School Of Basic And Applied Sciences
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Item Evaluation of amyloid beta (aβ)-induced Mitochondrial dysfunction: Neuroprotective role of Apurinic/apyrimidinic endonuclease (ape1) Via its interaction with cysteamine Dioxygenase (ado)(Central University of Punjab, 2020) Kaur,Navrattan; Mantha, Anil K.Oxidative stress and damage to mitochondrial DNA during the aging process can impair mitochondrial energy metabolism and ion homeostasis in neurons, ultimately leading to neurodegeneration. Themain pathway for repairing oxidative base lesions is base excision repair (BER), and such repair is crucial for neurons owing to high rate of oxygen metabolism. Apurinic/apyrimidinic endonuclease (APE1) is a protein of this pathway involved in DNA repair and also in the redox co-activating function of different transcription factors. Thus, manipulation of DNA repair mechanisms can be thought of as a putative approach to prevent neuronal loss in neurodegenerative disorders like Alzheimer’s disease (AD). Ginkgo biloba has been studied as a possible treatment for dementia and AD. The ginkgolides present in G. biloba possess antioxidant, neuroprotective and cholinergic activities. The aim of the study was to explore the repair and redox functions of APE1 and a detailed mechanism of association of APE1 with ADO (a thiol dioxygenase) and functional cross-talk between them has been studied. In the present study, we have standardized the differentiation of SH-SY5Y neuroblastoma cells into the cells possessing a mature neuron-like phenotype. The results of cell viability assay showed that differentiated cells are more sensitive/vulnerable to oxidative stress, which is elicited by Aβ. H2DCFDA and DAF- FM-based detection of ROS and RNS strongly advocates that under oxidative stress conditions elicited by Aβ, GB exerts ameliorating effect to render neuroprotection to the SH-SY5Y cells due to its antioxidant nature. Significant decrease in nNOS expression was seen, when cells were pre-treated with GB and then given Aβ treatment in whole cell, cytosol and nucleus. This shows that GB pre-treatment decreases the RNS (NO) levels due to its anti-oxidant property. Determination of DNA damage in terms of measurement of 8-oxo-dG was seen to be more pronounced in mitochondria. In response to DNA damage, pre-treatment with GB decreased the expression of DNA repair enzyme APE1 expression in mitochondria, showing that GB aids in lowering the oxidative stress generated by Aβ in the mitochondria. In the nuclear extracts, upon treatment with GB, there was a significant increase in ADO expression and Aβ treatment also increased the expression of ADO. Whereas, combination treatment of Aβ and GB led to lower expression of ADO. This points towards the possibility that ADO might be translocating to nucleus under oxidative stress and GB might be affecting APE1 – ADO interaction in lowering oxidative stress by the anti-oxidant action of GB, which was clearly observed by immunostaining using confocal microscopy. JC-1 assay points toward GB’s role in restoring the mitochondrial membrane potential against Aβ- challenge. Determination of apoptotic markers (Caspase 9 and AIF) showed that Aβ(25-35) induced oxidative stress caused initiation of apoptosis and GB treatment was able to rescue apoptosis. Our study elucidates activation of synaptic CaMKII and CREB exerting neuroprotective effects; and GB acting to restore the expression and active, phosphorylated state of CaMKII and CREB in presence of Aβ-induced oxidative stress in the SH-SY5Y neuroblastoma cells. This study points towards the use of phytochemicals like GB which will may prove to be beneficial for the enhancement of synaptic functionality and promote neuroprotection.Item Effect of amyloid beta (25-35) peptide on mitochondrial respiratiory function in neuroral cells over expressing ape1(Central University of Punjab, 2014) Kaur, Navrattam; Mantha, Anil K.Alzheimer's disease (AD) is an important public health problem which affects millions of people worldwide. The major pathological hallmarks associated with AD are the accumulation of amyloid beta (A?) in senile plaques and neurofibrillary tangles (NFTs) made up of hyperphosphorylated tau proteins. Accumulating evidences point towards the role of oxidative stress and mitochondrial dysfunction in the pathogenesis of AD. Aging is considered as one of the greatest risk factor for AD. In order to maintain genome integrity, base excision repair (BER) pathway is the predominant pathway for repairing oxidized base lesions in neuronal cells. APE1 is the central enzyme of the BER-pathway, having both repair and redox activities and shown to enhance neuronal survival after oxidative stress. In my study, effect of A?(25-35) on mitochondrial ROS/RNS levels and activities of respiratory complexes (I, III, & IV) in neuronal cells was studied with and without ectopic APE1 expression and the neuro- modulatory role of Ginkgolide B (from leaves of G. biloba) was evaluated. It was seen that A?(25-35) increases the ROS/RNS levels in these cells which was decreased when pre-treated with Ginkgolide B (G.B) before treating with A?(25-35). APE1 levels were found to be decreased on treating with A?(25-35) and were increased on pre- treatment with G.B and subsequent treatment with A?(25-35). These results indicate that ectopic APE1 expression in the mitochondria of the neuronal cells might overcome the oxidative damage caused by A?(25-35). Also, phytochemical G.B has shown to modulate the mitochondrial complex activity upon A?(25-35)-induced oxidative stress and modulate the ROS/RNS levels in the presence of APE1. Further studies are needed to understand the mechanism of action of APE1 in relation to the above results, which will be carried out during my Ph.D. work.