Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting

Biesemann, Christoph; Grønborg, Mads; Luquet, Elisa; Wichert, Sven P.; Bernard, Véronique; Bungers, Simon R.; Cooper, Benjamin H.; Varoqueaux, Frédérique; Li, Liyi; Byrne, Jennifer A.; Urlaub, Henning; Jahn, Olaf; Brose, Nils; Herzog, Étienne

doi:10.1002/embj.201386120

Cited by 135 publications

(183 citation statements)

References 72 publications

(105 reference statements)

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“…Nevertheless, the role of these proteins in synaptic function has not been studied. The development of new techniques improving the isolation of synaptosomes will contribute to a more precise study of their protein content and the newly synthesized proteins in response to different neuronal stimulus (30). Altogether, these findings support the notion that local protein synthesis has a role in synaptic plasticity, but how is this local translation regulated in response to synaptic activity?…”

Section: Local Protein Synthesismentioning

confidence: 53%

The Regulation of Synaptic Protein Turnover

Álvarez-Castelao

2015

Journal of Biological Chemistry

100

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Emerging evidence indicates that protein synthesis and degradation are necessary for the remodeling of synapses. These two processes govern cellular protein turnover, are tightly regulated, and are modulated by neuronal activity in time and space. The anisotropic anatomy of the neurons presents a challenge for the study of protein turnover, but the understanding of protein turnover in neurons and its modulation in response to activity can help us to unravel the fine-tuned changes that occur at synapses in response to activity. Here we review the key experimental evidence demonstrating the role of protein synthesis and degradation in synaptic plasticity, as well as the turnover rates of specific neuronal proteins.The human brain is composed of a trillion neurons with complex and intricate arbors, which are interconnected by synapses (1). Synapses are highly dynamic in number and shape as a consequence of the continuous remodeling of neural circuits. A change in synaptic transmission elicited by neural activity is collectively called "synaptic plasticity," and learning and memory rely, at least in part, on this process. Synapses are made up of proteins, including receptors for neurotransmitters, scaffolding molecules, and signaling molecules. All proteins have a finite lifetime: they are synthesized and degraded continuously to maintain cellular function and viability. This continuous process is called "protein turnover." However, why is protein turnover important for cells? Even when the cells are in a basal state, the protein pool is dynamic and the coordination between protein synthesis and degradation maintains a steady-state level of proteins that is constantly renewed (2). Protein turnover is not only important to maintain protein concentrations in the cell, but also to allow for changes. Modulation of the proteome is necessary for most cellular responses, involving modifications in general or specific protein turnover (3,4). Neurons also have the ability to modulate and adjust their proteome in response to specific cues, for example, synaptic remodeling in response to patterns of action potentials in neurons.One complication of protein turnover in neurons is that synapses can be located up to hundreds of microns from the cell body. Where are proteins synthesized and degraded? Are proteins transported over the long distance from the soma to dendrites or axons, and if so, how do they reach their specific location within the intricate axonal and dendritic arbors. In fact, to overcome these challenges, neurons have very effective transport mechanisms to deliver proteins to remote regions of axons or dendrites; this has been recently reviewed in Refs. 5 and 6. Moreover, some proteins, such as receptors or scaffolding proteins, are in continuous movement in and out of the synapse with rapid rates (reviewed in Refs. 7 and 8). In addition to protein movement, neurons use local translation and degradation in dendrites and axons, allowing for a fine-tuned local protein turnover. Here we present an overview focused on...

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Section: Local Protein Synthesismentioning

confidence: 53%

The Regulation of Synaptic Protein Turnover

Álvarez-Castelao

2015

Journal of Biological Chemistry

100

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“…Die Fraktionen enthielten Glutamatrezeptoren, eine Vielzahl an heteromeren G-Protein -Untereinheiten und kleine GTPasen, die mit perisynaptischen Funktionen in Verbindung stehen, während gängige synaptosomale Proteine (wie die präsynaptischen SNA-RE -Proteine und die postsynaptischen Proteine NR1 und PSD-95) in Gliosomefraktionen abgereichert waren. Durch die Kombination mit fluoreszenzaktivierter Sortierung, wie kürzlich durchgeführt für VGLUT1-VENUS-markierte Synaptosomen [ 2 ], könnten damit spezialisierte as-…”

Section: Proteomische Ansätze Zur Enträtseln Glialer Heterogenitätunclassified

Entschlüsselung glialer Funktionen mittels OMICs-Technologien

Dieterich

Rossner²

2015

E-Neuroforum

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ZusammenfassungSowohl neuronale als auch gliale Zellen tragen zu höheren Hirnfunktionen bei. Viele Beobachtungen zeigen, dass Neurone und Gliazellen nicht nur physische Berührungspunkte besitzen, sondern auch physiologisch eng verbunden sind und auf vielen Ebenen wechselseitig voneinander abhängen. Zudem stellen Macroglia wie Astrozyten, NG2 - Zellen und Oligodendrocyten keine homogenen Zellpopulationen dar, sondern besitzen, ahnlich wie Neurone, merkliche Unterschiede in verschiedenen Aspekten. Diese Vielzahl der Unterschiede in Morphologie, Funktionalität und zellularer Aktivität wurden jungst anerkannt und in ein generelles Konzept der Gehirnfunktion integriert, welches eine neurale, statt einer ausschließlich neuronalen Welt zeichnet. Mit den neusten Fortschritten der ‚OMICs‘ Technologie wird ein unvoreingenommener und explorativer Ansatz möglich, über welchen ein detailliertes Verständnis des glialen Heterogenitätsprofils in Reichweite gelangt. In diesem Artikel geben wir einen Überblick über aktuelle Transkriptomics-und Proteomicstechniken, die benutzt werden, um die gliale Heterogenität des Gehirns zu analysieren.

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“…While common synaptosomal proteins (such as the presynaptic SNARE proteins and postsynaptic proteins NR1 and PSD-95) were depleted in the gliosome fraction, the fraction contained glutamate receptors and a plethora of heteromeric Gprotein subunits and small GTPases related to perisynaptic function. In combination with fluorescence-activated sorting, such as recently done for VGLUT1-Venus-labeled synaptosomes [2], specialized astrocytic gliosomes could be investigated in unprecedented detail in the future. Another approach to decipher glial proteomic heterogeneity might employ bioorthogonal metabolic protein labeling (.…”

Section: Transcriptomic Approaches To Unravel Glial Heterogeneitymentioning

confidence: 99%

Dissecting the regional diversity of glial cells by applying -omic technologies

Dieterich

Rossner²

2015

E-Neuroforum

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With an estimated number of approximately 10,000 different proteins in each single mammalian cell [16], in-depth identification of a cell's entire proteome Dissecting the regional diversity of glial cells by applying -omic technologies Abstract Neuronal as well as glial cells contribute to higher order brain functions. Many observations show that neurons and glial cells are not only physically highly intermingled but are physiologically tightly connected and mutually depend at various levels on each other. Moreover, macroglia classes like astrocytes, NG2 cells and oligodendrocytes are not at all homogenous cell populations but do possess a markedly heterogeneity in various aspects similar to neurons. The diversity of differences in morphology, functionality and, cellular activity has been acknowledged recently and will be integrated into a concept of brain function that pictures a neural rather than a puristical neuronal world. With the recent progress in "omic" technologies, an unbiased and exploratory approach toward an enhanced understanding of glial heterogeneity has become possible. Here, we provide an overview on current technical transcriptomic and proteomic approaches used to dissect glial heterogeneity of the brain. is still a major challenge for modern proteomic technologies. In addition, the total number of different/unique proteins per synapse is estimated to be at approximately 2000-2500 [17], which are often under control of different activity-dependent turnover rates. While today's state-of-theart MS instruments routinely sequence single purified proteins with subfemtomolar sensitivity, the effective identification of low-abundance proteins is orders of magnitude lower in complex mixtures due to limited dynamic range and sequencing speed and due to the common, strong bias toward acquiring MS/MS data on higher abundance molecules. Hence, to tackle activity-dependent proteome alterations entire, functionally heterogeneous brain regions with different subtypes of neurons and macroglial cells are usually being sampled at the loss of cell-type specificity and spatial resolution. The characterization of a proteome is an even more difficult challenge if temporal and spatial aspects of a proteome or a subpopulation of the proteome have to be taken into consideration. Thus, the separation and enrichment of the subproteome in question is key for its successful characterization. To overcome the above mentioned limitations, several cellular and biochemical (subcellular) enrichment strategies combined with proteomics have been developed to increase sensitivity and selectivity for the analysis of neuronal and glial proteomes, and to finally dissolve cellular heterogeneity of neural cells in the brain (. Fig. 2a-b). KeywordsConcerning cellular selectivity, intracellular proteomes and secretomes from cultured primary astrocytes and astroglial cell lines have been characterized in detail by several labs as proteomic approaches as indicated above require a much larger quantity than transcriptomic approaches. Analys...

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Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting

Cited by 135 publications

References 72 publications

The Regulation of Synaptic Protein Turnover

The Regulation of Synaptic Protein Turnover

Entschlüsselung glialer Funktionen mittels OMICs-Technologien

Dissecting the regional diversity of glial cells by applying -omic technologies

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