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

Litwa, Karen (2009)

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


Abstract
Hermansky-Pudlak Complexes, AP-3 and BLOC-1, Regulate
the Molecular Architecture of the Synapse
By Karen A. (Newell) Litwa
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

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

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