Golgi-Dependent Mechanisms of Cellular Copper Homeostasis Open Access

Abstract

Copper is required for diverse cellular processes including pigment production, neuropeptide synthesis and mitochondrial function, yet possesses the capacity to inflict oxidative damage to cells. Cells possess a network of chaperones and transporters that maintain appropriate copper levels both for its provision to cuproenzymes and to avoid oxidative damage. Mutations in copper-binding proteins strongly associate with neuropathologies, and neurodegenerative diseases such as Alzheimer's and Parkinson's disease exhibit altered copper homeostasis. The cellular trafficking and regulation of copper has traditionally been described as occurring in a discrete, organelle-specific manner, yet emerging research supports a model of copper-sensing and communication taking place between organelles.

I hypothesize that genetic defects in molecules required for the subcellular localization of copper transporters will impair neuronal tissue viability. I define an interaction network for the copper transporter ATP7A and find it is enriched in genes associated with neuropathologies. Among these genes are those encoding subunits of the conserved oligomeric Golgi (COG) complex, a multimeric tethering complex required for retrograde intra-Golgi traffic. I present biochemical and genetic evidence of an interaction between ATP7A and COG, and establish a role for the COG complex in copper homeostasis at multiple cellular compartments. I find that COG null cells display decreased ATP7A levels and perturbed surface expression of both ATP7A and the copper importer CTR1. Further, both copper content and levels of copper-sensitive transcripts are altered in COG null cells. Reduced copper content, measured by inductively coupled plasma mass spectrometry, and impaired mitochondrial function, assayed by the activity of mitochondrial reductases, can be rescued by the addition of copper in conjunction with an ionophore. Finally, ATP7A and COG synthetically interact in Drosophila melanogaster to influence viability and the development of the neuromuscular junction.

These data support a model of global cellular copper homeostasis, and the altered copper homeostasis observed in COG null cells suggests a mechanism to account for neurodevelopmental phenotypes in patients bearing COG mutations. The ATP7A interactome offers avenues for future work to elucidate copper homeostasis mechanisms, and the observed interaction between copper homeostasis and mitochondrial function in this work illuminates a possible mechanism in the etiology of copper-related neuropathologies.

Table of Contents

Table of Contents

1. General Introduction / 1-66

Overview / 2

Organization of the Introduction / 3

Section 1: Copper Transport / 4-21

1.1 Uptake / 4

1.2 Delivery to and activity of copper varies by cell compartment / 6

1.2.1 Cytoplasm: SOD1 / 8

1.2.2 Mitochondria: cytochrome c oxidase (COX) / 9

1.2.3 Golgi: ATP7A & ATP7B / 13

1.2.4 Extracellular and cytosolic copper binding / 15

1.3 Copper sensing and response mechanisms / 16

1.3.1 Post-translational regulation / 18

1.3.2 Redox regulation / 18

1.3.3 Transcriptional regulation / 19

1.4 Summary / 20

Section 2: Copper Homeostasis and Neurodegenerative Disease / 21-32

2.1 Copper in the nervous system / 22

2.2 Genetic and environmental factors, such as metals, influence the development and progression of neuropathologies / 22

2.3 Genetic defects in copper binding proteins provide direct evidence of metal homeostasis mechanisms in neurodegenerative diseases / 23

2.3.1 ATP7A: Menkes disease / 24

2.3.2 ATP7B: Wilson's disease / 25

2.3.3 SOD1: Amyotrophic lateral sclerosis / 25

2.3.4 ATOX1 / 26

2.3.5 SCO1 / 27

2.4 Indirect role of metal homeostasis mechanisms in neurodegenerative diseases / 27

2.4.1 Alzheimer's disease / 28

2.4.2 Parkinson's disease / 30

2.4.3 Prion diseases / 31

2.5 Summary of current models for the role of copper in the development of neuropathologies / 31

Section 3: Utilizing ATP7A as an Entry Point to Study Copper-Related Neurodegeneration / 32-52

3.1 ATP7A expression and localization / 33

3.1.1. CNS / 33

3.2 Molecular Basis of Neurodegeneration and Neurodevelopmental Defects in Menkes Disease / 39

3.2.1 Introduction / 39

3.2.2 Clinical and Pathological Characteristics of Menkes Disease / 41

3.2.3 Menkes Disease Neuropathology / 42

3.2.4 Cell Biology of Menkes Disease / 44

3.2.5 The Oligoenzymatic Pathogenic Hypothesis of Menkes Disease/47

3.2.6 Proposed Revisions to the Oligoenzymatic Hypothesis / 50

3.2.7 Conclusions / 51

Section 4: Introduction Summary, Hypothesis, and Contribution of this Dissertation to the Field / 52-55

4.1 Outstanding questions in ATP7A and copper biology / 53

4.2 Hypothesis / 54

4.3 Contribution of this Dissertation to the field / 55

Figure 1 / 7

Figure 2 / 10-11

Figure 3 / 12

Figure 4 / 14

Figure 5 / 17-18

Figure 6 / 34-35

Figure 7 / 37-39

Figure 8 / 43-44

Table 1 / 56-63

Table 2 / 64-65

Table 3 / 66

2: The Interactome of the Copper Transporter ATP7A Belongs to a Network of Neurodevelopmental and Neurodegeneration Factors / 67-114

Abstract / 68

Introduction / 69-71

Results / 71-96

The ATP7A Interactome / 71

The COG Complex is Necessary for Copper Transporter Stability and

Cell Surface Expression / 81

COG Complex Genetic Defects Decrease Cellular Copper and Modify the Expression of ATP7A and Other Copper-Sensitive Transcripts / 85

COG-Dependent ATP7A and CTR1 Defects Impair Copper Homeostasis/ 89

Genetic Interactions Between ATP7A and COG Complex Subunits in the Drosophila Melanogaster / 93

Discussion / 96-103

Materials & Methods / 104-111

Acknowledgements / 112

Figure 1 / 73-74

Figure 2 / 76

Figure 3 / 78

Figure 4 / 78-79

Figure 5 / 80-81

Figure 6 / 83-84

Figure 7 / 87

Figure 8 / 91

Figure 9 / 95

Figure 10 / 96

Supplementary Table 1 / 113-114

3: Discussion / 115-140

Overview of Findings and Significance / 116-117

Revisiting the Central Hypothesis / 117

Addressing Questions in ATP7A and Copper Biology

1. What mechanisms underlie the contribution of copper to neurodegenerative and neurodevelopmental disorders? / 118 - 121

2. Does copper influence other neurodegenerative diseases? / 121-124

3. How is global copper homeostasis regulated? / 124-127

4. What proteins are required for the regulation of ATP7A localization and trafficking? / 128-131

Summary / 132

Figure 1 / 123

Figure 2 / 128-129

Table 1 / 133-138

References / 139-158

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	Biochemistry, Cell & Developmental Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry Biology, Neuroscience Biology, Cell
Keyword	Neurodegeneration Metal homeostasis Protein interactome Copper biology COG complex ATP7A
Committee Chair / Thesis Advisor	Faundez, Victor, Emory University
Committee Members	Kowalczyk, Andrew, Emory University Koval, Michael H, Emory University L'Hernault, Steven, Emory University Sale, Winfield S, Emory University

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