Immunoisolation of two synaptic vesicle pools from synaptosomes: a proteomics analysis

Morciano, Marco; Burré, Jacqueline; Corvey, Carsten; Karas, Michael; Zimmermann, Herbert; Volknandt, Walter

doi:10.1111/j.1471-4159.2005.03506.x

Cited by 149 publications

(207 citation statements)

References 79 publications

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“…Consistent with previous results of Morciano et al (31), Western blot analysis showed that the synaptic vesicle markers synaptogyrin-3, synaptophysin, SV2, and VMAT 2 were distributed throughout the entire gradient (Fig. 5A).…”

Section: Cytosolic Domains Of Vmatsupporting

confidence: 80%

A Biochemical and Functional Protein Complex Involving Dopamine Synthesis and Transport into Synaptic Vesicles

Cartier

Parra

Baust

et al. 2010

Journal of Biological Chemistry

115

103

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Synaptic transmission depends on neurotransmitter pools stored within vesicles that undergo regulated exocytosis. In the brain, the vesicular monoamine transporter-2 (VMAT 2 ) is responsible for the loading of dopamine (DA) and other monoamines into synaptic vesicles. Prior to storage within vesicles, DA synthesis occurs at the synaptic terminal in a two-step enzymatic process. First, the rate-limiting enzyme tyrosine hydroxylase (TH) converts tyrosine to di-OH-phenylalanine. Aromatic amino acid decarboxylase (AADC) then converts di-OH-phenylalanine into DA. Here, we provide evidence that VMAT 2 physically and functionally interacts with the enzymes responsible for DA synthesis. In rat striata, TH and AADC co-immunoprecipitate with VMAT 2 , whereas in PC 12 cells, TH co-immunoprecipitates with the closely related VMAT 1 and with overexpressed VMAT 2 . GST pull-down assays further identified three cytosolic domains of VMAT 2 involved in the interaction with TH and AADC. Furthermore, in vitro binding assays demonstrated that TH directly interacts with VMAT 2 . Additionally, using fractionation and immunoisolation approaches, we demonstrate that TH and AADC associate with VMAT 2 -containing synaptic vesicles from rat brain. These vesicles exhibited specific TH activity. Finally, the coupling between synthesis and transport of DA into vesicles was impaired in the presence of fragments involved in the VMAT 2 /TH/AADC interaction. Taken together, our results indicate that DA synthesis can occur at the synaptic vesicle membrane, where it is physically and functionally coupled to VMAT 2 -mediated transport into vesicles.Monoamines, including dopamine (DA), 3 norepinephrine (NE), and serotonin (5-HT), are neurotransmitters that play major roles in a variety of brain functions, including emotion, reward, cognition, memory, attention, locomotion, and stress control (1-6). In neurons and neuroendocrine cells, monoamines are stored in large dense core vesicles (LDCVs) and small synaptic vesicles (SVs) (7-11) that undergo regulated exocytosis through a complex network of protein-protein interactions (12). Loading of monoamines into LDCVs and SVs of neurons and neuroendocrine cells is mediated by two vesicular monoamine transporter isoforms: VMAT 1 (13) and VMAT 2 (14). These transporters contain 12 putative transmembrane domains with both the N and C termini facing the cytosolic side of the vesicle membrane. VMAT 1 is mostly present in LDCVs of neuroendocrine cells, including chromaffin and PC12 cells, whereas VMAT 2 is primarily expressed by monoaminergic neurons of the central nervous system (15). In midbrain DA neurons, VMAT 2 is sorted to LDCVs and SVs in axon terminals and to LDCVs and tubulo-vesicular structures in the somatodendritic compartment (7)(8)(9)(10)(11)15).It is generally accepted that VMAT 2 transports DA that has been previously synthesized in the cytosolic compartment of the presynaptic terminal (16). DA synthesis requires two enzymatic reactions. First, tyrosine hydroxylase (TH) converts tyrosine into DOP...

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Section: Cytosolic Domains Of Vmatsupporting

confidence: 80%

A Biochemical and Functional Protein Complex Involving Dopamine Synthesis and Transport into Synaptic Vesicles

Cartier

Parra

Baust

et al. 2010

Journal of Biological Chemistry

115

103

View full text Add to dashboard Cite

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“…Synaptosomes are small lipid vesicles that form from synapses and astrocytic process following mechanical disruption of brain tissue using a blender or homogenizer (Whittaker et al, 1963). Synaptosomes may contain pre-and postsynaptic elements; this is a potentially confounding problem, as many of the proteins that facilitate receptor localization, anchoring, and signaling may be present in both compartments (Carlin et al, 1980;Morciano et al, 2005). One recent study has made strides to address this problem.…”

Section: Biochemical Enrichment Of Postsynaptic Densitiesmentioning

confidence: 99%

Postmortem Brain: An Underutilized Substrate for Studying Severe Mental Illness

McCullumsmith

Hammond

Shan

et al. 2013

Neuropsychopharmacol

107

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We propose that postmortem tissue is an underutilized substrate that may be used to translate genetic and/or preclinical studies, particularly for neuropsychiatric illnesses with complex etiologies. Postmortem brain tissues from subjects with schizophrenia have been extensively studied, and thus serve as a useful vehicle for illustrating the challenges associated with this biological substrate. Schizophrenia is likely caused by a combination of genetic risk and environmental factors that combine to create a disease phenotype that is typically not apparent until late adolescence. The complexity of this illness creates challenges for hypothesis testing aimed at understanding the pathophysiology of the illness, as postmortem brain tissues collected from individuals with schizophrenia reflect neuroplastic changes from a lifetime of severe mental illness, as well as treatment with antipsychotic medications. While there are significant challenges with studying postmortem brain, such as the postmortem interval, it confers a translational element that is difficult to recapitulate in animal models. On the other hand, data derived from animal models typically provide specific mechanistic and behavioral measures that cannot be generated using human subjects. Convergence of these two approaches has led to important insights for understanding molecular deficits and their causes in this illness. In this review, we discuss the problem of schizophrenia, review the common challenges related to postmortem studies, discuss the application of biochemical approaches to this substrate, and present examples of postmortem schizophrenia studies that illustrate the role of the postmortem approach for generating important new leads for understanding the pathophysiology of severe mental illness.

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“…Although synaptic vesicles are well characterized trafficking organelles (3)(4)(5)(6)(7), to date, few attempts to purify and characterize axonally transported vesicular compartments at the morphological and biochemical level have been carried out. These include dense-core granulated vesicles transporting the presynaptic active zone components Piccolo and Bassoon to nascent synapses (8) and retrograde signaling endosomes (9 -11).…”

mentioning

confidence: 99%

Sunday Driver Interacts with Two Distinct Classes of Axonal Organelles

Abe

Almenar-Queralt

Lillo

et al. 2009

Journal of Biological Chemistry

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The extreme polarized morphology of neurons poses a challenging problem for intracellular trafficking pathways. The distant synaptic terminals must communicate via axonal transport with the cell soma for neuronal survival, function, and repair. Multiple classes of organelles transported along axons may establish and maintain the polarized morphology of neurons, as well as control signaling and neuronal responses to extracellular cues such as neurotrophic or stress factors. We reported previously that the motorbinding protein Sunday Driver (syd), also known as JIP3 or JSAP1, links vesicular axonal transport to injury signaling. To better understand syd function in axonal transport and in the response of neurons to injury, we developed a purification strategy based on anti-syd antibodies conjugated to magnetic beads to identify sydassociated axonal vesicles. Electron microscopy analyses revealed two classes of syd-associated vesicles of distinct morphology. To identify the molecular anatomy of syd vesicles, we determined their protein composition by mass spectrometry. Gene Ontology analyses of each vesicle protein content revealed their unique identity and indicated that one class of syd vesicles belongs to the endocytic pathway, whereas another may belong to an anterogradely transported vesicle pool. To validate these findings, we examined the transport and localization of components of syd vesicles within axons of mouse sciatic nerve. Together, our results lead us to propose that endocytic syd vesicles function in part to carry injury signals back to the cell body, whereas anterograde syd vesicles may play a role in axonal outgrowth and guidance.

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Immunoisolation of two synaptic vesicle pools from synaptosomes: a proteomics analysis

Cited by 149 publications

References 79 publications

A Biochemical and Functional Protein Complex Involving Dopamine Synthesis and Transport into Synaptic Vesicles

A Biochemical and Functional Protein Complex Involving Dopamine Synthesis and Transport into Synaptic Vesicles

Postmortem Brain: An Underutilized Substrate for Studying Severe Mental Illness

Sunday Driver Interacts with Two Distinct Classes of Axonal Organelles

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