calpain and Stroke

Postsynaptic density protein-95 (PSD-95) is a multidomain protein critical to the assembly of signaling complexes at excitatory synapses, required for neuronal survival and function. However, calpain-processing challenges PSD-95 function after overactivation of excitatory glutamate receptors (excitotoxicity) in stroke, a leading cause of death, disability and dementia in need of efficient pharmacological treatments. A promising strategy is neuroprotection of the infarct penumbra, a potentially recoverable area, by promotion of survival signaling. Interference of PSD-95 processing induced by excitotoxicity might thus be a therapeutic target for stroke and other excitotoxicity-associated pathologies.

Topics: Animals; Brain Ischemia; Calpain; Cell Survival; Cell-Penetrating Peptides; Cells, Cultured; Disease Models, Animal; Disks Large Homolog 4 Protein; Down-Regulation; Excitatory Amino Acid Agonists; HEK293 Cells; Humans; Male; Mice; Mice, Inbred BALB C; Movement Disorders; N-Methylaspartate; Neurons; Neuroprotection; Stroke

Ischemic stroke has become one of the leading causes of deaths and disabilities all over the world. In this study, we investigated the therapeutic effects of combined bone marrow stromal cells (BMSCs) and oxiracetam treatments on acute cerebral ischemia/reperfusion (I/R) injury. A rat model of middle cerebral artery occlusion (MCAO) followed by complete reperfusion, as well as a cortex neuron oxygen-glucose deprivation (OGD) model was established. When compared with BMSCs or oxiracetam monotherapy, combination therapy significantly improved functional restoration with decreased infarct volume in observed ischemic brain. We propose that it may occur through the transient receptor potential canonical (TRPC)6 neuron survival pathway. The increased expression of TRPC6 along with the reduction of neuronal cell death in the OGD cortex neurons and combination therapy group indicated that the TRPC6 neuron survival pathway plays an important role in the combined BMSCs and oxiracetam treatments. We further tested the activity of the calpain proteolytic system, and the results suggested that oxiracetam could protect the integrity of TRPC6 neuron survival pathway by inhibiting TRPC6 degradation. The protein levels of phospho-cAMP response element binding protein (p-CREB) were tested. It was found that BMSCs play a role in the activation of the TRPC6 pathway. Our study suggests that the TRPC6 neuron survival pathway plays a significant role in the protective effect of combined BMSCs and oxiracetam treatments on acute cerebral I/R injury. Combined therapy could inhibit the abnormal degradation of TRPC6 via decreasing the activity of calpain and increasing the activation of TRPC6 neuron survival pathway.

Topics: Animals; Bone Marrow Transplantation; Brain Ischemia; Calpain; Cerebral Cortex; Cyclic AMP Response Element-Binding Protein; Glucose; Infarction, Middle Cerebral Artery; Male; Mesenchymal Stem Cells; Neurons; Neuroprotective Agents; Oxygen; Pyrrolidines; Rats; Rats, Wistar; Reperfusion Injury; Stroke; Treatment Outcome; TRPC Cation Channels

Neuroprotective strategies aimed to pharmacologically treat stroke, a prominent cause of death, disability, and dementia, have remained elusive. A promising approach is restriction of excitotoxic neuronal death in the infarct penumbra through enhancement of survival pathways initiated by brain-derived neurotrophic factor (BDNF). However, boosting of neurotrophic signaling after ischemia is challenged by downregulation of BDNF high-affinity receptor, full-length tropomyosin-related kinase B (TrkB-FL), due to calpain-degradation, and, secondarily, regulated intramembrane proteolysis. Here, we have designed a blood-brain barrier (BBB) permeable peptide containing TrkB-FL sequences (TFL

Topics: Animals; Brain Ischemia; Calpain; Cyclic AMP Response Element-Binding Protein; Disease Models, Animal; Down-Regulation; Male; MEF2 Transcription Factors; Membrane Glycoproteins; Mice; Neurons; Neuroprotection; Peptides; Protein-Tyrosine Kinases; Proteolysis; Stroke

Enriched environment (EE) has been shown to promote neurogenesis and functional recovery after ischemic stroke. However, the underlying molecular mechanisms are not fully understood. In this study, C57BL/6 mice underwent middle cerebral artery occlusion (60 min) followed by reperfusion, after which mice were housed in either standard environment (SE) or EE and allowed to survive for 3, 4, 6 or 10 weeks. Ipsilateral subventricular zone (SVZ) or striatum cells were dissociated from ischemic hemispheric brains of enriched mice at 14 days post-ischemia (dpi) and cultured in vitro. Our data showed that post-ischemic EE inhibited calpain 1 activity, and increased the expression of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in the ischemic hemisphere of enriched mice at 21 dpi. Calpain 1-specific inhibitor PD151746 further increased p-STAT3 expression and augmented the promoting effects of EE on post-stroke SVZ neural precursor cells (NPCs) proliferation and functional recovery. EE also increased the expression of hypoxia-inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in the ischemic hemisphere at 21 dpi. Inhibition of the JAK/STAT3 pathway with AG490 decreased the expression of HIF-1α and VEGF. Furthermore, inhibition of HIF-1α with 2-methoxyestradiol robustly abolished EE-induced elevation of VEGF l expression. Furthermore, VEGF-A promoted the production and secretion of high mobility group box-1 protein (HMGB1) from reactive astrocytes in vitro. Culture supernatant from reactive astrocytes treated with VEGF-A promoted the proliferation and differentiation of NPCs. Glycyrrhizin reversed the promoting effects of EE on post-stroke neurorepair and functional recovery in vivo. Taken together, our data indicate that EE promotes post-stroke functional recovery through the inhibition of calpain 1 activity, and subsequent STAT3-HIF-1α-VEGF-mediated neurogenesis.

Topics: Acrylates; Analysis of Variance; Animals; Astrocytes; Bromodeoxyuridine; Calpain; Cells, Cultured; Disease Models, Animal; Housing; Hypoxia-Inducible Factor 1, alpha Subunit; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Neurogenesis; Psychomotor Performance; Recovery of Function; Signal Transduction; STAT3 Transcription Factor; Stroke; Swimming; Vascular Endothelial Growth Factor A

CDK5 activation promotes ischemic neuronal death in stroke, with the recognized activation mechanism being calpain-dependent p35 cleavage to p25. Here we reported that CDK5-Tyr15 phosphorylation by zinc induced CDK5 activation in brain ischemic injury. CDK5 activation and CDK5-Tyr15 phosphorylation were observed in the hippocampus of the rats that had been subjected to middle cerebral artery occlusion, both of which were reversed by pretreatment with zinc chelator; while p35 cleavage and calpain activation in ischemia were not reversed. Zinc incubation resulted in CDK5-Tyr15 phosphorylation and CDK5 activation, without increasing p35 cleavage in cultured cells. Site mutation experiment confirmed that zinc-induced CDK5 activation was dependent on Tyr15 phosphorylation. Further exploration showed that Src kinase contributed to zinc-induced Tyr15 phosphorylation and CDK5 activation. Src kinase inhibition or expression of an unphosphorylable mutant Y15F-CDK5 abolished Tyr15 phosphorylation, prevented CDK5 activation and protected hippocampal neurons from ischemic insult in rats. We conclude that zinc-induced CDK5-Tyr15 phosphorylation underlies CDK5 activation and promotes ischemic neuronal death in stroke.

Topics: Animals; Brain Ischemia; Calpain; Cell Death; Cyclin-Dependent Kinase 5; Hippocampus; Infarction, Middle Cerebral Artery; Male; Mice; Mice, Inbred C57BL; Neurons; Phosphorylation; Rats; Rats, Sprague-Dawley; src-Family Kinases; Stroke; Zinc

Obstructive sleep apnea-hypopnea syndrome (OSAHS) is a common chronic disorder which is followed by various complications. Calpain-10 belongs to a commonly expressed member of the Calpain-like cysteine protease family, which acts as risk marker for some diseases. The purpose of this study is to elucidate correlation between Calpain-10 single-nucleotide polymorphisms (SNPs) and the incidence of OSAHS followed by ischemic stroke (IS).. OSAHS patients were divided as OSAHS + IS, OSAHS, and control groups, respectively. Immunohistochemistry was performed for Calpain-10 protein expression, polymerase chain reaction (PCR)-restriction fragment length polymorphism for detection of gene polymorphisms of SNP 43 and SNP 19, and PCR-allele specific amplification for SNP 44. Polysomnography was conducted to check the nocturnal polysomnography indicators, and also Montreal Cognitive Assessment (MoCA), Scientific Data System scores cognition and anxiety of patients, respectively. Logistic analysis was used for the risky factors for OSAHS.. Calpain-10 protein expression was significantly increased in the OSAHS + IS and OSAHS groups compared with the control group. Significant differences in SNP 43 and SNP 44 genotype, and also allele frequency were observed in 3 groups, among which the OSAHS + IS group had higher SNP 43 and SNP 44 allele frequency than the control and OSAHS groups. There were differences regarding apnea-hypopnea index, minimum fingertip blood oxygen saturation (LSaO2 [%]), oxygen reduction index (ODI) between patients with different genotypes of SNP 43 and SNP 44 in OSAHS patients, and also GC and AT frequency in the OSAHS + IS and OSAHS groups. As compared with the OSAHS group, the MoCA scores and MoCA subitems in the OSAHS + IS group were declined, whereas the Scientific Data System scores were elevated. Additionally, GG 43 genotype, high apnea-hypopnea index, and body mass index were detected as the risk factors of OSAHS.. These findings indicate that the Calpain-10 SNP 43 may be related to OSAHS with IS, with SNP 43 GG genotype as a risk factor for OSAHS with IS.

Topics: Asian People; Calpain; Enzyme-Linked Immunosorbent Assay; Female; Gene Frequency; Humans; Male; Middle Aged; Oxygen; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Polysomnography; Sleep Apnea, Obstructive; Stroke

Topics: Adenosine Triphosphate; Animals; Apoptosis; Brain Ischemia; Calpain; Caspase 3; Cerebral Arteries; Cerebrovascular Disorders; Culture Media; Disks Large Homolog 4 Protein; Gene Expression Regulation; Glucose; Hippocampus; Humans; Intracellular Signaling Peptides and Proteins; Memantine; Membrane Proteins; Microtubule-Associated Proteins; Neurons; Neuroprotective Agents; Primary Cell Culture; Rats; Rats, Sprague-Dawley; Signal Transduction; Stroke

We previously reported that conventional protein kinase C (cPKC)β participated in hypoxic preconditioning-induced neuroprotection against cerebral ischemic injury, and collapsin response-mediated protein 2 (CRMP2) was identified as a cPKCβ interacting protein. In this study, we explored the regulation of CRMP2 phosphorylation and proteolysis by cPKCβ, and their role in ischemic injury of oxygen-glucose deprivation (OGD)-treated cortical neurons and brains of mice with middle cerebral artery occlusion-induced ischemic stroke. The results demonstrated that cPKCβ-mediated CRMP2 phosphorylation via the cPKCβ-selective activator 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) and inhibition of calpain-mediated CRMP2 proteolysis by calpeptin and a fusing peptide containing TAT peptide and the calpain cleavage site of CRMP2 (TAT-CRMP2) protected neurons against OGD-induced cell death through inhibiting CRMP2 proteolysis in cultured cortical neurons. The OGD-induced nuclear translocation of the CRMP2 breakdown product was inhibited by DOPPA, calpeptin, and TAT-CRMP2 in cortical neurons. In addition, both cPKCβ activation and CRMP2 proteolysis inhibition by hypoxic preconditioning and intracerebroventricular injections of DOPPA, calpeptin, and TAT-CRMP2 improved the neurological deficit in addition to reducing the infarct volume and proportions of cells with pyknotic nuclei in the peri-infact region of mice with ischemic stroke. These results suggested that cPKCβ modulates CRMP2 phosphorylation and proteolysis, and cPKCβ activation alleviates ischemic injury in the cultured cortical neurons and brains of mice with ischemic stroke through inhibiting CRMP2 proteolysis by phosphorylation. Focal cerebral ischemia induces a large flux of Ca(2+) to activate calpain which cleaves collapsin response mediator (CRMP) 2 into breakdown product (BDP). Inhibition of CRMP2 cleavage by calpeptin and TAT-CRMP2 alleviates ischemic injury. Conventional protein kinase C (cPKC)β-mediated phosphorylation could inhibit CRMP2 proteolysis and alleviate ischemic injury in cultured cortical neurons and ischemic stroke-induced mice.

Topics: Animals; Brain Ischemia; Calpain; Cells, Cultured; Cerebral Cortex; Glucose; Hypoxia, Brain; Injections, Intraventricular; Intercellular Signaling Peptides and Proteins; Ischemic Preconditioning; Male; Mice; Mice, Inbred BALB C; Nerve Tissue Proteins; Neurons; Phorbol Esters; Phosphorylation; Protein Kinase C beta; Stroke

The brain and the immune system interact in complex ways after ischemic stroke, and the long-term effects of immune response associated with stroke remain controversial. As a linkage between innate and adaptive immunity, interleukin-17 A (IL-17 A) secreted from gamma delta (γδ) T cells has detrimental roles in the pathogenesis of acute ischemic stroke. However, to date, the long-term actions of IL-17 A after stroke have not been investigated. Here, we found that IL-17 A showed two distinct peaks of expression in the ischemic hemisphere: the first occurring within 3 days and the second on day 28 after stroke. Our data also showed that astrocyte was the major cellular source of IL-17 A that maintained and augmented subventricular zone (SVZ) neural precursor cells (NPCs) survival, neuronal differentiation, and subsequent synaptogenesis and functional recovery after stroke. IL-17 A also promoted neuronal differentiation in cultured NPCs from the ischemic SVZ. Furthermore, our in vitro data revealed that in primary astrocyte cultures activated astrocytes released IL-17 A via p38 mitogen-activated protein kinase (MAPK). Culture media from reactive astrocytes increased neuronal differentiation of NSCs in vitro. Blockade of IL-17 A with neutralizing antibody prevented this effect. In addition, after screening for multiple signaling pathways, we revealed that the p38 MAPK/calpain 1 signaling pathway was involved in IL-17 A-mediated neurogenesis in vivo and in vitro. Thus, our results reveal a previously uncharacterized property of astrocytic IL-17 A in the maintenance and augment of survival and neuronal differentiation of NPCs, and subsequent synaptogenesis and spontaneous recovery after ischemic stroke.

Topics: Aging; Animals; Astrocytes; Axons; Calpain; Cell Differentiation; Cell Movement; Cell Survival; Interleukin-17; Male; Mice, Inbred C57BL; Nerve Regeneration; Neural Stem Cells; Neurogenesis; Neurons; p38 Mitogen-Activated Protein Kinases; Recombinant Proteins; Recovery of Function; Stroke; Synapses

Control of GABA neurotransmission at the pre-synaptic site occurs substantially through the activation of the glutamic acid decarboxylase (GAD) enzymes GAD65 and GAD67. Concentrations of GAD65 and GAD67 are controlled either by transcription or by mRNA splicing and importantly the activities of these key enzymes are regulated by post-translational mechanisms. Important post-translational modifications include proteolytic cleavage, phosphorylation and palmitoylation. A truncated form of GAD65 (tGAD65) is more active than full length GAD65 (fGAD65) whereas, by contrast, truncated GAD67 (tGAD67) is less active than full length GAD67 (fGAD67). The protein responsible for cleaving of fGAD65 and fGAD67 is mu-calpain. GABA neurotransmission is dependent upon whether GAD is associated with synaptic vesicles (SV) and calpain performs a vital role by generating the highly active tGAD65 resulting in augmented GABA synthesis and wrapping uptake into SV. Studies on GAD phosphorylation demonstrate that GAD65 is regulated through phosphorylation by PKC while GAD67 is inhibited through phosphorylation by PKA. Cysteine residues 455 and 446 in GAD67 and GAD65 individually are critical for full GAD regulation. Interaction with the cofactor pyridoxal 50-phosphate (PLP) at this these respective locations regulate the switch between PLP-bound active holoGAD and an unbound active apoGAD form. Transient switching to the PLP bound active holoGAD is integral to GABA neurotransmission. Specific to GAD65 but not GAD67 is palmitoylation by HIP14 which facilitates GAD65 anchoring to SV and enhances the contribution of vesicular GABA to neurotransmission. From studies on a rodent stroke model calpain-mediated cleavage of GAD enzyme has been shown to occur under pathological conditions resulting in less SV refilling and depletion of existing pools of SV releasable GABA.

Topics: Animals; Calpain; gamma-Aminobutyric Acid; Glutamate Decarboxylase; Humans; Phosphorylation; Protein Processing, Post-Translational; Stroke; Synaptic Transmission; Synaptic Vesicles

Salidroside is proven to be a neuroprotective agent of natural origin, and its analog, 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-d-pyranoside (named SalA-4g), has been synthesized in our lab. In this study, we showed that SalA-4g promoted neuronal survival and inhibited neuronal apoptosis in primary hippocampal neurons exposed to oxygen and glucose deprivation (OGD) and in rats subjected to ischemia by transient middle cerebral artery occlusion (MCAO), respectively, and that SalA-4g was more neuroprotective than salidroside. We further found that SalA-4g elevated glucose uptake in OGD-injured primary hippocampal neurons and increased the expression and recruitment of glucose transporter 3 (GLUT3) in ischemic brain. Signaling analysis revealed that SalA-4g triggered the phosphorylation of CREB, and increased the expression of PKA RII in primary hippocampal neurons exposed to OGD injury, while inhibition of PKA/CREB by H-89 alleviated the elevation in glucose uptake and GLUT3 expression, and blocked the protective effects of SalA-4g. Moreover, SalA-4g was noted to inhibit intracellular Ca(2+) influx and calpain1 activation in OGD-injured primary hippocampal neurons. Our results suggest that SalA-4g neuroprotection might be mediated by increased glucose uptake and elevated GLUT3 expression through calpain1/PKA/CREB pathway.

Topics: Acetylglucosamine; Animals; Brain Ischemia; Calpain; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Gene Expression Regulation; Glucose Transporter Type 3; Hippocampus; Mice; Molecular Structure; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Stroke

MicroRNAs (miRNAs) are highly conserved non-coding RNAs modulating gene expression via mRNA binding. Recent work suggests an involvement of miRNAs in cardiovascular diseases including stroke. As such, the brain-abundant miR-124 and its transcriptional repressor RE1-silencing transcription factor (REST) do not only have elementary roles in the developing and the adult brain, but also alter expression upon cerebral ischemia. However, the therapeutic potential of miR-124 against stroke and the mechanisms involved remain elusive. Here, we analyzed the therapeutic potential of ectopic miR-124 against stroke and its underlying mechanisms with regard to the interaction between miR-124 and REST. Our results show that viral vector-mediated miR-124 delivery increased the resistance of cultured oxygen-glucose-deprived cortical neurons in vitro and reduced brain injury as well as functional impairment in mice submitted to middle cerebral artery occlusion. Likewise, miR-124 induced enhanced neurovascular remodeling leading to increased angioneurogenesis 8 weeks post-stroke. While REST abundance increased upon stroke, the increase was prevented by miR-124 despite a so far unknown negative feedback loop between miR-124 and REST. Rather, miR-124 decreased the expression of the deubiquitinating enzyme Usp14, which has two conserved miR-124-binding sites in the 3'UTR of its mRNA, and thereby mediated reduced REST levels. The down-regulation of REST by miR-124 was also mimicked by the Usp14 inhibitor IU-1, suggesting that miR-124 promotes neuronal survival under ischemic conditions via Usp14-dependent REST degradation. Ectopic miR-124 expression, therefore, appears as an attractive and novel tool in stroke treatment, mediating neuroprotection via a hitherto unknown mechanism that involves Usp14-dependent REST degradation.

Topics: Animals; Brain Ischemia; Calpain; Cell Survival; Glucose; HEK293 Cells; Humans; Lipid Peroxidation; Male; Mice; Mice, Inbred C57BL; MicroRNAs; Neurons; Oxygen; Receptors, AMPA; Repressor Proteins; Stroke; Thiobarbituric Acid Reactive Substances; Ubiquitin Thiolesterase; Ubiquitination

Excitotoxicity resulting from overstimulation of glutamate receptors is a major cause of neuronal death in cerebral ischemic stroke. The overstimulated ionotropic glutamate receptors exert their neurotoxic effects in part by overactivation of calpains, which induce neuronal death by catalyzing limited proteolysis of specific cellular proteins. Here, we report that in cultured cortical neurons and in vivo in a rat model of focal ischemic stroke, the tyrosine kinase Src is cleaved by calpains at a site in the N-terminal unique domain. This generates a truncated Src fragment of ~52 kDa, which we localized predominantly to the cytosol. A cell membrane-permeable fusion peptide derived from the unique domain of Src prevents calpain from cleaving Src in neurons and protects against excitotoxic neuronal death. To explore the role of the truncated Src fragment in neuronal death, we expressed a recombinant truncated Src fragment in cultured neurons and examined how it affects neuronal survival. Expression of this fragment, which lacks the myristoylation motif and unique domain, was sufficient to induce neuronal death. Furthermore, inactivation of the prosurvival kinase Akt is a key step in its neurotoxic signaling pathway. Because Src maintains neuronal survival, our results implicate calpain cleavage as a molecular switch converting Src from a promoter of cell survival to a mediator of neuronal death in excitotoxicity. Besides unveiling a new pathological action of Src, our discovery of the neurotoxic action of the truncated Src fragment suggests new therapeutic strategies with the potential to minimize brain damage in ischemic stroke.

Topics: Animals; Brain Ischemia; Calpain; Cell Death; Cell Membrane; Gene Expression Regulation, Enzymologic; HEK293 Cells; Humans; Lentivirus; Male; Models, Biological; Mutation; Neurons; Peptides; Rats; Rats, Wistar; Recombinant Proteins; Signal Transduction; src-Family Kinases; Stroke

Taurine, an abundant amino acid in the nervous system, is reported to reduce ischemic brain injury in a dose-dependent manner. This study was designed to investigate whether taurine protected brain against experimental stroke through affecting mitochondria-mediated cell death pathway. Rats were subjected to 2-h ischemia by intraluminal filament, and then reperfused for 22 h. It was confirmed again that taurine (50 mg/kg) administered intravenously 1 h after ischemia markedly improved neurological function and decreased infarct volume at 22 h after reperfusion. In vehicle-treated rats, the levels of intracellular ATP and the levels of cytosolic and mitochondrial Bcl-xL in the penumbra and core were markedly reduced, while the levels of cytosolic Bax in the core and mitochondrial Bax in the penumbra and core were enhanced significantly. There was a decrease in cytochrome C in mitochondria and an increase in cytochrome C in the cytosol of the penumbra and core. These changes were reversed by taurine. Furthermore, taurine inhibited the activation of calpain and caspase-3, reduced the degradation of αII-spectrin, and attenuated the necrotic and apoptotic cell death in the penumbra and core. These data demonstrated that preserving the mitochondrial function and blocking the mitochondria-mediated cell death pathway may be one mechanism of taurine's action against brain ischemia.

Topics: Adenosine Triphosphate; Animals; bcl-2-Associated X Protein; bcl-X Protein; Brain Ischemia; Calpain; Caspase 3; Cell Death; Cerebral Infarction; Cytochromes c; Disease Models, Animal; Dose-Response Relationship, Drug; Male; Mitochondria; Nervous System Diseases; Rats; Rats, Sprague-Dawley; Stroke; Taurine

Recent studies in neonatal rodent stroke models suggest that recovery is due in part to upregulation of hypoxia-inducible factor-1-a and its downstream target, vascular endothelial growth factor. Vascular endothelial growth factor is upregulated after a hypoxic insult and is involved in neuronal survival, angiogenesis, and neurogenesis during the recovery process.. We performed a 1.5-hour transient middle cerebral artery occlusion in 10-day-old rats with injury verified by diffusion-weighted MRI during occlusion to determine the effects of vascular endothelial growth factor receptor-2 (VEGFR2) inhibition on injury, apoptosis, and angiogenesis. Two days after reperfusion, the pups received either the VEGFR inhibitor, SU5416 (10 mg/kg per dose) or vehicle (1% dimethyl sulfoxide) for 3 days.. VEGFR2 inhibition worsened injury 7 days after injury when compared with the vehicle-treated and injury-alone groups (P<0.01). Furthermore, receptor inhibition was associated with increased VEGFR2 expression 5 days after injury (P<0.05) and increased spectrin cleavage with a shift in favor of the calpain-mediated, caspase-3-independent cleavage (P<0.01). Increased areas of cleaved caspase-3 staining were seen in treated rats at 7 days (P<0.01) There were no differences in gliosis or macrophage recruitment as measured by glial fibrillary acidic protein and Iba-1 expression at this time point. Lastly, VEGFR2 inhibition did not affect the overall vessel surface area but reduced endothelial cell proliferation in injured caudate.. Inhibition of VEGFR2 signaling worsens injury, affects cell death, and reduces endothelial cell proliferation after neonatal stroke. Injury exacerbation may be in part due to a shift of cell fate from apoptosis to necrosis on the continuum spectrum of cell death as well as effects on angiogenesis in the injured brain.

Topics: Angiogenesis Inhibitors; Animals; Animals, Newborn; Apoptosis; Calpain; Caspase 3; Cell Proliferation; Cerebral Arteries; Disease Models, Animal; Endothelial Cells; Indoles; Infarction, Middle Cerebral Artery; Magnetic Resonance Imaging; Necrosis; Neovascularization, Physiologic; Pyrroles; Rats; Rats, Sprague-Dawley; Stroke; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Two intracellular cysteine proteases (calpains and caspases) and inducible nitric oxide synthase (iNOS) participate in the ischemic brain injury. In vitro nitric oxide (NO) regulates calpain and caspase-3 activation. The present study investigated whether aminoguanidine (AG), an iNOS inhibitor, protected brain against experimental stroke through inhibiting calpain and caspase-3 activation. Rats received 1h ischemia by intraluminal filament, then, reperfused for 23 h (R 23 h). AG (100 mg/kg) was administered intraperitoneally 5 min before ischemia. Our data showed that treatment with AG markedly improved neurological deficit, reduced brain swelling, decreased infarct volume, and attenuated the necrotic cell death in ischemic penumbra and core, and apoptotic cell death in penumbra at R 23 h. Enzymatic studies demonstrated the significant inhibition of the activities of mu- and m-calpain and caspase-3, and Western blot analysis revealed marked increases in the levels of MAP-2 and spectrin in penumbra and core in AG-treated rats versus vehicle-treated rats. AG also significantly enhanced the calpastatin levels in core, although it had no significant effects on that in penumbra. These data demonstrate that inhibiting calpain and caspase-3 activation is one mechanism of AG against experimental stroke, suggesting that NO produced by iNOS may be involved in calpain- and caspase-3-mediated ischemic cell death, at least in part.

Topics: Animals; Apoptosis; Biotransformation; Blotting, Western; Brain; Brain Edema; Calcium-Binding Proteins; Calpain; Caspase 3; Cerebral Infarction; Cytosol; Enzyme Activation; Guanidines; Male; Microtubule-Associated Proteins; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Recovery of Function; Spectrin; Stroke

Topics: Animals; Calcium; Calpain; Neurons; Protein Processing, Post-Translational; Rats; Sodium-Calcium Exchanger; Stroke

Preclinical studies have identified numerous neuroprotective drugs that attenuate brain damage and improve functional outcome after cerebral ischemia. Despite this success in animal models, neuroprotective therapies in the clinical setting have been unsuccessful. Identification of biochemical markers common to preclinical and clinical cerebral ischemia will provide a more sensitive and objective measure of injury severity and outcome to facilitate clinical management and treatment. However, there are currently no effective biomarkers available for assessment of stroke. Nonerythroid alphaII-spectrin is a cytoskeletal protein that is cleaved by calpain and caspase-3 proteases to signature alphaII-spectrin breakdown products (alphaII-SBDPs) after cerebral ischemia in rodents. This investigation examined accumulation of calpain- and caspase-3-cleaved alphaII-SBDPs in cerebrospinal fluid (CSF) of rodents subjected to 2 hours of transient focal cerebral ischemia produced by middle cerebral artery occlusion (MCAO) followed by reperfusion. After MCAO injury, full-length alphaII-spectrin protein was decreased in brain tissue and increased in CSF from 24 to 72 hours after injury. Whereas alphaII-SBDPs were undetectable in sham-injured control animals, calpain but not caspase-3 specific alphaII-SBDPs were significantly increased in CSF after injury. However, caspase-3 alphaII-SBDPS were observed in CSF of some injured animals. These results indicate that alphaII-SBDPs detected in CSF after injury, particularly those mediated by calpain, may be useful diagnostic indicators of cerebral infarction that can provide important information about specific neurochemical events that have occurred in the brain after acute stroke.

Topics: Animals; Biomarkers; Brain Chemistry; Calpain; Caspase 3; Caspases; Cerebral Cortex; Densitometry; Immunoblotting; Infarction, Middle Cerebral Artery; Middle Cerebral Artery; Peptide Fragments; Rats; Reperfusion Injury; Spectrin; Stroke

Several earlier studies have implicated platelet activation with the pathogenesis of thrombotic stroke. In this report we have studied the changes in membrane physical microenvironment and signal transduction in the platelets obtained from the patients with thrombotic stroke. Aggregation induced by the synthetic agonist thrombin receptor-activating peptide was significantly enhanced (p < 0.001) in the platelets obtained from the patients. Steady-state fluorescence anisotropy measurements using diphenylhexatriene reflected a significant increase in membrane microviscosity from 3.315 (+/- 0.103) in the control to 4.600 (+/- 0.119) in the stroke. Proteins of relative mobilities of 131, 100, 47 and 38 kDa were found to remain phosphorylated on tyrosine in the resting platelets obtained from thrombotic stroke patients while they were not phosphorylated in the control counter-parts. Besides, calpain, a calcium dependent thiol protease present in the platelets, was found to remain active in this disease as reflected from the proteolysis of calpain substrates. Taken together, these data indicated abnormal circulating platelets in the patients ofthrombotic stroke. which could contribute to the etiopathogenesis of this disease.

Topics: Adult; Blood Platelets; Calpain; Cell Membrane; Diphenylhexatriene; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Female; Fluorescence Polarization; Humans; Male; Membrane Fluidity; Middle Aged; Phosphorylation; Phosphotyrosine; Platelet Aggregation; Signal Transduction; Stroke; Thrombosis

The contributions of calpain and caspase-3 to apoptosis and necrosis after central nervous system (CNS) trauma are relatively unexplored. No study has examined concurrent activation of calpain and caspase-3 in necrotic or apoptotic cell death after any CNS insult. Experiments used a model of oxygen-glucose deprivation (OGD) in primary septo-hippocampal cultures and assessed cell viability, occurrence of apoptotic and necrotic cell death phenotypes, and protease activation. Immunoblots using an antibody detecting calpain and caspase-3 proteolysis of alpha-spectrin showed greater accumulation of calpain-mediated breakdown products (BDPs) compared with caspase-3-mediated BDPs. Administration of calpain and caspase-3 inhibitors confirmed that activation of these proteases contributed to cell death, as inferred by lactate dehydrogenase release. Oxygen-glucose deprivation resulted in expression of apoptotic and necrotic cell death phenotypes, especially in neurons. Immunocytochemical studies of calpain and caspase-3 activation in apoptotic cells indicated that these proteases are almost always concurrently activated during apoptosis. These data demonstrate that calpain and caspase-3 activation is associated with expression of apoptotic cell death phenotypes after OGD, and that calpain activation, in combination with caspase-3 activation, could contribute to the expression of apoptotic cell death by assisting in the degradation of important cellular proteins.

Topics: Animals; Apoptosis; Blotting, Western; Calpain; Caspase 3; Caspase Inhibitors; Caspases; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Fusobacterium Infections; Glucose; Hippocampus; Neuroglia; Neurons; Oxygen; Phenotype; Rats; Reperfusion Injury; Rosette Formation; Septum of Brain; Spectrin; Stroke