Calbindin-D28k deficiency mediates tau-driven hippocampal hyperexcitement and cognitive impairment

Calbindin-D28k deficiency mediates tau-driven hippocampal hyperexcitement and cognitive impairment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Calbindin-D28k deficiency mediates tau-driven hippocampal hyperexcitement and cognitive impairment yang gao, Xiaoqing Tao, yuying wang, yarong wang, Huan Li, yang Yu, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6470265/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background Medial temporal lobe hyperexcitation or seizures originating from hippocampus are frequently observed in Alzheimer's disease (AD) patients, contributing to accelerated cognitive decline. Given the hippocampus's role as an early vulnerable area of tau pathology, a hallmark of AD, the mechanisms by which abnormal tau aggregation promotes temporal lobe epilepsy (TLE) remain poorly understood. Methods We investigated the role and mechanisms of AD-like hippocampal tau aggregation in neuronal hyperexcitation using transgenic (Tg) tau-driven mice. Tau aggregation on intracellular calcium dynamics was assessed by calcium imaging. Neuronal/network hyperexcitability and seizure susceptibility were evaluated through patch-clamp electrophysiology, 18 F-FDG PET/CT, and optogenetic induction. A tetracycline-controlled (tet-on) system in Tg hTau368 mice enabled spatiotemporal induction of tau pathology to investigate interactions with calbindin-D28k (CB) and synaptic proteins. Adeno-associated virus (AAV)-mediated CB supplementation in hippocampal CA1 and dentate gyrus (DG) excitatory neurons was tested for rescuing hyperexcitability and cognitive deficits. Finally, the relationship between CB and disease progress was analyzed using AD public database. Results Tau accumulation in hippocampal CA1/DG CaMKII-positive excitatory neurons reduced CB expression with disrupted calcium homeostasis. This dysregulation heightened neuronal excitability, diminished synaptic protein levels, and increased seizure susceptibility and cognitive impairment. AAV-driven CB restoration in CA1/DG neurons attenuated both hyperexcitability and cognitive deficits. In the brain of AD patients, the reduced CB expression was associated with cognitive deterioration and the advanced disease stages. Conclusions Tau aggregation drives CB-dependent calcium dysregulation and hippocampal neuronal hyperexcitation. These results establish a potential mechanistic link between tauopathy and TLE pathogenesis in AD, providing with CB as a promising therapeutic target for mitigating seizure risk and related cognitive decline in AD. Alzheimer's disease temporal lobe epilepsy tau hyperphosphorylation calbindin-D28k cognitive impairment calcium homeostasis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Alzheimer's disease (AD) is the most common and devastating neurodegenerative disease, with lack of effective prevention or approved therapies yet [ 1 – 3 ] . A striking clinical feature of AD is higher prevalence of temporal lobe epilepsy (TLE) and 42–64% of AD patients exhibiting overt epileptic seizures or subclinical epileptic activity [ 4 – 7 ] . Notably, early-onset AD patients (aged 50–60 years) show an 87-fold increased incidence of seizures compared to age-matched healthy individuals [ 8 ] . Intracranial recordings reveal that silent hippocampal seizures and spikes emerge during the preclinical phase of AD, preceding overt cognitive symptoms [ 9 ] . Human studies demonstrated that increased neuronal network and brain activity start as early as a decade before the onset age of disease [ 10 – 12 ] . Hippocampal excitability follows an inverted U-shaped trajectory during AD progression, with hyperexcitability in early stages transitioning to hypoexcitability in later phases [ 4 , 13 ] . Critically, seizures in AD patients accelerate cognitive decline and increase mortality risk due to trauma from sudden loss of consciousness [ 14 , 15 ] . Extracellular amyloid plaque deposition and intraneuronal neurofibrillary tangles (NFTs) in the brain are two characteristic pathological hallmarks of AD. However, they demonstrate distinct spatiotemporal progression patterns that Aβ deposition predominates in medial parietal and frontal cortices, whereas tau pathology initially accumulates within the entorhinal-hippocampal network at prodromal stages [ 16 , 17 ] . Asymmetrically deposited tau in the epileptogenic hemisphere was the most striking finding, whereas asymmetries in amyloid deposition were less pronounced in early clinical stages of AD with TLE [ 7 ] .The entorhinal-hippocampal network contains key excitatory pathways: entorhinal cortex (EC) efferents directly innervate the dentate gyrus (DG), where granule cells extend mossy fiber axons to CA3. CA3 neurons subsequently relay signals to CA1 via Schaffer collaterals, while parallel EC projections establish direct monosynaptic connections with CA1 pyramidal neurons. Notably, the DG-CA1-EC-DG loop has been identified as one of principal circuit involved in seizures generation and propagation of TLE [ 18 ] . Functional neuroimaging reveals enhanced blood-oxygen-level-dependent (BOLD) activation in hippocampal-temporal circuits during memory tasks in early AD [ 11 , 12 ] . Glutamatergic neurotransmission, mediated by CA1 pyramidal neurons and DG granule cells, critically regulates learning and memory processes [ 18 , 19 ] . Of particular significance, the CA1 pyramidal layer demonstrates selective vulnerability to early tau deposition [ 20 , 21 ] . Nevertheless, the precise mechanisms through which tau pathology disrupts hippocampal excitatory-inhibitory balance remain to be elucidated. Calcium homeostasis is of utmost significance in maintaining excitatory-inhibitory balance within neural circuits, regarded as an important target for the treatment of AD recently [ 22 , 23 ] . Among the various elements involved in calcium regulation, Calbindin - D28k (CB), a calcium - binding protein, is highly expressed in the neurons of the hippocampal CA1 and DG neurons [ 24 – 26 ] . Functionally, CB serves as a key regulator of intracellular free calcium ion concentrations, actively participating in the modulation of intracellular calcium homeostasis, synaptic plasticity, and cognitive processes [ 25 , 27 ] . A decline in CB expression has been consistently associated with cognitive impairment in patients suffering from AD and TLE [ 27 ] . Evidences from animal models further supports this relationship that knockdown of CB in the excitatory neurons of hippocampal CA1 and DG leads to significant impairments in the spatial memory of mice [ 26 ] . Clinical observations have also revealed reduced CB expression in the hippocampal CA1 and DG regions of AD and TLE patients [ 27 ] . Notably, the question of whether replenishing CB in the excitatory neurons of hippocampal CA1 and DG could alleviate neuronal over - excitation and mitigate cognitive impairment remains an open and compelling area of investigation. Addressing this question could potentially unlock new therapeutic strategies for treating AD and TLE. To address the relationship between tau aggregation and hippocampal hyperexcitability, we employed a newly created tauopathy Tg hTau368 mice to investigate tau aggregation feature and calcium dysregulation. Neuronal hyperexcitability and seizure susceptibility were evaluated using patch-clamp electrophysiology, 18 F-FDG PET/CT and optogenetic activation. A tetracycline-controlled system (Tet-on) enabled spatiotemporal induction of tau pathology in Tg hTau368 mice to dissect its interplay with CB and synaptic proteins. Furthermore, adeno-associated virus (AAV)-mediated CB restoration in hippocampal CA1/DG excitatory neurons was tested for its therapeutic effects on hyperexcitability and cognition. Public AD-related databases were mined to validate clinical correlations between CB levels and cognitive outcomes. Our findings demonstrate that tau accumulation in CA1/DG CamKIIα-positive neurons suppresses CB expression, triggering calcium dyshomeostasis. This cascade enhances hippocampal excitability, reduces synaptic protein expression, and increases susceptibility to seizures and cognitive deficits. Critically, AAV-delivered CB rescues neuronal hyperexcitability and cognitive impairment. These results identify CB/calcium homeostasis as a pivotal mechanism linking tau pathology to TLE and cognitive decline in AD, highlighting its potential as a therapeutic target. Methods Animals and treatment All mice were housed in groups of three to four per cage, under a 12 h light/dark cycle at 23–25°C. Food and water were available ad libitum . doxycycline hyclate (Dox, Beyotime, Shanghai, China) was dissolved in drinking water (2 mg/l) and administered ad libitum for Tg hTau368 mice. Equal numbers of male and female mice were randomly assigned to groups. All experiments and data analyses were conducted by experimenters blind to the groupings. All animal experiments were conducted in accordance with relevant ethical regulations for animal testing and research, and were approved by institutional guidelines and the Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology. Tg hTau368 and PR5 mice The Tg hTau368 mice were generated jointly by our laboratory and Nanjing Biomedical Research Institute of Nanjing University that human MAPT gene encoding the hTau368 fragment (2N4R tau) was placed downstream the tetracycline-responsive element (TRE) promoter, which joined to assemble a tet-on system with a second module expressing the reverse tetracycline-controlled transactivator (rtTA) under the control of the neuron-specific Eno2 promoter [ 28 ] . In the presence of Dox (Dox-on), reverse tetracycline transactivator (rtTA) binds to the tetracycline-responsive element (TRE) to initiate hTau368 expression. In the absence of doxycycline (Dox-off), hTau368 expression stops, as rtTA cannot bind to TRE, and tauophathology gradually dismissed [ 28 ] . PR5 mice, which overexpress the longest human tau isoform (2N4R tau) together with the P301L mutation on the C57BL/6 background. PR5 mice transgenic expression of human P301L mutant tau under control of the murine Thy1.2 promoter leads to the formation of sarcosyl-insoluble, 15-nm-wide tau filaments. By comparison, tau filaments in frontotemporal dementia with parkinsonism linked to chromosome 17 patients with the P301L mutation [ 29 , 30 ] . Antibodies Antibodies used in the present study are summarized in Table 1 . Table 1 Antibodies used in this study. Antibody Type Specificity Species Source/reference Tau368 Mono Tau 1-368 N M Jointly developed with AtaGenix [ 28 ] Tau5 Mono Tau (a.a.210–230) M Abcam (ab80579) HT7 Mono Tau (a.a.159–163) M ThermoFisher (MN10000) Anti-pT205 Poly p-tau (T205) R SAB (11108) AT8 Mono p-tau (S202/T205) M ThermoScientific (MN1020) PSD-95 Poly PSD-95 R SAB (41365) SYN-1 Mono Synapsin1 M CST (D12G5) β-actin Mono β-actin M SAB (21800) Anti-Calbindin Mono Calbindin-D28k R Abcam (ab108404) Calbindin Mono Calbindin-D28k M Sigma (C9848) CamKII Mono CamKII R Cell signaling (3362) GAD67 Mono GAD67 M Sigma (MAB5406) Anti-Parvalbumin Mono Parvalbumin M Sigma (SAB4200545) Dylight 800 Dylight 800 R Immunoway (RS23920) Dylight 800 Dylight 800 M Immunoway (RS23910) Stereotaxic viral injection Mice were anesthetized using isoflurane and secured in a stereotaxic frame (RWD Life Science, Shenzhen, China). The scalp was surgically incised to expose the skull, and residual soft tissue on the skull surface was meticulously removed using sterile cotton swabs. The coordinate origin was defined as the intersection of the bilateral tangents at the bregma. The skull was leveled by aligning the anterior and posterior bregma, ensuring a height discrepancy of less than 0.03 mm between the left and right sides. The target injection site was identified based on stereotaxic coordinates and marked on the skull. A small craniotomy was performed at the marked location using a precision cranial drill. Viral solution was delivered at a rate of 100 nl/min using a microinjection system and retained for 5 min post-injection. Kainic acid-induced epilepsy model and in vivo electrophysiological recording Kainic acid (KA), an analog of glutamate, was used to induce TLE by activating kainate receptors, leading to seizure activity, neuronal damage, and chronic spontaneous epilepsy. KA (500 nl, 0.5 µg/µl, dissolved in saline) was stereotaxically injected into the hippocampal CA1 (-1.0 mm AP, + 1.8 mm ML, -1.5 mm DV) and DG (-1.0 mm AP, + 1.8 mm ML, -2.1 mm DV) of 4-month-old WT mice. A 8-channel electrode was implanted 0.1 mm above the injection sites to record electrophysiological signals when mice waken up. Local field potentials were recorded using a Recording System (Plexon, Hong Kong, China). Data were stored for offline analysis with 16-bit format, visualized in Neuro Explore. Amplitude and power spectral density (PSD) of LFPs were analyzed in default parameters by NeuroExplorer (Plexon, Hong Kong, China) : shift (0.5 s), number of frequency values (8,192), normalization (log of PSD), show frequency from 0 to 150 Hz. Optogenetic stimulation and seizures induction The operation method has been reported by us before [31]. To put it simply, pAAV-CaMKIIα-ChR2 (H134R) -mCherry was stereotaxically injected into the right dorsal hippocampal CA1 (-1.0 mm AP, + 1.8 mm ML, -1.1 mm DV, 500 nl) and DG (-1.0 mm AP, + 1.8 mm ML, -2.1 mm DV, 500 nl). pAAV-CaMKIIα-ChR2 (H134R) -mCherry was stereotaxically injected into the right ventral hippocampus CA1 (-3.2 mm AP, + 3.2 mm ML, -4.1 mm DV, 500 nl) and DG (-3.2 mm AP, + 3.2 mm ML, -4.8 mm DV, 500 nl). Following viral injection, a fiber optic cannula (200 µm core, NA = 0.37) was implanted above 0.4 mm of CA1. A 8-channel electrode was implanted into the ipsilateral M1 (+ 1.5 mm AP, + 1.5 mm ML, − 1.5 mm DV) for electrophysiological signals recording described above. Three to four anchoring screws (25 µm diameter) were fixed into the skull near the injection site. The cannula and screws were secured with dental cement (RWD Life Science, Shenzhen, China). Four weeks post-surgery, seizures were induced. Mice were anesthetized with isoflurane, and the cannula was connected to a fiber optic patch cable and laser source. Laser parameters were set to 472 nm blue light, 20 Hz frequency, 10% duty cycle, and pulsed waveform, delivering 2.8 mW/mm² at the fiber tip. Seizure latency and severity were assessed using a modified Racine scale [32]: Stage 1, Facial twitching and chewing; Stage 2, Chewing and head nodding; Stage 3, Unilateral forelimb clonus; Stage 4, Bilateral forelimb clonus with rearing; Stage 5, Bilateral clonus, rearing, and falling; Stage 6, Wild jumping. Stages 1–3 were classified as focal seizures (FS), and stages 4–6 as generalized seizures (GS). The latency to stage 4 (GS) was recorded as the primary metric in our study. Upon reaching stage 4, stimulation continued for 15 s before cessation. The maximum seizure stage was recorded, and mice were monitored until normal behavior resumed. The fiber optic cable was removed under anesthesia, and mice were returned to their cages. Seizures were induced once daily, with GS latency and maximum seizure stage recorded over consecutive days. In some mice, a 8-channel electrode was implanted into the ipsilateral M1 (+ 1.5 mm AP, + 1.5 mm ML, − 1.5 mm DV) for electrophysiological recording (Supplementary Video 1). Small animal PET/CT imaging Brain 18 F-FDG PET/CT imaging was conducted using a small animal PET/CT scanner (Novel Medical, Beijing, China) at Union Hospital, Wuhan. Mice were fasted for 12 h, weighed, and injected with 3–4 MBq of 18F-FDG via the tail vein. Anesthesia was induced and maintained with 2% isoflurane. After a 45-minute uptake period, whole-body PET/CT scans were performed with the following parameters: energy window of 350–650 keV, timing window of 1.2 ns, spatial resolution of 1.5 mm, and scan duration of 300 s. Acquired DICOM images were analyzed using PMOD 4.0 software. A standardized mouse brain template was used to segment the brain into 19 regions of interest (ROIs), and the average 18 F-FDG uptake intensity for each ROI was quantified as standardized uptake value normalized to body weight. Monitoring of mouse energy metabolism and activity The Oxymax CLAMS system (Columbus Instruments, Columbus, USA) was employed to monitor the 24-h energy metabolism and locomotor activity of mice. Prior to the experiment, mice were acclimated to the laboratory environment for 24 hs. Each mouse was singly housed in a cage, ensuring an adequate Supplementaryy of food and water. Subsequently, the oxygen consumption (VO₂), carbon dioxide production (VCO₂), respiratory quotient (RQ), and spontaneous activity counts of each mouse were recorded over a 24-h period (light cycle: 7:00–19:00; dark cycle: 19:00-next day 7:00). The energy expenditure of mice was determined using indirect calorimetry, with the calculation formula as follows: Total energy expenditure = (3.815 + 1.232 × RQ) × VO₂. This system utilizes infrared photocell technology to enable three-dimensional monitoring of animal movements. Mouse locomotion interrupts the infrared beam, generating a count to quantify spontaneous movement. Calcium imaging recording Four weeks prior to imaging, pAAV-CaMKIIα-GCaMP6f-eGFP (500 nl) was injected into the right hippocampal CA1 region (-1.0 mm AP, + 1.8 mm ML, -1.5 mm DV) of mice in the Dox and Veh groups (n = 4 per group). Ice-cold slicing solution was prepared, and artificial cerebrospinal fluid (aCSF) was oxygenated (95% O₂, 5% CO 2 ) in a incubation chamber at 35°C for at least 30 min. Mice were anesthetized with 1% sodium pentobarbital (intraperitoneal injection) and decapitated. Brains were quickly removed, trimmed, and affixed to a vibratome stage using cyanoacrylate glue. Coronal slices (300 µm thickness) were cut in ice-cold slicing solution and transferred to oxygenated aCSF at 35°C for 30 min, followed by 1- h incubation at room temperature before imaging. Brain slices were placed under a confocal microscope equipped with a 40× water-immersion objective. The green fluorescence signal (GCaMP6f) in the CA1 pyramidal cell layer was identified and focused for imaging. Neuronal depolarization was induced by perfusing the slices with 30 mM KCl. Fluorescence signals were excited at 488 nm and recorded within the 505–550 nm emission range. Fluorescence signals were analyzed using ImageJ. Calcium signals were calculated as ΔF/F using the formula: ΔF/F = [(F1- B1) - (F0- B0)] / (F0 – B0), where F1and F0 represent fluorescence intensities during and before stimulation, and B1 and B0 are background signals. Data were normalized, with the baseline signal set to 0%. In vitro patch - clamp electrophysiological recording All experiments were conducted using solutions prepared as follows: Cutting Solution contained choline chloride (110 mM), KCl (2.5 mM), NaH₂PO₄ (1.25 mM), NaHCO₃ (26.0 mM), CaCl₂ (0.5 mM), MgSO₄ (7 mM), D-glucose (10 mM), Na-ascorbate (11.6 mM), Na-pyruvate (3.1 mM), and atropine sulfate (0.01 mM); Recording Solution comprised NaCl (119 mM), KCl (2.5 mM), NaH₂PO₄ (1.25 mM), NaHCO₃ (26 mM), CaCl₂ (2.5 mM), MgCl₂ (1.3 mM), and D-glucose (10 mM); K⁺ Intracellular Solution was formulated with K-gluconate (128 mM), KCl (17.5 mM), Na₂ATP (5 mM), MgCl₂ (1 mM), EGTA (0.2 mM), and HEPES (10 mM), adjusted to pH 7.4 and osmolarity of 290–300 mOsm. Two-month-old homozygous Tg hTau368 mice were randomly divided into three groups (Veh + eGFP, Dox + eGFP, Dox + CB), with viral vectors (AAV-CaMKIIα-eGFP or AAV-CaMKIIα-Calb1-eGFP) bilaterally injected into hippocampal CA1 and DG 1.5 months prior to recording. Brain slices were prepared, transferred to a perfusion chamber, and stabilized under a microscope, where green-fluorescent CA1 pyramidal neurons in optimal physiological condition were selected for recording. For spontaneous excitatory postsynaptic currents (sEPSCs), a K⁺ intracellular solution-filled glass electrode (3–5 MΩ) was used to clamp neurons at − 70 mV after gigaseal formation and membrane rupture; sEPSCs were recorded for 120 s in voltage-clamp mode. Action potentials (APs) were recorded in current-clamp mode using the same electrode, applying step-current stimuli (0–140 pA, 20 pA increments, 10 s intervals) and counting APs within 600 ms windows. Data were acquired via a Multiclamp 700B amplifie and Digidata 1440A digitizer (sampling at 10 kHz, low-pass filtered at 2 kHz), with analysis performed using Clampfit 10.6 software for frequency, amplitude, and AP quantification (Molecular Devices, CA, USA). Western blotting The hippocampus was separately on ice and homogenized in a customized RIPA lysis buffer. This buffer was formulated with 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X − 100, 1% sodium deoxycholate, and 0.1% SDS (Beyotime, Shanghai, China). To prevent protein degradation and maintain phosphorylation states, a protease and phosphatase inhibitor cocktail (Thermo Scientific, Waltham, USA) was added at a ratio of 10 µl per mg of tissue. The protein concentration within the RIPA - soluble lysates (pun at 12,000×g for 20 min) was accurately determined using a BCA protein assay kit (Thermo Fisher, Waltham, USA). Equal amounts of protein from the soluble fraction were loaded onto 10% SDS - PAGE gels for separation. The proteins were electrophoretically transferred onto nitrocellulose membranes (Merck Millipore, Darmstadt, Germany). The membranes were then blocked with 5% bovine serum albumin (BSA) to prevent non - specific antibody binding. Subsequently, they were incubated sequentially with primary and secondary antibodies as listed in Table 1 . Visualization of the protein bands was achieved using an enhanced chemiluminescence substrate system (Santa Cruz Biotech, Dallas, USA). The membranes were imaged with an Odyssey Imaging System (LI - COR Biosciences, Lincoln, USA), and the band intensities were quantified using the Image J. β-actin was used as a loading control to ensure consistent protein loading across samples. Immunostaining and quantification Mice were anesthetized with 2% isoflurane (RWD Life Science, Shenzhen, China). Through the left ventricle, they were first perfused with 0.9% NaCl for 5 min to clear the blood, followed by perfusion with 4% paraformaldehyde in PBS for 1 day. The brains were treated with 25% sucrose for 1 day and then transferred to 30% for an additional day. Using a cryostat microtome (Leica, Wetzlar, Germany), the brains were cut into 30 µm - thick sections. For immunohistochemistry, the free - floating sections were first immersed in 3% H 2 O 2 in anhydrous methanol for 30 min to quench endogenous peroxidases. Non - specific binding sites were blocked with BSA for 30 min at room temperature. The brain slices were then incubated overnight at 4°C with primary antibodies. The immunoreactions were developed using a DAB - staining kit (ZSGB - BIO, Beijing, China). Images of the stained sections were captured at 20× magnification using an automatic slice scanning system (Olympus, Tokyo, Japan) and analyzed with ImageJ software. The areas of different brain regions were measured to assess the staining distribution. For immunofluorescence, the sections were thoroughly washed with PBST (PBS containing 0.1% Triton X − 100). They were incubated overnight at 4°C with primary antibodies. After the incubation, the sections were washed with PBST for 15 min and then incubated with the secondary antibody at 37°C for 1 h. Finally, the nuclei were counterstained with DAPI. Images were acquired at 20× magnification using both an automatic slice scanning system (Olympus, Tokyo, Japan) and a two - photon laser - scanning confocal microscope (Zeiss, Oberkochen, Germany). Image J software was used for analysis. Novel-location recognition test Before the test, mice were acclimated to handling. On day 1, each mouse was placed in the center of a plastic box. In the box, two identical objects (A and B) were positioned in two corners. The mouse was allowed to explore freely for 5 min. The mouse was re - introduced to the box after 24 h. Object A remained in its original corner, while object B was placed in a new location. Again, the mouse was given 5 min for exploration. The exploration time for objects A and B, denoted as TA and TB respectively, was recorded. A video tracking system (Anymaze Technology SA, Stoelting Co., IL, USA) was used to identify exploration, which was defined as the mouse's head being within 3 cm of an object. Mice with TA or TB less than 2 s were excluded from the analysis. The discrimination index was calculated as (TB - TA) / (TB + TA). A higher discrimination index indicated better spatial memory retention. Morris water maze test Mice were housed in the test room for 24 h prior to the test commencement. During the trainning phase, the mice were trained to locate a hidden platform within the Morris water maze. The trainning phase spanned 5 consecutive days, with 3 trials per day. The trials were conducted between 14:00 and 17:00, with a 30 min interval between each trial. In each trial, the mouse was placed in one of the three quadrants that did not contain the platform, facing the pool wall. If the mouse found the hidden platform within 60 s, it was allowed to remain on the platform for an additional 15 s for learning consolidation. If the platform was not found within 60 s, the mouse was gently guided to the platform and allowed to stay there for 15 s. The time taken to find the platform over the 5 - day training period was recorded as the escape latency. On day 6, a testing trial was carried out. The hidden platform was removed, and each mouse was placed in the quadrant opposite to the target quadrant. A video tracking system (Chengdu Taimeng Software Co., Ltd, China) was used to record and analyze the time, distance, and trajectory of each mouse's movement in the pool. Mice with any visual or limb impairments were excluded from the analysis. Open field test Mice were handled for 1 day before the test and were placed in the test room the day prior to the behavioral test to acclimatize to the environment. The open field apparatus was a white plastic box measuring 60×60×50 cm³. In the monitoring system, the floor of the box was virtually divided into 16 equal squares, with a central field consisting of the central 4 square regions and 12 peripheral fields. Each mouse was allowed to explore freely in the box for 5 min. The ANY - maze video tracking system (Stoelting Co., WoodDale, IL, USA) was used to record and analyze the time and distance each mouse traveled in different zones, providing insights into the mouse's locomotor activity and exploratory behavior. Statistical analyses All data were processed and visualized using GraphPad Prism 8 (La Jolla, CA). For comparisons between two groups, two - tailed unpaired Student’s t - tests were utilized. When comparing multiple groups, one - way, two - way, or repeated measures ANOVA was performed, followed by post - hoc tests for multiple comparisons. Statistical significance was set at P < 0.05. All values are presented as mean ± SEM. Results pTau accumulation in hippocampal excitatory neurons disrupts intracellular calcium homeostasis As truncated Tau368 fragements naturally exists in the brain and increases during aging and AD, which is more neurotoxic than other truncated and full-lengthen tau [ 33 , 34 ] . We have previously generated Tg hTau368 mouse model with a tetracycline-controlled expressing truncated human tau (hTau368) under the neuronal promoter Eno2 [ 28 ] . This Tg mice exhibited hippocampal-predominant tau phosphorylation (pTau) aggregation (Fig. 1 A-C) and cognitive impairments when treated with doxycycline hyclate (Dox) for 2 months as previously reported [ 28 ] . Immunofluorescence co-staining revealed pTau (AT8 or pTau205) co-localization with CamKIIα-positive excitatory neurons in hippocampal CA1 and DG regions, with no parvalbumin (PV)- or GAD67-positive inhibitory neurons (Fig. 1 D and E). Consistently, similar tau aggregation patterns were observed in Tg PR5 mice expressing P301L mutant human tau under control of the murine Thy1.2 (Supplementary Figure S1 ). To assess the impact of tau aggregation on calcium dynamics, we injected AAV-CaMKIIα-GCaMP6f-eGFP into CA1 of Tg hTau368 mice (Fig. 2 A and B). There was a concentration difference between intracellular and extracellular calcium ions in neurons, and when neurons were activated, extracellular calcium ions entered into the cell and rapidly increased the intracellular calcium ion concentration. Using a protein fluorescent probe (GCaMP6f-eGFP) as a calcium ion indicator, the calcium ion concentration in neurons is expressed by the fluorescence intensity, so as to detect the change of intracellular calcium ion concentration [ 35 ] . We found that the KCl-induced depolarization of CA1 CaMKIIα-GCaMP6f-eGFP -positive neurons (green) revealed significantly higher intracellular calcium peaks in Dox-treated mice (with tau pathology) compared to Veh group (without tau pathology) (Fig. 2 C-E), demonstrating tau-driven calcium dysregulation in CA1 excitatory neurons. Tau aggregation promotes TLE induced by hippocampal CA1 and DG hyperexcitability To investigate the role of hippocampal CA1 and DG in TLE, we injected kainic acid (KA), a natural excitatory neurotoxin derived from red algae and an agonist of ionotropic glutamate receptors (AMPA and KA receptors), into the CA1 and DG of aged wild-type (WT) mice respectively. KA injection in both regions induced epileptic seizures (Fig. 3 A), mimicking human TLE [ 36 ] . This suggests that KA-mediated hyperactivation of CA1 and DG contributes to TLE epileptogenesis. To further elucidate the role of excitatory neurons in CA1 and DG and the influence of tau pathology on epileptogenesis, we injected AAV-CaMKIIα-ChR2-mCherry into the CA1 and DG regions of aged Tg hTau368 mice. Four weeks post-injection, blue light stimulation was used to activate neurons in these regions. Both dorsal (dHipp) and ventral (vHipp) hippocampal stimulation induced seizures of varying severity and typical epileptic seizure waveforms occurred during electrophysiological recording in vivo (Fig. 3 B and C, Supplementary Video 1). Seizures ceased upon cessation of light stimulation, and mice returned to normal behavior gradually (Supplementary Video 1). We compared seizure latency and severity between the Dox group and the Veh group. The Dox group exhibited shorter seizure latency (Fig. 3 D-left and Supplementary Figure S2 ) and higher seizure severity (Fig. 3 D-right), indicating that tau pathology enhances susceptibility to TLE. The hippocampal - parahippocampal circuits, particularly the DG-CA1-EC-DG loop, play a critical role in TLE initiation and propagation [ 18 ] . Our findings suggest that abnormal activation of CA1 and DG excitatory neurons drives TLE, and that tau aggregation increases TLE susceptibility, potentially through hyperexcitability in the DG-CA1-EC-DG circuit. Tau pathology correlates with cerebral hypermetabolism, hyperexcitable behavioral phenotypes and cognitive deficits Increased neuronal and brain network excitability is age-dependent, and dysregulation of excitatory inhibition of neural microcircuits in aged mice is associated with cognitive impairment [ 37 ] . In order to simulate the effects of tau pathology on aging as AD is an age-dependent disease, we took advantage of the controllable induction of hTau expression in 14–15 months of Tg hTau368 mice by administering 1–2 months Dox treatment, and then FDG PET/CT scanning, energy metabolism, activity monitoring and cognitive tests were conducted (Fig. 4 A and B). 18 F-FDG-PET imaging, could reflect the intensity of glucose metabolism in the brain, and to a certain extent, the excitability state of the whole brain, which has a certain diagnostic value for AD progression [ 38 , 39 ] . 18 F-FDG PET/CT in aged Tg hTau368 mice (16 months) revealed elevated glucose metabolism in the hippocampus and olfactory bulb following 1–2 months of Dox treatment (Fig. 4 C and D), consistent with early-stage hypermetabolic states observed in young 3×Tg (7 months old, carrying the APP/ PS1/P301L tau mutation) and PR5 (3 months old, carrying the tau P301L mutation) mice [ 40 – 42 ] . The cerebral hypermetabolism paralleled with increased O2 consumption (Fig. 4 E and F), energy expenditure (Fig. 4 G), and spontaneous movements (Supplementary Figure S3 ), especially during the daytime in resting state of mice. Cognitive deficits were also found in Dox-treated Tg hTau368 mice, manifested as a lower novel object discrimination index and longer latency to platform, as detected by the novel object recognition test (Fig. 4 H and I) and Morris water maze test (Fig. 4 J and L), respectively. The results above suggest that tau pathology associated with elevated metabolism, hyperexcitable behavioral phenotype and cognitive deficits in aged mice simulating the early stage of tau pathology. Tau pathology induces CB reduction and synaptic dysfunction To explore the relationship of tau pathology with CB and synapse-associated proteins, we utilized controlled hTau expression via the Tet-on system in Tg hTau368, i.e., hTau was expressed when Dox was given (Dox-on), and when Dox was withdrawn, hTau expression ceased and was gradually cleared (Dox-on-off), the phenomenon has also been reported in other Tg lines [ 43 , 44 ] . As we expected, tau pathology in the Dox-on-off group of mice were largely cleared after 3 months, especially in CA1 pyramidal neurons and DG granule cells (Fig. 5 A-C), which was also accompanied by a rebound in CB expression (Fig. 5 D), and an increase in synaptic proteins expression (Supplementary Figure S3 ), such as postsynaptic density protein 95 (PSD95) and synapsin 1 (SYN-1). Strikingly, Dox withdrawal for 3 months reversed tau pathology and restored CB/synaptic proteins expression (Fig. 5 A-D and Supplementary Figure S4 ). Immunofluorescent staining and Western blotting data also confirmed CB reduction in Dox-on group compared with Veh group (Fig. 5 E, F, I and J), as well as AT8-positive neurons compared with AT8-negative neurons (Fig. 5 G and J). The results above linked tau pathology, CB reduction and synaptic dysfunction. Upregulation of CB alleviates tau pathology-induced neuronal hyperexcitability and cognitive impairment To investigate whether CB defects mediate tau -associated neuronal hyperexcitability, we recorded electrophysiological properties in CA1 CamkII-eGFP-positive pyramidal neurons of Tg hTau368 mice treated with Veh + AAV- CaMKIIa -eGFP (Veh + eGFP), Dox + AAV-CaMKIIa-eGFP (Dox + eGFP) and Dox + AAV-CaMKIIa-Calb1-eGFP (Dox + CB) (Fig. 6 A). For the 2 - month - old homozygous Tg hTau368 mice in the Veh + eGFP, Dox + eGFP, and Dox + CB groups, they received treatments of Veh, Dox, and Dox respectively, for a duration of 2 months. One and a half months prior to patch - clamp detection, the viruses AAV - CaMKIIa - eGFP, AAV - CaMKIIa - eGFP, and AAV - CaMKIIa - Calb1 - eGFP were injected into the hippocampal CA1 and DG regions respectively. CB expression was confirmed in CA1 and DG neurons in the Dox + CB group (Fig. 6 B). Tau accumulation (Dox + eGFP group) increased sEPSC amplitude and frequency, indicating elevated neuronal excitability, while CB Supplementaryementation reversed these effects (Fig. 6 C-H). Additionally, neurons in Dox + eGFP group exhibited higher resting membrane potentials, lower rheobase, and increased evoked action potential frequency, all of which were normalized by CB upregulation (Fig. 6 I-O). These findings suggest that CB upregulation alleviates tau accumulation induced hyperexcitability in CA1 neurons. Behavioral tests also revealed that CB upregulation improved cognitive performance in Tg hTau368 mice. In the Morris water maze, the Dox + CB group showed a tendency of reduced latency to find the platform (Fig. 7 A) and increased platform crossings compared to the Dox + eGFP group (Fig. 7 B and C). Similarly, in the novel object recognition test, the Dox + CB group demonstrated better recognition of novel object locations (Fig. 7 D and E). However, CB did not affect spontaneous behaviors in the open field test (Fig. 7 F and G). Overall, CB Supplementation in hippocampal excitatory neurons mitigates tau-induced cognitive dysfunction, highlighting its therapeutic potential. Reduced CB correlates with cognitive deterioration and an advanced disease stage in AD patients To investigate the association between CB and cognitive decline in AD patients, we analyzed CB expression using a publicly available AD database ( http://www.alzcode.xyz/ ) [ 45 ] . Compared to healthy controls, AD patients exhibited significantly decreased CB transcript levels in brain tissues (Fig. 8 A). To evaluate disease progression, patients were stratified by cognitive function (Clinical Dementia Rating [CDR] scale; Fig. 8 B) and neuropathological severity (Braak staging; Fig. 8 C). Strikingly, CB transcript levels progressively declined with worsening cognitive impairment (higher CDR scores) and advanced Braak stages. Consistent with transcriptional changes, total CB protein expression was also markedly reduced in AD patients (Fig. 8 D and E), and immunohistochemical staining confirmed diminished CB levels in the hippocampal regions of AD brains (Fig. 8 F). These findings collectively highlight a robust inverse correlation between CB depletion and both cognitive decline and AD progression. Discussion Emerging evidences suggest that hippocampal network hyperexcitability caused by excitation-inhibition imbalance occurs in early AD progression and exacerbates cognitive decline, though the underlying mechanisms remain elusive. Our study demonstrates that tau aggregation within hippocampal CA1 and DG excitatory neurons leads to reduced CB expression, elevated intracellular calcium transients, and enhanced neuronal network excitability, ultimately increasing susceptibility to TLE and cognitive impairment. Notably, CB Supplementation in hippocampal CA1 and DG excitatory neurons ameliorates tauopathy-induced hyperexcitability and cognitive deficits in mice (Fig. 9 ). Clinical correlation analysis from AD databases further reveals CB deficiency is associated with accelerated cognitive decline and disease severity. These findings elucidate CB-mediated mechanisms underlying tau-related epileptogenesis and cognitive dysfunction at both circuit and molecular levels, providing novel therapeutic targets for AD intervention. Tau, a microtubule-associated protein, plays a pivotal role in maintaining microtubule assembly and stability. Under physiological conditions, tau exists predominantly in an unstructured conformation with low intrinsic aggregation propensity. However, pathological hyperphosphorylation drives its misfolding into paired helical filaments (PHFs) and neurofibrillary tangles (NFTs), which coalesce into large aggregates—a defining feature of tauopathies. These pathologies are not exclusive to AD but are also implicated in a spectrum of neurodegenerative disorders, including progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic grain disease (AGD), Pick’s disease (PiD), Huntington’s disease (HD), and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) [ 46 , 47 ] . Notably, tau pathology has been identified in the hippocampus of TLE patients [ 4 , 48 ] , suggesting a shared mechanism between AD and TLE. Both disorders exhibit hippocampal network hyperexcitability during early disease stages [ 4 ] . To investigate the role of hippocampal microcircuits in epileptogenesis, we employed kainic acid (KA) to activate CA1 and DG subregions, coupled with selectively optogenetic stimulation of excitatory neurons. Our results revealed that overactivation of CA1/DG neurons reliably induced seizures in mice, underscoring the critical contribution of hippocampal hyperexcitability to epileptogenesis. In the optogenetic model, CaMKIIα promoter-driven expression of channelrhodopsin-2 (ChR2) enabled precise spatiotemporal control of neuronal activity. Blue light (475 nm) triggered rapid cation influx (e.g., Na⁺) through ChR2, eliciting action potentials and transient hippocampal overexcitation. Termination of photostimulation promptly closed the channels, allowing neuronal activity to return to baseline [31]. Dox-treated Tg hTau368 mice exhibited significantly reduced seizure thresholds and more severe phenotypes upon optogenetic induction, further supporting tau pathology as a driver of epileptogenic vulnerability. The phenomenon recapitulated in other Tg mouse models expressing mutant tau variants such as PS19 (P301S), Tau22 (G272V/P301S), and rTg4510 (P301L), which display exacerbated neuronal hyperexcitability, heightened seizure susceptibility, and increased mortality [ 49 , 50 ] .Complementary studies in Mapt⁻/⁻ mice demonstrated that tau ablation reduces action potential release, lowers cortical excitation-inhibition (E/I) ratios, and enhances inhibitory neuron recruitment, collectively mitigating neuronal hypersynchronization and seizure incidence [ 51 ] . These findings highlight tau-driven E/I imbalance as a central mechanism underlying circuit-level hyperexcitability in epilepsy. Conversely, some studies indicate tau pathology may also drive hypoexcitability [ 52 ] , and suppress Aβ-driven cortical hyperactivation [ 53 ] . Phosphorylation of different tau residues had different effect on firing frequency of primary hippocampal neurons [ 52 ] . The role of tau in mediating neuronal excitability changes may vary depending on tau features (mutation, phosphorylation, isoform), brain region and disease stage [ 10 ] . In our study, we uniquely establishes truncated hTau368 as a potent enhancer of CA1 excitatory neuron activity at early stage of tau pathology that hippocampus is in a hypermetabolic state detected by FDG-PET/CT, consistent with systemic hypermetabolic states and increased spontaneous movements. Head to head comparison in diverse tauopathy models (e.g., P301L, P301S) is needed to further validate the current hypotheses. Under resting conditions, extracellular calcium concentrations (1.1–1.4 mM) starkly contrast with intracellular levels (50–300 nM), yet neuronal activation rapidly elevates cytosolic calcium to micromolar ranges [ 54 ] . Calcium homeostasis is tightly regulated by influx through receptor channels, buffering via calcium-binding proteins, such as CB, and endoplasmic reticulum (ER) store release [ 55 ] . Electrophysiological recordings and GCaMP6f-mediated calcium imaging provided a robust methods to study the interplay between tau, calcium, and neuronal activity. Our data demonstrated that tau pathology exacerbated intracellular calcium transients and CA1 neuronal hyperexcitability, aligning with prior in vitro observations. Full-length tau overexpression similarly disrupts calcium handling and induces ER stress [ 56 ] . Knocking out PSEN1 and PSEN2 or expressing a FAD mutation in mouse hippocampal neurons increased ER Ca2 + levels and consequently elevated Ca2 + release into the cytoplasm in the presence of a stimulus [ 57 ] . Notably, CB overexpression in CA1 neurons rescued tau-induced hyperexcitability, suggesting CB deficiency as a key mediator. CB exhibits high calcium affinity and serves as a critical buffer against cytosolic calcium overload. Beyond its neuroprotective role, CB modulates action potential kinetics and synaptic transmission [ 24 , 58 ] . CB is enriched in CA1 pyramidal neurons and DG granule cells, with sparse expression in CA1 interneurons [ 26 , 59 , 60 ] . Anatomically, CB is localized within the axons and dendritic spines of neurons, where it exerts a dynamic regulatory effect on synaptic plasticity. At the presynaptic terminals, CB plays a promoting role in vesicle release and paired - pulse facilitation [ 29 ] . Conversely, in the postsynaptic context, CB is indispensable for the induction and maintenance of long - term potentiation in CA1 and DG excitatory neurons [ 30 ] . Spatiotemporal localization of CB-presynaptically facilitating vesicle release and paired-pulse facilitation [ 61 ] , and postsynaptically enabling long-term potentiation [ 62 ] —underscores its dual role in synaptic plasticity. By binding excess calcium ions, it effectively down - regulates the intracellular free calcium levels, thereby safeguarding neurons from the potentially lethal effects of calcium overload - induced cell death [ 28 ] . Moreover, CB's regulatory function extends to influencing the firing of neuronal action potentials and the intricate processes of synaptic neurotransmitter transport and release. In our study, tau pathology-associated calcium surges in CA1 neurons correlated with diminished CB expression, likely due to impaired buffering capacity. The pTau aggregates localized exclusively to CA1/DG excitatory neurons and coincided with CB downregulation, leading to calcium dysregulation, mitochondrial stress, oxidative damage, and apoptotic signaling [ 56 , 58 ] . Dysfunctional calcium homeostasis further alters neurotransmitter release and transcriptional programs, exacerbating E/I imbalance [ 22 ] . Remarkably, Dox withdrawal for three months reversed tau pathology, restored CB levels, normalized synaptic transmission, and cognition recovery. Similarly, AAV-mediated CB Supplementation in Tg hTau368 mice attenuated tau-induced cognitive deficits, implicating CB restoration as a viable therapeutic strategy. Our data established connections among tauopathy, CB deficiency, calcium homeostatic imbalance, synaptic dysfunction and cognitive deficits by patch-clamp electrophysiological recording, calcium imaging, and the Tet-on system in tau-driven Tg mice. Despite these advances, key limitations must be acknowledged. First, the molecular link between tau pathology and CB suppression remains unclear. Previous findings implicates ΔFosB-mediated epigenetic silencing of CB in AD/TLE [ 27 ] , and tau-induced JAK2–STAT1 activation [ 63 ] , wheher the pathway linked to Calb1 transcriptional repression warrants further investigation. Second, the role of CB in interneurons remains unexplored, despite evidence that interneuron-specific CB knockdown protects against stress-induced memory deficits [ 25 ] . Finally, whether there is an interaction between Aβ and tauopathy, along with its influence on the excitability of the hippocampal neural circuit, still requires further exploration. Conclusion In conclusion, our study demonstrates that tau selectively accumulate in hippocampal excitatory neurons, resulting in increased neuronal excitability and a heightened susceptibility to TLE. The concomitant reduction in CB expression within these neurons mediates the hyperexcitability and cognitive dysfunction associated with tau pathology. These findings underscore the potential of targeting CB-mediated calcium homeostasis as a therapeutic strategy for AD. Abbreviations AD Alzheimer’s disease TLE temporal lobe epilepsy CB calbindin-D28k Declarations Acknowledgements We thank Pro. Jianxin Wang (School of Computer Science and Engineering, Central South University, Changsha, Hunan 410083, China) and his colleagues for providing the raw data from AlzCode (http://www.alzcode.xyz/) and giving data analysis. We thank Dr. Jiuyang Ding (Department of Forensic Medicine, Zunyi Medical University,Guiyang, Zunyi 563000, China) for generously providing the human brain tissue sections. Competing interests The authors declare no competing interests. Author contributions J.Z.W., H.B.X.,Y.Z. and Y.G. designed this research; Y.G., X.Q.T., Y.Y.W., Y.R.W., H.L., Y.Y., M.Q.T. and J.Z.performed the experiments. W.W.,J.Z. and X.C.W. provided advices on this research. Y.G, X.Q.T., Y.Y.W. and J.Z. performed statistical analysis and data interpretation. J.Z.W., H.B.X., Y.Z. and Y.G. wrote the manuscript. All authors read and approved the final manuscript. J.Z.W. is the supervisor of this work who has full access to all the data in the study and takes responsibility for the data integrity and accuracy. Funding This study was supported in parts by the Natural Science Foundation of China (91949205, 31730035, 81721005), China Postdoctoral Science Foundation General Program (2024M762504) and Wuhan Health Commission Project (WZ21Q21). The funders neither played a role in the study design, conduct, data collection, analysis, interpretation, nor participated in the preparation, review, approval of the manuscript. Availability of data and materials All data provided in this paper are available from the leading contact, Prof Jian-Zhi Wang upon reasonable request. Ethics approval and consent to participate All animal studies had complied with all relevant ethical regulations for the animal testing and research, and were approved by institutional guidelines and the Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Long JM, Holtzman DM. Alzheimer Disease: An Update on Pathobiology and Treatment Strategies. Cell. 2019;179:312-39. Zhang Y, Chen H, Li R, Sterling K, Song W. Amyloid β-based therapy for Alzheimer's disease: challenges, successes and future. Signal transduction and targeted therapy. 2023;8:248. Heneka MT, Morgan D, Jessen F. Passive anti-amyloid β immunotherapy in Alzheimer's disease-opportunities and challenges. Lancet (London, England). 2024;404:2198-208. Zawar I, Kapur J. Does Alzheimer's disease with mesial temporal lobe epilepsy represent a distinct disease subtype? Alzheimer's & dementia : the journal of the Alzheimer's Association. 2023;19:2697-706. Cheng C, Liu C, Ou S, Yeh C, Chen T, Lin Y, et al. Incidence and risk of seizures in Alzheimer's disease: A nationwide population-based cohort study. Epilepsy research. 2015;115:63-6. Vossel K, Tartaglia M, Nygaard H, Zeman A, Miller B. Epileptic activity in Alzheimer's disease: causes and clinical relevance. The Lancet Neurology. 2017;16:311-22. Lam AD, Thibault EG, Mayblyum DV, Hsieh S, Pellerin KR, Sternberg EJ, et al. Association of Seizure Foci and Location of Tau and Amyloid Deposition and Brain Atrophy in Patients With Alzheimer Disease and Seizures. Neurology. 2024;103:e209920. Amatniek J, Hauser W, DelCastillo-Castaneda C, Jacobs D, Marder K, Bell K, et al. Incidence and predictors of seizures in patients with Alzheimer's disease. Epilepsia. 2006;47:867-72. Lam A, Deck G, Goldman A, Eskandar E, Noebels J, Cole A. Silent hippocampal seizures and spikes identified by foramen ovale electrodes in Alzheimer's disease. Nature medicine. 2017;23:678-80. Targa Dias Anastacio H, Matosin N, Ooi L. Neuronal hyperexcitability in Alzheimer's disease: what are the drivers behind this aberrant phenotype? Translational psychiatry. 2022;12:257. Celone KA, Calhoun VD, Dickerson BC, Atri A, Chua EF, Miller SL, et al. Alterations in memory networks in mild cognitive impairment and Alzheimer's disease: an independent component analysis. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2006;26:10222-31. Hämäläinen A, Pihlajamäki M, Tanila H, Hänninen T, Niskanen E, Tervo S, et al. Increased fMRI responses during encoding in mild cognitive impairment. Neurobiol Aging. 2007;28:1889-903. Gallego-Rudolf J, Wiesman AI, Pichet Binette A, Villeneuve S, Baillet S. Synergistic association of Aβ and tau pathology with cortical neurophysiology and cognitive decline in asymptomatic older adults. Nat Neurosci. 2024;27:2130-7. Vossel K, Ranasinghe KG, Beagle AJ, La A, Ah Pook K, Castro M, et al. Effect of Levetiracetam on Cognition in Patients With Alzheimer Disease With and Without Epileptiform Activity: A Randomized Clinical Trial. JAMA neurology. 2021;78:1345-54. Sainju RK, Dragon DN, Winnike HB, Ten Eyck P, Granner MA, Gehlbach BK, et al. Hypercapnic ventilatory response in epilepsy patients treated with VNS: A case-control study. Epilepsia. 2021;62:e140-e6. Villeneuve S, Rabinovici GD, Cohn-Sheehy BI, Madison C, Ayakta N, Ghosh PM, et al. Existing Pittsburgh Compound-B positron emission tomography thresholds are too high: statistical and pathological evaluation. Brain. 2015;138:2020-33. Giorgio J, Adams JN, Maass A, Jagust WJ, Breakspear M. Amyloid induced hyperexcitability in default mode network drives medial temporal hyperactivity and early tau accumulation. Neuron. 2024;112:676-86.e4. Lu Y, Zhong C, Wang L, Wei P, He W, Huang K, et al. Optogenetic dissection of ictal propagation in the hippocampal-entorhinal cortex structures. Nature communications. 2016;7:10962. King MV, Kurian N, Qin S, Papadopoulou N, Westerink BH, Cremers TI, et al. Lentiviral delivery of a vesicular glutamate transporter 1 (VGLUT1)-targeting short hairpin RNA vector into the mouse hippocampus impairs cognition. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology. 2014;39:464-76. Fu H, Possenti A, Freer R, Nakano Y, Hernandez Villegas N, Tang M, et al. A tau homeostasis signature is linked with the cellular and regional vulnerability of excitatory neurons to tau pathology. Nature neuroscience. 2019;22:47-56. Murdock MH, Tsai LH. Insights into Alzheimer's disease from single-cell genomic approaches. Nat Neurosci. 2023;26:181-95. Dridi H, Liu Y, Reiken S, Liu X, Argyrousi E, Yuan Q, et al. Heart failure-induced cognitive dysfunction is mediated by intracellular Ca leak through ryanodine receptor type 2. Nature neuroscience. 2023;26:1365-78. Princen K, Van Dooren T, van Gorsel M, Louros N, Yang X, Dumbacher M, et al. Pharmacological modulation of septins restores calcium homeostasis and is neuroprotective in models of Alzheimer's disease. Science (New York, NY). 2024;384:eadd6260. Lambers T, Mahieu F, Oancea E, Hoofd L, de Lange F, Mensenkamp A, et al. Calbindin-D28K dynamically controls TRPV5-mediated Ca2+ transport. The EMBO journal. 2006;25:2978-88. Berdal A, Hotton D, Saffar J, Thomasset M, Nanci A. Calbindin-D9k and calbindin-D28k expression in rat mineralized tissues in vivo. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 1996;11:768-79. Li J, Xie X, Yu J, Sun Y, Liao X, Wang X, et al. Suppressed Calbindin Levels in Hippocampal Excitatory Neurons Mediate Stress-Induced Memory Loss. Cell reports. 2017;21:891-900. You J, Muralidharan K, Park J, Petrof I, Pyfer M, Corbett B, et al. Epigenetic suppression of hippocampal calbindin-D28k by ΔFosB drives seizure-related cognitive deficits. Nature medicine. 2017;23:1377-83. Gao Y, Wang Y, Lei H, Xu Z, Li S, Yu H, et al. A novel transgenic mouse line with hippocampus-dominant and inducible expression of truncated human tau. Transl Neurodegener. 2023;12:51. Götz J, Chen F, Barmettler R, Nitsch RM. Tau filament formation in transgenic mice expressing P301L tau. The Journal of biological chemistry. 2001;276:529-34. Ren QG, Gong WG, Zhou H, Shu H, Wang YJ, Zhang ZJ. Spatial Training Ameliorates Long-Term Alzheimer's Disease-Like Pathological Deficits by Reducing NLRP3 Inflammasomes in PR5 Mice. Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics. 2019;16:450-64. Gao Y, Zheng J, Jiang T, Pi G, Sun F, Xiong R, et al. Targeted Reducing of Tauopathy Alleviates Epileptic Seizures and Spatial Memory Impairment in an Optogenetically Inducible Mouse Model of Epilepsy. Front Cell Dev Biol. 2020;8:633725. Lewczuk E, Joshi S, Williamson J, Penmetsa M, Shan S, Kapur J. Electroencephalography and behavior patterns during experimental status epilepticus. Epilepsia. 2018;59:369-80. Zhang Z, Song M, Liu X, Kang SS, Kwon IS, Duong DM, et al. Cleavage of tau by asparagine endopeptidase mediates the neurofibrillary pathology in Alzheimer's disease. Nat Med. 2014;20:1254-62. Li S, Zhou Q, Liu E, Du H, Yu N, Yu H, et al. Alzheimer-like tau accumulation in dentate gyrus mossy cells induces spatial cognitive deficits by disrupting multiple memory-related signaling and inhibiting local neural circuit. Aging Cell. 2022;21:e13600. Chen TW, Wardill TJ, Sun Y, Pulver SR, Renninger SL, Baohan A, et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature. 2013;499:295-300. Tzeng TT, Tsay HJ, Chang L, Hsu CL, Lai TH, Huang FL, et al. Caspase 3 involves in neuroplasticity, microglial activation and neurogenesis in the mice hippocampus after intracerebral injection of kainic acid. Journal of biomedical science. 2013;20:90. Radulescu CI, Doostdar N, Zabouri N, Melgosa-Ecenarro L, Wang X, Sadeh S, et al. Age-related dysregulation of homeostatic control in neuronal microcircuits. Nature Neuroscience. 2023;26:2158-70. Chiaravalloti A, Barbagallo G, Martorana A, Castellano AE, Ursini F, Schillaci O. Brain metabolic patterns in patients with suspected non-Alzheimer's pathophysiology (SNAP) and Alzheimer's disease (AD): is [(18)F] FDG a specific biomarker in these patients? European journal of nuclear medicine and molecular imaging. 2019;46:1796-805. Xie F, Peng F. Radiopharmaceuticals for Assessment of Altered Metabolism and Biometal Fluxes in Brain Aging and Alzheimer's Disease with Positron Emission Tomography. Journal of Alzheimer's disease : JAD. 2017;59:527-36. Sancheti H, Kanamori K, Patil I, Díaz Brinton R, Ross BD, Cadenas E. Reversal of metabolic deficits by lipoic acid in a triple transgenic mouse model of Alzheimer's disease: a 13C NMR study. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 2014;34:288-96. Sancheti H, Patil I, Kanamori K, Díaz Brinton R, Zhang W, Lin AL, et al. Hypermetabolic state in the 7-month-old triple transgenic mouse model of Alzheimer's disease and the effect of lipoic acid: a 13C-NMR study. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 2014;34:1749-60. Yin F, Sancheti H, Patil I, Cadenas E. Energy metabolism and inflammation in brain aging and Alzheimer's disease. Free radical biology & medicine. 2016;100:108-22. Santacruz K, Lewis J, Spires T, Paulson J, Kotilinek L, Ingelsson M, et al. Tau suppression in a neurodegenerative mouse model improves memory function. Science (New York, NY). 2005;309:476-81. Ozcelik S, Sprenger F, Skachokova Z, Fraser G, Abramowski D, Clavaguera F, et al. Co-expression of truncated and full-length tau induces severe neurotoxicity. Molecular psychiatry. 2016;21:1790-8. Lin CX, Li HD, Deng C, Erhardt S, Wang J, Peng X, et al. AlzCode: a platform for multiview analysis of genes related to Alzheimer's disease. Bioinformatics (Oxford, England). 2022;38:2030-2. Goedert M, Eisenberg DS, Crowther RA. Propagation of Tau Aggregates and Neurodegeneration. Annual review of neuroscience. 2017;40:189-210. Viney TJ, Sarkany B, Ozdemir AT, Hartwich K, Schweimer J, Bannerman D, et al. Spread of pathological human Tau from neurons to oligodendrocytes and loss of high-firing pyramidal neurons in aging mice. Cell Reports. 2022;41:111646. Witt J, Becker A, Helmstaedter C. The multifactorial etiology of cognitive deficits in epilepsy and the neuropathology of mesial temporal lobe epilepsy beyond hyperphosphorylated tau. Alzheimer's & dementia : the journal of the Alzheimer's Association. 2023;19:3231-2. Liu S, Shen Y, Shultz SR, Nguyen A, Hovens C, Adlard PA, et al. Accelerated kindling epileptogenesis in Tg4510 tau transgenic mice, but not in tau knockout mice. Epilepsia. 2017;58:e136-e41. Gomez-Murcia V, Sandau U, Ferry B, Parrot S, Laurent C, Basquin M, et al. Hyperexcitability and seizures in the THY-Tau22 mouse model of tauopathy. Neurobiology of Aging. 2020;94:265-70. Chang C, Evans M, Yu X, Yu G, Mucke L. Tau reduction affects excitatory and inhibitory neurons differently, reduces excitation/inhibition ratios, and counteracts network hypersynchrony. Cell reports. 2021;37:109855. Hatch R, Wei Y, Xia D, Götz J. Hyperphosphorylated tau causes reduced hippocampal CA1 excitability by relocating the axon initial segment. Acta neuropathologica. 2017;133:717-30. Busche M, Wegmann S, Dujardin S, Commins C, Schiantarelli J, Klickstein N, et al. Tau impairs neural circuits, dominating amyloid-β effects, in Alzheimer models in vivo. Nature neuroscience. 2019;22:57-64. Bagur R, Hajnóczky G. Intracellular Ca(2+) Sensing: Its Role in Calcium Homeostasis and Signaling. Mol Cell. 2017;66:780-8. Calvo-Rodriguez M, Kharitonova EK, Bacskai BJ. Therapeutic Strategies to Target Calcium Dysregulation in Alzheimer's Disease. Cells. 2020;9. Ye J, Yin Y, Yin Y, Zhang H, Wan H, Wang L, et al. Tau-induced upregulation of C/EBPβ-TRPC1-SOCE signaling aggravates tauopathies: A vicious cycle in Alzheimer neurodegeneration. Aging cell. 2020;19:e13209. Zhang H, Sun S, Herreman A, De Strooper B, Bezprozvanny I. Role of presenilins in neuronal calcium homeostasis. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2010;30:8566-80. Ge M, Zhang J, Chen S, Huang Y, Chen W, He L, et al. Role of Calcium Homeostasis in Alzheimer's Disease. Neuropsychiatric disease and treatment. 2022;18:487-98. Celio M. Calbindin D-28k and parvalbumin in the rat nervous system. Neuroscience. 1990;35:375-475. Yi S. Time-dependent changes of calbindin D-28K and parvalbumin immunoreactivity in the hippocampus of rats with streptozotocin-induced type 1 diabetes. Journal of veterinary science. 2013;14:373-80. Blatow M, Caputi A, Burnashev N, Monyer H, Rozov A. Ca2+ buffer saturation underlies paired pulse facilitation in calbindin-D28k-containing terminals. Neuron. 2003;38:79-88. Westerink R, Beekwilder J, Wadman W. Differential alterations of synaptic plasticity in dentate gyrus and CA1 hippocampal area of Calbindin-D28K knockout mice. Brain research. 2012;1450:1-10. Li XG, Hong XY, Wang YL, Zhang SJ, Zhang JF, Li XC, et al. Tau accumulation triggers STAT1-dependent memory deficits by suppressing NMDA receptor expression. EMBO reports. 2019;20. Supplementary Files SupplementaryVideo1.mp4 Westernblotrawdata.pdf supplementarymaterials.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 23 Apr, 2025 Reviewers invited by journal 22 Apr, 2025 Editor assigned by journal 22 Apr, 2025 First submitted to journal 21 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6470265","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":446632328,"identity":"1c3fe1dc-6e5c-44f3-8f8f-f440a0374daf","order_by":0,"name":"yang gao","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"yang","middleName":"","lastName":"gao","suffix":""},{"id":446632329,"identity":"8e337a49-8988-49a4-af12-93d43d47f927","order_by":1,"name":"Xiaoqing Tao","email":"","orcid":"","institution":"Jianghan University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoqing","middleName":"","lastName":"Tao","suffix":""},{"id":446632330,"identity":"10bbdd00-50ea-4faa-9947-deaf529f7386","order_by":2,"name":"yuying wang","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"yuying","middleName":"","lastName":"wang","suffix":""},{"id":446632331,"identity":"64ecb5e6-4122-450d-974f-a8e658ee1ccd","order_by":3,"name":"yarong wang","email":"","orcid":"","institution":"Huazhong University of Science and Technology Tongji Medical College","correspondingAuthor":false,"prefix":"","firstName":"yarong","middleName":"","lastName":"wang","suffix":""},{"id":446632332,"identity":"6f81d61f-1f7a-4a9f-a09f-b33ec24f28d1","order_by":4,"name":"Huan Li","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Huan","middleName":"","lastName":"Li","suffix":""},{"id":446632333,"identity":"656f72ec-fea4-4142-9f87-8475895f9e7d","order_by":5,"name":"yang Yu","email":"","orcid":"","institution":"Jianghan University","correspondingAuthor":false,"prefix":"","firstName":"yang","middleName":"","lastName":"Yu","suffix":""},{"id":446632334,"identity":"d217deac-8a62-4995-8f2a-802e212b735f","order_by":6,"name":"Mengqi Tu","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Mengqi","middleName":"","lastName":"Tu","suffix":""},{"id":446632335,"identity":"9396a7ff-358d-4e28-abb9-6c7e2d3f3831","order_by":7,"name":"Yanchao Liu","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Yanchao","middleName":"","lastName":"Liu","suffix":""},{"id":446632336,"identity":"fda1fc21-a1fd-45b3-9bb5-af8cc2a8c6a7","order_by":8,"name":"Jie Zhou","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Zhou","suffix":""},{"id":446632337,"identity":"df80656b-0c5a-4cfd-b1e9-0926c6e5a61f","order_by":9,"name":"Yuchen Li","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Yuchen","middleName":"","lastName":"Li","suffix":""},{"id":446632338,"identity":"79cbb675-583d-45c1-abcc-d8cd0697fd5a","order_by":10,"name":"Wei Wei","email":"","orcid":"","institution":"general hospital of Yangze River Shipping","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Wei","suffix":""},{"id":446632339,"identity":"cd65459e-2295-43f8-80f3-01ef00b2b556","order_by":11,"name":"Xiaochuang Wang","email":"","orcid":"","institution":"Huazhong University of Science and Technology Tongji Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xiaochuang","middleName":"","lastName":"Wang","suffix":""},{"id":446632340,"identity":"bb866a61-56b7-4ee2-8c1a-c2ba5be37a71","order_by":12,"name":"Jie Zheng","email":"","orcid":"","institution":"Peking University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Zheng","suffix":""},{"id":446632341,"identity":"f40eb117-3559-40b6-bc07-68eaba0fca2b","order_by":13,"name":"Yao Zhang","email":"","orcid":"","institution":"Huazhong University of Science and Technology Tongji Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Zhang","suffix":""},{"id":446632342,"identity":"dec78e99-546b-442c-b958-b520b6144424","order_by":14,"name":"Haibo Xu","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Haibo","middleName":"","lastName":"Xu","suffix":""},{"id":446632343,"identity":"ce228968-4d64-4876-83ba-16590c969c8a","order_by":15,"name":"Jian-Zhi Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYLACCQYbBgZm5gZmKN+AGC1pQC2MYC0SxGlhYDgMxMRqMTh+9vALiz/no/nbgVoK2+rqGNibt0kw1NzBreVMXpqFBM/t3BmHgVpmth2WYOA5VibBcOwZTi1mB3LMDCQkbuc2gLTwth2QYJDIMZNgbDiMW8v5N0AtBudy50O01EkwyL8hoOVGjvEDiYQDuRsgWpiBtvDg12J/440Zg8SB5NyNQC2Hec4dlmzjSSu2SDiGW4tkf47xZ4k/drnzzh8++JinrI6fn/3wxhsfanBrAQI2aQko6wCYCyIS8GkARvvHD/gVjIJRMApGwUgHAMGTUPMEWLqXAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-0181-4415","institution":"Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Key Laboratory of Ministry of Education of China for Neurological Disorders, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030; Co-innovation Center","correspondingAuthor":true,"prefix":"","firstName":"Jian-Zhi","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-04-17 09:27:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6470265/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6470265/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82120757,"identity":"1164a324-2203-4540-92f2-873436a63f7b","added_by":"auto","created_at":"2025-05-07 03:20:03","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1011504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe pTau is predominately accumulated in hippocampal excitatory neurons of Tg Tau-driven mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-C) 2-month doxycycline (Dox)-treated Tg hTau368 mice exhibited hippocampal-predominant phosphorylated tau (pTau) aggregated (detected by AT8 and pT205-tau antibodies), particularly in the CA1 pyramidal cell layer and DG granule cell layer.(A) Representative immunofluorescence (IF) staining images of hippocampal sections from Tg hTau368 mice treated with Veh/Dox for 2 months. (B) Western blotting analysis of hippocampal tissues from Tg hTau368 mice after 2-month Veh/Dox treatment. Unpaired Student’s t-test; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.7-month-old homozygous Tg hTau368 mice were used (3 mice per group).\u003c/p\u003e\n\u003cp\u003e(C) Representative immunohistochemical (IHC) staining images of phosphorylated tau (detected by AT8) aggregated in hippocampal sections from Tg hTau368 mice treated with Dox for 2 months.\u003c/p\u003e\n\u003cp\u003e(D-E) Representative IF staining images of hippocampal sections from Dox-treated hTau368 mice for 2 months demonstrating that pTau aggregates localized mainly to CamkII-positive excitatory neurons, with no colocalization observed in PV- or GAD67-positive inhibitory neurons.7-month-old homozygous Tg hTau368 mice were used.\u003c/p\u003e","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6470265/v1/b9180716c533238736056134.jpg"},{"id":82119883,"identity":"7e5424fd-ab57-476a-a60a-b4d946e449ab","added_by":"auto","created_at":"2025-05-07 03:12:03","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":419794,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTau aggregation in hippocampal CA1 excitatory neurons elevates the intracellular calcium peak.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of the experiment. 5-month - old Tg hTau368 mice were treated with Veh/Dox for 2 months respectively. 1 month before detecting the ex vivo calcium signals in the hippocampus, the virus (AAV - CamkII - GCamP6f - eGFP) was injected in CA1.\u003c/p\u003e\n\u003cp\u003e(B) Representative IF image of the injection site of AAV - CamkII - GCaMP6f – eGFP in CA1.\u003c/p\u003e\n\u003cp\u003e(C - D) Representative images of the ex vivo calcium signal recordings of baseline and KCl - induced action potentials in CA1 excitatory neurons in Tg hTau368 mice treated with Veh/Dox for 2 months, and (E) quantitative statistical analysis of the fluorescence signals. Unpaired Student’s t - test, *P \u0026lt; 0.05. Each point represents a single recording of the calcium signal within a field of view. 7 - month - old homozygous Tg hTau368 mice, 3 - 4 mice per group. Data are presented as Mean±SEM.\u003c/p\u003e","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6470265/v1/8b3ed43db5386aa3d8c189c1.jpg"},{"id":82120759,"identity":"2759147e-5990-4ef5-91c5-d790586181bd","added_by":"auto","created_at":"2025-05-07 03:20:03","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":434321,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTau aggregation promotes the occurrence of temporal lobe epilepsy induced by hyperexcitation of the hippocampal microcircuit.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) In 16-month - old aged WT mice, kainic acid (KA, 500 nl, 0.5 μg/μl) was locally injected into CA1 and DG of the hippocampus respectively, and a multi - channel electrode was implanted 0.1 mm above the injection site. Typical epileptic seizure waveforms were successfully recorded in mice. This experiment was repeated for three times.\u003c/p\u003e\n\u003cp\u003e(B) Schematic diagram of optogenetic activation of the hippocampal - related epileptic circuit (DG - CA1 - EC - DG) to induce epileptic seizures in 16-month - old aged Tg hTau368 mice. Four weeks after injecting the AAV - CamkII - ChR2 - mCherry virus into CA1 and DG of the hippocampus, blue light was used to over - activate excitatory neurons, which could induce epileptic seizures in aged mice. Typical epileptic seizure waves were recorded in cortex M1.\u003c/p\u003e\n\u003cp\u003e(C) A schematic diagram of the photo - activated area (left) and representative images of AAV - CamkII - ChR2 - mCherry injection in the ventral/dorsal hippocampus (right).\u003c/p\u003e\n\u003cp\u003e(D) Aged 16-month - old Tg hTau368 mice treated with Dox for 2 months (from fourteen - month - old) had a shorter latency of generalized seizures (GS) and a higher seizure grade. Unpaired Student’s t - test, *P \u0026lt; 0.05. Homozygous aged Tg mice were used, 3 mice per group, with 9 epileptic seizures in 3 days recorded. Data are presented as Mean±SEM.\u003c/p\u003e","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6470265/v1/1eac83e78e88fdc1b124c790.jpg"},{"id":82119887,"identity":"ddad22ac-83cb-4971-b3b9-7f11f392e8cf","added_by":"auto","created_at":"2025-05-07 03:12:03","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":808366,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe aged Tg Tau-driven mice show brain hypermetabolism, higher energy expenditure and cognitive deficits.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of the experiment. After 14 - and 15 - month - old hTau368 Tg aged mice were treated with Dox for 2 months and 1 month respectively, FDG-PET/CT scanning, energy metabolism, activity monitoring and cognitive tests were conducted.\u003c/p\u003e\n\u003cp\u003e(B) Schematic diagram of mouse energy metabolism and activity monitoring for a whole day.\u003c/p\u003e\n\u003cp\u003e(C) Representative images of brain FDG PET /CT examination and (D) the average \u003csup\u003e18\u003c/sup\u003eF-FDG uptake in multiple brain regions. Unpaired Student’s t - test was used, with *P \u0026lt; 0.05. 16-month - old homozygous hTau368 transgenic aged mice, 3 - 4 mice per group. Data were presented as Mean±SEM.\u003c/p\u003e\n\u003cp\u003e(E) The oxygen consumption every 5 min at different times within 24 h and quantitative statistics of (F) mean oxygen consumption and energy expenditure of aged Tg hTau368 mice treated with Veh/Dox for 2 months during night (Dark, 19:00-7：00) and day ( Light, 7:00-19：00) . Unpaired Student’s t - test was used, with **P \u0026lt; 0.01. 16-month - old homozygous mice, 7 - 8 mice per group. Data were presented as Mean±SEM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(G) Schematic illustration of the procedure of the novel-location recognition test. F stands for familiar; N stands for novel.\u003c/p\u003e\n\u003cp\u003e(H) Aged Tg hTau368 mice Dox-treated for 2 months showed poorer performance in discriminating the object removed to a new place in the novel-location recognition test.\u003c/p\u003e\n\u003cp\u003e(I) Schematic illustration of Morris-water maze test.\u003c/p\u003e\n\u003cp\u003e(J) Aged Tg hTau368 mice Dox-treated for 2 months showed longer latency to find the platform during training (Day 1-5) and comparable target quadrant crossings after training (Day 6) in Morris-water maze test. Repeated measures ANOVA followed by Tukey’s \u003cem\u003epost-hoc\u003c/em\u003e test for K.\u003cem\u003e \u003c/em\u003eUnpaired Student’s t - test was used for G and L. *P \u0026lt; 0.05. 16-month - old homozygous aged hTau368 mice, 12 mice per group. Data were presented as Mean±SEM.\u003c/p\u003e","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6470265/v1/51e7bff54af051b5394a5088.jpg"},{"id":82119889,"identity":"4b1ccf4d-eb3a-426b-a90e-7e62c4d2b809","added_by":"auto","created_at":"2025-05-07 03:12:03","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1035345,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTauopathy downregulates CB expression in the hippocampus.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of the experiment.\u003c/p\u003e\n\u003cp\u003e(B - D) In Tg hTau368 mice treated with Dox for 2 months (Dox - on), tau aggregation occurred in the hippocampal CA1 and DG regions, and the levels of calbindin - D28k (CB) in hippocampal CA1 and DG were reduced. However, after withdrawing Dox for 3 months (Dox - on - off), the tau pathology was cleared, accompanied by the recovery of CB expression levels. Representative images of IHC staining of AT8 (C) and calbindin-D28k (D). One - way analysis of variance was used for B, followed by Post hoc Tukey’s multiple comparison test. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 vs Veh group, #P \u0026lt; 0.05, ##P \u0026lt; 0.01, ###P \u0026lt; 0.001 vs Dox - on group.\u003c/p\u003e\n\u003cp\u003e(E) Representative images of IF co - staining of AT8 and CB in the DG region of the Dox - on group and the Veh group, and (F) Quantitative statistics of the average fluorescence density. Unpaired Student’s t - test, *P \u0026lt; 0.05. 3 mice per group.\u003c/p\u003e\n\u003cp\u003e(G) Representative magnified images of IF co - staining of AT8 and CB in the CA1 and DG regions of the Dox - on group, and (H) quantitative statistics of CB fluorescence intensity in AT8 - positive neurons and AT8 - negative neurons. The asterisk represents AT8-positive neuron, and the arrow represents AT8-negative neuron. Unpaired Student’s t - test, *P \u0026lt; 0.05. 3 mice per group.\u003c/p\u003e\n\u003cp\u003e(I) Western blot of hippocampal tissue in the Veh group and the Dox - on group, and (J) quantitative statistics. Unpaired Student’s t - test, *P \u0026lt; 0.05. 3 mice per group.\u003c/p\u003e\n\u003cp\u003eSeven - month - old homozygous Tg hTau368 mice were used. Data are presented as Mean±SEM.\u003c/p\u003e","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6470265/v1/875289371b11a14703f93374.jpg"},{"id":82119892,"identity":"06ca5704-e049-4142-8ad7-a57559c76009","added_by":"auto","created_at":"2025-05-07 03:12:03","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":675209,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExogenous expressing CB in hippocampal excitatory neurons alleviates neuronal hyperexcitation caused by tau aggregation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagrams of the virus injection sites in CA1/DG of three groups of Tg hTau368 mice treated with Veh + AAV- CaMKIIa -eGFP (Veh + eGFP), Dox + AAV-CaMKIIa-eGFP (Dox + eGFP) and Dox + AAV-CaMKIIa-Calb1-eGFP (Dox + CB).\u003c/p\u003e\n\u003cp\u003e(B) Representative images of the virus expression in the hippocampus and CB immunostaining in the Dox + CB group. The left side shows the representative images of hippocampal CB immunostaining and virus expression, and the right side shows the locally magnified images of the injection sites in CA1 and DG.\u003c/p\u003e\n\u003cp\u003e(C) Schematic diagram of patch - clamp recording of spontaneous excitatory postsynaptic potentials (sEPSCs) in excitatory neurons of the CA1 pyramidal cell layer, and (D) representative images of the three groups.\u003c/p\u003e\n\u003cp\u003e(E - H) Quantitative statistics of the (E) amplitude and (F) firing frequency of sEPSCs in the three groups, and the corresponding cumulative probability curves in (G, H).\u003c/p\u003e\n\u003cp\u003e(I) Schematic diagram of patch - clamp recording of evoked action potentials in the three groups, and (J - L) representative images. An input current with 20 pA (marked in red) could induced action potential in Dox + eGFP group (K), but not in Veh + eGFP (J) and Dox + CB group (L).\u003c/p\u003e\n\u003cp\u003e(M-O) Quantitative statistics of the (M) resting membrane potential, (N) rheobase, and (O) action potential firing frequency.\u003c/p\u003e\n\u003cp\u003eOne - way analysis of variance was performed, followed by Post hoc Tukey’s multiple comparison test. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. Data are presented as Mean±SEM. Each point represents a single recording of one neuron. Four - month - old homozygous Tg hTau368 mice, 4 - 5 mice per group.\u003c/p\u003e","description":"","filename":"fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6470265/v1/c46296c8a571c40db213769a.jpg"},{"id":82120760,"identity":"1a0148cd-a637-4902-ac39-83d9fb34f966","added_by":"auto","created_at":"2025-05-07 03:20:03","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":563270,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUpregulation of CB alleviates cognitive deficits caused by hippocampal tau aggregation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) In the Morris water maze test, the latency to find the platform during training was recorded for three groups of Tg hTau368 mice (Veh + eGFP, Dox + eGFP and Dox + CB).\u003c/p\u003e\n\u003cp\u003e(B) The number of platform-crossing times after training was statistically analyzed.\u003c/p\u003e\n\u003cp\u003e(C) Representative images of swimming tracks are shown.\u003c/p\u003e\n\u003cp\u003e(D) The discrimination ability of three groups of Tg hTau368 mice in the novel-location recognition test was assessed.\u003c/p\u003e\n\u003cp\u003e(E) Representative images of movement tracks are presented.\u003c/p\u003e\n\u003cp\u003e(F-G) In the open field test, (F) the total moving distance and (G) the number of times crossing the central area were measured.\u003c/p\u003e\n\u003cp\u003eTwo - way repeated - measures analysis of variance was used for A. one - way analysis of variance was used for B, D, F, and G, followed by the Post hoc Tukey’s multiple comparison test. Significant differences are indicated as *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. Data are presented as Mean ± SEM. Data are presented as Mean ±SEM. F stands for familiar; N stands for novel.The experiments were conducted using four-month-old homozygous Tg hTau368 mice, with the following group sizes: Veh + eGFP (n = 15), Dox + eGFP (n = 12), and Dox + CB (n = 12).\u003c/p\u003e","description":"","filename":"fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6470265/v1/e88e0d223681a79e6b872b0e.jpg"},{"id":82119894,"identity":"16dbd2a3-de22-4f6d-8cd0-598d2e35b992","added_by":"auto","created_at":"2025-05-07 03:12:03","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":412978,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCB deficiency is correlated with cognitive deterioration in AD patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The mRNA level of CB in the brain tissue of AD patients is decreased, and it is correlated with (B) the Clinical Dementia Rating Scale score and (C) the severity of the clinical Braak staging. (D) The decrease in expreession of CB in the brain tissue of AD patients is related to cognitive decline and (E) the severity of clinical symptoms. One - way analysis of variance was performed, followed by Post hoc Tukey’s multiple comparison test. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001. Data are presented as Mean±SEM. The raw data come from http://www.alzcode.xyz/.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(F) Immunohistochemical staining of hippocampal tissue in a 65 years old AD patient demonstrated a marked reduction of CB expression compared with an age-matched elderly individual without AD.\u003c/p\u003e\n\u003cp\u003eCon: normal control; Nor: normal cognitive function; MCI: mild cognitive impairment; Asym AD: AD patients in the early pre - clinical stage; AD: AD patients in the dementia stage; CDR: Clinical Dementia Rating Scale.\u003c/p\u003e","description":"","filename":"fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6470265/v1/db7abf45c8e6cfe3c511c8a2.jpg"},{"id":82122297,"identity":"a915f312-112c-4512-9282-ac5c672e2bb4","added_by":"auto","created_at":"2025-05-07 03:28:03","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":283119,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWorking model. \u003c/strong\u003eTau aggregation in hippocampal CA1 and DG excitatory neurons reduces CB expression, leading to the increased epileptic susceptibility and cognitive impairment in tau-driven mouse model. (The image was created by BioRender.com.)\u003c/p\u003e","description":"","filename":"fig9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6470265/v1/185a13dac849bd2055c5465d.jpg"},{"id":82123836,"identity":"08a0689f-4bc2-4212-bb27-e2d1b504a10b","added_by":"auto","created_at":"2025-05-07 03:36:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7250843,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6470265/v1/5169e835-c1af-4e4a-9a3f-55030e8cbaee.pdf"},{"id":82119900,"identity":"a5016040-f668-4c5f-b769-e443c0aa9eae","added_by":"auto","created_at":"2025-05-07 03:12:06","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":105060673,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryVideo1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6470265/v1/192f553129c567781f12b88b.mp4"},{"id":82119885,"identity":"437583f8-5857-4fac-9a1b-34b3cc31dee5","added_by":"auto","created_at":"2025-05-07 03:12:03","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":112801,"visible":true,"origin":"","legend":"","description":"","filename":"Westernblotrawdata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6470265/v1/02bca1a32f27f82ab161f3b8.pdf"},{"id":82120762,"identity":"b47faff5-080f-4060-b8d5-311b909e3bf1","added_by":"auto","created_at":"2025-05-07 03:20:03","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1075124,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6470265/v1/4f03f5e900b178527f27881c.docx"}],"financialInterests":"","formattedTitle":"Calbindin-D28k deficiency mediates tau-driven hippocampal hyperexcitement and cognitive impairment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlzheimer's disease (AD) is the most common and devastating neurodegenerative disease, with lack of effective prevention or approved therapies yet \u003csup\u003e[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. A striking clinical feature of AD is higher prevalence of temporal lobe epilepsy (TLE) and 42\u0026ndash;64% of AD patients exhibiting overt epileptic seizures or subclinical epileptic activity \u003csup\u003e[\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Notably, early-onset AD patients (aged 50\u0026ndash;60 years) show an 87-fold increased incidence of seizures compared to age-matched healthy individuals \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Intracranial recordings reveal that silent hippocampal seizures and spikes emerge during the preclinical phase of AD, preceding overt cognitive symptoms \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Human studies demonstrated that increased neuronal network and brain activity start as early as a decade before the onset age of disease \u003csup\u003e[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Hippocampal excitability follows an inverted U-shaped trajectory during AD progression, with hyperexcitability in early stages transitioning to hypoexcitability in later phases \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Critically, seizures in AD patients accelerate cognitive decline and increase mortality risk due to trauma from sudden loss of consciousness \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eExtracellular amyloid plaque deposition and intraneuronal neurofibrillary tangles (NFTs) in the brain are two characteristic pathological hallmarks of AD. However, they demonstrate distinct spatiotemporal progression patterns that Aβ deposition predominates in medial parietal and frontal cortices, whereas tau pathology initially accumulates within the entorhinal-hippocampal network at prodromal stages \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Asymmetrically deposited tau in the epileptogenic hemisphere was the most striking finding, whereas asymmetries in amyloid deposition were less pronounced in early clinical stages of AD with TLE \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.The entorhinal-hippocampal network contains key excitatory pathways: entorhinal cortex (EC) efferents directly innervate the dentate gyrus (DG), where granule cells extend mossy fiber axons to CA3. CA3 neurons subsequently relay signals to CA1 via Schaffer collaterals, while parallel EC projections establish direct monosynaptic connections with CA1 pyramidal neurons. Notably, the DG-CA1-EC-DG loop has been identified as one of principal circuit involved in seizures generation and propagation of TLE \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Functional neuroimaging reveals enhanced blood-oxygen-level-dependent (BOLD) activation in hippocampal-temporal circuits during memory tasks in early AD \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Glutamatergic neurotransmission, mediated by CA1 pyramidal neurons and DG granule cells, critically regulates learning and memory processes \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Of particular significance, the CA1 pyramidal layer demonstrates selective vulnerability to early tau deposition \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Nevertheless, the precise mechanisms through which tau pathology disrupts hippocampal excitatory-inhibitory balance remain to be elucidated.\u003c/p\u003e \u003cp\u003eCalcium homeostasis is of utmost significance in maintaining excitatory-inhibitory balance within neural circuits, regarded as an important target for the treatment of AD recently \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Among the various elements involved in calcium regulation, Calbindin - D28k (CB), a calcium - binding protein, is highly expressed in the neurons of the hippocampal CA1 and DG neurons \u003csup\u003e[\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Functionally, CB serves as a key regulator of intracellular free calcium ion concentrations, actively participating in the modulation of intracellular calcium homeostasis, synaptic plasticity, and cognitive processes \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. A decline in CB expression has been consistently associated with cognitive impairment in patients suffering from AD and TLE \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Evidences from animal models further supports this relationship that knockdown of CB in the excitatory neurons of hippocampal CA1 and DG leads to significant impairments in the spatial memory of mice \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Clinical observations have also revealed reduced CB expression in the hippocampal CA1 and DG regions of AD and TLE patients\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Notably, the question of whether replenishing CB in the excitatory neurons of hippocampal CA1 and DG could alleviate neuronal over - excitation and mitigate cognitive impairment remains an open and compelling area of investigation. Addressing this question could potentially unlock new therapeutic strategies for treating AD and TLE.\u003c/p\u003e \u003cp\u003eTo address the relationship between tau aggregation and hippocampal hyperexcitability, we employed a newly created tauopathy Tg hTau368 mice to investigate tau aggregation feature and calcium dysregulation. Neuronal hyperexcitability and seizure susceptibility were evaluated using patch-clamp electrophysiology, \u003csup\u003e18\u003c/sup\u003eF-FDG PET/CT and optogenetic activation. A tetracycline-controlled system (Tet-on) enabled spatiotemporal induction of tau pathology in Tg hTau368 mice to dissect its interplay with CB and synaptic proteins. Furthermore, adeno-associated virus (AAV)-mediated CB restoration in hippocampal CA1/DG excitatory neurons was tested for its therapeutic effects on hyperexcitability and cognition. Public AD-related databases were mined to validate clinical correlations between CB levels and cognitive outcomes. Our findings demonstrate that tau accumulation in CA1/DG CamKIIα-positive neurons suppresses CB expression, triggering calcium dyshomeostasis. This cascade enhances hippocampal excitability, reduces synaptic protein expression, and increases susceptibility to seizures and cognitive deficits. Critically, AAV-delivered CB rescues neuronal hyperexcitability and cognitive impairment. These results identify CB/calcium homeostasis as a pivotal mechanism linking tau pathology to TLE and cognitive decline in AD, highlighting its potential as a therapeutic target.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and treatment\u003c/h2\u003e \u003cp\u003eAll mice were housed in groups of three to four per cage, under a 12 h light/dark cycle at 23\u0026ndash;25\u0026deg;C. Food and water were available \u003cem\u003ead libitum\u003c/em\u003e. doxycycline hyclate (Dox, Beyotime, Shanghai, China) was dissolved in drinking water (2 mg/l) and administered \u003cem\u003ead libitum\u003c/em\u003e for Tg hTau368 mice. Equal numbers of male and female mice were randomly assigned to groups. All experiments and data analyses were conducted by experimenters blind to the groupings. All animal experiments were conducted in accordance with relevant ethical regulations for animal testing and research, and were approved by institutional guidelines and the Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTg hTau368 and PR5 mice\u003c/h3\u003e\n\u003cp\u003eThe Tg hTau368 mice were generated jointly by our laboratory and Nanjing Biomedical Research Institute of Nanjing University that human \u003cem\u003eMAPT\u003c/em\u003e gene encoding the hTau368 fragment (2N4R tau) was placed downstream the tetracycline-responsive element (TRE) promoter, which joined to assemble a tet-on system with a second module expressing the reverse tetracycline-controlled transactivator (rtTA) under the control of the neuron-specific Eno2 promoter \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. In the presence of Dox (Dox-on), reverse tetracycline transactivator (rtTA) binds to the tetracycline-responsive element (TRE) to initiate hTau368 expression. In the absence of doxycycline (Dox-off), hTau368 expression stops, as rtTA cannot bind to TRE, and tauophathology gradually dismissed \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePR5 mice, which overexpress the longest human tau isoform (2N4R tau) together with the P301L mutation on the C57BL/6 background. PR5 mice transgenic expression of human P301L mutant tau under control of the murine Thy1.2 promoter leads to the formation of sarcosyl-insoluble, 15-nm-wide tau filaments. By comparison, tau filaments in frontotemporal dementia with parkinsonism linked to chromosome 17 patients with the P301L mutation \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eAntibodies\u003c/h3\u003e\n\u003cp\u003eAntibodies used in the present study are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntibodies used in this study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntibody\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eType\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSpecificity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eSource/reference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTau368\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMono\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTau 1-368 N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eJointly developed with AtaGenix \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTau5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMono\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTau (a.a.210\u0026ndash;230)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAbcam (ab80579)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHT7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMono\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTau (a.a.159\u0026ndash;163)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eThermoFisher (MN10000)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-pT205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePoly\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep-tau (T205)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSAB (11108)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAT8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMono\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep-tau (S202/T205)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eThermoScientific (MN1020)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePSD-95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePoly\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePSD-95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSAB (41365)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSYN-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMono\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSynapsin1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCST (D12G5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-actin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMono\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eβ-actin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSAB (21800)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-Calbindin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMono\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCalbindin-D28k\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAbcam (ab108404)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCalbindin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMono\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCalbindin-D28k\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSigma (C9848)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCamKII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMono\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCamKII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCell signaling (3362)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAD67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMono\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGAD67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSigma (MAB5406)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-Parvalbumin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMono\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eParvalbumin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSigma (SAB4200545)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDylight 800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDylight 800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eImmunoway (RS23920)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDylight 800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDylight 800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eImmunoway (RS23910)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eStereotaxic viral injection\u003c/h3\u003e\n\u003cp\u003eMice were anesthetized using isoflurane and secured in a stereotaxic frame (RWD Life Science, Shenzhen, China). The scalp was surgically incised to expose the skull, and residual soft tissue on the skull surface was meticulously removed using sterile cotton swabs. The coordinate origin was defined as the intersection of the bilateral tangents at the bregma. The skull was leveled by aligning the anterior and posterior bregma, ensuring a height discrepancy of less than 0.03 mm between the left and right sides. The target injection site was identified based on stereotaxic coordinates and marked on the skull. A small craniotomy was performed at the marked location using a precision cranial drill. Viral solution was delivered at a rate of 100 nl/min using a microinjection system and retained for 5 min post-injection.\u003c/p\u003e\n\u003ch3\u003eKainic acid-induced epilepsy model and in vivo electrophysiological recording\u003c/h3\u003e\n\u003cp\u003eKainic acid (KA), an analog of glutamate, was used to induce TLE by activating kainate receptors, leading to seizure activity, neuronal damage, and chronic spontaneous epilepsy. KA (500 nl, 0.5 \u0026micro;g/\u0026micro;l, dissolved in saline) was stereotaxically injected into the hippocampal CA1 (-1.0 mm AP, +\u0026thinsp;1.8 mm ML, -1.5 mm DV) and DG (-1.0 mm AP, +\u0026thinsp;1.8 mm ML, -2.1 mm DV) of 4-month-old WT mice. A 8-channel electrode was implanted 0.1 mm above the injection sites to record electrophysiological signals when mice waken up. Local field potentials were recorded using a Recording System (Plexon, Hong Kong, China). Data were stored for offline analysis with 16-bit format, visualized in Neuro Explore. Amplitude and power spectral density (PSD) of LFPs were analyzed in default parameters by NeuroExplorer (Plexon, Hong Kong, China) : shift (0.5 s), number of frequency values (8,192), normalization (log of PSD), show frequency from 0 to 150 Hz.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eOptogenetic stimulation and seizures induction\u003c/h2\u003e \u003cp\u003eThe operation method has been reported by us before [31]. To put it simply, pAAV-CaMKIIα-ChR2 (H134R) -mCherry was stereotaxically injected into the right dorsal hippocampal CA1 (-1.0 mm AP, +\u0026thinsp;1.8 mm ML, -1.1 mm DV, 500 nl) and DG (-1.0 mm AP, +\u0026thinsp;1.8 mm ML, -2.1 mm DV, 500 nl). pAAV-CaMKIIα-ChR2 (H134R) -mCherry was stereotaxically injected into the right ventral hippocampus CA1 (-3.2 mm AP, +\u0026thinsp;3.2 mm ML, -4.1 mm DV, 500 nl) and DG (-3.2 mm AP, +\u0026thinsp;3.2 mm ML, -4.8 mm DV, 500 nl). Following viral injection, a fiber optic cannula (200 \u0026micro;m core, NA\u0026thinsp;=\u0026thinsp;0.37) was implanted above 0.4 mm of CA1. A 8-channel electrode was implanted into the ipsilateral M1 (+\u0026thinsp;1.5 mm AP, +\u0026thinsp;1.5 mm ML, \u0026minus;\u0026thinsp;1.5 mm DV) for electrophysiological signals recording described above. Three to four anchoring screws (25 \u0026micro;m diameter) were fixed into the skull near the injection site. The cannula and screws were secured with dental cement (RWD Life Science, Shenzhen, China). Four weeks post-surgery, seizures were induced. Mice were anesthetized with isoflurane, and the cannula was connected to a fiber optic patch cable and laser source. Laser parameters were set to 472 nm blue light, 20 Hz frequency, 10% duty cycle, and pulsed waveform, delivering 2.8 mW/mm\u0026sup2; at the fiber tip. Seizure latency and severity were assessed using a modified Racine scale [32]: Stage 1, Facial twitching and chewing; Stage 2, Chewing and head nodding; Stage 3, Unilateral forelimb clonus; Stage 4, Bilateral forelimb clonus with rearing; Stage 5, Bilateral clonus, rearing, and falling; Stage 6, Wild jumping. Stages 1\u0026ndash;3 were classified as focal seizures (FS), and stages 4\u0026ndash;6 as generalized seizures (GS). The latency to stage 4 (GS) was recorded as the primary metric in our study. Upon reaching stage 4, stimulation continued for 15 s before cessation. The maximum seizure stage was recorded, and mice were monitored until normal behavior resumed. The fiber optic cable was removed under anesthesia, and mice were returned to their cages. Seizures were induced once daily, with GS latency and maximum seizure stage recorded over consecutive days. In some mice, a 8-channel electrode was implanted into the ipsilateral M1 (+\u0026thinsp;1.5 mm AP, +\u0026thinsp;1.5 mm ML, \u0026minus;\u0026thinsp;1.5 mm DV) for electrophysiological recording (Supplementary Video 1).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSmall animal PET/CT imaging\u003c/h3\u003e\n\u003cp\u003eBrain \u003csup\u003e18\u003c/sup\u003eF-FDG PET/CT imaging was conducted using a small animal PET/CT scanner (Novel Medical, Beijing, China) at Union Hospital, Wuhan. Mice were fasted for 12 h, weighed, and injected with 3\u0026ndash;4 MBq of 18F-FDG via the tail vein. Anesthesia was induced and maintained with 2% isoflurane. After a 45-minute uptake period, whole-body PET/CT scans were performed with the following parameters: energy window of 350\u0026ndash;650 keV, timing window of 1.2 ns, spatial resolution of 1.5 mm, and scan duration of 300 s. Acquired DICOM images were analyzed using PMOD 4.0 software. A standardized mouse brain template was used to segment the brain into 19 regions of interest (ROIs), and the average \u003csup\u003e18\u003c/sup\u003eF-FDG uptake intensity for each ROI was quantified as standardized uptake value normalized to body weight.\u003c/p\u003e\n\u003ch3\u003eMonitoring of mouse energy metabolism and activity\u003c/h3\u003e\n\u003cp\u003eThe Oxymax CLAMS system (Columbus Instruments, Columbus, USA) was employed to monitor the 24-h energy metabolism and locomotor activity of mice. Prior to the experiment, mice were acclimated to the laboratory environment for 24 hs. Each mouse was singly housed in a cage, ensuring an adequate Supplementaryy of food and water. Subsequently, the oxygen consumption (VO₂), carbon dioxide production (VCO₂), respiratory quotient (RQ), and spontaneous activity counts of each mouse were recorded over a 24-h period (light cycle: 7:00\u0026ndash;19:00; dark cycle: 19:00-next day 7:00). The energy expenditure of mice was determined using indirect calorimetry, with the calculation formula as follows: Total energy expenditure = (3.815\u0026thinsp;+\u0026thinsp;1.232 \u0026times; RQ) \u0026times; VO₂. This system utilizes infrared photocell technology to enable three-dimensional monitoring of animal movements. Mouse locomotion interrupts the infrared beam, generating a count to quantify spontaneous movement.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCalcium imaging recording\u003c/h2\u003e \u003cp\u003eFour weeks prior to imaging, pAAV-CaMKIIα-GCaMP6f-eGFP (500 nl) was injected into the right hippocampal CA1 region (-1.0 mm AP, +\u0026thinsp;1.8 mm ML, -1.5 mm DV) of mice in the Dox and Veh groups (n\u0026thinsp;=\u0026thinsp;4 per group). Ice-cold slicing solution was prepared, and artificial cerebrospinal fluid (aCSF) was oxygenated (95% O₂, 5% CO\u003csub\u003e2\u003c/sub\u003e) in a incubation chamber at 35\u0026deg;C for at least 30 min. Mice were anesthetized with 1% sodium pentobarbital (intraperitoneal injection) and decapitated. Brains were quickly removed, trimmed, and affixed to a vibratome stage using cyanoacrylate glue. Coronal slices (300 \u0026micro;m thickness) were cut in ice-cold slicing solution and transferred to oxygenated aCSF at 35\u0026deg;C for 30 min, followed by 1- h incubation at room temperature before imaging. Brain slices were placed under a confocal microscope equipped with a 40\u0026times; water-immersion objective. The green fluorescence signal (GCaMP6f) in the CA1 pyramidal cell layer was identified and focused for imaging. Neuronal depolarization was induced by perfusing the slices with 30 mM KCl. Fluorescence signals were excited at 488 nm and recorded within the 505\u0026ndash;550 nm emission range. Fluorescence signals were analyzed using ImageJ. Calcium signals were calculated as ΔF/F using the formula: ΔF/F = [(F1- B1) - (F0- B0)] / (F0 \u0026ndash; B0), where F1and F0 represent fluorescence intensities during and before stimulation, and B1 and B0 are background signals. Data were normalized, with the baseline signal set to 0%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro patch - clamp electrophysiological recording\u003c/h2\u003e \u003cp\u003eAll experiments were conducted using solutions prepared as follows: Cutting Solution contained choline chloride (110 mM), KCl (2.5 mM), NaH₂PO₄ (1.25 mM), NaHCO₃ (26.0 mM), CaCl₂ (0.5 mM), MgSO₄ (7 mM), D-glucose (10 mM), Na-ascorbate (11.6 mM), Na-pyruvate (3.1 mM), and atropine sulfate (0.01 mM); Recording Solution comprised NaCl (119 mM), KCl (2.5 mM), NaH₂PO₄ (1.25 mM), NaHCO₃ (26 mM), CaCl₂ (2.5 mM), MgCl₂ (1.3 mM), and D-glucose (10 mM); K⁺ Intracellular Solution was formulated with K-gluconate (128 mM), KCl (17.5 mM), Na₂ATP (5 mM), MgCl₂ (1 mM), EGTA (0.2 mM), and HEPES (10 mM), adjusted to pH 7.4 and osmolarity of 290\u0026ndash;300 mOsm. Two-month-old homozygous Tg hTau368 mice were randomly divided into three groups (Veh\u0026thinsp;+\u0026thinsp;eGFP, Dox\u0026thinsp;+\u0026thinsp;eGFP, Dox\u0026thinsp;+\u0026thinsp;CB), with viral vectors (AAV-CaMKIIα-eGFP or AAV-CaMKIIα-Calb1-eGFP) bilaterally injected into hippocampal CA1 and DG 1.5 months prior to recording. Brain slices were prepared, transferred to a perfusion chamber, and stabilized under a microscope, where green-fluorescent CA1 pyramidal neurons in optimal physiological condition were selected for recording. For spontaneous excitatory postsynaptic currents (sEPSCs), a K⁺ intracellular solution-filled glass electrode (3\u0026ndash;5 MΩ) was used to clamp neurons at \u0026minus;\u0026thinsp;70 mV after gigaseal formation and membrane rupture; sEPSCs were recorded for 120 s in voltage-clamp mode. Action potentials (APs) were recorded in current-clamp mode using the same electrode, applying step-current stimuli (0\u0026ndash;140 pA, 20 pA increments, 10 s intervals) and counting APs within 600 ms windows. Data were acquired via a Multiclamp 700B amplifie and Digidata 1440A digitizer (sampling at 10 kHz, low-pass filtered at 2 kHz), with analysis performed using Clampfit 10.6 software for frequency, amplitude, and AP quantification (Molecular Devices, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eThe hippocampus was separately on ice and homogenized in a customized RIPA lysis buffer. This buffer was formulated with 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X \u0026minus;\u0026thinsp;100, 1% sodium deoxycholate, and 0.1% SDS (Beyotime, Shanghai, China). To prevent protein degradation and maintain phosphorylation states, a protease and phosphatase inhibitor cocktail (Thermo Scientific, Waltham, USA) was added at a ratio of 10 \u0026micro;l per mg of tissue. The protein concentration within the RIPA - soluble lysates (pun at 12,000\u0026times;g for 20 min) was accurately determined using a BCA protein assay kit (Thermo Fisher, Waltham, USA). Equal amounts of protein from the soluble fraction were loaded onto 10% SDS - PAGE gels for separation. The proteins were electrophoretically transferred onto nitrocellulose membranes (Merck Millipore, Darmstadt, Germany). The membranes were then blocked with 5% bovine serum albumin (BSA) to prevent non - specific antibody binding. Subsequently, they were incubated sequentially with primary and secondary antibodies as listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Visualization of the protein bands was achieved using an enhanced chemiluminescence substrate system (Santa Cruz Biotech, Dallas, USA). The membranes were imaged with an Odyssey Imaging System (LI - COR Biosciences, Lincoln, USA), and the band intensities were quantified using the Image J. β-actin was used as a loading control to ensure consistent protein loading across samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eImmunostaining and quantification\u003c/h2\u003e \u003cp\u003eMice were anesthetized with 2% isoflurane (RWD Life Science, Shenzhen, China). Through the left ventricle, they were first perfused with 0.9% NaCl for 5 min to clear the blood, followed by perfusion with 4% paraformaldehyde in PBS for 1 day. The brains were treated with 25% sucrose for 1 day and then transferred to 30% for an additional day. Using a cryostat microtome (Leica, Wetzlar, Germany), the brains were cut into 30 \u0026micro;m - thick sections. For immunohistochemistry, the free - floating sections were first immersed in 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in anhydrous methanol for 30 min to quench endogenous peroxidases. Non - specific binding sites were blocked with BSA for 30 min at room temperature. The brain slices were then incubated overnight at 4\u0026deg;C with primary antibodies. The immunoreactions were developed using a DAB - staining kit (ZSGB - BIO, Beijing, China). Images of the stained sections were captured at 20\u0026times; magnification using an automatic slice scanning system (Olympus, Tokyo, Japan) and analyzed with ImageJ software. The areas of different brain regions were measured to assess the staining distribution. For immunofluorescence, the sections were thoroughly washed with PBST (PBS containing 0.1% Triton X \u0026minus;\u0026thinsp;100). They were incubated overnight at 4\u0026deg;C with primary antibodies. After the incubation, the sections were washed with PBST for 15 min and then incubated with the secondary antibody at 37\u0026deg;C for 1 h. Finally, the nuclei were counterstained with DAPI. Images were acquired at 20\u0026times; magnification using both an automatic slice scanning system (Olympus, Tokyo, Japan) and a two - photon laser - scanning confocal microscope (Zeiss, Oberkochen, Germany). Image J software was used for analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eNovel-location recognition test\u003c/h2\u003e \u003cp\u003eBefore the test, mice were acclimated to handling. On day 1, each mouse was placed in the center of a plastic box. In the box, two identical objects (A and B) were positioned in two corners. The mouse was allowed to explore freely for 5 min. The mouse was re - introduced to the box after 24 h. Object A remained in its original corner, while object B was placed in a new location. Again, the mouse was given 5 min for exploration. The exploration time for objects A and B, denoted as TA and TB respectively, was recorded. A video tracking system (Anymaze Technology SA, Stoelting Co., IL, USA) was used to identify exploration, which was defined as the mouse's head being within 3 cm of an object. Mice with TA or TB less than 2 s were excluded from the analysis. The discrimination index was calculated as (TB - TA) / (TB\u0026thinsp;+\u0026thinsp;TA). A higher discrimination index indicated better spatial memory retention.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMorris water maze test\u003c/h2\u003e \u003cp\u003eMice were housed in the test room for 24 h prior to the test commencement. During the trainning phase, the mice were trained to locate a hidden platform within the Morris water maze. The trainning phase spanned 5 consecutive days, with 3 trials per day. The trials were conducted between 14:00 and 17:00, with a 30 min interval between each trial. In each trial, the mouse was placed in one of the three quadrants that did not contain the platform, facing the pool wall. If the mouse found the hidden platform within 60 s, it was allowed to remain on the platform for an additional 15 s for learning consolidation. If the platform was not found within 60 s, the mouse was gently guided to the platform and allowed to stay there for 15 s. The time taken to find the platform over the 5 - day training period was recorded as the escape latency. On day 6, a testing trial was carried out. The hidden platform was removed, and each mouse was placed in the quadrant opposite to the target quadrant. A video tracking system (Chengdu Taimeng Software Co., Ltd, China) was used to record and analyze the time, distance, and trajectory of each mouse's movement in the pool. Mice with any visual or limb impairments were excluded from the analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eOpen field test\u003c/h2\u003e \u003cp\u003eMice were handled for 1 day before the test and were placed in the test room the day prior to the behavioral test to acclimatize to the environment. The open field apparatus was a white plastic box measuring 60\u0026times;60\u0026times;50 cm\u0026sup3;. In the monitoring system, the floor of the box was virtually divided into 16 equal squares, with a central field consisting of the central 4 square regions and 12 peripheral fields. Each mouse was allowed to explore freely in the box for 5 min. The ANY - maze video tracking system (Stoelting Co., WoodDale, IL, USA) was used to record and analyze the time and distance each mouse traveled in different zones, providing insights into the mouse's locomotor activity and exploratory behavior.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eAll data were processed and visualized using GraphPad Prism 8 (La Jolla, CA). For comparisons between two groups, two - tailed unpaired Student\u0026rsquo;s t - tests were utilized. When comparing multiple groups, one - way, two - way, or repeated measures ANOVA was performed, followed by post - hoc tests for multiple comparisons. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All values are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003epTau accumulation in hippocampal excitatory neurons disrupts intracellular calcium homeostasis\u003c/h2\u003e \u003cp\u003eAs truncated Tau368 fragements naturally exists in the brain and increases during aging and AD, which is more neurotoxic than other truncated and full-lengthen tau \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. We have previously generated Tg hTau368 mouse model with a tetracycline-controlled expressing truncated human tau (hTau368) under the neuronal promoter Eno2 \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. This Tg mice exhibited hippocampal-predominant tau phosphorylation (pTau) aggregation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C) and cognitive impairments when treated with doxycycline hyclate (Dox) for 2 months as previously reported \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Immunofluorescence co-staining revealed pTau (AT8 or pTau205) co-localization with CamKIIα-positive excitatory neurons in hippocampal CA1 and DG regions, with no parvalbumin (PV)- or GAD67-positive inhibitory neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and E). Consistently, similar tau aggregation patterns were observed in Tg PR5 mice expressing P301L mutant human tau under control of the murine Thy1.2 (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess the impact of tau aggregation on calcium dynamics, we injected AAV-CaMKIIα-GCaMP6f-eGFP into CA1 of Tg hTau368 mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B). There was a concentration difference between intracellular and extracellular calcium ions in neurons, and when neurons were activated, extracellular calcium ions entered into the cell and rapidly increased the intracellular calcium ion concentration. Using a protein fluorescent probe (GCaMP6f-eGFP) as a calcium ion indicator, the calcium ion concentration in neurons is expressed by the fluorescence intensity, so as to detect the change of intracellular calcium ion concentration \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. We found that the KCl-induced depolarization of CA1 CaMKIIα-GCaMP6f-eGFP -positive neurons (green) revealed significantly higher intracellular calcium peaks in Dox-treated mice (with tau pathology) compared to Veh group (without tau pathology) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-E), demonstrating tau-driven calcium dysregulation in CA1 excitatory neurons.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eTau aggregation promotes TLE induced by hippocampal CA1 and DG hyperexcitability\u003c/h2\u003e \u003cp\u003eTo investigate the role of hippocampal CA1 and DG in TLE, we injected kainic acid (KA), a natural excitatory neurotoxin derived from red algae and an agonist of ionotropic glutamate receptors (AMPA and KA receptors), into the CA1 and DG of aged wild-type (WT) mice respectively. KA injection in both regions induced epileptic seizures (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), mimicking human TLE \u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. This suggests that KA-mediated hyperactivation of CA1 and DG contributes to TLE epileptogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further elucidate the role of excitatory neurons in CA1 and DG and the influence of tau pathology on epileptogenesis, we injected AAV-CaMKIIα-ChR2-mCherry into the CA1 and DG regions of aged Tg hTau368 mice. Four weeks post-injection, blue light stimulation was used to activate neurons in these regions. Both dorsal (dHipp) and ventral (vHipp) hippocampal stimulation induced seizures of varying severity and typical epileptic seizure waveforms occurred during electrophysiological recording in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and C, Supplementary Video 1). Seizures ceased upon cessation of light stimulation, and mice returned to normal behavior gradually (Supplementary Video 1). We compared seizure latency and severity between the Dox group and the Veh group. The Dox group exhibited shorter seizure latency (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-left and Supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) and higher seizure severity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-right), indicating that tau pathology enhances susceptibility to TLE. The hippocampal - parahippocampal circuits, particularly the DG-CA1-EC-DG loop, play a critical role in TLE initiation and propagation \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Our findings suggest that abnormal activation of CA1 and DG excitatory neurons drives TLE, and that tau aggregation increases TLE susceptibility, potentially through hyperexcitability in the DG-CA1-EC-DG circuit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eTau pathology correlates with cerebral hypermetabolism, hyperexcitable behavioral phenotypes and cognitive deficits\u003c/h2\u003e \u003cp\u003eIncreased neuronal and brain network excitability is age-dependent, and dysregulation of excitatory inhibition of neural microcircuits in aged mice is associated with cognitive impairment \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. In order to simulate the effects of tau pathology on aging as AD is an age-dependent disease, we took advantage of the controllable induction of hTau expression in 14\u0026ndash;15 months of Tg hTau368 mice by administering 1\u0026ndash;2 months Dox treatment, and then FDG PET/CT scanning, energy metabolism, activity monitoring and cognitive tests were conducted (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). \u003csup\u003e18\u003c/sup\u003eF-FDG-PET imaging, could reflect the intensity of glucose metabolism in the brain, and to a certain extent, the excitability state of the whole brain, which has a certain diagnostic value for AD progression \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. \u003csup\u003e18\u003c/sup\u003eF-FDG PET/CT in aged Tg hTau368 mice (16 months) revealed elevated glucose metabolism in the hippocampus and olfactory bulb following 1\u0026ndash;2 months of Dox treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D), consistent with early-stage hypermetabolic states observed in young 3\u0026times;Tg (7 months old, carrying the APP/ PS1/P301L tau mutation) and PR5 (3 months old, carrying the tau P301L mutation) mice \u003csup\u003e[\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe cerebral hypermetabolism paralleled with increased O2 consumption (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and F), energy expenditure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), and spontaneous movements (Supplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), especially during the daytime in resting state of mice. Cognitive deficits were also found in Dox-treated Tg hTau368 mice, manifested as a lower novel object discrimination index and longer latency to platform, as detected by the novel object recognition test (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH and I) and Morris water maze test (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ and L), respectively. The results above suggest that tau pathology associated with elevated metabolism, hyperexcitable behavioral phenotype and cognitive deficits in aged mice simulating the early stage of tau pathology.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eTau pathology induces CB reduction and synaptic dysfunction\u003c/h2\u003e \u003cp\u003eTo explore the relationship of tau pathology with CB and synapse-associated proteins, we utilized controlled hTau expression via the Tet-on system in Tg hTau368, i.e., hTau was expressed when Dox was given (Dox-on), and when Dox was withdrawn, hTau expression ceased and was gradually cleared (Dox-on-off), the phenomenon has also been reported in other Tg lines\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. As we expected, tau pathology in the Dox-on-off group of mice were largely cleared after 3 months, especially in CA1 pyramidal neurons and DG granule cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C), which was also accompanied by a rebound in CB expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), and an increase in synaptic proteins expression (Supplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), such as postsynaptic density protein 95 (PSD95) and synapsin 1 (SYN-1). Strikingly, Dox withdrawal for 3 months reversed tau pathology and restored CB/synaptic proteins expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-D and Supplementary Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Immunofluorescent staining and Western blotting data also confirmed CB reduction in Dox-on group compared with Veh group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F, I and J), as well as AT8-positive neurons compared with AT8-negative neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and J). The results above linked tau pathology, CB reduction and synaptic dysfunction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eUpregulation of CB alleviates tau pathology-induced neuronal hyperexcitability and cognitive impairment\u003c/h2\u003e \u003cp\u003eTo investigate whether CB defects mediate tau -associated neuronal hyperexcitability, we recorded electrophysiological properties in CA1 CamkII-eGFP-positive pyramidal neurons of Tg hTau368 mice treated with Veh\u0026thinsp;+\u0026thinsp;AAV- CaMKIIa -eGFP (Veh\u0026thinsp;+\u0026thinsp;eGFP), Dox\u0026thinsp;+\u0026thinsp;AAV-CaMKIIa-eGFP (Dox\u0026thinsp;+\u0026thinsp;eGFP) and Dox\u0026thinsp;+\u0026thinsp;AAV-CaMKIIa-Calb1-eGFP (Dox\u0026thinsp;+\u0026thinsp;CB) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). For the 2 - month - old homozygous Tg hTau368 mice in the Veh\u0026thinsp;+\u0026thinsp;eGFP, Dox\u0026thinsp;+\u0026thinsp;eGFP, and Dox\u0026thinsp;+\u0026thinsp;CB groups, they received treatments of Veh, Dox, and Dox respectively, for a duration of 2 months. One and a half months prior to patch - clamp detection, the viruses AAV - CaMKIIa - eGFP, AAV - CaMKIIa - eGFP, and AAV - CaMKIIa - Calb1 - eGFP were injected into the hippocampal CA1 and DG regions respectively. CB expression was confirmed in CA1 and DG neurons in the Dox\u0026thinsp;+\u0026thinsp;CB group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Tau accumulation (Dox\u0026thinsp;+\u0026thinsp;eGFP group) increased sEPSC amplitude and frequency, indicating elevated neuronal excitability, while CB Supplementaryementation reversed these effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-H). Additionally, neurons in Dox\u0026thinsp;+\u0026thinsp;eGFP group exhibited higher resting membrane potentials, lower rheobase, and increased evoked action potential frequency, all of which were normalized by CB upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI-O). These findings suggest that CB upregulation alleviates tau accumulation induced hyperexcitability in CA1 neurons.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBehavioral tests also revealed that CB upregulation improved cognitive performance in Tg hTau368 mice. In the Morris water maze, the Dox\u0026thinsp;+\u0026thinsp;CB group showed a tendency of reduced latency to find the platform (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA) and increased platform crossings compared to the Dox\u0026thinsp;+\u0026thinsp;eGFP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB and C). Similarly, in the novel object recognition test, the Dox\u0026thinsp;+\u0026thinsp;CB group demonstrated better recognition of novel object locations (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD and E). However, CB did not affect spontaneous behaviors in the open field test (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF and G). Overall, CB Supplementation in hippocampal excitatory neurons mitigates tau-induced cognitive dysfunction, highlighting its therapeutic potential.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eReduced CB correlates with cognitive deterioration and an advanced disease stage in AD patients\u003c/h2\u003e \u003cp\u003eTo investigate the association between CB and cognitive decline in AD patients, we analyzed CB expression using a publicly available AD database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.alzcode.xyz/\u003c/span\u003e\u003cspan address=\"http://www.alzcode.xyz/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) \u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. Compared to healthy controls, AD patients exhibited significantly decreased CB transcript levels in brain tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). To evaluate disease progression, patients were stratified by cognitive function (Clinical Dementia Rating [CDR] scale; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB) and neuropathological severity (Braak staging; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Strikingly, CB transcript levels progressively declined with worsening cognitive impairment (higher CDR scores) and advanced Braak stages. Consistent with transcriptional changes, total CB protein expression was also markedly reduced in AD patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD and E), and immunohistochemical staining confirmed diminished CB levels in the hippocampal regions of AD brains (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF). These findings collectively highlight a robust inverse correlation between CB depletion and both cognitive decline and AD progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eEmerging evidences suggest that hippocampal network hyperexcitability caused by excitation-inhibition imbalance occurs in early AD progression and exacerbates cognitive decline, though the underlying mechanisms remain elusive. Our study demonstrates that tau aggregation within hippocampal CA1 and DG excitatory neurons leads to reduced CB expression, elevated intracellular calcium transients, and enhanced neuronal network excitability, ultimately increasing susceptibility to TLE and cognitive impairment. Notably, CB Supplementation in hippocampal CA1 and DG excitatory neurons ameliorates tauopathy-induced hyperexcitability and cognitive deficits in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Clinical correlation analysis from AD databases further reveals CB deficiency is associated with accelerated cognitive decline and disease severity. These findings elucidate CB-mediated mechanisms underlying tau-related epileptogenesis and cognitive dysfunction at both circuit and molecular levels, providing novel therapeutic targets for AD intervention.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTau, a microtubule-associated protein, plays a pivotal role in maintaining microtubule assembly and stability. Under physiological conditions, tau exists predominantly in an unstructured conformation with low intrinsic aggregation propensity. However, pathological hyperphosphorylation drives its misfolding into paired helical filaments (PHFs) and neurofibrillary tangles (NFTs), which coalesce into large aggregates\u0026mdash;a defining feature of tauopathies. These pathologies are not exclusive to AD but are also implicated in a spectrum of neurodegenerative disorders, including progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic grain disease (AGD), Pick\u0026rsquo;s disease (PiD), Huntington\u0026rsquo;s disease (HD), and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17)\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. Notably, tau pathology has been identified in the hippocampus of TLE patients \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e, suggesting a shared mechanism between AD and TLE. Both disorders exhibit hippocampal network hyperexcitability during early disease stages \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo investigate the role of hippocampal microcircuits in epileptogenesis, we employed kainic acid (KA) to activate CA1 and DG subregions, coupled with selectively optogenetic stimulation of excitatory neurons. Our results revealed that overactivation of CA1/DG neurons reliably induced seizures in mice, underscoring the critical contribution of hippocampal hyperexcitability to epileptogenesis. In the optogenetic model, CaMKIIα promoter-driven expression of channelrhodopsin-2 (ChR2) enabled precise spatiotemporal control of neuronal activity. Blue light (475 nm) triggered rapid cation influx (e.g., Na⁺) through ChR2, eliciting action potentials and transient hippocampal overexcitation. Termination of photostimulation promptly closed the channels, allowing neuronal activity to return to baseline [31]. Dox-treated Tg hTau368 mice exhibited significantly reduced seizure thresholds and more severe phenotypes upon optogenetic induction, further supporting tau pathology as a driver of epileptogenic vulnerability. The phenomenon recapitulated in other Tg mouse models expressing mutant tau variants such as PS19 (P301S), Tau22 (G272V/P301S), and rTg4510 (P301L), which display exacerbated neuronal hyperexcitability, heightened seizure susceptibility, and increased mortality \u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e.Complementary studies in Mapt⁻/⁻ mice demonstrated that tau ablation reduces action potential release, lowers cortical excitation-inhibition (E/I) ratios, and enhances inhibitory neuron recruitment, collectively mitigating neuronal hypersynchronization and seizure incidence\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. These findings highlight tau-driven E/I imbalance as a central mechanism underlying circuit-level hyperexcitability in epilepsy.\u003c/p\u003e \u003cp\u003eConversely, some studies indicate tau pathology may also drive hypoexcitability\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e, and suppress Aβ-driven cortical hyperactivation\u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. Phosphorylation of different tau residues had different effect on firing frequency of primary hippocampal neurons\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. The role of tau in mediating neuronal excitability changes may vary depending on tau features (mutation, phosphorylation, isoform), brain region and disease stage \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. In our study, we uniquely establishes truncated hTau368 as a potent enhancer of CA1 excitatory neuron activity at early stage of tau pathology that hippocampus is in a hypermetabolic state detected by FDG-PET/CT, consistent with systemic hypermetabolic states and increased spontaneous movements. Head to head comparison in diverse tauopathy models (e.g., P301L, P301S) is needed to further validate the current hypotheses.\u003c/p\u003e \u003cp\u003eUnder resting conditions, extracellular calcium concentrations (1.1\u0026ndash;1.4 mM) starkly contrast with intracellular levels (50\u0026ndash;300 nM), yet neuronal activation rapidly elevates cytosolic calcium to micromolar ranges \u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e. Calcium homeostasis is tightly regulated by influx through receptor channels, buffering via calcium-binding proteins, such as CB, and endoplasmic reticulum (ER) store release\u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. Electrophysiological recordings and GCaMP6f-mediated calcium imaging provided a robust methods to study the interplay between tau, calcium, and neuronal activity. Our data demonstrated that tau pathology exacerbated intracellular calcium transients and CA1 neuronal hyperexcitability, aligning with prior in vitro observations. Full-length tau overexpression similarly disrupts calcium handling and induces ER stress\u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e. Knocking out PSEN1 and PSEN2 or expressing a FAD mutation in mouse hippocampal neurons increased ER Ca2\u0026thinsp;+\u0026thinsp;levels and consequently elevated Ca2\u0026thinsp;+\u0026thinsp;release into the cytoplasm in the presence of a stimulus \u003csup\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e. Notably, CB overexpression in CA1 neurons rescued tau-induced hyperexcitability, suggesting CB deficiency as a key mediator.\u003c/p\u003e \u003cp\u003eCB exhibits high calcium affinity and serves as a critical buffer against cytosolic calcium overload. Beyond its neuroprotective role, CB modulates action potential kinetics and synaptic transmission \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e. CB is enriched in CA1 pyramidal neurons and DG granule cells, with sparse expression in CA1 interneurons \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/sup\u003e. Anatomically, CB is localized within the axons and dendritic spines of neurons, where it exerts a dynamic regulatory effect on synaptic plasticity. At the presynaptic terminals, CB plays a promoting role in vesicle release and paired - pulse facilitation \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Conversely, in the postsynaptic context, CB is indispensable for the induction and maintenance of long - term potentiation in CA1 and DG excitatory neurons \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Spatiotemporal localization of CB-presynaptically facilitating vesicle release and paired-pulse facilitation\u003csup\u003e[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]\u003c/sup\u003e, and postsynaptically enabling long-term potentiation\u003csup\u003e[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]\u003c/sup\u003e\u0026mdash;underscores its dual role in synaptic plasticity. By binding excess calcium ions, it effectively down - regulates the intracellular free calcium levels, thereby safeguarding neurons from the potentially lethal effects of calcium overload - induced cell death \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Moreover, CB's regulatory function extends to influencing the firing of neuronal action potentials and the intricate processes of synaptic neurotransmitter transport and release. In our study, tau pathology-associated calcium surges in CA1 neurons correlated with diminished CB expression, likely due to impaired buffering capacity. The pTau aggregates localized exclusively to CA1/DG excitatory neurons and coincided with CB downregulation, leading to calcium dysregulation, mitochondrial stress, oxidative damage, and apoptotic signaling \u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e. Dysfunctional calcium homeostasis further alters neurotransmitter release and transcriptional programs, exacerbating E/I imbalance \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Remarkably, Dox withdrawal for three months reversed tau pathology, restored CB levels, normalized synaptic transmission, and cognition recovery. Similarly, AAV-mediated CB Supplementation in Tg hTau368 mice attenuated tau-induced cognitive deficits, implicating CB restoration as a viable therapeutic strategy. Our data established connections among tauopathy, CB deficiency, calcium homeostatic imbalance, synaptic dysfunction and cognitive deficits by patch-clamp electrophysiological recording, calcium imaging, and the Tet-on system in tau-driven Tg mice.\u003c/p\u003e \u003cp\u003eDespite these advances, key limitations must be acknowledged. First, the molecular link between tau pathology and CB suppression remains unclear. Previous findings implicates ΔFosB-mediated epigenetic silencing of CB in AD/TLE\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e, and tau-induced JAK2\u0026ndash;STAT1 activation\u003csup\u003e[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/sup\u003e, wheher the pathway linked to \u003cem\u003eCalb1\u003c/em\u003e transcriptional repression warrants further investigation. Second, the role of CB in interneurons remains unexplored, despite evidence that interneuron-specific CB knockdown protects against stress-induced memory deficits \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Finally, whether there is an interaction between Aβ and tauopathy, along with its influence on the excitability of the hippocampal neural circuit, still requires further exploration.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our study demonstrates that tau selectively accumulate in hippocampal excitatory neurons, resulting in increased neuronal excitability and a heightened susceptibility to TLE. The concomitant reduction in CB expression within these neurons mediates the hyperexcitability and cognitive dysfunction associated with tau pathology. These findings underscore the potential of targeting CB-mediated calcium homeostasis as a therapeutic strategy for AD.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlzheimer\u0026rsquo;s disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTLE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etemporal lobe epilepsy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecalbindin-D28k\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe thank Pro. Jianxin Wang (School of Computer Science and Engineering, Central South University, Changsha, Hunan 410083, China) and his colleagues for providing the raw data from AlzCode (http://www.alzcode.xyz/) and giving data analysis. We thank Dr. Jiuyang Ding (Department of Forensic Medicine, Zunyi Medical University,Guiyang, Zunyi 563000, China) for generously providing the human brain tissue sections.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting 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\u003eJ.Z.W., H.B.X.,Y.Z. and Y.G. designed this research; Y.G., X.Q.T., Y.Y.W., Y.R.W., H.L., Y.Y., M.Q.T. and J.Z.performed the experiments. W.W.,J.Z. and X.C.W. provided advices on this research. Y.G, X.Q.T., Y.Y.W. and J.Z. performed statistical analysis and data interpretation. J.Z.W., H.B.X., Y.Z. and Y.G. wrote the manuscript. All authors read and approved the final manuscript. J.Z.W. is the supervisor of this work who has full access to all the data in the study and takes responsibility for the data integrity and accuracy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported in parts by the Natural Science Foundation of China (91949205, 31730035, 81721005), China Postdoctoral Science Foundation General Program (2024M762504) and Wuhan Health Commission Project (WZ21Q21). The funders neither played a role in the study design, conduct, data collection, analysis, interpretation, nor participated in the preparation, review, approval of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data provided in this paper are available from the leading contact, Prof Jian-Zhi Wang upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal studies had complied with all relevant ethical regulations for the animal testing and research, and were approved by institutional guidelines and the Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology.\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\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLong JM, Holtzman DM. Alzheimer Disease: An Update on Pathobiology and Treatment Strategies. Cell. 2019;179:312-39.\u003c/li\u003e\n\u003cli\u003eZhang Y, Chen H, Li R, Sterling K, Song W. Amyloid \u0026beta;-based therapy for Alzheimer\u0026apos;s disease: challenges, successes and future. Signal transduction and targeted therapy. 2023;8:248.\u003c/li\u003e\n\u003cli\u003eHeneka MT, Morgan D, Jessen F. Passive anti-amyloid \u0026beta; immunotherapy in Alzheimer\u0026apos;s disease-opportunities and challenges. Lancet (London, England). 2024;404:2198-208.\u003c/li\u003e\n\u003cli\u003eZawar I, Kapur J. Does Alzheimer\u0026apos;s disease with mesial temporal lobe epilepsy represent a distinct disease subtype? Alzheimer\u0026apos;s \u0026amp; dementia : the journal of the Alzheimer\u0026apos;s Association. 2023;19:2697-706.\u003c/li\u003e\n\u003cli\u003eCheng C, Liu C, Ou S, Yeh C, Chen T, Lin Y, et al. Incidence and risk of seizures in Alzheimer\u0026apos;s disease: A nationwide population-based cohort study. Epilepsy research. 2015;115:63-6.\u003c/li\u003e\n\u003cli\u003eVossel K, Tartaglia M, Nygaard H, Zeman A, Miller B. Epileptic activity in Alzheimer\u0026apos;s disease: causes and clinical relevance. The Lancet Neurology. 2017;16:311-22.\u003c/li\u003e\n\u003cli\u003eLam AD, Thibault EG, Mayblyum DV, Hsieh S, Pellerin KR, Sternberg EJ, et al. Association of Seizure Foci and Location of Tau and Amyloid Deposition and Brain Atrophy in Patients With Alzheimer Disease and Seizures. Neurology. 2024;103:e209920.\u003c/li\u003e\n\u003cli\u003eAmatniek J, Hauser W, DelCastillo-Castaneda C, Jacobs D, Marder K, Bell K, et al. Incidence and predictors of seizures in patients with Alzheimer\u0026apos;s disease. Epilepsia. 2006;47:867-72.\u003c/li\u003e\n\u003cli\u003eLam A, Deck G, Goldman A, Eskandar E, Noebels J, Cole A. Silent hippocampal seizures and spikes identified by foramen ovale electrodes in Alzheimer\u0026apos;s disease. Nature medicine. 2017;23:678-80.\u003c/li\u003e\n\u003cli\u003eTarga Dias Anastacio H, Matosin N, Ooi L. Neuronal hyperexcitability in Alzheimer\u0026apos;s disease: what are the drivers behind this aberrant phenotype? Translational psychiatry. 2022;12:257.\u003c/li\u003e\n\u003cli\u003eCelone KA, Calhoun VD, Dickerson BC, Atri A, Chua EF, Miller SL, et al. Alterations in memory networks in mild cognitive impairment and Alzheimer\u0026apos;s disease: an independent component analysis. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2006;26:10222-31.\u003c/li\u003e\n\u003cli\u003eH\u0026auml;m\u0026auml;l\u0026auml;inen A, Pihlajam\u0026auml;ki M, Tanila H, H\u0026auml;nninen T, Niskanen E, Tervo S, et al. Increased fMRI responses during encoding in mild cognitive impairment. Neurobiol Aging. 2007;28:1889-903.\u003c/li\u003e\n\u003cli\u003eGallego-Rudolf J, Wiesman AI, Pichet Binette A, Villeneuve S, Baillet S. Synergistic association of A\u0026beta; and tau pathology with cortical neurophysiology and cognitive decline in asymptomatic older adults. Nat Neurosci. 2024;27:2130-7.\u003c/li\u003e\n\u003cli\u003eVossel K, Ranasinghe KG, Beagle AJ, La A, Ah Pook K, Castro M, et al. Effect of Levetiracetam on Cognition in Patients With Alzheimer Disease With and Without Epileptiform Activity: A Randomized Clinical Trial. JAMA neurology. 2021;78:1345-54.\u003c/li\u003e\n\u003cli\u003eSainju RK, Dragon DN, Winnike HB, Ten Eyck P, Granner MA, Gehlbach BK, et al. Hypercapnic ventilatory response in epilepsy patients treated with VNS: A case-control study. Epilepsia. 2021;62:e140-e6.\u003c/li\u003e\n\u003cli\u003eVilleneuve S, Rabinovici GD, Cohn-Sheehy BI, Madison C, Ayakta N, Ghosh PM, et al. Existing Pittsburgh Compound-B positron emission tomography thresholds are too high: statistical and pathological evaluation. Brain. 2015;138:2020-33.\u003c/li\u003e\n\u003cli\u003eGiorgio J, Adams JN, Maass A, Jagust WJ, Breakspear M. Amyloid induced hyperexcitability in default mode network drives medial temporal hyperactivity and early tau accumulation. Neuron. 2024;112:676-86.e4.\u003c/li\u003e\n\u003cli\u003eLu Y, Zhong C, Wang L, Wei P, He W, Huang K, et al. Optogenetic dissection of ictal propagation in the hippocampal-entorhinal cortex structures. Nature communications. 2016;7:10962.\u003c/li\u003e\n\u003cli\u003eKing MV, Kurian N, Qin S, Papadopoulou N, Westerink BH, Cremers TI, et al. Lentiviral delivery of a vesicular glutamate transporter 1 (VGLUT1)-targeting short hairpin RNA vector into the mouse hippocampus impairs cognition. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology. 2014;39:464-76.\u003c/li\u003e\n\u003cli\u003eFu H, Possenti A, Freer R, Nakano Y, Hernandez Villegas N, Tang M, et al. A tau homeostasis signature is linked with the cellular and regional vulnerability of excitatory neurons to tau pathology. Nature neuroscience. 2019;22:47-56.\u003c/li\u003e\n\u003cli\u003eMurdock MH, Tsai LH. Insights into Alzheimer\u0026apos;s disease from single-cell genomic approaches. Nat Neurosci. 2023;26:181-95.\u003c/li\u003e\n\u003cli\u003eDridi H, Liu Y, Reiken S, Liu X, Argyrousi E, Yuan Q, et al. Heart failure-induced cognitive dysfunction is mediated by intracellular Ca leak through ryanodine receptor type 2. Nature neuroscience. 2023;26:1365-78.\u003c/li\u003e\n\u003cli\u003ePrincen K, Van Dooren T, van Gorsel M, Louros N, Yang X, Dumbacher M, et al. Pharmacological modulation of septins restores calcium homeostasis and is neuroprotective in models of Alzheimer\u0026apos;s disease. Science (New York, NY). 2024;384:eadd6260.\u003c/li\u003e\n\u003cli\u003eLambers T, Mahieu F, Oancea E, Hoofd L, de Lange F, Mensenkamp A, et al. Calbindin-D28K dynamically controls TRPV5-mediated Ca2+ transport. The EMBO journal. 2006;25:2978-88.\u003c/li\u003e\n\u003cli\u003eBerdal A, Hotton D, Saffar J, Thomasset M, Nanci A. Calbindin-D9k and calbindin-D28k expression in rat mineralized tissues in vivo. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 1996;11:768-79.\u003c/li\u003e\n\u003cli\u003eLi J, Xie X, Yu J, Sun Y, Liao X, Wang X, et al. Suppressed Calbindin Levels in Hippocampal Excitatory Neurons Mediate Stress-Induced Memory Loss. Cell reports. 2017;21:891-900.\u003c/li\u003e\n\u003cli\u003eYou J, Muralidharan K, Park J, Petrof I, Pyfer M, Corbett B, et al. Epigenetic suppression of hippocampal calbindin-D28k by \u0026Delta;FosB drives seizure-related cognitive deficits. Nature medicine. 2017;23:1377-83.\u003c/li\u003e\n\u003cli\u003eGao Y, Wang Y, Lei H, Xu Z, Li S, Yu H, et al. A novel transgenic mouse line with hippocampus-dominant and inducible expression of truncated human tau. Transl Neurodegener. 2023;12:51.\u003c/li\u003e\n\u003cli\u003eG\u0026ouml;tz J, Chen F, Barmettler R, Nitsch RM. Tau filament formation in transgenic mice expressing P301L tau. The Journal of biological chemistry. 2001;276:529-34.\u003c/li\u003e\n\u003cli\u003eRen QG, Gong WG, Zhou H, Shu H, Wang YJ, Zhang ZJ. Spatial Training Ameliorates Long-Term Alzheimer\u0026apos;s Disease-Like Pathological Deficits by Reducing NLRP3 Inflammasomes in PR5 Mice. Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics. 2019;16:450-64.\u003c/li\u003e\n\u003cli\u003eGao Y, Zheng J, Jiang T, Pi G, Sun F, Xiong R, et al. Targeted Reducing of Tauopathy Alleviates Epileptic Seizures and Spatial Memory Impairment in an Optogenetically Inducible Mouse Model of Epilepsy. Front Cell Dev Biol. 2020;8:633725.\u003c/li\u003e\n\u003cli\u003eLewczuk E, Joshi S, Williamson J, Penmetsa M, Shan S, Kapur J. Electroencephalography and behavior patterns during experimental status epilepticus. Epilepsia. 2018;59:369-80.\u003c/li\u003e\n\u003cli\u003eZhang Z, Song M, Liu X, Kang SS, Kwon IS, Duong DM, et al. Cleavage of tau by asparagine endopeptidase mediates the neurofibrillary pathology in Alzheimer\u0026apos;s disease. Nat Med. 2014;20:1254-62.\u003c/li\u003e\n\u003cli\u003eLi S, Zhou Q, Liu E, Du H, Yu N, Yu H, et al. Alzheimer-like tau accumulation in dentate gyrus mossy cells induces spatial cognitive deficits by disrupting multiple memory-related signaling and inhibiting local neural circuit. Aging Cell. 2022;21:e13600.\u003c/li\u003e\n\u003cli\u003eChen TW, Wardill TJ, Sun Y, Pulver SR, Renninger SL, Baohan A, et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature. 2013;499:295-300.\u003c/li\u003e\n\u003cli\u003eTzeng TT, Tsay HJ, Chang L, Hsu CL, Lai TH, Huang FL, et al. Caspase 3 involves in neuroplasticity, microglial activation and neurogenesis in the mice hippocampus after intracerebral injection of kainic acid. Journal of biomedical science. 2013;20:90.\u003c/li\u003e\n\u003cli\u003eRadulescu CI, Doostdar N, Zabouri N, Melgosa-Ecenarro L, Wang X, Sadeh S, et al. Age-related dysregulation of homeostatic control in neuronal microcircuits. Nature Neuroscience. 2023;26:2158-70.\u003c/li\u003e\n\u003cli\u003eChiaravalloti A, Barbagallo G, Martorana A, Castellano AE, Ursini F, Schillaci O. Brain metabolic patterns in patients with suspected non-Alzheimer\u0026apos;s pathophysiology (SNAP) and Alzheimer\u0026apos;s disease (AD): is [(18)F] FDG a specific biomarker in these patients? European journal of nuclear medicine and molecular imaging. 2019;46:1796-805.\u003c/li\u003e\n\u003cli\u003eXie F, Peng F. Radiopharmaceuticals for Assessment of Altered Metabolism and Biometal Fluxes in Brain Aging and Alzheimer\u0026apos;s Disease with Positron Emission Tomography. Journal of Alzheimer\u0026apos;s disease : JAD. 2017;59:527-36.\u003c/li\u003e\n\u003cli\u003eSancheti H, Kanamori K, Patil I, D\u0026iacute;az Brinton R, Ross BD, Cadenas E. Reversal of metabolic deficits by lipoic acid in a triple transgenic mouse model of Alzheimer\u0026apos;s disease: a 13C NMR study. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 2014;34:288-96.\u003c/li\u003e\n\u003cli\u003eSancheti H, Patil I, Kanamori K, D\u0026iacute;az Brinton R, Zhang W, Lin AL, et al. Hypermetabolic state in the 7-month-old triple transgenic mouse model of Alzheimer\u0026apos;s disease and the effect of lipoic acid: a 13C-NMR study. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 2014;34:1749-60.\u003c/li\u003e\n\u003cli\u003eYin F, Sancheti H, Patil I, Cadenas E. Energy metabolism and inflammation in brain aging and Alzheimer\u0026apos;s disease. Free radical biology \u0026amp; medicine. 2016;100:108-22.\u003c/li\u003e\n\u003cli\u003eSantacruz K, Lewis J, Spires T, Paulson J, Kotilinek L, Ingelsson M, et al. Tau suppression in a neurodegenerative mouse model improves memory function. Science (New York, NY). 2005;309:476-81.\u003c/li\u003e\n\u003cli\u003eOzcelik S, Sprenger F, Skachokova Z, Fraser G, Abramowski D, Clavaguera F, et al. Co-expression of truncated and full-length tau induces severe neurotoxicity. Molecular psychiatry. 2016;21:1790-8.\u003c/li\u003e\n\u003cli\u003eLin CX, Li HD, Deng C, Erhardt S, Wang J, Peng X, et al. AlzCode: a platform for multiview analysis of genes related to Alzheimer\u0026apos;s disease. Bioinformatics (Oxford, England). 2022;38:2030-2.\u003c/li\u003e\n\u003cli\u003eGoedert M, Eisenberg DS, Crowther RA. Propagation of Tau Aggregates and Neurodegeneration. Annual review of neuroscience. 2017;40:189-210.\u003c/li\u003e\n\u003cli\u003eViney TJ, Sarkany B, Ozdemir AT, Hartwich K, Schweimer J, Bannerman D, et al. Spread of pathological human Tau from neurons to oligodendrocytes and loss of high-firing pyramidal neurons in aging mice. Cell Reports. 2022;41:111646.\u003c/li\u003e\n\u003cli\u003eWitt J, Becker A, Helmstaedter C. The multifactorial etiology of cognitive deficits in epilepsy and the neuropathology of mesial temporal lobe epilepsy beyond hyperphosphorylated tau. Alzheimer\u0026apos;s \u0026amp; dementia : the journal of the Alzheimer\u0026apos;s Association. 2023;19:3231-2.\u003c/li\u003e\n\u003cli\u003eLiu S, Shen Y, Shultz SR, Nguyen A, Hovens C, Adlard PA, et al. Accelerated kindling epileptogenesis in Tg4510 tau transgenic mice, but not in tau knockout mice. Epilepsia. 2017;58:e136-e41.\u003c/li\u003e\n\u003cli\u003eGomez-Murcia V, Sandau U, Ferry B, Parrot S, Laurent C, Basquin M, et al. Hyperexcitability and seizures in the THY-Tau22 mouse model of tauopathy. Neurobiology of Aging. 2020;94:265-70.\u003c/li\u003e\n\u003cli\u003eChang C, Evans M, Yu X, Yu G, Mucke L. Tau reduction affects excitatory and inhibitory neurons differently, reduces excitation/inhibition ratios, and counteracts network hypersynchrony. Cell reports. 2021;37:109855.\u003c/li\u003e\n\u003cli\u003eHatch R, Wei Y, Xia D, G\u0026ouml;tz J. Hyperphosphorylated tau causes reduced hippocampal CA1 excitability by relocating the axon initial segment. Acta neuropathologica. 2017;133:717-30.\u003c/li\u003e\n\u003cli\u003eBusche M, Wegmann S, Dujardin S, Commins C, Schiantarelli J, Klickstein N, et al. Tau impairs neural circuits, dominating amyloid-\u0026beta; effects, in Alzheimer models in vivo. Nature neuroscience. 2019;22:57-64.\u003c/li\u003e\n\u003cli\u003eBagur R, Hajn\u0026oacute;czky G. Intracellular Ca(2+) Sensing: Its Role in Calcium Homeostasis and Signaling. Mol Cell. 2017;66:780-8.\u003c/li\u003e\n\u003cli\u003eCalvo-Rodriguez M, Kharitonova EK, Bacskai BJ. Therapeutic Strategies to Target Calcium Dysregulation in Alzheimer\u0026apos;s Disease. Cells. 2020;9.\u003c/li\u003e\n\u003cli\u003eYe J, Yin Y, Yin Y, Zhang H, Wan H, Wang L, et al. Tau-induced upregulation of C/EBP\u0026beta;-TRPC1-SOCE signaling aggravates tauopathies: A vicious cycle in Alzheimer neurodegeneration. Aging cell. 2020;19:e13209.\u003c/li\u003e\n\u003cli\u003eZhang H, Sun S, Herreman A, De Strooper B, Bezprozvanny I. Role of presenilins in neuronal calcium homeostasis. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2010;30:8566-80.\u003c/li\u003e\n\u003cli\u003eGe M, Zhang J, Chen S, Huang Y, Chen W, He L, et al. Role of Calcium Homeostasis in Alzheimer\u0026apos;s Disease. Neuropsychiatric disease and treatment. 2022;18:487-98.\u003c/li\u003e\n\u003cli\u003eCelio M. Calbindin D-28k and parvalbumin in the rat nervous system. Neuroscience. 1990;35:375-475.\u003c/li\u003e\n\u003cli\u003eYi S. Time-dependent changes of calbindin D-28K and parvalbumin immunoreactivity in the hippocampus of rats with streptozotocin-induced type 1 diabetes. Journal of veterinary science. 2013;14:373-80.\u003c/li\u003e\n\u003cli\u003eBlatow M, Caputi A, Burnashev N, Monyer H, Rozov A. Ca2+ buffer saturation underlies paired pulse facilitation in calbindin-D28k-containing terminals. Neuron. 2003;38:79-88.\u003c/li\u003e\n\u003cli\u003eWesterink R, Beekwilder J, Wadman W. Differential alterations of synaptic plasticity in dentate gyrus and CA1 hippocampal area of Calbindin-D28K knockout mice. Brain research. 2012;1450:1-10.\u003c/li\u003e\n\u003cli\u003eLi XG, Hong XY, Wang YL, Zhang SJ, Zhang JF, Li XC, et al. Tau accumulation triggers STAT1-dependent memory deficits by suppressing NMDA receptor expression. EMBO reports. 2019;20.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"translational-neurodegeneration","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tneu","sideBox":"Learn more about [Translational Neurodegeneration](http://translationalneurodegeneration.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/tneu/default.aspx","title":"Translational Neurodegeneration","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Alzheimer's disease, temporal lobe epilepsy, tau hyperphosphorylation, calbindin-D28k, cognitive impairment, calcium homeostasis","lastPublishedDoi":"10.21203/rs.3.rs-6470265/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6470265/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cb\u003eBackground\u003c/b\u003e Medial temporal lobe hyperexcitation or seizures originating from hippocampus are frequently observed in Alzheimer's disease (AD) patients, contributing to accelerated cognitive decline. Given the hippocampus's role as an early vulnerable area of tau pathology, a hallmark of AD, the mechanisms by which abnormal tau aggregation promotes temporal lobe epilepsy (TLE) remain poorly understood.\u003c/p\u003e \u003cp\u003eMethods\u003c/p\u003e \u003cp\u003eWe investigated the role and mechanisms of AD-like hippocampal tau aggregation in neuronal hyperexcitation using transgenic (Tg) tau-driven mice. Tau aggregation on intracellular calcium dynamics was assessed by calcium imaging. Neuronal/network hyperexcitability and seizure susceptibility were evaluated through patch-clamp electrophysiology, \u003csup\u003e18\u003c/sup\u003eF-FDG PET/CT, and optogenetic induction. A tetracycline-controlled (tet-on) system in Tg hTau368 mice enabled spatiotemporal induction of tau pathology to investigate interactions with calbindin-D28k (CB) and synaptic proteins. Adeno-associated virus (AAV)-mediated CB supplementation in hippocampal CA1 and dentate gyrus (DG) excitatory neurons was tested for rescuing hyperexcitability and cognitive deficits. Finally, the relationship between CB and disease progress was analyzed using AD public database.\u003c/p\u003e \u003cp\u003eResults\u003c/p\u003e \u003cp\u003eTau accumulation in hippocampal CA1/DG CaMKII-positive excitatory neurons reduced CB expression with disrupted calcium homeostasis. This dysregulation heightened neuronal excitability, diminished synaptic protein levels, and increased seizure susceptibility and cognitive impairment. AAV-driven CB restoration in CA1/DG neurons attenuated both hyperexcitability and cognitive deficits. In the brain of AD patients, the reduced CB expression was associated with cognitive deterioration and the advanced disease stages.\u003c/p\u003e \u003cp\u003eConclusions\u003c/p\u003e \u003cp\u003eTau aggregation drives CB-dependent calcium dysregulation and hippocampal neuronal hyperexcitation. These results establish a potential mechanistic link between tauopathy and TLE pathogenesis in AD, providing with CB as a promising therapeutic target for mitigating seizure risk and related cognitive decline in AD.\u003c/p\u003e","manuscriptTitle":"Calbindin-D28k deficiency mediates tau-driven hippocampal hyperexcitement and cognitive impairment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 03:11:58","doi":"10.21203/rs.3.rs-6470265/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-04-23T06:14:34+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-23T02:47:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-22T06:39:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Translational Neurodegeneration","date":"2025-04-21T23:56:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"translational-neurodegeneration","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tneu","sideBox":"Learn more about [Translational Neurodegeneration](http://translationalneurodegeneration.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/tneu/default.aspx","title":"Translational Neurodegeneration","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1104ef46-8770-4baa-886b-71096ee01eb2","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-10T07:14:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-07 03:11:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6470265","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6470265","identity":"rs-6470265","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}