Investigating mitochondrial dysfunction in high-risk psychiatric-associated copy number variants in a stem cell model of neurodevelopment Restricted; Files Only

Abstract

Schizophrenia (SCZ) is a highly heritable psychiatric disorder, yet the cellular mechanisms linking genetic risk to disease remain unclear. Highly penetrant, recurrent copy number variants (CNVs) provide a tractable framework for identifying causal biological pathways. This dissertation tests the hypothesis that SCZ-associated CNVs converge on mitochondrial translation as a developmentally constrained point of vulnerability during human neurodevelopment. To investigate this, the 22q11.2 deletion (22qDel), one of the strongest genetic risk factors for SCZ, was examined using CRISPR-engineered isogenic induced pluripotent stem cell (iPSC) models differentiated into neural progenitor cells (NPCs) and cortical organoids. A targeted experimental framework assessed interval-encoded mitochondrial proteins, nuclear-encoded regulators of mitochondrial translation, mitochondrially encoded oxidative phosphorylation (OxPhos) subunits, and steady-state respiratory chain protein abundance. 22qDel models exhibited reductions in multiple interval-encoded mitochondrial proteins, accompanied by coordinated dysregulation of nuclear-encoded factors involved in mitochondrial RNA processing and mitoribosome function, including PDE12 and mitochondrial ribosomal proteins. These changes were associated with altered abundance of mitochondrially encoded OxPhos subunits across both early and late stages of cortical organoid development, consistent with disruption of mitochondrial protein synthesis and stoichiometric balance. Despite these molecular alterations, basal mitochondrial respiration remained largely preserved in NPCs; however, under conditions enforcing OxPhos dependence, 22qDel cells exhibited reduced viability, revealing a context-dependent bioenergetic vulnerability.

To determine whether these effects represent a shared disease mechanism, a second high-risk CNV, the 3q29 deletion, was analyzed in parallel. Integrative analyses revealed that NDD- and SCZ-associated genes are enriched within mitochondrial interaction networks, with convergence comparable to that observed for synaptic pathways and strongest at the level of mitochondrial translation. Consistent with this systems-level enrichment, TMT-based quantitative proteomic analyses of cortical organoids demonstrated highly similar profiles of dysregulated proteins in 22q11.2 and 3q29 deletion models, with significant convergence on the mitochondrial ribosome and translation machinery. These molecular alterations were accompanied by disrupted stoichiometry of OxPhos complexes and conserved neurodevelopmental phenotypes across CNVs. Functional perturbation of mitochondrial translation elicited similar proteomic and cellular responses across both models, and both CNVs exhibited increased sensitivity to metabolic stress, indicating that translational capacity becomes limiting under conditions of increased energetic demand. Together, these findings demonstrate that genetically distinct CNVs converge on mitochondrial translation as a shared cellular pathway linking gene dosage imbalance to impaired metabolic adaptability during neurodevelopment. This work advances a mechanistic framework in which diverse genetic risk factors impact a common mitochondrial process, providing a bridge between genetic heterogeneity and cellular dysfunction in SCZ.

Table of Contents

CHAPTER 1: GENERAL INTRODUCTION……………………………………………………...……1

1.1 Schizophrenia (SCZ)……………………………..……………………...................................2

1.1.1 Background and introduction …………………………………………………………….2

1.1.2 Epidemiology……………………………………………………………………………….3

1.1.3 Core symptoms and functional outcomes………………....…………………...............4

1.1.4 Treatment and current limitations………………………………………………………...5

1.2 Genetic architecture of SCZ…………………………………………………..........................6

     1.2.1 Heritability and polygenicity………………………………………………………………6

1.2.2 Common variation and cross-disorder overlap ………………………………………..7

1.2.3 Rare variants and CNVs………………………………………………………………….8

1.3 Recurrent copy number variants as mechanistic entry points………………………………8

     1.3.1 Mechanisms of CNV formation………………………………………………………..…8

     1.3.2 Experimental advantages and limitations of CNVs……………….……………..…….9

     1.3.3 Association with SCZ and other neurodevelopmental disorders…………………...10

1.4 22q11.2 deletion as a genetic model for SCZ……………………………………………….12

     1.4.1 Overview and SCZ-associated risk…………………………………………………….12

     1.4.2 Multigenic biology: genes and convergent pathways………………………………..13

     1.4.3 Model systems…………………………………………………………………………...17

                1.4.3.1 Animal models………………………………………………………………...17

                 1.4.3.2 Human iPSC/organoid models………………………………………………19

     1.4.4 Remaining mechanistic gap…………………………………………………………….21

1.5 The 3q29 deletion: A complementary high-risk CNV model for SCZ …………………….22

1.5.1 Genetic architecture and clinical risk…………………………………………………..23

1.5.2 Neurodevelopmental and circuit-level phenotypes…………………………………..24

1.5.3 Multigenic and network-level mechanisms……………………………………………25

1.5.4 Mitochondrial dysfunction in 3q29 deletion and convergence across CNVs……...25

1.6 Mitochondria as regulators of neurodevelopment…………………………………………..26

     1.6.1 Neurons as high-energy demand cells………………………………………………..26

     1.6.2 Mitochondria as regulators of neuronal function…………………………………......27

1.6.3 Mitochondria in synaptic development and plasticity………………………………...27

1.6.4 Evidence for mitochondrial dysfunction in SCZ………………………………………28

1.7 Mitochondrial gene expression and translation……………………………………………..30

     1.7.1 mtDNA and the mitoribosome………………………………………………………….31

1.7.2 Nuclear-mitochondrial coordination…………………………………………………....31

1.7.3 Stoichiometric assembly of OxPhos complexes……………………………………...32

1.7.4 Mitochondrial translation during neurodevelopment…………………………………33

1.8 Utilizing iPSC-derived neural models…………………………………………………………34

           1.8.1 Advantages and limitations of animal models……………………………………...…34

     1.8.2 Human iPSC-derived models…………………………………………………………..36

     1.8.3 Power of isogenic CNV systems……………………………………………………….37

1.9 Dissertation hypothesis and objectives………………………………………………………39

           1.9.1 Central hypothesis…………………………………………………………………….…39

           1.9.2 Objectives and overview of the dissertation………………………………………..…39

1.10 Figures…………………………………………………………………………………………43

CHAPTER 2: INVESTIGATING MITOCHONDRIAL PROTEIN DYSREGULATION IN A NEURAL MODEL OF THE SCZ-ASSOCIATED 22Q11.2 DELETION…………………………..45

2.1 Overview of the chapter……………………………………………………………………..…46

2.2 Introduction…………………..………………………………………………………………….47

2.3 Materials and methodology……………………………………………………………………51

2.4 Results………………………………………………………………………………………..…57

2.5 Discussion………………………………………………………………………………………65

2.6 Figures, Tables, & Supplementary Figures………………………………………………….72

CHAPTER 3: CONVERGENT EFFECTS OF NEURODEVELOPMENTAL DISORDER-ASSOCIATED VARIANTS AT MITOCHONDRIA…………………………………………………..88

3.1 Overview of the chapter………………………………………………………………………..89

3.2 Introduction……………………………………………………………………………………...90

3.3 Materials and methodology…………………………………………………………………....93

3.4 Results…………………………………………………………………………………………..99

3.5 Discussion……………………………………………………………………………………..107

3.6 Figures, Tables, & Supplemental Figures………………………………………………….112

CHAPTER 4: GENERAL DISCUSSION….…………………………………..…………………….126

4.1 Summary of major findings…………………………………………………………………..127

4.2 Human iPSC-based models for dissecting mechanisms of SCZ-associated CNVs…..129

4.3 From gene dosage to cellular mechanism: integrating 3q29 and 22q11.2 deletions….130

4.4 Therapeutic opportunities arising from mitochondrial and translational dysfunction…..134

4.5 Limitations and future remarks………………………………………………………………136

4.6 Concluding remarks…………………………………………………………………………..139

REFERENCES………………………………………………………………………………………...141

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience
Keyword	Schizophrenia Mitochondria Copy number variants Induced pluripotent stem cells (iPSCs)
Committee Chair / Thesis Advisor	Gary Bassell, PhD, Emory University
Committee Members	Shannon Gourley, PhD, Emory University Victor Faundez, MD, PhD, Emory University Ryan Purcell, PhD, Virginia Tech Jimena Andersen, PhD, Emory University Jennifer Mulle, PhD, Rutgers University

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