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Single cell RNA sequencing of P7 cortex from Top1 knockout mice

ABSTRACT: Single cell RNA sequencing of P7 cortex from Top1 knockout mice

PROVIDER: PRJNA611603 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11273532_1.fastq.gz	Fastqsanger.gz
	SRR11273532_2.fastq.gz	Fastqsanger.gz
	SRR11273533_1.fastq.gz	Fastqsanger.gz
	SRR11273533_2.fastq.gz	Fastqsanger.gz
	SRR11273534_1.fastq.gz	Fastqsanger.gz

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Single cell RNA sequencing of P7 cortex from Top1 knockout mice

Project description:Topoisomerase 1 (TOP1) relieves torsional stress in DNA during transcription and facilitates the expression of long (>100 kb) genes, many of which are important for neuronal functions. To evaluate how loss of Top1 affected neurons in vivo, we conditionally deleted (cKO) Top1 in postmitotic excitatory neurons in the mouse cerebral cortex and hippocampus. We found that Top1 cKO neurons developed properly but then showed biased transcriptional downregulation of long genes, signs of DNA damage, neuroinflammation, increased poly(ADP-ribose) polymerase-1 (PARP1) activity, single-cell somatic mutations, and ultimately degenerated. We used single-cell RNA-seq to verify cell-type decreases in long-gene expression as well as investigate how TOP1 loss may lead to premature neuronal death.

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Gene expression profiling of brain cortex pericytes from control and Rbpj-knockout mice at P7 and P10

Project description:Background: Pericytes are capillary-associated mural cells especially prominent in the central nervous system where they regulate vascular permeability and blood-brain barrier integrity. Despite their relevance for neurovascular unit homeostasis and their involvement in the pathobiology of neurodegenerative diseases, signalling mechanisms responsible for functional specialization and intercellular communication remain elusive. Objectives: The main goal of this study is to understand the relevance of Rbpj (the common downstream mediator of Notch signalling, an important mediator of cell-to-cell communication) in brain pericytes and to identify molecular signatures associated to Rbpj deletion. Methods: We used mouse genetic experiments to knockout Rbpj specifically in pericytes using a mural cell-specific Cre-driver (PdgfrbCreERT2). Inclusion of the Rpl22-HA (RiboTag) construct allowed Cre-mediated HA-labeling of ribosomal proteins and consequent isolation of actively translating mRNA from pericytes. PdgfrbCreERT2 transgenic mice were used to drive recombination of Rpl22-HA (RiboTag) allele and/or Rbpj-lox conditional knockout. Tamoxifen administration from postnatal day 1 (P1) to P3 allowed efficient recombination in pericytes. Brain cortices were collected at P7 or P10 and HA-tagged polyribosomes were immunoprecipitated using anti-HA coated beads. Total RNA was extracted and sequencing libraries prepared. Results: Pericyte-specific Rbpj deletion induces severe changes in the neurovascular unit characterized by defects in vascular morphogenesis, brain hemorrhaging, altered extracellular matrix composition and strong inflammatory responses. Analysis of pericytes translatome of Rbpj-KO mice compared to controls revealed profound changes in pericyte identity, increased expression of vascular smooth muscle cell markers and increased signaling through TGFbeta, among other alterations in mural cell behavior.

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