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Translational profiling of hypothalamic and midbrain neurons that project to the nucleus accumbens.

ABSTRACT: Neuroanatomical methods enable high-resolution mapping of neural circuitry, but do not allow systematic molecular profiling of neurons based on their connectivity. Here, we report the development of a novel approach for molecularly profiling projective neurons. We show that ribosomes can be labeled with a camelid nanobody raised against GFP and that this system can be engineered to selectively capture translating mRNAs from cells expressing GFP. We generated a transgenic mouse encoding a nanobody-ribosomal protein fusion (Syn-NBL10) and used a retrograde virus (CAV) encoding GFP to immunoprecipitate ribosomes from projection neurons. This enabled us to profile neurons projecting to the nucleus accumbens. The current method provides a new means for profiling neurons based on their projections.

ORGANISM(S): Mus musculus

PROVIDER: GSE55800 | GEO | 2014/05/22

SECONDARY ACCESSION(S): PRJNA241023

REPOSITORIES: GEO

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Translational profiling of hypothalamic and midbrain neurons that project to the nucleus accumbens.

Project description:Neuroanatomical methods enable high-resolution mapping of neural circuitry, but do not allow systematic molecular profiling of neurons based on their connectivity. Here, we report the development of a novel approach for molecularly profiling projective neurons. We show that ribosomes can be labeled with a camelid nanobody raised against GFP and that this system can be engineered to selectively capture translating mRNAs from cells expressing GFP. We generated a transgenic mouse encoding a nanobody-ribosomal protein fusion (Syn-NBL10) and used a retrograde virus (CAV) encoding GFP to immunoprecipitate ribosomes from projection neurons. This enabled us to profile neurons projecting to the nucleus accumbens. The current method provides a new means for profiling neurons based on their projections. Translating mRNAs immunoprecipitated from neurons projecting to the nucleus accumbens. Each Input and IP sample corrspond to a pooled group of 6 mice.

2014-05-22 | E-GEOD-55800 | biostudies-arrayexpress

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Identification of tissue-specific SPD-5 and PLK-1 proximity interactors by direct and indirect TurboID

Project description:In this set of proof of principle experiments we compare the proximity interactomes obtained for two C. elegans centrosomal proteins, SPD-5 and PLK-1, by direct TurboID and indirect, GFP nanobody-targeted, TurboID in a range of different tissues, embryos, germ cell precursors and ciliated neurons.

2022-04-01 | PXD031970 | Pride

Antibody RING-Mediated Destruction (ARMeD) of endogenous proteins

Project description:To understand the cellular function of a gene the encoding DNA or mRNA transcript can be manipulated and the consequences observed. However, these approaches do not have a direct effect on the protein product of the gene, which is either permanently abrogated or depleted at a rate defined by the half-life of the protein. Therefore, complete gene deletions and RNA interference of proteins with a very long half-life risk obscuring the direct consequences via compensatory or secondary cellular responses. We therefore sought to develop a single-component system that could induce the rapid degradation of the specific endogenous protein itself. A genetically introduced inducible construct of the RING domain of the ubiquitin E3 ligase RNF4 with a protein-specific camelid nanobody mediates destruction of the target by the ubiquitin proteasome system, a process we describe as Antibody RING-Mediated Destruction (ARMeD). The technique is acutely specific as we observed no off-target protein destruction. Furthermore, bacterially produced nanobody-RING fusion proteins electroporated into cells induce degradation of target within minutes. With the increasing availability of protein-specific nanobodies this method will allow the rapid and specific degradation of a wide range of endogenous proteins.

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Project description:We performed a BacTRAP-based ribosome profiling of PNOC-expressing cells in the hypothalamus of mice. Therefore, we employed mice, which express a fusion protein of the ribosomal L10a protein with EGFP (L10a-EGFP) under control of the PNOC promoter (Doyle et al., 2008). Precipitation of ribosomes of hypothalamic PNOC neurons with anti-GFP antibodies and subsequent mRNA sequencing of associated mRNAs allowed for assessment of an in-depth translational profile of these cells. Affinity purification of translating ribosomes was performed as described by (Heiman et al., 2014) with minor modifications.

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greenCUT&RUN: a rapid and efficient genome profiling approach for transcription and chromatin regulatory proteins

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Translation deficits drive C9orf72 associated poly-GR/PR toxicity

Project description:Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) patients with the C9orf72 mutation show predominantly cytoplasmic aggregates of poly-GR and poly-PR proteins that are acutely toxic in various model systems. To identify the molecular mediators of neurotoxicity of poly-GR/PR, we analyzed their interactomes in primary neurons. GFP-(GR)149 and (PR)175-GFP preferentially interacted with RNA-binding proteins, including stress granule-associated and nucleolar proteins, as well as ribosomes. Overexpression of the poly-GR/PR interactors Staufen 1/2 (STAU1/2) and YBX1 led to cytoplasmic aggregation of poly-GR/PR into large stress granule-like inclusions, while the poly-GR/PR interactor nucleophosmin (NPM1) recruited poly-GR into the nucleolus. In addition, poly-PR expression reduced ribosome levels and translation, which is consistent with the widespread reduction of synaptic proteins detected by proteomics. Surprisingly, only GFP-(GR)53, but not GFP-(GR)149, localized to the nucleolus and reduced ribosome levels and translation in neurons, suggesting impaired ribosome biogenesis is driving the acute toxicity commonly observed in vitro. In C9orf72 patient brains, we detected co-aggregation of poly-GR/PR inclusions with ribosomes, but not stress granules. Partial sequestration of ribosomes may chronically impair protein synthesis and contribute to C9orf72 ALS/FTD pathogenesis.

2018-06-27 | PXD008691 | Pride

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