Regulation of Drosophila Synaptic Function and Plasticity by a Schizophrenia Susceptibility Network. Pubblico

Abstract

Neurodevelopmental disorders (NDDs) are genetically complex, arising from single or multiple gene defects, and include schizophrenia, intellectual disability, and autism spectrum disorder. Many NDDs, particularly those associated with large chromosomal deletions, either share common genetic variations or it is postulated that the associated gene products converge into a common molecular or cellular pathway. However, the way multiple loci interact to modify phenotypes remains poorly understood. Current studies focus on monogenic NDDs because of their straightforward study and conceptualization, despite the involvement of multiple loci. No studies have explored the interactions of multiple genes or gene products associated with NDDs and their effects at the synapse. Here, I use a biochemically curated interaction network centered around the schizophrenia susceptibility gene dysbindin (dysb), the Drosophila ortholog of the human gene DTNBP1. I examined the phenotypes associated with mutations in dysbindin(dysb), in isolation or in combination with null alleles in the dysb network component Blos1. In humans, the Blos1 ortholog Bloc1s1 encodes a polypeptide that assembles, with dysbindin, into the octameric BLOC-1 complex. I biochemically confirmed BLOC-1 in Drosophila neurons, and measured synaptic output and complex adaptive behavior in response to BLOC-1 perturbation. Homozygous loss-of-function alleles of dysb, Blos1, or compound heterozygotes of these alleles impaired neurotransmitter release, synapse morphology, and homeostatic plasticity at the larval neuromuscular junction, and impaired olfactory habituation. This multiparameter assessment indicated that phenotypes were differentially sensitive to genetic dosages of loss-of-function BLOC-1 alleles. Further, I identified the N-Ethylmaleimide Sensitive Factor (NSF) as sensitive to BLOC-1 deficiency. I used NSF to test the hypothesis that molecular and genetic interactors converge into a functionally-defined pathway. My findings suggest that modification of a second genetic locus in a defined neurodevelopmental regulatory network does not follow strict additive genetic inheritance; rather, precise stoichiometry within the network determines phenotype. I demonstrate that a biochemically curated interactome can be used to direct investigation of pathways associated to complex genetic diseases, such as schizophrenia and related NDDs. Together, this work supports the investigation of NDDs through the assessment of multiple endophenotypes in response to polygenic experimental manipulations to better approximate complex disease states.

Table of Contents

Table of Contents

Page Number

Chapter I. General Introduction

Overview and Significance…………………………………………………………………………….2

Section 1. Neurodevelopmental Disorders: Mechanisms and Boundary

Definitions from Genomes, Interactomes, and Proteomes……………………………….7

NDD: Boundary Definitions from Genomes………………………………………….8

NDD: Boundary Definitions from Interactomes…………………………………..10

Figure 1. DTNBP1-dysbindin interactomes differ in their

constituents and topology………………………………………………….……13

NDD: "Guilty by Association" Mechanisms of Disease……………………...14

Creating Understanding from Genome Informed Proteomes-

Interactomes…………………………………………………………………………………….15

Figure 2. Models of cross-fertilization between genomes,

proteomes, and interactomes………………………….……………………...18

Creating a genome-independent nosology from proteomes-

interactomes………….………………………………………………………………………...19

Section 2. Cell Biology of the BLOC-1 Complex Subunit Dysbindin, a

Schizophrenia Susceptibility Gene……………………………………………………………….21

Association of dysbindin and schizophrenia: Genetic evidence.…………….21

Biochemical, anatomical, and functional consequences of

carrying DTNBP1 polymorphisms associated to disease……………………23

The Eight Musketeers: "all for one, one for all", that is

BLOC-1's motto.……………………………………………….………………………………26

Figure 3. Molecular Architecture of the BLOC-1 complex…………28

Figure 4. Levels of the BLOC-1 subunits pallidin and

dysbindin in brains of wild type and BLOC-1 or AP-3

mutant mice…………………………………………………………………………..31

What fundamental cellular processes are affected in

dysbindin/BLOC-1 loss-of-function?.......................................................32

Transcriptional and Cytoskeletal Regulation…………………………..32

Membrane Protein Sorting……………………………………………………..34

Figure 5. The stages of vesicle budding

and fusion………………………………………………………………...38

Membrane Fusion-Secretion…………………………………………………..39

Section 3. Organziation and Trafficking of Synaptic Vesicles…………………..46

Figure 6. Synaptic vesicle fusion cycle……………………………………………..48

Figure 7. Synaptic vesicle pool organization and endocytic

pathways…………………………………………….……………………………………….…..50

Section 4. Drosophila melanogaster as a model system…………………………….51

Table 1. Homologues of human BLOC-1 subunits encoded

by the fruit fly genome……………………………………………………………..………54

Figure 8. Schematic of the Drosophila neuromuscular junction………..56

Figure 9. GAL4/UAS system for targeted gene expression

in Drosophila…………………………………………………………………………………….57

Contributions of this dissertation research………………………………………………58

Chapter II. Gene Dosage in the Dysbindin Schizophrenia

Susceptibility Network Differentially Affect Synaptic

Function and Plasticity

Abstract…………………………………………………………………………………………………….62

Introduction………………………………………………………………………………….....……...63

Results………………………………………………………………………………….…………………..67

BLOC-1 assembles into an octameric complex in Drosophila neurons

BLOC-1 acts presynaptically to regulate quantal content at the NMJ

Normal synaptic growth and morphology require BLOC-1 function

Dysbindin and Blos1 are necessary for the function of synaptic

vesicle pools

Dysbindin and Blos1 are required for olfactory short-term habituation in Drosophila

Hierarchical Clustering Analysis of BLOC-1 Genotype and Associated Phenotype

Discussion…………………………………………………………………………………………………80

Materials and Methods……………………………………………………………………............86

Drosophila stocks, rearing, genetics, and electrophysiology

Immunohistochemistry and confocal microscopy

Immunoprecipitation and mass spectrometry

Short-term olfactory habituation

Statistical and cluster analysis

Figure 1. BLOC-1 assembles into an octameric complex in Drosophila

neurons…………………………………………………………………………….….……91

Figure 2. BLOC-1 presynaptically regulates quantal content at the

Drosophila NMJ....................................................................................92

Figure 3. Normal synaptic morphology requires BLOC-1 function……………..93

Figure 4. BLOC-1 gene-dosage regulates synaptic homeostasis………………......94

Figure 5. BLOC-1 gene-dosage regulates synaptic vesicle pool

properties…………………………………………………………………………………95

Figure 6. BLOC-1 is required at local interneurons and projection

neurons for short-term olfactory habituation in Drosophila……….…96

Figure 7. Hierarchical Clustering Analysis of BLOC-1 Genotype and

their Associated Phenotypes……………………………………………………….97

Figure 8. How do BLOC-1 mutations produce divergent synaptic

phenotypes?.........................................................................................98

Supplementary Table 1. Summary of results for olfactory short-term

habituation experimental data shown in Figure 6………………….……99

Chapter III. NSF Acts Downstream of the Schizophrenia Susceptibility

Factor, Dysbindin, to Regulate Synaptic Homeostasis

Abstract………………………………………………………………………………………………….. 102

Significance Statement………………………………………………………………………….…..103

Introduction………………………………………………………………………………………….... 104

Results……………………………………………………………………………………………………. 107

Discussion………………………………………………………………………………………………..111

Materials and Methods………………………………………………………………………………115

Cell culture

SILAC labeling and mass spectrometry analysis

Immunoprecipitation

Sucrose density sedimentation

Immunofluorescence

S2 Drosophila and HeLa cell secretion assay

Drosophila stocks, rearing, genetics, andbiochemical procedures.

Statistical analysis

Antibodies used

Figure 1. Fusion apparatus content is altered in BLOC-1 deficiency………..……126

Figure 2. BLOC-1 interacts with NSF or SNAREs……………………………….……… 127

Figure 3. NSF presynaptically rescues dysbindin synaptic homeostasis

defect………………………………………………………………………………………128

Supplementary Figure 1. Fusion apparatus content is altered in

BLOC-1 deficiency………………………………………………………………….…129

Supplementary Figure 2. Down-regulation of Bloc1s6 Pallidin in iPSC-

derived Human Neurons…………………………………………………………..130

Supplementary Figure 3. NSF and VAMP7 down-regulation phenotypes

are independent of each other…………………………………….……………..131

Supplementary Figure 4. Interaction of the BLOC-1 complex with

SNAP23, 25 and 29……………………………………………………………….... 132

Supplementary Figure 5. A pulse of constiutive secretion cargo is not

impaired by BLOC-1 deficiency……………………………………………….…133

Supplementary Figure 6. Model of BLOC-1-SNARE-NSF interactions……..134

Chapter IV. Discussion

Overview………………………………………………………………………………………………….136

Summary of Results………………………………………………………………………………….142

Hypothesis 1: Phenotypes arising from loss-of-function mutations to

members of the BLOC-1 complex are governed by the dosage balance

hypothesis……………………………………………………………………………………………..…144

Figure 1. Flowchart of Possible BLOC-1 Remnants Predicted

from the Dosage Balance Hypothesis…………………………....146

Figure 2. Cellular processes involving BLOC-1 are largely

governed by different subunits……………………………………..150

Figure 3. Potential steps of NSF and BLOC-1 involvement

and interaction during fast vesicle cycling at the

plasma membrane……………………………………………………….153

Hypothesis 2: Genotype-to-phenotype correlations observed in a trait

following a gene pair analysis can better, although not precisely, predict

how other traits may respond………………………………………………………………….…154

Hypothesis 3: Polypeptides that are associated with a disease, form

a biochemical network, and are all sensitive to genetic perturbation

of a common network constituent, converge in a defined functional

pathway where endophenotypes can be assessed………………………………………...156

Chapter V. References

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience
Parola chiave	Drosophila Neuromuscular Junction gene dosage Synaptic Plasticity Schizophrenia dysbindin BLOC-1
Committee Chair / Thesis Advisor	Faundez, Victor, Emory University
Committee Members	Li, Lian, Emory University Caspary, Tamara, Emory University Smith, Yoland, Emory University Kowalczyk, Andrew, Emory University

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