Lysosomal function is essential for cellular homeostasis. This is highlighted by the involvement of dysfunctional lysosomes in Alzheimer’s and Batten disease. Exploring the regulatory networks involved in neuronal function is crucial for understanding disease mechanisms. Recently, lncRNAs have emerged as a major regulator of cellular pathways, including autophagy — the degradation process reliant on lysosomes.
Our focus is on investigating the lncRNA DANCR and its impact on neuronal lysosomes. While DANCR is known to regulate autophagy, no studies have directly investigated the influence of DANCR on lysosomal regulation, particularly in human neurons.
Transcript variants of DANCR and LINC01618, a lncRNA in the same genomic locus, remain unexplored. To address this, a qPCR panel was developed to detect four DANCR and four LINC01618 transcripts. In induced pluripotent stem cells (iPSCs), five out of eight transcripts were detected. CRISPRi guides were designed to target DANCR or LINC01618 transcripts and reduced their expression by 99.6% (P=0.0088) and 99.2% (P=0.0694) respectively. The DANCR knockdown iPSCs showed almost 2-fold increase (P=0.0378) in proliferation compared to control iPSCs. Long-read RNAseq further resolved transcript variants of DANCR present in control iPSCs and confirmed the silencing of DANCR in the knockdown line. This data also revealed the molecular and functional pathways impacted by the loss of these transcripts.
Neurons derived from DANCR knockdown iPSC lines will be used to investigate the impact of DANCR on differentiation and lysosomal function. This will reveal the relationship between these lncRNAs and lysosomal regulation and point towards potential therapeutic strategies for neurodegenerative disease.