HEBP1 - An early trigger for neuronal cell death and circuit dysfunction in Alzheimer's disease.

Alzheimer's Disease (AD) is a progressive neurodegenerative disorder that gradually impairs memory, cognition and the ability to perform simple daily tasks. It is the most prevalent form of dementia in the elderly and its incidence increases exponentially with age. Neuronal and synapse loss, key hallmarks of the disorder, are widely regarded to occur early during the onset of AD, and the extent of this loss closely correlates with the progression of cognitive decline and dysfunction of the underlying neuronal circuity. Nevertheless, the mechanisms driving neuronal and synapse loss during early AD remains poorly understood. This review focuses on Heme-binding protein 1 (HEBP1), a mitochondrial-associated protein that has recently emerged as an important mediator of neuronal cell death during early AD pathogenesis. Acting downstream of Aß and heme, HEBP1-mediated apoptosis contributes to neuronal loss and neuronal circuit dysfunction. Deleting HEBP1 expression in neurons protects them from heme- and Aß-induced apoptosis, both of which are mechanisms implicated in neurodegeneration. HEBP1 participates in heme metabolism and binds to heme to modulate mitochondrial dynamics vital to the maintenance of neural circuitry that is affected in AD. HEBP1 elevation is also associated with AGE/RAGE-related neuronal damage, further implicating its involvement in neuronal loss during early AD. Moreover, F2L, a cleavage product of HEBP1 modulates inflammation. Collectively, these findings highlight the importance of HEBP1 in the disruption of neural circuits during early AD.

miR-449a mediated repression of the cell cycle machinery prevents neuronal apoptosis.

Aberrant activation of the cell cycle of terminally differentiated neurons results in their apoptosis and is known to contribute to neuronal loss in various neurodegenerative disorders like Alzheimer's Disease. However, the mechanisms that regulate cell cycle-related neuronal apoptosis are poorly understood. We identified several miRNA that are dysregulated in neurons from a transgenic APP/PS1 mouse model for AD (TgAD). Several of these miRNA are known to and/or are predicted to target cell cycle-related genes. Detailed investigation on miR-449a revealed the following: a, it promotes neuronal differentiation by suppressing the neuronal cell cycle; b, its expression in cortical neurons was impaired in response to amyloid peptide Aß42; c, loss of its expression resulted in aberrant activation of the cell cycle leading to apoptosis. miR-449a may prevent cell cycle-related neuronal apoptosis by targeting cyclin D1 and protein phosphatase CDC25A, which are important for G1-S transition. Importantly, the lentiviral-mediated delivery of miR-449a in TgAD mouse brain significantly reverted the defects in learning and memory, which are associated with AD.

Nrf2 dictates the neuronal survival and differentiation of embryonic zebrafish harboring compromised alanyl-tRNA synthetase.

tRNA synthetase deficiency leads to unfolded protein responses in neuronal disorders; however, its function in embryonic neurogenesis remains unclear. This study identified an aars1cq71/cq71 mutant zebrafish allele that showed increased neuronal apoptosis and compromised neurogenesis. aars1 transcripts were highly expressed in primary neural progenitor cells, and their aberration resulted in protein overloading and activated Perk. nfe2l2b, a paralog of mammalian Nfe2l2, which encodes Nrf2, is a pivotal executor of Perk signaling that regulates neuronal phenotypes in aars1cq71/cq71 mutants. Interference of nfe2l2b in nfe2l2bΔ1/Δ1 mutants did not affect global larval development. However, aars1cq71/cq71;nfe2l2bΔ1/Δ1 mutant embryos exhibited increased neuronal cell survival and neurogenesis compared with their aars1cq71/cq71 siblings. nfe2l2b was harnessed by Perk at two levels. Its transcript was regulated by Chop, an implementer of Perk. It was also phosphorylated by Perk. Both pathways synergistically assured the nuclear functions of nfe2l2b to control cell survival by targeting p53. Our study extends the understanding of tRNA synthetase in neurogenesis and implies that Nrf2 is a cue to mitigate neurodegenerative pathogenesis.

MicroRNA-29b-3p degenerates terminally differentiated dopaminergic SH-SY5Y cells by perturbation of mitochondrial functions.

Mitochondrial dysfunction is the main cause of gradual deterioration of structure and function of neuronal cells, eventually resulting in neurodegeneration. Studies have revealed a complex interrelationship between neurotoxicant exposure, mitochondrial dysfunction, and neurodegenerative diseases. Alteration in the expression of microRNAs (miRNAs) has also been linked with disruption in mitochondrial homeostasis and bioenergetics. In our recent research (Cellular and Molecular Neurobiology (2023) https://doi.org/10.1007/s10571-023-01362-4), we have identified miR-29b-3p as one of the most significantly up-regulated miRNAs in the blood of Parkinson's patients. The findings of the present study revealed that neurotoxicants of two different natures, that is, arsenic or rotenone, dramatically increased miR-29b-3p expression (18.63-fold and 12.85-fold, respectively) in differentiated dopaminergic SH-SY5Y cells. This dysregulation of miR-29b-3p intricately modulated mitochondrial morphology, induced oxidative stress, and perturbed mitochondrial membrane potential, collectively contributing to the degeneration of dopaminergic cells. Additionally, using assays for mitochondrial bioenergetics in live and differentiated SH-SY5Y cells, a reduction in oxygen consumption rate (OCR), maximal respiration, basal respiration, and non-mitochondrial respiration was observed in cells transfected with mimics of miR-29b-3p. Inhibition of miR-29b-3p by transfecting inhibitor of miR-29b-3p prior to exposure to neurotoxicants significantly restored OCR and other respiration parameters. Furthermore, we observed that induction of miR-29b-3p activates neuronal apoptosis via sirtuin-1(SIRT-1)/YinYang-1(YY-1)/peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α)-regulated Bcl-2 interacting protein 3-like-dependent mechanism. Collectively, our studies have shown the role of miR-29b-3p in dysregulation of mitochondrial bioenergetics during degeneration of dopaminergic neurons via regulating SIRT-1/YY-1/PGC-1α axis.

Extracellular vesicles in sepsis plasma mediate neuronal inflammation in the brain through miRNAs and innate immune signaling.

BACKGROUND: Neuroinflammation reportedly plays a critical role in the pathogenesis of sepsis-associated encephalopathy (SAE). We previously reported that circulating plasma extracellular vesicles (EVs) from septic mice are proinflammatory. In the current study, we tested the role of sepsis plasma EVs in neuroinflammation. METHODS: To track EVs in cells and tissues, HEK293T cell-derived EVs were labeled with the fluorescent dye PKH26. Cecal ligation and puncture (CLP) was conducted to model polymicrobial sepsis in mice. Plasma EVs were isolated by ultracentrifugation and their role in promoting neuronal inflammation was tested following intracerebroventricular (ICV) injection. miRNA inhibitors (anti-miR-146a, -122, -34a, and -145a) were applied to determine the effects of EV cargo miRNAs in the brain. A cytokine array was performed to profile microglia-released protein mediators. TLR7- or MyD88-knockout (KO) mice were utilized to determine the underlying mechanism of EVs-mediated neuroinflammation. RESULTS: We observed the uptake of fluorescent PKH26-EVs inside the cell bodies of both microglia and neurons. Sepsis plasma EVs led to a dose-dependent cytokine release in cultured microglia, which was partially attenuated by miRNA inhibitors against the target miRNAs and in TLR7-KO cells. When administered via the ICV, sepsis plasma EVs resulted in a marked increase in the accumulation of innate immune cells, including monocyte and neutrophil and cytokine gene expression, in the brain. Although sepsis plasma EVs had no direct effect on cytokine production or neuronal injury in vitro, the conditioned media (CM) of microglia treated with sepsis plasma EVs induced neuronal cell death as evidenced by increased caspase-3 cleavage and Annexin-V staining. Cytokine arrays and bioinformatics analysis of the microglial CM revealed multiple cytokines/chemokines and other factors functionally linked to leukocyte chemotaxis and migration, TLR signaling, and neuronal death. Moreover, sepsis plasma EV-induced brain inflammation in vivo was significantly dependent on MyD88. CONCLUSIONS: Circulating plasma EVs in septic mice cause a microglial proinflammatory response in vitro and a brain innate immune response in vivo, some of which are in part mediated by TLR7 in vitro and MyD88 signaling in vivo. These findings highlight the importance of circulating EVs in brain inflammation during sepsis.

Aerobic exercise mitigates hippocampal neuronal apoptosis by regulating DAPK1/CDKN2A/REDD1/FoXO1/FasL signaling pathway in D-galactose-induced aging mice.

Brain aging is the most important risk factor for neurodegenerative disorders, and abnormal apoptosis is linked to neuronal dysfunction. Specifically, studies have found that exercise effectively inhibits hippocampal neuronal apoptosis, while the molecular mechanism remains unclear. In the present study, we investigated the impact of aerobic exercise on hippocampal neuronal apoptosis in aging mice and the potential involvement of DAPK1 and its downstream pathways based on recent data that DAPK1 may be associated with neuronal death in neurodegenerative diseases. Senescent mice were subjected to 8 weeks of Aerobic training. Following behavioral testing, hippocampal samples were examined histologically and biochemically to detect pathological changes, neuronal apoptosis, and mRNA and protein levels. We found that the exercise intervention improved spatial memory and alleviated neuronal apoptosis in the brain. Notably, exercise down-regulated DAPK1 expression and inhibited Fas death receptor transactivation and the mitochondrial apoptotic pathway in the hippocampus. These results shed new light on the protective effect of regular exercise against brain aging though modulating the DAPK1 pathway.

Epidermal Neural Crest Stem Cell Conditioned Medium Enhances Spinal Cord Injury Recovery via PI3K/AKT-Mediated Neuronal Apoptosis Suppression.

This study aimed to assess the impact of conditioned medium from epidermal neural crest stem cells (EPI-NCSCs-CM) on functional recovery following spinal cord injury (SCI), while also exploring the involvement of the PI3K-AKT signaling pathway in regulating neuronal apoptosis. EPI-NCSCs were isolated from 10-day-old Sprague-Dawley rats and cultured for 48 h to obtain EPI-NCSC-CM. SHSY-5Y cells were subjected with H2O2 treatment to induce apoptosis. Cell viability and survival rates were evaluated using the CCK-8 assay and calcein-AM/PI staining. SCI contusion model was established in adult Sprague-Dawley rats to assess functional recovery, utilizing the Basso, Beattie and Bresnahan (BBB) scoring system, inclined test, and footprint observation. Neurological restoration after SCI was analyzed through electrophysiological recordings. Histological analysis included hematoxylin and eosin (H&E) staining and Nissl staining to evaluate tissue organization. Apoptosis and oxidative stress levels were assessed using TUNEL staining and ROS detection methods. Additionally, western blotting was performed to examine the expression of apoptotic markers and proteins related to the PI3K/AKT signaling pathway. EPI-NCSC-CM significantly facilitated functional and histological recovery in SCI rats by inhibiting neuronal apoptosis through modulation of the PI3K/AKT pathway. Administration of EPI-NCSCs-CM alleviated H2O2-induced neurotoxicity in SHSY-5Y cells in vitro. The use of LY294002, a PI3K inhibitor, underscored the crucial role of the PI3K/AKT signaling pathway in regulating neuronal apoptosis. This study contributes to the ongoing exploration of molecular pathways involved in spinal cord injury (SCI) repair, focusing on the therapeutic potential of EPI-NCSC-CM. The research findings indicate that EPI-NCSC-CM exerts a neuroprotective effect by suppressing neuronal apoptosis through activation of the PI3K/AKT pathway in SCI rats. These results highlight the promising role of EPI-NCSC-CM as a potential treatment strategy for SCI, emphasizing the significance of the PI3K/AKT pathway in mediating its beneficial effects.

SIRT1 restores mitochondrial structure and function in rats by activating SIRT3 after cerebral ischemia/reperfusion injury.

Mitochondrial dysfunction contributes to cerebral ischemia-reperfusion (CI/R) injury, which can be ameliorated by Sirtuin-3 (SIRT3). Under stress conditions, the SIRT3-promoted mitochondrial functional recovery depends on both its activity and expression. However, the approach to enhance SIRT3 activity after CI/R injury remains unelucidated. In this study, Sprague-Dawley (SD) rats were intracranially injected with either adeno-associated viral Sirtuin-1 (AAV-SIRT1) or AAV-sh_SIRT1 before undergoing transient middle cerebral artery occlusion (tMCAO). Primary cortical neurons were cultured and transfected with lentiviral SIRT1 (LV-SIRT1) and LV-sh_SIRT1 respectively before oxygen-glucose deprivation/reoxygenation (OGD/R). Afterwards, rats and neurons were respectively treated with a selective SIRT3 inhibitor, 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP). The expression, function, and related mechanism of SIRT1 were investigated by Western Blot, flow cytometry, immunofluorescence staining, etc. After CI/R injury, SIRT1 expression decreased in vivo and in vitro. The simulation and immune-analyses reported strong interaction between SIRT1 and SIRT3 in the cerebral mitochondria before and after CI/R. SIRT1 overexpression enhanced SIRT3 activity by increasing the deacetylation of SIRT3, which ameliorated CI/R-induced cerebral infarction, neuronal apoptosis, oxidative stress, neurological and motor dysfunction, and mitochondrial respiratory chain dysfunction, promoted mitochondrial biogenesis, and retained mitochondrial integrity and mitochondrial morphology. Meanwhile, SIRT1 overexpression alleviated OGD/R-induced neuronal death and mitochondrial bioenergetic deficits. These effects were reversed by AAV-sh_SIRT1 and the neuroprotective effects of SIRT1 were partially offset by 3-TYP. These results suggest that SIRT1 restores the structure and function of mitochondria by activating SIRT3, offering neuroprotection against CI/R injury, which signifies a potential approach for the clinical management of cerebral ischemia.

The P38MAPK/ATF2 signaling pathway is involved in PND in mice.

Accumulating evidence indicates that microglia-mediated neuroinflammation in the hippocampus contributes to the development of perioperative neurocognitive disorder (PND). P38MAPK, a point of convergence for different signaling processes involved in inflammation, can be activated by various stresses. This study aims to investigate the role of the P38MAPK/ATF2 signaling pathway in the development of PND in mice. Aged C57BL/6 mice were subjected to tibial fracture surgery under isoflurane anesthesia to establish a PND animal model. The open field test was used to evaluate the locomotor activity of the mice. Neurocognitive function was assessed with the Morris water maze (MWM) and fear conditioning test (FCT) on postoperative days 1, 3 and 7. The mice exhibited cognitive impairment accompanied by increased expression of proinflammatory factors (IL-1ß, TNF-α), proapoptotic molecules (caspase-3, bax) and microglial activation in the hippocampus 1, 3 and 7 days after surgery. Treatment with SB239063 (a P38MAPK inhibitor) decreased the expression of proinflammatory factors, proapoptotic molecules and Iba-1 in the CA1 region of the hippocampus. The number of surviving neurons was significantly increased. Inhibition of the P38MAPK/ATF2 signaling pathway attenuates hippocampal neuroinflammation and neuronal apoptosis in aged mice with PND, thus improving the perioperative cognitive function of the mice.

Long-Term Stimulation of the Left Dorsal Branch of the Thoracic Nerve Improves Ventricular Electrical Remodeling in a Canine Model of Chronic Myocardial Infarction.

PURPOSE: To evaluate the ventricular electrophysiologic effects of long-term stimulation of the left dorsal branch of thoracic nerve (LDTN) derived from the left stellate ganglion (LSG) in a canine model of chronic myocardial infarction (MI). METHODS: Seventeen adult male beagles were randomly divided into three groups: the sham group (sham operated, n = 6), the MI group (n = 6), and the MI + LDTN group (MI plus LDTN stimulation, n = 5). The canine model of chronic MI was induced by the occlusion of the left anterior descending artery (LADO). The LDTN was separated and intermittently stimulated immediately after LADO for 2 months. The heart rate variability (HRV) analysis, in vivo electrophysiology, the evaluation of LSG function and neural activity, histological staining, and western blotting (WB) assay were performed to evaluate the effect of LDTN stimulation on the heart. RESULTS: The canine MI model was successfully established by LADO, and the LDTN was separated and stimulated immediately after LADO. The HRV analysis showed that LDTN stimulation reversed the increased LF value and LF/HF ratio of the MI group. LDTN stimulation prolonged the shortening ERP and APD90, decreased the dispersion of ERP and APD90, and increased the VFT. Additionally, LDTN stimulation inhibits the LSG function and neural activity. Furthermore, LDTN stimulation suppressed the activation of Wnt/ß-catenin signaling, which contributed to the LSG neuronal apoptosis by upregulation of pro-apoptotic Bax and downregulation of anti-apoptotic Bcl-2. CONCLUSION: LDTN stimulation could attenuate cardiac sympathetic remodeling and improve ventricular electrical remodeling, which may be mediated by suppressing the activated Wnt/ß-catenin signaling pathway and then promoting the LSG neuronal apoptosis.

A novel anti-apoptotic role for Cdc42/ACK-1 signaling in neurons.

Neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's and Parkinson's disease are caused by a progressive and aberrant destruction of neurons in the brain and spinal cord. These disorders lack effective long-term treatments that impact the underlying mechanisms of pathogenesis and as a result, existing options focus primarily on alleviating symptomology. Dysregulated programmed cell death (i.e., apoptosis) is a significant contributor to neurodegeneration, and is controlled by a number of different factors. Rho family GTPases are molecular switches with recognized importance in proper neuronal development and migration that have more recently emerged as central regulators of apoptosis and neuronal survival. Here, we investigated a role for the Rho GTPase family member, Cdc42, and its downstream effectors, in neuronal survival and apoptosis. We initially induced apoptosis in primary cultures of rat cerebellar granule neurons (CGNs) by removing both growth factor-containing serum and depolarizing potassium from the cell medium. We then utilized both chemical inhibitors and adenoviral shRNA targeted to Cdc42 to block the function of Cdc42 or its downstream effectors under either control or apoptotic conditions. Our in vitro studies demonstrate that functional inhibition of Cdc42 or its downstream effector, activated Cdc42-associated tyrosine kinase-1 (ACK-1), had no adverse effects on CGN survival under control conditions, but significantly sensitized neurons to cell death under apoptotic conditions. In conclusion, our results suggest a key pro-survival role for Cdc42/ACK-1 signaling in neurons, particularly in regulating neuronal susceptibility to pro-apoptotic stress such as that observed in neurodegenerative disorders.

miR-128-3p is involved in aluminum-induced cognitive impairment by regulating the Sirt1-Keap1/Nrf2 pathway.

Aluminum (Al) is a common neurotoxicant in the environment, but the molecular mechanism of its toxic effects is still unclear. Studies have shown that aluminum exposure causes an increase in neuronal apoptosis. The aim of this study was to investigate the mechanism and signaling pathway of neuronal apoptosis induced by aluminum exposure. The rat model was established by intraperitoneal injection of maltol aluminum for 90 days. The results showed that the escape latency of the three groups exposed to maltol aluminum was higher than that of the control group on the 3rd, 4th and 5th days of the positioning cruise experiment (P < 0.05). On the 6th day of the space exploration experiment, compared with the control group(6.00 ± 0.71,15.33 ± 1.08) and the low-dose group(5.08 ± 1.69,13.67 ± 1.09), the number of times that the high-dose group crossed the platform(2.25 ± 0.76) and the platform quadrant(7.58 ± 1.43) was significantly reduced (P < 0.01). The relative expression levels of Sirt1 and Nrf2 in hippocampal tissues of all groups decreased gradually with increasing maltol aluminum exposure dose the relative expression levels of Sirt1 and Nrf2 in high-dose group (0.261 ± 0.094,0.325 ± 0.108) were significantly lower than those in control group (1.018 ± 0.222,1.009 ± 0.156)(P < 0.05). The relative expression level of Keap1 increased gradually with increasing maltol aluminum exposure dose (P < 0.05). The relative expression level of miR-128-3p in the high-dose group(1.520 ± 0.280) was significantly higher than that in the control group(1.000 ± 0.420) (P < 0.05). The content of GSH-Px in the hippocampus of rats decreased with increasing dose. ROS levels gradually increased. We speculated that subchronic aluminum exposure may lead to the activation of miR-128-3p in rat hippocampus of rats, thereby inhibiting the Sirt1-Keap1/Nrf2 pathway so that the Sirt1-Keap1/Nrf2 pathway could not be activated to exert antioxidant capacity, resulting in an imbalance in the antioxidant system of rats and the apoptosis of neurons, which caused reduced cognitive impairment in rats.

2,3-Diphosphoglyceric Acid Alleviating Hypoxic-Ischemic Brain Damage through p38 MAPK Modulation.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a critical condition characterized by significant brain damage due to insufficient blood flow and oxygen delivery at birth, leading to high rates of neonatal mortality and long-term neurological deficits worldwide. 2,3-Diphosphoglyceric acid (2,3-DPG), a small molecule metabolite prevalent in erythrocytes, plays an important role in regulating oxygen delivery, but its potential neuroprotective role in hypoxic-ischemic brain damage (HIBD) has yet to be fully elucidated. Our research reveals that the administration of 2,3-DPG effectively reduces neuron damage caused by hypoxia-ischemia (HI) both in vitro and in vivo. We observed a notable decrease in HI-induced neuronal cell apoptosis, attributed to the downregulation of Bax and cleaved-caspase 3, alongside an upregulation of Bcl-2 expression. Furthermore, 2,3-DPG significantly alleviates oxidative stress and mitochondrial damage induced by oxygen-glucose deprivation/reperfusion (OGD/R). The administration of 2,3-DPG in rats subjected to HIBD resulted in a marked reduction in brain edema and infarct volume, achieved through the suppression of neuronal apoptosis and neuroinflammation. Using RNA-seq analysis, we validated that 2,3-DPG offers protection against neuronal apoptosis under HI conditions by modulating the p38 MAPK pathway. These insights indicated that 2,3-DPG might act as a promising novel therapeutic candidate for HIE.

Epidermal Growth Factor Receptor Mediates Neuronal Apoptosis After Subarachnoid Hemorrhage in Mice.

BACKGROUND: Early brain injury including neuronal apoptosis is a main contributor to neurological deterioration after subarachnoid hemorrhage (SAH). This study was aimed to investigate whether EGFR (epidermal growth factor receptor)/NFκB (nuclear factor-kappa B) inducing kinase (NIK)/NFκB (p65 and p50) pathway is involved in the neuronal apoptosis after SAH in mice. METHODS: C57BL/6 adult male mice underwent endovascular perforation SAH modeling or sham-operation (n=286), and 86 mild SAH mice were excluded. In experiment 1, vehicle or an EGFR inhibitor (632.0 ng AG1478) was administered intraventricularly at 30 minutes postmodeling. At 24 or 72 hours, after neurological score was tested, brain water content, double immunolabeling with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and a neuronal marker antimicrotubule-associated protein-2 antibody, Western blotting using whole tissue lysate or nuclear protein extraction of the left cortex, and immunohistochemistry for cleaved caspase-3, phosphorylated (p-) EGFR, NIK, p-NFκB p65, and NFκB p105/50 were evaluated. In experiment 2, after sham or SAH modeling, AG1478+vehicle or AG1478+4.0 ng EGF was administered intraventricularly. The brain was used for TUNEL staining and immunohistochemistry after 24-hour observation. RESULTS: SAH group showed deteriorated neurological score (P<0.01, Mann-Whitney U test), more TUNEL- and cleaved caspase-3-positive neurons (P<0.01, ANOVA), and higher brain water content (P<0.01, Mann-Whitney U test), and these observations were improved in SAH-AG1478 group. Western blotting showed that expression levels of p-EGFR, p-p65, p50, and nuclear-NIK were increased after SAH (P<0.05, ANOVA), and decreased by AG1478 administration. Immunohistochemistry revealed these molecules localized in degenerating neurons. EGF administration resulted in neurological deterioration, increased TUNEL-positive neurons, and activation of EGFR, NIK, and NFκB. CONCLUSIONS: Activated EGFR, nuclear-NIK, and NFκB expressions were observed in cortical degenerating neurons after SAH, and were decreased by administration of AG1478, associated with suppression of TUNEL- and cleaved caspase-3-positive neurons. EGFR/NIK/NFκB pathway is suggested to be involved in neuronal apoptosis after SAH in mice.

Ethanol-induced ceramide production causes neuronal apoptosis by increasing MCL-1S-mediated ER-mitochondria contacts.

Heavy alcohol consumption causes neuronal cell death and cognitive impairment. Neuronal cell death induced by ethanol may result from increased production of the sphingolipid metabolite ceramide. However, the molecular mechanisms of neuronal cell death caused by ethanol-induced ceramide production have not been elucidated. Therefore, we investigated the mechanism through which ethanol-induced ceramide production causes neuronal cell apoptosis using human induced-pluripotent stem cell-derived neurons and SH-SY5Y cells and identified the effects of ceramide on memory deficits in C57BL/6 mice. First, we found that ethanol-induced ceramide production was decreased by inhibition of the de novo synthesis pathway, mediated by serine palmitoyltransferase (SPT). The associated alterations of the molecules related to the ceramide pathway suggest that the elevated level of ceramide activated protein phosphatase 1 (PP1), which inhibited the nuclear translocation of serine/arginine-rich splicing factor 1 (SRSF1). This led to aberrant splicing of myeloid cell leukemia 1 (MCL-1) pre-mRNA, which upregulated MCL-1S expression. Our results demonstrated that the interaction of MCL-1S with the inositol 1, 4, 5-trisphosphate receptor (IP3R) increases calcium release from the endoplasmic reticulum (ER) and then activated ER-bound inverted formin 2 (INF2). In addition, we discovered that F-actin polymerization through INF2 activation promoted ER-mitochondria contacts, which induced mitochondrial calcium influx and mitochondrial reactive oxygen species (mtROS) production. Markedly, MCL-1S silencing decreased mitochondria-associated ER membrane (MAM) formation and prevented mitochondrial calcium influx and mtROS accumulation, by inhibiting INF2-dependent actin polymerization interacting with mitochondria. Furthermore, the inhibition of ceramide production in ethanol-fed mice reduced MCL-1S expression, neuronal cell death, and cognitive impairment. In conclusion, we suggest that ethanol-induced ceramide production may lead to mitochondrial calcium overload through MCL-1S-mediated INF2 activation-dependent MAM formation, which promotes neuronal apoptosis.

Inhibition of neutrophil extracellular trap formation ameliorates neuroinflammation and neuronal apoptosis via STING-dependent IRE1α/ASK1/JNK signaling pathway in mice with traumatic brain injury.

BACKGROUND: Neuroinflammation is one of the most important pathogeneses in secondary brain injury after traumatic brain injury (TBI). Neutrophil extracellular traps (NETs) forming neutrophils were found throughout the brain tissue of TBI patients and elevated plasma NET biomarkers correlated with worse outcomes. However, the biological function and underlying mechanisms of NETs in TBI-induced neural damage are not yet fully understood. Here, we used Cl-amidine, a selective inhibitor of NETs to investigate the role of NETs in neural damage after TBI. METHODS: Controlled cortical impact model was performed to establish TBI. Cl-amidine, 2'3'-cGAMP (an activator of stimulating Interferon genes (STING)), C-176 (a selective STING inhibitor), and Kira6 [a selectively phosphorylated inositol-requiring enzyme-1 alpha [IRE1α] inhibitor] were administrated to explore the mechanism by which NETs promote neuroinflammation and neuronal apoptosis after TBI. Peptidyl arginine deiminase 4 (PAD4), an essential enzyme for neutrophil extracellular trap formation, is overexpressed with adenoviruses in the cortex of mice 1 day before TBI. The short-term neurobehavior tests, magnetic resonance imaging (MRI), laser speckle contrast imaging (LSCI), Evans blue extravasation assay, Fluoro-Jade C (FJC), TUNEL, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), western blotting, and quantitative-PCR were performed in this study. RESULTS: Neutrophils form NETs presenting in the circulation and brain at 3 days after TBI. NETs inhibitor Cl-amidine treatment improved short-term neurological functions, reduced cerebral lesion volume, reduced brain edema, and restored cerebral blood flow (CBF) after TBI. In addition, Cl-amidine exerted neuroprotective effects by attenuating BBB disruption, inhibiting immune cell infiltration, and alleviating neuronal death after TBI. Moreover, Cl-amidine treatment inhibited microglia/macrophage pro-inflammatory polarization and promoted anti-inflammatory polarization at 3 days after TBI. Mechanistically, STING ligand 2'3'-cGAMP abolished the neuroprotection of Cl-amidine via IRE1α/ASK1/JNK signaling pathway after TBI. Importantly, overexpression of PAD4 promotes neuroinflammation and neuronal death via the IRE1α/ASK1/JNK signaling pathway after TBI. However, STING inhibitor C-176 or IRE1α inhibitor Kira6 effectively abolished the neurodestructive effects of PAD4 overexpression after TBI. CONCLUSION: Altogether, we are the first to demonstrate that NETs inhibition with Cl-amidine ameliorated neuroinflammation, neuronal apoptosis, and neurological deficits via STING-dependent IRE1α/ASK1/JNK signaling pathway after TBI. Thus, Cl-amidine treatment may provide a promising therapeutic approach for the early management of TBI.

Cyanidin-3-O-glucoside protects the brain and improves cognitive function in APPswe/PS1ΔE9 transgenic mice model.

Cyanidin-3-O-glucoside (C3G) is a natural anthocyanin with antioxidant, anti-inflammatory, and antitumor properties. However, as the effects of C3G on the amyloidogenic pathway, autophagy, tau phosphorylation, neuronal cell death, and synaptic plasticity in Alzheimer's disease models have not been reported, we attempted to investigate the same in the brains of APPswe/PS1ΔE9 mice were analyzed. After oral administration of C3G (30 mg/kg/day) for 16 weeks, the cortical and hippocampal regions in the brains of APPswe/PS1ΔE9 mice were analyzed. C3G treatment reduced the levels of soluble and insoluble Aß (Aß40 and Aß42) peptides and reduced the protein expression of the amyloid precursor protein, presenilin-1, and ß-secretase in the cortical and hippocampal regions. And C3G treatment upregulated the expression of autophagy-related markers, LC3B-II, LAMP-1, TFEB, and PPAR-α and downregulated that of SQSTM1/p62, improving the autophagy of Aß plaques and neurofibrillary tangles. In addition, C3G increased the protein expression of phosphorylated-AMPK/AMPK and Sirtuin 1 and decreased that of mitogen-activated protein kinases, such as phosphorylated-Akt/Akt and phosphorylated-ERK/ERK, thus demonstrating its neuroprotective effects. Furthermore, C3G regulated the PI3K/Akt/GSK3ß signaling by upregulating phosphorylated-Akt/Akt and phosphorylated-GSK3ß/GSK3ß expression. C3G administration mitigated tau phosphorylation and improved synaptic function and plasticity by upregulating the expression of synapse-associated proteins synaptophysin and postsynaptic density protein-95. Although the potential of C3G in the APPswe/PS1ΔE9 mouse models has not yet been reported, oral administration of the C3G is shown to protect the brain and improve cognitive behavior.

Neuropilin-1 promotes mitochondrial structural repair and functional recovery in rats with cerebral ischemia.

OBJECTIVES: Available literature documents that ischemic stroke can disrupt the morphology and function of mitochondria and that the latter in other disease models can be preserved by neuropilin-1 (NRP-1) via oxidative stress suppression. However, whether NRP-1 can repair mitochondrial structure and promote functional recovery after cerebral ischemia is still unknown. This study tackled this very issue and explored the underlying mechanism. METHODS: Adeno-associated viral (AAV)-NRP-1 was stereotaxically inoculated into the cortex and ipsilateral striatum posterior of adult male Sprague-Dawley (SD) rats before a 90-min transient middle cerebral artery occlusion (tMCAO) and subsequent reperfusion. Lentivirus (LV)-NRP-1 was transfected into rat primary cortical neuronal cultures before a 2-h oxygen-glucose deprivation and reoxygenation (OGD/R) injury to neurons. The expression and function of NRP-1 and its specific protective mechanism were investigated by Western Blot, immunofluorescence staining, flow cytometry, magnetic resonance imaging, transmission electron microscopy, etc. The binding was detected by molecular docking and molecular dynamics simulation. RESULTS: Both in vitro and in vivo models of cerebral ischemia/reperfusion (I/R) injury presented a sharp increase in NRP-1 expression. The expression of AAV-NRP-1 markedly ameliorated the cerebral I/R-induced damage to the motor function and restored the mitochondrial morphology. The expression of LV-NRP-1 alleviated mitochondrial oxidative stress and bioenergetic deficits. AAV-NRP-1 and LV-NRP-1 treatments increased the wingless integration (Wnt)-associated signals and ß-catenin nuclear localization. The protective effects of NRP-1 were reversed by the administration of XAV-939. CONCLUSIONS: NRP-1 can produce neuroprotective effects against I/R injury to the brain by activating the Wnt/ß-catenin signaling pathway and promoting mitochondrial structural repair and functional recovery, which may serve as a promising candidate target in treating ischemic stroke.

ERK inhibition reduces neuronal death and ameliorates inflammatory responses in forebrain-specific Ppp2cα knockout mice.

It has been shown that PP2A is critical for apoptosis in neural progenitor cells. However, it remains unknown whether PP2A is required for neuronal survival. To address this question, we generated forebrain-specific Ppp2cα knockout (KO) mice. We show that Ppp2cα KO mice display robust neuronal apoptosis and inflammatory responses in the postnatal cortex. Previous evidence has revealed that PD98059 is a potent ERK inhibitor and may protect the brain against cell death after cardiac arrest. To study whether PD98059 may have any effects on Ppp2cα KO mice, the latter was treated with this inhibitor. We demonstrated that the total number of cleaved caspase3 positive (+) cells in the cortex was significantly reduced in Ppp2cα KO mice treated with PD98059 compared with those without PD98059 treatment. We observed that the total number of IBA1+ cells in the cortex was significantly decreased in Ppp2cα KO mice treated with PD98059. Mechanistic analysis reveals that deletion of PP2Aca causes DNA damage, which may be attenuated by PD98059. Together, this study suggests that inhibition of ERK may be an effective strategy to reduce cell death in brain diseases with abnormal neuronal apoptosis.

mTOR-dependent TFEB activation and TFEB overexpression enhance autophagy-lysosome pathway and ameliorate Alzheimer's disease-like pathology in diabetic encephalopathy.

BACKGROUND: Diabetic encephalopathy (DE) is a complication of type 2 diabetes mellitus (T2DM) that features Alzheimer's disease (AD)-like pathology, which can be degraded by the autophagy-lysosome pathway (ALP). Since transcription factor EB (TFEB) is a master regulator of ALP, TFEB-mediated ALP activation might have a therapeutic effect on DE, but this has yet to be investigated. METHODS: We established T2DM mouse models and cultured HT22 cells under high-glucose (HG) conditions to confirm the role of ALP in DE. To further investigate this, both mice and HT22 cells were treated with 3-methyladenine (3-MA). We also analyzed the content of TFEB in the nucleus and cytoplasm to evaluate its role in ALP. To confirm the effect of TFEB activation at the post-translational level in DE, we used rapamycin to inhibit the mechanistic target of rapamycin (mTOR). We transduced both mice and cells with TFEB vector to evaluate the therapeutic effect of TFEB overexpression on DE. Conversely, we conducted TFEB knockdown to verify its role in DE in another direction. RESULTS: We found that T2DM mice experienced compromised cognitive function, while HG-cultured HT22 cells exhibited increased cell apoptosis. Additionally, both T2DM mice and HG-cultured HT22 cells showed impaired ALP and heavier AD-like pathology. This pathology worsened after treatment with 3-MA. We also observed decreased TFEB nuclear translocation in both T2DM mice and HG-cultured HT22 cells. However, inhibiting mTOR with rapamycin or overexpressing TFEB increased TFEB nuclear translocation, enhancing the clearance of ALP-targeted AD-like pathology. This contributed to protection against neuronal apoptosis and alleviation of cognitive impairment. Conversely, TFEB knockdown lessened ALP-targeted AD-like pathology clearance and had a negative impact on DE. CONCLUSION: Our findings suggest that impaired ALP is responsible for the aggravation of AD-like pathology in T2DM. We propose that mTOR-dependent TFEB activation and TFEB overexpression are promising therapeutic strategies for DE, as they enhance the clearance of ALP-targeted AD-like pathology and alleviate neuronal apoptosis. Our study provides insight into the underlying mechanisms of DE and offers potential avenues for the development of new treatments for this debilitating complication of T2DM. Video abstract.