Generation of stable cell lines over-expressing OPA1 to study the impact of mitochondrial ultrastructure in the ALS syndrome

The aim of this thesis is the generation of stable cell lines over-expressing OPA1 protein, in order to verify mitochondrial effects in ALS syndrome patients’ cells. Amyotrophic lateral sclerosis (ASL) is a neurodegenerative disease that affects motor neurons; can be caused by mutated proteins, as in the case of TDP-43. In patient cells this protein moves from the nucleus to the cytoplasm, accumulating in the mitochondria and altering its functionality. OPA1 is a mitochondrial protein involved in many processes that affect the morphology of organelles and the proper functioning of metabolic mechanisms, thus ensuring the well-being of organelles and, as a result, long-term cell survival. To investigate the effect of this protein over-expression on cells, we used a lentiviral vector, through which Human OPA1 gene was conveyed within cells, so that it could be integrated stably into their genome. SH_5YSY cells were transduced with the viral particles, containing OPA1 plasmid in which the protein gene was under the control of a strong promoter. Then, cells were put in selection with growing concentration of Hygromycin, for different time periods. To investigate the correct integration of the gene and the protein over-expression, a combination of Western blot and PCR techniques was applied. Our results provide a protocol to generate stable cell lines that overpower the protein of interest. It could help clarify the molecular effects of OPA1 overexpression in cells, and the potential compensation of mitochondrial abnormalities in ALS patients' cells.