Panchanan Maiti

Hypobaric hypoxia induces oxidative stress in rat brain

High altitude exposure results in decreased partial pressure of oxygen and an increased formation of reactive oxygen and nitrogen species (RONS), which causes oxidative damage to lipids, proteins and DNA. Exposure to high altitude appears to decrease the activity and effectiveness of antioxidant enzyme system. The antioxidant system is very less in brain tissue and is very much susceptible to hypoxic stress. The aim of the present study was to investigate the time dependent and region specific changes in cortex, hippocampus and striatum on oxidative stress markers on chronic exposure to hypobaric hypoxia. The rats were exposed to simulated high altitude equivalent to 6100 m in animal decompression chamber for 3 and 7 days. Results indicate an increase in oxidative stress as seen by increase in free radical production, nitric oxide level, lipid peroxidation and lactate dehydrogenase levels. The magnitude of increase in oxidative stress was more in 7 days exposure group as compared to 3 days exposure group. The antioxidant defence system such as reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD) and reduced/oxidized glutathione (GSH/GSSG) levels were significantly decreased in all the three regions. The observation suggests that the hippocampus is more susceptible to hypoxia than the cortex and striatum. It may be concluded that hypoxia differentially affects the antioxidant status in the cortex, hippocampus and striatum.

Curcumin Suppresses Soluble Tau Dimers and Corrects Molecular Chaperone, Synaptic, and Behavioral Deficits in Aged Human Tau Transgenic Mice

Background: Various types of Tau aggregates in AD brains may differentially impact neurodegeneration. Results: Curcumin selectively suppresses soluble Tau dimers and corrects molecular chaperone, synaptic, and behavioral deficits. Conclusion: A drug increasing HSPs involved in Tau clearance reduced Tau dimers and improved cognition. Significance: Curcumin that reduced Tau dimers and increased molecular chaperones was efficacious without altering insoluble Tau. The mechanisms underlying Tau-related synaptic and cognitive deficits and the interrelationships between Tau species, their clearance pathways, and synaptic impairments remain poorly understood. To gain insight into these mechanisms, we examined these interrelationships in aged non-mutant genomic human Tau mice, with established Tau pathology and neuron loss. We also examined how these interrelationships changed with an intervention by feeding mice either a control diet or one containing the brain permeable beta-amyloid and Tau aggregate binding molecule curcumin. Transgene-dependent elevations in soluble and insoluble phospho-Tau monomer and soluble Tau dimers accompanied deficits in behavior, hippocampal excitatory synaptic markers, and molecular chaperones (heat shock proteins (HSPs)) involved in Tau degradation and microtubule stability. In human Tau mice but not control mice, HSP70, HSP70/HSP72, and HSP90 were reduced in membrane-enriched fractions but not in cytosolic fractions. The synaptic proteins PSD95 and NR2B were reduced in dendritic fields and redistributed into perikarya, corresponding to changes observed by immunoblot. Curcumin selectively suppressed levels of soluble Tau dimers, but not of insoluble and monomeric phospho-Tau, while correcting behavioral, synaptic, and HSP deficits. Treatment increased PSD95 co-immunoprecipitating with NR2B and, independent of transgene, increased HSPs implicated in Tau clearance. It elevated HSP90 and HSC70 without increasing HSP mRNAs; that is, without induction of the heat shock response. Instead curcumin differentially impacted HSP90 client kinases, reducing Fyn without reducing Akt. In summary, curcumin reduced soluble Tau and elevated HSPs involved in Tau clearance, showing that even after tangles have formed, Tau-dependent behavioral and synaptic deficits can be corrected.

Improvement of neuropathology and transcriptional deficits in CAG 140 knock-in mice supports a beneficial effect of dietary curcumin in Huntington's disease

BackgoundNo disease modifying treatment currently exists for Huntingtons disease (HD), a fatal neurodegenerative disorder characterized by the formation of amyloid-like aggregates of the mutated huntingtin protein. Curcumin is a naturally occurring polyphenolic compound with Congo red-like amyloid binding properties and the ability to cross the blood brain barrier. CAG140 mice, a knock-in (KI) mouse model of HD, display abnormal aggregates of mutant huntingtin and striatal transcriptional deficits, as well as early motor, cognitive and affective abnormalities, many months prior to exhibiting spontaneous gait deficits, decreased striatal volume, and neuronal loss. We have examined the ability of life-long dietary curcumin to improve the early pathological phenotype of CAG140 mice.ResultsKI mice fed a curcumin-containing diet since conception showed decreased huntingtin aggregates and increased striatal DARPP-32 and D1 receptor mRNAs, as well as an amelioration of rearing deficits. However, similar to other antioxidants, curcumin impaired rotarod behavior in both WT and KI mice and climbing in WT mice. These behavioral effects were also noted in WT C57Bl/6 J mice exposed to the same curcumin regime as adults. However, neither locomotor function, behavioral despair, muscle strength or food utilization were affected by curcumin in this latter study. The clinical significance of curcumins impairment of motor performance in mice remains unclear because curcumin has an excellent blood chemistry and adverse event safety profile, even in the elderly and in patients with Alzheimers disease.ConclusionTogether with this clinical experience, the improvement in several transgene-dependent parameters by curcumin in our study supports a net beneficial effect of dietary curcumin in HD.

Comparison of Three Amyloid Assembly Inhibitors: The Sugar scyllo- Inositol, the Polyphenol Epigallocatechin Gallate, and the Molecular Tweezer CLR01

Many compounds have been tested as inhibitors or modulators of amyloid β-protein (Aβ) assembly in hope that they would lead to effective, disease-modifying therapy for Alzheimers disease (AD). These compounds typically were either designed to break apart β-sheets or selected empirically. Two such compounds, the natural inositol derivative scyllo-inositol and the green-tea-derived flavonoid epigallocatechin gallate (EGCG), currently are in clinical trials. Similar to most of the compounds tested thus far, the mechanism of action of scyllo-inositol and EGCG is not understood. Recently, we discovered a novel family of assembly modulators, Lys-specific molecular tweezers, which act by binding specifically to Lys residues and modulate the self-assembly of amyloid proteins, including Aβ, into formation of nontoxic oligomers by a process-specific mechanism (Sinha, S., Lopes, D. H., Du, Z., Pang, E. S., Shanmugam, A., Lomakin, A., Talbiersky, P., Tennstaedt, A., McDaniel, K., Bakshi, R., Kuo, P. Y., Ehrmann, M., Benedek, G. B., Loo, J. A., Klarner, F. G., Schrader, T., Wang, C., and Bitan, G. (2011) Lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins. J. Am. Chem. Soc.133, 16958-16969). Here, we compared side-by-side the capability of scyllo-inositol, EGCG, and the molecular tweezer CLR01 to inhibit Aβ aggregation and toxicity. We found that EGCG and CLR01 had comparable activity whereas scyllo-inositol was a weaker inhibitor. Exploration of the binding of EGCG and CLR01 to Aβ using heteronuclear solution-state NMR showed that whereas CLR01 bound to the two Lys and single Arg residues in Aβ monomers, only weak, nonspecific binding was detected for EGCG, leaving the binding mode of the latter unresolved.

Hypobaric hypoxia damages the hippocampal pyramidal neurons in the rat brain

Hypobaric hypoxia (HH), a predisposing environmental condition at high altitude (HA), encountered by many mountaineers, jeopardizes their normal physiology like motor coordination and cognitive functions. A large body of evidence shows that HH has deleterious effect on cognitive functions. Among them the hippocampal dependent memory deficit is well known. However, our current understanding of the mechanistic details of cognitive deficits at HA remains largely unclear and hence limits a solution for this problem. Therefore, the present study was designed to investigate the temporal component of the hippocampal pyramidal neuron damage in the rat brain subjected to chronic HH exposure. Three groups (sham HH, 3 days HH and 7 days HH) of rats were exposed to simulated HH equivalent to 6100 m in an animal decompression chamber for 3 or 7 days. Later, the hippocampal (CA1 and CA3) neurons were analysed for the cell morphology, neurodegeneration and DNA fragmentation. The CA1 and CA3 neurons showed HH induced neuronal pyknosis, cell shrinkage, and consequent inter-cellular vacuolization in the CA1 and CA3 areas. In addition, the total neuron (intact) numbers and mean surface area were decreased. The number of dead neurons increased significantly following exposure to HH for 3 or 7 days. The neurodegenerative (Fluoro jade B) and apoptotic (TUNEL) markers were more positive in CA1 and CA3 neurons. The magnitude of morphological changes, neurodegeneration and apoptosis was enhanced in 7 days HH group than 3 days HH group. Our studies indicate that CA3 neurons are more vulnerable to HH than CA1 neurons, and that may destabilize the neural circuits in the hippocampus and thus cause memory dysfunction.

Clinical development of curcumin in neurodegenerative disease

Curcumin, a polyphenolic antioxidant derived from the turmeric root has undergone extensive preclinical development, showing remarkable efficacy in wound repair, cancer and inflammatory disorders. This review addresses the rationale for its use in neurodegenerative disease, particularly Alzheimer’s disease. Curcumin is a pleiotropic molecule, which not only directly binds to and limits aggregation of the β-sheet conformations of amyloid characteristic of many neurodegenerative diseases but also restores homeostasis of the inflammatory system, boosts the heat shock system to enhance clearance of toxic aggregates, scavenges free radicals, chelates iron and induces anti-oxidant response elements. Although curcumin corrects dysregulation of multiple pathways, it may exert many effects via a few molecular targets. Pharmaceutical development of natural compounds like curcumin and synthetic derivatives have strong scientific rationale, but will require overcoming various hurdles including; high cost of trials, concern about profitability and misconceptions about drug specificity, stability, and bioavailability.

Molecular Chaperone Dysfunction in Neurodegenerative Diseases and Effects of Curcumin

The intra- and extracellular accumulation of misfolded and aggregated amyloid proteins is a common feature in several neurodegenerative diseases, which is thought to play a major role in disease severity and progression. The principal machineries maintaining proteostasis are the ubiquitin proteasomal and lysosomal autophagy systems, where heat shock proteins play a crucial role. Many protein aggregates are degraded by the lysosomes, depending on aggregate size, peptide sequence, and degree of misfolding, while others are selectively tagged for removal by heat shock proteins and degraded by either the proteasome or phagosomes. These systems are compromised in different neurodegenerative diseases. Therefore, developing novel targets and classes of therapeutic drugs, which can reduce aggregates and maintain proteostasis in the brains of neurodegenerative models, is vital. Natural products that can modulate heat shock proteins/proteosomal pathway are considered promising for treating neurodegenerative diseases. Here we discuss the current knowledge on the role of HSPs in protein misfolding diseases and knowledge gained from animal models of Alzheimers disease, tauopathies, and Huntingtons diseases. Further, we discuss the emerging treatment regimens for these diseases using natural products, like curcumin, which can augment expression or function of heat shock proteins in the cell.

Photo-induced cross-linking of unmodified proteins (PICUP) applied to amyloidogenic peptides.

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimers, Parkinsons, and Huntingtons diseases, hereditary amyotrophic lateral sclerosis, and type 2 diabetes. Owing to the metastable nature of these protein assemblies, it is difficult to assess their oligomer size distribution quantitatively using classical methods, such as electrophoresis, chromatography, fluorescence, or dynamic light scattering. Oligomers of amyloidogenic proteins exist as metastable mixtures, in which the oligomers dissociate into monomers and associate into larger assemblies simultaneously. PICUP stabilizes oligomer populations by covalent cross-linking and when combined with fractionation methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), PICUP provides snapshots of the oligomer size distributions that existed before cross-linking. Hence, PICUP enables visualization and quantitative analysis of metastable protein populations and can be used to monitor assembly and decipher relationships between sequence modifications and oligomerization(1). Mechanistically, PICUP involves photo-oxidation of Ru(2+) in a tris(bipyridyl)Ru(II) complex (RuBpy) to Ru(3+) by irradiation with visible light in the presence of an electron acceptor. Ru(3+) is a strong one-electron oxidizer capable of abstracting an electron from a neighboring protein molecule, generating a protein radical(1,2). Radicals are unstable, highly-reactive species and therefore disappear rapidly through a variety of intra- and intermolecular reactions. A radical may utilize the high energy of an unpaired electron to react with another protein monomer forming a dimeric radical, which subsequently loses a hydrogen atom and forms a stable, covalently-linked dimer. The dimer may then react further through a similar mechanism with monomers or other dimers to form higher-order oligomers. Advantages of PICUP relative to other photo- or chemical cross-linking methods(3,4) include short (<or=1 s) exposure to non-destructive visible light, no need for pre facto modification of the native sequence, and zero-length covalent cross-linking. In addition, PICUP enables cross-linking of proteins within wide pH and temperature ranges, including physiologic parameters. Here, we demonstrate application of PICUP to cross-linking of three amyloidogenic proteins the 40- and 42-residue amyloid beta-protein variants (Abeta40 and Abeta42), and calcitonin, and a control protein, growth-hormone releasing factor (GRF).

Nitric oxide system is involved in hypobaric hypoxia-induced oxidative stress in rat brain

Oxidative stress is involved in memory impairment at high altitude (HA). The aim of the present study was to investigate the involvement of reactive nitrogen species in hippocampus, cortex and striatum of rat brain under simulated HA conditions. Rats were exposed to hypobaric hypoxia (HH) equivalent to 6100 m of HA in an animal decompression chamber for 3, 7, 14 and 21 days. Biochemical estimation of free radicals, nitric oxide (NO) level along with immunoreactivity, reverse transcriptase polymerase chain reaction (RT-PCR) and western blot of neuronal nitric oxide synthase (nNOS), neurodegeneration and DNA fragmentation were studied after HH exposure. The free radicals, NO level, nNOS immunoreactivity (nNOS-IR), nNOS expression, neurodegeneration and DNA fragmentation were increased in hippocampus, cortex and striatum after HH exposure. After 7 and 14 days of HH exposure, the nNOS-IR, nNOS expression, free radical, NO level, neurodegeneration and DNA fragmentation were increased in comparison to 3 or 21 days of HH. The NO system may be involved in increasing oxidative stress and neurodegeneration after HH.

Induction of methionine-sulfoxide reductases protects neurons from amyloid β-protein insults in vitro and in vivo.

Self-assembly of amyloid β-protein (Aβ) into toxic oligomers and fibrillar polymers is believed to cause Alzheimers disease (AD). In the AD brain, a high percentage of Aβ contains Met-sulfoxide at position 35, though the role this modification plays in AD is not clear. Oxidation of Met(35) to sulfoxide has been reported to decrease the extent of Aβ assembly and neurotoxicity, whereas surprisingly, oxidation of Met(35) to sulfone yields a toxicity similar to that of unoxidized Aβ. We hypothesized that the lower toxicity of Aβ-sulfoxide might result not only from structural alteration of the C-terminal region but also from activation of methionine-sulfoxide reductase (Msr), an important component of the cellular antioxidant system. Supporting this hypothesis, we found that the low toxicity of Aβ-sulfoxide correlated with induction of Msr activity. In agreement with these observations, in MsrA(-/-) mice the difference in toxicity between native Aβ and Aβ-sulfoxide was essentially eliminated. Subsequently, we found that treatment with N-acetyl-Met-sulfoxide could induce Msr activity and protect neuronal cells from Aβ toxicity. In addition, we measured Msr activity in a double-transgenic mouse model of AD and found that it was increased significantly relative to that of nontransgenic mice. Immunization with a novel Met-sulfoxide-rich antigen for 6 months led to antibody production, decreased Msr activity, and lowered hippocampal plaque burden. The data suggest an important neuroprotective role for the Msr system in the AD brain, which may lead to development of new therapeutic approaches for AD.