scholarly journals Mapping the Proteome of the Synaptic Cleft through Proximity Labeling Reveals New Cleft Proteins

Proteomes ◽  
2018 ◽  
Vol 6 (4) ◽  
pp. 48 ◽  
Author(s):  
Tony Cijsouw ◽  
Austin Ramsey ◽  
TuKiet Lam ◽  
Beatrice Carbone ◽  
Thomas Blanpied ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Functional Organization ◽  
Neuronal Cell ◽  
Super Resolution ◽  
Synaptic Cleft ◽  
Surface Proteins ◽  
Mammalian Brain ◽  
Label Free ◽  
Edge Zone ◽  
Cultured Cortical Neurons

Synapses are specialized neuronal cell-cell contacts that underlie network communication in the mammalian brain. Across neuronal populations and circuits, a diverse set of synapses is utilized, and they differ in their molecular composition to enable heterogenous connectivity patterns and functions. In addition to pre- and post-synaptic specializations, the synaptic cleft is now understood to be an integral compartment of synapses that contributes to their structural and functional organization. Aiming to map the cleft proteome, this study applied a peroxidase-mediated proximity labeling approach and used the excitatory synaptic cell adhesion protein SynCAM 1 fused to horseradish peroxidase (HRP) as a reporter in cultured cortical neurons. This reporter marked excitatory synapses as measured by confocal microcopy and was targeted to the edge zone of the synaptic cleft as determined using 3D dSTORM super-resolution imaging. Proximity labeling with a membrane-impermeant biotin-phenol compound restricted labeling to the cell surface, and Label-Free Quantitation (LFQ) mass spectrometry combined with ratiometric HRP tagging of membrane vs. synaptic surface proteins was used to identify the proteomic content of excitatory clefts. Novel cleft candidates were identified, and Receptor-type tyrosine-protein phosphatase zeta was selected and successfully validated. This study supports the robust applicability of peroxidase-mediated proximity labeling for synaptic cleft proteomics and its potential for understanding synapse heterogeneity in health and changes in diseases such as psychiatric disorders and addiction.

scholarly journals The schizophrenia susceptibility factor dysbindin and its associated complex sort cargoes from cell bodies to the synapse

2011 ◽  
Vol 22 (24) ◽  
pp. 4854-4867 ◽  
Author(s):  
Jennifer Larimore ◽  
Karine Tornieri ◽  
Pearl V. Ryder ◽  
Avanti Gokhale ◽  
Stephanie A. Zlatic ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Cortical Neurons ◽  
Neuronal Cells ◽  
Neuronal Cell ◽  
Adaptor Protein ◽  
Nerve Terminals ◽  
Endosome Trafficking ◽  
Neuronal Cell Bodies ◽  
Cultured Cortical Neurons ◽  
Lysosome Related Organelles

Dysbindin assembles into the biogenesis of lysosome-related organelles complex 1 (BLOC-1), which interacts with the adaptor protein complex 3 (AP-3), mediating a common endosome-trafficking route. Deficiencies in AP-3 and BLOC-1 affect synaptic vesicle composition. However, whether AP-3-BLOC-1–dependent sorting events that control synapse membrane protein content take place in cell bodies upstream of nerve terminals remains unknown. We tested this hypothesis by analyzing the targeting of phosphatidylinositol-4-kinase type II α (PI4KIIα), a membrane protein present in presynaptic and postsynaptic compartments. PI4KIIα copurified with BLOC-1 and AP-3 in neuronal cells. These interactions translated into a decreased PI4KIIα content in the dentate gyrus of dysbindin-null BLOC-1 deficiency and AP-3–null mice. Reduction of PI4KIIα in the dentate reflects a failure to traffic from the cell body. PI4KIIα was targeted to processes in wild-type primary cultured cortical neurons and PC12 cells but failed to reach neurites in cells lacking either AP-3 or BLOC-1. Similarly, disruption of an AP-3–sorting motif in PI4KIIα impaired its sorting into processes of PC12 and primary cultured cortical neuronal cells. Our findings indicate a novel vesicle transport mechanism requiring BLOC-1 and AP-3 complexes for cargo sorting from neuronal cell bodies to neurites and nerve terminals.

scholarly journals Protective effects of adenosine on apoptotic neuronal cell death in cultured cortical neurons

2000 ◽  
Vol 82 ◽  
pp. 182
Author(s):  
Yutaka Tamura ◽  
Taizo Fukui ◽  
Megumi Kajikawa ◽  
Mikiko Omoto ◽  
Hirohito Shiomi
Keyword(s):  
Cell Death ◽  
Cortical Neurons ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Protective Effects ◽  
Cultured Cortical Neurons

Neuroprotection by MAPK/ERK kinase inhibition with U0126 against oxidative stress in a mouse neuronal cell line and rat primary cultured cortical neurons

2000 ◽  
Vol 288 (2) ◽  
pp. 163-166 ◽  
Author(s):  
Takumi Satoh ◽  
Daisaku Nakatsuka ◽  
Yasuyoshi Watanabe ◽  
Izumi Nagata ◽  
Haruhiko Kikuchi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Line ◽  
Cortical Neurons ◽  
Neuronal Cell ◽  
Kinase Inhibition ◽  
Neuronal Cell Line ◽  
Cultured Cortical Neurons ◽  
Erk Kinase

scholarly journals Transgenic Silencing of Neurons in the Mammalian Brain by Expression of the Allatostatin Receptor (AlstR)

2009 ◽  
Vol 102 (4) ◽  
pp. 2554-2562 ◽  
Author(s):  
M. Wehr ◽  
U. Hostick ◽  
M. Kyweriga ◽  
A. Tan ◽  
A. P. Weible ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Cortical Neurons ◽  
Action Potentials ◽  
Neural Circuits ◽  
Hippocampal Slices ◽  
Neuronal Cell ◽  
Cell Types ◽  
Mammalian Brain ◽  
Specific Cell ◽  
Membrane Hyperpolarization

The mammalian brain is an enormously complex set of circuits composed of interconnected neuronal cell types. The analysis of central neural circuits will be greatly served by the ability to turn off specific neuronal cell types while recording from others in intact brains. Because drug delivery cannot be restricted to specific cell types, this can only be achieved by putting “silencer” transgenes under the control of neuron-specific promoters. Towards this end we have created a line of transgenic mice putting the Drosophila allatostatin (AL) neuropeptide receptor (AlstR) under the control of the tetO element, thus enabling its inducible expression when crossed to tet-transactivator lines. Mammals have no endogenous AL or AlstR, but activation of exogenously expressed AlstR in mammalian neurons leads to membrane hyperpolarization via endogenous G-protein-coupled inward rectifier K+ channels, making the neurons much less likely to fire action potentials. Here we show that this tetO/AlstR line is capable of broadly expressing AlstR mRNA in principal neurons throughout the forebrain when crossed to a commercially-available transactivator line. We electrophysiologically characterize this cross in hippocampal slices, demonstrating that bath application of AL leads to hyperpolarization of CA1 pyramidal neurons, making them refractory to the induction of action potentials by injected current. Finally, we demonstrate the ability of AL application to silence the sound-evoked spiking responses of auditory cortical neurons in intact brains of AlstR/tetO transgenic mice. When crossed to other transactivator lines expressing in defined neuronal cell types, this AlstR/tetO line should prove a very useful tool for the analysis of intact central neural circuits.

scholarly journals A role for c-Jun N-terminal kinase 1 (JNK1), but not JNK2, in the beta-amyloid-mediated stabilization of protein p53 and induction of the apoptotic cascade in cultured cortical neurons

2003 ◽  
Vol 371 (3) ◽  
pp. 789-798 ◽  
Author(s):  
Marie P. FOGARTY ◽  
Eric J. DOWNER ◽  
Veronica CAMPBELL
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Cortical Neurons ◽  
Caspase 3 ◽  
P53 Protein ◽  
Neuronal Cell ◽  
P53 Expression ◽  
Protein P53 ◽  
Cultured Cortical Neurons ◽  
Apoptotic Cascade

β-Amyloid (Aβ) peptide has been shown to induce neuronal apoptosis; however, the mechanisms underlying Aβ-induced neuronal cell death remain to be fully elucidated. The stress-activated protein kinase, c-Jun N-terminal kinase (JNK), is activated in response to cellular stress and has been identified as a proximal mediator of cell death. In the present study, expression of active JNK was increased in the nucleus and cytoplasm of Aβ-treated cells. Evaluation of the nature of the JNK isoforms activated by Aβ revealed a transient increase in JNK1 activity that reached its peak at 1 h and a later activation (at 24 h) of JNK2. The tumour suppressor protein, p53, is a substrate for JNK and can serve as a signalling molecule in apoptosis. In cultured cortical neurons, we found that Aβ increased p53 protein expression and phosphorylation of p53 at Ser15. Thus it appears that Aβ increases p53 expression via phosphorylation-mediated stabilization of the protein. Given the lack of availability of a JNK inhibitor that can distinguish between JNK1- and JNK2-mediated effects, we employed antisense technology to deplete cells of JNK1 or JNK2 selectively. Using this strategy, the respective roles of JNK1 and JNK2 on the Aβ-mediated activation of the apoptotic cascade (i.e. p53 stabilization, caspase 3 activation and DNA fragmentation) were examined. The results obtained demonstrate a role for JNK1 in the Aβ-induced stabilization of p53, activation of caspase 3 and DNA fragmentation. In contrast, depletion of JNK2 had no effect on the proclivity of Aβ to activate capase 3 or induce DNA fragmentation. These results demonstrate a significant role for JNK1 in Aβ-mediated induction of the apoptotic cascade in cultured cortical neurons.

scholarly journals The Bacterial Enzyme Cas13 Interferes with Neurite Outgrowth from Cultured Cortical Neurons

Toxins ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 262
Author(s):  
Qin-Wei Wu ◽  
Josef P. Kapfhammer
Keyword(s):  
Rna Editing ◽  
Cortical Neurons ◽  
High Efficiency ◽  
Primary Cultures ◽  
Cultured Neurons ◽  
Therapeutic Tool ◽  
Bacterial Enzyme ◽  
Rna Targeting ◽  
Cultured Cortical Neurons ◽  
New Generation

The CRISPR-Cas13 system based on a bacterial enzyme has been explored as a powerful new method for RNA manipulation. Due to the high efficiency and specificity of RNA editing/interference achieved by this system, it is currently being developed as a new therapeutic tool for the treatment of neurological and other diseases. However, the safety of this new generation of RNA therapies is still unclear. In this study, we constructed a vector expressing CRISPR-Cas13 under a constitutive neuron-specific promoter. CRISPR-Cas13 from Leptotrichia wadei was expressed in primary cultures of mouse cortical neurons. We found that the presence of CRISPR-Cas13 impedes the development of cultured neurons. These results show a neurotoxic action of Cas13 and call for more studies to test for and possibly mitigate the toxic effects of Cas13 enzymes in order to improve CRISPR-Cas13-based tools for RNA targeting.

scholarly journals Nanoscale chromatin imaging and analysis platform bridges 4D chromatin organization with molecular function

2021 ◽  
Vol 7 (1) ◽  
pp. eabe4310
Author(s):  
Yue Li ◽  
Adam Eshein ◽  
Ranya K.A. Virk ◽  
Aya Eid ◽  
Wenli Wu ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Super Resolution ◽  
Population Level ◽  
Average Diameter ◽  
Live Cells ◽  
Molecular Function ◽  
Label Free ◽  
Multiple Length Scales ◽  
Chromatin Packing ◽  
Analysis Platform

Extending across multiple length scales, dynamic chromatin structure is linked to transcription through the regulation of genome organization. However, no individual technique can fully elucidate this structure and its relation to molecular function at all length and time scales at both a single-cell level and a population level. Here, we present a multitechnique nanoscale chromatin imaging and analysis (nano-ChIA) platform that consolidates electron tomography of the primary chromatin fiber, optical super-resolution imaging of transcription processes, and label-free nano-sensing of chromatin packing and its dynamics in live cells. Using nano-ChIA, we observed that chromatin is localized into spatially separable packing domains, with an average diameter of around 200 nanometers, sub-megabase genomic size, and an internal fractal structure. The chromatin packing behavior of these domains exhibits a complex bidirectional relationship with active gene transcription. Furthermore, we found that properties of PDs are correlated among progenitor and progeny cells across cell division.

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