Alcohol and micronutrient-induced regulation of Aβ load and Tau hyperphosphorylation through modulation of mitochondrial fusion/fission pathways

Okoro, Grace U

Abstract

Introduction and aims: Alzheimer’s disease (AD) has for decades been associated with increased accumulation of brain amyloid β peptides (Aβ) and phosphorylated tau. Unfortunately, targeted therapy to Aβ in particular has not been successful resulting in a diversified approach to understanding the disease pathology. Mitochondrial dysfunction is among a range of cellular and molecular processes postulated to contribute to AD, which could be a target for lifestyle factors such as alcohol and micronutrients. Ethanol-mediated neurotoxicity and its contribution to AD has in part been associated with increased oxidative stress and Aβ levels. However, conflicting reports debate whether moderate levels offer protection whereas heavy alcohol consumption accelerate cognitive decline. Similarly, micronutrient, which are essential requirements for proper growth and development can become neurotoxic when taken or when accumulated in excess. Toxic levels of Zinc (Zn) and Manganese (Mn), the focus in this thesis have both been associated with neurotoxicity and implicated in AD. To address if different levels of alcohol evoke a switch from neuroprotection to neurotoxicity and the neurotoxic impact of excessive Zn and Mn, we investigated the contribution of ethanol, Zn, Mn-induced neurotoxicity in regulating protein aggregation, Aβ, p-tau accumulation. We hypothesize using the SH-SY5Y neuronal cell model, ethanol, Zn and Mn-induced regulation of protein aggregation, Aβ, p-tau accumulation is mediated by a dysregulation of mitochondrial function and fusion/fission dynamics.
Methods: Using Western blot analysis, antioxidant proteins, fusion/fission proteins, autophagy and apoptotic markers, ERK/AKT kinases, Aβ and p-tau were assessed. Mitochondrial membrane potential (MMP) was analyzed using the JC-1 MMP assay and SOD activity using enzyme activity assay. Confocal microscopy of live cells was used to determine changes in mitochondrial morphology while fixed cells were assessed for LC3 levels. Mitochondrial respiration and function were assessed using the Seahorse Mitostress Assay.
Results: In response to toxic ethanol exposure, SH-SY5Y cells show an increase in ROS, a targeted antioxidant response, dysregulated mitochondrial dynamics and function indicated by a significant increase in fission protein, Drp1, fragmented mitochondria while decreasing fusion proteins, Opa1 and Mfn1 and decrease in mitochondrial membrane potential, low basal mitochondrial respiration and low mitochondrial ATP production. This results in shift towards autophagy/mitophagy, a switch between cell survival and toxicity linking to ERK activation and mTORC1 suppression that leads to increased apoptosis and cell death. Despite protein aggregation and Aβ increase with toxic ethanol exposure, levels of p-tau showed a dramatic decrease indicating that ethanol regulation of AD supports Aβ production rather than p-tau increase. Similar response was observed following toxic Mn and Zn levels, however cells exposed to toxic levels of Zn undergo a non-apoptotic cell death process likely to be necrotic.
Discussion: Together these findings have identified new and novel mechanisms by which ethanol, Zn and Mn affects mitophagy and mitochondrial fusion/fission integrity through increased ROS generation and MMP dissipation and consequent cell survival pathway response impact modulation of Aβ and p-tau, offering insight into how these pathways become dysregulated under conditions that are associated with AD pathology.

Thesis Type	Thesis
Publicly Available Date	Jul 27, 2020
Public URL	https://uwe-repository.worktribe.com/output/1491050
Award Date	Jul 27, 2020

Alcohol and micronutrient-induced regulation of Aβ load and Tau hyperphosphorylation through modulation of mitochondrial fusion/fission pathways

Okoro, Grace U

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