Abstract
Lysosomes, the cell's recycling centres, are trafficked up and down neuronal projections, recycling and degrading cellular waste. Long noncoding RNAs (lncRNAs) have been associated with the dysregulation of autophagy, a pathway commonly disrupted in neurodegenerative disorders. In a childhood form of neurodegeneration, loss of a single lysosomal protein, CLN5, leads to lysosomal dysfunction. The connection between lncRNAs and lysosome function remains to be elucidated. In this project we aim to discover how novel lncRNAs regulate lysosome and neuron function.
Transcriptomic analysis of human induced pluripotent stem cell (iPSC)-derived CLN5-deficient cortical neurons revealed differentially expressed lncRNAs. Functional characterisation was performed using CRISPR inhibition of the novel lncRNA NL02 in Day 14 cortical neurons. Lysosome acidity was assessed via microscopy analysis of the lysotracker assay and neuron function was determined through confocal time lapse for calcium imaging.
CRISPRi inhibition of NL02 in Day 14 human cortical neurons showed a significant increase in lysosome acidity (1.9 fold increase, P < 0.05) compared to control neurons, indicating change in lysosomal health and function. NL02 inhibition impaired neuronal activity (measured using calcium transients, P < 0.01) compared to control neurons. Bioinformatic analysis revealed NL02 localises to the cytoplasm and comprises proposed binding site for several RNA interactions including both RNA and microRNA targets.
Our study reveals novel lncRNAs associated with lysosome and neuron function. Confirming RNA and protein partners in cortical neurons will assist us in understanding the mechanism behind NL02 induced lysosome changes. NL02 offers a new potential therapeutic target for the treatment of neurodegenerative disorders.