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Development of a multiplexed RNAi-coupled sensor assay to study neuronal function on the large-scale

dc.contributor.advisorRossner, Moritz Prof. Dr.
dc.contributor.authorHerholt, Alexander
dc.titleDevelopment of a multiplexed RNAi-coupled sensor assay to study neuronal function on the large-scalede
dc.contributor.refereeRossner, Moritz Prof. Dr.
dc.description.abstractengPsychiatric diseases such as schizophrenia, bipolar disorder and autism spectrum disorders are considered neurodevelopmental synaptopathies. Compelling evidence obtained from large-scale genome-wide association studies, identified a plethora of genetic variations within hundreds of genes encoding components of the post-synaptic compartment and calcium signaling mediating excitation-transcription-coupling. This synapse-to-nucleus signaling is thought to be particularly important for synaptic plasticity and learning and memory. In the last decade, tremendous progress has been made in neuroscience research when employing an array of molecular and cellular techniques to study the impact of individual genes on synaptic plasticity. Nevertheless, neuroscience methodology lags behind the field of cancer research in terms of large scale functional genetic screens, e.g. mediated by RNA interference (RNAi). The underlying cause is likely due to both the difficulties of culturing post-mitotic neurons and the phenotypic complexity. In this regard, comprehensively identifying genes involved in neuronal excitation and synapse-to-nucleus signaling may not only deepen our understanding of the corresponding biological processes, but might also be key in unearthing promising targets for psychiatric drug discovery. I have developed a functional genomics tool that is applicable to primary neurons and combines the throughput of a pooled RNAi screen with the sensitivity of a pathway reporter assay based on the synaptic activity-response element, modified from the Arc enhancer. This thesis describes a proof-of-concept study in which an AAV-based RNAi library was screened for regulators of neuronal excitation and synapse-to-nucleus signaling. The assay principle relies on molecular barcodes, which serve as quantitative reporters, while at the same time also functioning as unique identifiers of the targeted genes. Upon synaptic stimulation, the screen identified a multitude of known genes involved in glutamatergic synapse-to-nucleus signaling, as well as previously unknown candidates like the chemokine receptor XCR1. The technical approach’s reproducibility has been verified by substantial overlap of gene hits during three independent screens. Later in the thesis, I also present the principal applicability of CRISPR-Cas9 tools in neurons, which may improve performance for genetic interference screens in the near future. This assay seeks to enhance the analytic toolbox used for analyzing regulatory processes during neuronal signaling and for the identification of novel targets in psychiatric drug
dc.contributor.coRefereeBrose, Nils Prof. Dr.
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de

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