The Role of NMDA Receptor Partial Antagonist, Carbamathione, as a Therapeutic Agent for Transient Global Ischemia.

Carbamathione (Carb), an NMDA glutamate receptor partial antagonist, has potent neuroprotective functions against hypoxia- or ischemia-induced neuronal injury in cell- or animal-based stroke models. We used PC-12 cell cultures as a cell-based model and bilateral carotid artery occlusion (BCAO) for stroke. Whole-cell patch clamp recording in the mouse retinal ganglion cells was performed. Key proteins involved in apoptosis, endoplasmic reticulum (ER) stress, and heat shock proteins were analyzed using immunoblotting. Carb is effective in protecting PC12 cells against glutamate- or hypoxia-induced cell injury. Electrophysiological results show that Carb attenuates NMDA-mediated glutamate currents in the retinal ganglion cells, which results in activation of the AKT signaling pathway and increased expression of pro-cell survival biomarkers, e.g., Hsp 27, P-AKT, and Bcl2 and decreased expression of pro-cell death markers, e.g., Beclin 1, Bax, and Cleaved caspase 3, and ER stress markers, e.g., CHOP, IRE1, XBP1, ATF 4, and eIF2α. Using the BCAO animal stroke model, we found that Carb reduced the brain infarct volume and decreased levels of ER stress markers, GRP 78, CHOP, and at the behavioral level, e.g., a decrease in asymmetric turns and an increase in locomotor activity. These findings for Carb provide promising and rational strategies for stroke therapy.

The role of G-CSF neuroprotective effects in neonatal hypoxic-ischemic encephalopathy (HIE): current status.

Hypoxic-ischemic encephalopathy (HIE) is an important cause of permanent damage to central nervous system (CNS) that may result in neonatal death or manifest later as mental retardation, epilepsy, cerebral palsy, or developmental delay. The primary cause of this condition is systemic hypoxemia and/or reduced cerebral blood flow with long-lasting neurological disabilities and neurodevelopmental impairment in neonates. About 20 to 25% of infants with HIE die in the neonatal period, and 25-30% of survivors are left with permanent neurodevelopmental abnormalities. The mechanisms of hypoxia-ischemia (HI) include activation and/or stimulation of myriad of cascades such as increased excitotoxicity, oxidative stress, N-methyl-D-aspartic acid (NMDA) receptor hyperexcitability, mitochondrial collapse, inflammation, cell swelling, impaired maturation, and loss of trophic support. Different therapeutic modalities have been implicated in managing neonatal HIE, though translation of most of these regimens into clinical practices is still limited. Therapeutic hypothermia, for instance, is the most widely used standard treatment in neonates with HIE as studies have shown that it can inhibit many steps in the excito-oxidative cascade including secondary energy failure, increases in brain lactic acid, glutamate, and nitric oxide concentration. Granulocyte-colony stimulating factor (G-CSF) is a glycoprotein that has been implicated in stimulation of cell survival, proliferation, and function of neutrophil precursors and mature neutrophils. Extensive studies both in vivo and ex vivo have shown the neuroprotective effect of G-CSF in neurodegenerative diseases and neonatal brain damage via inhibition of apoptosis and inflammation. Yet, there are still few experimentation models of neonatal HIE and G-CSF's effectiveness, and extrapolation of adult stroke models is challenging because of the evolving brain. Here, we review current studies and/or researches of G-CSF's crucial role in regulating these cytokines and apoptotic mediators triggered following neonatal brain injury, as well as driving neurogenesis and angiogenesis post-HI insults.

Granulocyte-colony stimulating factor gene therapy as a novel therapeutics for stroke in a mouse model.

BACKGROUND: Global ischemia is the resulting effect of a cardiopulmonary arrest (CPA). Presently there is no effective treatment to address neurological deficits in patients who survived a CPA. Granulocyte-colony stimulating factor is a growth factor (G-CSF) with a plethora of beneficial effects, including neuroprotection. Clinical application of human G-CSF (hG-CSF) is limited due to its plasma half-life of 4 h. Therefore, novel approaches need to be investigated that would (1) enable prolonged manifestation of hG-CSF and (2) demonstrate G-CSF efficacy from studying the underlying protective mechanisms of hG-CSF. In our previous work, we used the self-complementary adeno-associated virus (stereotype2: scAAV2) as a vector to transfect the hG-CSF gene into the global ischemic brain of a mouse. As an extension of that work, we now seek to elucidate the protective mechanisms of hG-CSF gene therapy against endoplasmic reticulum induced stress, mitochondrial dynamics and autophagy in global ischemia. METHOD: A single drop of either AAV-CMV-hG-CSF or AAV-CMV-GFP was dropped into the conjunctival sac of the Swiss Webster mouse's left eye, 30-60 min after bilateral common artery occlusion (BCAO). The efficacy of the expressed hG-CSF gene product was analyzed by monitoring the expression levels of endoplasmic reticulum stress (ER), mitochondrial dynamics and autophagic proteins over 4- and 7-days post-BCAO in vulnerable brain regions including the striatum, overlying cortex (frontal brain regions) and the hippocampus (middle brain regions). Statistical analysis was performed using mostly One-Way Analysis of variance (ANOVA), except for behavioral analysis, which used Repeated Measures Two-Way ANOVA, post hoc analysis was performed using the Tukey test. RESULTS: Several biomarkers that facilitated cellular death, including CHOP and GRP78 (ER stress) DRP1 (mitochondrial dynamics) and Beclin 1, p62 and LC3-ll (autophagy) were significantly downregulated by hG-CSF gene transfer. hG-CSF gene therapy also significantly upregulated antiapoptotic Bcl2 while downregulating pro-apoptotic Bax. The beneficial effects of hG-CSF gene therapy resulted in an overall improvement in functional behavior. CONCLUSION: Taken together, this study has substantiated the approach of sustaining the protein expression of hG-CSF by eye drop administration of the hG-CSF gene. In addition, the study has validated the efficacy of using hG-CSF gene therapy against endoplasmic reticulum induced stress, mitochondrial dynamics and autophagy in global ischemia.

G-CSF attenuates neuroinflammation and neuronal apoptosis via the mTOR/p70SK6 signaling pathway in neonatal Hypoxia-Ischemia rat model.

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) is an important cause of permanent damage to the central nervous system, associated with long-lasting neurological disabilities and neurodevelopmental impairment in neonates. Granulocyte-colony stimulating factor (G-CSF) has been shown to have neuroprotective activity in a variety of experimental brain injury models and G-CSF is a standard treatment in chemotherapeutic-induced neutropenia. The underlying mechanisms are still unclear. The mTOR (mammalian target of rapamycin) signaling pathway is a master regulator of cell growth and proliferation in the nervous system. However, the effects of G-CSF treatment on the mTOR signaling pathway have not been elucidated in neonates with hypoxic-ischemic (HI) brain injury. Our study investigated the neuroprotective effect of G-CSF on neonates with hypoxic-ischemic (HI) brain injury and the possible mechanism involving the mTOR/p70S6K pathway. METHODS: Sprague-Dawley rat pups at postnatal day 7 (P7) were subjected to right unilateral carotid artery ligation followed by hypoxic (8% oxygen and balanced nitrogen) exposure for 2.5 h or sham surgery. Pups received normal saline, G-CSF, G-CSF combined with rapamycin or ethanol (vehicle for rapamycin) intraperitoneally. On postnatal day 9 (P9), TTC staining for infarct volume, and Nissl and TUNEL staining for neuronal cell injury were conducted. Activation of mTOR/p70S6K pathway, cleaved caspase-3 (CC3), Bax and Bcl-2 and cytokine expression levels were determined by western blotting. RESULTS: The G-CSF treated group was associated with significantly reduced infarction volume and decreased TUNEL positive neuronal cells compared to the HI group treated with saline. The expression levels of TNF-α and IL-1ß were significantly decreased in the G-CSF treated group, while IL-10 expression level was increased. The relative immunoreactivity of p-mTOR and p-p70S6K was significantly reduced in the HI group compared to sham. The HI group treated with G-CSF showed significant upregulated protein expression for p-mTOR and p-p70S6K levels compared to the HI group treated with saline. Furthermore, G-CSF treatment increased Bcl-2 expression levels and decreased CC3 and Bax expression levels in the ipsilateral hemispheres of the HI brain. The effects induced by G-CSF were all reversed by rapamycin. CONCLUSION: Treatment with G-CSF decreases inflammatory mediators and apoptotic factors, attenuating neuroinflammation and neuronal apoptosis via the mTOR/p70S6K signalling pathway, which represents a potential target for treating HI induced brain damage in neonatal HIE.

Mode of action of granulocyte-colony stimulating factor (G-CSF) as a novel therapy for stroke in a mouse model.

BACKGROUND: The FDA approved drug granulocyte-colony stimulating factor (G-CSF) displays anti-apoptotic and immunomodulatory properties with neurogenesis and angiogenic functions. It is known to demonstrate neuroprotective mechanisms against ischemic global stroke. Autophagy is a method for the degradation of intracellular components and in particular, unrestrained autophagy may lead to uncontrolled digestion of affected neurons as well as neuronal death in cerebral ischemia. Mitochondrial dynamics is vital for the regulation of cell survival and death after cerebral ischemia and an early upstream event in neuronal death is mitochondrial fission. We examined the pro-survival mechanisms of G-CSF against apoptosis resulting from autophagy, mitochondrial stress and endoplasmic reticulum (ER) stress. METHODS: Male Swiss Webster mice (20 weeks of age) were subjected to bilateral common carotid artery occlusion (BCAO) for 30 min. After occlusion, mice were injected with G-CSF (50 µg/kg) subcutaneously for 4 days. Behavioral analysis was carried out using the corner test and locomotor activity test before animals were sacrificed on day 4 or day 7. Key proteins in ER stress, autophagy and mitochondrial stress induced apoptosis were analyzed by immunoblotting. RESULTS: G-CSF improved neurological deficits and improved behavioral performance on corner and locomotor test. G-CSF binds to G-CSF receptors and its activation leads to upregulation of Akt phosphorylation (P-Akt) which in turn decreases levels of the ER stress sensor, GRP 78 and expression of proteins involved in ER stress apoptosis pathway; ATF6, ATF4, eIF2α, XBP1, Caspase 12 and CHOP. G-CSF treatment significantly decreased Beclin-1, an autophagy marker, and decreased mitochondrial stress biomarkers DRP1 and P53. G-CSF also up-regulated the mitochondrial fusion protein, OPA1 and anti-apoptotic protein Bcl-2 while down-regulating the pro-apoptotic proteins Bax, Bak and PUMA. CONCLUSIONS: G-CSF is an endogenous ligand in the CNS that has a dual activity that is beneficial both in reducing acute neuronal degeneration and adding to long-term plasticity after cerebral ischemia. G-CSF treatment exerts neuroprotective effects on damaged neurons through the suppression of the ER stress and mitochondrial stress and maintains cellular homeostasis by decreasing pro-apoptotic proteins and increasing of anti-apoptotic proteins.

A New Neural Pathway from the Ventral Striatum to the Nucleus Basalis of Meynert with Functional Implication to Learning and Memory.

The cholinergic neurons in the nucleus basalis of Meynert (NBM) are among the first group of neurons known to become degenerated in Alzheimer's disease, and thus the NBM is proposed to be involved in learning and memory. The marginal division (MrD) of the striatum is a newly discovered subdivision at the ventromedial border of the mammalian striatum and is considered to be one part of the ventral striatum involved in learning and memory. The present study provided evidence to support the hypothesis that the MrD and the NBM were structurally connected at cellular and subcellular levels with functional implications in learning and memory. First, when wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was stereotaxically injected into the NBM, fusiform neurons in the MrD were retrogradely labeled with WGA-HRP gray-blue particles and some of them were double stained in brown color by AchE staining method. Thus, cholinergic neurons of the MrD were shown to project to the neurons in the NBM. Second, in anterograde tract-tracing experiments where WGA-HRP was injected to the MrD, the labeled WGA-HRP was found to be anterogradely transported in axons from the MrD to the synaptic terminals with dendrites, axons, and perikaryons of the cholinergic neurons in the NBM when observed under an electronic microscope, indicating reciprocal structural connections between the MrD and the NBM. Third, when bilateral lesions of the MrD were injured with kainic acid in rats, degenerative terminals were observed in synapses of the NBM by an electronic microscope and severe learning and memory deficiency was found in these rats by the Y-maze behavioral test. Our results suggest reciprocal cholinergic connections between the MrD of the ventral striatum and the NBM, and implicate a role of the MrD-NBM pathway in learning and memory. The efferent fibers of cholinergic neurons in the NBM mainly project to the cortex, and severe reduction of the cholinergic innervation in the cortex is the common feature of Alzheimer's patients. The newly discovered cholinergic neural pathway between the MrD of the ventral striatum and the NBM is supposed involved in the memory circuitries of the brain and probably might play a role in the pathogenesis of the Alzheimer's disease.

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.

Role of Mitochondria and Endoplasmic Reticulum in Taurine-Deficiency-Mediated Apoptosis.

Taurine is a ubiquitous sulfur-containing amino acid found in high concentration in most tissues. Because of its involvement in fundamental physiological functions, such as regulating respiratory chain activity, modulating cation transport, controlling inflammation, altering protein phosphorylation and prolonging lifespan, taurine is an important nutrient whose deficiency leads to severe pathology and cell death. However, the mechanism by which taurine deficiency causes cell death is inadequately understood. Therefore, the present study examined the hypothesis that overproduction of reactive oxygen species (ROS) by complex I of the respiratory chain triggers mitochondria-dependent apoptosis in hearts of taurine transporter knockout (TauTKO) mice. In support of the hypothesis, a 60% decrease in mitochondrial taurine content of 3-month-old TauTKO hearts was observed, which was associated with diminished complex I activity and the onset of mitochondrial oxidative stress. Oxidative damage to stressed mitochondria led to activation of a caspase cascade, with stimulation of caspases 9 and 3 prevented by treatment of 3-month-old TauTKO mice with the mitochondria specific antioxidant, MitoTempo. In 12 month-old, but not 3-month-old, TauTKO hearts, caspase 12 activation contributes to cell death, revealing a pathological role for endoplasmic reticulum (ER) stress in taurine deficient, aging mice. Thus, taurine is a cytoprotective nutrient that ensures normal mitochondrial and ER function, which is important for the reduction of risk for apoptosis and premature death.

Neuroprotective Functions Through Inhibition of ER Stress by Taurine or Taurine Combination Treatments in a Rat Stroke Model.

Taurine, as a free amino acid, is found at high levels in many tissues including brain, heart and skeletal muscle and is known to demonstrate neuroprotective effects in a range of disease conditions including stroke and neurodegenerative disease. Using in vitro culture systems we have demonstrated that taurine can elicit protection against endoplasmic reticulum stress (ER stress) from glutamate excitotoxicity or from excessive reactive oxygen species in PC12 cells or rat neuronal cultures. In our current investigation we hypothesized that taurine treatment after stroke in the rat middle cerebral artery occlusion (MCAO) model would render protection against ER stress processes as reflected in decreased levels of expression of ER stress pathway components. We demonstrated that taurine elicited high level protection and inhibited both ATF-6 and IRE-1 ER stress pathway components. As ischemic stroke has a complex pathology it is likely that certain combination treatment approaches targeting multiple disease mechanisms may have excellent potential for efficacy. We have previously employed the partial NMDA antagonist DETC-MeSO to render protection against in vivo ischemic stroke using a rat cerebral ischemia model. Here we tested administration of subcutaneous administration of 0.56 mg/kg DETC-MeSO or 40 mg/kg of taurine separately or as combined treatment after a 120 min cerebral ischemia in the rat MCAO model. Neither drug alone demonstrated protection at the low doses employed. Remarkably however the combination of low dose DETC-MeSO plus low dose taurine conferred a diminished infarct size and an enhanced Neuroscore (reflecting decreased neurological deficit). Analysis of ER stress markers pPERK, peIF-2-alpha and cleaved ATF-6 all showed decreased expression demonstrating that all 3 ER stress pathways were inhibited concurrent with a synergistic protective effect by the post-stroke administration of this DETC-MeSO-taurine combination treatment.

Analysis of Neuroprotection by Taurine and Taurine Combinations in Primary Neuronal Cultures and in Neuronal Cell Lines Exposed to Glutamate Excitotoxicity and to Hypoxia/Re-oxygenation.

Ischemic stroke is one of the greatest contributors to death and long term disability in developed countries. Ischemia induced brain injury arises due to excessive release of glutamate and involves cell death due to apoptosis and endoplasmic reticulum (ER) stress responses. Despite major research efforts there are currently no effective treatments for stroke. Taurine, a free amino acid found in high concentrations in many invertebrate and vertebrate systems can provide protection against a range of neurological disorders. Here we demonstrate that taurine can combat ER stress responses induced by glutamate or by hypoxia/re-oxygenation in neuronal cell lines and primary neuronal cultures. Taurine decreased expression of ER stress markers GRP78, CHOP, Bim and caspase 12 in primary neuronal cultures exposed to hypoxia/re-oxygenation. In analyzing individual ER stress pathways we demonstrated that taurine treatment can result in reduced levels of cleaved ATF6 and decreased p-IRE1 levels. We hypothesized that because of the complex nature of stroke a combination therapy approach may be optimal. For this reason we proceeded to test combination therapies using taurine plus low dose administration of an additional drug: either granulocyte colony stimulating factor (G-CSF) or sulindac a non-steroidal anti-inflammatory drug with potent protective functions through signaling via ischemic preconditioning pathways. When primary neurons were pretreated with 25 mM taurine and 25 ng/mL G-CSF for I hour and then exposed to high levels of glutamate, the taurine/G-CSF combination increased the protective effect against glutamate toxicity to 88% cell survival compared to 75% cell survival from an individual treatment with taurine or G-CSF alone. Pre-exposure of PC12 cells to 5 mM taurine or 25 µM sulindac did not protect the cells from hypoxia/re-oxygenation stress whereas at these concentrations the combination of taurine plus sulindac provided significant protection. In summary we have demonstrated the protective effect of taurine in primary neuronal cultures against hypoxia with re-oxygenation through inhibition of ATF6 or p-IRE-1 pathway but not the PERK pathway of ER stress. Furthermore the combinations of taurine plus an additional drug (either G-CSF or sulindac) can show enhanced potency for protecting PC 12 cells from glutamate toxicity or hypoxia/re-oxygenation through inhibition of ER stress responses.

Dr. Eugene Roberts.

This is an obituary for Eugene Roberts (1920-2016), an outstanding neurobiochemist who was the first to report on the discovery of gamma-aminobutyric acid (GABA) in the brain, and whose research focused on the important role of GABA in human health and disease.

Preparation, Stimulation and Other Uses of Adult Rat Brain Synaptosomes.

In this paper, our protocol for preparation of brain synaptosomes is described. Synaptosomes are a valuable model system for analysis of structural components of the synapse as well as for investigation of synaptic function. Synaptosomal preparations are necessary for understanding molecular changes at synapses where critical post-translational modifications of synaptic proteins may occur. Not only are synaptosomes rich in synaptic proteins, but they can be used for analyzing uptake of neurotransmitters into synaptic vesicles and for analysis of the involvement of neurotransmitter synthesis and release. Synaptosomes can be stimulated with increased calcium influx to release neurotransmitters. Synaptosomal preparations have been used in characterizing calcium dependent phosphorylation and activation of the GABA synthesizing enzyme GAD65 (L-glutamic acid decarboxylase with molecular weight of 65 kDa). By examining protein complexes on the membrane of synaptic vesicles obtained from synaptosomal preparations, it was possible to characterize the role of GAD65 in the coupled synthesis and vesicular uptake of GABA (γ-aminobutyric acid) culminating in GABA vesicular release, which contributes in an important way to fine-tuning of GABAergic neurotransmission.

Activation of Brain L-glutamate Decarboxylase 65 Isoform (GAD65) by Phosphorylation at Threonine 95 (T95).

Protein phosphorylation plays an important role in regulating soluble L-glutamic acid decarboxylase (GAD) and membrane-associated GAD activity. Previously, we reported the effect of phosphorylation on the two well-defined GAD isoforms, namely, GAD65 and GAD67, using highly purified preparations of recombinant human brain GAD65 (hGAD65) and GAD67. GAD65 was activated by phosphorylation, while GAD67 was inhibited by phosphorylation. The effect of phosphorylation on GAD65 and GAD67 could be reversed by treatment with protein phosphatases. We further demonstrated that protein kinase A (PKA) and protein kinase C isoform ε were the protein kinases responsible for phosphorylation and regulation of GAD67 and GAD65, respectively. In the current study, using MALDI-TOF, a total of four potential phosphorylation sites were identified in GAD65, two of which (threonine-95 (T-95) and Ser-417) were not reported previously. We have identified one specific phosphorylation site, (T95), in hGAD65 that can be phosphorylated by kinase C ε (PKCε) using MALDITOF. When T95 is mutated to alanine, hGAD65 could no longer be phosphorylated by PKCε, and the effect of PKC-mediated activation on hGAD65 is abolished. However, when T95 is mutated to glutamic acid, which mimics the phosphorylation status of hGAD65, the activity was greatly increased. An increase of GAD65 activity by 55 % compared to the wild type hGAD65 was observed indicating that mutation of T95 to glutamic acid mimics the effect of phosphorylation. A model depicting the role of phosphorylation of GAD65 in regulation of GABA neurotransmission is presented.

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.

Regulation of GABA Neurotransmission by Glutamic Acid Decarboxylase (GAD).

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.

The marginal division of the striatum and hippocampus has different role and mechanism in learning and memory.

The memory function of the hippocampal formation (Hip) and the marginal division (MrD) of neostriatum was compared. Rats with bilateral lesions of the MrD either immediate or 24 h after training in Y-maze were found to have decrease in correct runs in both groups. However, animals with transected afferent and efferent nerve bundles to isolate the Hip immediately or 24 h after training in Y-maze were found to show a decrease in correct runs only in the group injured immediately after Y-maze training but not in the 24 h group suggesting that MrD is likely involved in the entire process of long-term memory consolidation whereas the Hip only contributes to memory in the early stage. In addition, animals treated with a NMDA receptor (NMDAR) blocker, e.g. MK-801, showed decreased correct runs in Y-maze test and in expression level of phosphorylated CREB (pCREB) in neurons of the MrD but not in the Hip. Furthermore, animals treated with okadaic acid (OA), a potent protein phosphatase 1 inhibitor, showed increased correct runs in the Y-maze test. The expression level of pCREB and c-Fos and c-Jun was found increased in neurons of the MrD and the Hip in response to OA treatment. In conclusion, NMDAR and pCREB are involved in memory functions of both the Hip and the MrD. NMDAR might regulate pCREB level in neurons of the MrD but not in the Hip. Hence, the processes and mechanism of learning and memory involved in the MrD and the Hip may be different.

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.

Mode of action of S-methyl-N, N-diethylthiocarbamate sulfoxide (DETC-MeSO) as a novel therapy for stroke in a rat model.

One approach for protecting neurons from excitotoxic damage in stroke is to attenuate receptor activity with specific antagonists. S-Methyl-N, N-diethylthiocarbamate sulfoxide (DETC-MeSO), the active metabolite of disulfiram, has been shown to be a partial antagonist of glutamate receptors and effective in reducing seizure. First, we investigated neuroprotective effect of DETC-MeSO on primary cortical neuronal culture under hypoxia/reoxygenation condition in vitro. Then, DETC-MeSO was administered subcutaneously for 4 and 8 days with the first injection occurring 1 h before or 24 h after reperfusion in the rat middle cerebral artery occlusion stroke model. Rats were subjected to the neuroscore test, and the brain was analyzed for infarct size. Monitoring neurotransmitter release was carried out by microdialysis. Heat shock proteins, key proteins involved in apoptosis and endoplasmic reticulum (ER) stress, were analyzed by immunoblotting. DETC-MeSO greatly reduced both cell death following hypoxia/reoxygenation and brain infarct size. It improved performance on the neuroscore test and attenuated proteolysis of αII-spectrin. The level of pro-apoptotic proteins declined, and anti-apoptotic and HSP27 protein expressions were markedly increased. Levels of the ER stress protein markers p-PERK, p-eIF2α, ATF4, JNK, XBP-1, GADD34, and CHOP significantly declined after DETC-MeSO administration. Microdialysis data showed that DETC-MeSO increased high potassium-induced striatal dopamine release indicating that more neurons were protected and survived under ischemic insult in the presence of DETC-MeSO. We also showed that DETC-MeSO can prevent gliosis. DETC-MeSO elicits neuroprotection through the preservation of ER resulting in reduction of apoptosis by increase of anti-apoptotic proteins and decrease of pro-apoptotic proteins.

Taurine and central nervous system disorders.

In the present era, investigators seek to find therapeutic interventions that are multifaceted in their mode of action. Such targets provide the most advantageous routes for addressing the multiplicity of pathophysiological avenues that lead to neuronal dysfunction and death observed in neurological disorders and neurodegenerative diseases. Taurine, an endogenous amino acid, exhibits a plethora of physiological functions in the central nervous system. In this review, we describe the mode of action of taurine and its clinical application in the neurological diseases: Alzheimer's disease, Parkinson's disease and Huntington's disease.