured. Nissl staining Hippocampal tissues were rapidly dissected, fixed, dehydrated, and embedded in paraffin. The paraffin blocks were dried overnight at 60°C. The tissue sections were rehydrated through a series of gradient ethanol solutions, stained with 0.1% cresyl violet (Nissl stain), and briefly differentiated in 95% ethanol. After progressive dehydration in gradient ethanol solutions, the sections were cleared in xylene and mounted with neutral balsamAfter subjecting rats to euthanasia, hippocampal tissues were rapidly dissected, fixed, dehydrated, and embedded in paraffin. Paraffin blocks were dried overnight at 60°C. The tissue sections were rehydrated through a series of gradient ethanol solutions, stained with 0.1% cresyl violet (Nissl stain), and briefly differentiated in 95% ethanol. After progressive dehydration in gradient ethanol solutions, the sections were cleared in xylene and mounted with neutral balsam. Immunohistochemistry For immunohistochemical analysis, hippocampal tissue sections and HT22 cells were fixed, permeabilized, and blocked, followed by incubation with appropriately diluted primary antibodies overnight at 4°C. The following day, samples were incubated with species-matched horseradish peroxidase (HRP)-conjugated secondary antibodies at 37°C for 60 min. After washing, the sections were developed using 3,3′-diaminobenzidine chromogen. Nuclei were counterstained with hematoxylin for 1 min, after which sections were dehydrated, cleared, and mounted. Images were captured using a light microscope. A list of the primary antibodies used in this study is presented in Supplementary Table 1. Immunofluorescence staining After appropriate processing, cells were fixed, permeabilized, and blocked with phosphate-buffered saline (PBS), and blocked for 1 h at room temperature. The cells were then incubated with primary antibodies diluted in blocking buffer overnight at 4°C. The next day, the cells were washed three times with PBS and then incubated with fluorophore-conjugated secondary antibodies for 30 min at 37°C. After three additional washes with PBS, nuclei were counterstained with 4′,6-diamidino-2-phenylindole for 10 min at room temperature. A list of the primary antibodies used in this study is presented in Supplementary Table 1. Flow cytometry Cells were collected and centrifuged, and the supernatant was carefully removed. The cell pellet was resuspended in sheath fluid, gently vortexed to form a homogeneous suspension, and centrifuged again. After removal of the supernatant, cells were adjusted to a final concentration of 2 × 10 4 cells per sample and resuspended in MitoSO™ Red staining working solution (1:2,000 dilution) to form a uniform single-cell suspension for flow cytometric analysis. Transmission electron microscope(TEM) Tissues or cells were fixed, dehydrated, infiltrated, and embedded in resin. Ultrathin sections (60–90 nm) were prepared using an ultramicrotome and stained with uranyl acetate and lead citrate at room temperature. Images were acquired using a JEM-1400FLASH TEM (JEOL Ltd., Tokyo, Japan). Quantitative Real - Time Polymerase Chain Reaction(RT-qPCR) Total RNA was extracted using TRIzol reagent. The purity and concentration of the extracted RNA were measured using a NanoDrop™ 2000 spectrophotometer. Two micrograms of total RNA were reverse - transcribed into complementary DNA (cDNA) using FastKing RT SuperMix according to the manufacturer's instructions. The cDNA samples were stored at − 80°C until use. Gene - specific primers were synthesized by Shanghai Sangon Biotech Co., Ltd. qPCR reactions were prepared using the Tiangen SuperReal PreMix Plus kit and performed on an ABI Prism™ Q5 Sequence Detection System.Total RNA was extracted using TRIzol reagent. The purity and concentration of the extracted RNA were measured using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Two micrograms of total RNA were reverse-transcribed into complementary DNA (cDNA) using FastKing RT SuperMix (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions. The cDNA samples were stored at − 80°C until use. Gene-specific primers were synthesized by Shanghai Sangon Biotech Co., Ltd. qPCR reactions were prepared using the Tiangen SuperReal PreMix Plus kit (Tiangen Biotech) and performed on an ABI Prism™ Q5 Sequence Detection System (Thermo Fisher Scientific). Western blot analysis Cells were resuspended in ice-cold radioimmunoprecipitation assay lysis buffer. The cell lysates were centrifuged, and the supernatants were collected. Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit, and samples were normalized to ensure equal total protein loading. Protein aliquots were either snap-frozen or resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST) and incubated with primary antibodies diluted in blocking buffer overnight at 4°C. The following day, the membranes were washed with TBST and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized using an enhanced chemiluminescence substrate. A list of the primary antibodies used in this study is presented in Supplementary Table 1. Proteomics Analysis Total protein was extracted from each sample and divided into two aliquots. One aliquot was used for quantification and SDS-PAGE analysis to assess protein integrity, while the remaining aliquot was subjected to in-solution tryptic digestion and isobaric or isotopic labeling. Equal amounts of labeled peptides were pooled and fractionated using high-pH reversed-phase chromatography. The resulting peptide fractions were analyzed using nanoflow liquid chromatography-tandem mass spectrometry. Differentially expressed proteins and signaling pathways were identified based on statistical significance and biological relevance to the study objectives and were subsequently prioritized for downstream validation. Co-Immunoprecipitation (Co-IP) Cells were lysed on ice, and protein concentrations were determined using the BCA assay. For immunoprecipitation, the cell lysates were transferred into spin columns with end caps, mixed with the appropriate primary antibody and incubation buffer, and rotated end-over-end for 2 h at 4°C. Following incubation, alkaline neutralization buffer and 5× loading buffer were added to the samples, which were then heated in a boiling water bath for 5 min. Each immunoprecipitation eluate (40 µL) was resolved by SDS-PAGE and analyzed using western blotting. Whole - Cell Patch - Clamp Recording Glass micropipettes were fabricated from a single piece of Farad glass and fire-polished to achieve a tip resistance of 5–8 MΩ. The seal resistance was rapidly increased to over 1 GΩ and allowed to stabilize for 2 min. Series resistance was compensated (typically > 70%) before initiating the voltage protocol. Outward potassium (K⁺) currents were elicited using a holding potential of − 80 mV for 50 ms, followed by a depolarization to − 40 mV for 750 ms, and then a hyperpolarization to − 120 mV for 500 ms. K⁺ currents were continuously recorded throughout the experiment. DNA Damage Assay Following routine washes and fixation, non-specific binding was blocked for 20 min at room temperature. Cells were then incubated with a γ-H2AX rabbit monoclonal antibody for 1 h at room temperature. After three washes with PBS, cells were incubated with an Alexa Fluor 488-conjugated anti-rabbit IgG for an additional hour at room temperature. Following further washes, nuclei were counterstained with DAPI for 5 min at room temperature, and the slides were mounted in anti-fade medium and examined under a fluorescence microscope. Mitochondrial Permeability Transition Pore(mPTP) Assay Cells were incubated in the dark at 37°C for 30 min. The medium was replaced with fresh, pre-warmed (37°C) culture medium. After another 30 min of incubation in the dark, the medium was removed, and the cells were washed twice with PBS. A detection buffer was added, and cell fluorescence was immediately measured to assess mPTP opening. ER-Tracker™ Red staining Cells were washed with Hank’s Balanced Salt Solution supplemented with Ca²⁺ and Mg²⁺ and incubated with ER-tracker™ Red (1:1,000 dilution in complete medium) at 37°C for 20 min. The staining solution was aspirated, the cells were gently rinsed once with fresh, pre-warmed culture medium, and ER fluorescence was immediately visualized using a fluorescence microscope. Mito-Tracker Green staining Cells were incubated with Mito-Tracker™ Green diluted 1:20,000 in pre-warmed medium at 37°C for 25 min. After staining, the solution was removed, and the cells were gently washed once with fresh, pre-warmed medium. Mitochondrial fluorescence was immediately visualized using a fluorescence microscope Calcium Colorimetric Assay Cells were lysed by adding ice-cold lysis buffer and gently pipetting to ensure complete lysis. Standards and samples were loaded into a 96-well plate, and 150 µL of detection working solution was added to each well. The plate was gently mixed and incubated in the dark at room temperature for 10 min. Absorbance at 575 nm was measured using a microplate reader, and Ca²⁺ concentrations were calculated based on a standard curve. MitoSO™ Red staining Culture medium was aspirated, and cells were rinsed once with pre-warmed PBS. Cells were then incubated with MitoSO™ Red working solution at 37°C for 30 min. After incubation, the dye was removed, and the cells were washed twice with PBS. Mitochondrial superoxide-associated fluorescence was immediately visualized in PBS using a fluorescence microscope. ATPase Activity Assay The cell pellet was homogenized in ice-cold 0.9% saline at a ratio of 1:5 (w/v). The homogenate was centrifuged at 10,000 × g at 4°C for 10 min, and an aliquot of the supernatant was reserved for protein concentration determination. ATPase activity was calculated using the following formula: $$\:U/g\:protein=\frac{\left(\varDelta\:A−B\right)}{a}÷{C}_{pr}÷T\times\:f\times\:1000$$ where ΔA is the absorbance difference between the sample and the blank, B is the blank absorbance, a is the slope of the standard curve, Cpr is the protein concentration of the sample, T is the reaction time, and f is the dilution factor. Statistical analysis All experiments were performed at least three times independently. Pairwise comparisons were analyzed using an unpaired two-tailed Student t-test. Normally distributed data were compared using one-way analysis of variance followed by Tukey's post-hoc test. For non-normally distributed data, the Kruskal–Wallis test with Dunn's correction was used. Data are presented as the mean ± standard deviation. P < 0.05 was considered statistically significant. Results Hypoxia impairs learning&memory Acute hypoxia disrupts hippocampal-dependent memory and higher-order cognitive functions(Chattopadhyaya et al., 2025b ). In this study, the Morris water maze test revealed a significant decrease in the number of platform crossings in the hypoxia group (H28) compared to that in the control group (C28) (Fig. 1 A), indicating impaired spatial learning and memory recall. To investigate the structural basis of this cognitive deficit, we subjected mice to high-resolution MRI, which revealed an indistinct hippocampal silhouette accompanied by a conspicuous “burr” phenomenon in the H28 group (Fig. 1 B). Consistently, immunostaining for the neuroblast marker doublecortin (DCX) demonstrated a significant downregulation in DCX expression within the hippocampal neurogenic niche of the H28 group (Fig. 1 C). Histological analysis using HE and Nissl staining revealed extensive vacuolization of pyramidal neurons in the CA1 stratum, accompanied by marked neuronal degeneration and loss of cellular architecture (Fig. 1 D, E). At the molecular level, we observed significant reductions in the expression levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and glial fibrillary acidic protein (GFAP) (Fig. 1 F). These results suggest that hypoxia elicited a global suppression of neurodevelopmental trophic signaling. Given that BDNF is indispensable for activity-dependent hippocampal plasticity, reduced BDNF signaling impairs synaptic adaptation (Chen et al., 2024b ). In the H28 group, TEM further confirmed a diffuse and poorly defined synaptic ultrastructure (Fig. 1 G), consistent with impaired synaptogenesis. Moreover, this group exhibited increased expression levels of postsynaptic density-95 (PSD-95), a key scaffold protein that maintains the integrity of excitatory synapses(Dar et al., 2025 ), but significantly decreased α-synuclein (α-SYN) expression (Fig. 1 H). These molecular changes were accompanied by significant neurodevelopmental retardation at the cellular level (Fig. 1 I–K) and a parallel erosion of synaptic plasticity (Fig. 1 L, M). Collectively, these data demonstrate that hypoxia triggers a cascade of events, including delayed neurodevelopment, neuronal vacuolization, and altered synaptic plasticity, ultimately leading to cognitive dysfunction in the hippocampus. Hypoxia triggers mitochondrial and ER dysfunction Depletion of BDNF induces abnormal mitochondrial reorganization and accumulation of dysfunctional mitochondria(Ahuja et al., 2022 ).Consistent with this notion, our quantitative proteomics analysis confirmed downregulation of mitochondrial-related proteins and DNA-binding proteins under hypoxic conditions (Fig. 2 A). Moreover, fluorescence imaging revealed enhanced mitochondrial fluorescence and perinuclear aggregation of mitochondria in hypoxic neurons (Fig. 2 B). Consistent with the increased opening of the mPTP, we detected a significant increase in mitochondrial ROS production and Ca²⁺ influx, which was further confirmed by subsequent biochemical assays (Fig. 2 D,E). Similarly, the expression levels of proteins involved in mitochondrial Ca²⁺ transport, including calmodulin, mitochondrial calcium uniporter (MCU), and mitochondrial calcium uptake 1 (MICU1), were also significantly altered under hypoxia (Fig. 2 F, G). As abnormal neurophysiological signaling is increasingly recognized as a driver of cognitive decline(Jiang et al., 2024 ), we conducted whole-cell patch-clamp recordings. We observed that outward K⁺ currents were significantly attenuated under hypoxia (Fig. 2 H), which may reduce the efficiency of neuronal repolarization and increase Ca²⁺ influx. Collectively, these data demonstrate that hypoxia impairs mitochondrial integrity, induces excessive ROS production, and causes intracellular Ca²⁺ overload, collectively contributing to the disruption of neuronal homeostasis. Excessive ROS results in cumulative mtDNA damage, establishing a self-propelling vicious cycle. While mitochondrial transcription factor A (TFAM) normally protects mtDNA from oxidative damage and facilitates the autophagic clearance of leaked mtDNA(Liu et al., 2024 a),, its protective capacity is overwhelmed under sustained oxidative stress. Consistent with impaired mitochondrial biogenesis, our results indicate that hypoxia markedly decreased the import receptor Tomm20 (Fig. 3 A), resulting in impaired mitochondrial protein import and concurrent suppression of mtDNA transcription, accompanied by severe mitochondrial ultrastructural damage (Fig. 3 B–E). When genotoxic stress overwhelms cellular DNA repair capacity, cells activate intrinsic DNA damage response (DDR) pathways. However, the effectiveness of these self-protective mechanisms is often compromised by exogenous insults(Huang and Zhou, 2021 ).Therefore, we quantified the expression levels of key mtDNA maintenance proteins and found that hypoxia significantly downregulated the expression of the architectural chromatin factor ( Satb1 ), the single-stranded DNA-binding protein ( Ssbp1 ), and the mitochondrial DNA polymerase accessory subunit (Polg2 ) (Fig. 3 F, G). Collectively, these data demonstrate that hypoxia suppresses mtDNA replication and mitochondrial biogenesis and impairs the DNA damage repair machinery, exacerbating ROS-mediated genotoxicity in the hippocampal mitochondrial compartment. Inter-organellar communication is essential for signal transduction and cellular homeostasis(Guan et al., 2024 ).In this study, high-resolution electron microscopy revealed that hypoxia significantly increased the minimum gap between mitochondria and the ER, indicating enhanced mitochondria–ER contact (Fig. 4 A, B). Concurrent with these changes, ER-tracker fluorescence was attenuated, and the ER reticular network adopted a fragmented, perinuclear-excluded distribution (Fig. 4 C). Elevated mitochondrial calcein fluorescence indicates increased Ca²⁺ efflux from the ER lumen. However, prolonged ER Ca²⁺depletion is a well-established trigger of ER stress(Zhang et al., 2024a ).The ER-resident chaperone BIP (immunoglobulin heavy-chain binding protein) is the master regulator of the ER stress response; its downregulation compromises the protein-folding capacity and accelerates cellular damage(Yang et al., 2025 ).Under physiological conditions, BIP associates with ER sensors, such as ATF6, to regulate proteostasis(Lim et al., 2023 ).In our hypoxic model, we observed downregulation of BIP levels, ATF6, and other unfolded protein response (UPR) transducers (Fig. 4 D–F). Moreover, the expression of the proapoptotic transcription factor CHOP, which translocates to the mitochondria to induce mitochondrial dysfunction(Xu et al., 2025 ), was significantly upregulated. These findings indicate that hypoxia-driven ER–mitochondrial Ca²⁺ flux and BIP depletion converge to initiate ER stress and propagate mitochondrial injury. Hypoxia suppresses the expression of Wnt5a Canonical WNT signaling is an essential regulatory pathway for neurodevelopment, governing synaptogenesis, synaptic maturation, and activity-dependent plasticity. Consistent with its role in cognitive function, our results indicate that hypoxia-induced cognitive decline is accompanied by a significant Wnt pathway downregulation(Soudy et al., 2025 )(Fig. 5 A-D). Hypoxia suppresses the expression of TRAP1 TRAP1 regulates mitochondrial bioenergetics and proteostasis (Li et al., 2024 b), and emerging evidence suggests that hypoxic stress suppresses TRAP1 expression, contributing to neuronal injury (Kim et al., 2024b , Liu et al., 2024 ). Consistently, our results revealed that hypoxia significantly inhibited TRAP1 expression (Fig. 6 A–E). Therefore, to further clarify the causal role of TRAP1 in hypoxia-induced neuronal dysfunction, we performed Trap1 knockdown and overexpression experiments in neurons. As illustrated in Fig. 6 F–H, we confirmed the efficiency of the knockdown and overexpression. Trap1 upregulation ameliorates hypoxia-induced neurodevelopmental deficits and restores impaired synaptic plasticity Our results indicated that hypoxia-induced Trap1 downregulation further suppressed the expression of neurodevelopmental markers (Fig. 7 A–D) and key synaptic plasticity-related proteins (Fig. 7 E, F). In contrast, lentivirus-mediated restoration of Trap1 expression completely reversed these deficits. These results highlight the protective role of TRAP1 against hypoxia-induced neuronal injury(Chen et al., 2025 ). Trap1 upregulation mitigates hypoxia-induced mitochondria and ER dysfunction Our results demonstrated that Trap1 overexpression normalized the hypoxia-induced ultrastructural abnormalities of mitochondria (Fig. 8 A, B). Moreover, genetic silencing of Trap1 exacerbated mPTP opening, whereas its re-expression significantly reduced mPTP opening, thereby attenuating both ROS overproduction and Ca²⁺ influx (Fig. 8 C–F). Disruption of mitochondrial redox homeostasis leads to excessive ROS production that exceeds antioxidant capacity, impairing oxidative metabolism and compromising cell viability. Consistent with this mechanism, Trap1 knockdown further increased mitochondrial ROS generation under hypoxia, while Trap1 up - regulation restored ROS levels to baseline (Fig. 8 G). Similarly, Trap1 depletion increased mitochondrial Ca²⁺ uptake by up - regulating Ca²⁺ transporters, an effect that was reversed by Trap1 overexpression (Fig. 8 H,I).Restoration of mitochondrial Ca²⁺ homeostasis is crucial for functional recovery and neuronal survival(Liiv et al., 2024 ). Electrophysiological recordings further revealed that Trap1 overexpression enhanced outward K⁺ currents, reducing voltage-gated Ca²⁺ entry and alleviating cellular Ca²⁺ overload (Fig. 8 J). Collectively, these data indicate that TRAP1 acts as a critical molecular regulator that decreases hypoxia-induced mitochondrial ROS production and alleviates Ca²⁺ dysregulation to preserve neuronal integrity under hypoxic conditions. Silencing of Trap1 further suppressed mitochondrial biogenesis, whereas its re-expression completely restored hypoxia-induced reduction in TFAM and TOMM20 levels (Fig. 9 A–D). Concurrently, the expression levels of nuclear and mitochondrial DNA damage markers returned to baseline (Fig. 9 E, F). Therefore, to investigate whether DNA damage further affects mtDNA, we evaluated mtDNA expression. Our results indicated that Trap1 overexpression significantly increased mtDNA levels under hypoxic conditions (Fig. 9 G–I), supporting the notion that enhancing mtDNA integrity can improve cognitive function(Li et al., 2024 ).We then investigated whether TRAP1 also promotes the DNA-binding events required for mtDNA maintenance using CO-IP. These experiments revealed that Trap1 overexpression significantly enhanced DNA binding under hypoxic conditions (Fig. 10 A, B). Collectively, these data demonstrate that TRAP1 is a critical factor that mitigates hypoxia-induced mtDNA damage and regulates the subsequent repair and transcriptional reactivation of mtDNA. Our results also indicated that Trap1 silencing decreased the distance between mitochondria and the ER, whereas its overexpression increased this inter-organellar spacing. These results indicate that TRAP1 positively regulates mitochondria–ER contact (Fig. 11 A–D). Moreover, Trap1 depletion exacerbated hypoxia-induced ER stress, whereas Trap1 overexpression inhibited UPR signaling and restored ER homeostasis (Fig. 11 E, F). Collectively, these findings indicate that TRAP1 regulates hypoxia-induced ER stress by stabilizing mitochondria–ER tethering, functioning as a critical regulator of inter-organellar communication under hypoxic conditions. TRAP1 interacts with Wnt5a and rescues its hypoxia-suppressed expression As our results revealed decreased WNT5a expression under hypoxic conditions, we further explored whether WNT5a interacts with TRAP1. Co-IP experiments revealed an interaction between TRAP1 and WNT5a, which was weakened under hypoxic conditions (Fig. 11 G). Wnt5a agonist up-regulates TRAP1 and attenuates neuronal injury under hypoxia TRAP1 maintains mitochondrial homeostasis and exerts cytoprotective effects through crosstalk with the WNT signaling pathway. As hypoxia suppresses TRAP1 expression, we further investigated whether WNT pathway activation could reverse this downregulation. Administration of a WNT5a-specific agonist restored TRAP1 protein levels under hypoxic conditions (Fig. 12 A). Moreover, the agonist upregulated the expression of neurodevelopmental markers, promoted mitochondrial biogenesis, reduced DNA damage foci, and alleviated mitochondrial Ca²⁺ overload (Fig. 12 A). Functional assays further revealed enhanced cell proliferation, increased outward K⁺ currents, and elevated ATPase activity. Overall, these findings suggest that TRAP1 upregulation through WNT5a activation effectively rescues hypoxia-induced mitochondrial dysfunction and neuronal injury. Discussion During hypoxic brain damage, long-term potentiation in hippocampal circuits is suppressed(Zhao et al., 2023 ), providing a physiological basis for the observed learning and memory deficits. In the present study, we confirmed previous reports that sustained hypoxia causes hippocampal injury and subsequent cognitive decline. Our morphometric and functional analyses revealed that chronic hypoxia induces: (i) significant remodeling of hippocampal architecture, (ii) downregulated expression of neurodevelopmental markers, (iii) ultrastructural changes in the synapses of CA1 pyramidal neurons, and (iv) increased death of pyramidal cells. These structural and molecular alterations directly impair hippocampus-dependent cognitive function, particularly during the early stages of hypoxic exposure(Geigenmuller et al., 2024 ). BDNF regulates neural development by activating distinct signaling cascades that control synaptogenesis and short-and long-term synaptic plasticity in a spatially and temporally restricted manner(Wang et al., 2022 ). Under physiological conditions, activity-dependent BDNF release from presynaptic terminals enhances synaptic connectivity and plasticity(Taylor et al., 2023 ), whereas pathological reductions in BDNF lead to cognitive dysfunction(Kang et al., 2024 ).Notably, this phenomenon was recapitulated in our hypoxic model. Although traditionally associated with T-cell development, NGF is also crucial for neuronal survival and axonal innervation(Pozzer et al., 2025 , Lian et al., 2024 ).Specifically, injured neurons disrupt network signaling by amplifying circuit-level damage in an NGF-dependent manner(Zhi et al., 2025 ). Moreover, GFAP downregulation results in abnormal mitochondrial morphology and accelerates cellular aging(Popov et al., 2023 ). DCX, a microtubule-associated phosphoprotein expressed by immature neurons, regulates cytoskeletal dynamics required for neuronal migration and hippocampal morphogenesis. Reduced DCX levels impair nuclear motility during early neurodevelopment(Sebastien et al., 2025 ). Consistent with these observations, our results indicated that hypoxia results in downregulated DCX expression, impaired neurodevelopment, and compromised synaptic plasticity. Bioenergetic failure disrupts inter-neuronal mitochondrial transfer, which is essential for maintaining metabolic homeostasis, leading to the accumulation of dysfunctional mitochondria and impaired neuronal energy utilization, a key pathological feature of neuronal injury(Cheng et al., 2022 ). Mitochondria function as central metabolic hubs whose dynamic cycles of fission and fusion actively regulate intracellular signal transduction(Konig and McBride, 2024 ).As synaptic transmission is energetically dependent on mitochondrial ATP production, any impairment of mitochondrial function directly affects neuronal survival. Our results confirmed that hypoxia causes rapid mitochondrial damage. Specifically, the loss of redox balance leads to an ROS burst that overwhelms the endogenous antioxidant system, disrupts oxidative phosphorylation, and impairs global cellular physiology(DiGiovanni et al., 2025 ). Excessive ROS and cytosolic Ca²⁺ lead to mitochondrial swelling, mPTP opening, and additional ROS releases into the cytoplasm; this amplifying loop that further exacerbates cellular injury(Yang et al., 2024 ).Mitochondrial function critically depends on the electrochemical gradient across the inner mitochondrial membrane, known as the mitochondrial membrane potential, which regulates all ionic fluxes. Therefore, disruption of ionic homeostasis across this membrane leads to the collapse of the mitochondrial membrane and subsequent cellular dysfunction(Szabo and Szewczyk, 2023 ). Mitochondrial permeability transition refers to the size-selective passage of solutes across the normally impermeable inner mitochondrial membrane through a pore formed at the interface between the cristae and the outer mitochondrial membrane(Bonora et al., 2022 ). Mitochondrial Ca²⁺ uptake and efflux are integral to metabolic regulation and intracellular signaling, and disruptions in this cycle are implicated in the pathogenesis of acquired disorders, including neurodegenerative diseases(Garbincius and Elrod, 2022 ). Ca²⁺ uptake through MCU buffers physiological Ca²⁺ transients required for synaptic memory formation(Gherardi et al., 2025 , Obara et al., 2024 ). However, prolonged Ca²⁺ sequestration, induced by stress or external stimuli, disturbs mitochondrial homeostasis and compromises cell viability(Cartes-Saavedra et al., 2025 ).This calcium overload induces oxidative stress and ATP depletion, ultimately leading to cellular injury(Wang et al., 2023 ).Moreover, elevated intracellular Ca²⁺ activates calmodulin-dependent kinase II, which is a pivotal signaling molecule that regulates activity-dependent synaptic plasticity, learning, and memory(Nicoll and Schulman, 2023 ). In the present study, using whole-cell patch-clamp recordings, we observed that hypoxia suppresses outward K⁺ currents, increasing Ca²⁺ influx. Collectively, these results indicate that the combined effects of ROS and Ca²⁺ overload indirectly disrupt the electrophysiological mechanisms underlying hippocampus-dependent learning and memory. The high replication rate of mtDNA increases the likelihood of replication errors, rendering the mitochondrial genome more susceptible to oxidative damage(Milano et al., 2024 ). Mitochondrial Ca²⁺ overload further impairs mitochondrial integrity and can trigger the release of mtDNA fragments into the cytoplasm; this process is called “mtDNA escape”(Zhao et al., 2025 ).In the nucleus, SATB1 recruits chromatin-remodeling complexes to specific DNA elements, shaping higher-order chromatin structure and regulating gene transcription[67](Zelenka et al., 2022 ). In contrast, mitochondrial SSBP1 stabilizes single-stranded mtDNA intermediates, preventing replication fork collapse and subsequent development of mitochondrial diseases(Riccio et al., 2024 , Nie et al., 2025 ).As single-strand breaks are the most common form of DNA damage (Caldecott, 2022 ), we investigated SSBP1 expression and found that it was significantly downregulated under hypoxic conditions. Moreover, we observed that hypoxia suppressed the entire transcriptional program that controls mitochondrial biogenesis. TFAM, which packages mtDNA into nucleoid structures, promotes the autolysosomal degradation of leaked cytosolic mtDNA(Liu et al., 2024 b).Therefore, TFAM depletion allows immunogenic mtDNA fragments to accumulate and activate the mitochondrial UPR. Notably, persistent activation of this response leads to an integrated stress response that, if unresolved, causes irreversible mitochondrial dysfunction, cellular damage, and ultimately programmed cell death(Bond et al., 2025 , Yang et al., 2024 ).In addition, impaired mitochondrial biogenesis further compromises the DNA-binding machinery required for genome maintenance. Upon detection of DNA lesions, nuclear sensors recruit DDR proteins to initiate repair(Chappidi et al., 2024 ). In mitochondria, TFAM packages mtDNA into nucleoids that serve as platforms for stress-induced DNA repair (Dai et al., 2023 , Urrutia et al., 2024 ).This process is essential for mitochondrial and cellular survival under adverse conditions (Lascaux et al., 2024 ). The accessory subunit POLG2 promotes mtDNA replication by enhancing DNA polymerase-γ processivity and facilitating stable primer-template binding(Valenzuela et al., 2025a ), and pathogenic mutations in POLG2 can cause mitochondrial disease(Yang et al., 2022 , Corra et al., 2025 ). In this study, using a 48h hypoxic challenge, we observed a marked attenuation of both nuclear and mtDNA repair capacity. Persistent hypoxia prevented efficient resolution of DNA damage, leading to cumulative damage that exceeds the cellular repair thresholds. As unrepaired DNA damage accumulates, proapoptotic signaling cascades are activated, reducing cell survival(Wheeler et al., 2024 ) and ultimately triggering programmed cell death(Huang et al., 2024 ). Organelle contact sites serve as signaling hubs that preserve intracellular homeostasis. Among these, MERCS are the most extensively studied. Compact MERCS function as metabolic platforms where tethering proteins on both membranes coordinate lipid transfer, Ca²⁺ signaling(Pihan et al., 2025 , Shiiba et al., 2025b ), ER stress, and mitochondrial homeostasis(Zhang et al., 2024b ). Conversely, excessive ROS can diffuse across MERCS and oxidize ER-resident proteins, leading to protein misfolding. The accumulation of these misfolded proteins can trigger ER stress cascades and alter the ER structure and luminal organization. Using high-resolution imaging, we found that hypoxia shortens the inter-organellar gap and enhances mitochondria–ER communication, suggesting a compensatory effort to redistribute metabolites and mitigate stress. Given that the ER is the largest intracellular Ca²⁺ reservoir, we observed that mitochondria transiently pump Ca²⁺ into the ER via MERCS. This flux of Ca²⁺ induces luminal swelling and cisternal expansion as the ER attempts to buffer the ionic load. When hypoxia persists, this delicate balance is disrupted: MERCS become dysfunctional, Ca²⁺ transfer is impaired, and cellular injury is exacerbated (Zhang et al., 2025 ).Overall, sustained hypoxia depletes ER luminal redox enzymes, impairs protein folding, and prevents the resolution of ER stress(Ugalde et al., 2022 ), thereby driving neurons toward pyroptotic death(Bhardwaj et al., 2025 ). In contrast, hypoxia also upregulates the transcription of BIP and other canonical sensors, driving the UPR(Zhao et al., 2023 , Wiseman et al. , 2022). However, ATF6-dependent release of mtDNA simultaneously amplifies ER stress(Rosing et al, 2024 ), and genetic ATF6 suppression is required to restore proteostasis, preserve hippocampal neuronal integrity, and rescue cognition(Chen et al., 2023 ). Notably, this apparent contradiction is resolved by our finding that severe hypoxia impairs the ER machinery at multiple levels. Specifically, we revealed that although stress-related transcripts accumulate, the protein folding and degradation machineries remain functionally inactive, preventing adaptive UPR and leading to irreversible cellular damage. The expression and functional significance of TRAP1 in the hypoxic brain remain largely unexplored. In this study, we provide the first evidence that TRAP1 expression is specifically downregulated in hippocampal neurons under chronic hypoxic stress. Our results indicated that, under these conditions, genetic Trap1 suppression further impaired neurodevelopment and synaptic plasticity, whereas restoration of its expression reversed hypoxia-induced dendritic simplification and LTP deficits. These findings demonstrate that TRAP1 acts as a critical regulatory molecule that links hypoxic stress to cognitive dysfunction by preserving neuronal maturation and synaptic adaptability. Moreover, Trap1 overexpression markedly suppressed mPTP opening, ROS generation, and mitochondrial Ca²⁺ load. The decrease in mitochondrial matrix Ca²⁺ levels subsequently inhibited mPTP activation, preserving inner mitochondrial membrane integrity and enhancing ATP synthesis(He et al, 2024 ). Whole-cell recordings further revealed that Trap1 overexpression restored the hypoxia-suppressed outward K⁺ current, reducing Ca²⁺ influx and stabilizing the transmembrane potential essential for synaptic reliability. In addition to its role in neurodevelopment, elevated TRAP1 expression strongly promotes mitochondrial biogenesis under hypoxia, accelerates mtDNA synthesis, and mitigates mtDNA damage. Enhanced mtDNA content increases oxidative phosphorylation capacity and restores efficient mitochondrial respiration(Valenzuela et al, 2025b ). Moreover, phosphorylation-dependent release of SSBP1 from mtDNA reduces its mitochondrial localization, preventing excessive replication fork stalling and the associated ROS burst, thus alleviating mitochondrial stress (Zhang et al, 2023 ).Our morphometric analysis revealed that Trap1 overexpression increased the distance between mitochondria and the ER under hypoxia, reducing inter-organellar cargo transfer, particularly that of ROS and Ca²⁺. Notably, this spatial separation limits ROS-mediated ER oxidation, prevents mitochondrial Ca²⁺ overload, and preserves mitochondrial matrix bioenergetics, thereby increasing neuronal ATP availability and improving cognitive performance(Korte et al, 2024 ). Consistently, the normalization of the expression of ER stress sensors reflects the comprehensive restoration of cellular homeostasis and synaptic function. Given our functional data and proteomic signatures of hypoxia-induced mitochondrial Ca²⁺ overload and ER stress, we investigated the WNT signaling pathway and identified it as a key modulator of TRAP1 expression. Based on our findings, we propose that TRAP1 interacts with WNT5a to: (i) restore mitochondrial and ER homeostasis, (ii) promote hippocampal neurodevelopment, and (iii) rescue synaptic plasticity, alleviating hypoxia-induced neuronal injury. Moreover, pharmacological WNT5a activation under hypoxic stress increased TRAP1 expression, suppressed DRP1-mediated mitochondrial fission, normalized mitochondrial Ca²⁺ handling, and upregulated BDNF and DCX levels. Mitochondria play a key role in cell growth and proliferation through ATP synthesis. Under hypoxic conditions, DRP1 is activated and promotes excessive mitochondrial fission, increasing MERCS formation(Duan et al., 2023b ). While DRP1 normally mediates lipid turnover in neuronal mitochondria to maintain energy balance(Haynes et al, 2024 ), its hyperactivation under hypoxia has detrimental effects. Inhibiting DRP1-mediated fission disrupts mitochondrial dynamics and reduces the generation of mitochondria enriched in ATP synthase(Ryu et al, 2024 ), ultimately disrupting oxidative phosphorylation and inducing oxidative stress(Hong et al, 2022 ). Notably, Trap1 upregulation counteracts these pathological changes by modulating mitochondrial–ER interactions. It also increases ATP production and globally restores neuronal function.Overall, these results indicate that WNT5a-driven Trap1 upregulation is sufficient to reverse hypoxia-induced mitochondrial-ER dysfunction and cognitive deficits. As a mitochondria-restricted molecular chaperone, the function of TRAP1 remains poorly understood and understudied. In this study, we reveal its previously unrecognized role in mitochondrial quality control and hypoxic brain injury. We not only identify TRAP1 as a central regulator of mitochondria–ER communication and hippocampal synaptic plasticity under hypoxic stress but also reveal a druggable WNT5a–TRAP1 axis. Based on our results, we propose two complementary therapeutic strategies for hypoxia-induced cognitive deficits: (i) direct upregulation of TRAP1 and (ii) indirect upregulation through WNT5 activation, suggesting a potential new strategy for the clinical treatment of hypoxia-associated neurodegenerative disorders. However, this study has certain limitations. First, this study lacked clinical specimens and supportive epidemiological data, limiting the translational relevance of our findings. Second, the precise subcellular localization of TRAP1, and, consequently, its compartment-specific functions, was not fully characterized. Finally, in vivo evidence that Trap1 overexpression rescues hypoxia-induced learning and memory deficits in intact animals remains limited, necessitating further investigations. Conclusion In summary, TRAP1 overexpression is sufficient to rescue hypoxia-induced deficits in learning and memory, neuronal development, and synaptic plasticity. Pharmacological activation of the WNT signaling pathway further increased TRAP1 expression, restoring MERCS integrity and alleviating downstream organellar dysfunction. Overall, our findings suggest TRAP1 as a potential therapeutic target for hypoxia-associated cognitive decline and related neurodegenerative diseases. Declarations Ethical approval This experiment met the ethical standards for animal experiments and was approved by the Ethics Committee of the Affiliated Hospital of Qinghai University (Clinical Medical College) (Ethics approval number: P-SL-2023-448). All methods were carried out in accordance with relevant guidelines and regulations. CONSENT FOR PUBLICATION Not applicable DATA AVAILABILITY The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. All methods are reported in accordance with ARRIVE guidelines. FUNDING We thanks the Science and Technology Department of Qinghai Talent Scientist Program（Grant No.2025-ZJ-748）and The major project of the National Natural Science Foundation of China（82130054） DECLARATION OF INTERESTS The authors declare no competing interests. AUTHOR CONTRIBUTIONS Guan Lu designed the study, performed most of the experiments, and drafted the manuscript. Hao Chengxiao assisted with animal experiments, such as the Morris Water Maze test. Cao Rui assisted with patch - clamp recordings. Guo Yanrong participated in the study design. Ma Shuang participated in the study design and provided financial support. Ge Rili revised the manuscript and also provided financial support. All authors read and approved the final manuscript. ACKNOWLEDGMENTS We would like to express our gratitude to Genechem Co.,Ltd. (Shanghai, China), Proteintech Group, Inc(Wuhan, China) and OE Biotech. Co., Ltd.(Shanghai, China) for their technical support, and to Bioreder.com for their assistance with mapping technology. AUTHOR’S INFORMATION Not applicable. References AHUJA, P., NG, C. F., PANG, B., CHAN, W. S., TSE, M., BI, X., KWAN, H. R., BROBST, D., HERLEA-PANA, O., YANG, X., Du G, SAENGNIPANTHKUL, S., NOH, H. L., JIAO, B., KIM, J. K., LEE, C. W., YE, K. & CHAN, C. B. 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(2025), "GPAT4 sustains endoplasmic reticulum homeostasis in endocardial cells and safeguards heart development", Nat Commun, Vol. 16 No. 1, pp. 3345. ZHAO, X., HSU, C. L. & SPRUSTON, N. (2022), "Rapid synaptic plasticity contributes to a learned conjunctive code of position and choice-related information in the hippocampus", Neuron, Vol. 110 No. 1, pp. 96-108.e4. ZHI, X., WU, F., QIAN, J., OCHIAI, Y., LIAN, G., MALAGOLA, E., ZHENG, B., TU, R., ZENG, Y., KOBAYASHI, H., XIA, Z., WANG, R., PENG, Y., SHI, Q., CHEN, D., RYEOM, S. W. & WANG, T. C. (2025), "Nociceptive neurons promote gastric tumour progression via a CGRP-RAMP1 axis", Nature, Vol. 640 No. 8059, pp. 802-810. ZHONG, C., NIU, Y., LIU, W., YUAN, Y., LI, K., SHI, Y., QIU, Z., LI, K., LIN, Z., HUANG, Z., ZUO, D., YANG, Z., LIAO, Y., ZHANG, Y., WANG, C., QIU, J., HE, W., YUAN, Y. & LI, B. (2022), "S100A9 Derived from Chemoembolization-Induced Hypoxia Governs Mitochondrial Function in Hepatocellular Carcinoma Progression", Adv Sci (Weinh), Vol. 9 No. 30, pp. e2202206. Additional Declarations No competing interests reported. Supplementary Files supplementaryWB.docx floatimage1.png Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 11 May, 2026 Reviews received at journal 08 May, 2026 Reviews received at journal 06 May, 2026 Reviewers agreed at journal 29 Apr, 2026 Reviewers agreed at journal 27 Apr, 2026 Reviewers invited by journal 12 Apr, 2026 Editor assigned by journal 12 Apr, 2026 Editor invited by journal 19 Mar, 2026 Submission checks completed at journal 18 Mar, 2026 First submitted to journal 18 Mar, 2026 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9067186","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":622652682,"identity":"0e42b040-d08d-49f5-a5b2-22bf86acd174","order_by":0,"name":"Guan Lu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIie3PsQqCUBTG8SM3dLntVwyf4QshGgQfpEUXpwKh1cG4UEvg3FvkGwgXbPEBHANfoAdwSGhp87oF3f98fhw+IpPpB7MkU/2AnEePQpOwi52CZ41Pba1JnJJD8BcLqIs1yVJyQMBOTre+6igPd5PElXb2BFaJ9NLjlpr0UEyRtWR3xOOXs7ffCKtQ0yRSBFGDJVe31SSWXMAtwAIhuDax04DQ+ODjllhni1Uq1dOQcziq6l55OE2+g4jnnH/IXGEymUz/0Rt+LDtFZKAGkAAAAABJRU5ErkJggg==","orcid":"","institution":"Qinghai University","correspondingAuthor":true,"prefix":"","firstName":"Guan","middleName":"","lastName":"Lu","suffix":""},{"id":622652683,"identity":"7149cbb4-aa17-4cca-abe0-8783fa16f8ef","order_by":1,"name":"Hao Chengxiao","email":"","orcid":"","institution":"Dingxi City People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Chengxiao","suffix":""},{"id":622652684,"identity":"a259147e-541e-40dd-8458-b29cd7a4b095","order_by":2,"name":"Cao Rui","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Cao","middleName":"","lastName":"Rui","suffix":""},{"id":622652685,"identity":"cd38400e-c2ec-4ec7-9f19-1a8a3b228a63","order_by":3,"name":"Guo Yanrong","email":"","orcid":"","institution":"Ankang City Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Guo","middleName":"","lastName":"Yanrong","suffix":""},{"id":622652686,"identity":"310a21cd-d2a9-4dd2-9d7d-5a539b884727","order_by":4,"name":"Ma Shuang","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Ma","middleName":"","lastName":"Shuang","suffix":""},{"id":622652687,"identity":"8affcb7e-191b-48c7-a74c-c02fa7d38058","order_by":5,"name":"Ge Rili","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Ge","middleName":"","lastName":"Rili","suffix":""}],"badges":[],"createdAt":"2026-03-09 01:23:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9067186/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9067186/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107448128,"identity":"788563cb-6885-4855-a72f-b85d3ea3ae63","added_by":"auto","created_at":"2026-04-21 14:56:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":521612,"visible":true,"origin":"","legend":"\u003cp\u003eHypoxia-induced impairments in hippocampal learning and memory. \u003cstrong\u003eA\u003c/strong\u003ePlatform-crossing frequencies in the Morris water maze (mean ± SD; n = 6 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003eB\u003c/strong\u003e Representative T2-weighted magnetic resonance imaging of hippocampal architecture. \u003cstrong\u003eC\u003c/strong\u003e Immunofluorescence micrographs of doublecortin (DCX) in the CA1 region. \u003cstrong\u003eD\u003c/strong\u003e Hematoxylin-eosin staining of CA1 pyramidal neurons. \u003cstrong\u003eE\u003c/strong\u003e Nissl staining of neuronal integrity in CA1. \u003cstrong\u003eF\u003c/strong\u003e Hippocampal protein levels of neurodevelopmental markers (mean ± SD; n = 3–4 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003eG\u003c/strong\u003eTransmission electron microscopy image of the ultrastructural synaptic morphology in CA1. \u003cstrong\u003eH\u003c/strong\u003e Quantitative real-time polymerase chain reaction analysis of the expression levels of synaptic plasticity-related genes in hippocampal tissue (mean ± SD; n = 3–6 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003eI, J\u003c/strong\u003eImmunofluorescence quantification of NeuN and GFAP in cultured cells (mean ± SD; n = 6 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003eK\u003c/strong\u003e Neurodevelopmental protein expression in cell lysates (mean ± SD; n = 3 per group; *P \u0026lt; 0.05). \u003cstrong\u003eL\u003c/strong\u003eImmunohistochemical density of PSD95 \u003cem\u003ein vitro\u003c/em\u003e (mean ± SD; n = 6 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003eM\u003c/strong\u003e mRNA expression of synaptic plasticity genes in cultured cells (mean ± SD; n = 3–6 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9067186/v1/0b13ae53f1e18e2fed0fc7df.png"},{"id":107448058,"identity":"4aca33bc-4c33-430c-ad3b-8d5b80d744ca","added_by":"auto","created_at":"2026-04-21 14:56:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":374351,"visible":true,"origin":"","legend":"\u003cp\u003eHypoxia-induced mitochondrial dysfunction.\u003cstrong\u003eA\u003c/strong\u003eVolcano plot and Gene Ontology enrichment analysis of differentially expressed genes between the control (C28) and hypoxia (H28) groups.\u003cstrong\u003eB, C\u003c/strong\u003eRepresentative images and quantitative analysis of mitochondrial marker staining and mitochondrial permeability-transition-pore (mPTP) opening (mean ± SD; n = 6 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05).\u003cstrong\u003eD\u003c/strong\u003e Flow-cytometric quantification of MitoSOX Red fluorescence indicating mitochondrial superoxide production (mean ± SD; n = 3 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05).\u003cstrong\u003eE\u003c/strong\u003e \u0026nbsp;\u003cem\u003eIn vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e Ca²⁺ content determinations (mean ± SD; n = 3 - 6 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05).\u003cstrong\u003eF,G\u003c/strong\u003eProtein expression levels of mitochondrial Ca²⁺-transport machinery (mean ± SD; n = 3–4 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003eH\u003c/strong\u003e Representative recordings and quantification of outward K⁺ currents in neurons.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9067186/v1/de06f57100c2a43d52a5470d.png"},{"id":107448158,"identity":"b1faa4b5-a674-4d2d-b067-c44cb6798a3c","added_by":"auto","created_at":"2026-04-21 14:56:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":321240,"visible":true,"origin":"","legend":"\u003cp\u003eHypoxia provokes quantifiable mtDNA damage. \u003cstrong\u003eA\u003c/strong\u003e Protein expression of mitochondrial biogenesis markers (mean ± SD; n = 3 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003eB–D\u003c/strong\u003e Relative mtDNA copy number assessed by qPCR and corresponding protein levels by Western blot(mean ± SD; n = 3–6 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003eE \u003c/strong\u003eDetection of DNA damage markers (mean ± SD; n = 6 per group; \u003cem\u003e*P\u003c/em\u003e\u0026lt; 0.05). \u003cstrong\u003eF, G \u003c/strong\u003eExpression of DNA binding proteins and DNA damage repair proteins (mean ± SD; n = 3–6 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9067186/v1/bbe7abb1ecf9271931e40478.png"},{"id":107448069,"identity":"1052985e-65d0-49e7-93d3-5e7ec5f1088b","added_by":"auto","created_at":"2026-04-21 14:56:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":367494,"visible":true,"origin":"","legend":"\u003cp\u003eHypoxia triggers ER stress via mitochondria–ER contact sites. \u003cstrong\u003eA \u003c/strong\u003eMitochondria and endoplasmic reticulum in hippocampus as shown by transmission electron microscopy. \u003cstrong\u003eB\u003c/strong\u003e Inter-organellar distance quantification (mean ± SD; n = 12 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05).\u003cstrong\u003eC\u003c/strong\u003eFluorescence intensity of ER-tracker dye in cells (mean ± SD; n = 9 rats per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003eD–F\u003c/strong\u003e Protein levels of ER-stress sensors (ATF6,CHOP,BIP,GRP94) in hippocampal tissue and neurons (mean ± SD; n = 3–4 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9067186/v1/d2808b87877afde0e4b9b6f0.png"},{"id":107448068,"identity":"a3e2245e-2703-49d2-8cc2-f9997e5327df","added_by":"auto","created_at":"2026-04-21 14:56:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":164546,"visible":true,"origin":"","legend":"\u003cp\u003eHypoxia resulted in decreased Wnt5a expression.\u003cstrong\u003eA\u003c/strong\u003eProteomics showed that Wnt signaling in H28 group was weaker than that in C28 group.\u003cstrong\u003eB-D\u003c/strong\u003e Western blot and immunofluorescence were used to detect the expression of Wnt5a(mean ± SD; n = 3-6 per group; \u003cem\u003e*P\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9067186/v1/a3897749ac58cd5ac686fe61.png"},{"id":107448070,"identity":"0901601a-eff3-4dda-b347-495bf1f41e29","added_by":"auto","created_at":"2026-04-21 14:56:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":275348,"visible":true,"origin":"","legend":"\u003cp\u003eHypoxia inhibited the expression of TRAP1.\u003cstrong\u003eA-E\u003c/strong\u003eRT-qPCR, Western blot and immunofluorescence were used to detect the expression level of TRAP1(mean ± SD; n = 3-6 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05).\u003cstrong\u003eF-H\u003c/strong\u003eExpression of TRAP1 after TRAP1 knockdown and overexpression(mean ± SD; n = 3-6 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-9067186/v1/4f9f8335384827bb6918a588.png"},{"id":107448134,"identity":"ac812d68-4ea6-4177-a1e3-9e0430a8e399","added_by":"auto","created_at":"2026-04-21 14:56:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":461013,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eTrap1\u003c/em\u003e overexpression alleviates hypoxia-induced neurodevelopmental damage and synaptic plasticity changes.\u003cstrong\u003eA\u003c/strong\u003e-\u003cstrong\u003eD\u003c/strong\u003eNeurodevelopmental protein expression upon knockdown and overexpression of TRAP1(mean ± SD; n = 3-6 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05) \u003cstrong\u003eE.F\u003c/strong\u003e Synaptic plasticity protein mRNA levels in response to TRAP1 knockdown and overexpression (mean ± SD; n = 3-6 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-9067186/v1/ea078119428d4c9b4a4ec8fc.png"},{"id":107448059,"identity":"bcf64cb5-37f3-4fd5-98f4-af6be5996a21","added_by":"auto","created_at":"2026-04-21 14:56:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":486427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eTrap1\u003c/em\u003e overexpression rescues hypoxia-induced mitochondrial dysfunction.\u003cstrong\u003eA.B\u003c/strong\u003e Detection of mitochondrial markers(mean ± SD; n = 3-6 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05).\u003cstrong\u003eC\u003c/strong\u003e.\u003cstrong\u003eD\u003c/strong\u003e Detection of mPTP (mean ± SD; n = 3-6 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05).\u003cstrong\u003eE\u003c/strong\u003e.\u003cstrong\u003eF\u003c/strong\u003e Detection of Detection of MitoSOX(mean ± SD; n = 3-6 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05).\u003cstrong\u003eG \u003c/strong\u003eThe content of calcium ion in cells was detected by biochemical method(mean ± SD; n = 3-6 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05).\u003cstrong\u003e H,I \u003c/strong\u003eProtein expression levels of mitochondrial Ca²⁺-transport machinery (mean ± SD; n = 3–4 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003eJ\u003c/strong\u003e Representative whole-cell patch-clamp recordings and quantification of outward K⁺ currents in neurons subjected to TRAP1 knockdown or overexpression under hypoxia.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-9067186/v1/b291de1e125fe64b5565669c.png"},{"id":107448056,"identity":"74589850-ab80-4c5a-910b-73ea36c85987","added_by":"auto","created_at":"2026-04-21 14:56:31","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":552287,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eTrap1\u003c/em\u003e overexpression alleviates hypoxia-induced mitochondrial DNA damage.\u003cstrong\u003eA-D \u003c/strong\u003eExpression levels of genes involved in mitochondrial biogenesis when TRAP1 was knocked down and overexpressed(Mean±SD , n=3-6 per group. \u003cem\u003e*P\u003c/em\u003e\u0026lt;0.05).\u003cstrong\u003eE.F\u003c/strong\u003e Detection of DNA damage upon \u003cem\u003eTrap1 \u003c/em\u003eknockdown and overexpression(Mean±SD , n=3-6 per group. \u003cem\u003e*P\u003c/em\u003e\u0026lt;0.05).\u003cstrong\u003eG\u003c/strong\u003e-\u003cstrong\u003eI \u003c/strong\u003emRNA and protein levels of mtDNA in response to \u003cem\u003eTrap1\u003c/em\u003e knockdown and overexpression(Mean±SD , n=3-6 per group. \u003cem\u003e*P\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-9067186/v1/01314c28e06d9c290029a2f6.png"},{"id":107448135,"identity":"d10b4a91-f2b6-431b-8b70-7254f3238b49","added_by":"auto","created_at":"2026-04-21 14:56:43","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":217158,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eTrap1 o\u003c/em\u003everexpression effectively restored mitochondrial DNA binding.\u003cstrong\u003eA,B\u003c/strong\u003e Expression of DNA binding proteins and DNA damage repair proteins (mean ± SD; n = 3–6 per group; \u003cem\u003e*P\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-9067186/v1/508770356dc81a0382427efb.png"},{"id":107448091,"identity":"c79c7541-c8e5-43d3-9080-d2fc0a02377a","added_by":"auto","created_at":"2026-04-21 14:56:38","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":451784,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eTrap1 \u003c/em\u003eoverexpression effectively alleviated endoplasmic reticulum dysfunction under hypoxia.\u003cstrong\u003eA.B\u003c/strong\u003e Electron microscopy was used to detect the distance between mitochondria and ER(mean ± SD; n = 12 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05).\u003cstrong\u003eC.D\u003c/strong\u003e ER indicator showed ER morphology under different treatments(mean ± SD; n = 3-6 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05).\u003cstrong\u003eE.F \u003c/strong\u003eDetection of ER stress signals during \u003cem\u003eTrap1\u003c/em\u003e knockdown and overexpression1(mean ± SD; n = 3-4 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05).\u003cstrong\u003eG\u003c/strong\u003eCo-immunoprecipitation analysis of the interaction between TRAP1 and WNT5a.\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-9067186/v1/b484c55bd1ea512bb7526bfb.png"},{"id":107448086,"identity":"4d6859b7-808e-4101-bca9-efceb8a5ae1d","added_by":"auto","created_at":"2026-04-21 14:56:37","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":259489,"visible":true,"origin":"","legend":"\u003cp\u003eWNT5a agonist improves cell damage caused by hypoxia.\u003cstrong\u003eA\u003c/strong\u003e Immunoblotting was used to detect neurodevelopmental proteins, mitochondrial biogenesis proteins, DNA-binding proteins, and calcium transporters(mean ± SD; n = 3-4 per group; \u003cem\u003e*P\u003c/em\u003e\u0026lt; 0.05).\u003cstrong\u003eB\u003c/strong\u003e Cell viability was detected by CCK8 assay(mean ± SD; n =9 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05).\u003cstrong\u003eC \u003c/strong\u003eThe outward potassium current was measured by patch clamp.\u003cstrong\u003eD\u003c/strong\u003eATPase activity was detected by ATPase colorimetric method(mean ± SD; n = 9 per group; \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-9067186/v1/db57031f6c7c71d8d5903f7e.png"},{"id":107490423,"identity":"51cd8a9f-6d48-4682-ae7e-fe2dc21677e5","added_by":"auto","created_at":"2026-04-22 02:52:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5108948,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9067186/v1/d952cefa-4a6c-4173-b202-b54d935d0f44.pdf"},{"id":107448133,"identity":"099172a7-38b9-4269-8260-1f67d232c9ca","added_by":"auto","created_at":"2026-04-21 14:56:43","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3086597,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryWB.docx","url":"https://assets-eu.researchsquare.com/files/rs-9067186/v1/4f3580fe1c09026722478fce.docx"},{"id":107448088,"identity":"687e186d-fe68-48a0-bc25-7dfcbf20f809","added_by":"auto","created_at":"2026-04-21 14:56:37","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":294697,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9067186/v1/81cd72977d7c76ef7f0e8d91.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hippocampal TRAP1 Overexpression Mitigates Hypoxia-Evoked Learning and Memory Impairments via MERCS * major project of the National Natural Science Foundation of China （82130054）","fulltext":[{"header":"Background","content":"\u003cp\u003eHigh-altitude environments impose persistent hypobaric hypoxia, triggering a series of systemic pathophysiological adaptations. Acute hypoxia initially induces compensatory vasodilation, leading to a transient increase in cerebral oxygen delivery. However, prolonged or severe hypoxic exposure leads to a pathological cascade that is strongly implicated in the development of neurological diseases(Midha et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).The brain, particularly the hippocampus, exhibits high sensitivity to changes in oxygen availability(Chattopadhyaya \u003cem\u003eet al.\u003c/em\u003e 2025). CA1 pyramidal neurons within the hippocampus integrate multi - modal spatial and contextual information to establish activity - dependent synaptic plasticityWithin the hippocampus, CA1 pyramidal neurons integrate multi-modal spatial and contextual information to establish activity-dependent synaptic plasticity (Jain et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Zhao \u003cem\u003eet al.\u003c/em\u003e, 2022).This synaptic plasticity is fundamentally reliant on normal hippocampal neurodevelopmental programming, which requires preservation of dendritic architecture and efficient dendritic signal transduction. These signal transduction processes are essential for acquiring new spatial memories and enabling long-term retention of episodic information. These functions are collectively referred to as the \u0026ldquo;cognitive map\u0026rdquo; (O'Hare et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2025\u003c/span\u003e, Ben-Simon et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Cone and Clopath, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) \u0026mdash;and constitute the neurobiological substrate for learning and memory. Consequently, hypoxia-induced synaptic loss and ultrastructural remodeling within the CA1 region are strongly correlated with measurable cognitive impairments(Vanderlinden et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2025\u003c/span\u003e, Han et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Chen et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to generating ATP to meet the energy demands of neuronal activity, mitochondria act as the primary cellular oxygen sensors. The dynamic homeostasis of mitochondria is highly sensitive to hypoxia. Mild hypoxia initially promotes mitochondrial fusion, forming elongated mitochondrial networks as an adaptive response (Hao et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, prolonged hypoxic stress reverses this protective response and results in mitochondrial dysfunction (Wu et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Hypoxia-induced bursts of reactive oxygen species (ROS) inhibit oxidative phosphorylation and suppress mitochondrial biogenesis, compromising cellular energy metabolism (Guo et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Wang et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Zhong et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Dynamin-related protein 1 (DRP1) is recruited to mitochondria\u0026ndash;endoplasmic reticulum (ER) contact sites (MERCS), where it mediates mitochondrial fission and amplifies organellar fragmentation. This excessive fission disrupts inter-organellar communication, accelerates energy failure, and promotes neuronal death(Janbandhu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Duan et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e)Notably, MERCS further facilitate pathological crosstalk: mitochondria-derived ROS, including superoxide and hydroxyl radicals, traverse MERCS to oxidize ER membranes and induce ER stress (Shiiba et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e). The resultant accumulation of misfolded or excessive proteins in the ER triggers ER stress. Suppression of the proapoptotic transcription factor C/EBP homologous protein (CHOP) can confer cytoprotection (Chen et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). To restore ER luminal homeostasis, the stressed ER redistributes Ca\u0026sup2;⁺ to mitochondria and other cellular compartments via MERCS. However, this compensatory Ca\u0026sup2;⁺ transfer imposes an additional burden on mitochondria, leading to mitochondrial Ca\u0026sup2;⁺ overload. When the mitochondrial Ca\u0026sup2;⁺ buffering capacity is exceeded, the mitochondrial permeability transition pore (mPTP) opens, releasing Ca\u0026sup2;⁺ into the cytoplasm. Although this process temporarily alleviates mitochondrial matrix Ca\u0026sup2;⁺ overload, it ultimately amplifies cellular Ca\u0026sup2;⁺ dysregulation and activates cell death signaling pathways(Xian et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMitochondria contain an organelle-specific chaperone tumor necrosis factor receptor-associated protein 1 (TRAP1). This ATP-dependent heat-shock protein 90 (HSP90) paralog plays a crucial role in regulating client protein folding and facilitating the proteolysis of hypoxia-inducible factor-1α (HIF-1α)(Li et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003ea, Yoon et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Despite extensive studies on TRAP1 in oncology (Kim et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e), its role in hypoxic brain injury, particularly in the associated learning and memory impairments, remains largely unknown. In this study, we provide the first evidence that TRAP1 is a critical determinant of hypoxia-induced cognitive dysfunction. Our results indicate that TRAP1 regulates hippocampal neurodevelopment and synaptic plasticity by fine-tuning the structure and function of MERCS and synaptic plasticity to preserve hippocampus-dependent memory processes under hypoxic stress.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eRat and cell line\u003c/h2\u003e \u003cp\u003eThe rats in this experiment were purchased from Beijing Weitong Lihua Company and were healthy male SD rats aged 6 weeks, weighing between 200 and 240g. Qualified certificate No. : SCXK (Beijing) 2021-0006. The rats in the hypoxia group were exposed to a hypobaric chamber simulating an altitude of 5,000 m, while the control group was maintained in the natural environment of Xining, Qinghai Province. The rats were sacrificed by intraperitoneal injection of 20% urethane solution at a dose of 1g/kg. The HT22 hippocampal neuron cell line(Cat NO.: CL-0697) was obtained from Procell Life Science \u0026amp; Technology Co., Ltd. and authenticated by short tandem repeat (STR) profiling. The cells were cultured in DMEM/F12 medium (supplemented with AML12) containing 10% fetal bovine serum. The hypoxic cell model was established by exposing the cells to an atmosphere of 1% O₂.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlasmid and transfection\u003c/h3\u003e\n\u003cp\u003eLentiviral vectors encoding Trap1 (Trap1 overexpression, Trap1 OE) and small interfering RNAs (siRNAs) targeting Trap1 were purchased from Genechem Co., Ltd. (Shanghai, China). The sequences of the Trap1-specific siRNAs used for Trap1 silencing in HT22 cells are provided in the Supplementary Materials.\u003c/p\u003e\n\u003ch3\u003eMorris Water Maze Test\u003c/h3\u003e\n\u003cp\u003eA black circular pool (150 cm in diameter, 60 cm in height) was filled with water to a depth of 40 cm and maintained at a temperature of 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. Rats were subjected to a 5-day acquisition training period, followed by a probe trial. All behavioral data were recorded and analyzed using the same video-tracking software.\u003c/p\u003e\n\u003ch3\u003eMagnetic resonance imaging(MRI)\u003c/h3\u003e\n\u003cp\u003eInhalation anesthesia with 5% isofluranewas used for small animal MRI, and 1% isoflurane was used for maintenance anesthesia during scanning. Their respiratory status was continuously monitored using a pneumatic pillow sensor (SA Instruments, Stony Brook, NY, USA). High-resolution T2-weighted images were acquired in the axial, coronal, and sagittal planes with the following parameters: repetition time (TR)\u0026thinsp;=\u0026thinsp;3,000 ms, echo time (TE)\u0026thinsp;=\u0026thinsp;33 ms, echo-train length\u0026thinsp;=\u0026thinsp;8, field of view (FOV)\u0026thinsp;=\u0026thinsp;30 \u0026times; 30 mm2, matrix size\u0026thinsp;=\u0026thinsp;256 \u0026times; 256, 30 contiguous slices, and slice thickness\u0026thinsp;=\u0026thinsp;1.0 mm.\u003c/p\u003e\n\u003ch3\u003eHematoxylin - Eosin (HE) Staining\u003c/h3\u003e\n\u003cp\u003eParaffin-embedded hippocampal tissue sections were deparaffinized by sequential immersion in xylene followed by graded ethanol solutions (100%, 90%, 80%, and 70%), and then air-dried. Sections were subsequently stained with H\u0026amp;E according to standard protocols, examined under a light microscope at 40\u0026times; magnification, and images were captured.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNissl staining\u003c/h2\u003e \u003cp\u003eHippocampal tissues were rapidly dissected, fixed, dehydrated, and embedded in paraffin. The paraffin blocks were dried overnight at 60\u0026deg;C. The tissue sections were rehydrated through a series of gradient ethanol solutions, stained with 0.1% cresyl violet (Nissl stain), and briefly differentiated in 95% ethanol. After progressive dehydration in gradient ethanol solutions, the sections were cleared in xylene and mounted with neutral balsamAfter subjecting rats to euthanasia, hippocampal tissues were rapidly dissected, fixed, dehydrated, and embedded in paraffin. Paraffin blocks were dried overnight at 60\u0026deg;C. The tissue sections were rehydrated through a series of gradient ethanol solutions, stained with 0.1% cresyl violet (Nissl stain), and briefly differentiated in 95% ethanol. After progressive dehydration in gradient ethanol solutions, the sections were cleared in xylene and mounted with neutral balsam.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eFor immunohistochemical analysis, hippocampal tissue sections and HT22 cells were fixed, permeabilized, and blocked, followed by incubation with appropriately diluted primary antibodies overnight at 4\u0026deg;C. The following day, samples were incubated with species-matched horseradish peroxidase (HRP)-conjugated secondary antibodies at 37\u0026deg;C for 60 min. After washing, the sections were developed using 3,3\u0026prime;-diaminobenzidine chromogen. Nuclei were counterstained with hematoxylin for 1 min, after which sections were dehydrated, cleared, and mounted. Images were captured using a light microscope. A list of the primary antibodies used in this study is presented in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence staining\u003c/h3\u003e\n\u003cp\u003eAfter appropriate processing, cells were fixed, permeabilized, and blocked with phosphate-buffered saline (PBS), and blocked for 1 h at room temperature. The cells were then incubated with primary antibodies diluted in blocking buffer overnight at 4\u0026deg;C. The next day, the cells were washed three times with PBS and then incubated with fluorophore-conjugated secondary antibodies for 30 min at 37\u0026deg;C. After three additional washes with PBS, nuclei were counterstained with 4\u0026prime;,6-diamidino-2-phenylindole for 10 min at room temperature. A list of the primary antibodies used in this study is presented in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eCells were collected and centrifuged, and the supernatant was carefully removed. The cell pellet was resuspended in sheath fluid, gently vortexed to form a homogeneous suspension, and centrifuged again. After removal of the supernatant, cells were adjusted to a final concentration of 2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per sample and resuspended in MitoSO\u0026trade; Red staining working solution (1:2,000 dilution) to form a uniform single-cell suspension for flow cytometric analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscope(TEM)\u003c/h2\u003e \u003cp\u003eTissues or cells were fixed, dehydrated, infiltrated, and embedded in resin. Ultrathin sections (60\u0026ndash;90 nm) were prepared using an ultramicrotome and stained with uranyl acetate and lead citrate at room temperature. Images were acquired using a JEM-1400FLASH TEM (JEOL Ltd., Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative Real - Time Polymerase Chain Reaction(RT-qPCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using TRIzol reagent. The purity and concentration of the extracted RNA were measured using a NanoDrop\u0026trade; 2000 spectrophotometer. Two micrograms of total RNA were reverse - transcribed into complementary DNA (cDNA) using FastKing RT SuperMix according to the manufacturer's instructions. The cDNA samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until use. Gene - specific primers were synthesized by Shanghai Sangon Biotech Co., Ltd. qPCR reactions were prepared using the Tiangen SuperReal PreMix Plus kit and performed on an ABI Prism\u0026trade; Q5 Sequence Detection System.Total RNA was extracted using TRIzol reagent. The purity and concentration of the extracted RNA were measured using a NanoDrop\u0026trade; 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Two micrograms of total RNA were reverse-transcribed into complementary DNA (cDNA) using FastKing RT SuperMix (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions. The cDNA samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until use. Gene-specific primers were synthesized by Shanghai Sangon Biotech Co., Ltd. qPCR reactions were prepared using the Tiangen SuperReal PreMix Plus kit (Tiangen Biotech) and performed on an ABI Prism\u0026trade; Q5 Sequence Detection System (Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eCells were resuspended in ice-cold radioimmunoprecipitation assay lysis buffer. The cell lysates were centrifuged, and the supernatants were collected. Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit, and samples were normalized to ensure equal total protein loading. Protein aliquots were either snap-frozen or resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST) and incubated with primary antibodies diluted in blocking buffer overnight at 4\u0026deg;C. The following day, the membranes were washed with TBST and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized using an enhanced chemiluminescence substrate. A list of the primary antibodies used in this study is presented in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eProteomics Analysis\u003c/h2\u003e \u003cp\u003eTotal protein was extracted from each sample and divided into two aliquots. One aliquot was used for quantification and SDS-PAGE analysis to assess protein integrity, while the remaining aliquot was subjected to in-solution tryptic digestion and isobaric or isotopic labeling. Equal amounts of labeled peptides were pooled and fractionated using high-pH reversed-phase chromatography. The resulting peptide fractions were analyzed using nanoflow liquid chromatography-tandem mass spectrometry. Differentially expressed proteins and signaling pathways were identified based on statistical significance and biological relevance to the study objectives and were subsequently prioritized for downstream validation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCo-Immunoprecipitation (Co-IP)\u003c/h2\u003e \u003cp\u003eCells were lysed on ice, and protein concentrations were determined using the BCA assay. For immunoprecipitation, the cell lysates were transferred into spin columns with end caps, mixed with the appropriate primary antibody and incubation buffer, and rotated end-over-end for 2 h at 4\u0026deg;C. Following incubation, alkaline neutralization buffer and 5\u0026times; loading buffer were added to the samples, which were then heated in a boiling water bath for 5 min. Each immunoprecipitation eluate (40 \u0026micro;L) was resolved by SDS-PAGE and analyzed using western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eWhole - Cell Patch - Clamp Recording\u003c/h2\u003e \u003cp\u003eGlass micropipettes were fabricated from a single piece of Farad glass and fire-polished to achieve a tip resistance of 5\u0026ndash;8 MΩ. The seal resistance was rapidly increased to over 1 GΩ and allowed to stabilize for 2 min. Series resistance was compensated (typically\u0026thinsp;\u0026gt;\u0026thinsp;70%) before initiating the voltage protocol. Outward potassium (K⁺) currents were elicited using a holding potential of \u0026minus;\u0026thinsp;80 mV for 50 ms, followed by a depolarization to \u0026minus;\u0026thinsp;40 mV for 750 ms, and then a hyperpolarization to \u0026minus;\u0026thinsp;120 mV for 500 ms. K⁺ currents were continuously recorded throughout the experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eDNA Damage Assay\u003c/h2\u003e \u003cp\u003eFollowing routine washes and fixation, non-specific binding was blocked for 20 min at room temperature. Cells were then incubated with a γ-H2AX rabbit monoclonal antibody for 1 h at room temperature. After three washes with PBS, cells were incubated with an Alexa Fluor 488-conjugated anti-rabbit IgG for an additional hour at room temperature. Following further washes, nuclei were counterstained with DAPI for 5 min at room temperature, and the slides were mounted in anti-fade medium and examined under a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial Permeability Transition Pore(mPTP) Assay\u003c/h2\u003e \u003cp\u003eCells were incubated in the dark at 37\u0026deg;C for 30 min. The medium was replaced with fresh, pre-warmed (37\u0026deg;C) culture medium. After another 30 min of incubation in the dark, the medium was removed, and the cells were washed twice with PBS. A detection buffer was added, and cell fluorescence was immediately measured to assess mPTP opening.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eER-Tracker\u0026trade; Red staining\u003c/h2\u003e \u003cp\u003eCells were washed with Hank\u0026rsquo;s Balanced Salt Solution supplemented with Ca\u0026sup2;⁺ and Mg\u0026sup2;⁺ and incubated with ER-tracker\u0026trade; Red (1:1,000 dilution in complete medium) at 37\u0026deg;C for 20 min. The staining solution was aspirated, the cells were gently rinsed once with fresh, pre-warmed culture medium, and ER fluorescence was immediately visualized using a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMito-Tracker Green staining\u003c/h2\u003e \u003cp\u003eCells were incubated with Mito-Tracker\u0026trade; Green diluted 1:20,000 in pre-warmed medium at 37\u0026deg;C for 25 min. After staining, the solution was removed, and the cells were gently washed once with fresh, pre-warmed medium. Mitochondrial fluorescence was immediately visualized using a fluorescence microscope\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eCalcium Colorimetric Assay\u003c/h2\u003e \u003cp\u003eCells were lysed by adding ice-cold lysis buffer and gently pipetting to ensure complete lysis. Standards and samples were loaded into a 96-well plate, and 150 \u0026micro;L of detection working solution was added to each well. The plate was gently mixed and incubated in the dark at room temperature for 10 min. Absorbance at 575 nm was measured using a microplate reader, and Ca\u0026sup2;⁺ concentrations were calculated based on a standard curve.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eMitoSO\u0026trade; Red staining\u003c/h2\u003e \u003cp\u003eCulture medium was aspirated, and cells were rinsed once with pre-warmed PBS. Cells were then incubated with MitoSO\u0026trade; Red working solution at 37\u0026deg;C for 30 min. After incubation, the dye was removed, and the cells were washed twice with PBS. Mitochondrial superoxide-associated fluorescence was immediately visualized in PBS using a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eATPase Activity Assay\u003c/h2\u003e \u003cp\u003eThe cell pellet was homogenized in ice-cold 0.9% saline at a ratio of 1:5 (w/v). The homogenate was centrifuged at 10,000 \u0026times; g at 4\u0026deg;C for 10 min, and an aliquot of the supernatant was reserved for protein concentration determination. ATPase activity was calculated using the following formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:U/g\\:protein=\\frac{\\left(\\varDelta\\:A\u0026minus;B\\right)}{a}\u0026divide;{C}_{pr}\u0026divide;T\\times\\:f\\times\\:1000$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere ΔA is the absorbance difference between the sample and the blank, B is the blank absorbance, a is the slope of the standard curve, Cpr is the protein concentration of the sample, T is the reaction time, and f is the dilution factor.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed at least three times independently. Pairwise comparisons were analyzed using an unpaired two-tailed Student t-test. Normally distributed data were compared using one-way analysis of variance followed by Tukey's post-hoc test. For non-normally distributed data, the Kruskal\u0026ndash;Wallis test with Dunn's correction was used. Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003eHypoxia impairs learning\u0026amp;memory\u003c/h2\u003e \u003cp\u003eAcute hypoxia disrupts hippocampal-dependent memory and higher-order cognitive functions(Chattopadhyaya et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025b\u003c/span\u003e). In this study, the Morris water maze test revealed a significant decrease in the number of platform crossings in the hypoxia group (H28) compared to that in the control group (C28) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), indicating impaired spatial learning and memory recall. To investigate the structural basis of this cognitive deficit, we subjected mice to high-resolution MRI, which revealed an indistinct hippocampal silhouette accompanied by a conspicuous \u0026ldquo;burr\u0026rdquo; phenomenon in the H28 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Consistently, immunostaining for the neuroblast marker doublecortin (DCX) demonstrated a significant downregulation in DCX expression within the hippocampal neurogenic niche of the H28 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Histological analysis using HE and Nissl staining revealed extensive vacuolization of pyramidal neurons in the CA1 stratum, accompanied by marked neuronal degeneration and loss of cellular architecture (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). At the molecular level, we observed significant reductions in the expression levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and glial fibrillary acidic protein (GFAP) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). These results suggest that hypoxia elicited a global suppression of neurodevelopmental trophic signaling.\u003c/p\u003e \u003cp\u003eGiven that BDNF is indispensable for activity-dependent hippocampal plasticity, reduced BDNF signaling impairs synaptic adaptation (Chen et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). In the H28 group, TEM further confirmed a diffuse and poorly defined synaptic ultrastructure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), consistent with impaired synaptogenesis. Moreover, this group exhibited increased expression levels of postsynaptic density-95 (PSD-95), a key scaffold protein that maintains the integrity of excitatory synapses(Dar et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), but significantly decreased α-synuclein (α-SYN) expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). These molecular changes were accompanied by significant neurodevelopmental retardation at the cellular level (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI\u0026ndash;K) and a parallel erosion of synaptic plasticity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL, M). Collectively, these data demonstrate that hypoxia triggers a cascade of events, including delayed neurodevelopment, neuronal vacuolization, and altered synaptic plasticity, ultimately leading to cognitive dysfunction in the hippocampus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eHypoxia triggers mitochondrial and ER dysfunction\u003c/h2\u003e \u003cp\u003eDepletion of BDNF induces abnormal mitochondrial reorganization and accumulation of dysfunctional mitochondria(Ahuja et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).Consistent with this notion, our quantitative proteomics analysis confirmed downregulation of mitochondrial-related proteins and DNA-binding proteins under hypoxic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Moreover, fluorescence imaging revealed enhanced mitochondrial fluorescence and perinuclear aggregation of mitochondria in hypoxic neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Consistent with the increased opening of the mPTP, we detected a significant increase in mitochondrial ROS production and Ca\u0026sup2;⁺ influx, which was further confirmed by subsequent biochemical assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD,E). Similarly, the expression levels of proteins involved in mitochondrial Ca\u0026sup2;⁺ transport, including calmodulin, mitochondrial calcium uniporter (MCU), and mitochondrial calcium uptake 1 (MICU1), were also significantly altered under hypoxia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, G).\u003c/p\u003e \u003cp\u003eAs abnormal neurophysiological signaling is increasingly recognized as a driver of cognitive decline(Jiang et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), we conducted whole-cell patch-clamp recordings. We observed that outward K⁺ currents were significantly attenuated under hypoxia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH), which may reduce the efficiency of neuronal repolarization and increase Ca\u0026sup2;⁺ influx.\u003c/p\u003e \u003cp\u003eCollectively, these data demonstrate that hypoxia impairs mitochondrial integrity, induces excessive ROS production, and causes intracellular Ca\u0026sup2;⁺ overload, collectively contributing to the disruption of neuronal homeostasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExcessive ROS results in cumulative mtDNA damage, establishing a self-propelling vicious cycle. While mitochondrial transcription factor A (TFAM) normally protects mtDNA from oxidative damage and facilitates the autophagic clearance of leaked mtDNA(Liu et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2024\u003c/span\u003ea),, its protective capacity is overwhelmed under sustained oxidative stress. Consistent with impaired mitochondrial biogenesis, our results indicate that hypoxia markedly decreased the import receptor Tomm20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), resulting in impaired mitochondrial protein import and concurrent suppression of mtDNA transcription, accompanied by severe mitochondrial ultrastructural damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u0026ndash;E).\u003c/p\u003e \u003cp\u003eWhen genotoxic stress overwhelms cellular DNA repair capacity, cells activate intrinsic DNA damage response (DDR) pathways. However, the effectiveness of these self-protective mechanisms is often compromised by exogenous insults(Huang and Zhou, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).Therefore, we quantified the expression levels of key mtDNA maintenance proteins and found that hypoxia significantly downregulated the expression of the architectural chromatin factor (\u003cem\u003eSatb1\u003c/em\u003e), the single-stranded DNA-binding protein (\u003cem\u003eSsbp1\u003c/em\u003e), and the mitochondrial DNA polymerase accessory subunit \u003cem\u003e(Polg2\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G).\u003c/p\u003e \u003cp\u003eCollectively, these data demonstrate that hypoxia suppresses mtDNA replication and mitochondrial biogenesis and impairs the DNA damage repair machinery, exacerbating ROS-mediated genotoxicity in the hippocampal mitochondrial compartment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInter-organellar communication is essential for signal transduction and cellular homeostasis(Guan et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).In this study, high-resolution electron microscopy revealed that hypoxia significantly increased the minimum gap between mitochondria and the ER, indicating enhanced mitochondria\u0026ndash;ER contact (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Concurrent with these changes, ER-tracker fluorescence was attenuated, and the ER reticular network adopted a fragmented, perinuclear-excluded distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eElevated mitochondrial calcein fluorescence indicates increased Ca\u0026sup2;⁺ efflux from the ER lumen. However, prolonged ER Ca\u0026sup2;⁺depletion is a well-established trigger of ER stress(Zhang et al., \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e).The ER-resident chaperone BIP (immunoglobulin heavy-chain binding protein) is the master regulator of the ER stress response; its downregulation compromises the protein-folding capacity and accelerates cellular damage(Yang et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).Under physiological conditions, BIP associates with ER sensors, such as ATF6, to regulate proteostasis(Lim et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).In our hypoxic model, we observed downregulation of BIP levels, ATF6, and other unfolded protein response (UPR) transducers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u0026ndash;F). Moreover, the expression of the proapoptotic transcription factor CHOP, which translocates to the mitochondria to induce mitochondrial dysfunction(Xu et al., \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), was significantly upregulated. These findings indicate that hypoxia-driven ER\u0026ndash;mitochondrial Ca\u0026sup2;⁺ flux and BIP depletion converge to initiate ER stress and propagate mitochondrial injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eHypoxia suppresses the expression of Wnt5a\u003c/h2\u003e \u003cp\u003eCanonical WNT signaling is an essential regulatory pathway for neurodevelopment, governing synaptogenesis, synaptic maturation, and activity-dependent plasticity. Consistent with its role in cognitive function, our results indicate that hypoxia-induced cognitive decline is accompanied by a significant Wnt pathway downregulation(Soudy et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2025\u003c/span\u003e)(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHypoxia suppresses the expression of TRAP1\u003c/h3\u003e\n\u003cp\u003eTRAP1 regulates mitochondrial bioenergetics and proteostasis (Li et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003eb), and emerging evidence suggests that hypoxic stress suppresses TRAP1 expression, contributing to neuronal injury (Kim et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e, Liu et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Consistently, our results revealed that hypoxia significantly inhibited TRAP1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;E). Therefore, to further clarify the causal role of TRAP1 in hypoxia-induced neuronal dysfunction, we performed Trap1 knockdown and overexpression experiments in neurons. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF\u0026ndash;H, we confirmed the efficiency of the knockdown and overexpression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTrap1\u003c/b\u003e \u003cb\u003eupregulation ameliorates hypoxia-induced neurodevelopmental deficits and restores impaired synaptic plasticity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur results indicated that hypoxia-induced Trap1 downregulation further suppressed the expression of neurodevelopmental markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;D) and key synaptic plasticity-related proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, F). In contrast, lentivirus-mediated restoration of \u003cem\u003eTrap1\u003c/em\u003e expression completely reversed these deficits. These results highlight the protective role of TRAP1 against hypoxia-induced neuronal injury(Chen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTrap1\u003c/b\u003e \u003cb\u003eupregulation mitigates hypoxia-induced mitochondria and ER dysfunction\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur results demonstrated that Trap1 overexpression normalized the hypoxia-induced ultrastructural abnormalities of mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, B). Moreover, genetic silencing of \u003cem\u003eTrap1\u003c/em\u003e exacerbated mPTP opening, whereas its re-expression significantly reduced mPTP opening, thereby attenuating both ROS overproduction and Ca\u0026sup2;⁺ influx (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC\u0026ndash;F).\u003c/p\u003e \u003cp\u003eDisruption of mitochondrial redox homeostasis leads to excessive ROS production that exceeds antioxidant capacity, impairing oxidative metabolism and compromising cell viability. Consistent with this mechanism, \u003cem\u003eTrap1\u003c/em\u003e knockdown further increased mitochondrial ROS generation under hypoxia, while \u003cem\u003eTrap1\u003c/em\u003e up - regulation restored ROS levels to baseline (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG). Similarly, \u003cem\u003eTrap1\u003c/em\u003e depletion increased mitochondrial Ca\u0026sup2;⁺ uptake by up - regulating Ca\u0026sup2;⁺ transporters, an effect that was reversed by \u003cem\u003eTrap1\u003c/em\u003e overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH,I).Restoration of mitochondrial Ca\u0026sup2;⁺ homeostasis is crucial for functional recovery and neuronal survival(Liiv et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Electrophysiological recordings further revealed that \u003cem\u003eTrap1\u003c/em\u003e overexpression enhanced outward K⁺ currents, reducing voltage-gated Ca\u0026sup2;⁺ entry and alleviating cellular Ca\u0026sup2;⁺ overload (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eJ). Collectively, these data indicate that TRAP1 acts as a critical molecular regulator that decreases hypoxia-induced mitochondrial ROS production and alleviates Ca\u0026sup2;⁺ dysregulation to preserve neuronal integrity under hypoxic conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSilencing of \u003cem\u003eTrap1\u003c/em\u003e further suppressed mitochondrial biogenesis, whereas its re-expression completely restored hypoxia-induced reduction in TFAM and TOMM20 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA\u0026ndash;D). Concurrently, the expression levels of nuclear and mitochondrial DNA damage markers returned to baseline (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE, F). Therefore, to investigate whether DNA damage further affects mtDNA, we evaluated mtDNA expression. Our results indicated that \u003cem\u003eTrap1\u003c/em\u003e overexpression significantly increased mtDNA levels under hypoxic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eG\u0026ndash;I), supporting the notion that enhancing mtDNA integrity can improve cognitive function(Li et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).We then investigated whether TRAP1 also promotes the DNA-binding events required for mtDNA maintenance using CO-IP. These experiments revealed that \u003cem\u003eTrap1\u003c/em\u003e overexpression significantly enhanced DNA binding under hypoxic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA, B).\u003c/p\u003e \u003cp\u003eCollectively, these data demonstrate that TRAP1 is a critical factor that mitigates hypoxia-induced mtDNA damage and regulates the subsequent repair and transcriptional reactivation of mtDNA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur results also indicated that \u003cem\u003eTrap1\u003c/em\u003e silencing decreased the distance between mitochondria and the ER, whereas its overexpression increased this inter-organellar spacing. These results indicate that TRAP1 positively regulates mitochondria\u0026ndash;ER contact (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eA\u0026ndash;D). Moreover, \u003cem\u003eTrap1\u003c/em\u003e depletion exacerbated hypoxia-induced ER stress, whereas \u003cem\u003eTrap1\u003c/em\u003e overexpression inhibited UPR signaling and restored ER homeostasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eE, F).\u003c/p\u003e \u003cp\u003eCollectively, these findings indicate that TRAP1 regulates hypoxia-induced ER stress by stabilizing mitochondria\u0026ndash;ER tethering, functioning as a critical regulator of inter-organellar communication under hypoxic conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eTRAP1 interacts with Wnt5a and rescues its hypoxia-suppressed expression\u003c/h2\u003e \u003cp\u003eAs our results revealed decreased WNT5a expression under hypoxic conditions, we further explored whether WNT5a interacts with TRAP1. Co-IP experiments revealed an interaction between TRAP1 and WNT5a, which was weakened under hypoxic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eG).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eWnt5a agonist up-regulates TRAP1 and attenuates neuronal injury under hypoxia\u003c/h2\u003e \u003cp\u003eTRAP1 maintains mitochondrial homeostasis and exerts cytoprotective effects through crosstalk with the WNT signaling pathway. As hypoxia suppresses TRAP1 expression, we further investigated whether WNT pathway activation could reverse this downregulation. Administration of a WNT5a-specific agonist restored TRAP1 protein levels under hypoxic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eA). Moreover, the agonist upregulated the expression of neurodevelopmental markers, promoted mitochondrial biogenesis, reduced DNA damage foci, and alleviated mitochondrial Ca\u0026sup2;⁺ overload (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eA). Functional assays further revealed enhanced cell proliferation, increased outward K⁺ currents, and elevated ATPase activity. Overall, these findings suggest that TRAP1 upregulation through WNT5a activation effectively rescues hypoxia-induced mitochondrial dysfunction and neuronal injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDuring hypoxic brain damage, long-term potentiation in hippocampal circuits is suppressed(Zhao et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), providing a physiological basis for the observed learning and memory deficits. In the present study, we confirmed previous reports that sustained hypoxia causes hippocampal injury and subsequent cognitive decline. Our morphometric and functional analyses revealed that chronic hypoxia induces: (i) significant remodeling of hippocampal architecture, (ii) downregulated expression of neurodevelopmental markers, (iii) ultrastructural changes in the synapses of CA1 pyramidal neurons, and (iv) increased death of pyramidal cells. These structural and molecular alterations directly impair hippocampus-dependent cognitive function, particularly during the early stages of hypoxic exposure(Geigenmuller et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBDNF regulates neural development by activating distinct signaling cascades that control synaptogenesis and short-and long-term synaptic plasticity in a spatially and temporally restricted manner(Wang et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Under physiological conditions, activity-dependent BDNF release from presynaptic terminals enhances synaptic connectivity and plasticity(Taylor et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), whereas pathological reductions in BDNF lead to cognitive dysfunction(Kang et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).Notably, this phenomenon was recapitulated in our hypoxic model. Although traditionally associated with T-cell development, NGF is also crucial for neuronal survival and axonal innervation(Pozzer et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2025\u003c/span\u003e, Lian et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).Specifically, injured neurons disrupt network signaling by amplifying circuit-level damage in an NGF-dependent manner(Zhi et al., \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Moreover, GFAP downregulation results in abnormal mitochondrial morphology and accelerates cellular aging(Popov et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). DCX, a microtubule-associated phosphoprotein expressed by immature neurons, regulates cytoskeletal dynamics required for neuronal migration and hippocampal morphogenesis. Reduced DCX levels impair nuclear motility during early neurodevelopment(Sebastien et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Consistent with these observations, our results indicated that hypoxia results in downregulated DCX expression, impaired neurodevelopment, and compromised synaptic plasticity.\u003c/p\u003e \u003cp\u003eBioenergetic failure disrupts inter-neuronal mitochondrial transfer, which is essential for maintaining metabolic homeostasis, leading to the accumulation of dysfunctional mitochondria and impaired neuronal energy utilization, a key pathological feature of neuronal injury(Cheng et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Mitochondria function as central metabolic hubs whose dynamic cycles of fission and fusion actively regulate intracellular signal transduction(Konig and McBride, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).As synaptic transmission is energetically dependent on mitochondrial ATP production, any impairment of mitochondrial function directly affects neuronal survival. Our results confirmed that hypoxia causes rapid mitochondrial damage. Specifically, the loss of redox balance leads to an ROS burst that overwhelms the endogenous antioxidant system, disrupts oxidative phosphorylation, and impairs global cellular physiology(DiGiovanni et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Excessive ROS and cytosolic Ca\u0026sup2;⁺ lead to mitochondrial swelling, mPTP opening, and additional ROS releases into the cytoplasm; this amplifying loop that further exacerbates cellular injury(Yang et al., \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).Mitochondrial function critically depends on the electrochemical gradient across the inner mitochondrial membrane, known as the mitochondrial membrane potential, which regulates all ionic fluxes. Therefore, disruption of ionic homeostasis across this membrane leads to the collapse of the mitochondrial membrane and subsequent cellular dysfunction(Szabo and Szewczyk, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Mitochondrial permeability transition refers to the size-selective passage of solutes across the normally impermeable inner mitochondrial membrane through a pore formed at the interface between the cristae and the outer mitochondrial membrane(Bonora et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMitochondrial Ca\u0026sup2;⁺ uptake and efflux are integral to metabolic regulation and intracellular signaling, and disruptions in this cycle are implicated in the pathogenesis of acquired disorders, including neurodegenerative diseases(Garbincius and Elrod, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Ca\u0026sup2;⁺ uptake through MCU buffers physiological Ca\u0026sup2;⁺ transients required for synaptic memory formation(Gherardi et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2025\u003c/span\u003e, Obara et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, prolonged Ca\u0026sup2;⁺ sequestration, induced by stress or external stimuli, disturbs mitochondrial homeostasis and compromises cell viability(Cartes-Saavedra et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).This calcium overload induces oxidative stress and ATP depletion, ultimately leading to cellular injury(Wang et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).Moreover, elevated intracellular Ca\u0026sup2;⁺ activates calmodulin-dependent kinase II, which is a pivotal signaling molecule that regulates activity-dependent synaptic plasticity, learning, and memory(Nicoll and Schulman, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In the present study, using whole-cell patch-clamp recordings, we observed that hypoxia suppresses outward K⁺ currents, increasing Ca\u0026sup2;⁺ influx. Collectively, these results indicate that the combined effects of ROS and Ca\u0026sup2;⁺ overload indirectly disrupt the electrophysiological mechanisms underlying hippocampus-dependent learning and memory.\u003c/p\u003e \u003cp\u003eThe high replication rate of mtDNA increases the likelihood of replication errors, rendering the mitochondrial genome more susceptible to oxidative damage(Milano et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Mitochondrial Ca\u0026sup2;⁺ overload further impairs mitochondrial integrity and can trigger the release of mtDNA fragments into the cytoplasm; this process is called \u0026ldquo;mtDNA escape\u0026rdquo;(Zhao et al., \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).In the nucleus, SATB1 recruits chromatin-remodeling complexes to specific DNA elements, shaping higher-order chromatin structure and regulating gene transcription[67](Zelenka et al., \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In contrast, mitochondrial SSBP1 stabilizes single-stranded mtDNA intermediates, preventing replication fork collapse and subsequent development of mitochondrial diseases(Riccio et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Nie et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).As single-strand breaks are the most common form of DNA damage (Caldecott, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), we investigated SSBP1 expression and found that it was significantly downregulated under hypoxic conditions. Moreover, we observed that hypoxia suppressed the entire transcriptional program that controls mitochondrial biogenesis. TFAM, which packages mtDNA into nucleoid structures, promotes the autolysosomal degradation of leaked cytosolic mtDNA(Liu et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2024\u003c/span\u003eb).Therefore, TFAM depletion allows immunogenic mtDNA fragments to accumulate and activate the mitochondrial UPR. Notably, persistent activation of this response leads to an integrated stress response that, if unresolved, causes irreversible mitochondrial dysfunction, cellular damage, and ultimately programmed cell death(Bond et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e, Yang et al., \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).In addition, impaired mitochondrial biogenesis further compromises the DNA-binding machinery required for genome maintenance. Upon detection of DNA lesions, nuclear sensors recruit DDR proteins to initiate repair(Chappidi et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In mitochondria, TFAM packages mtDNA into nucleoids that serve as platforms for stress-induced DNA repair (Dai et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Urrutia et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).This process is essential for mitochondrial and cellular survival under adverse conditions (Lascaux et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The accessory subunit POLG2 promotes mtDNA replication by enhancing DNA polymerase-γ processivity and facilitating stable primer-template binding(Valenzuela et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e), and pathogenic mutations in POLG2 can cause mitochondrial disease(Yang et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Corra et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In this study, using a 48h hypoxic challenge, we observed a marked attenuation of both nuclear and mtDNA repair capacity. Persistent hypoxia prevented efficient resolution of DNA damage, leading to cumulative damage that exceeds the cellular repair thresholds. As unrepaired DNA damage accumulates, proapoptotic signaling cascades are activated, reducing cell survival(Wheeler et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and ultimately triggering programmed cell death(Huang et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOrganelle contact sites serve as signaling hubs that preserve intracellular homeostasis. Among these, MERCS are the most extensively studied. Compact MERCS function as metabolic platforms where tethering proteins on both membranes coordinate lipid transfer, Ca\u0026sup2;⁺ signaling(Pihan et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2025\u003c/span\u003e, Shiiba et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2025b\u003c/span\u003e), ER stress, and mitochondrial homeostasis(Zhang et al., \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). Conversely, excessive ROS can diffuse across MERCS and oxidize ER-resident proteins, leading to protein misfolding. The accumulation of these misfolded proteins can trigger ER stress cascades and alter the ER structure and luminal organization. Using high-resolution imaging, we found that hypoxia shortens the inter-organellar gap and enhances mitochondria\u0026ndash;ER communication, suggesting a compensatory effort to redistribute metabolites and mitigate stress. Given that the ER is the largest intracellular Ca\u0026sup2;⁺ reservoir, we observed that mitochondria transiently pump Ca\u0026sup2;⁺ into the ER via MERCS. This flux of Ca\u0026sup2;⁺ induces luminal swelling and cisternal expansion as the ER attempts to buffer the ionic load. When hypoxia persists, this delicate balance is disrupted: MERCS become dysfunctional, Ca\u0026sup2;⁺ transfer is impaired, and cellular injury is exacerbated (Zhang et al., \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).Overall, sustained hypoxia depletes ER luminal redox enzymes, impairs protein folding, and prevents the resolution of ER stress(Ugalde et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), thereby driving neurons toward pyroptotic death(Bhardwaj et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In contrast, hypoxia also upregulates the transcription of BIP and other canonical sensors, driving the UPR(Zhao et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Wiseman \u003cem\u003eet al.\u003c/em\u003e, 2022). However, ATF6-dependent release of mtDNA simultaneously amplifies ER stress(Rosing et al, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and genetic ATF6 suppression is required to restore proteostasis, preserve hippocampal neuronal integrity, and rescue cognition(Chen et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Notably, this apparent contradiction is resolved by our finding that severe hypoxia impairs the ER machinery at multiple levels. Specifically, we revealed that although stress-related transcripts accumulate, the protein folding and degradation machineries remain functionally inactive, preventing adaptive UPR and leading to irreversible cellular damage.\u003c/p\u003e \u003cp\u003eThe expression and functional significance of TRAP1 in the hypoxic brain remain largely unexplored. In this study, we provide the first evidence that TRAP1 expression is specifically downregulated in hippocampal neurons under chronic hypoxic stress. Our results indicated that, under these conditions, genetic \u003cem\u003eTrap1\u003c/em\u003e suppression further impaired neurodevelopment and synaptic plasticity, whereas restoration of its expression reversed hypoxia-induced dendritic simplification and LTP deficits. These findings demonstrate that TRAP1 acts as a critical regulatory molecule that links hypoxic stress to cognitive dysfunction by preserving neuronal maturation and synaptic adaptability. Moreover, \u003cem\u003eTrap1\u003c/em\u003e overexpression markedly suppressed mPTP opening, ROS generation, and mitochondrial Ca\u0026sup2;⁺ load. The decrease in mitochondrial matrix Ca\u0026sup2;⁺ levels subsequently inhibited mPTP activation, preserving inner mitochondrial membrane integrity and enhancing ATP synthesis(He et al, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Whole-cell recordings further revealed that Trap1 overexpression restored the hypoxia-suppressed outward K⁺ current, reducing Ca\u0026sup2;⁺ influx and stabilizing the transmembrane potential essential for synaptic reliability.\u003c/p\u003e \u003cp\u003eIn addition to its role in neurodevelopment, elevated TRAP1 expression strongly promotes mitochondrial biogenesis under hypoxia, accelerates mtDNA synthesis, and mitigates mtDNA damage. Enhanced mtDNA content increases oxidative phosphorylation capacity and restores efficient mitochondrial respiration(Valenzuela et al, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2025b\u003c/span\u003e). Moreover, phosphorylation-dependent release of SSBP1 from mtDNA reduces its mitochondrial localization, preventing excessive replication fork stalling and the associated ROS burst, thus alleviating mitochondrial stress (Zhang et al, \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).Our morphometric analysis revealed that Trap1 overexpression increased the distance between mitochondria and the ER under hypoxia, reducing inter-organellar cargo transfer, particularly that of ROS and Ca\u0026sup2;⁺. Notably, this spatial separation limits ROS-mediated ER oxidation, prevents mitochondrial Ca\u0026sup2;⁺ overload, and preserves mitochondrial matrix bioenergetics, thereby increasing neuronal ATP availability and improving cognitive performance(Korte et al, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Consistently, the normalization of the expression of ER stress sensors reflects the comprehensive restoration of cellular homeostasis and synaptic function.\u003c/p\u003e \u003cp\u003eGiven our functional data and proteomic signatures of hypoxia-induced mitochondrial Ca\u0026sup2;⁺ overload and ER stress, we investigated the WNT signaling pathway and identified it as a key modulator of TRAP1 expression. Based on our findings, we propose that TRAP1 interacts with WNT5a to: (i) restore mitochondrial and ER homeostasis, (ii) promote hippocampal neurodevelopment, and (iii) rescue synaptic plasticity, alleviating hypoxia-induced neuronal injury. Moreover, pharmacological WNT5a activation under hypoxic stress increased TRAP1 expression, suppressed DRP1-mediated mitochondrial fission, normalized mitochondrial Ca\u0026sup2;⁺ handling, and upregulated BDNF and DCX levels. Mitochondria play a key role in cell growth and proliferation through ATP synthesis. Under hypoxic conditions, DRP1 is activated and promotes excessive mitochondrial fission, increasing MERCS formation(Duan et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). While DRP1 normally mediates lipid turnover in neuronal mitochondria to maintain energy balance(Haynes et al, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), its hyperactivation under hypoxia has detrimental effects. Inhibiting DRP1-mediated fission disrupts mitochondrial dynamics and reduces the generation of mitochondria enriched in ATP synthase(Ryu et al, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), ultimately disrupting oxidative phosphorylation and inducing oxidative stress(Hong et al, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Notably, \u003cem\u003eTrap1\u003c/em\u003e upregulation counteracts these pathological changes by modulating mitochondrial\u0026ndash;ER interactions. It also increases ATP production and globally restores neuronal function.Overall, these results indicate that WNT5a-driven \u003cem\u003eTrap1\u003c/em\u003e upregulation is sufficient to reverse hypoxia-induced mitochondrial-ER dysfunction and cognitive deficits.\u003c/p\u003e \u003cp\u003eAs a mitochondria-restricted molecular chaperone, the function of TRAP1 remains poorly understood and understudied. In this study, we reveal its previously unrecognized role in mitochondrial quality control and hypoxic brain injury. We not only identify TRAP1 as a central regulator of mitochondria\u0026ndash;ER communication and hippocampal synaptic plasticity under hypoxic stress but also reveal a druggable WNT5a\u0026ndash;TRAP1 axis. Based on our results, we propose two complementary therapeutic strategies for hypoxia-induced cognitive deficits: (i) direct upregulation of TRAP1 and (ii) indirect upregulation through WNT5 activation, suggesting a potential new strategy for the clinical treatment of hypoxia-associated neurodegenerative disorders.\u003c/p\u003e \u003cp\u003eHowever, this study has certain limitations. First, this study lacked clinical specimens and supportive epidemiological data, limiting the translational relevance of our findings. Second, the precise subcellular localization of TRAP1, and, consequently, its compartment-specific functions, was not fully characterized. Finally, in vivo evidence that Trap1 overexpression rescues hypoxia-induced learning and memory deficits in intact animals remains limited, necessitating further investigations.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, TRAP1 overexpression is sufficient to rescue hypoxia-induced deficits in learning and memory, neuronal development, and synaptic plasticity. Pharmacological activation of the WNT signaling pathway further increased TRAP1 expression, restoring MERCS integrity and alleviating downstream organellar dysfunction. Overall, our findings suggest TRAP1 as a potential therapeutic target for hypoxia-associated cognitive decline and related neurodegenerative diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis experiment met the ethical standards for animal experiments and was approved by the Ethics Committee of the Affiliated Hospital of Qinghai University (Clinical Medical College) (Ethics approval number: P-SL-2023-448). All methods were carried out in accordance with relevant guidelines and regulations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT FOR PUBLICATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA \u0026nbsp; AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request. \u0026nbsp;All methods are reported in accordance with ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thanks the Science and Technology Department of Qinghai Talent Scientist Program（Grant No.2025-ZJ-748）and The major project of the National Natural Science Foundation of China（82130054）\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDECLARATION OF INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGuan Lu designed the study, performed most of the experiments, and drafted the manuscript. \u0026nbsp;Hao Chengxiao assisted with animal experiments, such as the Morris Water Maze test. Cao Rui assisted with patch - clamp recordings. Guo Yanrong participated in the study design. Ma Shuang participated in the study design and provided financial support. Ge Rili revised the manuscript and also provided financial support. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our gratitude to Genechem Co.,Ltd. (Shanghai, China), Proteintech Group, Inc(Wuhan, China) and OE Biotech. Co., Ltd.(Shanghai, China) for their technical support, and to Bioreder.com for their assistance with mapping technology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR\u0026rsquo;S INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAHUJA, P., NG, C. F., PANG, B., CHAN, W. S., TSE, M., BI, X., KWAN, H. 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(2022), \u0026quot;S100A9 Derived from Chemoembolization-Induced Hypoxia Governs Mitochondrial Function in Hepatocellular Carcinoma Progression\u0026quot;, \u003cem\u003eAdv Sci (Weinh),\u003c/em\u003e Vol. 9 No. 30, pp. e2202206.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"TRAP1, hypoxia, Learning and Memory, Mitochondria, ROS, ER, Wnt","lastPublishedDoi":"10.21203/rs.3.rs-9067186/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9067186/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChronic exposure to high-altitude hypoxia impairs hippocampus-dependent learning and memory. However, the upstream mitochondrial mechanisms by which oxygen deficiency triggers synaptic dysfunction remain incompletely understood. Therefore, the present study aimed to investigate the role of tumor necrosis factor receptor-associated protein 1 (TRAP1), a mitochondria-restricted chaperone, in this pathological process. In vivo, adult male Sprague-Dawley rats were exposed to a hypobaric chamber simulating an altitude of 5,000 m for 28 days. In vitro, HT22 hippocampal neurons were exposed to 1% oxygen (O₂) for 48 h to establish hypoxic cell models. TRAP1 was overexpressed using lentivirus or pharmacologically activated by a selective WNT5a agonist. Hippocampal structure and cognitive function were evaluated using the Morris Water Maze test for cognitive function assessment, high-resolution magnetic resonance imaging (MRI) for hippocampal volumetric and structural analysis, and whole-cell patch-clamp for neuronal recording. Moreover, the following series of experiments was conducted: transmission electron microscopy (TEM) for ultrastructural characterization of mitochondria\u0026ndash;endoplasmic reticulum (ER) contact sites (MERCS), fluorometric assays for intracellular calcium (Ca\u0026sup2;⁺) and reactive oxygen species (ROS) detection, reverse transcription quantitative PCR for mitochondrial DNA (mtDNA) integrity and gene expression quantification, and western blotting and co-immunoprecipitation analysis to assess the expression of synaptic-related proteins and their interactions. Hypoxia exposure led to a significant downregulation of hippocampal TRAP1 and WNT5a expression. It also caused a reduction in the MERCS gap width, a three-fold increase in mitochondrial ROS production, a two-fold elevation in mitochondrial matrix Ca\u0026sup2;⁺ concentration, a 50% decrease in mtDNA copy number, and significant reductions in the expression of brain-derived neurotrophic factor, doublecortin, and postsynaptic density protein 95. These pathological changes were accompanied by decreased platform crossings in the Morris water maze test and loss of hippocampal definition on MRI. Notably, Trap1 overexpression reversed these hypoxia-induced deficits: it restored MERCS gap width, normalized redox balance and intracellular Ca\u0026sup2;⁺levels, promoted transcription factor A mitochondrial-dependent mtDNA synthesis, recovered synaptic protein expression levels, and ultimately ameliorated neurodevelopmental impairment. In contrast, Trap1 knockdown recapitulated the hypoxic injury phenotypes. Additionally, treatment with a WNT5a agonist significantly upregulated TRAP1 expression, inhibited dynamin-related protein 1-mediated mitochondrial fission, and exerted a neuroprotective effect against hypoxia-induced neuronal damage. TRAP1 downregulation is an early and reversible event that links hypoxic stress to mitochondria ER dysfunction and cognitive impairment. Direct Trap1 overexpression or indirect upregulation via WNT5a agonism represents a promising dual therapeutic strategy for hypoxia-induced neurodegeneration and memory deficits.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Hippocampal TRAP1 Overexpression Mitigates Hypoxia-Evoked Learning and Memory Impairments via MERCS * major project of the National Natural Science Foundation of China （82130054）","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-21 14:54:52","doi":"10.21203/rs.3.rs-9067186/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-11T08:05:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-08T11:09:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-06T14:01:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"255517563335704122994195993697616187516","date":"2026-04-29T08:33:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"254463017227933745630366070970036467739","date":"2026-04-27T12:47:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-13T03:01:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-13T02:59:15+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-19T11:44:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-18T21:54:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-18T13:27:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f72900a0-4030-4254-a066-25a104173b12","owner":[],"postedDate":"April 21st, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-11T08:05:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-08T11:09:35+00:00","index":96,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-06T14:01:34+00:00","index":94,"fulltext":""},{"type":"reviewerAgreed","content":"255517563335704122994195993697616187516","date":"2026-04-29T08:33:17+00:00","index":93,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":66257743,"name":"Biological sciences/Cell biology"},{"id":66257744,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-05-13T08:57:23+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-21 14:54:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9067186","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9067186","identity":"rs-9067186","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}