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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 Design, Synthesis and Evaluation of Donepezil-Rasagiline Based Compounds as Multipotent Inhibitors for the Treatment of Alzheimer’s Disease(Central University of Punjab, 2019) Kumar, Bhupinder; Kumar, VinodAlzheimer’s disease (AD) is multifactorial in nature and different enzymes including MAO, AChE, and amyloid beta are implicated in its pathogenesis. The pathomechanism of AD is complex in nature and single target drugs proved to be ineffective for the treatment of the disease. With an aim of developing dual/multipotent inhibitors, 4,6- diphenylpyrimidines were optionally substituted with propargyl group and an ethyl chain containing a cyclic or acyclic tertiary nitrogen atom (piperidine/morpholine/pyrrolidine/N,N-dimethyl) as potential pharmacophores for MAO and AChE enzymes. Compound VB1 was found to be the most potent MAO-A (IC50 value of 18.34 ± 0.38 nM) inhibitor and VB8 was found to be the most potent AChE (IC50 value of 9.54 ± 0.07 nM) inhibitor. Compound VB3 was another promising compound in series-I with IC50 values of 28.33 ± 3.22 nM and 18.92 ± 0.29 nM against MAO-A and AChE, respectively and displayed very high selectivity index (103) for AChE over BuChE. These compounds were found to be reversible inhibitors of MAO and AChE enzymes and non-toxic to the human neuroblastoma SH-SY5Y cells. Based on structure-activity relationship analysis of the first series of compounds, second series of the compounds were designed by fixing the position of piperidine/morpholine ethyl chain at the para position of one of the phenyl rings. In the second series, compound VP15 v was found to be a multi-potent inhibitor of MAO-B and AChE with IC50 values of 0.37 ± 0.03 μM and 0.04 ± 0.003 μM, respectively. VP15 was found to be selective for MAOB with selectivity index of 270 over MAO-A. It also displayed SI of 625 for AChE over BuChE. VP15 was found to be irreversible inhibitor of MAO-B. In the third series of target compounds, both the phenyl rings of diphenylpyrimidines were substituted with O-propargyl groups. Different derivatives have been synthesized with O-propargyl groups substituted at ortho, meta and para positions of the phenyl rings. In the third series of compounds, AVB1 and AVB4 were found to be the most potent inhibitors of AChE and MAO-B with IC50 values of 1.35 ±0.03 μM and 1.49 ± 0.09 μM, respectively. In the reversible inhibition studies, the lead compounds were found to be reversible inhibitors of MAO-B and AChE enzymes. In the ROS protection inhibition studies, AVB1 and AVB4 displayed good activity in SH-SY5Y cells and AVB1 reduced the ROS levels up to 30% at 5 μM. This series of compounds were also found to be non-toxic to the SH-SY5Y cells in the cytotoxicity studies. Thus, from the present study it can be concluded that 4,6-diphenylpyrimidine derivatives can act as potential lead for the development of effective drug candidates for the treatment of AD. Compound VB3 and VP15 were found to be the most potent dual inhibitors of MAO and AChE.Item Curcumin revitalizes Amyloid beta (25–35)-induced and organophosphate pesticides pestered neurotoxicity in SH-SY5Y and IMR-32 cells via activation of APE1 and Nrf2(Springer, 2017) Sarkar, Bibekananda; Dhiman, Monisha; Mittal, Sunil; Mantha, Anil K.Amyloid beta (Aβ) peptide deposition is the primary cause of neurodegeneration in Alzheimer’s disease (AD) pathogenesis. Several reports point towards the role of pesticides in the AD pathogenesis, especially organophosphate pesticides (OPPs). Monocrotophos (MCP) and Chlorpyrifos (CP) are the most widely used OPPs. In this study, the role of MCP and CP in augmenting the Aβ-induced oxidative stress associated with the neurodegeneration in AD has been assessed in human neuroblastoma IMR-32 and SH-SY5Y cell lines. From the cell survival assay, it was observed that MCP and CP reduced cell survival both dose- and time-dependently. Nitro blue tetrazolium (NBT) based assay for determination of intracellular reactive oxygen species (ROS) demonstrated that Aβ(25–35), MCP or CP produce significant oxidative stress alone or synergistically in IMR-32 and SH-SY5Y cells, while pretreatment of curcumin reduced ROS levels significantly in all treatment combinations. In this study, we also demonstrate that treatment of Aβ(25–35) and MCP upregulated inducible nitric oxide synthase (iNOS/NOS2) whereas, no change was observed in neuronal nitric oxide synthase (nNOS/NOS1), but down-regulation of the nuclear factor erythroid 2-related factor 2 (Nrf2) level was observed. While curcumin pretreatment resulted in upregulation of iNOS and Nrf2 proteins. Also, the expression of key DNA repair enzymes APE1, DNA polymerase beta (Pol β), and PARP1 were found to be downregulated upon treatment with MCP, Aβ(25–35) and their combinations at 24 h and 48 h time points. In this study, pretreatment of curcumin to the SH-SY5Y cells enhanced the expression of DNA repair enzymes APE1, pol β, and PARP1 enzymes to counter the oxidative DNA base damage via base excision repair (BER) pathway, and also activated the antioxidant element (ARE) via Nrf2 upregulation. Furthermore, the immunofluorescent confocal imaging studies in SH-SY5Y and IMR-32 cells treated with Aβ(25–35) and MCP-mediated oxidative stress and their combinations at different time periods suggesting for cross-talk between the two proteins APE1 and Nrf2. The APE1’s association with Nrf2 might be associated with the redox function of APE1 that might be directly regulating the ARE-mediated neuronal survival mechanisms.Item Ginkgolide B Revamps Neuroprotective Role of Apurinic/Apyrimidinic Endonuclease 1 and Mitochondrial Oxidative Phosphorylation Against Ab 25–35 -Induced Neurotoxicity in Human Neuroblastoma Cells(Wiley, 2015) Kaur, Navrattan; Dhiman, Monisha; Perez-Polo, J. Regino; Mantha, Anil K.Accumulating evidence points to roles for oxidative stress, amyloid beta (Aβ), and mitochondrial dysfunction in the pathogenesis of Alzheimer's disease (AD). In neurons, the base excision repair pathway is the predominant DNA repair (BER) pathway for repairing oxidized base lesions. Apurinic/apyrimidinic endonuclease 1 (APE1), a multifunctional enzyme with DNA repair and reduction–oxidation activities, has been shown to enhance neuronal survival after oxidative stress. This study seeks to determine 1) the effect of Aβ25–35 on reactive oxygen species (ROS)/reactive nitrogen species (RNS) levels, 2) the activities of respiratory complexes (I, III, and IV), 3) the role of APE1 by ectopic expression, and 4) the neuromodulatory role of ginkgolide B (GB; from the leaves of Ginkgo biloba). The pro-oxidant Aβ25–35 peptide treatment increased the levels of ROS/RNS in human neuroblastoma IMR-32 and SH-SY5Y cells, which were decreased after pretreatment with GB. Furthermore, the mitochondrial APE1 level was found to be decreased after treatment with Aβ25–35 up to 48 hr, and the level was increased significantly in cells pretreated with GB. The oxidative phosphorylation (OXPHOS; activities of complexes I, III, and IV) indicated that Aβ25–35 treatment decreased activities of complexes I and IV, and pretreatment with GB and ectopic APE1 expression enhanced these activities significantly compared with Aβ25–35 treatment. Our results indicate that ectopic expression of APE1 potentiates neuronal cells to overcome the oxidative damage caused by Aβ25–35. In addition, GB has been shown to modulate the mitochondrial OXPHOS against Aβ25–35-induced oxidative stress and also to regulate the levels of ROS/RNS in the presence of ectopic APE1. This study presents findings from a new point of view to improve therapeutic potential for AD via the synergistic neuroprotective role played by APE1 in combination with the phytochemical GB. © 2015 Wiley Periodicals, Inc.