Regulation of Drosophila Synaptic Function and Plasticity by a Schizophrenia Susceptibility Network. Open Access
Mullin, Ariana Paone (2014)
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
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