Ischemic Neuronal Cell Death Research Articles

LDHB-deficient brain exhibits resistance to ischemic neuronal cell death due to increased vasodilation

Ischemic stroke triggers a cascade of metabolic and inflammatory events leading to neuronal death, particularly in the hippocampus. Here, we investigate the role of lactate metabolism in ischemic resistance using LDHB-deficient mice, which exhibit impaired lactate utilization. Contrary to expectations of severe neuronal damage due to metabolic defects, LDHB-deficient mice displayed significantly increased neuronal survival following ischemic insult. Magnetic resonance spectroscopy revealed elevated lactate levels in LDHB-deficient brains, which correlated with enhanced vasodilation of the posterior communicating artery (PComA) and increased extracellular PGE2 levels. These findings suggest that elevated lactate inhibits PGE2 reabsorption, promoting vasodilation and neuronal protection. Our results highlight lactate's potential role in neuroprotection and its therapeutic promise for ischemic stroke.

Intranasal Delivery of Anti-Apoptotic siRNA Complexed with Fas-Signaling Blocking Peptides Attenuates Cellular Apoptosis in Brain Ischemia.

Ischemic stroke-induced neuronal cell death leads to the permanent impairment of brain function. The Fas-mediating extrinsic apoptosis pathway and the cytochrome c-mediating intrinsic apoptosis pathway are two major molecular mechanisms contributing to neuronal injury in ischemic stroke. In this study, we employed a Fas-blocking peptide (FBP) coupled with a positively charged nona-arginine peptide (9R) to form a complex with negatively charged siRNA targeting Bax (FBP9R/siBax). This complex is specifically designed to deliver siRNA to Fas-expressing ischemic brain cells. This complex enables the targeted inhibition of Fas-mediating extrinsic apoptosis pathways and cytochrome c-mediating intrinsic apoptosis pathways. Specifically, the FBP targets the Fas/Fas ligand signaling, while siBax targets Bax involved in mitochondria disruption in the intrinsic pathway. The FBP9R carrier system enables the delivery of functional siRNA to hypoxic cells expressing the Fas receptor on their surface-a finding validated through qPCR and confocal microscopy analyses. Through intranasal (IN) administration of FBP9R/siCy5 to middle cerebral artery occlusion (MCAO) ischemic rat models, brain imaging revealed the complex specifically localized to the Fas-expressing infarcted region but did not localize in the non-infarcted region of the brain. A single IN administration of FBP9R/siBax demonstrated a significant reduction in neuronal cell death by effectively inhibiting Fas signaling and preventing the release of cytochrome c. The targeted delivery of FBP9R/siBax represents a promising alternative strategy for the treatment of brain ischemia.

The MT1 receptor as the target of ramelteon neuroprotection in ischemic stroke.

Stroke is the leading cause of death and disability worldwide. Novel and effective therapies for ischemic stroke are urgently needed. Here, we report that melatonin receptor 1A (MT1) agonist ramelteon is a neuroprotective drug candidate as demonstrated by comprehensive experimental models of ischemic stroke, including a middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia in vivo, organotypic hippocampal slice cultures ex vivo, and cultured neurons in vitro; the neuroprotective effects of ramelteon are diminished in MT1-knockout (KO) mice and MT1-KO cultured neurons. For the first time, we report that the MT1 receptor is significantly depleted in the brain of MCAO mice, and ramelteon treatment significantly recovers the brain MT1 losses in MCAO mice, which is further explained by the Connectivity Map L1000 bioinformatic analysis that shows gene-expression signatures of MCAO mice are negatively connected to melatonin receptor agonist like Ramelteon. We demonstrate that ramelteon improves the cerebral blood flow signals in ischemic stroke that is potentially mediated, at least, partly by mechanisms of activating endothelial nitric oxide synthase. Our results also show that the neuroprotection of ramelteon counteracts reactive oxygen species-induced oxidative stress and activates the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway. Ramelteon inhibits the mitochondrial and autophagic death pathways in MCAO mice and cultured neurons, consistent with gene set enrichment analysis from a bioinformatics perspective angle. Our data suggest that Ramelteon is a potential neuroprotective drug candidate, and MT1 is the neuroprotective target for ischemic stroke, which provides new insights into stroke therapy. MT1-KO mice and cultured neurons may provide animal and cellular models of accelerated ischemic damage and neuronal cell death.

Effect of woohwangchungsimwon and donepezil co-treatment on cognitive function and serum metabolic profiles in a scopolamine-induced model of Alzheimer's disease

Ethnopharmacological relevanceWoohwangchungsimwon (WCW) is a traditional medicine used in East Asian countries to treat central nervous system disorders. Reported pharmacological properties include antioxidant effects, enhanced learning and memory, and protection against ischemic neuronal cell death, supporting its use in treating neurodegenerative diseases like Alzheimer's disease (AD). Aim of the studyThe study aims to assess the effects of co-treatment with WCW and donepezil on cognitive functions and serum metabolic profiles in a scopolamine-induced AD model. Materials and methodsCell viability and reactive oxygen species (ROS) levels were measured in amyloid β-peptide25–35 (Aβ25–35)-induced SH-SY5Y cells. An AD model was established in ICR mice by intraperitoneal scopolamine administration. Animals underwent the step-through passive avoidance test (PAT) and Morris water maze (MWM) test. Hippocampal tissues were collected to examine specific protein expression. Serum metabolic profiles were analyzed using nuclear magnetic resonance (NMR) spectroscopy. ResultsCo-treatment with WCW and donepezil increased cell viability and reduced ROS production in Aβ25–35-induced SH-SY5Y cells compared to that with donepezil treatment alone. Co-treatment improved cognitive functions and was comparable to donepezil treatment alone in the PAT and MWM tests. Pathways related to tyrosine, phenylalanine, and tryptophan biosynthesis, phenylalanine metabolism, and cysteine and methionine metabolism were altered by co-treatment. Levels of tyrosine and methionine, major serum metabolites in these pathways, were significantly reduced after co-treatment. ConclusionsCo-treatment with WCW and donepezil shows promise as a therapeutic strategy for AD and is comparable to donepezil alone in improving cognitive function. Reduced tyrosine and methionine levels after co-treatment may enhance cognitive function by mitigating hypertyrosinemia and hyperhomocysteinemia, known risk factors for AD. The serum metabolic profiles obtained in this study can serve as a foundation for developing other bioactive compounds using a scopolamine-induced mouse model.

The COP9 signalosome reduces neuroinflammation and attenuates ischemic neuronal stress in organotypic brain slice culture model

The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) is a deNEDDylase controlling ubiquitination activity of cullin-RING-E3 ligases (CRLs) and thus the levels of key cellular proteins. While the CSN and its catalytic subunit CSN5 have been extensively studied in cancer, its role in inflammatory and neurological diseases is less understood. Following verification that CSN5 is expressed in mouse and human brain, here we studied the role of the CSN in neuroinflammation and ischemic neuronal damage employing models of relevant brain-resident cell types, an ex vivo organotypic brain slice culture model, and the CRL NEDDylation state-modifying drugs MLN4924 and CSN5i-3, which mimic and inhibit, respectively, CSN5 deNEDDylase activity. Untargeted mass spectrometry-based proteomics revealed that MLN4924 and CSN5i-3 substantially alter the microglial proteome, including inflammation-related proteins. Applying these drugs and mimicking microglial and endothelial inflammation as well as ischemic neuronal stress by TNF and oxygen-glucose-deprivation/reoxygenation (OGD/RO) treatment, respectively, we could link CSN5/CSN-mediated cullin deNEDDylation to reduction of microglial inflammation, attenuated cerebral endothelial inflammation, improved barrier integrity, as well as protection from ischemic stress-induced neuronal cell death. Specifically, MLN4924 reduced phagocytic activity, motility, and inflammatory cytokine expression of microglial cells, and this was linked to inhibition of inflammation-induced NF-κB and Akt signaling. Inversely, Csn5 knockdown and CSN5i-3 increased NF-κB signaling. Moreover, MLN4924 abrogated TNF-induced NF-κB signaling in cerebral microvascular endothelial cells (hCMECs) and rescued hCMEC monolayers from OGD/RO-triggered barrier leakage, while CSN5i-3 exacerbated permeability. In an ex vivo organotypic brain slice model of ischemia/reperfusion stress, MLN4924 protected from neuronal death, while CSN5i-3 impaired neuronal survival. Neuronal damage was attributable to microglial activation and inflammatory cytokines, as indicated by microglial shape tracking and TNF-blocking experiments. Our results indicate a protective role of the CSN in neuroinflammation via brain-resident cell types involved in ischemic brain disease and implicate CSN activity-mimicking deNEDDylating drugs as potential therapeutics.

Association of Serum Calcium With Infarct Size and Severity in Acute Ischemic Stroke: A Rural Hospital-Based Cross-Sectional Study.

Background One of the major mediators of ischemic neuronal cell death is calcium. It has been found that elevated serum calcium is associated with a better prognosis in patients with ischemic stroke. This study highlights the association of serum calcium, albumin-corrected calcium, and ionic calcium with the size of acute ischemic stroke as well as severity outcome in terms of the National Institutes of Health Stroke Scale (NIHSS) score and Barthel Index. Methods This cross-sectional study was conducted on 85 cases of acute ischemic stroke (based on a computerized tomography scan of the brain) from September 2019 to October 2021. All included patients had undergone complete clinical history, systemic examination, as well as estimation of serum total calcium, albumin corrected calcium, and ionic calcium.NIHSS score and Barthel Index were used to access the severity of each subject. Results A significant positive correlation was seen between infarct size with NIHSS with a correlation coefficient of 0.35. A significant negative correlation was seen between infarct size with serum calcium, albumin-corrected calcium, and Barthel Index with a correlation coefficient of -0.483, -0.354, and -0.365 respectively. No correlation was seen between infarct size and ionic calcium with a correlation coefficient of 0.082. Conclusion It can be concluded that higher normal levels of serum calcium and albumin-corrected calcium are associated with a smaller-sized infarct and had less severity index among patients with acute ischemic stroke.

Preserving and enhancing mitochondrial function after stroke to protect and repair the neurovascular unit: novel opportunities for nanoparticle-based drug delivery.

The neurovascular unit (NVU) is composed of vascular cells, glia, and neurons that form the basic component of the blood brain barrier. This intricate structure rapidly adjusts cerebral blood flow to match the metabolic needs of brain activity. However, the NVU is exquisitely sensitive to damage and displays limited repair after a stroke. To effectively treat stroke, it is therefore considered crucial to both protect and repair the NVU. Mitochondrial calcium (Ca2+) uptake supports NVU function by buffering Ca2+ and stimulating energy production. However, excessive mitochondrial Ca2+ uptake causes toxic mitochondrial Ca2+ overloading that triggers numerous cell death pathways which destroy the NVU. Mitochondrial damage is one of the earliest pathological events in stroke. Drugs that preserve mitochondrial integrity and function should therefore confer profound NVU protection by blocking the initiation of numerous injury events. We have shown that mitochondrial Ca2+ uptake and efflux in the brain are mediated by the mitochondrial Ca2+ uniporter complex (MCUcx) and sodium/Ca2+/lithium exchanger (NCLX), respectively. Moreover, our recent pharmacological studies have demonstrated that MCUcx inhibition and NCLX activation suppress ischemic and excitotoxic neuronal cell death by blocking mitochondrial Ca2+ overloading. These findings suggest that combining MCUcx inhibition with NCLX activation should markedly protect the NVU. In terms of promoting NVU repair, nuclear hormone receptor activation is a promising approach. Retinoid X receptor (RXR) and thyroid hormone receptor (TR) agonists activate complementary transcriptional programs that stimulate mitochondrial biogenesis, suppress inflammation, and enhance the production of new vascular cells, glia, and neurons. RXR and TR agonism should thus further improve the clinical benefits of MCUcx inhibition and NCLX activation by increasing NVU repair. However, drugs that either inhibit the MCUcx, or stimulate the NCLX, or activate the RXR or TR, suffer from adverse effects caused by undesired actions on healthy tissues. To overcome this problem, we describe the use of nanoparticle drug formulations that preferentially target metabolically compromised and damaged NVUs after an ischemic or hemorrhagic stroke. These nanoparticle-based approaches have the potential to improve clinical safety and efficacy by maximizing drug delivery to diseased NVUs and minimizing drug exposure in healthy brain and peripheral tissues.

Pharmacokinetics of hydrogen administered intraperitoneally as hydrogen-rich saline and its effect on ischemic neuronal cell death in the brain in gerbils.

Intraperitoneal administration of hydrogen (H2)-containing saline inhibited neuronal cell death in ischemic stroke in a number of animal models, but it is unknown whether H2 is absorbed from the abdominal cavity into the blood and reaches the brain. In this study, we investigated whether intraperitoneal administration of saline containing H2 inhibits neuronal cell death caused by cerebral ischemia and measured the concentration of H2 in the carotid artery and inferior vena cava (IVC). Gerbils were subjected to transient unilateral cerebral ischemia twice, and saline or H2-rich saline was administered intraperitoneally three or seven times every 12 hours. We evaluated the number of apoptotic cells in the hippocampus and cerebral cortex on day 3 and the number of viable neurons in the hippocampus and cerebral cortex on day 7. In addition, a single dose of saline or H2-rich saline was administered intraperitoneally, and blood H2 levels in the carotid artery and IVC were measured. On day 3 of ischemia/reperfusion, the number of neurons undergoing apoptosis in the cortex was significantly lower in the H2-rich saline group than in the saline group, and on day 7, the number of viable neurons in the hippocampus and cerebral cortex was significantly higher in the H2-rich saline group. Intraperitoneal administration of H2-rich saline resulted in large increases in H2 concentration in the IVC ranging from 0.00183 mg/L (0.114%) to 0.00725 mg/L (0.453%). In contrast, carotid H2 concentrations remained in the range of 0.00008 mg/L (0.0049%) to 0.00023 (0.0146%). On average, H2 concentrations in carotid artery were 0.04 times lower than in IVC. These results indicate that intraperitoneal administration of H2-rich saline significantly suppresses neuronal cell death after cerebral ischemia, even though H2 hardly reaches the brain.

Rapamycin ameliorates brain damage and maintains mitochondrial dynamic balance in diabetic rats subjected to middle cerebral artery occlusion.

To investigate the effect of rapamycin on mitochondrial dynamic balance in diabetic rats subjected to cerebral ischemia-reperfusion injury. Male Sprague Dawley (SD) rats (n = 78) were treated with high fat diet combined with streptozotocin injection to construct diabetic model in rats. Transient middle cerebral artery occlusion (MCAO) of 2hours was induced and the brains were harvested after 1 and 3days of reperfusion. Rapamycin was injected intraperitoneally for 3days prior to and immediately after operation, once a day. The neurological function was assessed, infarct volumes were measured and HE staining as well as immunohistochemistry were performed. The protein of hippocampus was extracted and Western blotting were performed to detect the levels of mTOR, mitochondrial dynamin related proteins (DRP1, p-DRP1, OPA1), SIRT3, and Nix/BNIP3L. Diabetic hyperglycemia worsened the neurological function performance (p < 0.01), enlarged infarct size (p < 0.01) and increased ischemic neuronal cell death (p < 0.01). The increased damage was associated with elevations of p-mTOR, p-S6, and p-DRP1; and suppressions of SIRT3 and Nix/BNIP3L. Rapamycin ameliorated diabetes-enhanced ischemic brain damage and reversed the biomarker alterations caused by diabetes. High glucose activated mTOR pathway and caused mitochondrial dynamics toward fission. The protective effect of rapamycin against diabetes-enhanced ischemic brain damage was associated with inhibiting mTOR pathway, redressing mitochondrial dynamic imbalance, and elevating SIRT3 and Nix/BNIP3L expression.

Stepwise disassembly of GABAergic synapses during pathogenic excitotoxicity.

SUMMARYGABAergic synaptic inhibition controls neuronal firing, excitability, and synaptic plasticity to regulate neuronal circuits. Following an acute excitotoxic insult, inhibitory synapses are eliminated, reducing synaptic inhibition, elevating circuit excitability, and contributing to the pathophysiology of brain injuries. However, mechanisms that drive inhibitory synapse disassembly and elimination are undefined. We find that inhibitory synapses are disassembled in a sequential manner following excitotoxicity: GABAARs undergo rapid nanoscale rearrangement and are dispersed from the synapse along with presynaptic active zone components, followed by the gradual removal of the gephyrin scaffold, prior to complete elimination of the presynaptic terminal. GABAAR nanoscale reorganization and synaptic declustering depends on calcineurin signaling, whereas disassembly of gephyrin relies on calpain activation, and blockade of both enzymes preserves inhibitory synapses after excitotoxic insult. Thus, inhibitory synapse disassembly occurs rapidly, with nanoscale precision, in a stepwise manner and most likely represents a critical step in the progression of hyperexcitability following excitotoxicity.

CaMKIIα knockout protects from ischemic neuronal cell death after resuscitation from cardiac arrest

CaMKIIα plays a dual role in synaptic plasticity, as it can mediate synaptic changes in opposing directions. We hypothesized that CaMKIIα plays a similar dual role also in neuronal cell death and survival. Indeed, the CaMKII inhibitor tatCN21 is neuroprotective when added during or after excitotoxic/ischemic insults, but was described to cause sensitization when applied long-term prior to such insult. However, when comparing long-term CaMKII inhibition by several different inhibitors in neuronal cultures, we did not detect any sensitization. Likewise, in a mouse in vivo model of global cerebral ischemia (cardiac arrest followed by cardiopulmonary resuscitation), complete knockout of the neuronal CaMKIIα isoform did not cause sensitization but instead significant neuroprotection.

Emerging role of PARP-1 and PARthanatos in ischemic stroke.

Cell death is a key feature of neurological diseases, including stroke and neurodegenerative disorders. Studies in a variety of ischemic/hypoxic mouse models demonstrate that poly(ADP-ribose) polymerase 1 (PARP-1)-dependent cell death, also named PARthanatos, plays a pivotal role in ischemic neuronal cell death and disease progress. PARthanatos has its unique triggers, processors, and executors that convey a highly orchestrated and programmed signaling cascade. In addition to its role in gene transcription, DNA damage repair, and energy homeostasis through PARylation of its various targets, PARP-1 activation in neuron and glia attributes to brain damage following ischemia/reperfusion. Pharmacological inhibition or genetic deletion of PARP-1 reduces infarct volume, eliminates inflammation, and improves recovery of neurological functions in stroke. Here, we reviewed the role of PARP-1 and PARthanatos in stroke and their therapeutic potential.

Serum Levels of Soluble Triggering Receptor Expressed on Myeloid Cells-1 Associated with the Severity and Outcome of Acute Ischemic Stroke.

Stroke is a neurological emergency, where the mechanism of the blood supply to the brain is impaired, resulting in brain cell ischemia and death. Neuroinflammation is a key component in the ischemic cascade that results in cell damage and death after cerebral ischemia. The triggering receptor expressed on myeloid cells-1 (TREM-1) modulates neuroinflammation after acute ischemic stroke. In the present study, 60 patients with acute ischemic stroke, who had been subjected to neurological examinations and National Institutes of Health Stroke Scale (NIHSS) and brain magnetic resonance imaging studies, were enrolled in the emergency room of Kaohsiung Chang Gung Memorial Hospital. Twenty-four healthy volunteers were recruited as controls. The serum levels of soluble TREM-1 (sTREM-1), human S100 calcium-binding protein B (S100B), and proinflammatory cytokines and chemokines, including tumor necrosis α (TNF-α), interleukin 1β, interleukin 6 (IL-6), interleukin 8, and interferon-γ were measured immediately after acute ischemic stroke. The serum levels of sTREM-1, TNFα, IL-6, and S100B were correlated with the stroke volume and NIHSS, after acute ischemic stroke. Additionally, the serum levels of sTREM-1 were significantly positively correlated with S100B. The functional outcomes were evaluated 6 months after ischemic stroke by the Barthel index, which was correlated with the age and levels of sTREM-1 and S100B. We suggest that acute ischemic stroke induces neuroinflammation by the activation of the TREM-1 signaling pathway and the downstream inflammatory machinery that modulates the inflammatory response and ischemic neuronal cell death. From a translational perspective, our results may allow for the development of a new therapeutic strategy for acute ischemic stroke by targeting the TREM-1 signaling pathway.

Waixenicin A, a marine-derived TRPM7 inhibitor: a promising CNS drug lead.

Ion channels are the third largest class of targets for therapeutic drugs. The pharmacology of ion channels is an important research area for identifying new treatment options for human diseases. The past decade or so has seen increasing interest in an ion channel protein belonging to the transient receptor potential (TRP) family, namely the melastatin subfamily member 7 (TRPM7), as an emerging drug target. TRPM7 is a bifunctional protein with amagnesium and calcium-conducting divalent ion channel fused with an active kinase domain. TRPM7 is ubiquitously expressed in human tissues, including the brain, and regulates various cell biology processes such as magnesium and calcium homeostasis, cell growth and proliferation, and embryonic development. TRPM7 provides a link between cellular metabolic status and intracellular calcium homeostasis in neurons due to TRPM7's unique sensitivity to fluctuating intracellular Mg·ATP levels. Thus, the protein plays a key role in ischemic and hypoxic neuronal cell death and brain injury, and is one of the key nonglutamate mechanisms in cerebral ischemia and stroke. Currently, the most potent and specific TRPM7 inhibitor is waixenicin A, a xenicane diterpenoid from the Hawaiian soft coral Sarcothelia edmondsoni. Using waixenicin A as a pharmacological tool, we demonstrated that TRPM7 is involved in promoting neurite outgrowth in vitro. Most recently, we found that waixenicin A reduced hypoxic-ischemic brain injury and preserved long-term behavioral outcomes in mouse neonates. We here suggest that TRPM7 is an emerging drug target for CNS diseases and disorders, and waixenicin A is a viable drug lead for these disorders.

Galectin-3 protects against ischemic stroke by promoting neuro-angiogenesis via apoptosis inhibition and Akt/Caspase regulation

Post-stroke neurological deficits and mortality are often associated with vascular disruption and neuronal apoptosis. Galectin-3 (Gal3) is a potent pro-survival and angiogenic factor. However, little is known about its protective role in the cerebral ischemia/reperfusion (I/R) injury. We have previously shown significant up-regulation of Gal3 in the post-stroke rat brain, and that blocking of Gal3 with neutralizing antibody decreases the cerebral blood vessel density. Our current study demonstrates that intracerebral local delivery of the Gal3 into rat brain at the time of reperfusion exerts neuroprotection. Ischemic lesion volume and neuronal cell death were significantly reduced as compared with the vehicle-treated MCAO rat brains. Gal3 increased vessel density and neuronal survival after I/R in rat brains. Importantly, Gal3-treated groups showed significant improvement in motor and sensory functional recovery. Gal3 increased neuronal cell viability under in vitro oxygen–glucose deprivation conditions in association with increased phosphorylated-Akt, decreased phosphorylated-ERK1/2, and reduced caspase-3 activity. Gene expression analysis showed down regulation of pro-apoptotic and inflammatory genes including Fas-ligand, and upregulation of pro-survival and pro-angiogenic genes including Bcl-2, PECAM, and occludin. These results indicate a key role for Gal3 in neuro-vascular protection and functional recovery following ischemic stroke through modulation of angiogenic and apoptotic pathways.

TRPM7 Mediates Neuronal Cell Death Upstream of Calcium/Calmodulin-Dependent Protein Kinase II and Calcineurin Mechanism in Neonatal Hypoxic-Ischemic Brain Injury.

Transient receptor potential melastatin 7 (TRPM7), a calcium-permeable, ubiquitously expressed ion channel, is critical for axonal development, and mediates hypoxic and ischemic neuronal cell death in vitro and in vivo. However, the downstream mechanisms underlying the TRPM7-mediated processes in physiology and pathophysiology remain unclear. In this study, we employed a mouse model of hypoxic-ischemic brain cell death which mimics the pathophysiology of hypoxic-ischemic encephalopathy (HIE). HIE is a major public health issue and an important cause of neonatal deaths worldwide; however, the available treatments for HIE remain limited. Its survivors face life-long neurological challenges including mental retardation, cerebral palsy, epilepsy and seizure disorders, motor impairments, and visual and auditory impairments. Through a proteomic analysis, we identified calcium/calmodulin-dependent protein kinase II (CaMKII) and phosphatase calcineurin as potential mediators of cell death downstream from TRPM7 activation. Further analysis revealed that TRPM7 mediates cell death through CaMKII, calmodulin, calcineurin, p38, and cofilin cascade. In vivo, we found a significant reduction of brain injury and improvement of short- and long-term functional outcomes after HI after administration of specific TRPM7 blocker waixenicin A. Our data demonstrate a molecular mechanism of TRPM7-mediated cell death and identifies TRPM7 as a promising therapeutic and drug development target for HIE.

Novel Targets for Stroke Therapy: Special Focus on TRPC Channels and TRPC6.

Stroke remains a leading cause of death, disability, and medical care burden worldwide. However, transformation from laboratory findings toward effective pharmacological interventions for clinical stroke has been unsatisfactory. Novel evidence has been gained on the underlying mechanisms and therapeutic potential related to the transient receptor potential (TRP) channels in several disorders. The TRP superfamily consists of a diverse group of Ca2+ permeable non-selective cation channels. In particular, the members of TRP subfamilies, TRP canonical (TRPC) channels and TRPC6, have been found in different cell types in the whole body and have high levels of expression in the central nervous system (CNS). Notably, the TRPCs and TRPC6 channel have been implicated in neurite outgrowth and neuronal survival during normal development and in a range of CNS pathological conditions. Recent studies have shown that suppression of TRPC6 channel degradation prevents ischemic neuronal cell death in experimental stroke. Accumulating evidence supports the important functions of TRPC6 in brain ischemia. We have highlighted some crucial advancement that points toward an important involvement of TRPCs and TRPC6 in ischemic stroke. This review will make an overview of the TRP and TRPC channels due to their roles as targets for clinical trials and CNS disorders. Besides, the primary goal is to discuss and update the critical role of TRPC6 channels in stroke and provide a promising target for stroke prevention and therapy.

CaMKII versus DAPK1 Binding to GluN2B in Ischemic Neuronal Cell Death after Resuscitation from Cardiac Arrest

SUMMARYDAPK1 binding to GluN2B was prominently reported to mediate ischemic cell death in vivo. DAPK1 and CaMKII bind to the same GluN2B region, and their binding is mutually exclusive. Here, we show that mutating the binding region on GluN2B (L1298A/ R1300Q) protected against neuronal cell death induced by cardiac arrest followed by resuscitation. Importantly, the GluN2B mutation selectively abolished only CaMKII, but not DAPK1, binding. During ischemic or excitotoxic insults, CaMKII further accumulated at excitatory synapses, and this accumulation was mediated by GluN2B binding. Interestingly, extra-synaptic GluN2B decreased after ischemia, but its relative association with DAPK1 increased. Thus, ischemic neuronal death requires CaMKII binding to synaptic GluN2B, whereas any potential role for DAPK1 binding is restricted to a different, likely extra-synaptic population of GluN2B.

Deletion of Chitinase-3-like 1 accelerates stroke development through enhancement of Neuroinflammation by STAT6-dependent M2 microglial inactivation in Chitinase-3-like 1 knockout mice

Chitinase 3-like 1 (Chi3L1) plays a major role in the pathogenesis of inflammatory diseases. We investigated the effect of Chi3L1 knockout on stroke development. Ischemia/reperfusion was induced by middle cerebral artery occlusion (MCAO) in Chi3L1 knockout and wildtype mice. Significantly increased infarct volume and decreased neurological deficit scores at 24 h after ischemia/reperfusion were found in Chi3L1 knockout mice compared to wildtype mice. Moreover, ischemic neuronal cell death was increased in Chi3L1 knockout mice through increased oxidative stress and release of IL-6 and IL-1β but IL-10 and IL-4 were reduced. Furthermore, expression of inflammation-related proteins (iNOS, COX-2, Iba-1, and GFAP) was significantly increased in Chi3L1 knockout mice compared to wildtype. In microglia isolated from MCAO-injured Chi3L1 knockout mice, expression of M1 markers (iNOS, CD86, IL-1β, and IL-6) was increased and M2 markers (Arg1, Mrc1, IL-10, and IL-4Ra) was decreased. In BV-2 cells, knockdown of Chi3L1 increased TNF-α- and INF-γ-induced expression of iNOS, COX-2, and Iba-1, but decreased the expression of Arg1, MRC1, and IL-4 receptor-alpha (IL-4Rα). Expression of IL-4Rα, an important factor of M2 polarization, and its downstream signals p-JAK1, p-JAK3, and p-STAT6, was much reduced in the knockout mice. Additionally, in BV-2 cells, knockdown of Chi3L1 by siRNA Chi3L1 decreased rhTNF-α- and INF-γ-induced expression of IL-4Rα, p-JAK1, p-JAK3, and p-STAT6. Furthermore, treatment with AS1517499 abolished Chi3L1 knockdown-induced reduced IL-4Rα and Arg1 but not CD86 expression. Our results indicate that deletion of Chi3L1 accelerates stroke development through enhancement of neuroinflammation by markedly decreasing STAT6-dependent M2 macrophage polarization.

Preconditioning exercise reduces brain damage and neuronal apoptosis through enhanced endogenous 14-3-3γ after focal brain ischemia in rats.

14-3-3γ is an important early ischemia-inducible protective factor against ischemic cell death in cerebral cortical neurons. We investigated the anti-apoptosis mechanism of enhanced 14-3-3γ mediated by preconditioning exercise-induced brain ischemic tolerance after stroke. Rats were assigned to four groups: exercise and ischemia (Ex group), ischemia and no exercise (No-Ex group), exercise and no ischemia (Ex-only group), and no exercise and ischemia (control group). Rats were trained on a treadmill for 5days a week for 3weeks (running speed, 25m/min; running duration, 30min/day). After the exercise program, stroke was induced by left middle cerebral artery occlusion. The infarct volume, neurological deficits, and motor function, as well as expression levels of hypoxia-induced factor-1α (HIF-1α), 14-3-3γ, P2X7 receptors, p-β-catenin Ser37, Bax, and caspase 3 were evaluated by immunohistochemistry and western blotting. The expression of HIF-1α and 14-3-3γ significantly increased in neurons and astrocytes in the Ex-only group. HIF-1α was co-expressed with P2X7 receptor- and GFAP-positive astrocytes. After stroke, the Ex group had significantly reduced brain infarction. HIF-1α and 14-3-3γ significantly increased in the Ex group compared to the No-Ex group. In addition, p-β-catenin Ser37 significantly increased following elevated 14-3-3γ; in contrast, Bax and caspase 3 were significantly reduced in the Ex group. Our findings suggest that preconditioning exercise prior to ischemia induces neuron- and astrocyte-mediated brain ischemic tolerance through increased expression of HIF-1α and 14-3-3γ, which are intrinsic protective factors; the upregulated 14-3-3γ induced by preconditioning exercise reduces ischemic neuronal cell death through the 14-3-3γ/p-β-catenin Ser37/Bax/caspase 3 anti-apoptotic pathway.