Mouse Hippocampal Neurons Research Articles

Non-motor behavioral disorders and the structure of hippocampal neurons in experimental parkinsonism and after administration of human umbilical cord-derived multipotent mesenchymal stromal cells and melatonin

Background. The neuroprotective effect of human umbilical cord-derived multipotent mesenchymal stromal cells (hUC-MMSCs) in Parkinson’s disease can depend on the genotype of the recipient and change under the influence of biologically active factors. The purpose was to investigate the effects of transplantation of the hUC-MMSCs as well as their combination with melatonin on indicators of non-motor activity and the structure of hippocampal neurons in mice with an experimental model of parkinsonism, which differ by the H-2 genotype (analogue of human leukocyte antigen). Materials and methods. Adult FVB/N (genotype H-2q) and 129/Sv (genotype H-2b) male mice have received one injection of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin at a dose of 30 mg/kg. Seven days after, the hUC-MMSCs were injected into the tail vein at a dose of 500,000, and from the next day — intraperitoneal melatonin (Sigma, USA) at a dose of 1 mg/kg daily, the course of 14 injections, at 6 p.m. We have evaluated the indicators of non-motor behavior in open field tests (emotional and orientation-exploratory activity), the development of the conditioned reaction of passive avoidance (cognitive function) and the structure of hippocampal neurons. Results. In mice of both strains under the influence of MPTP, the orientation-exploratory and cognitive activities have been suppressed, the emotional activity has been increased and the structure of neurons in the CA1 region and the dentate gyrus has been disturbed. Transplantation of hUC-MMSCs has improved the indicators of orientation-exploratory and cognitive functions in FVB/N mice and the emotional activity in 129/Sv mice. An activating effect of cells on some indicators of emotional behavior (the number of acts of grooming) in mice of both strains has been revealed. The number of pathologically changed neurons in the CA1 region and dentate gyrus in mice of both strains has decreased after transplantation of hUC-MMSCs. Injections of melatonin after the administration of cells have led to the strengthening of their positive effect on the cognitive function in FVB/N mice and on the emotional activity in 129/Sv mice and have also neutralized their negative effects on the number of acts of grooming in mice of both strains. In the hippocampus of such mice, there was a marked restoration of the cytoarchitectonics and morphometric indicators. At the same time, the positive effect of a combination of hUC-MMSCs and melatonin has been more pronounced in 129/Sv mice. Conclusions. Manifestations of the influence of transplanted hUC-MMSCs and their combination with melatonin on the functional state of the nervous system and the structure of hippocampal neurons of mice with a model of parkinsonism largely depend on their H-2 genotype. The results can be the basis for the development of personalized cell therapy for this pathology using hUC-MMSCs.

Neuroprotective effects of chitinase-1 and calcitonin gene-related peptide on Alzheimer's disease by promoting lysosomal function.

The amyloid cascade hypothesis still dominates in Alzheimer's disease (AD), and the acceleration of the clearance efficiency of amyloid-β (Aβ) has been always considered as an effective treatment option to slow the occurrence and progression of AD. This study aims to explore the role of zkscan3 and its related pathways in AD of the microglia-mediated pathogenesis, and whether the combined effect of drugs can exert neuroprotective function. N9 mouse microglia and HT-22 mouse hippocampal neurons were randomly divided into 6 groups, qRT-PCR technique was used to detect the gene expression level of zkscan3 and the genes related to lysosome generation and function. Fourteen C57 mice were randomly divided into two groups, and drug intervention model mice were randomly selected to establish from the AD group. Transmission electron microscope was used to detect the cell status and lysosome function in the hippocampus together with the other two groups. Compared with the AD model group, the gene expression of zkscan3 in the drug intervention group was downregulated, and the degree of neuronal injury in the hippocampus was reduced, the structure and number of synapses were improved, and the function of intracellular lysosome was enhanced. Zkscan3 and its related genes play a vital role in the development of AD. CGRP and CHIT-1, as a combined intervention, imparts effects through zkscan3-related pathways to improve lysosomal function and exert certain neuroprotective effects.

Bimolecular Fluorescence Complementation (BiFC) Technique for Exocytic Proteins in Murine Hippocampal Neurons.

The bimolecular fluorescence complementation (BiFC) technique is a powerful tool for visualizing protein-protein interactions in vivo. It involves genetically fused nonfluorescent fragments of green fluorescent protein (GFP) or its variants to the target proteins of interest. When these proteins interact, the GFP fragments come together, resulting in the reconstitution of a functional fluorescent protein complex that can be observed using fluorescence microscopy. In this chapter, we provide a detailed overview of the BiFC method and its application in studying protein-protein interactions in mouse hippocampal neurons. We discuss experimental procedures, including virus construct design, neuronal transduction, and imaging optimization. Additionally, we explore complementary assays for result validation and address potential challenges associated with BiFC experiments in the neuronal system. Overall, the BiFC offers researchers a valuable approach for investigating the spatial and temporal dynamics of protein interactions in living neuronal cells.

Chlorinated Phenolic, Benzenoid, and Phenylethanoid Glucosides From the Mangrove Endophytic Fungus Fuscoporia sp. A2A6.

A total of 12 new compounds, named fuscoposides A-L (1-12), including 2 phenolic, 9 benzenoid, and 1 phenylethanoid glucosides, were isolated from the mangrove endophytic fungus Fuscoporia sp. A2A6. The structures of these compounds were established by HRESIMS, NMR spectroscopic data, single-crystal x-ray diffraction analysis, and chemical methods. Most notably, 10 compounds, namely, fuscoposides A-I (1-9) and fuscoposide L (12), are mono- or di-chlorinated. Fuscoposide C (3) exhibited moderate neuroprotective effects against H2O2-induced oxidative damage in mouse hippocampal neuron HT-22 cells in a dose-dependent manner at the concentration range of 2.0-6.0µM, whereas fuscoposide G (7) decreased the expression of COX2 in lipopolysaccharide-stimulated mouse microglia BV2 cells at the concentration of 5.0µM, thereby displaying anti-inflammatory activity.

The deacetylase SIRT6 reduces amyloid pathology and supports cognition in mice by reducing the stability of APP in neurons.

Alzheimer's disease (AD) is an aging-related neurodegenerative disorder that results in progressively impaired memory and is often associated with amyloid plaques. Previous studies implicate the deacetylases SIRT1 and SIRT2 in regulating the processing of amyloid precursor protein (APP). Here, we investigated whether APP is regulated by the related deacetylase SIRT6, which shows aging-associated decreases in activity. We found that the abundance of SIRT6 was reduced in the cortex and hippocampus of aged and AD model mice and negatively correlated with that of APP. In mouse hippocampal neurons and transfected human cells, SIRT6 interacted with and deacetylated APP at three consecutive Lys residues (Lys649, Lys650, and Lys651). This deacetylation, in turn, increased the ubiquitylation of APP, leading to its proteasomal degradation. SIRT6 abundance in neurons was reduced by oxidative stress and DNA damage, both of which are implicated in neurodegenerative pathology. Systemic pharmacological activation of SIRT6 ameliorated both amyloid pathology and cognitive deficits in APP/PS1 mice, a mouse model of AD. The findings demonstrate that the activity of SIRT6 destabilizes APP and suggest that activating SIRT6 has therapeutic potential to reduce amyloid-associated pathology in patients with AD.

Attenuating Oxidative Damage with Macelignan in Glutamate-Induced HT22 Hippocampal Cells

Macelignan, from Myristica fragrans (nutmeg), is a bioactive compound with various pharmacological properties, including anti-inflammatory and neuroprotective activities. The purpose of this work was to investigate the antioxidant and anti-apoptotic effects of macelignan in glutamate-treated HT22 mouse hippocampal neurons. Macelignan was extracted and identified in a methanol extract of M. fragrans seeds. The DPPH was used to assess the antioxidative activity of macelignan. Glutamate (5 mM) was used to induce neurotoxicity in the HT22 cells. Neuroprotective effects were measured using relevant biochemical and imaging assays, including cell viability, ROS production, nuclear staining, apoptotic cell death, and protein expression. Macelignan markedly and concentration-dependently enhanced DPPH radical scavenging activity. In the HT22 cell model, glutamate induced cell damage by decreasing cell viability, promoting ROS generation, and increasing apoptotic cell death according to cell morphological changes. However, macelignan treatment restored cell viability, inhibited ROS generation concentration-dependently, and reduced apoptosis. Moreover, glutamate significantly up-regulated the phosphorylation of MAPK-pathway-related proteins, which was reversed by macelignan treatment. In conclusion, macelignan shows notable neuroprotective effects on oxidative stress and apoptotic cell death in glutamate-induced cells, and this study provides useful information on its potential therapeutic implications in neurological disorders.

To explore the protective mechanism of promethazine against hippocampal neuron injury based on network pharmacology and experimental verification.

This study aims to investigate the effect of promethazine (PMZ) on hippocampal neuronal injury through network pharmacology and in vivo experiments. Network pharmacology: The intersection genes of PMZ and Alzheimer Disease (AD) were obtained, and the core genes of PMZ in AD were screened. The intersection genes were enriched by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. In the in vitro experiment, mouse hippocampal neurons (HT22) were divided into control, glutamate (GLU) model, and GLU + PMZ treatment groups. The control group was given a complete culture medium, the model group was given GLU for 24 hours, the treatment group was given PMZ pretreatment for 3 hours, and then GLU was administered for 24 hours. Cell viability was determined, cell morphology was observed by microscopy, reactive oxygen species levels and glutathione content were detected, and protein expression of P53, PTGS2, SLC7A11, and GPX4 was detected by western blotting. Network pharmacology: A total of 317 PMZ targets, 1934 AD genes, 125 intersection genes, and 18 core genes, including P53 and PTGS2. Gene Ontology enrichment analysis showed that the effect of PMZ on AD was mainly related to cell proliferation, inflammation, hypoxia, synaptic structure, plasma membrane, and oxidoreductase activity. Kyoto Encyclopedia of Genes and Genomes results showed neuroactive ligand-receptor interaction, cell senescence, cancer pathway, PI3K-AKT signal pathway, neurodegeneration, and HIF-1 signal pathway. In vitro experiments: PMZ improved the GLU-induced decrease in cell viability and morphological changes in hippocampal neurons. PMZ inhibited reactive oxygen species levels and increased glutathione content in injured hippocampal neurons. Up-regulated of P53, SLC7A11 and GPX4 expression, and inhibited expression of PTGS2. PMZ regulates the SLC7A11-GPX4 antioxidant system to protect hippocampal neurons from oxidative stress injury.

TRPC6 is a mechanosensitive channel essential for ultrasound neuromodulation in the mammalian brain

Ultrasound neuromodulation has become an innovative technology that enables noninvasive intervention in mammalian brain circuits with high spatiotemporal precision. Despite the expanding utility of ultrasound neuromodulation in the neuroscience research field and clinical applications, the molecular and cellular mechanisms by which ultrasound impacts neural activity in the brain are still largely unknown. Here, we report that transient receptor potential canonical 6 (TRPC6), a mechanosensitive nonselective cation channel, is essential for ultrasound neuromodulation of mammalian neurons in vitro and in vivo. We first demonstrated that ultrasound irradiation elicited rapid and robust Ca2+ transients mediated via extracellular Ca2+ influx in cultured mouse cortical and hippocampal neurons. Ultrasound-induced neuronal responses were massively diminished by blocking either the generation of action potential or synaptic transmission. Importantly, both pharmacological inhibition and genetic deficiency of TRPC6 almost completely abolished neuronal responses to ultrasound. Furthermore, we found that intracerebroventricular administration of a TRPC6 blocker significantly attenuated the number of neuronal firings in the cerebral cortex evoked by transcranial ultrasound irradiation in mice. Our findings indicate that TRPC6 is an indispensable molecule of ultrasound neuromodulation in intact mammalian brains, providing fundamental understanding of biophysical molecular mechanisms of ultrasound neuromodulation as well as insight into its future feasibility in neuroscience and translational research in humans.

Impact of Chronic Intermittent Hypoxia on Cognitive Function and Hippocampal Neurons in Mice: A Study of Inflammatory and Oxidative Stress Pathways.

Chronic intermittent hypoxia (CIH) is considered one of the main pathophysiological mechanisms of obstructive sleep apnea (OSA). CIH can further lead to cognitive dysfunction by inducing processes such as neuroinflammation and oxidative stress. The hippocampus is primarily associated with cognitive functions such as learning and memory. This study aimed to explore the effects of CIH on cognitive function and hippocampal neurons in mice and to reveal its potential molecular mechanisms. SPF-grade C57BL/6J mice (n=36) were selected as subjects and divided into control, mild CIH, and severe CIH groups (12 mice per group). Cognitive function was assessed using the Morris water maze test, and hippocampal neuron numbers and morphological changes were observed using HE staining and Nissl staining. Additionally, differential genes and pathways were revealed through RNA sequencing (RNA-seq) and bioinformatics analysis. We examined oxidative stress-related biochemical markers in the hippocampal tissue and used Western Blot to verify changes in the expression of potential key genes. Statistical analyses were performed using ANOVA and post hoc tests to ensure robust comparisons between groups. CIH mice exhibited significant cognitive impairment, including decreased learning and memory abilities. The severe CIH group had a longer escape latency compared to the mild CIH group (p < 0.001) and the control group (p < 0.01), while the mild CIH group took longer than the control group (p < 0.01). In the probe test, the severe CIH group showed a significant decrease in platform crossings (p < 0.01) and target quadrant dwell time (p < 0.05), while the mild CIH group exhibited a reduction in target quadrant dwell time (p < 0.05). Abnormal hippocampal neuron morphology was observed, with a significant reduction in hippocampal neurons (p < 0.05). RNA-seq analysis revealed numerous differentially expressed genes, mainly enriched in biological processes such as inflammation and oxidative stress, as well as multiple signaling pathways. Specifically, downregulated LepR, SIRT1, and Nrf2 genes were found to exacerbate oxidative stress and neuroinflammation, impairing neuronal integrity and cognitive function. Further validation showed increased oxidative stress levels in hippocampal tissue and downregulation of key gene expression. Western blot analysis confirmed significantly reduced expression of LepR (p < 0.01), SIRT1 (p < 0.001), and Nrf2 (p < 0.001) in the severe CIH group. While oxidative stress and inflammation are well-established mechanisms in CIH-induced cognitive impairment, our study provides novel insights by identifying the specific roles of LepR, SIRT1, and Nrf2 in this process. The downregulation of these key genes suggests potential new targets for therapeutic intervention. Importantly, the differential expression patterns observed in varying degrees of hypoxia severity highlight the potential for tailored therapeutic strategies that modulate these pathways in response to the intensity of hypoxic exposure. These findings offer unique opportunities for developing targeted therapies aimed at mitigating CIH-related cognitive decline and neural damage. However, a key limitation of this study is the exclusive use of animal models, which may not fully replicate human pathophysiology. Further studies are needed to validate these findings in clinical settings and to explore the regulatory relationships between the key genes involved.

Latent learning drives sleep-dependent plasticity in distinct CA1 subpopulations

Latent learning is a process that enables the brain to transform experiences into "cognitive maps," a form of implicit memory, without requiring reinforced training. To investigate its neural mechanisms, we record from hippocampal neurons in mice during latent learning of spatial maps and observe that the high-dimensional neural state space gradually transforms into a low-dimensional manifold that closely resembles the physical environment. This transformation process is associated with the neural reactivation of navigational experiences during sleep. Additionally, we identify a subset of hippocampal neurons that, rather than forming place fields in a novel environment, maintain weak spatial tuning but gradually develop correlated activity with other neurons. The elevated correlation introduces redundancy into the ensemble code, transforming the neural state space into a low-dimensional manifold that effectively links discrete place fields of place cells into a map-like structure. These results suggest a potential mechanism for latent learning of spatial maps in the hippocampus.

Novel off-context experience constrains hippocampal representational drift.

The hippocampus forms unique neural representations for distinct experiences, supporting the formation of different memories.1,2,3,4,5,6 Hippocampal representations gradually change over time as animals repeatedly visit the same familiar environment ("representational drift").7,8,9,10,11,12 Such drift has also been observed in other brain areas, such as the parietal,13,14 visual,15,16,17 auditory,18,19 and olfactory20 cortices. While the underlying mechanisms of representational drift remain unclear, a leading hypothesis suggests that it results from ongoing learning processes.20,21,22 According to this hypothesis, because the brain uses the same neural substrates to support multiple distinct representations, learning of novel stimuli or environments leads to changes in the neuronal representation of a familiar one. If this is true, we would expect drift in a given environment to increase following new experiences in other, unrelated environments (i.e., off-context experiences). To test this hypothesis, we longitudinally recorded large populations of hippocampal neurons in mice while they repeatedly visited a familiar linear track over weeks. We introduced off-context experiences by placing mice in a novel environment for 1h after each visit to the familiar track. Contrary to our expectations, these novel episodes decreased place cells' representational drift. Our findings are consistent with a model in whichrepresentations of distinct memories occupy different areas within the neuronal activity space,and the drift of each of them within that space is constrained by the area occupied by the others.

Generate and Analyze Three-Dimensional Dendritic Spine Morphology Datasets With SpineTool Software.

Dendritic spine morphology is associated with the current state of the synapse and neuron, and changes during synaptic plasticity in response to stimulus. At the same time, dendritic spine alterations are reported during various neurodegenerative and neurodevelopmental disorders and other brain states. Accurate and informative analysis of spine shape has an urgent need for studying the synaptic processes and molecular pathways in normal and pathological conditions, and for testing synapto-protective strategies during preclinical studies. Primary neuronal cultures enable high quality imaging of dendritic spines and offer a wide spectrum of accessible experimental manipulations. This article outlines the protocol for isolating, culturing, fluorescent labeling, and imaging of mouse primary hippocampal neurons by three-dimensional (3D) confocal microscopy in a normal state and in conditions of low amyloid toxicity-an in vitro model of Alzheimer's disease. An alternate protocol describes the neuronal morphology analysis using the EGFP expressing neurons in line-M transgenic mouse brain slices. Since the dendritic spines are relatively small structures lying close to the confocal microscope resolution limit, their proper segmentation on the images is challenging. This protocol highlights the image-preprocessing steps, including generation of theoretical point spread function and deconvolution, which enhances resolution and removes noise, thereby enhancing the 3D spine reconstruction results. SpineTool, an open source Python-based script, enables 3D segmentation of dendrites and spines and numerical metric calculation, including key measures, such as spine length, volume, and surface area, with a new feature, the chord length distribution histogram, improving clustering results. SpineTool supports both manual and machine learning spine classification (i.e., mushroom, thin, stubby, filopodia) and automated clustering using k-means and DBSCAN methods. This protocol provides detailed instructions for using SpineTool to analyze and classify dendritic spines in control and experimental groups, enhancing our understanding of spine morphology across different experimental conditions. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Obtaining 3D confocal dendritic spine images of hippocampal neuronal culture in normal state and conditions of low amyloid toxicity Alternate Protocol: Obtaining confocal dendritic spine images of mice hippocampal neurons from fixed brain slices Support Protocol: Post-processing deconvolution of confocal images Basic Protocol 2: Segmentation of dendritic spines with SpineTool Basic Protocol 3: Spine dataset preparation using SpineTool Basic Protocol 4: Clustering of dendritic spines with SpineTool Basic Protocol 5: Machine classification of dendritic spines with SpineTool.

Neuronal protective effect of Artemisinin in ischemic stroke: Achieved by blocking lysine demethylase 1A-mediated demethylation of sphingosine kinase 2.

Artemisinin (ART), a natural product isolated from the traditional Chinese plant Artemisia annua L., has shown neuroprotective properties in addition to its well-established antimalarial activities. This study investigates the therapeutic effect of ART in ischemic stroke (IS) and delves into its functional mechanism. Bioinformatics analyses revealed lysine demethylase 1A (KDM1A) as a promising target of ART aberrantly overexpressed in the context of IS. Increased KDM1A expression was detected in oxygen-glucose deprivation/reoxygenation (OGD/R)-treated hippocampal neurons and transient middle cerebral artery occlusion (tMCAO)-challenged mice. Treatment with ART reduced KDM1A protein level, thus protecting mouse hippocampal neurons from OGD/R-induced oxidative stress and apoptosis. In vivo, ART reduced infarct size, reduced brain content, enhanced neurological function, and enhanced neuronal survival in tMCAO. Regarding the downstream cascade, KDM1A was found to repress transcription of sphingosine kinase 2 (SPHK2) by removing H3K4me2 modification near the SPHK2 promoter. Either KDM1A overexpression or SPHK2 knockdown abrogated the neuroprotective effects of ART. The ample evidence of this study suggests that ART fulfills neuroprotective functions in the context of IS by protecting SPHK2 from KDM1A-mediated transcription repression, highlighting ART as a promising regimen for the treatment of IS.

The role of FAM171A2-GRN-NF-κB pathway in TBBPA induced oxidative stress and inflammatory response in mouse-derived hippocampal neuronal HT22 cells

Tetrabromobisphenol A (TBBPA) is one of the brominated flame retardants (BFRs) widely used in industry, which has a broad toxic effect on organisms. However, there is still limited research on the neurotoxic mechanism of TBBPA. Using mouse hippocampal neurons (HT22) cells, the toxicity of TBBPA was evaluated, especially focusing on its alteration on the key molecules in FAM171A2-GRN-NF-κB signaling pathway. The results showed that TBBPA exposure could lead to an increase in the production of inflammation-related genes IL-6, iNOS, TGF-β1, COX2, and TNF-α in both HT22 cells and HT22-AD-model, intensifying the inflammatory response; it inhibits the mRNA expression of antioxidative enzymes genes Sod1, Cat, Gpx1, and Gsta1, resulting in reduced antioxidant enzyme activities of SOD, CAT, and GSH-Px/GPX. Mechanistically, TBBPA caused the upregulation of FAM171A2 expression level, alongside increased GRN, IκBα and p65 levels; whereas the expression of GRN, IκBα and p65 was decreased after FAM171A2 knockdown, demonstrating TBBPA-induced upregulation of FAM171A2 should be responsible for the increased GRN, IκBα and p65 expression. Therefore, for the first time, our data indicate that TBBPA-induced oxidative stress and inflammatory response is closely related to the FAM171A2-GRN-NF-κB pathway.

Biochanin A inhibits excitotoxicity-triggered ferroptosis in hippocampal neurons

Excitatory neurotransmitter-induced neuronal ferroptosis has been implicated in multiple neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Although there are several reports pertaining to the pharmacological activities of biochanin A, the effects of this isoflavone on excitotoxicity-triggered neuronal ferroptosis remain unclear. In this study, we demonstrate that biochanin A inhibits ferroptosis of mouse hippocampal neurons induced by glutamate or the glutamate analog, kainic acid. Biochanin A significantly inhibited accumulation of intracellular iron and lipid peroxidation in glutamate- or kainic acid-treated mouse hippocampal neurons. Furthermore, biochanin A regulated the level of glutathione peroxidase 4, a master regulator of ferroptosis, by modulating its autophagy-dependent degradation. We observed that biochanin A reduced the glutamate-induced accumulation of intracellular iron by regulating expression of iron metabolism-related proteins including ferroportin-1, divalent metal transferase 1, and transferrin receptor 1. Taken together, these results indicate that biochanin A effectively inhibits hippocampal neuronal death triggered by glutamate or kainic acid. Our study is the first to report that biochanin A has therapeutic potential for the treatment of diseases associated with hippocampal neuronal death, particularly ferroptosis induced by excitatory neurotransmitter.

Prolactin protects hippocampal neurons against H2O2-induced neurotoxicity by suppressing BAX and NOX4 via the NF-κB signaling pathway.

Reactive oxygen species (ROS) are physiological byproducts of neuronal metabolism. However, an imbalance between ROS generation and antioxidant capacity, often driven by dysregulated pro-oxidant enzymes like nicotinamide adenine dinucleotide phosphate oxidases (NOX), can result in deleterious oxidative stress. This oxidative stress is a critical factor in the pathogenesis of neurodegenerative diseases. While interventions with broad-spectrum antioxidants have demonstrated limited efficacy, the modulation of endogenous antioxidant mechanisms presents a promising therapeutic avenue. Here, we investigated the potential of the neuroprotective hormone prolactin to mitigate oxidative stress and subsequent neuronal cell death. Prolactin protected primary mouse hippocampal neurons from hydrogen peroxide (H2O2)-induced oxidative damage. Prolactin reduced ROS levels, lipid peroxidation, and apoptosis, and its effects were occluded by a specific prolactin receptor antagonist (G129R-hPRL). Mechanistically, prolactin suppressed H2O2-induced mRNA upregulation of pro-oxidative Nox4 and pro-apoptotic Bax. Moreover, prolactin induced nuclear factor kappa B (NF-κB) nuclear translocation, and the inhibition of the NF-κB signaling pathway abolished the neuroprotective and transcriptional effects of prolactin, indicating its central role in prolactin-mediated protection. Our findings indicate that prolactin exerts potent antioxidant and neuroprotective effects by modulating the expression of Nox4 and Bax, thereby reducing ROS generation and neuronal apoptosis. This study underscores the therapeutic potential of prolactin in attenuating oxidative stress and suggests a possible role in the treatment of neurodegenerative diseases.

Microglia depletion reduces human neuronal APOE4-related pathologies in a chimeric Alzheimer's disease model.

Despite strong evidence supporting the important roles of both apolipoprotein E4 (APOE4) and microglia in Alzheimer's disease (AD) pathogenesis, the effects of microglia on neuronal APOE4-related AD pathogenesis remain elusive. To examine such effects, we utilized microglial depletion in a chimeric model with induced pluripotent stem cell (iPSC)-derived human neurons in mouse hippocampus. Specifically, we transplanted homozygous APOE4, isogenic APOE3, and APOE-knockout (APOE-KO) iPSC-derived human neurons into the hippocampus of human APOE3 or APOE4 knockin mice and then depleted microglia in half of the chimeric mice. We found that both neuronal APOE and microglial presence were important for the formation of Aβ and tau pathologies in an APOE isoform-dependent manner (APOE4>APOE3). Single-cell RNA sequencing analysis identified two pro-inflammatory microglial subtypes with elevated MHC-II gene expression enriched in chimeric mice with human APOE4 neuron transplants. These findings highlight the concerted roles of neuronal APOE, especially APOE4, and microglia in AD pathogenesis.

Taxifolin ameliorates the D-galactose-induced aging of mouse hippocampal neurons HT-22 cells through modulating SIRT1/p53 and PI3K/AKT signaling pathways

By establishing an in vitro model of D-Gal-induced brain neuronal cell (HT-22) senescence, it was found that TAX treatment significantly increased the activities of SOD and GSH, while decreasing MDA levels in aging HT-22 cells, indicating that TAX effectively restored the total antioxidant capacity and antioxidant enzyme activity of aging HT-22 cells induced by D-Gal, and attenuated cellular oxidative stress injury. In addition, taxifolin could also protect HT-22 cells from aging by up-regulating SIRT1 while reducing the expression of Ac-p53, indicating that TAX may be an active substance that can effectively delay cell aging.

The deubiquitinase OTUD3 stabilizes IRP2 expression to reduce hippocampal neuron ferroptosis via the p53/PTGS2 pathway to ameliorate cerebral ischemia–reperfusion injury

BackgroundIschemic stroke (IS) is known for its high morbidity, disability and mortality rates, and studies designed to explore its pathophysiological mechanisms and identify novel therapeutic strategies are urgently needed. We aimed to probe the effects of the deubiquitinase OTUD3-IRP2-p53/PTGS2 pathway on cerebral ischemia‒reperfusion (I/R) injury and hippocampal neuron ferroptosis.MethodsA cerebral I/R mouse model was established. Furthermore, lentiviral vectors that overexpressed OTUD3 and knocked down IRP2 were constructed, and a series of assays were performed to probe the OTUD3/IRP2/p53/PTGS2 mechanism. An oxygen‒glucose deprivation and reoxygenation (OGD/R) model of mouse hippocampal neurons was constructed. Then, OTUD3 and IRP2 were knocked down and overexpressed, and p53 was overexpressed to explore the mechanism of the OTUD3/IRP2/p53/PTGS2 pathway.ResultsOTUD3 and IRP2 were expressed at low levels in cerebral I/R models. OTUD3 promoted IRP2 expression to protect damaged hippocampal neurons. Moreover, IRP2 affected ferroptosis in hippocampal neurons. In addition, IRP2 inhibited p53. After IRP2 and p53 were overexpressed, IRP2 regulated the p53/PTGS2 pathway and affected ferroptosis in hippocampal neurons. In vivo, after overexpressing OTUD3 and knocking down IRP2, we found that overexpression of OTUD3 promoted IRP2 expression to reduce ferroptosis in hippocampal neurons and improve cerebral I/R injury via the inhibition of the p53/PTGS2 pathway.ConclusionsThe deubiquitinase OTUD3 stabilized IRP2 expression to reduce hippocampal neuron ferroptosis via the p53/PTGS2 pathway to subsequently ameliorate cerebral I/R injury.

Rabphilin-3A negatively regulates neuropeptide release, through its SNAP25 interaction

Neuropeptides and neurotrophins are stored in and released from dense core vesicles (DCVs). While DCVs and synaptic vesicles (SVs) share fundamental SNARE/SM proteins for exocytosis, a detailed understanding of DCV exocytosis remains elusive. We recently identified the RAB3-RIM1 pathway to be essential for DCV, but not SV exocytosis, highlighting a significant distinction between the SV and DCV secretory pathways. Whether RIM1 is the only RAB3 effector that is essential for DCV exocytosis is currently unknown. In this study, we show that rabphilin-3A (RPH3A), a known downstream effector of RAB3A, is a negative regulator of DCV exocytosis. Using live-cell imaging at single-vesicle resolution with RPH3A deficient hippocampal mouse neurons, we show that DCV exocytosis increased threefold in the absence of RPH3A. RAB3A-binding deficient RPH3A lost its punctate distribution, but still restored DCV exocytosis to WT levels when re-expressed. SNAP25-binding deficient RPH3A did not rescue DCV exocytosis. In addition, we show that RPH3A did not travel with DCVs, but remained stationary at presynapses. RPH3A null neurons also had longer neurites, which was partly restored when ablating all regulated secretion with tetanus neurotoxin. Taken together, these results show that RPH3A negatively regulates DCV exocytosis, potentially also affecting neuron size. Furthermore, RAB3A interaction is required for the synaptic enrichment of RPH3A, but not for limiting DCV exocytosis. Instead, the interaction of RPH3A with SNAP25 is relevant for inhibiting DCV exocytosis.