Adaptive response of mTOR regulation in cellular models of copper depletion 公开

Abstract

Intracellular copper homeostasis is a critical cellular function required for a variety of physiological purposes including detoxification of reactive oxygen species, mitochondrial electron transport chain function, and more. To maintain a physiological balance of intracellular copper concentrations the copper importer CTR1, encoded by the SLC31A1 gene, is localized at the plasma membrane, allowing proper influx of copper. Genetic diseases that cause brain copper depletion such as Neurodegeneration and Seizures due to Copper Transport defect (NSCT, caused by mutations in CTR1) and Menkes disease (caused by mutations in the copper transporter ATP7A) cause neuronal dysfunction and neurodegeneration. These rare genetic diseases impair activities required for the function of copper-dependent enzymes, including components of the respiratory chain and enzymes required for the synthesis of neurotransmitters and neuromodulators. We introduced a mutation which abolishes CTR1 expression, causing intracellular copper depletion similar to the phenotype of NSCT and Menkes disease. From our proteomics data, we found that the CTR1 mutant cells increased activity of the mTOR pathway, which regulates cellular functions such as gene transcription, protein synthesis, cell division and growth, and differentiation. Using the CTR1 mutant and control cell lines, we analyzed protein expression, cell viability, and bioenergetic and metabolic variability in response to pharmacological manipulations of mTOR activity. We have found that copper deficient neuroblastoma cells increase cell survival by modulating the activity of the mTOR pathway. As these pathways become clear, we will have a comprehensible understanding of the cellular response to dysfunctional copper transport and how they can be controlled to improve neurodegeneration.

Table of Contents

Introduction 1

Materials & Methods 6

Cell lines, gene editing, and culture conditions 6

Antibodies 6

Drugs 7

Western blotting 8

Cell survival and Synergy analysis 9

Seahorse metabolic oximetry 10

Results 13

CTR1 and ATP7A-LA mutant cell lines are fundamentally depleted of copper 13

Cell viability in response to elesclomol and bathocuproine disulphonate (BCS) 14

Increased activation of mTOR-Raptor-S6K signaling and protein synthesis in CTR1 proteome and phosphoproteome 16

mTOR signaling activation and upregulation is necessary for CTR1 KO cell survival 18

Increased activity of the mTOR pathway 21

Adaptive metabolic processes of CTR1 mutants in response to copper depletion and increased mTOR signaling 22

Discussion 26

References 32

Supplemental Information 38

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Cell Biology, General Biology, Neuroscience
关键词	Neuroscience Copper mTOR Neurodegeneration Protein synthesis
Committee Chair / Thesis Advisor	Victor Faundez, MD, PhD, Emory University
Committee Members	Jennifer Kwong, PhD, Emory University Arri Eisen, PhD, Emory University

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