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Title
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Systematic image-based profiling of neuronal connectivity : from the synapse to the nucleus
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Author
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Abstract
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| Long-term adaptations of the brain, including learning, memory formation, addiction and chronic pain sensation, rely on a delicate balance of tunable connections between neurons. In numerous neurological diseases this neuronal connectivity is perturbed. Neuronal connectivity is classically assessed by means of morphological correlates such as the density of synapses or functional readouts such as synchronous calcium spiking activity. However, such approaches operate with blinders as they do not account for multifaceted effects. Furthermore, recent reports have inferred an important role for the nucleus in neuronal connectivity. As yet, nuclear morphology has not been regarded as a potential readout of neuronal connectivity and limited tools are available to capture it with sufficient sensitivity. We established a detailed interrogation of morphological and functional correlates of neuronal connectivity in one single microscopy-based assay to screen for its modulators in primary neuronal cultures. The integrative approach proved to be more sensitive than more classical single-readout approaches. Using this pipeline, we found that inhibition of dual leucine zipper kinase (DLK) increased neuronal connectivity in primary cortical cultures, albeit within a specific time and dose range. This neuroprotective effect was not only observed in basal conditions but also in cultures depleted from antioxidants and in cultures in which microtubule stability was genetically perturbed, suggesting it has broad-spectrum therapeutic relevance. In addition to this integrated assessment of neuronal connectivity, we optimized an image analysis algorithm that accurately detects nuclei in fluorescence images and captures their morphology by precise delineation of their contour and by extraction of robust, quantitative descriptors. Having established a method to interrogate nuclear morphology and to connect it with neuronal connectivity, we next sought to evaluate the influence of targeted perturbations to nuclear morphology. Given their crucial role in nuclear architecture and function, we thereby focused on the nuclear lamins. A significant decrease in lamin B1 levels with culture time and brain aging, drove us to interrogate the effect of its selective depletion in human neuroblastoma model cells using shotgun proteomics. In-depth analysis revealed that lamin B1 acts as a molecular switch controlling differentiation, by tempering integrated stress levels. The results that have been generated in this thesis, illustrate that profiling microscopy images with deep coverage enables sensitive interrogation of neuronal connectivity and allows exposing a pharmacological window for targeted treatments. Our work also proves that nuclear lamins have a crucial role in steering neuronal cell function and may be considered as a putative target for novel modulators of neuronal connectivity. |
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Language
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English
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Publication
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Antwerp
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University of Antwerp, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences
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2021
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Volume/pages
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202 p.
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Note
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:
De Vos, Winnok H. [Supervisor]
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Full text (publisher's version - intranet only)
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