| dc.description.abstract | Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease, which primarily affects motor neurons. Years of research have led to the identification of multiple genes involved in ALS. However, the non-coding genome has been largely unexplored. By intersecting open chromatin profiles of healthy human motor cortex with whole genome sequencing data from ALS patients, the neurocalcin delta (NCALD) enhancer region was found to be associated with ALS. An increase in rare variants was found in a 300 base pair region of the NCALD enhancer in healthy individuals compared to ALS patients. This suggests that the rare variants are involved in a protective mechanism towards ALS by increasing motor neuron function and survival. Here, we aim to elucidate the impact of rare variants on NCALD enhancer activity and the role of NCALD enhancer activity on cell morphology and viability in an ALS model. Four rare variants were predicted in silico to impact enhancer activity based on their location in the 300 base pair region and the presence of known transcription factor binding sites. We validated experimentally that three of these rare variants altered NCALD enhancer activity, indicating that they may also affect regulation of gene transcription. To study the effects of gene expression changes in the context of the disease, we set up a simple ALS in vitro model. We found this experimental framework to be robust, replicable, and suitable to use in fundamental ALS research with opportunity of high scalability. Neuroblastoma-derived SH-sy5y cells were differentiated, as they possess neuronal-like properties, are easy to use and to genetically manipulate. We edited the genome of SH-sy5y cells to study effects of gene expression changes in differentiated cells. In basal conditions, neurite length and cell viability were unchanged. In order to mimic ALS, we induced stress in the differentiated cells and aimed to investigate cell morphology and viability. Stress assays were performed to establish glutamate as a stressor for differentiated SH-sy5y cells, which was then implemented into our in vitro ALS model. Overall, our results show rare variants impact NCALD enhancer activity and this impact can be studied in differentiated SH-sy5y cells. Furthermore, our experimental setup can be used in a wide variety of fundamental neurodegenerative disease research. | |
