Supriya Swarnkar

Mechanism of Oxidative Stress and Synapse Dysfunction in the Pathogenesis of Alzheimer's Disease: Understanding the Therapeutics Strategies

Synapses are formed by interneuronal connections that permit a neuronal cell to pass an electrical or chemical signal to another cell. This passage usually gets damaged or lost in most of the neurodegenerative diseases. It is widely believed that the synaptic dysfunction and synapse loss contribute to the cognitive deficits in patients with Alzheimer’s disease (AD). Although pathological hallmarks of AD are senile plaques, neurofibrillary tangles, and neuronal degeneration which are associated with increased oxidative stress, synaptic loss is an early event in the pathogenesis of AD. The involvement of major kinases such as mitogen-activated protein kinase (MAPK), extracellular receptor kinase (ERK), calmodulin-dependent protein kinase (CaMKII), glycogen synthase-3β (GSK-3β), cAMP response element-binding protein (CREB), and calcineurin is dynamically associated with oxidative stress-mediated abnormal hyperphosphorylation of tau and suggests that alteration of these kinases could exclusively be involved in the pathogenesis of AD. N-methyl-d-aspartate (NMDA) receptor (NMDAR) activation and beta amyloid (Aβ) toxicity alter the synapse function, which is also associated with protein phosphatase (PP) inhibition and tau hyperphosphorylation (two main events of AD). However, the involvement of oxidative stress in synapse dysfunction is poorly understood. Oxidative stress and free radical generation in the brain along with excitotoxicity leads to neuronal cell death. It is inferred from several studies that excitotoxicity, free radical generation, and altered synaptic function encouraged by oxidative stress are associated with AD pathology. NMDARs maintain neuronal excitability, Ca2+ influx, and memory formation through mechanisms of synaptic plasticity. Recently, we have reported the mechanism of the synapse redox stress associated with NMDARs altered expression. We suggest that oxidative stress mediated through NMDAR and their interaction with other molecules might be a driving force for tau hyperphosphorylation and synapse dysfunction. Thus, understanding the oxidative stress mechanism and degenerating synapses is crucial for the development of therapeutic strategies designed to prevent AD pathogenesis.

Astrocytes and Microglia: Responses to Neuropathological Conditions

ABSTRACT Activated astrocytes and microglia, hallmark of neurodegenerative diseases release different factors like array of pro and anti-inflammatory cytokines, free radicals, anti-oxidants, and neurotrophic factors during neurodegeneration which further contribute to neuronal death as well as in survival mechanisms. Astrocytes act as double-edged sword exerting both detrimental and neuroprotective effects while microglial cells are attributed more in neurodegenerative mechanisms. The dual and insufficient knowledge about the precise role of glia in neurodegeneration showed the need for further investigations and thorough review of the function of glia in neurodegeneration. In this review, we consolidate and categorize the glia-released factors which contribute in degenerative and protective mechanisms during neuropathological conditions.

A study to correlate rotenone induced biochemical changes and cerebral damage in brain areas with neuromuscular coordination in rats

Rotenone induces neurotoxicity but its correlation with biochemical and cerebral changes in rat brain regions are not well defined. In the present study rotenone was administered (3, 6 and12mug/mul) intranigrally in adult male SD rats and its effect was assessed on neuromuscular coordination and in different brain areas viz. striatum (STR), mid-brain (MB), frontal cortex (FC) and hippocampus (HP) cerebral and biochemical changes on 1st and 7th day after treatment. All the doses of rotenone significantly impaired neuromuscular coordination performance on Rota rod test on 1st and 7th day. TTC staining showed significant increase in cerebral injury volume on 1st and 7th day after rotenone treatment indicating mitochondrial enzyme deficiency but increase after 7th day was less that after 1st day. Rotenone treated rats showed significant decrease in GSH and increase in MDA in different brain regions though the pattern was varied. After 1 day of rotenone (6 and 12mug) treatment significant decrease in GSH was observed in STR and MB while MDA was significantly increased only in MB. The maximal effect on GSH and MDA was obtained in STR and MB on 7th day after treatment with 12mug dose of rotenone. Thus, based on the occurrence of changes, it may be suggested that impairment of neuromuscular coordination is inked to oxidative stress rather than mitochondrial enzyme deficiency, all the processes are correlated with each other with the progression of time. MB appeared as most sensitive brain area towards rotenone toxicity.

OKADAIC ACID-INDUCED TAU PHOSPHORYLATION IN RAT BRAIN: ROLE OF NMDA RECEPTOR

Okadaic acid (OKA) is a potent inhibitor of protein phosphatases 1/2A (PP2A). Inhibition of PP2A leads to hyperphosphorylation of Tau protein. Hyperphosphorylated Tau protein is present in intraneuronal neurofibrillary tangles a characteristic feature of neuropathology of Alzheimers disease. Intracerebroventricular (ICV) administration of OKA causes neurotoxicity, which is associated with increased intracellular Ca(2+) level, oxidative stress, and mitochondrial dysfunction in the brain areas. The present study explored Tau phosphorylation in OKA-treated rats in relation to memory function, PP2A activity, intracellular Ca(2+), glycogen synthase kinase-3β (GSK-3β) and N-methyl-d-aspartate (NMDA) receptor after 13days of OKA (200ng, ICV) administration in rats, memory was found impaired in the water maze test. OKA-induced memory-impaired rats showed increased mRNA and protein expression of Tau, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), Calpain and GSK3β in the hippocampus and cerebral cortex. On the other hand, mRNA expression and activity of PP2A was reduced in these brain areas. OKA treatment also, resulted in decrease in mRNA expression of C and N terminals of Tau. Treatment with NMDA antagonist, MK801 (0.05mg/kg, i.p.) for 13days significantly prevented OKA-induced changes in the expression of PP2A, Tau, GSK3β, CaMKII and Calpain. Further, daily administration of anticholinergic drug, donepezil (5mg/kg, p.o.), and the NMDA receptor antagonist, memantine (10mg/kg, p.o.) initiated after OKA administration for 13days significantly attenuated OKA-induced variation in Tau, Tau-C terminal, Tau-N terminal CaMKII, Calpain, PP2A and GSK3β. These results infer that NMDA antagonist MK801 and memantine are effective against OKA-induced neurotoxicity. Therefore, the present study clearly indicates the involvement of NMDA receptor in OKA (ICV)-induced Tau hyperphosphorylation.

Huntingtin promotes mTORC1 signaling in the pathogenesis of Huntington’s disease

Mouse models indicate that mutant huntingtin promotes anabolic signaling to contribute to Huntington’s disease. Hunting the Pathways in Huntington’s Disease Huntington’s disease (HD) is caused by a mutant form of the protein huntingtin (Htt), which causes neurodegeneration in the striatum. HD-associated symptoms are alleviated by inhibition of the kinase mTOR, which is a key regulator of protein synthesis and is normally activated by amino acids. In primary mouse striatal neuronal cells, Pryor et al. found that wild-type Htt enhanced mTOR signaling in response to amino acids, and mutant Htt further potentiated mTOR activity. Mutant Htt bound more effectively than wild-type Htt to the guanosine triphosphatase (GTPase) Rheb and facilitated the activating interaction between Rheb and mTOR. Striatum-specific deletion of the gene encoding TSC1, an inhibitor of mTOR, accelerated the onset of HD phenotypes in mice, consistent with excessive mTOR activity contributing to HD. The findings identify a pathway by which mutant Htt contributes to HD pathology. In patients with Huntington’s disease (HD), the protein huntingtin (Htt) has an expanded polyglutamine (poly-Q) tract. HD results in early loss of medium spiny neurons in the striatum, which impairs motor and cognitive functions. Identifying the physiological role and molecular functions of Htt may yield insight into HD pathogenesis. We found that Htt promotes signaling by mTORC1 [mechanistic target of rapamycin (mTOR) complex 1] and that this signaling is potentiated by poly-Q–expanded Htt. Knocking out Htt in mouse embryonic stem cells or human embryonic kidney cells attenuated amino acid–induced mTORC1 activity, whereas overexpressing wild-type or poly-Q–expanded Htt in striatal neuronal cells increased basal mTOR activity. Striatal cells expressing endogenous poly-Q–expanded Htt showed an increase in the number and size of mTOR puncta on the perinuclear regions compared to cells expressing wild-type Htt. Pull-down experiments indicated that amino acids stimulated the interaction of Htt and the guanosine triphosphatase (GTPase) Rheb (a protein that stimulates mTOR activity), and that Htt forms a ternary complex with Rheb and mTOR. Pharmacologically inhibiting PI3K (phosphatidylinositol 3-kinase) or knocking down Rheb abrogated mTORC1 activity induced by expression of a poly-Q–expanded amino-terminal Htt fragment. Moreover, striatum-specific deletion of TSC1, encoding tuberous sclerosis 1, a negative regulator of mTORC1, accelerated the onset of motor coordination abnormalities and caused premature death in an HD mouse model. Together, our findings demonstrate that mutant Htt contributes to the pathogenesis of HD by enhancing mTORC1 activity.

A study on neuroinflammatory marker in brain areas of okadaic acid (ICV) induced memory impaired rats.

AIMS The aim of the present study is to investigate the status of proinflammatory cytokine in the brain of intracerebroventricular (i.c.v.) okadaic acid (OKA) induced memory impaired rat. MAIN METHODS OKA (200 ng) intracerebroventricular (i.c.v.) was administered in rats. Memory was assessed by Morris water maze test. Biochemical marker of neuroinflammation (TNF-α, IL-β), total nitrite, mRNA (RT PCR) and protein expression (WB) of iNOS and nNOS were estimated in rat brain areas. KEY FINDINGS OKA caused memory-impairment in rats with increased expression of proinflammatory cytokine TNF-α and IL-1β and total nitrite in brain regions hippocampus and cortex. The expression of mRNA and protein of iNOS was increased while; the expressions were decreased in case of nNOS. Pretreatment with antidementic drugs donepezil (5 mg/kg, p.o.) and memantine (10 mg/kg, p.o) for 13 days protected i.c.v. OKA induced memory impairment and changes in level of TNF-α, IL-β, total nitrite and expressions of iNOS and nNOS in OKA treated rat. SIGNIFICANCE This study suggests that neuroinflammation may play a vital role in OKA induced memory impairment.

Rotenone induced neurotoxicity in rat brain areas: A histopathological study☆

Rotenone a pesticide is known to induce neurotoxicity. In earlier study we correlated rotenone induced biochemical changes and cerebral damage in brain areas with neuromuscular coordination in rats. The present study involves investigation of rotenone induced histopathological changes in the brain areas, viz. striatum (STR) and substantia nigra (SN) using HE (hematoxylin and eosin) and CV (Cresyl Violet) staining after 1, 7, and 14 day of unilateral intranigral administration of rotenone (3, 6 and 12 μg/5 μl) in adult male SD rats. Significant morphological changes in cell area or shape were shown by HE staining. The neuronal degeneration was shown by distorted neuronal cells, shrinkage of nuclei, dark staining in the regions of rotenone treated animals by CV staining. Rota rod test demonstrated significant impairment in motor coordination after 14 days of treatment along with decreased GSH and increased MDA in STR and mid brain (MB). The study inferred rotenone causes neuronal damage which is evident by histopathological changes, impaired neuromuscular coordination and biochemical changes. The pattern of histopathological alterations corresponds with behavioral and biochemical manifestations.

Molecular and Cellular Mechanism of Okadaic Acid (OKA)-Induced Neurotoxicity: A Novel Tool for Alzheimer’s Disease Therapeutic Application

Okadaic acid (OKA), a polyether C38 fatty acid toxin extracted from a black sponge Hallichondria okadaii, is a potent and selective inhibitor of protein phosphatase, PP1 and PP2A. OKA has been proved to be a powerful probe for studying the various regulatory mechanisms and neurotoxicity. Because of its property to inhibit phosphatase activity, OKA is associated with protein phosphorylation; it is implicated in hyperphosphorylation of tau and in later stages causes Alzhiemer’s disease (AD)-like pathology. AD is a progressive neurodegenerative disorder, pathologically characterized by extracellular amyloid beta (Aβ) plaques and intracellular neurofibrillary tangles (NFTs). The density of tau tangles in AD pathology is associated with cognitive dysfunction. Recent studies have highlighted the importance of serine/threonine protein phosphatases in many processes including apoptosis and neurotoxicity. Although OKA causes neurotoxicity by various pathways, the exact mechanism is still not clear. The activation of major kinases, such as Ser/Thr, MAPK, ERK, PKA, JNK, PKC, CaMKII, Calpain, and GSK3β, in neurons is associated with AD pathology. These kinases, associated with abnormal hyperphosphorylation of tau, suggest that the cascade of these kinases could exclusively be involved in the pathogenesis of AD. The activity of serine/threonine protein phosphatases needs extensive study as these enzymes are potential targets for novel therapeutics with applications in many diseases including cancer, inflammatory diseases, and neurodegeneration. There is a need to pay ample attention on MAPK kinase pathways in AD, and OKA can be a better tool to study cellular and molecular mechanism for AD pathology. This review elucidates the regulatory mechanism of PP2A and MAPK kinase and their possible mechanisms involved in OKA-induced apoptosis, neurotoxicity, and AD-like pathology.

A comparative study on oxidative stress induced by LPS and rotenone in homogenates of rat brain regions

Lipopolysaccharide (LPS) and rotenone induced oxidative stress was investigated in homogenates of rat brain regions - striatum, mid brain, frontal cortex and hippocampus. LPS at concentration 1, 25 and 50μg and rotenone 1, 2 and 4mM was incubated with the brain homogenates and caused decrease in reduced glutathione (GSH) and rise in malondialdehyde (MDA) in different brain regions but in a varied manner. Anti-oxidants melatonin and nimesulide (0.75, 1.5 and 3mM) were incubated concurrently with LPS (50μg) and rotenone (4mM) in the homogenates. Melatonin as well as nimesulide (3mM) suppressed the LPS and rotenone induced increase in MDA but their effect on GSH differed. Lack of uniform response by different brain areas to LPS, rotenone and antioxidants indicate that sensitivity to oxidative stress may differ among the brain areas; this variability in sensitivity may be of significance in relation to free radicals induced selective neuronal degeneration.

Mechanism of synapse redox stress in Okadaic acid (ICV) induced memory impairment: Role of NMDA receptor

The N-methyl-D-aspartate (NMDA) receptor is a subtype of ionotropic glutamate receptor that is involved in synaptic mechanisms of learning and memory, and mediates excitotoxic neuronal injury. In this study, we tested the hypothesis that NMDA receptor subunit gene expression is altered in cortex and hippocampus of OKA induced memory impairment. Therefore in the present study, we checked the effect of OKA (ICV) on NMDA receptor regulation and synapse function. The memory function anomalies and synaptosomal calcium ion (Ca(2+)) level were increased in OKA treated rats brain; which was further protected by MK801 (0.05mg/kg. i.p) treatment daily for 13days. To elucidate the involvement of NMDA receptor, we estimated NR1, NR2A and NR2B (subunits) expression in rat brain. Results showed that expression of NR1 and NR2B were significantly increased, but expression of NR2A had no significant change in OKA treated rat brain. We also observed decrease in synapsin-1 mRNA and protein expression which indicates synapse dysfunction. In addition, we detected an increase in MDA and nitrite levels and a decrease in GSH level in synapse preparation which indicates synapse altered redox stress. Moreover, neuronal loss was also confirmed by nissl staining in periventricular cortex and hippocampus. Altered level of oxidative stress markers along with neuronal loss confirmed neurotoxicity. Further, MK801 treatment restored the level of NR1, NR2B and synapsin-1 expression, and protected from neuronal loss and synapse redox stress. In conclusion, Okadaic acid (OKA) induced expression of NR1 and NR2B deteriorates synapse function in rat brain which was confirmed by the neuroprotective effect of MK801.