Inhibition of cystathionine-γ-lyase leads to loss of glutathione and aggravation of mitochondrial dysfunction mediated by excitatory amino acid in the CNS

Oxidative stress has been implicated in the pathogenesis and progression of neurodegenerative disorders and antioxidants potentially have a major role in neuroprotection. Optimum levels of glutathione (γ-glutamylcysteinyl glycine), an endogenous thiol antioxidant are required for the maintenance of the redox status of cells. Cystathionine γ-lyase is the rate-limiting enzyme for the synthesis of cysteine from methionine and availability of cysteine is a critical factor in glutathione synthesis. In the present study, we have examined the role of cystathionine γ-lyase in maintaining the redox homeostasis in brain, particularly with reference to mitochondrial function since the complex I of the electron transport chain is sensitive to redox perturbation. Inhibition of cystathionine γ-lyase by L-propargylglycine caused loss of glutathione and decrease in complex I activity in the brain although the enzyme activity in mouse brain was 1% of the corresponding hepatic activity. We then examined the effect of this inhibition on the neurotoxicity mediated by the excitatory amino acid, L-β-oxalyl amino-L-alanine, which is the causative factor of a type of motor neuron disease, neurolathyrism. L-β-oxalyl amino-L-alanine toxicity was exacerbated by L-propargylglycine measured as loss of complex I activity indicating the importance of cystathionine γ-lyase in maintaining glutathione levels and in turn the mitochondrial function during excitotoxicity. Oxidative stress generated by L-β-oxalyl amino-L-alanine itself inhibited cystathionine γ-lyase, which could be prevented by prior treatment with thiol antioxidant. Thus, cystathionine γ-lyase itself is susceptible to inactivation by oxidative stress and this can potentially exacerbate oxidant-induced damage. Cystathionine γ-lyase is present in neuronal cells in human brain and its activity is several-fold higher compared to mouse brain. It could potentially play an important role in maintaining glutathione and protein thiol homeostasis in brain and hence afford neuroprotection

Estrogen and neuroprotection: higher constitutive expression of glutaredoxin in female mice offers protection against MPTP-mediated neurodegeneration

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SYNTHESIS AND IN-VITRO STUDY OF NOVEL (Z)-1-BENZHYDRYL-4-CINNAMYLPIPERAZINE DERIVATIVES AS POTENTIAL ANTICANCER AGENTS

Objective: The objective of this study was to synthesize Z- 1-benzhydryl-4-cinnamylpiperazines by novel stereo selective synthetic method and evaluation of their anticancer properties.Methods: A series of novel (Z)-1-benzhydryl-4-cinnamylpiperazine derivatives (9a-j) were synthesized, starting from benzophenones in six steps. Wittig condensation of appropriate benzyltriphenyl phosphonium halides with various 1-benzhydryl- 4-(2-ethanal) piperazines (3a-j), and column purification over silica gel afforded pure Z- 1-benzhydryl-4-cinnamylpiperazines.Results: The structures of newly synthesized compounds 9a-j were established by 1H & 13C NMR and mass spectral analysis. The anticancer potential (MTT assay) of synthesized compounds was tested against human cervical cancer (HeLa) and murine microglial (BV-2) cell lines. Results indicated that the most of the Z-derivatives exhibited moderate to good anticancer activity on both the cell lines over their E- antipodes.Conclusion: Compound 9i (cis- flunarizine) exhibited exceptionally superior activity against both HeLa and BV-2 cell lines with IC50 value of 13.23±3.51 µM and 23.1±4.12 µM respectively. Hence, this compound may be considered to be a potential lead molecule for further developmentÂ

Thioltransferase (glutaredoxin) mediates recovery of motor neurons from excitotoxic mitochondrial injury

Mitochondrial dysfunction involving electron transport components is implicated in the pathogenesis of several neurodegenerative disorders and is a critical event in excitotoxicity. Excitatory amino acid L-β-N-oxalylamino-L-alanine (L-BOAA), causes progressive corticospinal neurodegeneration in humans. In mice, L-BOAA triggers glutathione loss and protein thiol oxidation that disrupts mitochondrial complex I selectively in motor cortex and lumbosacral cord, the regions affected in humans. We examined the factors regulating postinjury recovery of complex I in CNS regions after a single dose of L-BOAA. The expression of thioltransferase (glutaredoxin), a protein disulfide oxidoreductase regulated through AP1 transcription factor was upregulated within 30 min of L-BOAA administration, providing the first evidence for functional regulation of thioltransferase during restoration of mitochondrial function. Regeneration of complex I activity in motor cortex was concurrent with increase in thioltransferase protein and activity, 1 hr after the excitotoxic insult. Pretreatment with α-lipoic acid, a thiol delivery agent that protects motor neurons from L-BOAA-mediated toxicity prevented the upregulation of thioltransferase and AP1 activation, presumably by maintaining thiol homeostasis. Downregulation of thioltransferase using antisense oligonucleotides prevented the recovery of complex I in motor cortex and exacerbated the mitochondrial dysfunction in lumbosacral cord, providing support for the critical role for thioltransferase in maintenance of mitochondrial function in the CNS

Down-regulation of glutaredoxin by estrogen receptor antagonist renders female mice susceptible to excitatory amino acid mediated complex I inhibition in CNS

β-N-oxalyl-amino-L-alanine, (L-BOAA), an excitatory amino acid, acts as an agonist of the AMPA subtype of glutamate receptors. It inhibits mitochondrial complex I in motor cortex and lumbosacral cord of male mice through oxidation of critical thiol groups, and glutaredoxin, a thiol disulfide oxido-reductase, helps maintain integrity of complex I. Since incidence of neurolathyrism is less common in women, we examined the mechanisms underlying the gender-related effects. Inhibition of complex I activity by L-BOAA was seen in male but not female mice. Pretreatment of female mice with estrogen receptor antagonist ICI 182,780 or tamoxifen sensitizes them to L-BOAA toxicity, indicating that the neuroprotection is mediated by estrogen receptors. L-BOAA triggers glutathione (GSH) loss in male mice but not in female mice, and only a small but significant increase in oxidized glutathione (GSSG) was seen in females. As a consequence, up-regulation of γ-glutamyl cysteinyl synthase (the rate-limiting enzyme in glutathione synthesis) was seen only in male mouse CNS but not in females. Both glutathione reductase and glutaredoxin that reduce oxidized glutathione and protein glutathione mixed disulfides, respectively, were constitutively expressed at higher levels in females. Furthermore, glutaredoxin activity in female mice was down-regulated by estrogen antagonist indicating its regulation by estrogen receptor. The higher constitutive expression of glutathione reductase and glutaredoxin could potentially confer neuroprotection to female mice

Thioltransferase (glutaredoxin) mediates recovery of motor neurons from excitotoxic mitochondrial injury

Mitochondrial dysfunction involving electron transport components is implicated in the pathogenesis of several neurodegenerative disorders and is a critical event in excitotoxicity. Excitatory amino acid L-␤-N-oxalylamino-L-alanine (L-BOAA), causes progressive corticospinal neurodegeneration in humans. In mice, L-BOAA triggers glutathione loss and protein thiol oxidation that disrupts mitochondrial complex I selectively in motor cortex and lumbosacral cord, the regions affected in humans. We examined the factors regulating postinjury recovery of complex I in CNS regions after a single dose of L-BOAA. The expression of thioltransferase (glutaredoxin), a protein disulfide oxidoreductase regulated through AP1 transcription factor was upregulated within 30 min of L-BOAA administration, providing the first evidence for functional regulation of thioltransferase during restoration of mitochondrial function. Regeneration of complex I activity in motor cortex was concurrent with increase in thioltransferase protein and activity, 1 hr after the excitotoxic insult. Pretreatment with ␣-lipoic acid, a thiol delivery agent that protects motor neurons from L-BOAA-mediated toxicity prevented the upregulation of thioltransferase and AP1 activation, presumably by maintaining thiol homeostasis. Downregulation of thioltransferase using antisense oligonucleotides prevented the recovery of complex I in motor cortex and exacerbated the mitochondrial dysfunction in lumbosacral cord, providing support for the critical role for thioltransferase in maintenance of mitochondrial function in the CNS

Selective neurodegeneration of hippocampus and entorhinal cortex correlates with spatial learning impairments in rats with bilateral ibotenate lesions of ventral subiculum

Rats with bilateral ibotenic acid lesions of ventral subiculum were tested in an eight-arm radial maze task for spatial learning and memory functions. The performance of the lesioned rats was severely impaired relative to control rats in both acquisition and retention of the spatial task. Following subicular lesions, profound neurodegeneration of the CA1 and CA3 sub sectors of hippocampus and entorhinocortical layers I, II, III, V and VI was observed. These results support the concept that neurons in the ventral subiculum are a part of the neural network along with the above neurons, which could be involved in the processing of spatial information

Protein Glutathionylation and Glutaredoxin: Role in Neurodegenerative Diseases

Oxidative stress has been implicated in the pathogenesis and progression of many neurodegenerative disorders including Parkinson’s disease and Alzheimer’s disease. One of the major enzyme systems involved in the defense against reactive oxygen species are the tripeptide glutathione and oxidoreductase glutaredoxin. Glutathione and glutaredoxin system are very important in the brain because of the oxidative modification of protein thiols to protein glutathione mixed disulfides with the concomitant formation of oxidized glutathione during oxidative stress. Formation of Pr-SSG acts as a sink in the brain and is reduced back to protein thiols during recovery, thus restoring protein functions. This is unlike in the liver, which has a high turnover of glutathione, and formation of Pr-SSG is very minimal as liver is able to quickly quench the prooxidant species. Given the important role glutathione and glutaredoxin play in the brain, both in normal and pathologic states, it is necessary to study ways to augment the system to help maintain the protein thiol status. This review details the importance of glutathione and glutaredoxin systems in several neurodegenerative disorders and emphasizes the potential augmentation of this system as a target to effectively protect the brain during aging