The proteome of the presynaptic active zone from mouse brain

Weingarten, Jens; Laßek, Melanie; Mueller, Benjamin F.; Rohmer, Marion; Lunger, Ilaria; Baeumlisberger, Dominic; Dudek, Simone; Gogesch, Patricia; Karas, Michael; Volknandt, Walter

doi:10.1016/j.mcn.2014.02.003

Cited by 49 publications

(68 citation statements)

References 75 publications

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“…Synapse-enriched fractions containing the pre-synaptic terminal and the apposing post-synaptic membrane (synaptoneurosomes) were prepared according to a modification of published protocols (Rao and Steward, 1991, An et al, 2008, Weingarten et al, 2014, Wilhelm et al, 2014) from Wt cerebral cortical neurons (DIV 15-17) treated during 60 seconds with 5 nM of tPA prepared on a solution containing HBSS, B27 and 10 mM of glucose, or with HBSS, B27 and glucose alone (herein referred to as vehicle-control). Cells were homogenized and centrifuged at 2000 x g for 5 min.…”

Section: 0 Experimental Proceduresmentioning

confidence: 99%

Tissue-type plasminogen activator induces synaptic vesicle endocytosis in cerebral cortical neurons

Yepes

Torre

et al. 2016

Neuroscience

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The release of the serine proteinase tissue-type plasminogen activator (tPA) from the presynaptic terminal of cerebral cortical neurons plays a central role in the development of synaptic plasticity, adaptation to metabolic stress and neuronal survival. Our earlier studies indicate that by inducing the recruitment of the cytoskeletal protein βII-spectrin and voltage-gated calcium channels to the active zone (AZ), tPA promotes Ca2+-dependent translocation of synaptic vesicles (SVs) to the synaptic release site where they release their load of neurotransmitters into the synaptic cleft. Here we used a combination of in vivo and in vitro experiments to investigate whether this effect leads to depletion of SVs in the presynaptic terminal. Our data indicate that tPA promotes SVs endocytosis via a mechanism that does not require the conversion of plasminogen into plasmin. Instead, we show that tPA induces calcineurin (CaN) - mediated dynamin I dephosphorylation, which is followed by dynamin I-induced recruitment of the actin binding protein profilin II to the presynaptic membrane, and profilin II-induced F-actin formation. We report that this tPA-induced sequence of events leads to the association of newly formed SVs with F-actin clusters in the endocytic zone. In summary, the data presented here indicate that following the exocytotic release of neurotransmitters tPA activates the mechanism whereby SVs are retrieved from the presynaptic membrane and endocytosed to replenish the pool of vesicles available for a new cycle of exocytosis. Together, these results indicate that in cerebral cortical neurons tPA plays a central role coupling SVs exocytosis and endocytosis.

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Section: 0 Experimental Proceduresmentioning

confidence: 99%

Tissue-type plasminogen activator induces synaptic vesicle endocytosis in cerebral cortical neurons

Yepes

Torre

et al. 2016

Neuroscience

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“…Synaptic vesicles were isolated from synaptosomes according to the protocol guidelines of Whittaker [16]. The protocol has previously been adapted to the fractionation of individual mouse brains [5] and now downscaled for individual mouse brain regions (downscaling II). The following modifications were applied: individual brain regions (olfactory bulb, hippocampus, and cerebellum) were homogenized each in 0.4 mL of preparation buffer (5 mM Tris-HCl, 320 mM sucrose, pH 7.4) containing the protease inhibitors antipain, leupeptin, chymostatin (2 µg/mL each), pepstatin (1 µg/mL) and benzamidine (1 mM).…”

Section: Methodsmentioning

confidence: 99%

“…In contrast to the complex dynamics of postsynaptic densities [3,4], the dynamic composition of the PAZs is less well characterized, but now arouses increasing interest as a molecular platform of presynaptic plasticity (reviewed in [1]). The structural and functional dynamics at synaptic contacts in the adult CNS are reflected by presynaptic rearrangements of the proteinaceous inventory [5]. The detailed understanding of regional specializations requires the analysis of the proteomes of the presynaptic active zone (PAZ) from specific brain regions.…”

Section: Introductionmentioning

confidence: 99%

“…The detailed understanding of regional specializations requires the analysis of the proteomes of the presynaptic active zone (PAZ) from specific brain regions. Based on our experimental expertise for immopurifying the PAZ from rat [6,7] and mouse brain [5,8], we developed a novel experimental approach for the purification of subregion-specific PAZ proteomes. By combining stringent subcellular fractionation with subsequent immunopurification, we obtain a highly purified native PAZ proteome.…”

Section: Introductionmentioning

confidence: 99%

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Regional Specializations of the PAZ Proteomes Derived from Mouse Hippocampus, Olfactory Bulb and Cerebellum

et al. 2015

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Neurotransmitter release as well as structural and functional dynamics at the presynaptic active zone (PAZ) comprising synaptic vesicles attached to the presynaptic plasma membrane are mediated and controlled by its proteinaceous components. Here we describe a novel experimental design to immunopurify the native PAZ-complex from individual mouse brain regions such as olfactory bulb, hippocampus, and cerebellum with high purity that is essential for comparing their proteome composition. Interestingly, quantitative immunodetection demonstrates significant differences in the abundance of prominent calcium-dependent PAZ constituents. Furthermore, we characterized the proteomes of the immunoisolated PAZ derived from the three brain regions by mass spectrometry. The proteomes of the release sites from the respective regions exhibited remarkable differences in the abundance of a large variety of PAZ constituents involved in various functional aspects of the release sites such as calcium homeostasis, synaptic plasticity and neurogenesis. On the one hand, our data support an identical core architecture of the PAZ for all brain regions and, on the other hand, demonstrate that the proteinaceous composition of their presynaptic active zones vary, suggesting that changes in abundance of individual proteins strengthen the ability of the release sites to adapt to specific functional requirements.

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“…500 (190,192,193,43) question. Novel approaches will circumvent this problem at least in part (see below).…”

Section: Figmentioning

confidence: 99%

Proteomics of the Synapse – A Quantitative Approach to Neuronal Plasticity

Dieterich

Kreutz

2016

Molecular & Cellular Proteomics

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The advances in mass spectrometry based proteomics in the past 15 years have contributed to a deeper appreciation of protein networks and the composition of functional synaptic protein complexes. However, research on protein dynamics underlying core mechanisms of synaptic plasticity in brain lag far behind. In this review, we provide a synopsis on proteomic research addressing various aspects of synaptic function. We discuss the major topics in the study of protein dynamics of the chemical synapse and the limitations of current methodology. We highlight recent developments and the future importance of multidimensional proteomics and metabolic labeling. Finally, emphasis is given on the conceptual framework of modern proteomics and its current shortcomings in the quest to gain a deeper understanding of synaptic plasticity. Molecular & Cellular Proteomics

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The proteome of the presynaptic active zone from mouse brain

Cited by 49 publications

References 75 publications

Tissue-type plasminogen activator induces synaptic vesicle endocytosis in cerebral cortical neurons

Tissue-type plasminogen activator induces synaptic vesicle endocytosis in cerebral cortical neurons

Regional Specializations of the PAZ Proteomes Derived from Mouse Hippocampus, Olfactory Bulb and Cerebellum

Proteomics of the Synapse – A Quantitative Approach to Neuronal Plasticity

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