Hermansky-Pudlak Complexes, AP-3 and BLOC-1, Regulate the MolecularArchitecture of the Synapse Pubblico

Abstract

Mechanisms that underlie pre-synaptic membrane composition are essential for proper neurotransmission. My thesis research examined how two endosomal sorting adaptors, AP-3 and BLOC-1, regulate synaptic vesicle biogenesis. Neurons express two AP-3 isoforms with distinct vesicle traffic functions: ubiquitous AP-3 in conjunction with BLOC-1 sorts membrane proteins (cargoes) to lysosomes, while neuronal AP-3 creates synaptic vesicles. However, recent proteomes of synaptic vesicles revealed the presence of AP-3-sorted lysosomal cargoes, leading me to hypothesize that these divergent endosomal sorting pathways coordinately regulate synaptic vesicle composition by sorting similar membrane proteins from a shared early endosome compartment. In support of this hypothesis, my dissertation demonstrates by both biochemical and immunomicroscopy techniques that synaptic vesicle and AP-3-sorted lysosomal cargoes co-localize in early endosomes and synaptic vesicles. Furthermore, AP-3 isoform-specific pathways competitively regulate synaptic vesicle content, with neuronal AP-3 deficiency decreasing and either ubiquitous AP-3 or BLOC-1 deficiencies increasing the content of similar cargoes in synaptic vesicle fractions. AP-3-dependent synaptic vesicle biogenesis mechanisms also contribute to brain region-specific differences in synaptic architecture through differential regulation of membrane protein content and synaptic vesicle size in pre-synaptic compartments of the striatum and dentate gyrus of the hippocampus. BLOC-1 selectively modifies AP-3-dependent traffic in the dentate gyrus, but not the striatum. Overall, my work leads me to propose the concept that rather than a unitary synaptic vesicle being produced by all neurons, as is currently believed, neurons assemble diverse pre-synaptic vesicles using lysosomal vesicle biogenesis mechanisms that contribute to pre-synaptic organelle biogenesis.

Table of Contents

Chapter I. General Introduction………………………….………………….1-42

Section 1.01 Summary………………………………………………………………….2

Section 1.02 Introduction to Vesicle-Mediated Transport……………..2-4

Section 1.03 Genetics of the AP-3 Pathway in Vertebrates…………….4-5

Section 1.04 Mutations Affecting Vertebrate AP-3 Subunits…………..5-6

Section 1.05 Regulation of AP-3 Function…………………………………..7-10

Section 1.06 The Elusive Role of Clathrin in AP-3 Vesiculation……10-12

Section 1.07 Not All AP-3s are Created Equal: Neuronal Versus Ubiquitous Adaptors…………………………………………………………12-14

Section 1.08 The Function(s) of AP-3 Complexes in Neuronal Cells…………………………………………………………………………………14-17

Section 1.09 Neuronal Functions for the ‘Non-Neuronal' AP-3 Complexes………………………………………………………………………..18-19

Section 1.10 Interactions between AP-3 and Other Hermansky-Pudlak Gene Products………………………………………………………...20-22

Section 1.11 The BLOC-1-AP-3 über-complex: A Possible Connection to Schizophrenia………………………………………………………22-24

Section 1.12 Conclusions and Perspectives……………………………….24-26

Section 1.13 Challenging Prevailing Paradigms: How this Dissertation Contributes to Our Understanding of Endosomal Sorting in Neurons…26-29

Section 1.14 Summary and General Overview……………………….…29-30

Section 1.15 Figures…………………………………………………………..…..31-42

(a) Figure 1: Adaptor-Mediated Vesicle Transport……………………..31

(b) Figure 2: Regulators of Vesicle Formation and Fusion……….…32

(c) Figure 3: Nomenclature and Structure of AP-3 subunit isoforms…………………………………………………………………………..33

(d) Figure 4: Subunit Composition and Intramolecular Interactions of the BLOC-1 Complex………………………………………………………34

(e) Figure 5: Molecular Interactions of the Adaptor Complex AP-3…………………………………………………………………………….35-36

(f) Figure 6: AP-3 Sorting Mechanisms in Neuronal Cells……..37-38

(g) Figure 7: Models of BLOC-1-AP-3 Sorting Functions………..39-40

(h) Figure 8: Intermolecular Interactions of the BLOC-1 Complex……………………………………………………………………………41

(i) Figure 9: Models of Endosomal Sorting in Neurons………………42

Chapter II. Roles of BLOC-1 and Adaptor Protein-3 Complexes in Cargo Sorting to Synaptic Vesicles………………..43-102

Section 2.01 Abstract…………………………………………………………………..44

Section 2.02 Introduction……………………………………………………….45-49

Section 2.03 Materials and Methods………………………………………..49-55

Section 2.04 Results………………………………………………………………..55-67

Section 2.05 Discussion…………………………………………………………..67-73

Section 2.06 Acknowledgements…………………………………………………..73

Section 2.07 Figures………………………………………………………………74-102

(a) Figure 1: AP-3 synaptic vesicle and lysosomal cargoes selectively colocalize in PC12 Cells………………………………………………….74-75

(b) Figure 2: Synaptic Vesicle and AP-3 lysosomal cargoes are present in rab5Q79L early endosomes…………………………………76

(c) Figure 3: Internalized EGF labels early endosomes positive for VAMP2 and AP-3 lysosomal cargoes……………………………….77-78

(d) Figure 4: Synaptic vesicle and lysosomal AP-3 cargoes colocalization in mouse primary neocortical neurons……..79-80

(e) Figure 5: Synaptic vesicles and synaptic-like microvesicles contain AP-3 lysosomal cargoes…………………………………….81-82

(f) Figure 6: AP-3 and BLOC-1 form a complex in PC12 cells and mouse primary neurons……………………………………………….83-84

(g) Figure 7: Ubiquitous and neuronal AP-3 isoforms regulate synaptic vesicle protein content…………………………………….85-86

(h) Figure 8: BLOC-1 selectively regulates synaptic vesicle levels of PI4KIIα and VAMP7-TI……………………………………………………..87

(i) Supplemental Figure 1: Characterization of VAMP7-TI Monoclonal Antibody……………………………………………………88-89

(j) Supplemental Figure 2: Synaptic Vesicle SNARE, VAMP2, Co-localizes with AP-3-Sorted Synaptic Vesicle and Lysosomal Proteins……90-91

(k) Supplemental Figure 3: Syntaxin 8 Colocalization with Synaptic Vesicle and Lysosomal SNAREs……………………….…………………92

(l) Supplemental Figure 4: Negligible Co-Localization of VAMP7-TI and VAMP2 with Golgi and Late Endosomal Markers………93-94

(m) Supplemental Figure 5: Quantification of Synaptic Vesicle and Lysosomal AP-3 Cargoes Co-Localization in Mouse Primary Neurons…95-96

(n) Supplemental Figure 6: Synaptic Vesicle Fractions from Mouse Brains do not Contain Late and Recycling Endosome Markers………..97

(o) Supplemental Figure 7: Glycerol Gradient Velocity Sedimentation of Synaptic Vesicle Fractions from AP-3-deficient Mouse Brains…98-99

(p) Supplemental Figure 8: Glycerol Gradient Velocity Sedimentation of Synaptic Vesicle Fractions from BLOC-1-deficient (muted) Mouse Brains. 100-101

(q) Supplemental Figure 9: AP-3 and BLOC-1 Deficiency Do Not Globally Affect Synaptic Vesicle and AP-3 Sorted Lysosomal Proteins in Mouse Brain..102

Chapter III. Hermansky-Pudlak Complexes, AP-3 and BLOC-1, Differentially Regulate Synaptic Composition in the Striatum and Hippocampus…………………………………………………………………….103-154

Section 3.01 Abstract…………………………………………………………………104

Section 3.02 Introduction…………………………………………………….105-107

Section 3.03 Materials and Methods……………………………………..108-114

Section 3.04 Results……………………………………………………………..115-125

Section 3.05 Discussion……………………………………………………....125-128

Section 3.06 Acknowledgements………………………………………………...129

Section 3.07 Figures………………………………………........................130-154

(a) Figure 1: AP-3 is expressed throughout the brain with prominent expression in the hippocampus and striatum……………130-131

(b) Figure 2: AP-3 localizes to synaptic terminals of the dentate gyrus………………………………………………………………………..132-133

(c) Figure 3: Sub-synaptic localization of AP-3 by immunoelectron microscopy……………………………………………………………….134-135

(d) Figure 4: The majority of AP-3 partially labels axons in the striatum and hippocampus…………………………………………………………….136-137

(e) Figure 5: AP-3 differentially regulates synaptic vesicle size in asymmetric excitatory synapses of the striatum and dentate gyrus…..138-139

(f) Figure 6: Quantification of synaptic vesicle size in asymmetric excitatory synapses from the striatum and dentate gyrus of Ap3d+/+ and Ap3dmh/mh mouse brain…140-141

(g) Figure 7: AP-3 and BLOC-1 form a complex in PC12 cells and synaptosome-enriched rat brain fractions…………………...142-143

(h) Figure 8: Deficiencies of AP-3 and BLOC-1 selectively reduce VAMP7-TI, but not synaptophysin, expression in the dentate gyrus……..144-146

(i) Figure 9: BLOC-1 deficiencies reduce AP-3 expression in the dentate gyrus…………………………………………………………….147-148

(j) Figure 10: BLOC-1 deficiency reduces axonal AP-3 in the dentate gyrus………………………………………………………………………..149-150

(k) Supplemental Figure 1: Schematic Representation of Identified Elements……………………...................................................151-152

(l) S. Figure 2: Mice deficient for ubiquitous AP-3 or BLOC-1 exhibit motor coordination defects……………………………..153-154

Chapter IV. Discussion………………………………………………………..155-181

Section 4.01 Synopsis of Findings and Their Significance……….156-157

Section 4.02 Traditional Conception of Endosomal Sorting in Neurons………………………………………………………………………..157-158

Section 4.03 Challenges to the Traditional Model…………………..158-159

Section 4.04 Experimental Evidence Demonstrates a Competitive Endosomal Sorting Mechanism for Synaptic Vesicle Regulation….159-165

Section 4.05 Contributions to a New Model of Endosomal Sorting in Neurons………………………………………………………………………..165-167

Section 4.06 Future Questions………………………………………………167-176

Section 4.07 Summary………………………………………………………….176-177

Section 4.08 Figures……………………………………………………..……..178-181

(a) Figure 1: Traditional Model of Endosomal Sorting in Neurons……………………………………………………………………178-179

(b) Figure 2: Novel Endosomal Mechanism for Synaptic Regulation in Neurons: The ‘See-Saw' Model………………………………………180

(c) Figure 3: Hypothetical BLOC-1-mediated Association of AP-3 and an Axonal Kinesin……………………………………………………..181

Chapter V. References………………………………………………………182-197

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience Biology, Molecular Biology, Cell
Parola chiave	AP-3 endosome synaptic vesicle lysosome BLOC-1 dentate gyrus
Committee Chair / Thesis Advisor	Faundez, Victor, Emory University
Committee Members	Kowalczyk, Andrew, Emory University Chen, Ping, Emory University Sale, Winfield S, Emory University Bassell, Gary, Emory University L'Hernault, Steven, Emory University

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