Functional genomic screens to identify and validate neuro-developmental modulators of immediate early genes, Arc and Bdnf

Sahoo, Vivek Kumar (2024): Functional genomic screens to identify and validate neuro-developmental modulators of immediate early genes, Arc and Bdnf. Dissertation, LMU München: Medizinische Fakultät

Abstract

This study aims to address the scalability challenges in functional genomics research on neuronal cells by using TargetFinder and shRNA-Perturb-seq to explore the molecular mechanisms of neuronal plasticity and survival within the context of neurodevelopment, focusing on the immediate early genes (IEGs), Arc and Bdnf. The TargetFinder assay was utilized as the initial screening tool, focusing on identifying potential modulators of the pathway sensor under investigation. Through systematic perturbation of gene expression, TargetFinder allowed for the exploration of a wide array of genetic factors that may influence the activity or output of the pathway sensor. Complementing this, Perturb-seq provided a deeper examination of the functional consequences of genetic perturbations identified through the initial screen. Perturb-seq enabled a closer look at the transcriptomic changes induced by these modulators, offering mechanistic insights into their effects at the molecular level. In this study, we identified neuronal modulators of the E-SARE and BDNF-E840 genetic sensors using the TargetFinder assay. To characterize modulators of the E-SARE sensor, we developed an shRNA-Perturb-seq assay, successfully adapting Perturb-Seq methodologies from previous studies to primary mouse cortical neurons. To address the unique challenges posed by in vitro primary neuron cultures, we devised novel strategies, including direct gene expression capture through robust Pert-BC expression and a protocol for in-suspension transduction and cell pooling. We created a marker gene list for cell-type cluster annotation, which will serve as a template for future primary mouse cortical neurons snRNA-seq experiments. By effectively capturing primary and delayed response genes, we observed distinct transcriptional profiles following BIC cocktail and AMPA stimulation. The perturbation effect induced by AMPA showed negative enrichment in pathways related to long-term potentiation, axon guidance, synaptic membrane regions, and post-synaptic signal transmission. Furthermore, studying the combined effects of perturbation and treatment revealed positive enrichment in nervous system developmental pathways, indicating that all perturbations result in neuron development delay. This supports our initial hypothesis of capturing near-developmental modulators of the E-SARE sensor. Further empirical validation is required to gain a deeper mechanistic understanding of both sensors.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Functional Genomics, Single-Cell Transcriptomics, Primary cortical neurons, NGS, Arc, Bdnf, PertubSeq assay, TargetFinder assay, shRNA
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 17. Dezember 2024
• 1. Bericht­erstatter:in: Rossner, Moritz