Krishna Banaudha

AMPA protects cultured neurons against glutamate excitotoxicity through a phosphatidylinositol 3-kinase-dependent activation in extracellular signal-regulated kinase to upregulate BDNF gene expression

The signal transduction and molecular mechanisms underlying α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA)‐mediated neuroprotection are unknown. In the present study, we determined a major AMPA receptor‐mediated neuroprotective pathway. Exposure of cerebellar granule cells to AMPA (500 µm) + aniracetam (1 µm), a known blocker of AMPA receptor desensitization, evoked an accumulation of brain‐derived neurotropic factor (BDNF) in the culture medium and enhanced TrkB‐tyrosine phosphorylation following the release of BDNF. AMPA also activated the src‐family tyrosine kinase, Lyn, and the downstream target of the phosphatidylinositol 3‐kinase (PI3‐K) pathway, Akt. Extracellular signal regulated kinase (ERK), a component of the mitogen‐activated protein kinase (MAPK) pathway, was also activated. K252a, a selective inhibitor of neurotrophin signaling, blocked the AMPA‐mediated neuroprotection. The involvement of BDNF release in protecting neurons by AMPA was confirmed using a BDNF‐blocking antibody. AMPA‐mediated neuroprotection is blocked by PP1, an inhibitor of src family kinases, LY294002, a PI3‐K inhibitor, or U0126, a MAPK kinase (MEK) inhibitor. Neuroprotective concentrations of AMPA increased BDNF mRNA levels that was blocked by the AMPA receptor antagonist, 1,2,3,4‐tetrahydro‐6‐nitro‐2,3‐dioxo‐benzo[f]quinoxaline‐7‐sulfonamide (NBQX). The increase in BDNF gene expression appeared to be the downstream target of the PI3‐K‐dependent activation of the MAPK cascade since MEK or the PI3‐K inhibitor blocked the AMPA receptor‐mediated increase in BDNF mRNA. Thus, AMPA receptors protect neurons through a mechanism involving BDNF release, TrkB receptor activation, and a signaling pathway involving a PI3‐K dependent activation of MAPK that increases BDNF expression.

N-Methyl-D-aspartate and TrkB Receptor Activation in Cerebellar Granule Cells: An In Vitro Model of Preconditioning to Stimulate Intrinsic Survival Pathways in Neurons

Abstract: Delineating the mechanisms of survival pathways that exist in neurons will provide important insight into how neurons utilize intracellular proteins as neuroprotectants against the causes of acute neurodegeneration. We have employed cultured rat cerebellar granule cells as a model for determining the mechanisms of these intraneuronal survival pathways. Glutamate has long been known to kill neurons by an N‐methyl‐d‐aspartate (NMDA) receptor‐mediated mechanism. Paradoxically, subtoxic concentrations of NMDA protect neurons against glutamate‐mediated excitotoxicity. Because NMDA protects neurons in physiologic concentrations of glucose and oxygen, we refer to this phenomenon as physiologic preconditioning. One of the major mechanisms of NMDA neuroprotection involves the activation of NMDA receptors leading to the rapid release of brain‐derived neurotrophic factor (BDNF). BDNF then binds to and activates its cognate receptor, receptor tyrosine kinase B (TrkB). The efficient utilization of these two receptors confers remarkable resistance against millimolar concentrations of glutamate that kill more than eighty percent of the neurons in the absence of preconditioning the neurons with a subtoxic concentration of NMDA. Exactly how the neurons mediate neuroprotection by activation of both receptors is just beginning to be understood. Both NMDA and TrkB receptors activate nuclear factor kappaB (NF‐κB), a transcription factor known to be involved in protecting neurons against many different kinds of toxic insults. By converging on survival transcription factors, such as NF‐κB, NMDA and TrkB receptors protect neurons. Thus, crosstalk between these very different receptors provides a rapid means of neuronal communication to upregulate survival proteins through release and transcriptional activation of messenger RNA.

N‐methyl‐D‐aspartate and TrkB receptors protect neurons against glutamate excitotoxicity through an extracellular signal‐regulated kinase pathway

N‐Methyl‐D‐aspartate (NMDA) at a subtoxic concentration (100 μM) promotes neuronal survival against glutamate‐mediated excitotoxicity via a brain‐derived neurotrophic factor (BDNF) autocrine loop in cultured cerebellar granule cells. The signal transduction mechanism(s) underlying NMDA neuroprotection, however, remains elusive. The mitogen‐activated protein kinase (MAPK) and phosphatidylinositol‐3 kinase (PI3‐K) pathways alter gene expression and are involved in synaptic plasticity and neuronal survival. This study tested whether neuroprotective activation of NMDA receptors, together with TrkB receptors, coactivated the MAPK or PI3‐K pathways to protect rat cerebellar neurons. NMDA receptor activation caused a concentration‐ and time‐dependent activation of MAPK lasting 24 hr. This activation was blocked by the NMDA receptor antagonist MK‐801 but was attenuated only partially by the tyrosine kinase inhibitor k252a, suggesting that activation of both NMDA and TrkB receptors are required for maximal neuroprotection. The MAPK kinase (MEK) inhibitor U0126 (10 μM) partially blocked NMDA neuroprotection, whereas LY294002, a selective inhibitor of the PI3‐K pathway, did not affect the neuroprotective activity of NMDA. Glutamate excitotoxicity decreased bcl‐2, bcl‐XL, and bax mRNA levels,. NMDA increases Bcl‐2 and Bcl‐XL protein levels and decreases Bax protein levels. NMDA and TrkB receptor activation thus converge on the extracellular signal‐regulated kinase (ERK) 1/2 signaling pathway to protect neurons against glutamate‐mediated excitotoxicity. By increasing antiapoptotic proteins of the Bcl‐2 family, NMDA receptor activation may also promote neuronal survival by preventing apoptosis.

Prevention of renal ischemia-reperfusion-induced injury in rats by picroliv

Picroliv is a potent antioxidant extracted from the roots and rhizome of Picrorhiza kurrooa. It has been shown to impart significant hepatoprotective activities, partly by modulation of free radical-induced lipid peroxidation. Lipid peroxidation and reactive oxygen species are associated with tissue injury in post-ischemic acute renal failure. The efficacy of picroliv was assessed in an in vivo model of renal ischemia-reperfusion injury (IRI) in rats at a dose of 12 mg/kg orally for 7 days. The animals were killed at various times after reperfusion. Increased lipid peroxidation and apoptotic cell number reflected the oxidative damage following renal IRI. Picroliv-pretreated rats exhibited lower lipid peroxidation, improved antioxidant status, and reduced apoptosis, indicating better viability of renal cells. Immunohistochemical studies revealed that picroliv pretreatment attenuated the expression of intercellular adhesion molecule-1 in the glomerular region. These results suggested that picroliv pretreatment protects rat kidneys from IRI, perhaps by modulation of free radical damage and adhesion molecules.

Non-Linear Effects of Cycloheximide in Glutamate-Treated Cultured Rat Cerebellar Neurons ☆

Multiple cell types and organisms across a wide array of phyla and a variety of toxins demonstrate non-linear dose responses to low-level chemical exposures with high doses inhibiting cellular function and low doses stimulating function. We tested whether such non-linear responses to low and ultra-low dose N-methyl-D-aspartate (NMDA), 1-methyl-4-phenylpyridinium (MPP+) or cycloheximide moderated toxic glutamate exposure in cultured cerebellar granule cells. Neurons were incubated over 72 h with successive NMDA, MPP+ iodide or cycloheximide additions producing specified low (10(-5), 10(-7), 10(-9), 10(-11), and 10(-13) M) and ultra-low (10(-27),10(-29), 10(-63), and 10(-65) M) concentrations. Subsequently these neuronal cells were exposed to a 50% excitotoxic concentration of glutamate for 24 h. Neuronal viability was significantly reduced in neurons treated with micromolar (10(-5) M) cycloheximide whereas viability was enhanced in neurons treated with an ultra-low dose exposure of 10(-27) M cycloheximide. Neither NMDA nor MPP+ elicited harmful or protective responses. This is the first report demonstrating non-linear dose-response effects of cycloheximide in low and ultra-low concentration ranges.

NONLINEAR EFFECTS OF GLUTAMATE AND KCl ON GLUTAMATE TOXICITY IN CULTURED RAT CEREBELLAR NEURONS

Nonlinear responses to toxin exposure have been observed in multiple cell types and organisms across a wide array of phyla. High dose toxin exposures inhibit or kill biological systems, while low dose exposures can stimulate survival mechanisms. We examined the effects of low (10-3, 10-5, 10-7, and 10-9M) and ultra-low (10-25 and 10-61M) KCl and glutamate pretreatment (72 h) against glutamate toxicity in rat cerebellar neurons. Ultra-low dilutions (10-31, 10-61, and 10-401) of an Arnica montana mother tincture were also investigated for their neuroprotective potentials. Viability was significantly enhanced in neurons pretreated with either 10-3M glutamate (10.6%) or 10-9M KCl (6.3%). None of the toxins evaluated displayed significant toxicity at the concentrations indicated. The protective effect of glutamate is likely mediated through activation of N-methyl-D- aspartate receptors, whereas low dose KCl might confer neuroprotection through enhanced alteration of Na+/K+ receptor dynamics. This is the first time high dose glutamate tolerance has been shown along with low dose KCl, and is consistent with previous reports demonstrating tolerance induced by low dose toxin exposure.

Enhancement of Wound Healing by Shikonin Analogue 93/637 in Normal and Impaired Healing

Wound healing is a complicated biological process, which involves interactions of multiple cell types, various growth factors, their mediators and the extracellular matrix proteins. In this study, we evaluated the effects of shikonin analogue 93/637 (SA), derived from the plant Arnebia nobilis, on normal and hydrocortisone-induced impaired healing in full thickness cutaneous punch wounds in rats. SA (0.1%) was applied topically daily as an ointment in polyethylene glycol base on wounds. SA treatment significantly accelerated healing of wounds, as measured by wound contraction compared to controls in hydrocortisone-impaired animals. SA treatment promoted formation of granulation tissue including cell migration and neovascularization, collagenization and reepithelialization. The expression of basic fibroblast growth factor (bFGF) was higher as revealed by immunohistochemistry in treated wounds compared to controls. However, the expression of transforming growth factor-β1 was not affected by SA treatment. Since bFGF is known to accelerate wound healing, the increased expression of bFGF by SA may be partly responsible for the enhancement of wound healing. These studies suggest that SA could be further studied for clinical use to enhance wound healing.

Picroliv modulates the expression of insulin-like growth factor (IGF)-I, IGF-II and IGF-I receptor during hypoxia in rats.

Abstract. The insulin-like growth factors (IGFs), IGF-I and IGF-II, play important roles in normal growth and differentiation. In recent studies, IGFs have been implicated in tissue repair and regeneration after hypoxic-ischemic injury. The growth effects of these genes are exerted primarily through IGF-I receptor (IGF-IR). We have earlier shown that picroliv, obtained from the roots of Picrorhiza kurrooa, reduces cellular damage caused by hypoxia in vitro. We have now studied the modulation of IGF-I, IGF-II and IGF-IR in hypoxia and the ability of picroliv to modify their expression in vivo. Male Sprague-Dawley rats, placed in l0% oxygen for 4 days, were sacrificed, and the expression of IGF-I, IGF-II and IGF-IR was determined by immunohistochemistry, in situ hybridization and reverse transcriptase polymerase chain reaction (RT-PCR) in brain, liver and lung. One group of animals was pretreated with picroliv and the other served as control. IGF-I and IGF-IR were expressed in distinct regions of the brain but not in liver or lung. IGF-I was mainly expressed in the hippocampus and cerebellum, whereas IGF-IR expression was also observed in the cortex. A significant reduction in the messenger RNA (mRNA) level of these genes was observed in response to hypoxia. Pretreatment with picroliv not only prevented such downregulation but more importantly resulted in increased levels of IGF-I and IGF-IR. These observations correlated with reduced neuronal cell death observed in these animals. The mRNA of IGF-II was constitutively expressed and was not altered by hypoxia. Modulation of IGF-I and IGF-II expression by picroliv, a novel pharmacological agent, could benefit in similar clinical settings such as myocardial ischemia and certain cerebral injuries.

AMPA prevents glutamate-induced neurotoxicity and apoptosis in cultured cerebellar granule cell neurons.

Exposure of cultured cerebellar neurons to ±-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) in the presence of aniracetam protects all of the vulnerable neurons against the excitotoxic actions of glutamate acting on N-methyl-D-aspartate receptors. The protective effect of AMPA was both time- and concentration-dependent. Aniracetam alone did not protect the neurons against the excitotoxic effects of glutamate. Pretreatment of cerebellar neurons with the AMPA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione blocked the neuroprotective effect mediated by AMPA indicating that the neuroprotective effect is mediated specifically by AMPA receptors. An excitotoxic concentration of glutamate, which killed between 60–80% of granule cell neurons on day 8in vitro, mediated its toxic effect via a time-dependent apoptotic pathway. Pretreatment of cerebellar granule cell neurons with AMPA (500 μM) completely blocked glutamate-mediated apoptosis. Our results suggest that AMPA receptors may play an important role in neuronal survival.