Evaluation of amyloid beta (aβ)-induced Mitochondrial dysfunction: Neuroprotective role of Apurinic/apyrimidinic endonuclease (ape1) Via its interaction with cysteamine Dioxygenase (ado)

Abstract

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.