Neuritin suppresses GABAergic neurons ferroptosis to improve cognitive impairment in diabetes mellitus

Neuritin suppresses GABAergic neurons ferroptosis to improve cognitive impairment in diabetes mellitus | 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 Neuritin suppresses GABAergic neurons ferroptosis to improve cognitive impairment in diabetes mellitus Hongli Zhou, Zuo Zhang, Jianyun Zhou, Jiyin Zhou This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5965662/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aims : Alterations in iron homeostasis are associated with several neurodegenerative diseases. Cognitive dysfunction has become an important concomitant symptom in people with type 2 diabetes mellitus. Therefore, we investigated the role of neuritin in ameliorating cognitive dysfunction resulting from ferroptosis in diabetic neurons using a model of neuritin overexpression in GABAergic. Methods : The constructed transgenic mice were used to observe memory function changes using the Morris water maze. The ferroptosis in GABAergic in hippocampus and AMPK/Nrf2 signaling pathway were detected by Western blot, transmission electron microscopy, and immunofluorescence. High glucose was used to induce ferroptosis in HT22 cells in vitro, and neuritin was further confirmed to reduce ferroptosis in HT22 cells through AMPK/Nrf2 signaling pathway by chemical assays and Western blot assays. Results : Neuritin overexpression in GABAergic of db/db mice significantly ameliorated cognitive dysfunction, mitochondrial dysfunction, reversed ferroptosis-associated symbolic changes and reduced ferroptosis in the hippocampus. And also increased the co-localisation coefficient of GAD65 and AMPK in the hippocampus. Neuritin activates the AMPK/Nrf2 signaling pathway to inhibit high glucose induced ferroptosis in HT22 cells. Neuritin was observed to regulate the AMPK/Nrf2 signaling pathway in HT22 cells and promote Nrf2 expression to inhibit HT22 cell ferroptosis and ameliorate diabetic cognitive dysfunction. Conclusions : These findings suggest that neuritin may attenuate diabetes associated cognitive dysfunction by modulating neuronal ferroptosis, at least partly via AMPK/Nrf2 signaling pathway. Diabetes mellitus cognitive impairment neuritin GABAergic neurons ferroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Introduction Type 2 diabetes mellitus (T2DM) is the most common type of diabetes mellitus, which is characterized by hyperglycemia. It has been found that patients with T2DM are more likely to develop cognitive impairment(Cai et al., 2018 ). And the development of this cognitive dysfunction is usually latent and not easily detectable, eventually leading to dementia(Moheet et al., 2015 ). The hippocampus is a key region of the brain for learning and memory, and it is also one of the most sensitive regions to high glucose stimulation(Kim et al., 2021 ). Numerous studies have demonstrated that changes in hippocampal plasticity and function are the main cause of cognitive impairment in diabetes, but the specific molecular mechanisms behind diabetic cognitive dysfunction remain unclear. In hippocampus, GABAergic inhibitory interneurons account for 10–15% of the total neuronal cell population(Pelkey et al., 2017 ). GABAergic interneurons are the primary inhibitory system of the mammalian brain and are associated with cognitive deficits(Yuan et al., 2018 ; Mederos et al., 2021 ). Patients with T2DM experience episodic memory loss when GABA levels in the medial prefrontal cortex are abnormal(Thielen et al., 2019 ). Inhibition of GABAB receptors effectively promotes hippocampal neurogenesis and improves spatial learning and memory after cerebral ischemia in adult mice(Song et al., 2021 ). Our previous publication indicated that GABAergic interneurons play an important role in diabetic cognitive deficits by showing significant changes in GABA receptor density and function in certain brain regions associated with cognitive function, such as the hippocampus, implying that spatial learning and memory are related in part to altered GABA receptors and function(Zhou et al., 2023 ). Neuritin is a neurotrophic factor that is mainly expressed in the brain(Zhou and Zhou, 2014 ). Studies have shown that neuritin improves the recovery of neuritin growth in hippocampal neurons after cerebral ischemia in mice(Zhao et al., 2017 ) and attenuates cognitive dysfunction in a mouse model of Alzheimer's disease(Choi et al., 2014 ; Wan et al., 2020 ). One of our previous studies also showed that neuritin ameliorating cognitive dysfunction in diabetic patients(Zhang et al., 2021 ). Importantly, our current study found a significant increase in the number of GABAergic neurons in the hippocampal tissue of mice overexpressing neuritin and a corresponding improvement in learning memory function. This suggests that the effect of neuritin in improving diabetic cognitive dysfunction may be related to the promotion of survival or proliferation of GABAergic nerves in the hippocampus. However, the specific mechanism by which neuritin protects GABAergic nerve survival has not yet been elucidated. Ferroptosis is a regulated form of cell death dependent on iron and reactive oxygen species and characterized by lipid peroxidation(Latunde-Dada, 2017 ). Ferroptosis has been found to characterize several diabetes models. Moreover, it is noteworthy that reduces iron overload improved hippocampal neuronal and synaptic plasticity and ultimately restores cognitive function(An et al., 2022 ; Abdul et al., 2021 ). Ferroptosis inducer RAS-selective lethal 3 inhibits GABAergic currents(Giustizieri et al., 2023 ), and reduced GABAergic neuron activity can reduce information processing in cortical microcircuits, leading to cognitive impairment(Prévot and Sibille, 2021 ). In our previous study, we found that overexpression of neuritin ameliorated mitochondrial atrophy, reduction of mitochondrial ridges, and increase in membrane density in hippocampal area cells of diabetic mice by transmission electron microscopy. This suggests that the protective effect of neuritin on GABAergic nerves may be related to the inhibition of their iron death. The study showed that these effects may closely relate to the activation of the AMPK signaling pathway(Xie et al., 2023 ), which regulates the expression of a range of biologically important genes in ferroptosis. For example, activated AMPK inhibits indicators of ferroptosis (e.g., reactive oxygen species, malondialdehyde, reduced glutathione, and mitochondrial membrane potential)(Zhang et al., 2023 ). Specifically, the activation of AMPK signaling pathway is implicated in cognitive impairment due to diabetic neuronal ferroptosis. In the present study, we found that hippocampal GABAergic neurons are more susceptible to hyperglycemia under T2DM conditions, inducing GABAergic neuronal ferroptosis and leading to cognitive dysfunction, which can be reversed by neuritin. The present study further investigated whether neuritin could play a role in ameliorating T2DM memory dysfunction by modulating the AMPK/Nrf2 signaling pathway to counteract iron oxidation in hippocampal GABAergic neurons. 2 Methods 2.1 Establishment of the GABAergic neurons with neuritin overexpression Transgenic mice expressing CMV-LoxP-STOP-LoxP-Nrn1(Zhang et al., 2022b ) were bred with Calb2tm1-Cre (B6(Cg)-Calb2 tm1(cre)Zjh /J) mice obtained from The Jackson Laboratory, resulting in the generation of neuritin-Cre transgenic mice. Non-diabetic (db/m, C57BL/6J-Leprdb/+) mice were purchased from The Jackson Laboratory. The db/m mice were crossed with the neuritin-Cre transgenic mice to generate db/m/neuritin/Calb2tm1-Cre transgenic mice. Subsequently, the db/m/neuritin/Calb2tm1-Cre transgenic mice were crossbred with each other, giving rise to db/db/neuritin/Calb2tm1-Cre triple transgenic mice. The evaluation of Cre-mediated neuritin expression was performed through polymerase chain reaction (PCR) analysis using genomic DNA extracted from the tail. All mice were housed in a specific pathogen-free environment with controlled temperature and humidity, adhering to a 12-hour light: 12-hour dark cycle. The mice were provided ad libitum access to food and water. Ethical approval for the animal experiments was obtained from the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Army Medical University, ensuring compliance with established guidelines and standards for the ethical treatment of animals in research. Drug treatment: Mice were divided into (1) db/m control group (2) db/db group, (3) db/db mice with overexpression neuritin group. 2.3 Morris water maze test Morris water maze experiment was used to assess learning and memory functions in mice. The day before the test, the inner wall of the swimming pool was decorated with white wallpaper (black mice and white environment), the escape platform was fixed, water at 22 ~ 24°C was filled into the pool and mice were allowed to swim freely for 60 s to familiarize themselves with the environment. Titanium dioxide (appropriate amount) was added to make the water white and the quadrants were marked as quadrants I, II, III and IV according to the position of the platform. Acquisition training (4 days): Mice were gently placed into the pool from the middle wall of the four quadrants I, II, III and IV. The experiment lasts for 60 s. Mice are found and landed on the platform (the platform is placed in the center of one quadrant) and allowed to remain on the platform for 10 s. If the platform is still not found within 60 s, mice are guided to land on the platform and remain there for 10 s. If the animal leaves, it is restarted until the rest period is 10 s and the latency period is recorded for 60s. At the end of each training session, the mice are dried using a dry rag. Each mouse was trained 4 times per day (once per quadrant, randomly distributed into quadrants each day) at 5–10 min intervals and trained for 4 consecutive days. The mean of the 4 latency periods was calculated as the score for that day. Exploratory training: After the last acquired training session, the underwater platform was removed and a 60 s exploratory training session was started. Mice were placed in the water from the quadrant opposite the platform and the time spent in the target quadrant (original platform quadrant) and the number of times the mice entered the quadrant were recorded. 2.4 Tissue preparation After the Morris water maze test, six mice of each group were anesthetized with sodium pentobarbital (30 mg/kg i.p.) blood was collected from heart, perfused with PBS at a rate of 5 mL/min for 10 min. The brains were then dissected and fixed in 4% paraformaldehyde at 4°C for 24 h, followed by dipping in 20% and 30% sucrose solution at 4°C successively until the tissues were sunk. Coronal sections were prepared in a thickness of 20 µM with a freezing microtome (Leica) when the brain tissues were completely frozen in the Optimum Cutting Temperature compound at -20°C. Hippocampus tissue was taken from six additional mice and stored frozen in liquid nitrogen for subsequent biochemical markers. 2.5 Biochemical index analysis Iron content, glutathione peroxidase 4 (GPX4), glutathione (GSH) and malondialdehyde (MDA) were determined using mouse hippocampal tissue homogenates or HT22 cell lysates according to the manufacturer's instructions. Assay kits were obtained from NanJing JianCheng Bioengineering Institute (NanJing, China). Protein concentrations in the samples were determined using the BCA protein assay kit (Beyotime, Shanghai, China). To ensure comparability of results between different groups GSH and MDA levels and GPX4 activity were normalized to protein content. 2.6 Immunofluorescence Frozen sections were repaired with modified sodium citrate antigen repair solution (Beyotime Biotechnology, P0083) at 95°C for 5 min. PBS was washed three times for 5 min each. Then incubated with PBS blocking solution containing 0.1% Triton and 10% goat serum for 1 h at room temperature. The primary antibody was then incubated overnight at 4°C. The following day, the sections were washed 3 times with PBS for 5 min each, followed by incubation with the corresponding fluorescently labelled secondary antibody for 1 h at room temperature, 3 times with PBS for 5 min each, staining with DAPI (Beyotime Biotechnology, C1002) for 10 min, and finally a drop of intifada mounting medium (Beyotime Biotechnology, P0126) on the tissue sections, cover with cover slips. The negative controls received the same procedures except that the primary antibodies were omitted and no unspecific staining was observed. The immunofluorescent staining signals were observed under a fluorescent microscope or a confocal laser-scanning microscopy (TCS SP8, Leica)(Li et al., 2020 ). Mouse neuritin antibody (Sc-365538, Santa, 1:300), rabbit GABAB2 (ab52248, abcam, 1:300), rabbit GAD65 antibody (Bioss antibodies, bs-0325R, 1:300) were used as the primary antibodies. Goat anti-rabbit IgG-Alexa Fluor 488 (Thermo, A-11008, 1:400), goat anti-mouse IgG-Alexa Fluor 647 (Thermo, A-11008, 1:400) were used as the second antibodies. Three sections of each mouse were obtained under a fluorescence microscope (Olympus, Tokyo, Japan) with three random fields of view (×40) in the DG region and the images were analyzed using ImageJ 1.50. 2.7 Transmission electron microscope Fresh mouse hippocampus tissue (CA1 area, 1 mm3) was fixed with 2% (v/v) glutaraldehyde fixative in groups of 3 mice each. After complete fixation, the tissues were rinsed with PBS buffer 4 times for 10 min, ultrapure water was rinsed for 3 min. The tissues were then dehydrated in graded alcohols of 50%, 70%, 80%, 90% and 100% for 10 min in each step. After dehydration, the sections were treated with ethanol: propylene oxide (1:1), pure propylene oxide for 10 min each time, then with propylene oxide: epoxy resin (2:1), propylene oxide: epoxy resin (1:2), pure epoxy resin for 1 h each time at 30°C. Finally, each sample was examined by transmission electron microscopy. Images of each section were taken for mitochondrial morphometry analysis. 2.8 Cell culture HT22 cells were obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mmol/L glutamine (Gibco, Grand Island, NY, USA), penicillin (100 U/mL), and streptomycin (100 µg/mL), and maintained at 37°C and 5% CO 2 in a humid environment. The medium was replaced twice each week(Wang et al., 2018 ). HT22 cells were treated with glucose, recombinant human neuritin (1000 ng/mL), dorsomorphin (25 µM) or ANA-12 (400 nM). Glucose, recombinant human neuritin, or dorsomorphin were prepared in complete medium and sterilized by using 0.2 µm syringe filter (Shanghai Fuxin, China). After drug treatment HT22 cells are lysed using ultrasonic fragmentation for subsequent biochemical assays or lysed using RIPA lysis solution for subsequent western blot analysis. 2.9 Cell viability by the CCK8 assay The cell viability of HT22 cells was determined using the Cell Counting Kit-8 (CCK-8, Dojindo, Tokyo, Japan). Briefly, HT22 cells were seeded at a density of 1 × 10 4 /well into 96-well plates for 24 h. Cells were pretreated with dorsomorphin or ANA-12. Cells were then treated with high glucose solution (75 mmol/L) or high glucose solution containing neuritin for 48 h. Cell viability was assessed using Cell CCK-8 according to the instructions. 100 µL of medium containing 10 µL of CCK-8 solution was applied. The plates were then incubated at 37°C for 2 h, after which the optical density at 450 nm was measured. 2.10 Prussian blue staining To assess iron deposition in hippocampus tissue, the Prussian Blue Iron Stain Kit (Enhance With DAB) (Solarbio, G1428) was used for the assay. Slices were routinely dewaxed and rehydrated. Perls Working Solution was added dropwise to the slices and incubated in a wet box at 37°C for 20 min. Slices were removed and rinsed gently with distilled water 3 times for 10 s each. Slices were incubated dropwise with Incubation Solution and placed in a wet box at 37°C for 20 min. Slices were removed and washed 3 times with 1× PBS for 60 s each. Slices were incubated dropwise with enhanced Working Solution and placed in a wet box at 37°C for 15 min. The sections were washed 3 times with 1× PBS for 5 s. The slices were stained for 1.5 min with dropwise addition of Redying Solution. Soak in distilled water for 10 min, dehydrated with gradient ethanol, transparent by xylene and seal with resinene. 2.11 Western blot analysis Western blot was conducted according to previously reported protocols(Cheng et al., 2019 ). Briefly, equal amounts of mouse hippocampal tissue RIPA lysates or HT22 cell RIPA lysates (25 µg protein/lane) were loaded onto 10% SDS-PAGE, proteins were separated and transferred to PVDF membranes. The membrane was blocked with 5% bovine serum albumin. Then incubated with antibody neuritin (santa, sc-365538, 1:500), GABAB receptor (abcam, ab52248, 1:500), rabbit anti-glutathione peroxidase 4 (GPX4, 52455S, CST, 1:1000), AMP-activated protein kinase α (AMPKα, 5831, CST, 1:1000), phospho AMP-activated protein kinase α (p-AMPKα, 2535, CST, 1:1000), nuclear factor-like 2 (Nrf2, 12721, CST, 1:1000) for overnight at 4°C. The TBST solution was washed 3 times for 10 min. After washing, the membranes were incubated for 1 h at room temperature with the appropriate secondary antibody (horseradish peroxidase [HRP] coupled, dilution 1:2000). Pre-stained molecular weight markers were manipulated in parallel to determine the molecular weight of the target protein. For chemiluminescence detection, membranes were treated with enhanced chemiluminescence reagents and images were obtained using ImageQuant LAS4010 (GE Healthcare). Samples were taken in duplicate for each experiment. Images were analysed for densitometry using ImageJ 1.50. 2.12 Statistical analysis The SPSS 20.0 software (IBM CORP) was applied to perform the statistical analyses. Behavioural data were presented as mean ± standard deviation (SD) and analyzed by a two-way analysis of variance (ANOVA) with repeated measures followed by Bonferroni post hoc comparisons. Results of the iron content assay, lipid peroxidation assay and western blot were presented as mean ± SD and analyzed by a one-way ANOVA followed by Bonferroni post hoc comparisons. 3 Results 3.1 Cognitive impairment in db/db mice Immunofluorescence staining was performed on the brain tissues of db/db mice, and the results revealed a significant reduction in neuritin levels and the number of GABAergic neurons in the hippocampal CA1 region (Fig. 1 A). Further confirmation using Western blot analysis yielded consistent results (Fig. 1 B- 1 C). Morris water maze experiments demonstrated cognitive abnormalities in db/db mice (Fig. 1 D). To investigate whether the cognitive impairment in db/db mice is related to neuritin and GABAergic neurons, we generated a triple transgenic mouse model, db/db/neuritin/Calb2tm1-Cre, characterized by high expression of neuritin in GABAergic neurons (Fig. 1 E- 1 F). Figure 1 Cognitive impairment in db/db mice. Immunofluorescence was used to observe the expression of neuritin and GABAergic neurons in the CA1 region of the hippocampus (A). Changes in neuritin and GABA content in hippocampal tissue homogenates were detected using western blot (B) and (C). Path diagram of mice in the Morris water maze experiment (D). Flow charts of constructing transgenic mice (E) and (F). Mean ± standard deviation (s.d.), n = 3–6. *P < 0.05, compared to db/m mice. 3.2. GABAergic overexpression of neuritin improves learning memory in db/db mice Firstly, we performed PCR testing on the tail DNA of the generated mice, selecting mice with positive identification of Calb2tm1 cre at 175 bp or 175 bp and 125 bp, LoxP at 234 bp, and Misty at 207 bp (Fig. 2 A) for further experiments. To confirm the success of the construct, immunofluorescence was conducted on the hippocampal tissue of the mice. The results revealed a significant increase in neuritin fluorescence levels in the CA1 region of mice overexpressing neuritin (Fig. 2 B). Western blot analysis also confirmed the elevated expression of neuritin (Fig. 2 C- 2 D). Additionally, to investigate the impact of neuritin overexpression on GABAergic neurons, immunofluorescence observation revealed a significant increase in the number of GABAergic neurons after neuritin overexpression (Fig. 2 F). To determine whether the overexpression of neuritin in GABAergic neurons could improve memory deficits in db/db mice, we utilized the Morris water maze test to measure mice learning abilities. There were no significant differences in latency on the first and second days for all animals. However, during the third to fifth days of training, db/db mice exhibited longer escape latencies compared to db/m mice, indicating lower platform-searching abilities. Following neuritin overexpression, db/db mice showed reduced escape latencies (Fig. 2 G). On the fifth day, when the platform was removed, the analysis of navigation paths revealed that neuritin-overexpressing db/db mice reached the original platform location in less time compared to db/db mice (Fig. 2 H). Analysis of swimming distances on the fifth day indicated a significant increase in swimming distance for db/db mice compared to db/m mice. However, after neuritin overexpression, the swimming distance of db/db mice was reduced. These results suggest that neuritin overexpression improves cognitive and memory functions in db/db mice. Figure 2 GABAergic overexpression of neuritin improves learning memory in db/db mice. Results of PCR gene identification in transgenic mice (A). Fluorescence intensity of hippocampal neuritin (B). Western blot detection of changes in neuritin protein content in hippocampal tissue homogenates (C) and (D). Immunofluorescence detection of changes in GABAergic neurons after overexpression of neurtin (F). Path diagram of mice in the Morris water maze (G). Mean latency to reach the plateau in mice (H). Mean ± s.d., n = 3–6. * P < 0.05. 3.3 Neuritin improves GABAergic neuronal ferroptosis Due to the pivotal role of iron imbalance in the development of several endocrine disorders(Miao et al., 2023 ), we investigated whether iron dysregulation is involved in the mechanism of T2DM-related cognitive impairment. Prussian blue staining and assay kits revealed a significant increase in iron deposition in db/db mice, while neuritin overexpression markedly reduced this iron deposition (Fig. 3 A- 3 B). Ultrastructural analysis indicated mitochondrial shrinkage, increased membrane density, decreased or vanished mitochondrial ridges in db/db mice, indicating the onset of ferroptosis, whereas neuritin overexpression ameliorated these changes in mitochondrial structure in db/db mice (Fig. 3 A). This suggests that neuritin may ameliorate ferroptosis in db/db mice. To further delineate the impact of neuritin on neuronal ferroptosis, we employed the HT22 cell line, a hippocampal neuron cell line. Results demonstrated that high glucose significantly reduced HT22 cell viability compared to the control group (Fig. 3 C), increased iron deposition (Fig. 3 D), and reduced GSH levels (Fig. 3 E). Western blot analysis revealed that high glucose significantly reduced GPX4 protein levels (Fig. 3 F). Exogenous neuritin notably increased cell viability (Fig. 3 G), reduced iron deposition (Fig. 3 H), and increased GSH levels (Fig. 3 J) compared to the control group. Particularly, the effect of 1000 ng/mL of neuritin was more pronounced; therefore, in subsequent experiments, we used a neuritin dosage of 1000 ng/mL. Figure 3 Effect of neuritin on db/db or high glucose-induced neuronal ferroptosis. Prussian blue staining was used to observe the iron deposition phenomenon in hippocampal tissues of db/db mice, and projection electron microscopy was used to observe the ultrastructural changes (A). Chemical kit to detect iron content in homogenate of hippocampal tissue of db/db mice (B).CCK8 to detect cell viability of HT22 cells (C) and (G). Chemistry kit for detection of iron content in HT22 cells (D) and (H) and Chemistry kit for detection of GSH content in HT22 cells (E) and (J). Mean ± s.d., n = 6. * P < 0.05, compared with db/m mice. # P < 0.05, compared with db/db mice. 3.4 Neuritin improves ferroptosis by promoting the AMPK/Nrf2 signaling pathway A wealth of research indicates that the AMPK/Nrf2 signaling pathway is involved in the regulation of ferroptosis(Lu et al., 2023 ; Wan et al., 2023 ). To investigate whether neuritin can inhibit GABAergic neuronal ferroptosis via the AMPK/Nrf2 pathway, we conducted validation using the AMPK inhibitor Dorsomorphin and the Trkb inhibitor ANA-12. The results revealed that when Dorsomorphin inhibited AMPK, it failed to ameliorate the suppression of HT22 cell survival induced by high glucose, even in the presence of neuritin (Fig. 4 A). Furthermore, compared to the neuritin group, administration of Dorsomorphin significantly increased the levels of iron ions and MDA in cell lysates, consistent with the trend induced by high glucose (Fig. 4 B- 4 C). However, notably, following Dorsomorphin treatment, the levels of GSH in cell lysates significantly increased compared to the neuritin group (Fig. 4 D). In contrast, inhibition of Trkb did not significantly affect neuritin's function. To determine if the activation of Nrf2 is induced by AMPK, we utilized Western blot analysis to assess the expression of AMPK, p-AMPK, Nrf2, and GPX4 proteins. The results indicated that inhibiting AMPK led to decreased expression levels of AMPK, Nrf2, and GPX4 proteins, suggesting an upstream augmentation of AMPK, which is necessary for neuritin-mediated Nrf2 activation (Fig. 4 E- 4 J). These findings indicate that neuritin partially improves GABAergic neuronal ferroptosis through the AMPK/Nrf2 signaling pathway. Figure 4 Neuritin promotes activation of AMPK/Nrf2 signaling. cCK8 assay for HT22 cell viability (A). Chemical kits were used to detect iron ion content (B), MDA protein level (C) and GSH protein content (D). Detection of AMPK, p-AMPK, Nrf2 and GPX4 protein expression using Western blot(E). p-AMPK protein statistics (F). AMPK protein statistics(G). GPX4 protein statistics(H). Nrf2 protein statistics(J).Mean ± s.d., n = 6. *P < 0.05, compared with db/m mice. #P < 0.05, compared with db/db mice. 3.5 neuritin exert protective effects through the AMPK/Nrf2 signaling pathway in GABAergic To further elucidate the regulatory role of the AMPK/Nrf2 signaling pathway on GABAergic neurons, we conducted immunofluorescence double labeling. Our findings indicate a reduction in AMPK and GAD65 expression in the hippocampal DG of db/db mice compared to db/m mice. However, the overexpression of neuritin effectively ameliorated this condition in db/db mice (Fig. 5 A). Additionally, the treatment with neuritin overexpression resulted in an increased area ratio, count ratio, and intden ratio of the double-labeled fraction of GAD65 and AMPK relative to the total GAD65-labeled fraction in db/db mice (Fig. 5 B- 5 D).These results collectively suggest that the activation of the AMPK/Nrf2 signaling pathway contributes to the enhanced survival of GABAergic neurons. Figure 5 Protective effect of AMPK/NRF2 pathway on ferroptosis in db/db mice. Immunofluorescence observation of AMPK and GAD65 fluorescence intensity (A). ratio of AMPK and GAD65 co-localization area to total GAD65 positive area (B). ratio of AMPK and GAD65 co-localization number to total GAD65 positive number (C). ratio of AMPK and GAD65 co-localization fluorescence intensity to total GAD65 fluorescence intensity (D). Mean ± s.d., n = 3. *P < 0.05, compared with db/m mice. #P < 0.05, compared with db/db mice. 4 Discussion This study demonstrates the crucial role of neuritin in the development of concurrent cognitive impairment in db/db mice. Neuritin plays a pivotal role in inhibiting lipid peroxidation by activating the AMPK/Nrf2 pathway, thereby mitigating the detrimental effects of GABAergic neuronal ferroptosis. Moreover, neuritin proves indispensable in averting high glucose-induced HT22 cell ferroptosis by leveraging its activation of the AMPK/Nrf2 pathway. The pathophysiological mechanisms behind the deleterious effects of T2DM in the brain have not been fully determined. T2DM is associated with insulin resistance, hyperglycemia, oxidative stress and inflammation(Biessels and Reagan, 2015 ; De Felice and Ferreira, 2014 ; Greenwood and Winocur, 2005 ; Gault et al., 2015 ). In addition, T2DM can also lead to neuronal damage(Larsson et al., 2016 ). All these factors may be associated with brain damage, impaired cognitive function and increased neurodegenerative processes in T2DM. The link between T2DM and cognitive impairment is well known(Dove et al., 2021 ). Several longitudinal studies have reported an increased risk of developing cognitive impairment(Rawlings et al., 2019 ; Srikanth et al., 2020 ) and cognitive impairment progressing to diabetic dementia(Velayudhan et al., 2010 ; Dove et al., 2021 ). We found that db/db mice exhibited longer transfer latency than db/m mice, and that transfer latency were reduced in mice in which GABAergic overexpression neuritin, consistent with previous findings that neuritin improved transfer latency in db/db mice(Zhang et al., 2021 ). The normal function of the GABAergic system is compromised in diabetes. Patients with T2DM exhibit lower glutamate and GABA(d'Almeida et al., 2020 ). Diabetic encephalopathy impairs the excitability and synaptic transmission of GABAergic neurons mediated by GABA, and progression of diabetic encephalopathy can be prevented by protecting GABAergic neurons(Wang et al., 2020 ). The loss of GABAergic neurons also exacerbated cognitive impairment in T2DM(Zhou et al., 2022 ). Activation of GABAB receptors improves spatial cognitive function and hippocampal neurons in T2DM rats(Liu et al., 2018 ). GAD65 is the key enzyme responsible for GABA synthesis in the central nervous system, as a potential marker for cognitive performance in an adult population with prediabetes(Tsai et al., 2020 ). Our study found that db/db mice had lower densities of GAD65 positive neurons and significantly reduced GABA content, but db/db mice with overexpressing neuritin had higher densities of GAD65 positive neurons and GABA levels in the hippocampus, as well as greater learning and memory abilities. These results suggest that the improvement of learning and memory functions in diabetic mice by neuritin may be related to the GABAergic system. The pathogenesis of cognitive impairment in patients with diabetes includes impaired insulin signaling pathways, toxic effects of hyperglycaemia, oxidative stress, vascular factors, and genetic factors(Biessels et al., 2008 ). Evidence confirms that ferroptosis occurs in brain tissue because fatty acids accumulate in the brain and, therefore, the brain has high levels of lipid peroxidation(Bazinet and Layé, 2014 ). It has been suggested that ferroptosis is involved in the pathogenesis of diabetes and diabetes-related complications(Yang and Yang, 2022 ). Our study also showed that ferroptosis is closely associated with diabetes. Hippocampus of T2DM mice or HT22 cells in the high glucose state showed a marked increase in iron content, mitochondrial contraction, increased membrane density and reduced or even absent mitochondrial ridges, marking the onset of ferroptosis. And neuritin rescued this phenomenon. Ferroptosis is a non-apoptotic form of regulated cell death that is induced by the overproduction of phospholipid hydroperoxides in an iron-dependent manner(Xie et al., 2016 ; Cao and Dixon, 2016 ; Stockwell et al., 2017 ; Dixon et al., 2012 ). Ferroptosis is characterized by an iron-dependent accumulation of lipid peroxides accompanied by a deficiency of oxidoreductases, particularly GPX4(Sha et al., 2021 ). GPX4 is a major scavenger of lipid peroxides in cells(Flohe et al., 1973 ; Rotruck et al., 1973 ). GSH is a cofactor for GPX4 and is synthesized from glutamate, cysteine and glycine(Dixon et al., 2012 ). If the supply of cysteine is inadequate, leading to reduced production of cysteine and depletion of GSH, this will ultimately inhibit the normal activity of GPX4 in preventing ferroptosis(Kagan et al., 2017 ). The antioxidant enzyme GPX4 uses the reducing substance GSH to convert phospholipid hydroperoxides into lipid alcohols and to inhibit ferroptosis(Yang et al., 2014 ; Friedmann Angeli et al., 2014 ). There is growing evidence that abnormalities in brain iron homeostasis are associated with pathological cell death in neurodegenerative disorders, such as Parkinson’s and Alzheimer’s(Ishii et al., 2019 ). Iron plays a fundamental role in the development of the CNS as well as in several neuronal functions including synaptic plasticity(Codazzi et al., 2015 ). Our results show that neuritin, a potent regulator of ferroptosis, can downregulate iron levels and reduce GPX4, GSH and malonic dialdehyde levels, attenuate lipid peroxidation, inhibit the onset of ferroptosis and ultimately rescue brain cognitive function in neuritin overexpression db/db mice. Thus, neuritin overexpression inhibits ferroptosis primarily through activation of Nrf2-regulated antioxidants to counteract lipid peroxidation. This is the first study to reveal a role for neuritin in the inhibition of ferroptosis. AMPK/Nrf2 signaling pathway is closely associated with ferroptosis(Wang et al., 2022a ; Liu et al., 2022 ). AMPK deficiency affects all ferroptosis inducer induced ferroptosis(Lee et al., 2020 ). Sulforaphane (an isothiocyanate ) reduces the risk of diabetic cardiomyopathy by preventing iron death through AMPK-mediated activation of Nrf2(Wang et al., 2022b ). As AMPK activity is impaired in diabetes, stimulation of AMPK has been shown to improve blood glucose level in animal models(Joshi et al., 2019 ). Furthermore, our results show that the protective effect of neuritin against ferroptosis imposed by Nrf2 activation is AMPK-dependent in db/db mice. AMPK acts as a central regulator of cell survival in response to stress stimuli and is upregulated in neuritin-treated cells in HT22 and neuritin overexpression db/db mice. AMPK is a pivotal enzyme regulating energy metabolism, altered functionality of AMPK has been associated with multiple metabolic disorders, such as obesity, T2DM, and cardiovascular disorders(Ng et al., 2016 ; Brynildsen et al., 2018 ). Recently, AMPK signaling is reported to protect neurons under pathologic conditions, AMPK mediated restoration of the CNS energy balance is critical to protect the brain under pathologic conditions(Paintlia et al., 2013 ). Metformin (AMPK agonist) increases angiogenesis and neuroregeneration in ischemic encephalopathy and reduces neuronal apoptosis in rats, which is associated with decreased levels of AMPK signaling pathway and oxidative stress(Jin et al., 2014 ). Our results show that neuritin increased AMPK and phosphorylated AMPK expression in db/db mice. Activated AMPK also mediate Nrf2-GPX4 redox homeostasis to restore mitochondrial function, reduce reactive oxygen species production and improve oxidative stress (Li et al. , 2021) . Also, our results show that neuritin overexpression increased the expression of Nrf2, GPX4 and GSH in db/db mice. The protein expressions of p-AMPK, Nrf2 and GPX4 were significantly reduced after inhibition of AMPK using dorsomorphin in vitro. Immunofluorescence results showed that phosphorylation of AMPK was significantly inhibited in db/db mice, and both total and nuclear expression of Nrf2 was reduced. Overexpression of neuritin treatment increased p-AMPK and Nrf2 nuclear translocation. It has been shown that activation of AMPK contributes to neuronal survival in high-fat diet mice(Zhuang et al., 2019 ). AMPK activation can also reduce oxidative stress through Nrf2, thereby reducing neuronal apoptosis in rats with diabetes peripheral neuropathy(Zhang et al., 2022a ). Our results also showed that neuritin overexpression increased the co-localization coefficient of GAD65 with AMPK. Similar results were found in in vitro studies, where neuritin reduced high glucose induced intracellular iron content in HT22 cells, increased intracellular GSH content and increased AMPK, p-AMPK, Nrf2, and GPX4 protein expressions. The effect of neuritin was abolished by the use of dorsomorphin. The above results suggest that neuritin can improve oxidative stress and promote GABAergic neuronal survival in T2DM mice via the AMPK/Nrf2 pathway. 5 Conclusions Neuritin overexpression in hippocampal GABAergic neurons of diabetic mice significantly improved cognitive dysfunction, inhibited reactive oxygen species/lipid peroxidation/GSH depletion through modulation of the AMPK/Nrf2 signaling pathway, thereby attenuating ferroptosis in GABAergic neurons. Neuritin modulates the AMPK/Nrf2 signaling pathway to inhibit high glucose induced ferroptosis in HT22 cells. Thus, neuritin may inhibit ferroptosis in GABAergic neurons and improve cognitive dysfunction in diabetes, at least in part by modulating the AMPK/Nrf2 signaling pathway. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Ethics approval This study has been approved by the Laboratory Animal Welfare and Ethics Committee of the Army Medical University, with the approval number AMUWEC2020308. Funding This study is supported by grants from the Chongqing Natural Science Foundation of China (cstc2021jcyj-msxmX0249). Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Hongli Zhou, Zuo Zhang, Jianyun Zhou and Jiyin Zhou. The first draft of the manuscript was written by Hongli Zhou and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data Availability The datasets generated during and/or analysed during the current study are not publicly available due to [REASON(S) WHY DATA ARE NOT PUBLIC] but are available from the corresponding author on reasonable request. References Abdul Y, Li W, Ward R, Abdelsaid M, Hafez S, Dong G, Jamil S, Wolf V, Johnson MH, Fagan SC, Ergul A (2021) Deferoxamine Treatment Prevents Post-Stroke Vasoregression and Neurovascular Unit Remodeling Leading to Improved Functional Outcomes in Type 2 Male Diabetic Rats: Role of Endothelial Ferroptosis. Transl Stroke Res 12(4):615–630. 10.1007/s12975-020-00844-7 An JR, Su JN, Sun GY, Wang QF, Fan YD, Jiang N, Yang YF, Shi Y (2022) Liraglutide Alleviates Cognitive Deficit in db/db Mice: Involvement in Oxidative Stress, Iron Overload, and Ferroptosis. Neurochem Res 47(2):279–294. 10.1007/s11064-021-03442-7 Bazinet RP, Layé S (2014) Polyunsaturated fatty acids and their metabolites in brain function and disease. Nat Rev Neurosci 15(12):771–785. 10.1038/nrn3820 Biessels GJ, Deary IJ, Ryan CM (2008) Cognition and diabetes: a lifespan perspective. Lancet Neurol 7(2):184–190. 10.1016/s1474-4422(08)70021-8 Biessels GJ, Reagan LP (2015) Hippocampal insulin resistance and cognitive dysfunction. Nat Rev Neurosci 16(11):660–671. 10.1038/nrn4019 Brynildsen JK, Lee BG, Perron IJ, Jin S, Kim SF, Blendy JA (2018) Activation of AMPK by metformin improves withdrawal signs precipitated by nicotine withdrawal. Proc Natl Acad Sci U S A 115(16):4282–4287. 10.1073/pnas.1707047115 Cai XJ, Wang L, Hu CM (2018) Effects of GABAB receptor activation on spatial cognitive function and hippocampal neurones in rat models of type 2 diabetes mellitus. Biosci Rep 38(1). 10.1042/BSR20171184 Cao JY, Dixon SJ (2016) Mechanisms of ferroptosis. Cell Mol Life Sci 73(11–12):2195–2209. 10.1007/s00018-016-2194-1 Cheng N, Zhang Z, Guo Y, Qiu ZL, Du JY, Hei ZQ, Li X (2019) Weighted gene co-expression network analysis reveals specific modules and hub genes related to neuropathic pain in dorsal root ganglions. Biosci Rep 39(11). 10.1042/bsr20191511 Choi Y, Lee K, Ryu J, Kim HG, Jeong AY, Woo RS, Lee JH, Hyun JW, Hahn S, Kim JH, Kim HS (2014) Neuritin attenuates cognitive function impairments in tg2576 mouse model of Alzheimer's disease. PLoS ONE 9(8):e104121. 10.1371/journal.pone.0104121 Codazzi F, Pelizzoni I, Zacchetti D, Grohovaz F (2015) Iron entry in neurons and astrocytes: a link with synaptic activity. Front Mol Neurosci 8:18. 10.3389/fnmol.2015.00018 d'Almeida OC, Violante IR, Quendera B, Moreno C, Gomes L, Castelo-Branco M (2020) The neurometabolic profiles of GABA and Glutamate as revealed by proton magnetic resonance spectroscopy in type 1 and type 2 diabetes. PLoS ONE 15(10):e0240907. 10.1371/journal.pone.0240907 De Felice FG, Ferreira ST (2014) Inflammation, defective insulin signaling, and mitochondrial dysfunction as common molecular denominators connecting type 2 diabetes to Alzheimer disease. Diabetes 63(7):2262–2272. 10.2337/db13-1954 Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN, Bauer AJ, Cantley AM, Yang WS, Morrison B, Stockwell BR (2012) Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149(5):1060–1072. 10.1016/j.cell.2012.03.042 Dove A, Shang Y, Xu W, Grande G, Laukka EJ, Fratiglioni L, Marseglia A (2021) The impact of diabetes on cognitive impairment and its progression to dementia. Alzheimers Dement 17(11):1769–1778. 10.1002/alz.12482 Flohe L, Günzler WA, Schock HH (1973) Glutathione peroxidase: a selenoenzyme. FEBS Lett 32(1):132–134. 10.1016/0014-5793(73)80755-0 Friedmann Angeli JP, Schneider M, Proneth B, Tyurina YY, Tyurin VA, Hammond VJ, Herbach N, Aichler M, Walch A, Eggenhofer E, Basavarajappa D, Rådmark O, Kobayashi S, Seibt T, Beck H, Neff F, Esposito I, Wanke R, Förster H, Yefremova O, Heinrichmeyer M, Bornkamm GW, Geissler EK, Thomas SB, Stockwell BR, O'Donnell VB, Kagan VE, Schick JA, Conrad M (2014) Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice. Nat Cell Biol 16(12):1180–1191. 10.1038/ncb3064 Gault VA, Lennox R, Flatt PR (2015) Sitagliptin, a dipeptidyl peptidase-4 inhibitor, improves recognition memory, oxidative stress and hippocampal neurogenesis and upregulates key genes involved in cognitive decline. Diabetes Obes Metab 17(4):403–413. 10.1111/dom.12432 Giustizieri M, Petrillo S, D'Amico J, Torda C, Quatrana A, Vigevano F, Specchio N, Piemonte F, Cherubini E (2023) The ferroptosis inducer RSL3 triggers interictal epileptiform activity in mice cortical neurons. Front Cell Neurosci 17:1213732. 10.3389/fncel.2023.1213732 Greenwood CE, Winocur G (2005) High-fat diets, insulin resistance and declining cognitive function. Neurobiol Aging 26:42–45. 10.1016/j.neurobiolaging.2005.08.017 Ishii T, Warabi E, Mann GE (2019) Circadian control of BDNF-mediated Nrf2 activation in astrocytes protects dopaminergic neurons from ferroptosis. Free Radic Biol Med 133:169–178. 10.1016/j.freeradbiomed.2018.09.002 Jin Q, Cheng J, Liu Y, Wu J, Wang X, Wei S, Zhou X, Qin Z, Jia J, Zhen X (2014) Improvement of functional recovery by chronic metformin treatment is associated with enhanced alternative activation of microglia/macrophages and increased angiogenesis and neurogenesis following experimental stroke. Brain Behav Immun 40:131–142. 10.1016/j.bbi.2014.03.003 Joshi T, Singh AK, Haratipour P, Sah AN, Pandey AK, Naseri R, Juyal V, Farzaei MH (2019) Targeting AMPK signaling pathway by natural products for treatment of diabetes mellitus and its complications. J Cell Physiol 234(10):17212–17231. 10.1002/jcp.28528 Kagan VE, Mao G, Qu F, Angeli JP, Doll S, Croix CS, Dar HH, Liu B, Tyurin VA, Ritov VB, Kapralov AA, Amoscato AA, Jiang J, Anthonymuthu T, Mohammadyani D, Yang Q, Proneth B, Klein-Seetharaman J, Watkins S, Bahar I, Greenberger J, Mallampalli RK, Stockwell BR, Tyurina YY, Conrad M, Bayır H (2017) Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis. Nat Chem Biol 13(1):81–90. 10.1038/nchembio.2238 Kim SJ, Saeidi S, Cho NC, Kim SH, Lee HB, Han W, Noh DY, Surh YJ (2021) Interaction of Nrf2 with dimeric STAT3 induces IL-23 expression: Implications for breast cancer progression. Cancer Lett 500:147–160. 10.1016/j.canlet.2020.11.047 Larsson M, Lietzau G, Nathanson D, Östenson CG, Mallard C, Johansson ME, Nyström T, Patrone C, Darsalia V (2016) Diabetes negatively affects cortical and striatal GABAergic neurons: an effect that is partially counteracted by exendin-4. Biosci Rep 36(6). 10.1042/bsr20160437 Latunde-Dada GO (2017) Ferroptosis: Role of lipid peroxidation, iron and ferritinophagy. Biochim Biophys Acta Gen Subj 1861(8):1893–1900. 10.1016/j.bbagen.2017.05.019 Lee H, Zandkarimi F, Zhang Y, Meena JK, Kim J, Zhuang L, Tyagi S, Ma L, Westbrook TF, Steinberg GR, Nakada D, Stockwell BR, Gan B (2020) Energy-stress-mediated AMPK activation inhibits ferroptosis. Nat Cell Biol 22(2):225–234. 10.1038/s41556-020-0461-8 Li C-J, Li J-J, Jiang Y, Mu Y-W, Lu D-X, Xiao Z-Y, Jiang H-Y, Zhao J-J, Chen X-H (2020) Decreased cpg15 augments oxidative stress in sleep deprived mouse brain. Biochem Biophys Res Commun 522(3):749–756. 10.1016/j.bbrc.2019.11.132 Li Z, Li J, Miao X, Cui W, Miao L, Cai L (2021) A minireview: Role of AMP-activated protein kinase (AMPK) signaling in obesity-related renal injury. Life Sci 265:118828. 10.1016/j.lfs.2020.118828 Liu W, Cui Y, Sun J, Cai L, Xie J, Zhou X (2018) Transforming growth factor-β1 up-regulates connexin43 expression in osteocytes via canonical Smad-dependent signaling pathway. Biosci Rep 38(6). 10.1042/bsr20181678 Liu Y, Huang P, Li Z, Xu C, Wang H, Jia B, Gong A, Xu M (2022) Vitamin C Sensitizes Pancreatic Cancer Cells to Erastin-Induced Ferroptosis by Activating the AMPK/Nrf2/HMOX1 Pathway. Oxid Med Cell Longev 2022: 5361241. 10.1155/2022/5361241 Lu Q, Yang L, Xiao JJ, Liu Q, Ni L, Hu JW, Yu H, Wu X, Zhang BF (2023) Empagliflozin attenuates the renal tubular ferroptosis in diabetic kidney disease through AMPK/NRF2 pathway. Free Radic Biol Med 195:89–102. 10.1016/j.freeradbiomed.2022.12.088 Mederos S, Sánchez-Puelles C, Esparza J, Valero M, Ponomarenko A, Perea G (2021) GABAergic signaling to astrocytes in the prefrontal cortex sustains goal-directed behaviors. Nat Neurosci 24(1):82–92. 10.1038/s41593-020-00752-x Miao R, Fang X, Zhang Y, Wei J, Zhang Y, Tian J (2023) Iron metabolism and ferroptosis in type 2 diabetes mellitus and complications: mechanisms and therapeutic opportunities. Cell Death Dis 14(3):186. 10.1038/s41419-023-05708-0 Moheet A, Mangia S, Seaquist ER (2015) Impact of diabetes on cognitive function and brain structure. Ann N Y Acad Sci 1353:60–71. 10.1111/nyas.12807 Ng RC, Cheng OY, Jian M, Kwan JS, Ho PW, Cheng KK, Yeung PK, Zhou LL, Hoo RL, Chung SK, Xu A, Lam KS, Chan KH (2016) Chronic adiponectin deficiency leads to Alzheimer's disease-like cognitive impairments and pathologies through AMPK inactivation and cerebral insulin resistance in aged mice. Mol Neurodegener 11(1):71. 10.1186/s13024-016-0136-x Paintlia AS, Paintlia MK, Mohan S, Singh AK, Singh I (2013) AMP-activated protein kinase signaling protects oligodendrocytes that restore central nervous system functions in an experimental autoimmune encephalomyelitis model. Am J Pathol 183(2):526–541. 10.1016/j.ajpath.2013.04.030 Pelkey KA, Chittajallu R, Craig MT, Tricoire L, Wester JC, McBain CJ (2017) Hippocampal GABAergic Inhibitory Interneurons. Physiol Rev 97(4):1619–1747. 10.1152/physrev.00007.2017 Prévot T, Sibille E (2021) Altered GABA-mediated information processing and cognitive dysfunctions in depression and other brain disorders. Mol Psychiatry 26(1):151–167. 10.1038/s41380-020-0727-3 Rawlings AM, Sharrett AR, Albert MS, Coresh J, Windham BG, Power MC, Knopman DS, Walker K, Burgard S, Mosley TH, Gottesman RF, Selvin E (2019) The Association of Late-Life Diabetes Status and Hyperglycemia With Incident Mild Cognitive Impairment and Dementia: The ARIC Study. Diabetes Care 42(7):1248–1254. 10.2337/dc19-0120 Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG (1973) Selenium: biochemical role as a component of glutathione peroxidase. Science 179(4073):588–590. 10.1126/science.179.4073.588 Sha W, Hu F, Xi Y, Chu Y, Bu S (2021) Mechanism of Ferroptosis and Its Role in Type 2 Diabetes Mellitus. Journal of diabetes research 2021: 9999612. 10.1155/2021/9999612 Song D, Chen Y, Chen C, Chen L, Cheng O (2021) GABA(B) receptor antagonist promotes hippocampal neurogenesis and facilitates cognitive function recovery following acute cerebral ischemia in mice. Stem Cell Res Ther 12(1):22. 10.1186/s13287-020-02059-x Srikanth V, Sinclair AJ, Hill-Briggs F, Moran C, Biessels GJ (2020) Type 2 diabetes and cognitive dysfunction-towards effective management of both comorbidities. Lancet Diabetes Endocrinol 8(6):535–545. 10.1016/s2213-8587(20)30118-2 Stockwell BR, Angeli F, Bayir JP, Bush H, Conrad AI, Dixon M, Fulda SJ, Gascón S, Hatzios S, Kagan SK, Noel VE, Jiang K, Linkermann X, Murphy A, Overholtzer ME, Oyagi M, Pagnussat A, Park GC, Ran J, Rosenfeld Q, Salnikow CS, Tang K, Torti D, Torti FM, Toyokuni SV, Woerpel S, K. A., Zhang DD (2017) Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. Cell 171(2):273–285. 10.1016/j.cell.2017.09.021 Thielen JW, Gancheva S, Hong D, Rohani Rankouhi S, Chen B, Apostolopoulou M, Anadol-Schmitz E, Roden M, Norris DG, Tendolkar I (2019) Higher GABA concentration in the medial prefrontal cortex of Type 2 diabetes patients is associated with episodic memory dysfunction. Hum Brain Mapp 40(14):4287–4295. 10.1002/hbm.24702 Tsai CK, Kao TW, Lee JT, Wang CC, Chou CH, Liang CS, Yang FC, Chen WL (2020) GAD65 as a potential marker for cognitive performance in an adult population with prediabetes. QJM 113(2):108–114. 10.1093/qjmed/hcz239 Velayudhan L, Poppe M, Archer N, Proitsi P, Brown RG, Lovestone S (2010) Risk of developing dementia in people with diabetes and mild cognitive impairment. Br J Psychiatry 196(1):36–40. 10.1192/bjp.bp.109.067942 Wan K, Mao F, Li Q, Wang L, Wei Z, Wang P, Liao X, Xu M, Huang J, Pan Z, Wang C, Luo J, Gao R, Gan S (2020) Neuritin-overexpressing transgenic mice demonstrate enhanced neuroregeneration capacity and improved spatial learning and memory recovery after ischemia-reperfusion injury. Aging 13(2):2681–2699. 10.18632/aging.202318 Wan Y, Shen K, Yu H, Fan W (2023) Baicalein limits osteoarthritis development by inhibiting chondrocyte ferroptosis. Free Radic Biol Med 196:108–120. 10.1016/j.freeradbiomed.2023.01.006 Wang C, Li J, Zhao S, Huang L (2020) Diabetic encephalopathy causes the imbalance of neural activities between hippocampal glutamatergic neurons and GABAergic neurons in mice. Brain Res 1742:146863. 10.1016/j.brainres.2020.146863 Wang HF, Wang ZQ, Ding Y, Piao MH, Feng CS, Chi GF, Luo YN, Ge PF (2018) Endoplasmic reticulum stress regulates oxygen-glucose deprivation-induced parthanatos in human SH-SY5Y cells via improvement of intracellular ROS. CNS Neurosci Ther 24(1):29–38. 10.1111/cns.12771 Wang S, Yi X, Wu Z, Guo S, Dai W, Wang H, Shi Q, Zeng K, Guo W, Li C (2022a) CAMKK2 Defines Ferroptosis Sensitivity of Melanoma Cells by Regulating AMPK–NRF2 Pathway. J Invest Dermatol 142(1):189–200e188. 10.1016/j.jid.2021.05.025 Wang X, Chen X, Zhou W, Men H, Bao T, Sun Y, Wang Q, Tan Y, Keller BB, Tong Q, Zheng Y, Cai L (2022b) Ferroptosis is essential for diabetic cardiomyopathy and is prevented by sulforaphane via AMPK/NRF2 pathways. Acta Pharm Sin B 12(2):708–722. 10.1016/j.apsb.2021.10.005 Xie Y, Hou W, Song X, Yu Y, Huang J, Sun X, Kang R, Tang D (2016) Ferroptosis: process and function. Cell Death Differ 23(3):369–379. 10.1038/cdd.2015.158 Xie Z, Wang X, Luo X, Yan J, Zhang J, Sun R, Luo A, Li S (2023) Activated AMPK mitigates diabetes-related cognitive dysfunction by inhibiting hippocampal ferroptosis. Biochem Pharmacol 207:115374. 10.1016/j.bcp.2022.115374 Yang WS, SriRamaratnam R, Welsch ME, Shimada K, Skouta R, Viswanathan VS, Cheah JH, Clemons PA, Shamji AF, Clish CB, Brown LM, Girotti AW, Cornish VW, Schreiber SL, Stockwell BR (2014) Regulation of ferroptotic cancer cell death by GPX4. Cell 156(1–2):317–331. 10.1016/j.cell.2013.12.010 Yang XD, Yang YY (2022) Ferroptosis as a Novel Therapeutic Target for Diabetes and Its Complications. Front Endocrinol (Lausanne) 13:853822. 10.3389/fendo.2022.853822 Yuan F, Chen X, Fang KH, Wang Y, Lin M, Xu SB, Huo HQ, Xu M, Ma L, Chen Y, He S, Liu Y (2018) Induction of human somatostatin and parvalbumin neurons by expressing a single transcription factor LIM homeobox 6. Elife 7. 10.7554/eLife.37382 Zhang T, Zhang D, Zhang Z, Tian J, An J, Zhang W, Ben Y (2022a) Alpha-lipoic acid activates AMPK to protect against oxidative stress and apoptosis in rats with diabetic peripheral neuropathy. Horm (Athens). 10.1007/s42000-022-00413-7 Zhang W, Lu J, Wang Y, Sun P, Gao T, Xu N, Zhang Y, Xie W (2023) Canagliflozin Attenuates Lipotoxicity in Cardiomyocytes by Inhibiting Inflammation and Ferroptosis through Activating AMPK Pathway. Int J Mol Sci 24(1). 10.3390/ijms24010858 Zhang Z, Liu Y, Zhou J (2022b) Neuritin Promotes Bone Marrow-Derived Mesenchymal Stem Cell Migration to Treat Diabetic Peripheral Neuropathy. Mol Neurobiol 59(11):6666–6683. 10.1007/s12035-022-03002-2 Zhang Z, Zhou H, Zhou J (2021) Neuritin inhibits astrogliosis to ameliorate diabetic cognitive dysfunction. J Mol Endocrinol 66(4):259–272. 10.1530/jme-20-0321 Zhao JJ, Hu JX, Lu DX, Ji CX, Qi Y, Liu XY, Sun FY, Huang F, Xu P, Chen XH (2017) Soluble cpg15 from Astrocytes Ameliorates Neurite Outgrowth Recovery of Hippocampal Neurons after Mouse Cerebral Ischemia. J Neurosci 37(6):1628–1647. 10.1523/jneurosci.1611-16.2016 Zhou H, Rao Z, Zhang Z, Zhou J (2022) Function of the GABAergic System in Diabetic Encephalopathy. Cell Mol Neurobiol. 10.1007/s10571-022-01214-7 Zhou H, Rao Z, Zhang Z, Zhou J (2023) Function of the GABAergic System in Diabetic Encephalopathy. Cell Mol Neurobiol 43(2):605–619. 10.1007/s10571-022-01214-7 Zhou S, Zhou J (2014) Neuritin, a neurotrophic factor in nervous system physiology. Curr Med Chem 21(10):1212–1219. 10.2174/0929867321666131218093327 Zhuang J, Lu J, Wang X, Wang X, Hu W, Hong F, Zhao XX, Zheng YL (2019) Purple sweet potato color protects against high-fat diet-induced cognitive deficits through AMPK-mediated autophagy in mouse hippocampus. J Nutr Biochem 65:35–45. 10.1016/j.jnutbio.2018.10.015 Statements & Declarations Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-5965662","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":412338916,"identity":"008a9aa2-9ba1-4178-bc45-1ebfb8fd1b0a","order_by":0,"name":"Hongli Zhou","email":"","orcid":"","institution":"Clinical Medical Research Center, The Second Affiliated Hospital, Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hongli","middleName":"","lastName":"Zhou","suffix":""},{"id":412338917,"identity":"f24b9ed4-b8f1-44f6-8bfc-9956d950f1af","order_by":1,"name":"Zuo Zhang","email":"","orcid":"","institution":"Clinical Medical Research Center, The Second Affiliated Hospital, Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zuo","middleName":"","lastName":"Zhang","suffix":""},{"id":412338918,"identity":"5fe99ff6-aa84-4ffd-8635-0d2a825d717c","order_by":2,"name":"Jianyun Zhou","email":"","orcid":"","institution":"Clinical Medical Research Center, The Second Affiliated Hospital, Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jianyun","middleName":"","lastName":"Zhou","suffix":""},{"id":412338920,"identity":"972be740-0e9b-4ec0-ae03-448062431884","order_by":3,"name":"Jiyin Zhou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYDACCRA2YOBhYG+AihwgWgvPYVK0QBjJRGqRn918TMKi4LCMueT7o5tutjHI8d1IYPzwMQe3FsY5x9IkJAwO81jOTma7ndvGYCx5I4FZcuY23FqYJXLMwFoMbkO0JG64kcDGzItHCxtcy83DYC31BLXwwLXcYAZrSTAgpEVCIi3ZQsIgncfgTLLZ7ZxzEoYzzzxsxusX+RnJB29L/LG2Nzh+8NntnDIbeb7jyQc/fMSjBRIEDM1wW4GYsQG/epCSDwx1BBWNglEwCkbBCAYAYchMCmraAy4AAAAASUVORK5CYII=","orcid":"","institution":"Clinical Medical Research Center, The Second Affiliated Hospital, Army Medical University","correspondingAuthor":true,"prefix":"","firstName":"Jiyin","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2025-02-05 12:23:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5965662/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5965662/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75884935,"identity":"10403e0b-b2a1-4969-9015-e44216690765","added_by":"auto","created_at":"2025-02-10 09:05:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":662362,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCognitive impairment in db/db mice.\u003c/strong\u003e Immunofluorescence was used to observe the expression of neuritin and GABAergic neurons in the CA1 region of the hippocampus (A). Changes in neuritin and GABA content in hippocampal tissue homogenates were detected using western blot (B) and (C). Path diagram of mice in the Morris water maze experiment (D). Flow charts of constructing transgenic mice (E) and (F). Mean ± standard deviation (s.d.), n =3-6. *P\u0026lt;0.05, compared to db/m mice.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5965662/v1/7dc7b84d36db8b620c3d7ba5.png"},{"id":75884577,"identity":"ff02b4a9-ead0-420c-aea0-d3ab804ad27f","added_by":"auto","created_at":"2025-02-10 08:57:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":775669,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGABAergic overexpression of neuritin improves learning memory in db/db mice.\u003c/strong\u003e Results of PCR gene identification in transgenic mice (A). Fluorescence intensity of hippocampal neuritin (B). Western blot detection of changes in neuritin protein content in hippocampal tissue homogenates (C) and (D). Immunofluorescence detection of changes in GABAergic neurons after overexpression of neurtin (F). Path diagram of mice in the Morris water maze (G). Mean latency to reach the plateau in mice (H). Mean ± s.d., n =3-6. \u003csup\u003e*\u003c/sup\u003eP \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5965662/v1/9f8b3cf9255a947f9434fd0a.png"},{"id":75884939,"identity":"bc605e40-c657-4d4e-9efa-c301e1f2a1bf","added_by":"auto","created_at":"2025-02-10 09:05:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":774324,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of neuritin on db/db or high glucose-induced neuronal ferroptosis.\u003c/strong\u003e Prussian blue staining was used to observe the iron deposition phenomenon in hippocampal tissues of db/db mice, and projection electron microscopy was used to observe the ultrastructural changes (A). Chemical kit to detect iron content in homogenate of hippocampal tissue of db/db mice (B).CCK8 to detect cell viability of HT22 cells (C) and (G). Chemistry kit for detection of iron content in HT22 cells (D) and (H) and Chemistry kit for detection of GSH content in HT22 cells (E) and (J). Mean ± s.d., n = 6. \u003csup\u003e*\u003c/sup\u003eP \u0026lt; 0.05, compared with db/m mice. \u003csup\u003e#\u003c/sup\u003eP \u0026lt; 0.05, compared with db/db mice.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5965662/v1/0354f76a19b82156e0ded582.png"},{"id":75884933,"identity":"afb693b5-7c9e-49d7-b2d1-d9fb68b067d1","added_by":"auto","created_at":"2025-02-10 09:05:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":268884,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeuritin promotes activation of AMPK/Nrf2 signaling.\u003c/strong\u003e cCK8 assay for HT22 cell viability (A). Chemical kits were used to detect iron ion content (B), MDA protein level (C) and GSH protein content (D). Detection of AMPK, p-AMPK, Nrf2 and GPX4 protein expression using Western blot(E). p-AMPK protein statistics (F). AMPK protein statistics(G). GPX4 protein statistics(H). Nrf2 protein statistics(J).Mean ± s.d., n = 6. *P \u0026lt; 0.05, compared with db/m mice. #P \u0026lt; 0.05, compared with db/db mice.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5965662/v1/8c4c8e0a0ab85ef692c7d955.png"},{"id":75884579,"identity":"940cd0a8-1e5a-43db-9231-46d283e1452a","added_by":"auto","created_at":"2025-02-10 08:57:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":843641,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtective effect of AMPK/NRF2 pathway on ferroptosis in db/db mice.\u003c/strong\u003e Immunofluorescence observation of AMPK and GAD65 fluorescence intensity (A). ratio of AMPK and GAD65 co-localization area to total GAD65 positive area (B). ratio of AMPK and GAD65 co-localization number to total GAD65 positive number (C). ratio of AMPK and GAD65 co-localization fluorescence intensity to total GAD65 fluorescence intensity (D). Mean ± s.d., n = 3. *P \u0026lt; 0.05, compared with db/m mice. #P \u0026lt; 0.05, compared with db/db mice.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5965662/v1/5f1943d5a487be09642346f9.png"},{"id":83797882,"identity":"3ca7f486-fbf9-4fe1-8bea-ce38d253f619","added_by":"auto","created_at":"2025-06-03 01:31:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3975624,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5965662/v1/7547cc9b-155b-47c9-9df0-4c0d1d075580.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Neuritin suppresses GABAergic neurons ferroptosis to improve cognitive impairment in diabetes mellitus","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eType 2 diabetes mellitus (T2DM) is the most common type of diabetes mellitus, which is characterized by hyperglycemia. It has been found that patients with T2DM are more likely to develop cognitive impairment(Cai et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). And the development of this cognitive dysfunction is usually latent and not easily detectable, eventually leading to dementia(Moheet et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The hippocampus is a key region of the brain for learning and memory, and it is also one of the most sensitive regions to high glucose stimulation(Kim et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Numerous studies have demonstrated that changes in hippocampal plasticity and function are the main cause of cognitive impairment in diabetes, but the specific molecular mechanisms behind diabetic cognitive dysfunction remain unclear.\u003c/p\u003e \u003cp\u003eIn hippocampus, GABAergic inhibitory interneurons account for 10\u0026ndash;15% of the total neuronal cell population(Pelkey et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). GABAergic interneurons are the primary inhibitory system of the mammalian brain and are associated with cognitive deficits(Yuan et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Mederos et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Patients with T2DM experience episodic memory loss when GABA levels in the medial prefrontal cortex are abnormal(Thielen et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Inhibition of GABAB receptors effectively promotes hippocampal neurogenesis and improves spatial learning and memory after cerebral ischemia in adult mice(Song et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Our previous publication indicated that GABAergic interneurons play an important role in diabetic cognitive deficits by showing significant changes in GABA receptor density and function in certain brain regions associated with cognitive function, such as the hippocampus, implying that spatial learning and memory are related in part to altered GABA receptors and function(Zhou et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNeuritin is a neurotrophic factor that is mainly expressed in the brain(Zhou and Zhou, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Studies have shown that neuritin improves the recovery of neuritin growth in hippocampal neurons after cerebral ischemia in mice(Zhao et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and attenuates cognitive dysfunction in a mouse model of Alzheimer's disease(Choi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wan et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). One of our previous studies also showed that neuritin ameliorating cognitive dysfunction in diabetic patients(Zhang et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Importantly, our current study found a significant increase in the number of GABAergic neurons in the hippocampal tissue of mice overexpressing neuritin and a corresponding improvement in learning memory function. This suggests that the effect of neuritin in improving diabetic cognitive dysfunction may be related to the promotion of survival or proliferation of GABAergic nerves in the hippocampus. However, the specific mechanism by which neuritin protects GABAergic nerve survival has not yet been elucidated.\u003c/p\u003e \u003cp\u003eFerroptosis is a regulated form of cell death dependent on iron and reactive oxygen species and characterized by lipid peroxidation(Latunde-Dada, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Ferroptosis has been found to characterize several diabetes models. Moreover, it is noteworthy that reduces iron overload improved hippocampal neuronal and synaptic plasticity and ultimately restores cognitive function(An et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Abdul et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ferroptosis inducer RAS-selective lethal 3 inhibits GABAergic currents(Giustizieri et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and reduced GABAergic neuron activity can reduce information processing in cortical microcircuits, leading to cognitive impairment(Pr\u0026eacute;vot and Sibille, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In our previous study, we found that overexpression of neuritin ameliorated mitochondrial atrophy, reduction of mitochondrial ridges, and increase in membrane density in hippocampal area cells of diabetic mice by transmission electron microscopy. This suggests that the protective effect of neuritin on GABAergic nerves may be related to the inhibition of their iron death. The study showed that these effects may closely relate to the activation of the AMPK signaling pathway(Xie et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), which regulates the expression of a range of biologically important genes in ferroptosis. For example, activated AMPK inhibits indicators of ferroptosis (e.g., reactive oxygen species, malondialdehyde, reduced glutathione, and mitochondrial membrane potential)(Zhang et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Specifically, the activation of AMPK signaling pathway is implicated in cognitive impairment due to diabetic neuronal ferroptosis.\u003c/p\u003e \u003cp\u003eIn the present study, we found that hippocampal GABAergic neurons are more susceptible to hyperglycemia under T2DM conditions, inducing GABAergic neuronal ferroptosis and leading to cognitive dysfunction, which can be reversed by neuritin. The present study further investigated whether neuritin could play a role in ameliorating T2DM memory dysfunction by modulating the AMPK/Nrf2 signaling pathway to counteract iron oxidation in hippocampal GABAergic neurons.\u003c/p\u003e"},{"header":"2 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Establishment of the GABAergic neurons with neuritin overexpression\u003c/h2\u003e \u003cp\u003eTransgenic mice expressing CMV-LoxP-STOP-LoxP-Nrn1(Zhang et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e) were bred with Calb2tm1-Cre (B6(Cg)-Calb2\u003csup\u003e\u003cem\u003etm1(cre)Zjh\u003c/em\u003e\u003c/sup\u003e/J) mice obtained from The Jackson Laboratory, resulting in the generation of neuritin-Cre transgenic mice. Non-diabetic (db/m, C57BL/6J-Leprdb/+) mice were purchased from The Jackson Laboratory. The db/m mice were crossed with the neuritin-Cre transgenic mice to generate db/m/neuritin/Calb2tm1-Cre transgenic mice. Subsequently, the db/m/neuritin/Calb2tm1-Cre transgenic mice were crossbred with each other, giving rise to db/db/neuritin/Calb2tm1-Cre triple transgenic mice. The evaluation of Cre-mediated neuritin expression was performed through polymerase chain reaction (PCR) analysis using genomic DNA extracted from the tail. All mice were housed in a specific pathogen-free environment with controlled temperature and humidity, adhering to a 12-hour light: 12-hour dark cycle. The mice were provided ad libitum access to food and water. Ethical approval for the animal experiments was obtained from the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Army Medical University, ensuring compliance with established guidelines and standards for the ethical treatment of animals in research.\u003c/p\u003e \u003cp\u003eDrug treatment: Mice were divided into (1) db/m control group (2) db/db group, (3) db/db mice with overexpression neuritin group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Morris water maze test\u003c/h2\u003e \u003cp\u003eMorris water maze experiment was used to assess learning and memory functions in mice. The day before the test, the inner wall of the swimming pool was decorated with white wallpaper (black mice and white environment), the escape platform was fixed, water at 22\u0026thinsp;~\u0026thinsp;24\u0026deg;C was filled into the pool and mice were allowed to swim freely for 60 s to familiarize themselves with the environment. Titanium dioxide (appropriate amount) was added to make the water white and the quadrants were marked as quadrants I, II, III and IV according to the position of the platform.\u003c/p\u003e \u003cp\u003eAcquisition training (4 days): Mice were gently placed into the pool from the middle wall of the four quadrants I, II, III and IV. The experiment lasts for 60 s. Mice are found and landed on the platform (the platform is placed in the center of one quadrant) and allowed to remain on the platform for 10 s. If the platform is still not found within 60 s, mice are guided to land on the platform and remain there for 10 s. If the animal leaves, it is restarted until the rest period is 10 s and the latency period is recorded for 60s. At the end of each training session, the mice are dried using a dry rag. Each mouse was trained 4 times per day (once per quadrant, randomly distributed into quadrants each day) at 5\u0026ndash;10 min intervals and trained for 4 consecutive days. The mean of the 4 latency periods was calculated as the score for that day.\u003c/p\u003e \u003cp\u003eExploratory training: After the last acquired training session, the underwater platform was removed and a 60 s exploratory training session was started. Mice were placed in the water from the quadrant opposite the platform and the time spent in the target quadrant (original platform quadrant) and the number of times the mice entered the quadrant were recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Tissue preparation\u003c/h2\u003e \u003cp\u003eAfter the Morris water maze test, six mice of each group were anesthetized with sodium pentobarbital (30 mg/kg i.p.) blood was collected from heart, perfused with PBS at a rate of 5 mL/min for 10 min. The brains were then dissected and fixed in 4% paraformaldehyde at 4\u0026deg;C for 24 h, followed by dipping in 20% and 30% sucrose solution at 4\u0026deg;C successively until the tissues were sunk. Coronal sections were prepared in a thickness of 20 \u0026micro;M with a freezing microtome (Leica) when the brain tissues were completely frozen in the Optimum Cutting Temperature compound at -20\u0026deg;C. Hippocampus tissue was taken from six additional mice and stored frozen in liquid nitrogen for subsequent biochemical markers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Biochemical index analysis\u003c/h2\u003e \u003cp\u003eIron content, glutathione peroxidase 4 (GPX4), glutathione (GSH) and malondialdehyde (MDA) were determined using mouse hippocampal tissue homogenates or HT22 cell lysates according to the manufacturer's instructions. Assay kits were obtained from NanJing JianCheng Bioengineering Institute (NanJing, China). Protein concentrations in the samples were determined using the BCA protein assay kit (Beyotime, Shanghai, China). To ensure comparability of results between different groups GSH and MDA levels and GPX4 activity were normalized to protein content.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Immunofluorescence\u003c/h2\u003e \u003cp\u003eFrozen sections were repaired with modified sodium citrate antigen repair solution (Beyotime Biotechnology, P0083) at 95\u0026deg;C for 5 min. PBS was washed three times for 5 min each. Then incubated with PBS blocking solution containing 0.1% Triton and 10% goat serum for 1 h at room temperature. The primary antibody was then incubated overnight at 4\u0026deg;C. The following day, the sections were washed 3 times with PBS for 5 min each, followed by incubation with the corresponding fluorescently labelled secondary antibody for 1 h at room temperature, 3 times with PBS for 5 min each, staining with DAPI (Beyotime Biotechnology, C1002) for 10 min, and finally a drop of intifada mounting medium (Beyotime Biotechnology, P0126) on the tissue sections, cover with cover slips. The negative controls received the same procedures except that the primary antibodies were omitted and no unspecific staining was observed. The immunofluorescent staining signals were observed under a fluorescent microscope or a confocal laser-scanning microscopy (TCS SP8, Leica)(Li et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Mouse neuritin antibody (Sc-365538, Santa, 1:300), rabbit GABAB2 (ab52248, abcam, 1:300), rabbit GAD65 antibody (Bioss antibodies, bs-0325R, 1:300) were used as the primary antibodies. Goat anti-rabbit IgG-Alexa Fluor 488 (Thermo, A-11008, 1:400), goat anti-mouse IgG-Alexa Fluor 647 (Thermo, A-11008, 1:400) were used as the second antibodies. Three sections of each mouse were obtained under a fluorescence microscope (Olympus, Tokyo, Japan) with three random fields of view (\u0026times;40) in the DG region and the images were analyzed using ImageJ 1.50.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Transmission electron microscope\u003c/h2\u003e \u003cp\u003eFresh mouse hippocampus tissue (CA1 area, 1 mm3) was fixed with 2% (v/v) glutaraldehyde fixative in groups of 3 mice each. After complete fixation, the tissues were rinsed with PBS buffer 4 times for 10 min, ultrapure water was rinsed for 3 min. The tissues were then dehydrated in graded alcohols of 50%, 70%, 80%, 90% and 100% for 10 min in each step. After dehydration, the sections were treated with ethanol: propylene oxide (1:1), pure propylene oxide for 10 min each time, then with propylene oxide: epoxy resin (2:1), propylene oxide: epoxy resin (1:2), pure epoxy resin for 1 h each time at 30\u0026deg;C. Finally, each sample was examined by transmission electron microscopy. Images of each section were taken for mitochondrial morphometry analysis. 2.8 Cell culture\u003c/p\u003e \u003cp\u003eHT22 cells were obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mmol/L glutamine (Gibco, Grand Island, NY, USA), penicillin (100 U/mL), and streptomycin (100 \u0026micro;g/mL), and maintained at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e in a humid environment. The medium was replaced twice each week(Wang et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHT22 cells were treated with glucose, recombinant human neuritin (1000 ng/mL), dorsomorphin (25 \u0026micro;M) or ANA-12 (400 nM). Glucose, recombinant human neuritin, or dorsomorphin were prepared in complete medium and sterilized by using 0.2 \u0026micro;m syringe filter (Shanghai Fuxin, China). After drug treatment HT22 cells are lysed using ultrasonic fragmentation for subsequent biochemical assays or lysed using RIPA lysis solution for subsequent western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Cell viability by the CCK8 assay\u003c/h2\u003e \u003cp\u003eThe cell viability of HT22 cells was determined using the Cell Counting Kit-8 (CCK-8, Dojindo, Tokyo, Japan). Briefly, HT22 cells were seeded at a density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e/well into 96-well plates for 24 h. Cells were pretreated with dorsomorphin or ANA-12. Cells were then treated with high glucose solution (75 mmol/L) or high glucose solution containing neuritin for 48 h. Cell viability was assessed using Cell CCK-8 according to the instructions. 100 \u0026micro;L of medium containing 10 \u0026micro;L of CCK-8 solution was applied. The plates were then incubated at 37\u0026deg;C for 2 h, after which the optical density at 450 nm was measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Prussian blue staining\u003c/h2\u003e \u003cp\u003eTo assess iron deposition in hippocampus tissue, the Prussian Blue Iron Stain Kit (Enhance With DAB) (Solarbio, G1428) was used for the assay. Slices were routinely dewaxed and rehydrated. Perls Working Solution was added dropwise to the slices and incubated in a wet box at 37\u0026deg;C for 20 min. Slices were removed and rinsed gently with distilled water 3 times for 10 s each. Slices were incubated dropwise with Incubation Solution and placed in a wet box at 37\u0026deg;C for 20 min. Slices were removed and washed 3 times with 1\u0026times; PBS for 60 s each. Slices were incubated dropwise with enhanced Working Solution and placed in a wet box at 37\u0026deg;C for 15 min. The sections were washed 3 times with 1\u0026times; PBS for 5 s. The slices were stained for 1.5 min with dropwise addition of Redying Solution. Soak in distilled water for 10 min, dehydrated with gradient ethanol, transparent by xylene and seal with resinene.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Western blot analysis\u003c/h2\u003e \u003cp\u003eWestern blot was conducted according to previously reported protocols(Cheng et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Briefly, equal amounts of mouse hippocampal tissue RIPA lysates or HT22 cell RIPA lysates (25 \u0026micro;g protein/lane) were loaded onto 10% SDS-PAGE, proteins were separated and transferred to PVDF membranes. The membrane was blocked with 5% bovine serum albumin. Then incubated with antibody neuritin (santa, sc-365538, 1:500), GABAB receptor (abcam, ab52248, 1:500), rabbit anti-glutathione peroxidase 4 (GPX4, 52455S, CST, 1:1000), AMP-activated protein kinase α (AMPKα, 5831, CST, 1:1000), phospho AMP-activated protein kinase α (p-AMPKα, 2535, CST, 1:1000), nuclear factor-like 2 (Nrf2, 12721, CST, 1:1000) for overnight at 4\u0026deg;C. The TBST solution was washed 3 times for 10 min. After washing, the membranes were incubated for 1 h at room temperature with the appropriate secondary antibody (horseradish peroxidase [HRP] coupled, dilution 1:2000). Pre-stained molecular weight markers were manipulated in parallel to determine the molecular weight of the target protein. For chemiluminescence detection, membranes were treated with enhanced chemiluminescence reagents and images were obtained using ImageQuant LAS4010 (GE Healthcare). Samples were taken in duplicate for each experiment. Images were analysed for densitometry using ImageJ 1.50.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe SPSS 20.0 software (IBM CORP) was applied to perform the statistical analyses. Behavioural data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and analyzed by a two-way analysis of variance (ANOVA) with repeated measures followed by Bonferroni post hoc comparisons. Results of the iron content assay, lipid peroxidation assay and western blot were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and analyzed by a one-way ANOVA followed by Bonferroni post hoc comparisons.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Cognitive impairment in db/db mice\u003c/h2\u003e \u003cp\u003eImmunofluorescence staining was performed on the brain tissues of db/db mice, and the results revealed a significant reduction in neuritin levels and the number of GABAergic neurons in the hippocampal CA1 region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Further confirmation using Western blot analysis yielded consistent results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Morris water maze experiments demonstrated cognitive abnormalities in db/db mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). To investigate whether the cognitive impairment in db/db mice is related to neuritin and GABAergic neurons, we generated a triple transgenic mouse model, db/db/neuritin/Calb2tm1-Cre, characterized by high expression of neuritin in GABAergic neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e Cognitive impairment in db/db mice. Immunofluorescence was used to observe the expression of neuritin and GABAergic neurons in the CA1 region of the hippocampus (A). Changes in neuritin and GABA content in hippocampal tissue homogenates were detected using western blot (B) and (C). Path diagram of mice in the Morris water maze experiment (D). Flow charts of constructing transgenic mice (E) and (F). Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (s.d.), n\u0026thinsp;=\u0026thinsp;3\u0026ndash;6. *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, compared to db/m mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. GABAergic overexpression of neuritin improves learning memory in db/db mice\u003c/h2\u003e \u003cp\u003eFirstly, we performed PCR testing on the tail DNA of the generated mice, selecting mice with positive identification of Calb2tm1 cre at 175 bp or 175 bp and 125 bp, LoxP at 234 bp, and Misty at 207 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) for further experiments. To confirm the success of the construct, immunofluorescence was conducted on the hippocampal tissue of the mice. The results revealed a significant increase in neuritin fluorescence levels in the CA1 region of mice overexpressing neuritin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Western blot analysis also confirmed the elevated expression of neuritin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Additionally, to investigate the impact of neuritin overexpression on GABAergic neurons, immunofluorescence observation revealed a significant increase in the number of GABAergic neurons after neuritin overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine whether the overexpression of neuritin in GABAergic neurons could improve memory deficits in db/db mice, we utilized the Morris water maze test to measure mice learning abilities. There were no significant differences in latency on the first and second days for all animals. However, during the third to fifth days of training, db/db mice exhibited longer escape latencies compared to db/m mice, indicating lower platform-searching abilities. Following neuritin overexpression, db/db mice showed reduced escape latencies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). On the fifth day, when the platform was removed, the analysis of navigation paths revealed that neuritin-overexpressing db/db mice reached the original platform location in less time compared to db/db mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Analysis of swimming distances on the fifth day indicated a significant increase in swimming distance for db/db mice compared to db/m mice. However, after neuritin overexpression, the swimming distance of db/db mice was reduced. These results suggest that neuritin overexpression improves cognitive and memory functions in db/db mice.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e GABAergic overexpression of neuritin improves learning memory in db/db mice. Results of PCR gene identification in transgenic mice (A). Fluorescence intensity of hippocampal neuritin (B). Western blot detection of changes in neuritin protein content in hippocampal tissue homogenates (C) and (D). Immunofluorescence detection of changes in GABAergic neurons after overexpression of neurtin (F). Path diagram of mice in the Morris water maze (G). Mean latency to reach the plateau in mice (H). Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;s.d., n\u0026thinsp;=\u0026thinsp;3\u0026ndash;6. \u003csup\u003e*\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Neuritin improves GABAergic neuronal ferroptosis\u003c/h2\u003e \u003cp\u003eDue to the pivotal role of iron imbalance in the development of several endocrine disorders(Miao et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), we investigated whether iron dysregulation is involved in the mechanism of T2DM-related cognitive impairment. Prussian blue staining and assay kits revealed a significant increase in iron deposition in db/db mice, while neuritin overexpression markedly reduced this iron deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Ultrastructural analysis indicated mitochondrial shrinkage, increased membrane density, decreased or vanished mitochondrial ridges in db/db mice, indicating the onset of ferroptosis, whereas neuritin overexpression ameliorated these changes in mitochondrial structure in db/db mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). This suggests that neuritin may ameliorate ferroptosis in db/db mice. To further delineate the impact of neuritin on neuronal ferroptosis, we employed the HT22 cell line, a hippocampal neuron cell line. Results demonstrated that high glucose significantly reduced HT22 cell viability compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), increased iron deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), and reduced GSH levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Western blot analysis revealed that high glucose significantly reduced GPX4 protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Exogenous neuritin notably increased cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), reduced iron deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH), and increased GSH levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ) compared to the control group. Particularly, the effect of 1000 ng/mL of neuritin was more pronounced; therefore, in subsequent experiments, we used a neuritin dosage of 1000 ng/mL.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e Effect of neuritin on db/db or high glucose-induced neuronal ferroptosis. Prussian blue staining was used to observe the iron deposition phenomenon in hippocampal tissues of db/db mice, and projection electron microscopy was used to observe the ultrastructural changes (A). Chemical kit to detect iron content in homogenate of hippocampal tissue of db/db mice (B).CCK8 to detect cell viability of HT22 cells (C) and (G). Chemistry kit for detection of iron content in HT22 cells (D) and (H) and Chemistry kit for detection of GSH content in HT22 cells (E) and (J). Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;s.d., n\u0026thinsp;=\u0026thinsp;6. \u003csup\u003e*\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05, compared with db/m mice. \u003csup\u003e#\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05, compared with db/db mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Neuritin improves ferroptosis by promoting the AMPK/Nrf2 signaling pathway\u003c/h2\u003e \u003cp\u003eA wealth of research indicates that the AMPK/Nrf2 signaling pathway is involved in the regulation of ferroptosis(Lu et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wan et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). To investigate whether neuritin can inhibit GABAergic neuronal ferroptosis via the AMPK/Nrf2 pathway, we conducted validation using the AMPK inhibitor Dorsomorphin and the Trkb inhibitor ANA-12. The results revealed that when Dorsomorphin inhibited AMPK, it failed to ameliorate the suppression of HT22 cell survival induced by high glucose, even in the presence of neuritin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Furthermore, compared to the neuritin group, administration of Dorsomorphin significantly increased the levels of iron ions and MDA in cell lysates, consistent with the trend induced by high glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). However, notably, following Dorsomorphin treatment, the levels of GSH in cell lysates significantly increased compared to the neuritin group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In contrast, inhibition of Trkb did not significantly affect neuritin's function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine if the activation of Nrf2 is induced by AMPK, we utilized Western blot analysis to assess the expression of AMPK, p-AMPK, Nrf2, and GPX4 proteins. The results indicated that inhibiting AMPK led to decreased expression levels of AMPK, Nrf2, and GPX4 proteins, suggesting an upstream augmentation of AMPK, which is necessary for neuritin-mediated Nrf2 activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). These findings indicate that neuritin partially improves GABAergic neuronal ferroptosis through the AMPK/Nrf2 signaling pathway.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e Neuritin promotes activation of AMPK/Nrf2 signaling. cCK8 assay for HT22 cell viability (A). Chemical kits were used to detect iron ion content (B), MDA protein level (C) and GSH protein content (D). Detection of AMPK, p-AMPK, Nrf2 and GPX4 protein expression using Western blot(E). p-AMPK protein statistics (F). AMPK protein statistics(G). GPX4 protein statistics(H). Nrf2 protein statistics(J).Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;s.d., n\u0026thinsp;=\u0026thinsp;6. *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, compared with db/m mice. #P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, compared with db/db mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5 neuritin exert protective effects through the AMPK/Nrf2 signaling pathway in GABAergic\u003c/h2\u003e \u003cp\u003eTo further elucidate the regulatory role of the AMPK/Nrf2 signaling pathway on GABAergic neurons, we conducted immunofluorescence double labeling. Our findings indicate a reduction in AMPK and GAD65 expression in the hippocampal DG of db/db mice compared to db/m mice. However, the overexpression of neuritin effectively ameliorated this condition in db/db mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Additionally, the treatment with neuritin overexpression resulted in an increased area ratio, count ratio, and intden ratio of the double-labeled fraction of GAD65 and AMPK relative to the total GAD65-labeled fraction in db/db mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).These results collectively suggest that the activation of the AMPK/Nrf2 signaling pathway contributes to the enhanced survival of GABAergic neurons.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e Protective effect of AMPK/NRF2 pathway on ferroptosis in db/db mice. Immunofluorescence observation of AMPK and GAD65 fluorescence intensity (A). ratio of AMPK and GAD65 co-localization area to total GAD65 positive area (B). ratio of AMPK and GAD65 co-localization number to total GAD65 positive number (C). ratio of AMPK and GAD65 co-localization fluorescence intensity to total GAD65 fluorescence intensity (D). Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;s.d., n\u0026thinsp;=\u0026thinsp;3. *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, compared with db/m mice. #P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, compared with db/db mice.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThis study demonstrates the crucial role of neuritin in the development of concurrent cognitive impairment in db/db mice. Neuritin plays a pivotal role in inhibiting lipid peroxidation by activating the AMPK/Nrf2 pathway, thereby mitigating the detrimental effects of GABAergic neuronal ferroptosis. Moreover, neuritin proves indispensable in averting high glucose-induced HT22 cell ferroptosis by leveraging its activation of the AMPK/Nrf2 pathway.\u003c/p\u003e \u003cp\u003eThe pathophysiological mechanisms behind the deleterious effects of T2DM in the brain have not been fully determined. T2DM is associated with insulin resistance, hyperglycemia, oxidative stress and inflammation(Biessels and Reagan, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; De Felice and Ferreira, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Greenwood and Winocur, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Gault et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In addition, T2DM can also lead to neuronal damage(Larsson et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). All these factors may be associated with brain damage, impaired cognitive function and increased neurodegenerative processes in T2DM. The link between T2DM and cognitive impairment is well known(Dove et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Several longitudinal studies have reported an increased risk of developing cognitive impairment(Rawlings et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Srikanth et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and cognitive impairment progressing to diabetic dementia(Velayudhan et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Dove et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We found that db/db mice exhibited longer transfer latency than db/m mice, and that transfer latency were reduced in mice in which GABAergic overexpression neuritin, consistent with previous findings that neuritin improved transfer latency in db/db mice(Zhang et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The normal function of the GABAergic system is compromised in diabetes. Patients with T2DM exhibit lower glutamate and GABA(d'Almeida et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Diabetic encephalopathy impairs the excitability and synaptic transmission of GABAergic neurons mediated by GABA, and progression of diabetic encephalopathy can be prevented by protecting GABAergic neurons(Wang et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The loss of GABAergic neurons also exacerbated cognitive impairment in T2DM(Zhou et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Activation of GABAB receptors improves spatial cognitive function and hippocampal neurons in T2DM rats(Liu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). GAD65 is the key enzyme responsible for GABA synthesis in the central nervous system, as a potential marker for cognitive performance in an adult population with prediabetes(Tsai et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Our study found that db/db mice had lower densities of GAD65 positive neurons and significantly reduced GABA content, but db/db mice with overexpressing neuritin had higher densities of GAD65 positive neurons and GABA levels in the hippocampus, as well as greater learning and memory abilities. These results suggest that the improvement of learning and memory functions in diabetic mice by neuritin may be related to the GABAergic system.\u003c/p\u003e \u003cp\u003eThe pathogenesis of cognitive impairment in patients with diabetes includes impaired insulin signaling pathways, toxic effects of hyperglycaemia, oxidative stress, vascular factors, and genetic factors(Biessels et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Evidence confirms that ferroptosis occurs in brain tissue because fatty acids accumulate in the brain and, therefore, the brain has high levels of lipid peroxidation(Bazinet and Lay\u0026eacute;, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). It has been suggested that ferroptosis is involved in the pathogenesis of diabetes and diabetes-related complications(Yang and Yang, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Our study also showed that ferroptosis is closely associated with diabetes. Hippocampus of T2DM mice or HT22 cells in the high glucose state showed a marked increase in iron content, mitochondrial contraction, increased membrane density and reduced or even absent mitochondrial ridges, marking the onset of ferroptosis. And neuritin rescued this phenomenon.\u003c/p\u003e \u003cp\u003eFerroptosis is a non-apoptotic form of regulated cell death that is induced by the overproduction of phospholipid hydroperoxides in an iron-dependent manner(Xie et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Cao and Dixon, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Stockwell et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Dixon et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Ferroptosis is characterized by an iron-dependent accumulation of lipid peroxides accompanied by a deficiency of oxidoreductases, particularly GPX4(Sha et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). GPX4 is a major scavenger of lipid peroxides in cells(Flohe et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1973\u003c/span\u003e; Rotruck et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1973\u003c/span\u003e). GSH is a cofactor for GPX4 and is synthesized from glutamate, cysteine and glycine(Dixon et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). If the supply of cysteine is inadequate, leading to reduced production of cysteine and depletion of GSH, this will ultimately inhibit the normal activity of GPX4 in preventing ferroptosis(Kagan et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The antioxidant enzyme GPX4 uses the reducing substance GSH to convert phospholipid hydroperoxides into lipid alcohols and to inhibit ferroptosis(Yang et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Friedmann Angeli et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). There is growing evidence that abnormalities in brain iron homeostasis are associated with pathological cell death in neurodegenerative disorders, such as Parkinson\u0026rsquo;s and Alzheimer\u0026rsquo;s(Ishii et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Iron plays a fundamental role in the development of the CNS as well as in several neuronal functions including synaptic plasticity(Codazzi et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Our results show that neuritin, a potent regulator of ferroptosis, can downregulate iron levels and reduce GPX4, GSH and malonic dialdehyde levels, attenuate lipid peroxidation, inhibit the onset of ferroptosis and ultimately rescue brain cognitive function in neuritin overexpression db/db mice. Thus, neuritin overexpression inhibits ferroptosis primarily through activation of Nrf2-regulated antioxidants to counteract lipid peroxidation. This is the first study to reveal a role for neuritin in the inhibition of ferroptosis. AMPK/Nrf2 signaling pathway is closely associated with ferroptosis(Wang et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). AMPK deficiency affects all ferroptosis inducer induced ferroptosis(Lee et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Sulforaphane (an isothiocyanate ) reduces the risk of diabetic cardiomyopathy by preventing iron death through AMPK-mediated activation of Nrf2(Wang et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). As AMPK activity is impaired in diabetes, stimulation of AMPK has been shown to improve blood glucose level in animal models(Joshi et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Furthermore, our results show that the protective effect of neuritin against ferroptosis imposed by Nrf2 activation is AMPK-dependent in db/db mice. AMPK acts as a central regulator of cell survival in response to stress stimuli and is upregulated in neuritin-treated cells in HT22 and neuritin overexpression db/db mice.\u003c/p\u003e \u003cp\u003eAMPK is a pivotal enzyme regulating energy metabolism, altered functionality of AMPK has been associated with multiple metabolic disorders, such as obesity, T2DM, and cardiovascular disorders(Ng et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Brynildsen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Recently, AMPK signaling is reported to protect neurons under pathologic conditions, AMPK mediated restoration of the CNS energy balance is critical to protect the brain under pathologic conditions(Paintlia et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Metformin (AMPK agonist) increases angiogenesis and neuroregeneration in ischemic encephalopathy and reduces neuronal apoptosis in rats, which is associated with decreased levels of AMPK signaling pathway and oxidative stress(Jin et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Our results show that neuritin increased AMPK and phosphorylated AMPK expression in db/db mice. Activated AMPK also mediate Nrf2-GPX4 redox homeostasis to restore mitochondrial function, reduce reactive oxygen species production and improve oxidative stress\u003csup\u003e(Li \u003cem\u003eet al.\u003c/em\u003e, 2021)\u003c/sup\u003e. Also, our results show that neuritin overexpression increased the expression of Nrf2, GPX4 and GSH in db/db mice. The protein expressions of p-AMPK, Nrf2 and GPX4 were significantly reduced after inhibition of AMPK using dorsomorphin in vitro. Immunofluorescence results showed that phosphorylation of AMPK was significantly inhibited in db/db mice, and both total and nuclear expression of Nrf2 was reduced. Overexpression of neuritin treatment increased p-AMPK and Nrf2 nuclear translocation. It has been shown that activation of AMPK contributes to neuronal survival in high-fat diet mice(Zhuang et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). AMPK activation can also reduce oxidative stress through Nrf2, thereby reducing neuronal apoptosis in rats with diabetes peripheral neuropathy(Zhang et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur results also showed that neuritin overexpression increased the co-localization coefficient of GAD65 with AMPK. Similar results were found in in vitro studies, where neuritin reduced high glucose induced intracellular iron content in HT22 cells, increased intracellular GSH content and increased AMPK, p-AMPK, Nrf2, and GPX4 protein expressions. The effect of neuritin was abolished by the use of dorsomorphin. The above results suggest that neuritin can improve oxidative stress and promote GABAergic neuronal survival in T2DM mice via the AMPK/Nrf2 pathway.\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eNeuritin overexpression in hippocampal GABAergic neurons of diabetic mice significantly improved cognitive dysfunction, inhibited reactive oxygen species/lipid peroxidation/GSH depletion through modulation of the AMPK/Nrf2 signaling pathway, thereby attenuating ferroptosis in GABAergic neurons. Neuritin modulates the AMPK/Nrf2 signaling pathway to inhibit high glucose induced ferroptosis in HT22 cells. Thus, neuritin may inhibit ferroptosis in GABAergic neurons and improve cognitive dysfunction in diabetes, at least in part by modulating the AMPK/Nrf2 signaling pathway.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthics approval\u003c/h2\u003e \u003cp\u003e This study has been approved by the Laboratory Animal Welfare and Ethics Committee of the Army Medical University, with the approval number AMUWEC2020308.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study is supported by grants from the Chongqing Natural Science Foundation of China (cstc2021jcyj-msxmX0249).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Hongli Zhou, Zuo Zhang, Jianyun Zhou and Jiyin Zhou. The first draft of the manuscript was written by Hongli Zhou and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe datasets generated during and/or analysed during the current study are not publicly available due to [REASON(S) WHY DATA ARE NOT PUBLIC] but are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e \u003cli\u003e\u003cspan\u003eAbdul Y, Li W, Ward R, Abdelsaid M, Hafez S, Dong G, Jamil S, Wolf V, Johnson MH, Fagan SC, Ergul A (2021) Deferoxamine Treatment Prevents Post-Stroke Vasoregression and Neurovascular Unit Remodeling Leading to Improved Functional Outcomes in Type 2 Male Diabetic Rats: Role of Endothelial Ferroptosis. Transl Stroke Res 12(4):615\u0026ndash;630. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s12975-020-00844-7\u003c/span\u003e\u003cspan address=\"10.1007/s12975-020-00844-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAn JR, Su JN, Sun GY, Wang QF, Fan YD, Jiang N, Yang YF, Shi Y (2022) Liraglutide Alleviates Cognitive Deficit in db/db Mice: Involvement in Oxidative Stress, Iron Overload, and Ferroptosis. Neurochem Res 47(2):279\u0026ndash;294. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11064-021-03442-7\u003c/span\u003e\u003cspan address=\"10.1007/s11064-021-03442-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBazinet RP, Lay\u0026eacute; S (2014) Polyunsaturated fatty acids and their metabolites in brain function and disease. Nat Rev Neurosci 15(12):771\u0026ndash;785. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nrn3820\u003c/span\u003e\u003cspan address=\"10.1038/nrn3820\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBiessels GJ, Deary IJ, Ryan CM (2008) Cognition and diabetes: a lifespan perspective. Lancet Neurol 7(2):184\u0026ndash;190. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s1474-4422(08)70021-8\u003c/span\u003e\u003cspan address=\"10.1016/s1474-4422(08)70021-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBiessels GJ, Reagan LP (2015) Hippocampal insulin resistance and cognitive dysfunction. Nat Rev Neurosci 16(11):660\u0026ndash;671. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nrn4019\u003c/span\u003e\u003cspan address=\"10.1038/nrn4019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrynildsen JK, Lee BG, Perron IJ, Jin S, Kim SF, Blendy JA (2018) Activation of AMPK by metformin improves withdrawal signs precipitated by nicotine withdrawal. Proc Natl Acad Sci U S A 115(16):4282\u0026ndash;4287. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.1707047115\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1707047115\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai XJ, Wang L, Hu CM (2018) Effects of GABAB receptor activation on spatial cognitive function and hippocampal neurones in rat models of type 2 diabetes mellitus. Biosci Rep 38(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1042/BSR20171184\u003c/span\u003e\u003cspan address=\"10.1042/BSR20171184\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao JY, Dixon SJ (2016) Mechanisms of ferroptosis. Cell Mol Life Sci 73(11\u0026ndash;12):2195\u0026ndash;2209. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00018-016-2194-1\u003c/span\u003e\u003cspan address=\"10.1007/s00018-016-2194-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng N, Zhang Z, Guo Y, Qiu ZL, Du JY, Hei ZQ, Li X (2019) Weighted gene co-expression network analysis reveals specific modules and hub genes related to neuropathic pain in dorsal root ganglions. Biosci Rep 39(11). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1042/bsr20191511\u003c/span\u003e\u003cspan address=\"10.1042/bsr20191511\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi Y, Lee K, Ryu J, Kim HG, Jeong AY, Woo RS, Lee JH, Hyun JW, Hahn S, Kim JH, Kim HS (2014) Neuritin attenuates cognitive function impairments in tg2576 mouse model of Alzheimer's disease. PLoS ONE 9(8):e104121. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0104121\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0104121\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCodazzi F, Pelizzoni I, Zacchetti D, Grohovaz F (2015) Iron entry in neurons and astrocytes: a link with synaptic activity. Front Mol Neurosci 8:18. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fnmol.2015.00018\u003c/span\u003e\u003cspan address=\"10.3389/fnmol.2015.00018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ed'Almeida OC, Violante IR, Quendera B, Moreno C, Gomes L, Castelo-Branco M (2020) The neurometabolic profiles of GABA and Glutamate as revealed by proton magnetic resonance spectroscopy in type 1 and type 2 diabetes. PLoS ONE 15(10):e0240907. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0240907\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0240907\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Felice FG, Ferreira ST (2014) Inflammation, defective insulin signaling, and mitochondrial dysfunction as common molecular denominators connecting type 2 diabetes to Alzheimer disease. Diabetes 63(7):2262\u0026ndash;2272. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2337/db13-1954\u003c/span\u003e\u003cspan address=\"10.2337/db13-1954\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN, Bauer AJ, Cantley AM, Yang WS, Morrison B, Stockwell BR (2012) Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149(5):1060\u0026ndash;1072. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cell.2012.03.042\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2012.03.042\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDove A, Shang Y, Xu W, Grande G, Laukka EJ, Fratiglioni L, Marseglia A (2021) The impact of diabetes on cognitive impairment and its progression to dementia. Alzheimers Dement 17(11):1769\u0026ndash;1778. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/alz.12482\u003c/span\u003e\u003cspan address=\"10.1002/alz.12482\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlohe L, G\u0026uuml;nzler WA, Schock HH (1973) Glutathione peroxidase: a selenoenzyme. FEBS Lett 32(1):132\u0026ndash;134. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/0014-5793(73)80755-0\u003c/span\u003e\u003cspan address=\"10.1016/0014-5793(73)80755-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFriedmann Angeli JP, Schneider M, Proneth B, Tyurina YY, Tyurin VA, Hammond VJ, Herbach N, Aichler M, Walch A, Eggenhofer E, Basavarajappa D, R\u0026aring;dmark O, Kobayashi S, Seibt T, Beck H, Neff F, Esposito I, Wanke R, F\u0026ouml;rster H, Yefremova O, Heinrichmeyer M, Bornkamm GW, Geissler EK, Thomas SB, Stockwell BR, O'Donnell VB, Kagan VE, Schick JA, Conrad M (2014) Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice. Nat Cell Biol 16(12):1180\u0026ndash;1191. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/ncb3064\u003c/span\u003e\u003cspan address=\"10.1038/ncb3064\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGault VA, Lennox R, Flatt PR (2015) Sitagliptin, a dipeptidyl peptidase-4 inhibitor, improves recognition memory, oxidative stress and hippocampal neurogenesis and upregulates key genes involved in cognitive decline. Diabetes Obes Metab 17(4):403\u0026ndash;413. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/dom.12432\u003c/span\u003e\u003cspan address=\"10.1111/dom.12432\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiustizieri M, Petrillo S, D'Amico J, Torda C, Quatrana A, Vigevano F, Specchio N, Piemonte F, Cherubini E (2023) The ferroptosis inducer RSL3 triggers interictal epileptiform activity in mice cortical neurons. Front Cell Neurosci 17:1213732. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fncel.2023.1213732\u003c/span\u003e\u003cspan address=\"10.3389/fncel.2023.1213732\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreenwood CE, Winocur G (2005) High-fat diets, insulin resistance and declining cognitive function. Neurobiol Aging 26:42\u0026ndash;45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.neurobiolaging.2005.08.017\u003c/span\u003e\u003cspan address=\"10.1016/j.neurobiolaging.2005.08.017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIshii T, Warabi E, Mann GE (2019) Circadian control of BDNF-mediated Nrf2 activation in astrocytes protects dopaminergic neurons from ferroptosis. Free Radic Biol Med 133:169\u0026ndash;178. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.freeradbiomed.2018.09.002\u003c/span\u003e\u003cspan address=\"10.1016/j.freeradbiomed.2018.09.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin Q, Cheng J, Liu Y, Wu J, Wang X, Wei S, Zhou X, Qin Z, Jia J, Zhen X (2014) Improvement of functional recovery by chronic metformin treatment is associated with enhanced alternative activation of microglia/macrophages and increased angiogenesis and neurogenesis following experimental stroke. Brain Behav Immun 40:131\u0026ndash;142. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbi.2014.03.003\u003c/span\u003e\u003cspan address=\"10.1016/j.bbi.2014.03.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoshi T, Singh AK, Haratipour P, Sah AN, Pandey AK, Naseri R, Juyal V, Farzaei MH (2019) Targeting AMPK signaling pathway by natural products for treatment of diabetes mellitus and its complications. J Cell Physiol 234(10):17212\u0026ndash;17231. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/jcp.28528\u003c/span\u003e\u003cspan address=\"10.1002/jcp.28528\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKagan VE, Mao G, Qu F, Angeli JP, Doll S, Croix CS, Dar HH, Liu B, Tyurin VA, Ritov VB, Kapralov AA, Amoscato AA, Jiang J, Anthonymuthu T, Mohammadyani D, Yang Q, Proneth B, Klein-Seetharaman J, Watkins S, Bahar I, Greenberger J, Mallampalli RK, Stockwell BR, Tyurina YY, Conrad M, Bayır H (2017) Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis. Nat Chem Biol 13(1):81\u0026ndash;90. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nchembio.2238\u003c/span\u003e\u003cspan address=\"10.1038/nchembio.2238\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim SJ, Saeidi S, Cho NC, Kim SH, Lee HB, Han W, Noh DY, Surh YJ (2021) Interaction of Nrf2 with dimeric STAT3 induces IL-23 expression: Implications for breast cancer progression. Cancer Lett 500:147\u0026ndash;160. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.canlet.2020.11.047\u003c/span\u003e\u003cspan address=\"10.1016/j.canlet.2020.11.047\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLarsson M, Lietzau G, Nathanson D, \u0026Ouml;stenson CG, Mallard C, Johansson ME, Nystr\u0026ouml;m T, Patrone C, Darsalia V (2016) Diabetes negatively affects cortical and striatal GABAergic neurons: an effect that is partially counteracted by exendin-4. Biosci Rep 36(6). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1042/bsr20160437\u003c/span\u003e\u003cspan address=\"10.1042/bsr20160437\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLatunde-Dada GO (2017) Ferroptosis: Role of lipid peroxidation, iron and ferritinophagy. Biochim Biophys Acta Gen Subj 1861(8):1893\u0026ndash;1900. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbagen.2017.05.019\u003c/span\u003e\u003cspan address=\"10.1016/j.bbagen.2017.05.019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee H, Zandkarimi F, Zhang Y, Meena JK, Kim J, Zhuang L, Tyagi S, Ma L, Westbrook TF, Steinberg GR, Nakada D, Stockwell BR, Gan B (2020) Energy-stress-mediated AMPK activation inhibits ferroptosis. Nat Cell Biol 22(2):225\u0026ndash;234. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41556-020-0461-8\u003c/span\u003e\u003cspan address=\"10.1038/s41556-020-0461-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi C-J, Li J-J, Jiang Y, Mu Y-W, Lu D-X, Xiao Z-Y, Jiang H-Y, Zhao J-J, Chen X-H (2020) Decreased cpg15 augments oxidative stress in sleep deprived mouse brain. Biochem Biophys Res Commun 522(3):749\u0026ndash;756. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbrc.2019.11.132\u003c/span\u003e\u003cspan address=\"10.1016/j.bbrc.2019.11.132\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Z, Li J, Miao X, Cui W, Miao L, Cai L (2021) A minireview: Role of AMP-activated protein kinase (AMPK) signaling in obesity-related renal injury. Life Sci 265:118828. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.lfs.2020.118828\u003c/span\u003e\u003cspan address=\"10.1016/j.lfs.2020.118828\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu W, Cui Y, Sun J, Cai L, Xie J, Zhou X (2018) Transforming growth factor-β1 up-regulates connexin43 expression in osteocytes via canonical Smad-dependent signaling pathway. Biosci Rep 38(6). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1042/bsr20181678\u003c/span\u003e\u003cspan address=\"10.1042/bsr20181678\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Huang P, Li Z, Xu C, Wang H, Jia B, Gong A, Xu M (2022) Vitamin C Sensitizes Pancreatic Cancer Cells to Erastin-Induced Ferroptosis by Activating the AMPK/Nrf2/HMOX1 Pathway. \u003cem\u003eOxid Med Cell Longev\u003c/em\u003e 2022: 5361241. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2022/5361241\u003c/span\u003e\u003cspan address=\"10.1155/2022/5361241\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu Q, Yang L, Xiao JJ, Liu Q, Ni L, Hu JW, Yu H, Wu X, Zhang BF (2023) Empagliflozin attenuates the renal tubular ferroptosis in diabetic kidney disease through AMPK/NRF2 pathway. Free Radic Biol Med 195:89\u0026ndash;102. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.freeradbiomed.2022.12.088\u003c/span\u003e\u003cspan address=\"10.1016/j.freeradbiomed.2022.12.088\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMederos S, S\u0026aacute;nchez-Puelles C, Esparza J, Valero M, Ponomarenko A, Perea G (2021) GABAergic signaling to astrocytes in the prefrontal cortex sustains goal-directed behaviors. Nat Neurosci 24(1):82\u0026ndash;92. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41593-020-00752-x\u003c/span\u003e\u003cspan address=\"10.1038/s41593-020-00752-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiao R, Fang X, Zhang Y, Wei J, Zhang Y, Tian J (2023) Iron metabolism and ferroptosis in type 2 diabetes mellitus and complications: mechanisms and therapeutic opportunities. Cell Death Dis 14(3):186. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41419-023-05708-0\u003c/span\u003e\u003cspan address=\"10.1038/s41419-023-05708-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoheet A, Mangia S, Seaquist ER (2015) Impact of diabetes on cognitive function and brain structure. Ann N Y Acad Sci 1353:60\u0026ndash;71. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/nyas.12807\u003c/span\u003e\u003cspan address=\"10.1111/nyas.12807\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNg RC, Cheng OY, Jian M, Kwan JS, Ho PW, Cheng KK, Yeung PK, Zhou LL, Hoo RL, Chung SK, Xu A, Lam KS, Chan KH (2016) Chronic adiponectin deficiency leads to Alzheimer's disease-like cognitive impairments and pathologies through AMPK inactivation and cerebral insulin resistance in aged mice. Mol Neurodegener 11(1):71. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13024-016-0136-x\u003c/span\u003e\u003cspan address=\"10.1186/s13024-016-0136-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePaintlia AS, Paintlia MK, Mohan S, Singh AK, Singh I (2013) AMP-activated protein kinase signaling protects oligodendrocytes that restore central nervous system functions in an experimental autoimmune encephalomyelitis model. Am J Pathol 183(2):526\u0026ndash;541. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ajpath.2013.04.030\u003c/span\u003e\u003cspan address=\"10.1016/j.ajpath.2013.04.030\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePelkey KA, Chittajallu R, Craig MT, Tricoire L, Wester JC, McBain CJ (2017) Hippocampal GABAergic Inhibitory Interneurons. Physiol Rev 97(4):1619\u0026ndash;1747. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1152/physrev.00007.2017\u003c/span\u003e\u003cspan address=\"10.1152/physrev.00007.2017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePr\u0026eacute;vot T, Sibille E (2021) Altered GABA-mediated information processing and cognitive dysfunctions in depression and other brain disorders. Mol Psychiatry 26(1):151\u0026ndash;167. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41380-020-0727-3\u003c/span\u003e\u003cspan address=\"10.1038/s41380-020-0727-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRawlings AM, Sharrett AR, Albert MS, Coresh J, Windham BG, Power MC, Knopman DS, Walker K, Burgard S, Mosley TH, Gottesman RF, Selvin E (2019) The Association of Late-Life Diabetes Status and Hyperglycemia With Incident Mild Cognitive Impairment and Dementia: The ARIC Study. Diabetes Care 42(7):1248\u0026ndash;1254. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2337/dc19-0120\u003c/span\u003e\u003cspan address=\"10.2337/dc19-0120\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG (1973) Selenium: biochemical role as a component of glutathione peroxidase. Science 179(4073):588\u0026ndash;590. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.179.4073.588\u003c/span\u003e\u003cspan address=\"10.1126/science.179.4073.588\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSha W, Hu F, Xi Y, Chu Y, Bu S (2021) Mechanism of Ferroptosis and Its Role in Type 2 Diabetes Mellitus. \u003cem\u003eJournal of diabetes research\u003c/em\u003e 2021: 9999612. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2021/9999612\u003c/span\u003e\u003cspan address=\"10.1155/2021/9999612\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong D, Chen Y, Chen C, Chen L, Cheng O (2021) GABA(B) receptor antagonist promotes hippocampal neurogenesis and facilitates cognitive function recovery following acute cerebral ischemia in mice. Stem Cell Res Ther 12(1):22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13287-020-02059-x\u003c/span\u003e\u003cspan address=\"10.1186/s13287-020-02059-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSrikanth V, Sinclair AJ, Hill-Briggs F, Moran C, Biessels GJ (2020) Type 2 diabetes and cognitive dysfunction-towards effective management of both comorbidities. Lancet Diabetes Endocrinol 8(6):535\u0026ndash;545. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s2213-8587(20)30118-2\u003c/span\u003e\u003cspan address=\"10.1016/s2213-8587(20)30118-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStockwell BR, Angeli F, Bayir JP, Bush H, Conrad AI, Dixon M, Fulda SJ, Gasc\u0026oacute;n S, Hatzios S, Kagan SK, Noel VE, Jiang K, Linkermann X, Murphy A, Overholtzer ME, Oyagi M, Pagnussat A, Park GC, Ran J, Rosenfeld Q, Salnikow CS, Tang K, Torti D, Torti FM, Toyokuni SV, Woerpel S, K. A., Zhang DD (2017) Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. Cell 171(2):273\u0026ndash;285. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cell.2017.09.021\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2017.09.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThielen JW, Gancheva S, Hong D, Rohani Rankouhi S, Chen B, Apostolopoulou M, Anadol-Schmitz E, Roden M, Norris DG, Tendolkar I (2019) Higher GABA concentration in the medial prefrontal cortex of Type 2 diabetes patients is associated with episodic memory dysfunction. Hum Brain Mapp 40(14):4287\u0026ndash;4295. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/hbm.24702\u003c/span\u003e\u003cspan address=\"10.1002/hbm.24702\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsai CK, Kao TW, Lee JT, Wang CC, Chou CH, Liang CS, Yang FC, Chen WL (2020) GAD65 as a potential marker for cognitive performance in an adult population with prediabetes. QJM 113(2):108\u0026ndash;114. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/qjmed/hcz239\u003c/span\u003e\u003cspan address=\"10.1093/qjmed/hcz239\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVelayudhan L, Poppe M, Archer N, Proitsi P, Brown RG, Lovestone S (2010) Risk of developing dementia in people with diabetes and mild cognitive impairment. Br J Psychiatry 196(1):36\u0026ndash;40. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1192/bjp.bp.109.067942\u003c/span\u003e\u003cspan address=\"10.1192/bjp.bp.109.067942\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan K, Mao F, Li Q, Wang L, Wei Z, Wang P, Liao X, Xu M, Huang J, Pan Z, Wang C, Luo J, Gao R, Gan S (2020) Neuritin-overexpressing transgenic mice demonstrate enhanced neuroregeneration capacity and improved spatial learning and memory recovery after ischemia-reperfusion injury. Aging 13(2):2681\u0026ndash;2699. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18632/aging.202318\u003c/span\u003e\u003cspan address=\"10.18632/aging.202318\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan Y, Shen K, Yu H, Fan W (2023) Baicalein limits osteoarthritis development by inhibiting chondrocyte ferroptosis. Free Radic Biol Med 196:108\u0026ndash;120. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.freeradbiomed.2023.01.006\u003c/span\u003e\u003cspan address=\"10.1016/j.freeradbiomed.2023.01.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang C, Li J, Zhao S, Huang L (2020) Diabetic encephalopathy causes the imbalance of neural activities between hippocampal glutamatergic neurons and GABAergic neurons in mice. Brain Res 1742:146863. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.brainres.2020.146863\u003c/span\u003e\u003cspan address=\"10.1016/j.brainres.2020.146863\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang HF, Wang ZQ, Ding Y, Piao MH, Feng CS, Chi GF, Luo YN, Ge PF (2018) Endoplasmic reticulum stress regulates oxygen-glucose deprivation-induced parthanatos in human SH-SY5Y cells via improvement of intracellular ROS. CNS Neurosci Ther 24(1):29\u0026ndash;38. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/cns.12771\u003c/span\u003e\u003cspan address=\"10.1111/cns.12771\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang S, Yi X, Wu Z, Guo S, Dai W, Wang H, Shi Q, Zeng K, Guo W, Li C (2022a) CAMKK2 Defines Ferroptosis Sensitivity of Melanoma Cells by Regulating AMPK\u0026ndash;NRF2 Pathway. J Invest Dermatol 142(1):189\u0026ndash;200e188. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jid.2021.05.025\u003c/span\u003e\u003cspan address=\"10.1016/j.jid.2021.05.025\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Chen X, Zhou W, Men H, Bao T, Sun Y, Wang Q, Tan Y, Keller BB, Tong Q, Zheng Y, Cai L (2022b) Ferroptosis is essential for diabetic cardiomyopathy and is prevented by sulforaphane via AMPK/NRF2 pathways. Acta Pharm Sin B 12(2):708\u0026ndash;722. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.apsb.2021.10.005\u003c/span\u003e\u003cspan address=\"10.1016/j.apsb.2021.10.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie Y, Hou W, Song X, Yu Y, Huang J, Sun X, Kang R, Tang D (2016) Ferroptosis: process and function. Cell Death Differ 23(3):369\u0026ndash;379. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/cdd.2015.158\u003c/span\u003e\u003cspan address=\"10.1038/cdd.2015.158\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie Z, Wang X, Luo X, Yan J, Zhang J, Sun R, Luo A, Li S (2023) Activated AMPK mitigates diabetes-related cognitive dysfunction by inhibiting hippocampal ferroptosis. Biochem Pharmacol 207:115374. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bcp.2022.115374\u003c/span\u003e\u003cspan address=\"10.1016/j.bcp.2022.115374\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang WS, SriRamaratnam R, Welsch ME, Shimada K, Skouta R, Viswanathan VS, Cheah JH, Clemons PA, Shamji AF, Clish CB, Brown LM, Girotti AW, Cornish VW, Schreiber SL, Stockwell BR (2014) Regulation of ferroptotic cancer cell death by GPX4. Cell 156(1\u0026ndash;2):317\u0026ndash;331. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cell.2013.12.010\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2013.12.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang XD, Yang YY (2022) Ferroptosis as a Novel Therapeutic Target for Diabetes and Its Complications. Front Endocrinol (Lausanne) 13:853822. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fendo.2022.853822\u003c/span\u003e\u003cspan address=\"10.3389/fendo.2022.853822\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan F, Chen X, Fang KH, Wang Y, Lin M, Xu SB, Huo HQ, Xu M, Ma L, Chen Y, He S, Liu Y (2018) Induction of human somatostatin and parvalbumin neurons by expressing a single transcription factor LIM homeobox 6. \u003cem\u003eElife\u003c/em\u003e 7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7554/eLife.37382\u003c/span\u003e\u003cspan address=\"10.7554/eLife.37382\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang T, Zhang D, Zhang Z, Tian J, An J, Zhang W, Ben Y (2022a) Alpha-lipoic acid activates AMPK to protect against oxidative stress and apoptosis in rats with diabetic peripheral neuropathy. Horm (Athens). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s42000-022-00413-7\u003c/span\u003e\u003cspan address=\"10.1007/s42000-022-00413-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang W, Lu J, Wang Y, Sun P, Gao T, Xu N, Zhang Y, Xie W (2023) Canagliflozin Attenuates Lipotoxicity in Cardiomyocytes by Inhibiting Inflammation and Ferroptosis through Activating AMPK Pathway. Int J Mol Sci 24(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms24010858\u003c/span\u003e\u003cspan address=\"10.3390/ijms24010858\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Z, Liu Y, Zhou J (2022b) Neuritin Promotes Bone Marrow-Derived Mesenchymal Stem Cell Migration to Treat Diabetic Peripheral Neuropathy. Mol Neurobiol 59(11):6666\u0026ndash;6683. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s12035-022-03002-2\u003c/span\u003e\u003cspan address=\"10.1007/s12035-022-03002-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Z, Zhou H, Zhou J (2021) Neuritin inhibits astrogliosis to ameliorate diabetic cognitive dysfunction. J Mol Endocrinol 66(4):259\u0026ndash;272. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1530/jme-20-0321\u003c/span\u003e\u003cspan address=\"10.1530/jme-20-0321\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao JJ, Hu JX, Lu DX, Ji CX, Qi Y, Liu XY, Sun FY, Huang F, Xu P, Chen XH (2017) Soluble cpg15 from Astrocytes Ameliorates Neurite Outgrowth Recovery of Hippocampal Neurons after Mouse Cerebral Ischemia. J Neurosci 37(6):1628\u0026ndash;1647. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1523/jneurosci.1611-16.2016\u003c/span\u003e\u003cspan address=\"10.1523/jneurosci.1611-16.2016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou H, Rao Z, Zhang Z, Zhou J (2022) Function of the GABAergic System in Diabetic Encephalopathy. Cell Mol Neurobiol. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10571-022-01214-7\u003c/span\u003e\u003cspan address=\"10.1007/s10571-022-01214-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou H, Rao Z, Zhang Z, Zhou J (2023) Function of the GABAergic System in Diabetic Encephalopathy. Cell Mol Neurobiol 43(2):605\u0026ndash;619. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10571-022-01214-7\u003c/span\u003e\u003cspan address=\"10.1007/s10571-022-01214-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou S, Zhou J (2014) Neuritin, a neurotrophic factor in nervous system physiology. Curr Med Chem 21(10):1212\u0026ndash;1219. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2174/0929867321666131218093327\u003c/span\u003e\u003cspan address=\"10.2174/0929867321666131218093327\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhuang J, Lu J, Wang X, Wang X, Hu W, Hong F, Zhao XX, Zheng YL (2019) Purple sweet potato color protects against high-fat diet-induced cognitive deficits through AMPK-mediated autophagy in mouse hippocampus. J Nutr Biochem 65:35\u0026ndash;45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jnutbio.2018.10.015\u003c/span\u003e\u003cspan address=\"10.1016/j.jnutbio.2018.10.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStatements \u0026amp; Declarations\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Diabetes mellitus, cognitive impairment, neuritin, GABAergic neurons, ferroptosis","lastPublishedDoi":"10.21203/rs.3.rs-5965662/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5965662/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eAims\u003c/strong\u003e: Alterations in iron homeostasis are associated with several neurodegenerative diseases. Cognitive dysfunction has become an important concomitant symptom in people with type 2 diabetes mellitus. Therefore, we investigated the role of neuritin in ameliorating cognitive dysfunction resulting from ferroptosis in diabetic neurons using a model of neuritin overexpression in GABAergic.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: The constructed transgenic mice were used to observe memory function changes using the Morris water maze. The ferroptosis in GABAergic in hippocampus and AMPK/Nrf2 signaling pathway were detected by Western blot, transmission electron microscopy, and immunofluorescence. High glucose was used to induce ferroptosis in HT22 cells in vitro, and neuritin was further confirmed to reduce ferroptosis in HT22 cells through AMPK/Nrf2 signaling pathway by chemical assays and Western blot assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Neuritin overexpression in GABAergic of db/db mice significantly ameliorated cognitive dysfunction, mitochondrial dysfunction, reversed ferroptosis-associated symbolic changes and reduced ferroptosis in the hippocampus. And also increased the co-localisation coefficient of GAD65 and AMPK in the hippocampus. Neuritin activates the AMPK/Nrf2 signaling pathway to inhibit high glucose induced ferroptosis in HT22 cells. Neuritin was observed to regulate the AMPK/Nrf2 signaling pathway in HT22 cells and promote Nrf2 expression to inhibit HT22 cell ferroptosis and ameliorate diabetic cognitive dysfunction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: These findings suggest that neuritin may attenuate diabetes associated cognitive dysfunction by modulating neuronal ferroptosis, at least partly via AMPK/Nrf2 signaling pathway.\u003c/p\u003e","manuscriptTitle":"Neuritin suppresses GABAergic neurons ferroptosis to improve cognitive impairment in diabetes mellitus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-10 08:57:15","doi":"10.21203/rs.3.rs-5965662/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8c1e6f4d-1e99-4407-b206-2681234946e3","owner":[],"postedDate":"February 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-03T01:23:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-10 08:57:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5965662","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5965662","identity":"rs-5965662","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}