Signaling mechanisms upregulating protein synthesis ameliorate neuronal phenotypes caused by copper deficiency Público

Abstract

Copper homeostasis is essential for neuronal functions from neurotransmitter synthesis to oxidative phosphorylation (the main supplier of energy for the nervous system). Copper is required to help the brain meet its high energy demands and plays a particularly important role in neurodevelopment, as there is a shift after birth toward mitochondrial respiration over glycolysis. Rare inherited diseases caused by mutations in the copper transporters SLC31A1 (CTR1) or ATP7A induce copper deficiency in the brain and throughout the body, causing seizures and neurodegeneration in infancy through poorly understood disease mechanisms. In this dissertation, I describe a method for quantifying trace metals in biological samples and characterize three model systems to investigate the molecular mechanisms downstream of copper deficiency in neuronal cells. CTR1-null human neuroblastoma cells exhibit copper-dependent impairments in mitochondrial enzymes, leading to decreases in oxidative phosphorylation and increased glycolysis. Multiomics analysis of these cells at the protein and RNA level and corroborative immunoblots identified elevated mTORC1 and S6K activation, reduced PERK signaling, and downstream increased protein synthesis in CTR1-null cells. Pharmacological manipulation of these cells suggests that mTOR activation and increased protein synthesis is a pro-survival response to copper deficiency. These findings were corroborated in vivo in mouse and Drosophila. Spatial transcriptomic profiling of Atp7a^flx/Y :: Vil1^Cre/+ mice, which model brain copper deficiency due to impaired absorption of dietary copper, identified upregulated protein synthesis machinery and mTORC1-S6K pathway genes in mutant Purkinje neurons before the onset of neurodegeneration. Pathological epidermis phenotypes in Drosophila caused by ATP7 overexpression (inducing copper extrusion) are intensified by RNAi against the mTOR pathway genes Akt, S6k, or raptor. Dendritic phenotypes in copper deficient class IV neurons are partially rescued by either of two genetic mTOR pathway manipulations (S6k overexpression or Thor RNAi) that ultimately increase protein synthesis. Together, this body of work points to increased mTORC1-S6K pathway activation and protein synthesis as an adaptive mechanism by which neuronal cells respond to copper deficiency. More broadly, these data demonstrate the interconnectivity of copper homeostasis, metabolism, and bioenergetics in neuronal cells and the particular importance of these processes during critical time periods in early neurodevelopment.

Table of Contents

Abstract   iv

Dedication   vi

Acknowledgments   vii

Table of Contents   x

List of Figures   xiii

List of Tables   xv

List of Symbols and Abbreviations   xvi

Chapter 1. General Introduction   1

1.1 Conceptual background and the goals of this thesis   1

1.2 Copper biology   2

1.2.1 Copper functions   2

1.2.2 Systemic and cellular copper homeostasis   3

1.2.3 Copper in the nervous system   5

1.2.4 Copper and metabolism during neurodevelopment   8

1.3 Neurological and neurodegenerative diseases of copper dyshomeostasis   10

1.3.1 ATP7A-related diseases   10

1.3.2 SLC31A1-related diseases   12

1.3.3 ATP7B-related diseases   14

1.3.4 Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and schizophrenia   14

1.4 Significance of this dissertation research and overview of findings   15

1.4.1 Model Systems Utilized in this Dissertation   16

1.4.2 Overview of Findings   18

1.4.3 Summary   21

Chapter 2. Sulfur- and phosphorus-standardized metal quantification of biological specimens using inductively coupled plasma mass spectrometry   22

2.1 Abstract   23

2.2 Protocol Overview   24

2.3 Before you begin   24

2.3.1 Culture cell lines   25

2.3.2 Prepare for D. melanogaster sample collection   26

2.3.3 Prepare buffers for ICP-MS   26

2.4 Materials and equipment   30

2.4.1 Final buffer/chemical concentrations and volumes   30

2.5 Step-by-step method details   32

2.5.1 Cell sample preparation   32

2.5.2 D. melanogaster larvae sample preparation   37

2.5.3 ICP mass spectrometry   40

2.6 Expected outcomes   46

2.7 Limitations   52

2.8 Troubleshooting   52

2.8.1 Problem 1   52

2.8.2 Problem 2   53

2.8.3 Problem 3   53

2.8.4 Problem 4   54

2.8.5 Problem 5   54

2.9 Resource availability   55

2.10 Acknowledgments   55

2.11 Contributor Information   56

2.11.1 Data and code availability   56

Chapter 3. Adaptive protein synthesis in genetic models of copper deficiency and childhood neurodegeneration   57

3.1 Abstract   58

3.2 Introduction   59

3.3 Results   61

3.3.1 Metabolic phenotypes in a cell-autonomous model of copper deficiency   61

3.3.2 Unbiased discovery of copper deficiency mechanisms using proteomics and NanoString transcriptomics   67

3.3.3 Increased steady-state activity of the mTOR-S6K pathway in CTR1 KO cells   76

3.3.4 Activation of the mTOR-S6K signaling pathway is necessary for CTR1 KO cell survival   78

3.3.5 Interaction between mitochondrial respiration and increased protein synthesis in CTR1 KO cells   83

3.3.6 Upregulation of protein synthesis machinery in a mouse model of copper deficiency   87

3.3.7 Genetic modulation of mTOR pathway-dependent protein synthesis activity modifies copper deficiency phenotypes in Drosophila   97

3.4 Discussion   101

3.5 Materials and Methods   106

3.5.1 Cell lines, gene editing, and culture conditions   106

3.5.2 Mouse husbandry   107

3.5.3 Antibodies   107

3.5.4 Drugs   108

3.5.5 Immunoblotting and puromycin pulse   109

3.5.6 Mitochondrial isolation and blue native gel electrophoresis   110

3.5.7 Cell survival and Synergy analysis   110

3.5.8 Seahorse metabolic oximetry   111

3.5.9 Resipher   113

3.5.10 Total RNA extraction and NanoString mRNA Quantification   114

3.5.11 ICP mass spectrometry   115

3.5.12 Preparation of brain tissue for proteomics, immunoblots, or Luminex analysis   116

3.5.13 TMT mass spectrometry for proteomics   116

3.5.14 Metabolite quantification by LC mass spectrometry   119

3.5.15 Insulin receptor phosphorylation quantification   120

3.5.16 Digital spatial profiling of mouse brain tissue   120

3.5.17 Drosophila husbandry and genotypes   122

3.5.18 Drosophila dendritic imaging and analysis   124

3.5.19 Data availability   125

3.5.20 Bioinformatic analyses and statistical analyses   125

Chapter 4. Discussion   126

4.1 Summary of findings   126

4.2 Cell type-specific responses to copper deficiency in the brain   128

4.3 Resilience mechanisms to copper deficiency during neurodevelopment   129

4.3.1 Adaptive and maladaptive proteostasis in copper deficiency   131

4.4 Future directions for this research   133

4.5 Conclusions   134

References   135

About this Dissertation

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• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Cell Biology, Neuroscience
Palavra-chave	Menkes Copper mTOR ATP7A Neurodegeneration CTR1
Committee Chair / Thesis Advisor	Victor Faundez, Emory University
Committee Members	Mike Caudle, Emory University Jennifer Kwong, Emory University Peter Wenner, Emory University Steven Sloan, Emory University

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