Jigar Modi

Mechanisms of Neuronal Protection against Excitotoxicity, Endoplasmic Reticulum Stress, and Mitochondrial Dysfunction in Stroke and Neurodegenerative Diseases

In stroke and neurodegenerative disease, neuronal excitotoxicity, caused by increased extracellular glutamate levels, is known to result in calcium overload and mitochondrial dysfunction. Mitochondrial deficits may involve a deficiency in energy supply as well as generation of high levels of oxidants which are key contributors to neuronal cell death through necrotic and apoptotic mechanisms. Excessive glutamate receptor stimulation also results in increased nitric oxide generation which can be detrimental to cells as nitric oxide interacts with superoxide to form the toxic molecule peroxynitrite. High level oxidant production elicits neuronal apoptosis through the actions of proapoptotic Bcl-2 family members resulting in mitochondrial permeability transition pore opening. In addition to apoptotic responses to severe stress, accumulation of misfolded proteins and high levels of oxidants can elicit endoplasmic reticulum (ER) stress pathways which may also contribute to induction of apoptosis. Two categories of therapeutics are discussed that impact major pro-death events that include induction of oxidants, calcium overload, and ER stress. The first category of therapeutic agent includes the amino acid taurine which prevents calcium overload and is also capable of preventing ER stress by inhibiting specific ER stress pathways. The second category involves N-methyl-D-aspartate receptor (NMDA receptor) partial antagonists illustrated by S-Methyl-N, N-diethyldithiocarbamate sulfoxide (DETC-MeSO), and memantine. DETC-MeSO is protective through preventing excitotoxicity and calcium overload and by blocking specific ER stress pathways. Another NMDA receptor partial antagonist is memantine which prevents excessive glutamate excitation but also remarkably allows maintenance of physiological neurotransmission. Targeting of these major sites of neuronal damage using pharmacological agents is discussed in terms of potential therapeutic approaches for neurological disorders.

The Mechanism of Taurine Protection Against Endoplasmic Reticulum Stress in an Animal Stroke Model of Cerebral Artery Occlusion and Stroke-Related Conditions in Primary Neuronal Cell Culture

Taurine is an inhibitory neurotransmitter and is one of the most abundant amino acids present in the mammalian nervous system. Taurine has been shown to provide protection against neurological diseases, such as Huntingtons disease, Alzheimers disease, and stroke. Ischemic stroke is one of the leading causes of death and disability in the world. It is generally believed that ischemia-induced brain injury is largely due to excessive release of glutamate resulting in excitotoxicity and cell death. Despite extensive research, there are still no effective interventions for stroke. Recently, we have shown that taurine can provide effective protection against endoplasmic reticulum (ER) stress induced by excitotoxicity or oxidative stress in PC12 cell line or primary neuronal cell cultures. In this study, we employed hypoxia/reoxygenation conditions for primary cortical neuronal cell cultures as an in vitro model of stroke as well as the in vivo model of rat focal middle cerebral artery occlusion (MCAO). Our data showed that when primary neuronal cultures were first subjected to hypoxic conditions (0.3%, 24 h) followed by reoxygenation (21%, 24-48 h), the cell viability was greatly reduced. In the animal model of stroke (MCAO), we found that 2 h ischemia followed by 4 days reperfusion resulted in an infarct of 47.42 ± 9.86% in sections 6 mm from the frontal pole. Using taurine greatly increased cell viability in primary neuronal cell culture and decreased the infarct area of sections at 6 mm to 26.76 ± 6.91% in the MCAO model. Furthermore, levels of the ER stress protein markers GRP78, caspase-12, CHOP, and p-IRE-1 which were markedly increased in both the in vitro and in vivo models significantly declined after taurine administration, suggesting that taurine may exert neuroprotection functions in both models. Moreover, taurine could downregulate the ratio of cleaved ATF6 and full-length ATF6 in both models. In the animal model of stroke, taurine induced an upregulation of the Bcl-2/Bax ratio and downregulation of caspase-3 protein activity indicating that it attenuates apoptosis in the core of the ischemic infarct. Our results show not only taurine elicits neuroprotection through the activation of the ATF6 and the IRE1 pathways, but also it can reduce apoptosis in these models.

Calpain Cleavage of Brain Glutamic Acid Decarboxylase 65 Is Pathological and Impairs GABA Neurotransmission

Previously, we have shown that the GABA synthesizing enzyme, L-glutamic acid decarboxylase 65 (GAD65) is cleaved to form its truncated form (tGAD65) which is 2–3 times more active than the full length form (fGAD65). The enzyme responsible for cleavage was later identified as calpain. Calpain is known to cleave its substrates either under a transient physiological stimulus or upon a sustained pathological insult. However, the precise role of calpain cleavage of fGAD65 is poorly understood. In this communication, we examined the cleavage of fGAD65 under diverse pathological conditions including rats under ischemia/reperfusion insult as well as rat brain synaptosomes and primary neuronal cultures subjected to excessive stimulation with high concentration of KCl. We have shown that the formation of tGAD65 progressively increases with increasing stimulus concentration both in rat brain synaptosomes and primary rat embryo cultures. More importantly, direct cleavage of synaptic vesicle - associated fGAD65 by calpain was demonstrated and the resulting tGAD65 bearing the active site of the enzyme was detached from the synaptic vesicles. Vesicular GABA transport of the newly synthesized GABA was found to be reduced in calpain treated SVs. Furthermore, we also observed that the levels of tGAD65 in the focal cerebral ischemic rat brain tissue increased corresponding to the elevation of local glutamate as indicated by microdialysis. Moreover, the levels of tGAD65 was also proportional to the degree of cell death when the primary neuronal cultures were exposed to high KCl. Based on these observations, we conclude that calpain-mediated cleavage of fGAD65 is pathological, presumably due to decrease in the activity of synaptic vesicle - associated fGAD65 resulting in a decrease in the GABA synthesis - packaging coupling process leading to reduced GABA neurotransmission.

Comparison between single and combined post-treatment with S-Methyl-N,N-diethylthiolcarbamate sulfoxide and taurine following transient focal cerebral ischemia in rat brain

We have recently reported on the efficacy of an N-methyl-d-aspartate (NMDA) receptor partial antagonist, S-Methyl-N,N-diethylthiolcarbamate sulfoxide (DETC-MeSO), in improving outcome following stroke, including reduced infarct size and calcium influx, suppressing the endoplasmic reticulum (ER) stress-induced apoptosis as well as improving behavioral outcome. DETC-MeSO was shown to suppress the protein kinase R-like endoplasmic reticulum kinase (PERK) pathway, one of the major ER stress pathways. Several studies including ours have provided evidence that taurine also has neuroprotective effects through reducing apoptosis and inhibiting activating transcription factor 6 (ATF6) and inositol requiring enzyme 1 (IRE-1) pathways. We hypothesized that a combined treatment with DETC-MeSO and taurine would ameliorate ischemia-induced brain injury by inhibiting all three ER stress pathways. Twenty four hours following reperfusion of a 2-h ischemic stroke, rats received either 0.56-mg/kg DETC-MeSO or 40-mg/kg of taurine, either alone or in combination, subcutaneously for 4days. Our study showed that combined DETC-MeSO and taurine, but not DETC-MeSO alone at the dose used, greatly reduced the infarct size, improved performance on the neuro-score test and attenuated proteolysis of αII-spectrin. Meanwhile, the level of the pro-apoptotic protein, Bax, declined and the anti-apoptotic protein, B-cell lymphoma 2 (BCL-2), expression was markedly increased. Combination therapy decreased both caspase-12 and caspase-3 activation by preventing the release of Cytochrome-c from mitochondria, indicating attenuation of apoptosis in ischemic infarct. Glucose-regulated protein (GRP)78 as a marker of the unfolded protein response decreased and levels of the key ER stress protein markers p-PERK-ATF4, p-eIF2α and cleaved-ATF-6 were found to significantly decline. NeuN expression levels indicated that more neurons were protected in the presence of DETC-MeSO and taurine. We also showed that combined treatment can prevent gliosis and increase p-AKT a pro-survival marker in the penumbra. Therefore, we conclude that combined treatment with both DETC-MeSO and taurine synergistically inhibits all three ER stress pathways and apoptosis and therefore can be a novel and effective treatment after ischemic stroke.

Protective mechanism of sulindac in an animal model of ischemic stroke

BACKGROUND AND PURPOSE The present study analyzed whether administration of sulindac, a non-steroidal anti-inflammatory drug (NSAID) would prevent, attenuate or repair ischemia induced brain injury and reverse functional impairment in a focal ischemia model of stroke. METHODS Male Sprague-Dawley rats (weight 250-300 g) were subjected to middle cerebral artery occlusion (MCAO). Sulindac was given 2 days before and 24 h after ischemia at 0.2 mg/day with daily injections until sacrifice on day 3 or day 11. Infarct size was measured by TTC staining and western immunoblot was employed. RESULTS TTC analysis of brain slices indicated a decrease in infarct size in sulindac treated animals. Western blot results indicated that sulindac induced expression of Hsp 27, a marker of cell stress, in the ischemic penumbra and core on days 3 and 11. Hsp 27 is important as a protective molecular chaperone. Increases were also found in the protective molecules Akt and Bcl-2 in the ischemic penumbra and core following sulindac administration. CONCLUSION Our data indicate that administration of sulindac results in decreased infarct size and that there is a central role for the molecular chaperone Hsp 27, the pro-survival kinase Akt and the anti-apoptotic component Bcl-2 in mediating these protective effects.

Granulocyte-colony stimulating factor protects against endoplasmic reticulum stress in an experimental model of stroke

Granulocyte-colony stimulating factor (G-CSF) is an endogenous growth factor that exhibits a diverse range of neuroprotective mechanisms against a variety of neurological disorders including ischemic stroke. We investigated the anti-apoptotic mechanisms of G-CSF against endoplasmic reticulum (ER) stress induced apoptosis. Sprague-Dawley rats were subjected to transient occlusion of the middle cerebral artery (MCAO) for 90 min. Rats were injected with G-CSF (n = 15; 50 μg/kg body weight s.c.) for 4 days, starting 24 h post-MCAO and brains were harvested after 4 days reperfusion (n = 16). Key proteins in ER stress apoptosis were analyzed by immunoblotting. G-CSF reduced infarct volume to 53% and improved neurological deficits. G-CSF treatment significantly (P < .05) attenuated the expression of proteins involved in ER stress apoptosis pathway; ATF4, ATF6, p-p38MAPK, pJNK and CHOP. G-CSF treatment also re-established ER homeostasis evident by the reduction of the intraluminal ER stress sensor, GRP78 as well as reducing the overall cellular stress level protein, HSP27. G-CSF also up-regulated anti-apoptotic proteins pAKT and Bcl-2 while down-regulated the pro-apoptotic protein Bax. G-CSF exerts neuroprotection from cerebral ischemia through the preservation of the ER, resulting in the attenuation of pro-apoptotic proteins and the potentiation of anti-apoptotic proteins.

Sulindac for stroke treatment: neuroprotective mechanism and therapy.

Sulindac, a widely used nonsteroidal anti-inflammatory drug (NSAID) is a prodrug that is reduced by methionine sulfoxide reductase to its active form as an inhibitor of cyclooxygenase 1 and 2. The drug has been shown to elicit tissue protection by processes that may include at least three functions: antioxidant, preconditioning and anti-inflammatory. Sulindac demonstrates neuroprotection that involves inhibition of mitochondrial calcium overload or a decrease in protein oxidation. We have demonstrated the induction by sulindac treatment of pro-survival proteins Hsp27, Akt and Bcl-2 in the ischemic penumbra and core of the central nervous system (CNS) infarct in a rat model of ischemic stroke. Our findings point to sulindac acting on the endoplasmic reticulum (ER) to decrease ATF-6 and on the mitochondrion to increase Bcl-2 as well as decrease pro-apoptotic components BAK and PUMA. The resulting decrease in ER stress and reduction in apoptosis underlies the protective effect of sulindac in reducing infarct size following transient focal brain ischemia. The potent neuroprotective effect of sulindac in the stroke model is obtained with low-dose administration of the drug pointing to the potential of sulindac as a valuable neuroprotective agent against oxidative stress in cerebral ischemia.