CRISPR-TRAPSeq identifies the QKI RNA binding protein as important for astrocytic maturation and control of thalamocortical synapses
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE147121 | GEO | 2021/01/21
REPOSITORIES: GEO
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Project description:Quaking RNA binding protein(QKI) is essential for oligodendrocyte development as myelination requires MBP mRNA regulation and localization to distal processes by its cytoplasmic isoforms(e.g. QKI-6). QKI-6 is also highly expressed in astrocytes, which we and others recently demonstrated have regulated mRNA localization. Here, we show via CLIPseq that QKI-6 binds 3’UTRs of a subset of astrocytic mRNAs, including many enriched in peripheral processes. Binding is enriched near stop codons, which is mediated partially by QKI binding motifs(QBMs) yet spreads to adjacent sequences. We developed CRISPR TRAPseq: a viral approach for mosaic, cell-type specific gene mutation with simultaneous translational profiling. This enabled study of QKI-6 CLIP targets in QKI-deleted astrocytes in an otherwise normal brain. Astrocyte-targeted QKI deletion altered translation and maturation, while also increasing synaptic density within the astrocyte's territory. Overall, our data indicate QKI is required for astrocyte maturation and demonstrate an approach for a highly targeted translational assessment of gene knockout in specific cell-types in vivo.
2021-01-21 | GSE146935 | GEO
Project description:We immunoprecipitated QK-6 from P21 mouse brains to identify which mRNAs QK-6 binds to in vivo.
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Project description:CRISPR-TRAPSeq identifies the QKI RNA binding protein as important for astrocytic maturation and control of thalamocortical synapses
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Project description:CRISPR-TRAPSeq identifies the QKI RNA binding protein as important for astrocytic maturation and control of thalamocortical synapses (CLIP-Seq dataset)
| PRJNA613063 | ENA
Project description:CRISPR-TRAPSeq identifies the QKI RNA binding protein as important for astrocytic maturation and control of thalamocortical synapses (TRAP-Seq dataset)
| PRJNA612470 | ENA
Project description:We have previously shown that the physiological size of postsynaptic currents maximises energy efficiency rather than information transfer across the retinothalamic relay synapse. Here, we investigate information transmission and postsynaptic energy use at the next synapse along the visual pathway: from relay neurons in the thalamus to spiny stellate cells in layer 4 of the primary visual cortex (L4SS). Using both multicompartment Hodgkin-Huxley-type simulations and electrophysiological recordings in rodent brain slices, we find that increasing or decreasing the postsynaptic conductance of the set of thalamocortical inputs to one L4SS cell decreases the energy efficiency of information transmission from a single thalamocortical input. This result is obtained in the presence of random background input to the L4SS cell from excitatory and inhibitory corticocortical connections, which were simulated (both excitatory and inhibitory) or injected experimentally using dynamic-clamp (excitatory only). Thus, energy efficiency is not a unique property of strong relay synapses: even at the relatively weak thalamocortical synapse, each of which contributes minimally to the output firing of the L4SS cell, evolutionarily-selected postsynaptic properties appear to maximise the information transmitted per energy used.
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Project description:Although it is considered to be the most complex organ in the body, the brain can be broadly classified into two major types of cells, neuronal cells and glial cells. Glia is a general term that encompasses multiple types of non-neuronal cells that function to maintain homeostasis, form myelin, and provide support and protection for neurons. Astrocytes, a major class of glial cell, have historically been viewed as passive support cells, but recently it has been discovered that astrocytes participate in signalling activities both with the vasculature and with neurons at the synapse. These cells have been shown to release D-serine, TNF-?, glutamate, atrial natriuretic peptide (ANP) and ATP among other signalling molecules. ATP and its metabolites are well established as important signalling molecules, and astrocytes represent a major source of ATP release in the nervous system. Novel molecular and genetic tools have recently shown that astrocytic release of ATP and other signalling molecules has a major impact on synaptic transmission. Via actions at the synapse, astrocytes have now been shown to regulate complex network signalling in the whole organism with impacts on respiration and the sleep-wake cycle. In addition, new roles for astrocytes are being uncovered in psychiatric disorders, and astrocyte signalling mechanisms represents an attractive target for novel therapeutic agents.
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Project description:Astrocytic Ca2+ signals can be fast and local, supporting the idea that astrocytes have the ability to regulate single synapses. However, the anatomical basis of such specific signaling remains unclear, owing to difficulties in resolving the spongiform domain of astrocytes where most tripartite synapses are located. Using 3D-STED microscopy in living organotypic brain slices, we imaged the spongiform domain of astrocytes and observed a reticular meshwork of nodes and shafts that often formed loop-like structures. These anatomical features were also observed in acute hippocampal slices and in barrel cortex in vivo. The majority of dendritic spines were contacted by nodes and their sizes were correlated. FRAP experiments and Ca2+ imaging showed that nodes were biochemical compartments and Ca2+ microdomains. Mapping astrocytic Ca2+ signals onto STED images of nodes and dendritic spines showed they were associated with individual synapses. Here, we report on the nanoscale organization of astrocytes, identifying nodes as a functional astrocytic component of tripartite synapses that may enable synapse-specific communication between neurons and astrocytes.
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Project description:Proper establishment of synapses is critical for constructing functional circuits. Interactions between presynaptic neurexins and postsynaptic neuroligins coordinate the formation of synaptic adhesions. An isoform code determines the direct interactions of neurexins and neuroligins across the synapse. However, whether extracellular linker proteins can expand such a code is unknown. Using a combination of in vitro and in vivo approaches, we found that hevin, an astrocyte-secreted synaptogenic protein, assembles glutamatergic synapses by bridging neurexin-1alpha and neuroligin-1B, two isoforms that do not interact with each other. Bridging of neurexin-1alpha and neuroligin-1B via hevin is critical for the formation and plasticity of thalamocortical connections in the developing visual cortex. These results show that astrocytes promote the formation of synapses by modulating neurexin/neuroligin adhesions through hevin secretion. Our findings also provide an important mechanistic insight into how mutations in these genes may lead to circuit dysfunction in diseases such as autism.
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Project description:BackgroundThe quaking viable (qk(v)) mice have uncompacted myelin in their central and peripheral nervous system (CNS, PNS). The qk gene encodes 3 major alternatively spliced isoforms that contain unique sequence at their C-terminus dictating their cellular localization. QKI-5 is a nuclear isoform, whereas QKI-6 and QKI-7 are cytoplasmic isoforms. The qk(v) mice harbor an enhancer/promoter deletion that prevents the expression of isoforms QKI-6 and QKI-7 in myelinating cells resulting in a dysmyelination phenotype. It was shown that QKI regulates the differentiation of oligodendrocytes, the myelinating cells of the CNS, however, little is known about the role of the QKI proteins, or RNA binding proteins in PNS myelination.Methodology/principal findingsTo define the role of the QKI proteins in PNS myelination, we ectopically expressed QKI-6 and QKI-7 in primary rat Schwann cell/neuron from dorsal root ganglia cocultures. We show that the QKI isoforms blocked proliferation and promoted Schwann cell differentiation and myelination. In addition, these events were coordinated with elevated proteins levels of p27(KIP1) and myelin basic protein (MBP), markers of Schwann cell differentiation. QKI-6 and QKI-7 expressing co-cultures contained myelinated fibers that had directionality and contained significantly thicker myelin, as assessed by electron microscopy. Moreover, QKI-deficient Schwann cells had reduced levels of MBP, p27(KIP1) and Krox-20 mRNAs, as assessed by quantitative RT-PCR.Conclusions/significanceOur findings suggest that the QKI-6 and QKI-7 RNA binding proteins are positive regulators of PNS myelination and show that the QKI RNA binding proteins play a key role in Schwann cell differentiation and myelination.
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