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Background/Objectives: Recent studies, typically using patient cerebrospinal fluid (CSF), have suggested that different autoantibodies (Aabs) acting on their respective receptors, may underlie neuropsychiatric disorders. The GluN1 (NR1) subunit of the N-methyl-D-aspartate receptor (NMDAR) has been identified as a target of anti-NMDAR Aabs in a number of central nervous system (CNS) diseases, including encephalitis and autoimmune epilepsy. However, the role or the nature of Aabs responsible for effects on neuronal excitability and synaptic plasticity is yet to be established fully. Methods: Peptide immunisation was used to generate Aabs against selected specific GluN1 extracellular sequences based on patient-derived anti-NMDAR Aabs that have been shown to bind to specific regions within the GluN1 subunit. 'Protein A' purification was used to obtain the total IgG, and further peptide purification was used to obtain a greater percentage of NMDAR-target specific IgG Aabs. The binding and specificity of these anti-NMDAR Aabs were determined using a range of methodologies including enzyme-linked immunosorbent assays, immunocytochemistry and immunoblotting. Functional effects were determined using different in vitro electrophysiology techniques: two-electrode voltage-clamps in Xenopus oocytes and measures of long-term potentiation (LTP) in ex vivo hippocampal brain slices using multi-electrode arrays (MEAs). Results: We show that anti-NMDAR Aabs generated from peptide immunisation had specificity for GluN1 immunisation peptides as well as target-specific binding to the native protein. Anti-NMDAR Aabs had no clear effect on isolated NMDARs in an oocyte expression system. However, peptide-purified anti-NMDAR Aabs prevented the induction of LTP at Schaffer collateral-CA1 synapses in ex vivo brain slices, consistent with causing synaptic NMDAR hypofunction at a network level. Conclusions: This work provides a solid basis to address outstanding questions regarding anti-NMDAR Aab mechanisms of action and, potentially, the development of therapies against CNS diseases. [ABSTRACT FROM AUTHOR]

Hippocampal neurons can be the first to be impaired with neurodegenerative disorders, including Alzheimer's disease (AD). Most drug candidates for causal therapy of AD cannot either enter the brain or accumulate around hippocampal neurons. Here, we genetically engineered insulin-fusion proteins, called hippocampal neuron-targeting (Ht) proteins, for targeting protein drugs to hippocampal neurons because insulin tends to accumulate in the neuronal cell layers of the hippocampus. In vitro examinations clarified that insulin and Ht proteins were internalized into the cultured hippocampal neurons through insulin receptor-mediated macropinocytosis. Cysteines were key determinants of the delivery of Ht proteins to hippocampal neurons, and insulin B chain mutant was most potent in delivering cargo proteins. In vivo accumulation of Ht proteins to hippocampal neuronal layers occurred after intracerebroventricular administration. Thus, hippocampal neuron-targeting technology can provide great help for developing protein drugs against neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum-associated degradation (ERAD) is a principal qualitycontrol mechanism responsible for targeting misfolded ER proteins for cytosolic degradation. Evidence suggests that impairment of ERAD contributes to neuron dysfunction and death in neurodegenerative diseases, many of which are characterized by accumulation and aggregation of misfolded proteins. However, the physiological role of ERAD in neurons remains unclear. The Sel1LHrd1 complex consisting of the E3 ubiquitin ligase Hrd1 and its adaptor protein Sel1L is the best-characterized ERAD machinery. Herein, we showed that Sel1L deficiency specifically in neurons of adult mice impaired the ERAD activity of the Sel1L-Hrd1 complex and led to disruption of ER homeostasis, ER stress and activation of the unfold protein response (UPR). Adult mice with Sel1L deficiency in neurons exhibited weight loss and severe motor dysfunction, and rapidly succumbed to death. Interestingly, Sel1L deficiency in neurons caused global brain atrophy, particularly cerebellar and hippocampal atrophy, in adult mice. Moreover, we found that cerebellar and hippocampal atrophy in these mice resulted from degeneration of Purkinje neurons and hippocampal neurons, respectively. These findings indicate that ERAD is required for maintaining ER homeostasis and the viability and function of neurons in adults under physiological conditions. [ABSTRACT FROM AUTHOR]

Sorting nexin 4 (SNX4) is an evolutionary conserved organizer of membrane recycling. In neurons, SNX4 accumulates in synapses, but how SNX4 affects synapse function remains unknown. We generated a conditional SNX4 knock-out mouse model and report that SNX4 cKO synapses show enhanced neurotransmission during train stimulation, while the first evoked EPSC was normal. SNX4 depletion did not affect vesicle recycling, basic autophagic flux, or the levels and localization of SNARE-protein VAMP2/synaptobrevin-2. However, SNX4 depletion affected synapse ultrastructure: an increase in docked synaptic vesicles at the active zone, while the overall vesicle number was normal, and a decreased active zone length. These effects together lead to a substantially increased density of docked vesicles per release site. In conclusion, SNX4 is a negative regulator of synaptic vesicle docking and release. These findings suggest a role for SNX4 in synaptic vesicle recruitment at the active zone.

ObjectiveTo investigate the effect of Crocin (CRO) on rats with hippocampal neuron cerebral ischemia reperfusion (CI/R) injury and explore its potential mechanism.MethodsA total of 144 male SD rats were selected and randomly divided into sham group, model (CI/R) group, CRO low-, medium-, high-dose (CRO-L, CRO-M, CRO-H) group and Nimodipine (NMP) group, with 24 rats in each group. The CI/R rat models were established by suture method. Rats in each group were administered by intrabitoneal injection (ip) once a day starting 7 days before modeling (the CRO-L, CRO-M, CRO-H groups were given 10, 20, 40 mg/kg CRO by ip respectively; the NMP group was given 1 mg/kg NMP by ip; and the sham group and CI/R group were given normal saline 5 mL/kg by ip). After 24 hours of reperfusion, the learning and memory ability of rats was detected by Morris water maze test. The cerebral infarction rate was detected using TTC staining. The neuron pathological changes of hippocampal CA1 and CA3 were observed using HE staining, and the neuronal apoptosis of hippocampal CA1 and CA3 was examined by TUNEL staining. The levels of interleukin (IL)-1β, IL-8, tumor necrosis factor-α (TNF-α) in hippocampal tissue were detected by ELISA. The expression of Janus kinase 2/signal transduction and activator of transcription 3 (JAK2/STAT3) signaling pathway related proteins in hippocampal tissue were detected by Western blot.ResultsCompared with the sham group, the learning and memory ability of the rats in the CI/R group was significantly decreased, and the cerebral infarction rate was significantly increased (PPPPPPPPP

Convulsive status epilepticus (CSE) is a common critical neurological condition that can lead to irreversible hippocampal neuron damage and cognitive dysfunction. Multiple studies have demonstrated the critical roles that long non-coding RNA Mir155hg plays in a variety of diseases. However, less is known about the function and mechanism of Mir155hg in CSE. Here we investigate and elucidate the mechanism underlying the contribution of Mir155hg to CSE-induced hippocampal neuron injury. By applying high-throughput sequencing, we examined the expression of differentially expressed genes in normal and CSE rats. Subsequent RT-qPCR enabled us to measure the level of Mir155hg in rat hippocampal tissue. Targeted knockdown of Mir155hg was achieved by the AAV9 virus. Additionally, we utilized HE and Tunel staining to evaluate neuronal injury. Immunofluorescence (IF), Golgi staining, and brain path clamping were also used to detect the synaptic plasticity of hippocampal neurons. Finally, through IF staining and Sholl analysis, we assessed the degree of microglial phagocytic function. It was found that the expression of Mir155hg was elevated in CSE rats. HE and Tunel staining results showed that Mir155hg knockdown suppressed the hippocampal neuron loss and apoptosis followed CSE. IF, Golgi staining and brain path clamp data found that Mir155hg knockdown enhanced neuronal synaptic plasticity. The results from IF staining and Sholl analysis showed that Mir155hg knockdown enhanced microglial phagocytosis. Our findings suggest that Mir155hg promotes CSE-induced hippocampal neuron injury by inhibiting microglial phagocytosis. [ABSTRACT FROM AUTHOR]

As a noninvasive technique, ultrasound stimulation is known to modulate neuronal activity both in vitro and in vivo. The latest explanation of this phenomenon is that the acoustic wave can activate the ion channels and further impact the electrophysiological properties of targeted neurons. However, the underlying mechanism of low-intensity pulsed ultrasound (LIPUS)-induced neuro-modulation effects is still unclear. Here, we characterize the excitatory effects of LIPUS on spontaneous activity and the intracellular Ca2+ homeostasis in cultured hippocampal neurons. By whole-cell patch clamp recording, we found that 15 min of 1-MHz LIPUS boosts the frequency of both spontaneous action potentials and spontaneous excitatory synaptic currents (sEPSCs) and also increases the amplitude of sEPSCs in hippocampal neurons. This phenomenon lasts for > 10 min after LIPUS exposure. Together with Ca2+ imaging, we clarified that LIPUS increases the [Ca2+]cyto level by facilitating L-type Ca2+ channels (LTCCs). In addition, due to the [Ca2+]cyto elevation by LIPUS exposure, the Ca2+-dependent CaMKII-CREB pathway can be activated within 30 min to further regulate the gene transcription and protein expression. Our work suggests that LIPUS regulates neuronal activity in a Ca2+-dependent manner via LTCCs. This may also explain the multi-activation effects of LIPUS beyond neurons. LIPUS stimulation potentiates spontaneous neuronal activity by increasing Ca2+ influx. [ABSTRACT FROM AUTHOR]

Although radiotherapy is the most effective treatment modality for brain tumors, it always injures the central nervous system, leading to potential sequelae such as cognitive dysfunction. Radiation induces molecular, cellular, and functional changes in neuronal and glial cells. The hippocampus plays a critical role in learning and memory; therefore, concerns about radiation‐induced injury are widespread. Multiple studies have focused on this complex problem, but the results have not been fully elucidated. Naked mole rat brains were irradiated with 60Co at a dose of 10 Gy. On 7 days, 14 days, and 28 days after irradiation, hippocampi in the control groups were obtained for next‐generation sequencing. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were subsequently performed. Venn diagrams revealed 580 differentially expressed genes (DEGs) that were common at different times after irradiation. GO and KEGG analyses revealed that the 580 common DEGs were enriched in molecular transducer activity. In particular, CACNA1B mediated regulatory effects after irradiation. CACNA1B expression increased significantly after irradiation. Downregulation of CACNA1B led to a reduction in apoptosis and reactive oxygen species levels in hippocampal neurons. This was due to the interaction between CACNA1B and Nrf2, which disturbed the normal nuclear localization of Nrf2. In addition, CACNA1B downregulation led to a decrease in the cognitive functions of naked mole rats. These findings reveal the pivotal role of CACNA1B in regulating radiation‐induced brain injury and will lead to the development of a novel strategy to prevent brain injury after irradiation. [ABSTRACT FROM AUTHOR]

Arsenic, a neurotoxic metalloid, poses significant health risks. However, ellagic acid, renowned for its antioxidant properties, has shown potential in neuroprotection. This study aimed to investigate the neuroprotective effects of ellagic acid against arsenic-induced neuronal ferroptosis and cognitive impairment and elucidate the underlying mechanisms. Using an arsenic-exposed Wistar rat model and an arsenic-induced HT22 cells model, we assessed cognitive ability, measured serum and brain arsenic levels, and evaluated pathological damage through histological analysis and transmission electron microscopy. Additionally, we examined oxidative stress and iron ion levels using GSH, MDA, ROS and tissue iron biochemical kits, and analyzed the expression of ferroptosis-related markers using western blot and qRT-PCR. Our results revealed that arsenic exposure increased both serum and brain arsenic levels, resulting in hippocampal pathological damage and subsequent decline in learning and memory abilities. Arsenic-induced neuronal ferroptosis was mediated by the inhibition of the xCT/GSH/GPX4/Nrf2 signaling axis and disruption of iron metabolism. Notably, ellagic acid intervention effectively reduced serum and brain arsenic levels, ameliorated neuronal damage, and improved oxidative stress, ferroptosis, and cognitive impairment. These beneficial effects were associated with the activation of the Nrf2/Keap1 signaling pathway, upregulation of GPX4 expression, and enhanced iron ion excretion. In conclusion, ellagic acid demonstrates promising neuroprotective effects against arsenic-induced neurotoxicity by mitigating neuronal ferroptosis and cognitive impairment.

Sevoflurane is one of the most widely used inhaled anesthetics. MicroRNAs (miRNAs) have been demonstrated to affect sevoflurane anesthesia-induced neuron damage. The purpose of this study was to investigate the role and mechanism of miR-181a-5p in sevoflurane-induced hippocampal neuronal injury. Primary hippocampal neurons were identified using microscopy and immunofluorescence. The viability and apoptosis of sevoflurane anesthesia-induced neurons were detected by cell counting kit-8 (CCK-8) assay and terminal-deoxynucleoitidyl transferase-mediated nick end-labeling (TUNEL) staining assay, respectively. The levels of apoptosis- and oxidative stress-related proteins as well as the markers in the Wnt/β-catenin signaling pathway were examined by immunoblotting. Enzyme-linked immuno-sorbent assays were performed to examine the levels of inflammatory cytokines. Luciferase reporter assay was conducted to validate the combination between miR-181a-5p and DEAD-box helicase 3, X-linked (DDX3X). Sevoflurane exposure led to significantly inhibited hippocampal neuron viability and elevated miR-181a-5p expression. Knockdown of miR-181a-5p alleviated sevoflurane-induced neuron injury by reducing cell apoptosis, inflammatory response, and oxidative stress. Additionally, DDX3X was targeted and negatively regulated by miR-181a-5p. Moreover, miR-181a-5p inhibitor activated the Wnt/β-catenin pathway via DDX3X in sevoflurane-treated cells. Rescue experiments revealed that DDX3X knockdown or overexpression of Wnt antagonist Dickkopf-1 (DKK1) reversed the suppressive effects of miR-181a-5p inhibitor on cell apoptosis, inflammatory response, and oxidative stress in sevoflurane-treated neuronal cells. MiR-181a-5p ameliorated sevoflurane-triggered neuron injury by regulating the DDX3X/Wnt/β-catenin axis, suggesting the potential of miR-181a-5p as a novel and promising therapeutic target for the treatment of sevoflurane-evoked neurotoxicity. [ABSTRACT FROM AUTHOR]

N6-methyladenosine (m6A) constitutes the paramount post-transcriptional modification within eukaryotic mRNA. This modification is subjected to stimulus-dependent regulation within the central nervous system of mammals, being influenced by sensory experiences, learning processes, and injuries. The patterns of m6A methylation within the hippocampal region of diabetes cognitive impairment (DCI) has not been investigated. A DCI model was established by feeding a high-fat diet to C57BL/6J mice. m6A and RNA sequencing was conducted to profile the m6A-tagged transcripts in the hippocampus. Methylated RNA immunoprecipitation with next-generation sequencing and RNA sequencing analyses yielded differentially m6A-modified and expressed genes in the hippocampus of DCI mice, which were enriched in pathways involving synaptic transmission and axonal guidance. Mechanistic analyses revealed a remarkable change in m6A modification levels through alteration of the mRNA expression of m6A methyltransferases (METTL3 and METTL14) and demethylase (FTO) in the hippocampus of DCI mice. We identified a co-mediated specific RNA regulatory strategy that broadens the epigenetic regulatory mechanism of RNA-induced neurodegenerative disorders associated with metabolic and endocrine diseases. [ABSTRACT FROM AUTHOR]

Ferroptosis is a distinct peroxidation-driven form of cell death tightly involved in subarachnoid hemorrhage (SAH). This study delved into the mechanism of deferoxamine (DFO, an iron chelator) in SAH-induced ferroptosis and inflammation. SAH mouse models were established by endovascular perforation method and injected intraperitoneally with DFO, or intraventricularly injected with the Nrf2 pathway inhibitor ML385 before SAH, followed by detection of neurological function, blood–brain barrier (BBB) permeability, and brain water content. Apoptotic level of hippocampal neurons, symbolic changes of ferroptosis, and levels of pro-inflammatory cytokines were assessed using TUNEL staining, Western blotting, colorimetry, and ELISA. The localization and expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) were detected. HT22 cells were exposed to Hemin as in vitro SAH models and treated with FIN56 to induce ferroptosis, followed by evaluation of the effects of DFO on FIN56-treated HT22 cells. The regulation of Nrf2 in thioredoxin reductase 1 (TXNRD1) was analyzed by co-immunoprecipitation and Western blotting. Moreover, HT22 cells were treated with DFO and ML385 to identify the role of DFO in the Nrf2/TXNRD1 axis. DFO extenuated brain injury, and ferroptosis and inflammation in hippocampal neurons of SAH mice. Nrf2 localized at the CA1 region of hippocampal neurons, and DFO stimulated nuclear translocation of Nrf2 protein in hippocampal neurons of SAH mice. Additionally, DFO inhibited ferroptosis and inflammatory responses in FIN56-induced HT22 cells. Nrf2 positively regulated TXNRD1 protein expression. Indeed, DFO alleviated FIN56-induced ferroptosis and inflammation via activation of the Nrf2/TXNRD1 axis. DFO alleviated neurological deficits, BBB disruption, brain edema, and brain injury in mice after SAH by inhibiting hippocampal neuron ferroptosis via the Nrf2/TXNRD1 axis. DFO ameliorates SAH-induced ferroptosis and inflammatory responses in hippocampal neurons by activating the Nrf2/TXNRD1 axis. [ABSTRACT FROM AUTHOR]

Regulation of the oxidative stress response is crucial for the management and prognosis of traumatic brain injury (TBI). The copper chaperone Antioxidant 1 (Atox1) plays a crucial role in regulating intracellular copper ion balance and impacting the antioxidant capacity of mitochondria, as well as the oxidative stress state of cells. However, it remains unknown whether Atox1 is involved in modulating oxidative stress following TBI. Here, we investigated the regulatory role of Atox1 in oxidative stress on neurons both in vivo and in vitro, and elucidated the underlying mechanism through culturing hippocampal HT-22 cells with Atox1 mutation. The expression of Atox1 was significantly diminished following TBI, while mice with overexpressed Atox1 exhibited a more preserved hippocampal structure and reduced levels of oxidative stress post-TBI. Furthermore, the mice displayed notable impairments in learning and memory functions after TBI, which were ameliorated by the overexpression of Atox1. In the stretch injury model of HT-22 cells, overexpression of Atox1 mitigated oxidative stress by preserving the normal morphology and network connectivity of mitochondria, as well as facilitating the elimination of damaged mitochondria. Mechanistically, co-immunoprecipitation and mass spectrometry revealed the binding of Atox1 to DJ-1. Knockdown of DJ-1 in HT-22 cells significantly impaired the antioxidant capacity of Atox1. Mutations in the copper-binding motif or sequestration of free copper led to a substantial decrease in the interaction between Atox1 and DJ-1, with overexpression of DJ-1 failing to restore the antioxidant capacity of Atox1 mutants. The findings suggest that DJ-1 mediates the ability of Atox1 to withstand oxidative stress. And targeting Atox1 could be a potential therapeutic approach for addressing post-traumatic neurological dysfunction.

ABSTRACTDopamine (DA) plays a significant role in regulating hippocampal function, particularly in modulating synaptic plasticity. Despite this, a comprehensive understanding of the molecular mechanisms involved in neuroplasticity-related signaling influenced by DA remains incomplete. This study aimed to elucidate the changes in the expression of key molecules related to hippocampal neuroplasticity following DA depletion in rats. To induce DA depletion, unilateral striatal infusions of 6-hydroxydopamine (6-OHDA) were administered to adult Sprague-Dawley rats. The subsequent loss of nigrostriatal DAergic signaling in these 6-OHDA-lesioned rats was confirmed using an apomorphine-induced rotation test at 4 weeks post-infusion and by assessing the expression levels of tyrosine hydroxylase (TH) through immunohistochemistry and western blotting at 7 weeks post-infusion. A decrease in DAergic signaling, evidenced by reduced TH-positive immunoreactivity, was also noted in the ipsilateral hippocampus of the lesioned rats. Interestingly, 6-OHDA infusion led to increased phosphorylation of pivotal hippocampal plasticity-related proteins, including extracellular signal-regulated kinase (ERK), protein kinase B (Akt), glycogen synthase kinase 3β (GSK3β), and cAMP response element-binding protein (CREB), in the ipsilateral hippocampus 7 weeks following the infusion. To extend these findings, in vitro experiments were conducted on primary hippocampal neurons exposed to DA and/or the active D1/D2 DA receptor antagonist, flupentixol (Flux). DA inhibited the constitutive phosphorylation of ERK, Akt, GSK3, and CREB, while Flux restored these phosphorylation levels. Taken together, these findings indicate that DA depletion triggers an increase in plasticity-related signaling in the hippocampus, suggesting a possible compensatory mechanism that promotes activity-independent neuroplasticity following DA depletion.

The hypothalamic–pituitary–adrenal (HPA) system plays an important role in stress response. Chronic stress is thought to induce neuronal damage and contribute to the pathogenesis of psychiatric disorders by causing dysfunction of the HPA system and promoting the production and release of glucocorticoids, including corticosterone and cortisol. Several clinical studies have demonstrated the efficacy of herbal medicines in treating psychiatric disorders; however, their effects on corticosterone-induced neuronal cell death remain unclear. Here, we used HT22 cells to evaluate the neuroprotective potential of herbal medicines used in neuropsychiatry against corticosterone-induced hippocampal neuronal cell death. Cell death was assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) reduction and Live/Dead assays. Hangekobokuto, Kamikihito, Saikokaryukotsuboreito, Kamishoyosan, and Yokukansan were supplied in the form of water-extracted dried powders. Exposure of HT22 cells to ≥ 100 μM corticosterone decreased MTT values. Exposure to 500 μM corticosterone alone reduced MTT values to 18%, while exposure to 10 μM Mifepristone (RU486)—a glucocorticoid receptor antagonist—restored values to 36%. Corticosterone-induced cell death was partially suppressed by treatment with RU486. At 100 μg/mL, Hangekobokuto significantly suppressed the decrease in MTT values (15–32%) and increase in the percentage of ethidium homodimer-1-positive dead cells caused by corticosterone exposure (78–36%), indicating an inhibitory effect on cell death. By contrast, Kamikihito, Saikokaryukotsuboreito, Kamishoyosan, and Yokukansan did not affect corticosterone-induced cell death. Therefore, our results suggest that Hangekobokuto may ameliorate the onset and progression of psychiatric disorders by suppressing neurological disorders associated with increased levels of glucocorticoids. [ABSTRACT FROM AUTHOR]

Uncovering mechanisms underlying epileptogenesis aids in preventing further epilepsy progression and to lessen seizure severity and frequency. The purpose of this study is to explore the antiepileptogenic and neuroprotective mechanisms of EGR1 in neuron injuries encountered in epilepsy. Bioinformatics analysis was conducted to identify the key genes related to epilepsy. The mice were rendered epileptic using the kainic acid protocol, followed by measurement of seizure severity, high amplitude and frequency, pathological changes of hippocampal tissues and neuron apoptosis. Furthermore, an in vitro epilepsy model was constructed in the neurons isolated from newborn mice, which was then subjected to loss- and gain-of-function investigations, followed by neuron injury and apoptosis assessment. Interactions among EGR1, METTL3, and VIM were analyzed by a series of mechanistic experiments. In the mouse and cell models of epilepsy, VIM was robustly induced. However, its knockdown reduced hippocampal neuron injury and apoptosis. Meanwhile, VIM knockdown decreased inflammatory response and neuron apoptosis in vivo. Mechanistic investigations indicated that EGR1 transcriptionally activated METTL3, which in turn downregulated VIM expression through m6A modification. EGR1 activated METTL3 and reduced VIM expression, thereby impairing hippocampal neuron injury and apoptosis, preventing epilepsy progression. Taken together, this study demonstrates that EGR1 alleviates neuron injuries in epilepsy by inducing METTL3-mediated inhibition of VIM, which provides clues for the development of novel antiepileptic treatments. [ABSTRACT FROM AUTHOR]

Ketamine, a noncompetitive N-methyl D-aspartate (NMDA) receptor antagonist, is widely used in pediatric clinical practice. The neuroprotective and neurotoxic effects of ketamine on brain neurons during development remain controversial. The reason may be related to the different concentrations of ketamine used in practice and the small range of concentrations used in previous studies. In this study, cultured hippocampal neurons were treated with ketamine in a wide range of concentrations to comprehensively observe the effects of different concentrations of ketamine on neurons. We demonstrated that low concentrations of ketamine (10 μM, 100 μM and 1000 μM) promoted neuronal survival (p < 0.05) and reduced neuronal apoptosis (p < 0.05) compared with those of the control group. High concentrations of ketamine (2000 μM, 2500 μM and 3000 μM) reduced neuronal survival (p < 0.05) and promoted neuronal apoptosis (p < 0.05). The p38 MAPK inhibitor SB203580 reduced neuronal apoptosis induced by high concentrations of ketamine (2500 μM) (p < 0.05). Our findings indicate that ketamine exerts a dual effect on the apoptosis of primary cultured fetal rat hippocampal neurons in vitro and that the neurotoxic effects of ketamine are related to activation of the p38 MAPK signaling pathway. [ABSTRACT FROM AUTHOR]

Lysosomal dysfunction is a pathogenic link that may explain the causal relationship between diabetes and Alzheimer disease; however, there is no information about the regulation of hyperglycemia in neuronal lysophagy modulating lysosomal function. We examined the effect and related mechanisms of action of high glucose on lysophagy impairment and subsequent Aβ accumulation in human induced pluripotent stem cell (hiPSC)-derived neurons, mouse hippocampal neurons, and streptozotocin (STZ)-induced diabetic mice. High-glucose induced neuronal lysosomal dysfunction through reactive oxygen species-mediated lysosomal membrane permeabilization and lysophagy impairment. Among lysophagy-related factors, the expression of TRIM16 (tripartite motif containing 16) was reduced in high-glucose-treated neuronal cells and the diabetic hippocampus through MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1)-mediated inhibition of TFEB (transcription factor EB) activity. TRIM16 overexpression recovered lysophagy through the recruitment of MAP1LC3/LC3 (microtubule associated protein 1 light chain 3), SQSTM1/p62, and ubiquitin to damaged lysosomes, which inhibited the high-glucose-induced accumulation of Aβ and p-MAPT/tau. In the diabetic mice model, TFEB enhancer recovered lysophagy in the hippocampus, resulting in the amelioration of cognitive impairment. In conclusion, TRIM16-mediated lysophagy is a promising candidate for the inhibition of diabetes-associated Alzheimer disease pathogenesis. Aβ: amyloid β; AD: Alzheimer disease; AMPK: 5' adenosine monophosphate-activated protein kinase; CTSB: cathepsin B; CTSD: cathepsin D; DM: diabetes mellitus; ESCRT: endosomal sorting complex required for transport; FBXO27: F-box protein 27; iPSC-NDs: induced pluripotent stem cell-derived neuronal differentiated cells; LAMP1: lysosomal-associated membrane protein 1; LMP: lysosomal membrane permeabilization; LRSAM1: leucine rich repeat and sterile alpha motif containing 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; p-MAPT/tau: phosphorylated microtubule associated protein tau; ROS: reactive oxygen species; STZ: streptozotocin; TFE3: transcription factor E3; TFEB: transcription factor EB; TRIM16: tripartite motif containing 16; UBE2QL1: ubiquitin conjugating enzyme E2 Q family like 1; VCP: valosin containing protein. [ABSTRACT FROM AUTHOR]

Abstract Background Alzheimer's disease presents an abnormal cognitive behavior. TgCtwh6 is one of the predominant T. gondii strains prevalent in China. Although T. gondii type II strain infection can cause host cognitive behavioral abnormalities, we do not know whether TgCtwh6 could also cause host cognitive behavioral changes. So, in this study, we will focus on the effect of TgCtwh6 on mouse cognitive behavior and try in vivo and in vitro to explore the underlying mechanism by which TgCtwh6 give rise to mice cognitive behavior changes at the cellular and molecular level. Methods C57BL/6 mice were infected orally with TgCtwh6 cysts. From day 90 post-infection on, all mice were conducted through the open field test and then Morris water maze test to evaluate cognitive behavior. The morphology and number of cells in hippocampus were examined with hematoxylin-eosin (H&E) and Nissl staining; moreover, Aβ protein in hippocampus was determined with immunohistochemistry and thioflavin S plaque staining. Synaptotagmin 1, apoptosis-related proteins, BACE1 and APP proteins and genes from hippocampus were assessed by western blotting or qRT-PCR. Hippocampal neuronal cell line or mouse microglial cell line was challenged with TgCtwh6 tachyzoites and then separately cultured in a well or co-cultured in a transwell device. The target proteins and genes were analyzed by immunofluorescence staining, western blotting and qRT-PCR. In addition, mouse microglial cell line polarization state and hippocampal neuronal cell line apoptosis were estimated using flow cytometry assay. Results The OFT and MWMT indicated that infected mice had cognitive behavioral impairments. The hippocampal tissue assay showed abnormal neuron morphology and a decreased number in infected mice. Moreover, pro-apoptotic proteins, as well as BACE1, APP and Aβ proteins, increased in the infected mouse hippocampus. The experiments in vitro showed that pro-apoptotic proteins and p-NF-κBp65, NF-κBp65, BACE1, APP and Aβ proteins or genes were significantly increased in the infected HT22. In addition, CD80, pro-inflammatory factors, notch, hes1 proteins and genes were enhanced in the infected BV2. Interestingly, not only the APP and pro-apoptotic proteins in HT22, but also the apoptosis rate of HT22 increased after the infected BV2 were co-cultured with the HT22 in a transwell device. Conclusions Neuron apoptosis, Aβ deposition and neuroinflammatory response involved with microglia polarization are the molecular and cellular mechanisms by which TgCtwh6 causes mouse cognitive behavioral abnormalities. Graphical Abstract

Context Sevoflurane (Sev) is a commonly used surgical anaesthetic; it has neurotoxic effects on the brain. Echinatin (Ech) is reported to have anti-inflammatory and antioxidant activity.Objective This research confirms the effect of Ech on Sev-induced neurotoxicity and cognitive deficits.Materials and methods Primary rat hippocampal neurons were treated with 4.1% Sev for 6 h in the presence of Ech (5, 10, and 20 μM) or vehicle, followed by a further 42 h of culture. Male Sprague-Dawley aged rats were divided into 6 groups (n = 6): control, Sev, Sev + Ech (20 mg/kg;), Sev + Ech (40 mg/kg), and Sev + Ech (80 mg/kg). Rats were intraperitoneally injected with Ech or vehicle 1 h before Sev exposure (2% Sev for 5 h).Results We found that Ech (5, 10, and 20 μM) elevated cell viability (1.29-, 1.51-, 1.68-fold) but mitigated apoptosis (23.87% vs. 16.48%, 12.72%, 9.02%), oxidative stress, and ferroptosis in hippocampal neurons with Sev treatment. Ech activated the Nrf2 expression in Sev-induced in vitro and in vivo models of anaesthetic neurotoxicity. Ech also weakened neurotoxicity in hippocampal neurons with Sev treatment by increasing Nrf2 expression level. Moreover, Ech alleviated hippocampus neurons apoptosis (19.38% vs. 16.05%, 11.71%, 8.88%), oxidative stress, and ferroptosis in rats with Sev treatment. Ech improved Sev-induced cognitive deficits in rats.Conclusions Ech alleviates Sev-induced neurotoxicity and cognitive deficits by mitigation of ferroptosis and oxidative stress. Ech may be developed as a new promising therapeutic drug for treatment of cerebral nerve injury caused by surgical anaesthesia.

Vascular dementia (VaD) is the second most common subtype of dementia, but the precise mechanism underlying VaD is not fully understood. Long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) can act as a key regulator in physiological and pathological processes, including neurological disorders, but whether it is correlated with VaD has not been elucidated. In this study, we established a mouse model of VaD by the transient bilateral common carotid artery occlusion surgery. As expected, the Morris water maze showed that VaD mice had significant deficits in spatial learning and memory. MALAT1 was elevated in the hippocampus of VaD mice. Additionally, we found that microRNA (miR)-9-3p was downregulated in the VaD hippocampus. By performing a dual-luciferase report assay, we verified the binding relationship between MALAT1 and miR-9-3p. Interestingly, synapse-associated protein-97 (SAP97), a well-known gene related to synaptic functions, was found upregulated in the hippocampus of VaD mice. In vitro experiments performed on hippocampal neurons demonstrated that miR-9-3p negatively regulated SAP97 expression. The downregulation of MALAT1 in hippocampal neurons increased miR-9-3p and reduced SAP97, whereas miR-9-3p inhibition rescued the MALAT1 downregulation-mediated SAP97 reduction. In conclusion, the present study reported the alterations in the expression levels of MALAT1, miR-9-3p, and SAP97 in the hippocampus of VaD mice, suggesting that MALAT1 targets miR-9-3p to upregulate SAP97 in the hippocampus of mice with VaD. This work will be helpful for understanding the molecular mechanisms of VaD. [ABSTRACT FROM AUTHOR]

Introduction: Growth-associated protein 43 (GAP-43) is known as a neuronal plasticity protein because it is widely expressed at high levels in neuronal growth cones during axonal regeneration. GAP-43 expressed in mature adult neurons is functionally important for the neuronal communication of synapses in learning and memory. Brain-derived neurotrophic factor (BDNF) is closely related to neurodegeneration and synaptic plasticity during the aging process. However, the molecular mechanisms regulating neurodegeneration and synaptic plasticity underlying the pathogenesis and progression of Alzheimer's disease (AD) still remain incompletely understood. Methods: Remarkably, the expressions of GAP-43 and BDNF perfectly match in various neurons in the Human Brain Atlas database. Moreover, GAP-43 and BDNF are highly expressed in a healthy adults' hippocampus brain region and are inversely correlated with the amyloid beta (Ab), which is the pathological peptide of amyloid plaques found in the brains of patients with AD. Results: These data led us to investigate the impact of the direct molecular interaction between GAP-43 and BDNF in hippocampal neuron fate. In this study, we show that GAP-43 and BDNF are inversely associated with pathological molecules for AD (Tau and Ab). In addition, we define the three-dimensional protein structure for GAP-43 and BDNF, including the predictive direct binding sites via analysis using ClusPro 2.0, and demonstrate that the deprivation of GAP-43 and BDNF triggers hippocampal neuronal death and memory dysfunction, employing the GAP-43 or BDNF knock-down cellular models and 5XFAD mice. Conclusion: These results showthatGAP-43 and BDNF are direct binding partners in hippocampal neurons and that their molecular signaling might be potential therapeutic targets for AD. [ABSTRACT FROM AUTHOR]

BACKGROUND: In recent years, primary neuronal cell models have played an important role in brain diseases. The characteristics of such cell models are closer to disease cells and can be used to simulate various neurological diseases. OBJECTIVE: To establish a convenient and practical culture method that can simultaneously extract primary cortical and hippocampal neurons. METHODS: The SD rats within 24 hours of the newborn were sacrificed by chiropractic method, and then sterilized with 75% ethanol. After separating the skull and meninges using forceps, the whole brain was dissected out. The cerebrovascular membrane was stripped, and the cerebral cortex and hippocampus were dissociated. The sequential digestion protocol of papain and appropriate amount of DNase was used. After pipetting, centrifugation, and filtration, the samples were inoculated into L-polylysine-coated six-well plates and slides. DMEM-F12 medium containing 10% fetal bovine serum was used as the seeding medium. 6 hours later, the serum-free special medium containing Neurobasal B27 was used. After culturing for 7 days, the cell morphology and growth state were observed under the microscope. The cortical and hippocampal neurons were identified by β-Tubulin immunofluorescence method and Neun antibody immunohistochemistry. The marker MAP2 immunofluorescence method was applied to identify the purity. RESULTS AND CONCLUSION: (1) 24 hours after inoculation, the volume of cells became clear, presented irregular circles, surrounded by halos, and a few cells had small protrusions, all of which had grown adherently to the wall. After 3 days of continuous culture, the cell bodies gradually increased; some of them grew in clusters; synapses were elongated; and cross-links appeared between cells. After 7 days of continuous culture, the cell body was mature and full; the cytoplasm was significantly increased; the halo was enhanced; and a denser neuronal network was formed. (2) Neurons were identified by Neun antibody immunohistochemistry and β-Tubulin immunofluorescence method. The purities of cortical and hippocampal neurons were (94.00±0.34)% and (91.00±0.26)%, respectively detected using neuronal marker MAP2 immunofluorescence method. Neuronal cells could be used for experiments. (3) These results suggest that the cerebral cortex and hippocampus were isolated from the same batch of neonatal SD rats within 24 hours. After sequential digestion with papain and DNase, high-quality hippocampal and cortical neurons can be extracted. The neurons obtained by this protocol have high purity and simplified operation, and can be used as the basis for various neurological disease cell models. [ABSTRACT FROM AUTHOR]

Dopamine (DA) plays a significant role in regulating hippocampal function, particularly in modulating synaptic plasticity. Despite this, a comprehensive understanding of the molecular mechanisms involved in neuroplasticity-related signaling influenced by DA remains incomplete. This study aimed to elucidate the changes in the expression of key molecules related to hippocampal neuroplasticity following DA depletion in rats. To induce DA depletion, unilateral striatal infusions of 6-hydroxydopamine (6-OHDA) were administered to adult Sprague-Dawley rats. The subsequent loss of nigrostriatal DAergic signaling in these 6-OHDA-lesioned rats was confirmed using an apomorphine-induced rotation test at 4 weeks post-infusion and by assessing the expression levels of tyrosine hydroxylase (TH) through immunohistochemistry and western blotting at 7 weeks post-infusion. A decrease in DAergic signaling, evidenced by reduced TH-positive immunoreactivity, was also noted in the ipsilateral hippocampus of the lesioned rats. Interestingly, 6-OHDA infusion led to increased phosphorylation of pivotal hippocampal plasticity-related proteins, including extracellular signal-regulated kinase (ERK), protein kinase B (Akt), glycogen synthase kinase 3β (GSK3β), and cAMP response element-binding protein (CREB), in the ipsilateral hippocampus 7 weeks following the infusion. To extend these findings, in vitro experiments were conducted on primary hippocampal neurons exposed to DA and/or the active D1/D2 DA receptor antagonist, flupentixol (Flux). DA inhibited the constitutive phosphorylation of ERK, Akt, GSK3, and CREB, while Flux restored these phosphorylation levels. Taken together, these findings indicate that DA depletion triggers an increase in plasticity-related signaling in the hippocampus, suggesting a possible compensatory mechanism that promotes activity-independent neuroplasticity following DA depletion. [ABSTRACT FROM AUTHOR]

Panax ginseng (P. ginseng) C. A. Meyer. has been used extensively globally as a medicine. It has a therapeutic effect on sleep and is an attractive alternative for patients with insomnia. The United States Patent of Invention has approved the use of P. ginseng alcohol extract (GAE) in nutraceuticals or food to improve sleep. It has shown promise as an effective therapeutic agent for improving sleep and cognition. However, its mechanism of action is not yet fully understood. To investigate the therapeutic benefits of GAE on sleep and cognition and its underlying mechanism in aged sleep-deprived rats, with a focus on orexin-mediated autophagy function. We conducted in vivo tests in an aged sleep-deprivation rat model produced using p-chlorophenylalanine (PCPA) coupled with modified multi-platform method to examine the therapeutic effects and mechanisms of GAE. A pentobarbital sodium-induced sleep test and water maze were used to assess sleep and cognitive performance, respectively. An enzyme-linked immunosorbent assay was used to determine orexin levels and aging and sleep markers in serum and hypothalamic tissues. Hematoxylin-eosin staining and Nissl staining were used to assess histopathological changes, and autophagy levels were assessed using transmission electron microscopy, immunofluorescence. Western blot and immunohistochemical staining were performed to detect the levels of orexin, orexin-receptor proteins, and autophagy-associated proteins to study the effects of GAE on hippocampal neurons, and the underlying mechanisms. In aged sleep-deprived rats, GAE treatment prolonged sleep duration, improved cognitive function, prevented hippocampal neuronal damage, increased the number of Nissl bodies, improved aging and sleep markers, and enhanced the LC3A/B expression in autophagosomes and neurons. The amount of orexin in serum and hypothalamic tissue and OX1R, OX2R, and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) proteins also reduced, which resulted in the inhibition of the PI3K/Akt/mTOR pathway and activation of the autophagy process. GAE may reduce hypothalamic orexin secretion and interact with orexin receptors to inhibit the PI3K/Akt/mTOR signalling network and activate autophagy. This may be a potential mechanism of action of GAE in regulating sleep-related cognitive function. [Display omitted] • GAE improved sleep duration and cognitive function in aged sleep-deprived rats. • GAE in aged sleep-deprived rats prevents hippocampal neuronal damage. • GAE promotes orexin mediated autophagy. [ABSTRACT FROM AUTHOR]

Most neurons are influenced by multiple neuromodulatory inputs that converge on common effectors. Mechanisms that route these signals are key to selective neuromodulation but are poorly understood. G protein-gated inwardly rectifying K+ (GIRK or Kir3) channels mediate postsynaptic inhibition evoked by G protein-coupled receptors (GPCRs) that signal via inhibitory G proteins. GIRK-dependent signaling is modulated by Regulator of G protein Signaling proteins RGS6 and RGS7, but their selectivity for distinct GPCR-GIRK signaling pathways in defined neurons is unclear. We compared how RGS6 and RGS7 impact GIRK channel regulation by the GABA B receptor (GABA B R), 5HT 1A receptor (5HT 1A R), and A 1 adenosine receptor (A 1 R) in hippocampal neurons. Our data show that RGS6 and RGS7 make non-redundant contributions to GABA B R- and 5HT 1A R-GIRK signaling and compartmentalization and suggest that GPCR-G protein preferences and the substrate bias of RGS proteins, as well as receptor-dependent differences in Gα o engagement and effector access, shape GPCR-GIRK signaling dynamics in hippocampal neurons. • GIRK channels are a common effector in G protein signaling pathways in neurons. • Our understanding of GPCR-GIRK signaling mechanisms in neurons is incomplete. • RGS6 and RGS7 regulate neuronal GIRK channels in a GPCR-dependent manner. • Inhibitory GPCRs engage Gα o to varying extents to activate GIRK channels in neurons. • G protein use, RGS substrate bias, and effector access shape GPCR-GIRK signaling. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease presents an abnormal cognitive behavior. TgCtwh6 is one of the predominant T. gondii strains prevalent in China. Although T. gondii type II strain infection can cause host cognitive behavioral abnormalities, we do not know whether TgCtwh6 could also cause host cognitive behavioral changes. So, in this study, we will focus on the effect of TgCtwh6 on mouse cognitive behavior and try in vivo and in vitro to explore the underlying mechanism by which TgCtwh6 give rise to mice cognitive behavior changes at the cellular and molecular level. Methods: C57BL/6 mice were infected orally with TgCtwh6 cysts. From day 90 post-infection on, all mice were conducted through the open field test and then Morris water maze test to evaluate cognitive behavior. The morphology and number of cells in hippocampus were examined with hematoxylin-eosin (H&E) and Nissl staining; moreover, Aβ protein in hippocampus was determined with immunohistochemistry and thioflavin S plaque staining. Synaptotagmin 1, apoptosis-related proteins, BACE1 and APP proteins and genes from hippocampus were assessed by western blotting or qRT-PCR. Hippocampal neuronal cell line or mouse microglial cell line was challenged with TgCtwh6 tachyzoites and then separately cultured in a well or co-cultured in a transwell device. The target proteins and genes were analyzed by immunofluorescence staining, western blotting and qRT-PCR. In addition, mouse microglial cell line polarization state and hippocampal neuronal cell line apoptosis were estimated using flow cytometry assay. Results: The OFT and MWMT indicated that infected mice had cognitive behavioral impairments. The hippocampal tissue assay showed abnormal neuron morphology and a decreased number in infected mice. Moreover, pro-apoptotic proteins, as well as BACE1, APP and Aβ proteins, increased in the infected mouse hippocampus. The experiments in vitro showed that pro-apoptotic proteins and p-NF-κBp65, NF-κBp65, BACE1, APP and Aβ proteins or genes were significantly increased in the infected HT22. In addition, CD80, pro-inflammatory factors, notch, hes1 proteins and genes were enhanced in the infected BV2. Interestingly, not only the APP and pro-apoptotic proteins in HT22, but also the apoptosis rate of HT22 increased after the infected BV2 were co-cultured with the HT22 in a transwell device. Conclusions: Neuron apoptosis, Aβ deposition and neuroinflammatory response involved with microglia polarization are the molecular and cellular mechanisms by which TgCtwh6 causes mouse cognitive behavioral abnormalities. [ABSTRACT FROM AUTHOR]

The GluR3 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) has been identified as a target for autoantibodies (Aabs) in autoimmune encephalopathy and other diseases. Recent studies have proposed mechanisms by which these Aabs act, but their exact role in neuronal excitability is yet to be established. Patient Aabs have been shown to bind to specific regions within the GluR3 subunit. GLUR3B peptides were designed based on described (ELISA) immunogenic epitopes for Aabs and an immunisation strategy was used to generate novel anti-AMPAR Aabs. Target-specific binding and specificity of affinity-purified anti-AMPAR Aabs was confirmed using enzyme-linked immunosorbent assay, immunocytochemistry and Western blot. Functional anti-AMPAR Aab effects were determined on excitatory postsynaptic currents (EPSCs) from primary hippocampal neurons using whole-cell patch-clamp electrophysiology. Acute (10 or 30 min) or longer-term (24 h) application of anti-AMPAR Aabs caused a significant reduction in the mean frequency of spontaneous and miniature EPSCs in hippocampal neurons. Our data demonstrate that anti-AMPAR Aabs targeting peptides linked to auto-immune diseases mediate inhibitory effects on neuronal excitability at the synaptic level, such effects may lead to disruption of the excitatory/inhibitory balance at a network level. [ABSTRACT FROM AUTHOR]

Naked mole‐rats (NMRs) (Heterocephalus glaber) are highly social and subterranean rodents with large communal colonies in burrows containing low oxygen levels. The inhibition of severe hypoxic conditions is of particular interest to this study. To understand the mechanisms that facilitate neuronal preservation during hypoxia, we investigated the proteins regulating hypoxia tolerance in NMR hippocampal neurons. Caveolin‐1 (Cav‐1), a transmembrane scaffolding protein, confers prosurvival signalling in the central nervous system. The present study aimed to investigate the role of Cav‐1 in hypoxia‐induced neuronal injury. Western blotting analysis and immunocytochemistry showed that Cav‐1 expression was significantly upregulated in NMR hippocampal neurons under 8% O2 conditions for 8 h. Cav‐1 alleviates apoptotic neuronal death from hypoxia. Downregulation of Cav‐1 by lentiviral vectors suggested damage to NMR hippocampal neurons under hypoxic conditions in vitro and in vivo. Overexpression of Cav‐1 by LV‐Cav‐1 enhanced hypoxic tolerance of NMR hippocampal neurons in vitro and in vivo. Mechanistically, the levels of hypoxia inducible factor‐1α (HIF‐1α) are also increased under hypoxic conditions. After inhibiting the binding of HIF‐1α to hypoxia response elements in the DNA by echinomycin, Cav‐1 levels were downregulated significantly. Furthermore, chromatin immunoprecipitation assays showed the direct role of HIF1α in regulating the expression levels of Cav‐1 in NMR hippocampal neurons under hypoxic conditions. These findings suggest that Cav‐1 plays a critical role in modulating the apoptosis of NMR hippocampal neurons and warrant further studies targeting Cav‐1 to treat hypoxia‐associated brain diseases. [ABSTRACT FROM AUTHOR]

[Display omitted] • Camk2b-induced differential proteomic profiling by iTRAQ-based quantitative approach. • Several candidate target proteins were directly affected by Camk2b dysexpression. • Camk2b-Mapk3 as a new potential pathway affected expression of the synaptic proteins and thereby synaptogenesis. Recent studies have demonstrated that Camk2b expression is modified in neuropsychiatric illnesses and potentially affects synaptic plasticity. However, the molecular events arising from Camk2b dysregulation are not fully elucidated and need to be comprehensively explored. In the present study, we first induced over-expression and under-expression of Camk2b in cultured rat hippocampal neurons through transfection with lentivirus plasmids. Then isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics followed by bioinformatics analyses were carried out to explore the impacts of Camk2b dysexpression on the proteome of the neurons. Compared with the respective controls, a total of 270 proteins in the Camk2b-overexpression group and 209 proteins in the Camk2b-underexpression group were experienced a divergence in expression. Gene ontology and pathway analyses indicated that Camk2b overexpression and under-expression respectively induced two different change profiles of protein expressions and functions, reflecting the potential differences in cellular processes and biological events. Through cross comparison, several candidate target proteins regulated directly by Camk2b were revealed. Further network and immunoblot analyses demonstrated that Mapk3 could be an important linker and Camk2b-Mapk3 might serve as a new potential pathway affecting the expression of synaptic proteins in hippocampal neurons. Collectively, the present results offer a new comprehension of the regulatory molecular mechanism of Camk2b and thereby increase our understanding of Camk2b-mediated synaptogenesis in synaptic plasticity. [ABSTRACT FROM AUTHOR]

Vascular dementia (VaD) is characterized by a time-dependent memory deficit and essentially combined with evidence of neuroinflammation. Thus, polyphenol-rich natural plants, which possess anti-inflammatory properties, have received much scientific attention. This study investigated whether Perilla frutescens leaf extract (PFL) exerts therapeutic efficacy against VaD. Sprague Dawley rats were divided into five groups: SO, sham-operated and vehicle treatment; OP, operated and vehicle treatment; PFL-L, operated and low-dose (30 mg/kg) PFL treatment; PFL-M, operated and medium-dose (60 mg/kg) PFL treatment; and PFL-H, operated and high-dose (90 mg/kg) PFL treatment. Two-vessel occlusion and hypovolemia (2VO/H) were employed as a surgical model of VaD, and PFL was given orally perioperatively for 23 days. The rats underwent the Y-maze, Barnes maze, and passive avoidance tests and their brains were subjected to histologic studies. The OP group showed VaD-associated memory deficits, hippocampal neuronal death, and microglial activation; however, the PFL-treated groups showed significant attenuations in all of the above parameters. Using lipopolysaccharide (LPS)-stimulated BV-2 cells, a murine microglial cell line, we measured PFL-mediated changes on the production of nitric oxide (NO), TNF-α, and IL-6, and the activities of their upstream MAP kinases (MAPKs)/NFκB/inducible NO synthase (iNOS). The LPS-induced upregulations of NO, TNF-α, and IL-6 production and MAPKs/NFκB/iNOS activities were globally and significantly reversed by 12-h pretreatment of PFL. This suggests that PFL can counteract VaD-associated structural and functional deterioration through the attenuation of neuroinflammation.

Abstract Dopamine (DA) and norepinephrine (NE) are pivotal neuromodulators that regulate a broad range of brain functions, often in concert. Despite their physiological importance, untangling the relationship between DA and NE in the fine control of output function is currently challenging, primarily due to a lack of techniques to allow the observation of spatiotemporal dynamics with sufficiently high selectivity. Although genetically encoded fluorescent biosensors have been developed to detect DA, their poor selectivity prevents distinguishing DA from NE. Here, we report the development of a red fluorescent genetically encoded GPCR (G protein-coupled receptor)-activation reporter for DA termed ‘R-GenGAR-DA’. More specifically, a circular permutated red fluorescent protein (cpmApple) was replaced by the third intracellular loop of human DA receptor D1 (DRD1) followed by the screening of mutants within the linkers between DRD1 and cpmApple. We developed two variants: R-GenGAR-DA1.1, which brightened following DA stimulation, and R-GenGAR-DA1.2, which dimmed. R-GenGAR-DA1.2 demonstrated a reasonable dynamic range (ΔF/F 0 = − 43%), DA affinity (EC50 = 0.92 µM) and high selectivity for DA over NE (66-fold) in HeLa cells. Taking advantage of the high selectivity of R-GenGAR-DA1.2, we monitored DA in presence of NE using dual-color fluorescence live imaging, combined with the green-NE biosensor GRABNE1m, which has high selectivity for NE over DA (> 350-fold) in HeLa cells and hippocampal neurons grown from primary culture. Thus, this is a first step toward the multiplex imaging of these neurotransmitters in, for example, freely moving animals, which will provide new opportunities to advance our understanding of the high spatiotemporal dynamics of DA and NE in normal and abnormal brain function.

Botulinum neurotoxins (BoNTs) are extremely potent naturally occurring poisons that act by silencing neurotransmission. Intriguingly, in addition to preventing presynaptic vesicle fusion, BoNT serotype A (BoNT/A) can also promote axonal regeneration in preclinical models. Here we report that the non-toxic C-terminal region of the receptor-binding domain of heavy chain BoNT/A (HCC/A) activates the small GTPase Rac1 and ERK pathway to potentiate axonal outgrowth, dendritic protrusion formation and synaptic vesicle release in hippocampal neurons. These data are consistent with HCC/A exerting neurotrophic properties, at least in part, independent of any BoNT catalytic activity or toxic effect.

Arsenic, a neurotoxic metalloid, poses significant health risks. However, ellagic acid, renowned for its antioxidant properties, has shown potential in neuroprotection. This study aimed to investigate the neuroprotective effects of ellagic acid against arsenic-induced neuronal ferroptosis and cognitive impairment and elucidate the underlying mechanisms. Using an arsenic-exposed Wistar rat model and an arsenic-induced HT22 cells model, we assessed cognitive ability, measured serum and brain arsenic levels, and evaluated pathological damage through histological analysis and transmission electron microscopy. Additionally, we examined oxidative stress and iron ion levels using GSH, MDA, ROS and tissue iron biochemical kits, and analyzed the expression of ferroptosis-related markers using western blot and qRT-PCR. Our results revealed that arsenic exposure increased both serum and brain arsenic levels, resulting in hippocampal pathological damage and subsequent decline in learning and memory abilities. Arsenic-induced neuronal ferroptosis was mediated by the inhibition of the xCT/GSH/GPX4/Nrf2 signaling axis and disruption of iron metabolism. Notably, ellagic acid intervention effectively reduced serum and brain arsenic levels, ameliorated neuronal damage, and improved oxidative stress, ferroptosis, and cognitive impairment. These beneficial effects were associated with the activation of the Nrf2/Keap1 signaling pathway, upregulation of GPX4 expression, and enhanced iron ion excretion. In conclusion, ellagic acid demonstrates promising neuroprotective effects against arsenic-induced neurotoxicity by mitigating neuronal ferroptosis and cognitive impairment. • Arsenic exposure induces neuronal ferroptosis and cognitive impairment. • Ellagic acid ameliorated ferroptosis by promoting excretion of arsenic-induced iron accumulation. • Ellagic acid balanced arsenic-induced oxidative stress through activation of the Nrf2/Keap1 signaling pathway. [ABSTRACT FROM AUTHOR]

• Ferroptosis was involved in the pathophysiological progression of epilepsy. • SS-31 can inhibit PILO-induced seizures by preventing ferroptosis. • SS-31 can alleviate ferroptosis-induced oxidative injury by targeting p38 MAPK signaling. Ferroptosis is a newly identified form of non-apoptotic regulated cell death (RCD) and plays an important role in epileptogenesis. The p38 mitogen-activated protein kinase (p38 MAPK) pathway has been confirmed to be involved in ferroptosis. The mitochondria-targeting antioxidant Elamipretide (SS-31) can reduce the generation of lipid peroxidation and the buildup of reactive oxygen species (ROS). Collectively, our present study was to decipher whether SS-31 inhibits ferroptosis via the p38 MAPK signaling pathway in the rat epilepsy model induced by pilocarpine (PILO). Adult male Wistar rats were randomly divided into four groups: control group (CON group), epilepsy group (EP group), SS-31 treatment group (SS group), and p38 MAPK inhibitor (SB203580) treatment group (SB group). Our results demonstrated that the rat hippocampal neurons after epilepsy were followed by accumulated iron and malondialdehyde (MDA) content, upregulated phosphorylated p38 MAPK protein (P-p38) and nuclear factor erythroid 2-related factor 2 (Nrf2) levels, reduced glutathione peroxidase 4 (Gpx4) content, and depleted glutathione (GSH) activity. Morphologically, mitochondrial ultrastructural damage under electron microscopy was manifested by a partial increase in outer membrane density, disappearance of mitochondrial cristae, and mitochondrial shrinkage. SS-31 and SB203580 treatment blocked the initiation and progression of ferroptosis in the hippocampus of epileptic rats via reducing the severity of epileptic seizures, reversing the expression of Gpx4, P-p38 , decreasing the levels of iron and MDA, as well as increasing the activity of GSH and Nrf2. To summarize, our findings proved that ferroptosis was coupled with the pathology of epilepsy, and SS-31 can inhibit PILO-induced seizures by preventing ferroptosis, which may be connected to the inhibition of p38 MAPK phosphorylation, highlighting the potential therapeutic value for targeting ferroptosis process in individuals with seizure-related diseases. [ABSTRACT FROM AUTHOR]

Neonatal hypoxic ischemic encephalopathy (Neonatal HIE) is a common but serious disease caused by perinatal asphyxia injury in newborns. Elevated neuronal apoptosis plays an important role in the injury process post hypoxia ischemia of the brain, which accurate mechanism is still worthy to be studied. Cellular repressor of E1A‐stimulated genes (CREG) possesses the protective effect in ischemia‐reperfusion in multiple organs, including livers and hearts. The main purpose of this work was to investigate whether CREG was involved in alleviating neonatal HIE and explore the possible mechanisms. We found that CREG expression was downregulated in the hippocampus of neonatal HIE rats as well as oxygen‐glucose deprivation/reperfusion (OGD/R)‐treated hippocampal neurons. Besides, CREG overexpression promoted survival while inhibited apoptosis in OGD/R‐induced hippocampal neurons accompanied by AKT signaling activation, which could be reversed by CREG silence. In addition, the protective effects of CREG overexpression could be antagonized by AKT deactivation, indicating the function of CREG was attributed by regulating AKT pathway. Collectedly, we demonstrated that CREG protected hippocampal neurons from hypoxic ischemia‐induced injury through regulating survival and apoptosis via activating AKT signaling pathway. [ABSTRACT FROM AUTHOR]

Alpha-tocotrienol (α-TCT) is a member of the vitamin E family. It has been reported to protect the brain against various pathologies including cerebral ischemia and neurodegeneration. However, it is still unclear if α-TCT exhibits beneficial effects during brain development. We hypothesized that treatment with α-TCT improves intracellular redox homeostasis supporting normal development of neurons. We found that primary hippocampal neurons isolated from rat feti grown in α-TCT-containing media achieved greater levels of neurite complexity compared to ethanol-treated control neurons. Neurons were treated with 1 μM α-TCT for 3 weeks, and media were replaced with fresh α-TCT every week. Treatment with α-TCT increased α-TCT levels (26 pmol/mg protein) in the cells, whereas the control neurons did not contain α-TCT. α-TCT-treated neurons produced adenosine triphosphate (ATP) at a higher rate and increased ATP retention at neurites, supporting formation of neurite branches. Although treatment with α-TCT alone did not change neuronal viability, neurons grown in α-TCT were more resistant to death at maturity. We further found that messenger RNA and protein levels of B-cell lymphoma-extra large (Bcl-xL) are increased by α-TCT treatment without inducing posttranslational cleavage of Bcl-xL. Bcl-xL is known to enhance mitochondrial energy production, which improves neuronal function including neurite outgrowth and neurotransmission. Therefore α-TCT-mediated Bcl-xL upregulation may be the central mechanism of neuroprotection seen in the α-TCT-treated group. In summary, treatment with α-TCT upregulates Bcl-xL and increases ATP levels at neurites. This correlates with increased neurite branching during development and with protection of mature neurons against oxidative stress. [Display omitted] [ABSTRACT FROM AUTHOR]

Aims: Hypoxia and inflammation may lead to BDNF/TrkB dysregulation and neurological disorders. Propofol is an anesthetic with neuroprotective properties. We wondered whether and how propofol affected BDNF/TrkB pathway in hippocampal neurons and astrocytes. Methods: Primary rat hippocampal neurons and astrocytes were cultured and exposed to propofol followed by hypoxia or TNF‐α treatment. The expression of BDNF and the expression/truncation/phosphorylation of TrkB were measured. The underlying mechanisms were investigated. Results: Hypoxia and TNF‐α reduced the expression of BDNF, which was reversed by pretreatment of 25 μM propofol in hippocampal neurons. Furthermore, hypoxia and TNF‐α increased the phosphorylation of ERK and phosphorylation of CREB at Ser142, while reduced the phosphorylation of CREB at Ser133, which were all reversed by 25 μM propofol and 10 μM ERK inhibitor. In addition, hypoxia or TNF‐α did not affect TrkB expression, truncation, or phosphorylation in hippocampal neurons and astrocytes. However, in hippocampal neurons, 50 μM propofol induced TrkB phosphorylation, which may be mediated by p35 expression and Cdk5 activation, as suggested by the data showing that blockade of p35 or Cdk5 expression mitigated propofol‐induced TrkB phosphorylation. Conclusions: Propofol modulated BDNF/TrkB pathway in hippocampal neurons via ERK/CREB and p35/Cdk5 under the condition of hypoxia or TNF‐α exposure. [ABSTRACT FROM AUTHOR]

Neuroinflammation is a key mechanism underlying the cognitive impairment induced by PM2.5, and activated microglia plays an important role in this process. However, the mechanisms by which activated microglia induced by PM2.5 impair hippocampal neurons have not been fully elucidated. In this study, we focused on the role of HMGB1-NLRP3-P2X7R pathway which mediated the microglia activation in hippocampal neurons impairment induced by PM2.5 using a co-culture model of microglia and hippocampal neurons. We found that PM2.5 resulted in activated microglia and HMGB1-NLRP3 inflammatory pathway, and elevated proinflammatory cytokines of IL-18 and IL-1β in a dose-dependent manner. Notably, we next utilized previously reported pharmacological inhibitors or siRNA for HMGB1 and found that they significantly inhibited the activation of downstream NLRP3 and MAPK pathways derived from PM2.5 exposure, and down-regulated IL-18 and IL-1β in microglia. Furthermore, we employed co-cultured hippocampal neurons and microglia and found that reducing HMGB1 significantly decreased neuron impairment, apoptosis related protein of cl-caspase3, synaptic damage, and neurotransmitter receptor of 5-HT2A, along with notably elevated presynaptic and postsynaptic proteins of SYP and PSD-95, as well as learning and memory related proteins of p-CREB and BDNF. The neuronal impairment induced by PM2.5 could not be prevented in the case of simultaneous employment of HMGB1 siRNA and NLRP3 agonist. After silencing NLRP3 alone in microglia, hippocampal neurons demonstrated decreased excessive autophagy and up-regulated synaptic protein of GAP43 as well as learning and memory related protein of NCAM1. Therefore, we further studied how hippocampal neurons affected microglia under PM2.5 exposure, Further investigation indicated that silencing HMGB1 could affect the activation of P2X7R and reduce the release of ATP from hippocampal neurons, thus protecting the interaction between microglia and hippocampal neurons. The present work suggests that regulation of HMGB1-NLRP3-P2X7R pathway can inhibit the microglia activation induced by PM2.5 to alleviate hippocampal neuron impairment and stabilize the microenvironment between microglia and neurons. This contributes to maintaining the normal function of hippocampal neurons and alleviating the cognitive impairment derived from PM2.5 exposure.

Cerebral ischemia/reperfusion (I/R) injury contributes considerably to the poor prognosis in patients with ischemic stroke. This study is aimed to delineate the molecular mechanistic actions by which sevoflurane protects against cerebral I/R injury. A rat model of cerebral I/R injury was established and pre-treated with sevoflurane, in which hippocampal neuron apoptosis was found to be repressed and the level of E2F transcription factor 1 (E2F1) was observed to be down-regulated. Then, the up-regulated expression of E2F1 was validated in rats with cerebral I/R injury, responsible for stimulated neuron apoptosis. Further, the binding of E2F1 to enhancer of zeste homolog 2 (EZH2) and EZH2 to tissue inhibitor of metalloproteinases-2 (TIMP2) was identified. The stimulative effect of the E2F1/EZH2/TIMP2 regulatory axis on neuron apoptosis was subsequently demonstrated through functional assays. After that, it was substantiated in vivo that sevoflurane suppressed the apoptosis of hippocampal neurons in rats with cerebral I/R injury by down-regulating E2F1 to activate the EZH2/TIMP2 axis. Taken together, our data elucidated that sevoflurane reduced neuron apoptosis through mediating the E2F1/EZH2/TIMP2 regulatory axis, thus protecting rats against cerebral I/R injury. [ABSTRACT FROM AUTHOR]

Abstract Lead (Pb) exhibits serious adverse effects on the central nervous system, and the major pathogenic mechanism of Pb toxicity is oxidative stress. As one of the carotenoid family members with potent antioxidant properties, lycopene has shown its protections by inhibiting oxidative stress damage in numerous models of neurotoxicity. The current study was designed to explore the possible protective property in primary cultured rat hippocampal neurons challenged with Pb. We observed that 5 μM lycopene pretreatment for 4 h efficiently ameliorated Pb‐caused damage in cell viability, accumulation of reactive oxygen species (ROS), and apoptosis in a dose‐dependent manner. Moreover, lycopene (5 μM) attenuated the 50 μM Pb‐induced mitochondrial ROS production, improved the activities of mitochondrial respiratory chain enzymes and ATP production, and ameliorated the 50 μM Pb‐induced depolarization of mitochondrial membrane potential as well as opening of mitochondrial permeability transition pores. In addition, 5 μM lycopene restored the imbalance of Bax/Bcl‐2, inhibited translocation of cytochrome c, and reduced caspase‐3 activation. Taken together, these findings indicate that lycopene antagonizes against Pb‐induced neurotoxicity and the underlying mechanism probably involves reduction of mitochondrial oxidative damage and mitochondria‐mediated apoptosis.

The protective effects of blueberry anthocyanin extracts against damage induced by extremely low-frequency electromagnetic field (ELF-EMF) were investigated in a rat model. Wistar rats were exposed to ELF-EMF with or without the administration of blueberry anthocyanin extracts (50, 100, and 200 mg/kg per day intragastrically once a day) for 30 days. Blueberry anthocyanin extracts supplementation inhibited the decrease in Nissl substance levels, cell membrane integrity, and mitochondrial membrane potential induced by ELF-EMF; prevented the increase in nitric oxide, malondialdehyde, and Ca2+ concentrations; suppressed superoxide dismutase and glutathione depletion; and enhanced the cognitive ability of the rats exposed to ELF-EMF. The protective effects of blueberry anthocyanin extracts against hippocampal neuron injury caused by ELF-EMF were dose-dependent. These results demonstrated that blueberry anthocyanin extracts suppress hippocampal neuron injury caused by ELF-EMF by inhibiting cell membrane damage and oxidative stress pathways, and suggested that blueberry anthocyanin treatment potentially prevents hippocampal neuron injury.

Dopamine (DA) and norepinephrine (NE) are pivotal neuromodulators that regulate a broad range of brain functions, often in concert. Despite their physiological importance, untangling the relationship between DA and NE in the fine control of output function is currently challenging, primarily due to a lack of techniques to allow the observation of spatiotemporal dynamics with sufficiently high selectivity. Although genetically encoded fluorescent biosensors have been developed to detect DA, their poor selectivity prevents distinguishing DA from NE. Here, we report the development of a red fluorescent genetically encoded GPCR (G protein-coupled receptor)-activation reporter for DA termed 'R-GenGAR-DA'. More specifically, a circular permutated red fluorescent protein (cpmApple) was replaced by the third intracellular loop of human DA receptor D1 (DRD1) followed by the screening of mutants within the linkers between DRD1 and cpmApple. We developed two variants: R-GenGAR-DA1.1, which brightened following DA stimulation, and R-GenGAR-DA1.2, which dimmed. R-GenGAR-DA1.2 demonstrated a reasonable dynamic range (ΔF/F0 = − 43%), DA affinity (EC50 = 0.92 µM) and high selectivity for DA over NE (66-fold) in HeLa cells. Taking advantage of the high selectivity of R-GenGAR-DA1.2, we monitored DA in presence of NE using dual-color fluorescence live imaging, combined with the green-NE biosensor GRABNE1m, which has high selectivity for NE over DA (> 350-fold) in HeLa cells and hippocampal neurons grown from primary culture. Thus, this is a first step toward the multiplex imaging of these neurotransmitters in, for example, freely moving animals, which will provide new opportunities to advance our understanding of the high spatiotemporal dynamics of DA and NE in normal and abnormal brain function. [ABSTRACT FROM AUTHOR]

Aims: This study aimed to investigate whether electroacupuncture (EA) promotes the survival and synaptic plasticity of hippocampal neurons by activating brain‐derived neurotrophic factor (BDNF)/tyrosine receptor kinase (TrkB)/extracellular signal‐regulated kinase (Erk) signaling, thereby improving spatial memory deficits in rats under SD. Methods: In vivo, Morris water maze (MWM) was used to detect the effect of EA on learning and memory, at the same time Western blotting (WB), immunofluorescence (IF), and transmission electron microscopy (TEM) were used to explore the plasticity of hippocampal neurons and synapses, and the expression of BDNF/TrkB/Erk signaling. In vitro, cultured hippocampal neurons were treated with exogenous BDNF and the TrkB inhibitor K252a to confirm the relationship between BDNF/TrkB/Erk signaling and synaptic plasticity. Results: Our results showed that EA mitigated the loss of hippocampal neurons and synapses, stimulated hippocampal neurogenesis, and improved learning and memory of rats under SD accompanied by upregulation of BDNF and increased phosphorylation of TrkB and Erk. In cultured hippocampal neurons, exogenous BDNF enhanced the expression of synaptic proteins, the frequency of the postsynaptic currents, and the phosphorylation of TrkB and Erk; these effects were reversed by treatment with K252a. Conclusions: Electroacupuncture alleviates SD‐induced spatial memory impairment by promoting hippocampal neurogenesis and synaptic plasticity via activation of BDNF/TrkB/Erk signaling, which provided evidence for EA as a therapeutic strategy for countering the adverse effects of SD on cognition. [ABSTRACT FROM AUTHOR]

Most patients that resuscitate successfully from cardiac arrest (CA) suffer from poor neurological prognosis. DL‐3‐n‐butylphthalide (NBP) is known to have neuroprotective effects via multiple mechanisms. This study aimed to investigate whether NBP can decrease neurological impairment after CA. We studied the protective role of NBP in the hippocampus of a rat model of cardiac arrest induced by asphyxia. Thirty‐nine rats were divided randomly into sham, control, and NBP groups. Rats in control and NBP groups underwent cardiopulmonary resuscitation (CPR) 6 min after asphyxia. NBP or vehicle (saline) was administered intravenously 10 min after the return of spontaneous circulation (ROSC). Ultrastructure of hippocampal neurons was observed under transmission electron microscope. NBP treatment improved neurological function up to 72 h after CA. The ultrastructural lesion in mitochondria recovered in the NBP‐treated CA model. In conclusion, our study demonstrated multiple therapeutic benefits of NBP after CA. [ABSTRACT FROM AUTHOR]

Studies have found that salidroside, isolated from Rhodiola rosea L, has various pharmacological activities, but there have been no studies on the effects of salidroside on brain hippocampal senescence. The purpose of this study was to investigate the mechanistic role of salidroside in hippocampal neuron senescence and injury. In this study, long-term cultured primary rat hippocampal neurons and naturally aged C57 mice were treated with salidroside. The results showed that salidroside increased the viability and MAP2 expression, reduced β-galactosidase (β-gal) levels of rat primary hippocampal neurons. Salidroside also improved cognition dysfunction in ageing mice and alleviated neuronal degeneration in the ageing mice CA1 region. Moreover, salidroside decreased the levels of oxidative stress and p21, p16 protein expressions of hippocampal neurons and ageing mice. Salidroside promoted telomerase reverse transcriptase (TERT) protein expression via the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) pathway. In conclusion, our findings suggest that salidroside has the potential to be used as a therapeutic strategy for anti-ageing and ageing-related disease treatment. [ABSTRACT FROM AUTHOR]

Ceriporia lacerata (CL) is a species of white rot fungi. In this study, we have examined the beneficial effect of CL on scopolamine-induced memory impairment in mice. A freeze-dried CL mycelial culture broth was dissolved and orally administered to scopolamine-treated C57BL/6J mice followed by behavioral tests using the Y-maze, passive avoidance, and Morris water maze tasks. CL administration at a daily dose of 200 mg/kg body weight resulted in restoration of exploration reduction and improvement of associative and spatial learning and memory impairment in scopolamine-treated mice. Concomitantly, heme oxygenase-1 was highly expressed in the hippocampal region of CL-administered mice. Moreover, the ethanolic extract of CL significantly increased the transcriptional activity of antioxidant response element and attenuated the glutamate-induced cytotoxicity in HT22 mouse hippocampal neuronal cells. These findings suggest that the CL intake can confer a beneficial effect on learning and memory presumably through protecting hippocampal neuronal cells from oxidative stress-induced damage. [ABSTRACT FROM AUTHOR]