Latha Diwakar

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)

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 (gamma-glutamylcysteinyl glycine), an endogenous thiol antioxidant are required for the maintenance of the redox status of cells. Cystathionine gamma-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 gamma-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 gamma-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-beta-oxalyl amino-l-alanine, which is the causative factor of a type of motor neuron disease, neurolathyrism. l-beta-Oxalyl amino-l-alanine toxicity was exacerbated by l-propargylglycine measured as loss of complex I activity indicating the importance of cystathionine gamma-lyase in maintaining glutathione levels and in turn the mitochondrial function during excitotoxicity. Oxidative stress generated by l-beta-oxalyl amino-l-alanine itself inhibited cystathionine gamma-lyase, which could be prevented by prior treatment with thiol antioxidant. Thus, cystathionine gamma-lyase itself is susceptible to inactivation by oxidative stress and this can potentially exacerbate oxidant-induced damage. Cystathionine gamma-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

Incidence of Parkinsons disease is lower in women as compared with men. Although neuroprotective effect of estrogen is recognized, the underlying molecular mechanisms are unclear. MPTP (1‐methyl‐4‐phenyl‐1, 2, 3, 6, tetrahydro‐pyridine), a neurotoxin that causes Parkinsons disease‐like symptoms acts through inhibition of mitochondrial complex I. Administration of MPTP to male mice results in loss of dopaminergic neurons in substantia nigra, whereas female mice are unaffected. Oxidation of critical thiol groups by MPTP disrupts mitochondrial complex I, and up‐regulation of glutaredoxin (a thiol disulfide oxidoreductase) is essential for recovery of complex I. Early events following MPTP exposure, such as increased AP1 transcription, loss of glutathione, and up‐regulation of glutaredoxin mRNA is seen only in male mice, indicating that early response to neurotoxic insult does not occur in females. Pretreatment of female mice with ICI 182,780, estrogen receptor (ER) antagonist sensitizes them to MPTP‐mediated complex I dysfunction. Constitutive expression of glutaredoxin is significantly higher in female mice as compared with males. ICI 182,780 down‐regulates glutaredoxin activity in female mouse brain regions (midbrain and striatum), indicating that glutaredoxin expression is regulated through estrogen receptor signaling. Higher constitutive expression of glutaredoxin could potentially contribute to the neuroprotection seen in female mouse following exposure to neurotoxins, such as MPTP.

Synthesis, anticancer and antioxidant activities of 2,4,5-trimethoxy chalcones and analogues from asaronaldehyde: Structure–activity relationship

2,4,5-Trimethoxy chalcones and analogues were synthesized from asaronaldehyde derived from β-asarone. These novel compounds when tested against three human tumour cell lines (MCF-7, SW-982 and HeLa) using MTT assay, revealed that chalcones possessing electron donor groups in para position to carbonyl moiety of phenyl ring A, showed better inhibitory activity (2, 3, 4, 6, 7, 10, 17). When evaluated for antioxidant activities, compound 15 exhibited better free radical scavenging property in DPPH assay while compounds 2, 3, 5, 7, 9, 10, 11, 16, and 18 showed significant NO scavenging activity. All compounds exhibited very good phenyl hydrazine induced haemolysis of erythrocytes in phenylhydrazine assay. Structure-activity relationship (SAR) study using in-silico analysis matched well with in-vitro tumour cell inhibitory activity.

Downregulation of glutaredoxin but not glutathione loss leads to mitochondrial dysfunction in female mice CNS: Implications in excitotoxicity

Oxidative stress, excitotoxicity and mitochondrial dysfunction play synergistic roles in neurodegeneration. Maintenance of thiol homeostasis is important for normal mitochondrial function and dysregulation of protein thiol homeostasis by oxidative stress leads to mitochondrial dysfunction and neurodegeneration. We examined the critical roles played by the antioxidant, non-protein thiol, glutathione and related enzyme, glutaredoxin in maintaining mitochondrial function during excitotoxicity caused by beta-N-oxalyl amino-L-alanine (L-BOAA), the causative factor of neurolathyrism, a motor neuron disease involving the pyramidal system. L-BOAA causes loss of GSH and inhibition of mitochondrial complex I in lumbosacral cord of male mice through oxidation of thiol groups, while female mice are resistant. Reducing GSH levels in female mice CNS by pretreatment with diethyl maleate or L-propargyl glycine did not result in inhibition of complex I activity, unlike male mice. Further, treatment of female mice depleted of GSH with L-BOAA did not induce inhibition of complex I indicating that GSH levels were not critical for maintaining complex I activity in female mice unlike their male counterpart. Glutaredoxin, a thiol disulfide oxidoreductase helps maintain redox status of proteins and downregulation of glutaredoxin results in loss of mitochondrial complex I activity. Female mice express higher levels of glutaredoxin in certain CNS regions and downregulation of glutaredoxin using antisense oligonucleotides sensitizes them to L-BOAA toxicity seen as mitochondrial complex I loss. Ovariectomy downregulates glutaredoxin and renders female mice vulnerable to L-BOAA toxicity as evidenced by activation of AP1, loss of GSH and complex I activity indicating the important role of glutaredoxin in neuroprotection. Estrogen protects against mitochondrial dysfunction caused by excitotoxicity by maintaining cellular redox status through higher constitutive expression of glutaredoxin in the CNS. Therapeutic interventions designed to upregulate glutaredoxin may offer neuroprotection against excitotoxicity in motor neurons.

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

beta-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 gamma-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.

Synthesis and biological evaluation of boswellic acid-NSAID hybrid molecules as anti-inflammatory and anti-arthritic agents

Methyl esters of the β-boswellic acid (BA) and 11-keto-β-boswellic acid (KBA) obtained from Boswellia serrata resin were subjected to Steglich esterification with the different non-steroidal anti-inflammatory drugs (NSAID) viz., ibuprofen, naproxen, diclophenac and indomethacin. The novel hybrids of methyl boswellate (5-8) and that of methyl 11-keto boswellate (9-12) were evaluated for anti-inflammatory activity by carrageenan-induced rat hind paw edema model and anti-arthritic activity by Complete Freunds Adjuvant (CFA) induced arthritis in Wister albino rat. Significant inhibition on carrageenan-induced paw edema has been observed with 5, 6 and 10 where as in CFA induced rats, hybrids 5, 8, 9 and 12 exhibited pronounced antiarthritic activity. Hybrid molecules 5 and 9 have been found to be more effective in inhibiting in-vivo COX-2 than ibuprofen by itself, thus showing the synergistic effect. Hybrid 5 and 9 tested for in-vitro lipoxygenase and cyclooxygenase-2 (LOX/COX-2) inhibitory activity. The studies revealed that both 5 and 9 inhibited COX-2 relatively better than LOX enzyme.

Nitro Derivatives of Naturally Occurring β-Asarone and Their Anticancer Activity

β-Asarone (2, 4, 5-trimethoxy-(Z)-1-propenylbenzene) was obtained from Acorus calamus. Nitration of β-asarone with AgNO2/I2 in ether yielded 1-(2, 4, 5-trimethoxy phenyl)-2-nitropropene (1) but with NaNO2/I2 in ethylene glycol obtained 1-(2, 4, 5-trimethoxy phenyl)-1-nitropropene (2). Compound 2 was prepared for the first time and characterized using IR, 1H-NMR, 13C-NMR, and GC-MS spectra and it was converted into 1-(2, 4, 5-trimethoxy) phenyl-1-propanone (3) using modified Nef reaction. Based on 1D NOESY experiments, compounds 1 and 2 have been assigned E configuration. Compounds 1 and 2 were subjected to cytotoxic activity using five human cancer cell lines, namely, MCF-7, SW-982, HeLa, PC-3, and IMR-32 by MTT assay. Except in breast cancer line (MCF-7) compound 2 exhibited five- to tenfold increase in activity compared to β-asarone and twofold increase over compound 1.

MOUSE MODEL OF VASCULAR DEMENTIA

been previously reported that human apoE might play a role in the inflammatory response of cells. Since inflammation and neurodegeneration are closely linked, we aimed at analyzing the role of apoE in modulating the inflammatory response of isogenic astrocytes. Indeed, stimulating these astrocytes with a cytokine cocktail triggered a differential inflammatory response depending on the apoE genotype. ApoE4 carrying astrocytes displayed stronger pro-inflammatory responses compared to apoE3 cells indicating a potential role for neuro-inflammation in AD. In this study the analysis of these differences was extended to additional apoE genotypes, like apoE2/2 and also included the heterozygote form apoE3/4. ApoE has also been reported to influence the complexity of neuronal networks denoted by the neuron’s ability to grow and extend neurites. However, this has not been tested in humanderived cells. Therefore, we investigated the impact of the various apoE isoforms on the differentiation and maturation of cortical neurons under basal conditions and in the presence of various stressors. Conclusions:Since apoE4 is a significant factor in the development of late onset AD, we believe that our mechanistic studies help understand its role in the disease course, and ultimately bring us closer to developing impactful treatments.

Complex I Assay in Mitochondrial Preparations from CNS

Mammalian NADH:ubiquinone oxidoreductase (complex I) is made up of at least 46 subunits and is one of the largest enzyme complexes known. It catalyzes the first step of the respiratory electron transport chain through the oxidation of NADH, providing two electrons for the reduction of ubiquinone to ubiquinol, thus propelling protons across the inner membrane of the mitochondria, which subsequently drive ATP synthesis. Dysfunction of complex I has been implicated in various neurodegenerative disorders, and it is probably the most vulnerable component of the electron transport chain to inhibition by reactive oxygen species. We describe a simple spectrophotometric method for estimating the activity of complex I from mitochondria isolated from regions of the central nervous system of mice. Curr. Protoc. Toxicol. 38:17.10.1‐17.10.7.