Abstract
Parkinson’s disease (PD) is the world’s second most common neurodegenerative disease that mainly affects the motor system, leading to impaired body movement and coordination. The substantia nigra, located in the mid-brain region, hosts dopaminergic neurons, which gradually degenerate in PD, followed by a decline in dopamine levels. Major hallmarks of PD include the accumulation of Lewy bodies, which are protein aggregates of alpha-synuclein (α-syn), along with lysosomal and mitochondrial dysfunction. Recent research has identified long non-coding RNAs (lncRNAs) as genetic markers associated with PD pathologies, but how lncRNAs affect the health of dopaminergic neurons in PD is poorly understood. This research explored the role of a brain-enriched lncRNA, NL02, in PD, which was previously shown to be decreased and hypermethylated in PD patients. The main aim of this project was to investigate the effects of NL02 knockout in induced pluripotent stem cells (iPSCs), and iPSC-derived dopaminergic neurons (iDAs). CRISPR-Cas9 gene editing was used to delete exon 4 (Deletion 2) of NL02, to generate NL02-deficient iPSCs. For Deletion 2, exon 4 was chosen as it is the longest NL02 exon, and in silico data analysis showed maximum mRNA/protein interactions of NL02 at exon 4. PCR screening revealed only heterozygous deletion, and no homozygous deletion of NL02 in iPSCs for Deletion 2 from three independent CRISPR-Cas9 gene editing experiments. Hence, for all assays, wild type (WT) and NL02 heterozygous Deletion 2 (NL02-del2-het) cells were used. NL02-del2-het iPSCs and induced neuronal progenitor cells (iNPCs) showed a significant decrease in proliferation compared to WT. However, immunocytochemistry (ICC) testing for cell pluripotency and proliferation indicated no difference. To investigate the effects of heterozygous NL02 deletion in dopaminergic neurons, both WT and NL02-del2-het iPSCs were induced into iDAs. A decrease in LysoTracker signal, a dye that binds to acidic lysosomes, was detected for the NL02-del2-het iDAs compared to WT iDAs, indicating potential impairment in lysosomal function, while no evident changes were observed for mitochondria. ICC staining for α-syn, a key hallmark of PD, indicated α-syn upregulation in NL02-del2-het iDAs compared to WT. These results indicate that even heterozygous NL02 knockout impacts lysosomal function, and leads to potential build-up of α-syn at the iDA level, ultimately contributing to PD-like characteristics. Overall, this study provides additional insight into how NL02 may be involved in neurodegenerative processes linked to PD.