Phenotypic Screening Following Transcriptomic Deconvolution to Identify Transcription Factors Mediating Axon Growth Induced by a Kinase Inhibitor
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA982769 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR24898208_1.fastq.gz | Fastqsanger.gz | |||
| SRR24898208_2.fastq.gz | Fastqsanger.gz | |||
| SRR24898209_1.fastq.gz | Fastqsanger.gz | |||
| SRR24898209_2.fastq.gz | Fastqsanger.gz | |||
| SRR24898210_1.fastq.gz | Fastqsanger.gz |
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Project description:After injury to the central nervous system (CNS), both neuron-intrinsic limitations on regenerative responses and inhibitory factors in the injured CNS environment restrict regenerative axon growth. Instances of successful axon regrowth offer opportunities to identify features that differentiate these situations from that of the normal adult CNS. One such opportunity is provided by the kinase inhibitor RO48, which dramatically enhances neurite outgrowth of neurons in vitro and substantially increased contralateral sprouting of corticospinal tract neurons when infused intraventricularly following unilateral pyramidotomy. The authors present here a transcriptomic deconvolution of RO48-associated axon growth, with the goal of identifying transcriptional regulators associated with axon growth in the CNS. Through the use of RNA sequencing (RNA-seq) and transcription factor binding site enrichment analysis, the authors identified a list of transcription factors putatively driving differential gene expression during RO48-induced neurite outgrowth of rat hippocampal neurons in vitro. The 82 transcription factor motifs identified in this way included some with known association to axon growth regulation, such as Jun, Klf4, Myc, Atf4, Stat3, and Nfatc2, and many with no known association to axon growth. A phenotypic loss-of-function screen was carried out to evaluate these transcription factors for their roles in neurite outgrowth; this screen identified several potential outgrowth regulators. Subsequent validation suggests that the Forkhead box (Fox) family transcription factor Foxp2 restricts neurite outgrowth, while FoxO subfamily members Foxo1 and Foxo3a promote neurite outgrowth. The authors’ combined transcriptomic-phenotypic screening strategy therefore allowed identification of novel transcriptional regulators of neurite outgrowth downstream of a multitarget kinase inhibitor.
2023-06-19 | GSE234696 | GEO
Project description:After injury to the central nervous system (CNS), both neuron-intrinsic limitations on regenerative responses and inhibitory factors in the injured CNS environment restrict regenerative axon growth. Instances of successful axon regrowth offer opportunities to identify features that differentiate these situations from that of the normal adult CNS. One such opportunity is provided by the kinase inhibitor RO48, which dramatically enhances neurite outgrowth of neurons in vitro and substantially increased contralateral sprouting of corticospinal tract neurons when infused intraventricularly following unilateral pyramidotomy. The authors present here a transcriptomic deconvolution of RO48-associated axon growth, with the goal of identifying transcriptional regulators associated with axon growth in the CNS. Through the use of RNA sequencing (RNA-seq) and transcription factor binding site enrichment analysis, the authors identified a list of transcription factors putatively driving differential gene expression during RO48-induced neurite outgrowth of rat hippocampal neurons in vitro. The 82 transcription factor motifs identified in this way included some with known association to axon growth regulation, such as Jun, Klf4, Myc, Atf4, Stat3, and Nfatc2, and many with no known association to axon growth. A phenotypic loss-of-function screen was carried out to evaluate these transcription factors for their roles in neurite outgrowth; this screen identified several potential outgrowth regulators. Subsequent validation suggests that the Forkhead box (Fox) family transcription factor Foxp2 restricts neurite outgrowth, while FoxO subfamily members Foxo1 and Foxo3a promote neurite outgrowth. The authors' combined transcriptomic-phenotypic screening strategy therefore allowed identification of novel transcriptional regulators of neurite outgrowth downstream of a multitarget kinase inhibitor.
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Project description:SEMA3B switches axon-axon to axon-glia interactions required for unmyelinated axon envelopment and integrity
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2023-07-13 | PXD035196 | Pride
Project description:During peripheral nerve (PN) development, unmyelinated axons (nmAs) initially form tight fascicles before being separated and enveloped by non-myelinating Schwann cells (nmSCs), which are essential for maintaining nmA integrity. The mechanism underlying this transition from axon-axon to axon-glia interactions remains unclear. Here, we found that inactivating nmSC-derived SEMA3B or its axonal receptor components in mice led to incomplete nmA separation and envelopment by nmSCs, leading to nmA dysfunction and inducing hyperalgesia and allodynia. Conversely, increasing SEMA3B levels in nmSCs accelerated nmA separation and envelopment. SEMA3B transiently promoted nmA defasciculation accompanied by cell adhesion molecule (CAM) endocytosis, thus facilitating subsequent CAM-mediated nmA-mSC association. Restoring SEMA3B expression post-PN injury promoted nmA-nmSC re-association and alleviated hyperalgesia and allodynia. We propose that SEMA3B-induced CAM turnover facilitates the switch from axon-axon to axon-glia interactions, promoting nmA envelopment by nmSCs, which may be exploited to alleviate PN injury-induced pain by accelerating the restoration of nmA integrity.
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Project description:Phenotypic screening is a valuable tool to both understand and engineer complex biological systems. We demonstrate the functionality of this approach in the development of cell-free protein synthesis (CFPS) technology. Phenotypic screening identified numerous compounds that enhanced protein production in yeast lysate CFPS reactions. Notably, many of these were competitive ATP kinase inhibitors, with exploitation of their inherent substrate promiscuity redirecting ATP flux towards heterologous protein expression. Chemoproteomic- guided strain engineering partially phenocopied drug effects, with a 30% increase in protein yield observed upon deletion of the ATP-consuming SSA1 component of the HSP70 chaperone. Moreover, drug-mediated metabolic rewiring coupled with template optimization generated the highest protein yields in yeast CFPS to date using a hitherto less efficient, but more cost-effective glucose energy regeneration system. Our approach highlights the utility of target-agnostic phenotypic screening and target identification to deconvolute cell-lysate complexity, adding to the expanding repertoire of strategies for improving CFPS.
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