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

Litwa, Karen (2009)

Permanent URL: https://etd.library.emory.edu/concern/etds/xk81jk698?locale=en
Published

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

Rights statement
  • 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
Department
Degree
Submission
Language
  • English
Research field
Keyword
Committee Chair / Thesis Advisor
Committee Members
Last modified

Primary PDF

Supplemental Files