Dataset Information

The effects of Sindbis viral vectors on neuronal function

ABSTRACT: Viral vectors are attractive tools to express genes in neurons. Transduction of neurons with a recombinant, replication-deficient Sindbis viral vector is a method of choice for studying the effects of short-term protein overexpression on neuronal function. However, to which extent Sindbis by itself may affect neurons is not fully understood. We assessed effects of neuronal transduction with a Sindbis viral vector on the transcriptome and proteome in organotypic hippocampal slice cultures, and analyzed the electrophysiological properties of individual CA1 neurons, at 24h and 72h after viral vector injection. Whereas Sindbis caused substantial gene expression alterations, changes at the protein level were less pronounced. Alterations in transcriptome and proteome were predominantly limited to proteins involved in mediating anti-viral innate immune responses. Sindbis transduction did not affect the electrophysiological properties of individual neurons: the membrane potential, excitability and synaptic currents were similar between transduced and nontransduced CA1 neurons up to 72h after Sindbis injection. We conclude that Sindbis viral vectors are suitable for studying short-term effects of a protein of interest on electrophysiological properties of neurons, but not for studies on the regulation of gene expression.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Sophie van der Spek

LAB HEAD: Ka wan Li

PROVIDER: PXD013634 | Pride | 2019-08-26

REPOSITORIES: Pride

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The Effects of Sindbis Viral Vectors on Neuronal Function.

Uyaniker Seçil S van der Spek Sophie J F SJF Reinders Niels R NR Xiong Hui H Li Ka Wan KW Bossers Koen K Smit August B AB Verhaagen Joost J Kessels Helmut W HW

Frontiers in cellular neuroscience 20190808

Viral vectors are attractive tools to express genes in neurons. Transduction of neurons with a recombinant, replication-deficient Sindbis viral vector is a method of choice for studying the effects of short-term protein overexpression on neuronal function. However, to which extent Sindbis by itself may affect neurons is not fully understood. We assessed effects of neuronal transduction with a Sindbis viral vector on the transcriptome and proteome in organotypic hippocampal slice cultures, and an ...[more]

PMID: 31440143

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Project description:Background Local translation at synapses is important for rapidly remodeling the synaptic proteome to sustain long-term plasticity and memory. While the regulatory mechanisms underlying memory-associated local translation have been widely elucidated in the postsynaptic/dendritic region, there is no direct evidence for which RNA-binding protein (RBP) in axons controls target-specific mRNA translation to promote long-term potentiation (LTP) and memory. We previously reported that translation controlled by cytoplasmic polyadenylation element binding protein 2 (CPEB2) is important for postsynaptic plasticity and memory. Here, we investigated whether CPEB2 regulates axonal translation to support presynaptic plasticity. Methods Behavioral and electrophysiological assessments were conducted in mice with pan neuron/glia- or AQ2glutamatergic neuron-specific knockout of CPEB2. Hippocampal Schaffer collateral (SC)-CA1 and temporoammonic (TA)-CA1 pathways were electro-recorded to monitor synaptic transmission and LTP evoked by 4 trains of high-frequency stimulation. RNA immunoprecipitation, coupled with bioinformatics analysis, were used to unveil CPEB2-binding axonal RNA candidates associated with learning, which were further validated by Western blotting and luciferase reporter assays. Adeno-associated viruses expressing Cre recombinase were stereotaxically delivered to the pre- or post-synaptic region of the TA circuit to ablate Cpeb2 for further electrophysiological investigation. Biochemically isolated synaptosomes and axotomized neurons cultured on a microfluidic platform were applied to measure axonal protein synthesis and FM4-64FX-loaded synaptic vesicles. Results Electrophysiological analysis of hippocampal CA1 neurons detected abnormal excitability and vesicle release probability in CPEB2-depleted SC and TA afferents, so we cross-compared the CPEB2-immunoprecipitated transcriptome with a learning-induced axonal translatome in the adult cortex to identify axonal targets possibly regulated by CPEB2. We validated that Slc17a6, encoding vesicular glutamate transporter 2 (VGLUT2), is translationally upregulated by CPEB2. Conditional knockout of CPEB2 in VGLUT2-expressing glutamatergic neurons impaired consolidation of hippocampus-dependent memory in mice. Presynaptic-specific ablation of Cpeb2 in VGLUT2-dominated TA afferents was sufficient to attenuate protein synthesis-dependent LTP. Moreover, blocking activity-induced axonal Slc17a6 translation by CPEB2 deficiency or cycloheximide diminished the releasable pool of VGLUT2-containing synaptic vesicles. Conclusions We identified 272 CPEB2-binding transcripts with altered axonal translation post-learning and established a causal link between CPEB2-driven axonal synthesis of VGLUT2 and presynaptic translation-dependent LTP. These findings extend our understanding of memory-related translational control mechanisms in the presynaptic compartment.

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