Vijayalakshmi Ravindranath

Grand challenges in global mental health

A consortium of researchers, advocates and clinicians announces here research priorities for improving the lives of people with mental illness around the world, and calls for urgent action and investment.

Evidence for generation of oxidative stress in brain by MPTP: in vitro and in vivo studies in mice

The role of oxidative stress in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated neurotoxicity is as yet unclear and the evidence for generation of oxygen free radicals as a primary event in the neurotoxicity is yet to be demonstrated. The present study was undertaken to ascertain the potential role of oxidative damage, and the protective role, if any, of the antioxidant, glutathione (GSH), in MPTP-induced neurotoxicity. Exposure of sagittal slices of mouse brain to MPTP resulted in significant increases of reactive oxygen species (ROS) and malondialdehyde (MDA, the product of lipid peroxidation) and decreases in GSH content. Pretreatment of mouse brain slices, in vitro, with GSH or GSH isopropyl ester attenuated MPTP toxicity as assessed by the tissue activity of the mitochondrial enzyme, NADH-dehydrogenase (NADH-DH), and by leakage of the cytosolic enzyme, lactate dehydrogenase (LDH), from the slice into the medium. In vivo administration of MPTP (30 mg/kg body weight, s.c.), to mice resulted in significant lowering of GSH in the striatum and midbrain, 2 h after dosage; ROS levels in the striatum and midbrain increased after 4 and 8 h, respectively. In the striatum significant inhibition of rotenone-sensitive NADH ubiquinone-1 oxido-reductase (Complex 1) was observed transiently 1 h after MPTP administration. The enzyme activity recovered thereafter; significant inhibition of mitochondrial Complex I was observed in the striatum only 18 h after MPTP dose. In the midbrain, mitochondrial Complex I was inhibited only 18 h after MPTP dose; no change was observed at the early time points examined. Thus, the depletion of GSH and increased ROS formation preceded the inhibition of the mitochondrial enzyme in the midbrain. Evidence presented herein from both in vitro and in vivo studies support that MPTP exposure generates ROS resulting in oxidative stress.

α-Lipoic acid protects against reperfusion injury following cerebral ischemia in rats

Ischemic-reperfusion injury in humans occurs in conditions such as stroke, cardiac arrest, subarachnoid hemorrhage or head trauma. Maximal tissue damage is observed during reperfusion, which is primarily attributed to oxidative injury resulting from production of oxygen free radicals. One of the major consequences of such damage is the depletion of the cellular antioxidant, glutathione (GSH) leading to oxidation of protein thiols to disulfides and the loss of activity of critical enzymes having active thiol group(s). Thus, the maintenance of thiol homeostasis is an important factor in cell survival. The effect of thiol antioxidants like alpha-lipoic acid and the isopropyl ester of GSH was examined on the morbidity and mortality of rats subjected to reperfusion following cerebral ischemia induced by bilateral carotid artery occlusion and hypotension. While the GSH isopropyl ester had no significant protective effect; after pretreatment of rats, alpha-lipoic acid was detected in the rat brain and it dramatically reduced the mortality rate from 78% to 26% during 24 h of reperfusion. The natural thiol antioxidant, alpha-lipoic acid is effective in improving survival and protecting the rat brain against reperfusion injury following cerebral ischemia.

Withania somnifera reverses Alzheimer's disease pathology by enhancing low-density lipoprotein receptor-related protein in liver

A 30-d course of oral administration of a semipurified extract of the root of Withania somnifera consisting predominantly of withanolides and withanosides reversed behavioral deficits, plaque pathology, accumulation of β-amyloid peptides (Aβ) and oligomers in the brains of middle-aged and old APP/PS1 Alzheimers disease transgenic mice. It was similarly effective in reversing behavioral deficits and plaque load in APPSwInd mice (line J20). The temporal sequence involved an increase in plasma Aβ and a decrease in brain Aβ monomer after 7 d, indicating increased transport of Aβ from the brain to the periphery. Enhanced expression of low-density lipoprotein receptor-related protein (LRP) in brain microvessels and the Aβ-degrading protease neprilysin (NEP) occurred 14–21 d after a substantial decrease in brain Aβ levels. However, significant increase in liver LRP and NEP occurred much earlier, at 7 d, and were accompanied by a rise in plasma sLRP, a peripheral sink for brain Aβ. In WT mice, the extract induced liver, but not brain, LRP and NEP and decreased plasma and brain Aβ, indicating that increase in liver LRP and sLRP occurring independent of Aβ concentration could result in clearance of Aβ. Selective down-regulation of liver LRP, but not NEP, abrogated the therapeutic effects of the extract. The remarkable therapeutic effect of W. somnifera mediated through up-regulation of liver LRP indicates that targeting the periphery offers a unique mechanism for Aβ clearance and reverses the behavioral deficits and pathology seen in Alzheimers disease models.

Activation of apoptosis signal regulating kinase 1 (ASK1) and translocation of death-associated protein, Daxx, in substantia nigra pars compacta in a mouse model of Parkinson’s disease: protection by α-lipoic acid

Parkinsons disease (PD), a neurodegenerative disorder, causes severe motor impairment due to loss of dopaminergic neurons in substantia nigra pars compacta (SNpc). MPTP, a neurotoxin that causes dopaminergic cell loss in mice, was used in an animal model to study the pathogenic mechanisms leading to neurodegeneration. We observed the activation of apoptosis signal regulating kinase (ASK1, MAPKKK) and phosphorylation of its downstream targets MKK4 and JNK, 12 h after administration of a single dose of MPTP. Further, Daxx, the death‐associated protein, translocated to the cytosol selectively in SNpc neurons seemingly due to MPTP mediated down‐regulation of DJ‐1, the redox‐sensitive protein that binds Daxx in the nucleus. Coadministration of a‐lipoic acid (ALA), a thiol antioxidant, abolished the activation of ASK1 and phosphorylation of downstream kinases, MKK4, and JNK and prevented the down‐regulation of DJ‐1 and translocation of Daxx to the cytosol seen after MPTP. ALA also attenuated dopaminergic cell loss in SNpc seen after subchronic MPTP treatment. Our studies demonstrate for the first time that MPTP triggers death signaling pathway by activating ASK1 and translocating Daxx, in vivo, in dopaminergic neurons in SNpc of mice and thiol antioxidants, such as ALA terminate this cascade and afford neuroprotection.–Karunakaran S., Diwakar, L., Saeed, U., Agarwal, V., Ramakrishnan, S., Iyengar, S., Ravindranath V. Activation of apoptosis signal regulating kinase 1 (ASK1) and translocation of death associated protein, Daxx in substantia nigra pars compacta in a mouse model of Parkinsons disease: Protection by α‐lipoic acid. FASEB J. 21, 2226–2236 (2007)

Selective Activation of p38 Mitogen-Activated Protein Kinase in Dopaminergic Neurons of Substantia Nigra Leads to Nuclear Translocation of p53 in 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Treated Mice

Parkinsons disease (PD) is a progressive neurodegenerative disease characterized by the degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNpc). Activation of the mixed lineage kinase and c-Jun N-terminal kinase (JNK) has been reported in models of PD. Our focus was to discern whether distinct pathways were activated in cell-specific manner within the SNpc. We now demonstrate the selective phosphorylation of p38 MAP kinase within the dopaminergic neurons, whereas JNK activation occurs predominantly in the microglia. p38 activation results in downstream phosphorylation of p53 and increased p53 mediated transcription of Bax and Puma in the ventral midbrain. Treatment with p38 inhibitor, SB239063 protected primary dopaminergic neurons derived from human progenitor cells from MPP+ mediated cell death and prevented the downstream phosphorylation of p53 and its translocation to the nucleus in vivo, in the ventral midbrain. The increased staining of phosphorylated p38 in the surviving neurons of SNpc in human brain sections from patients with PD and in MPTP treated mice but not in the ventral tegmental area provides further evidence suggesting a role for p38 in the degeneration of dopaminergic neurons of SNpc. We thus demonstrate the cell specific activation of MAP kinase pathways within the SNpc after MPTP treatment emphasizing the role of multiple signaling cascades in the pathogenesis and progression of the disease. Selective inhibitors of p38 may therefore, help preserve the surviving neurons in PD and slow down the disease progression.

Low glutathione levels in brain regions of aged rats

Glutathione (GSH) was measured in 6 regions of brain and liver of young adult, middle-aged and aged rats. GSH levels were significantly lower in cortex, cerebellum, striatum, thalamus and hippocampus of aged rats, while no changes were observed in liver as compared to young adult rats. On the other hand, lipid peroxidation as measured by thiobarbituric acid-reactive products increased significantly in all the regions of brain examined and in the liver of aged rats. Since GSH plays an important role as a cellular protectant against oxygen radical-mediated injury, decreased levels of GSH in aged rat brain are indicative of the vulnerability of the aged cerebral tissue to oxidative injury.

Thiol Oxidation and Loss of Mitochondrial Complex I Precede Excitatory Amino Acid-Mediated Neurodegeneration

Human ingestion of “chickling peas” from the plantLathyrus sativus, which contains an excitatory amino acid, l-BOAA (l-β-N-oxalylamino-l-alanine), leads to a progressive corticospinal neurodegenerative disorder, neurolathyrism. Exposure to l-BOAA, but not its optical enantiomer d-BOAA, causes mitochondrial dysfunction as evidenced by loss of complex I activity in vitro in male mouse brain slices and in vivo in selected regions of mouse CNS (lumbosacral cord and motor cortex). Loss of complex I activity in lumbosacral cord after l-BOAA administration to mice was accompanied by concurrent loss of glutathione. The inhibited complex I activity in mitochondria isolated from lumbosacral cord of animals treated with l-BOAA rebounded after incubation with the thiol-reducing agent dithiothreitol, indicating that oxidation of protein thiols to disulfides was responsible for enzyme inhibition. The inhibition of complex I could be abolished by pretreatment with antioxidant thiols such as glutathione ester and α-lipoic acid. Chronic treatment of male mice, but not female mice, withl-BOAA resulted in loss of complex I activity and vacuolation and dendritic swelling of neurons in the motor cortex and lumbar cord, paralleling the regionality of the aforementioned biochemical effects on CNS mitochondria. These results support the view that thiol oxidation and concomitant mitochondrial dysfunction (also implicated in other neurodegenerative disorders), occurring downstream of glutamate receptor activation by l-BOAA, are primary events leading to neurodegeneration. Maintenance of protein thiol homeostasis by thiol delivery agents could potentially offer protection against excitotoxic insults such as those seen withl-BOAA.

Induction of brain cytochrome P-450IIE1 by chronic ethanol treatmen

Cytochrome P-450 mediated metabolism is potentially involved in the expression of the pharmacological and/or toxicological effects of a wide variety of drugs and environmental chemicals upon tissues which contain this metabolic system. In the present investigation, the presence of cytochrome P-450IIE1 and associated mono-oxygenase activities in brain and the effect of chronic ethanol treatment on brain cytochrome P-450 (P-450) were studied. Aniline hydroxylase, N-nitroso-dimethylamine N-demethylase and p-nitrophenol hydroxylase activities (known to be mediated by P-450IIE1) were detectable in brain microsomes from untreated rats and were about 5%, 125% and 8.3%, respectively, of the corresponding hepatic levels. Chronic ethanol treatment resulted in induction of the above enzyme activities in brain microsomes by 243%, 496% and 155%, respectively. Intake of ethanol for a prolonged period also resulted in the induction of total P-450 in the brain (150% of the control). Addition of the antisera raised against rat liver cytochrome P-450IIE1 markedly inhibited brain microsomal p-nitrophenol hydroxylase activity. Immunoblot analysis of rat brain microsomes using the above antisera also revealed the induction of brain cytochrome P-450IIE1 following chronic ethanol administration. Immunocytochemical localization of cytochrome P-450IIE1 using the above antisera, revealed the preferential localization of the enzyme in the neuronal cell bodies in the cortex, hippocampus, basal ganglia, hypothalamic nuclei and reticular nuclei in the brainstem of rats treated chronically with ethanol. Based upon these studies, it is conceivable that chronic alcohol ingestion could enhance the sensitivity of certain regions of the brain to environmental chemicals that are metabolized to more toxic derivatives by the P-450 system.

Glutaredoxin is essential for maintenance of brain mitochondrial complex I: studies with MPTP

Mitochondrial complex I dysfunction is implicated in the pathogenesis of neurodegenerative disorders such as Parkinsons disease. Identification of factors involved in maintenance and restoration of complex I function could potentially help to develop prophylactic and therapeutic strategies for treatment of this class of disorders. Down‐regulation of glutaredoxin (thioltransferase, a thiol disulfide oxido‐reductase) using antisense oligonucleotides results in the loss of mitochondrial complex I activity in mouse brain. 1‐Methyl‐4‐phenyl‐1,2,3,6,tetrahydro‐pyridine (MPTP), the neurotoxin that causes Parkinsons disease‐like symptoms in primates and dopaminergic cell loss in mice, acts through the inhibition of complex I. Regeneration of complex I activity in the striatum occurs concurrently with increase in glutaredoxin activity, 4 h after the neurotoxic insult, and is mediated through activation of activating protein‐1. Down‐regulation of glutaredoxin using anti‐sense oligonucleotides prevents recovery of complex I in the striatum after MPTP treatment, providing support for the critical role for glutaredoxin in recovery of mitochondrial function in brain. Maintenance and restoration of protein thiol homeostasis by glutaredoxin may be important factors in preventing complex I dysfunction.