Effect of CLN3-loss-of-function on cellular metabolism and signalling

Since a coordinated function of all organelles is essential for the proper health of a eukaryotic cell, a defect in a single organelle can affect the condition of other organelles. For instance, mitochondrial dysfunction was reported in most of the lysosomal storage diseases and abnormal mitochondria-lysosome crosstalk was observed in several neurodegenerative disorders. Batten disease is a lysosomal storage disease and the most frequent cause of dementia in children, although, its precise mechanism remains unclear. Thereby, we aimed to advance the understanding of the role of organelle crosstalk in the disease progression and identify signaling pathways that might be later tested as potential therapeutic targets in Batten disease. Analysis of our transcriptome data indicates a lack of significant effect of CLN3 KO on organelle biogenesis in HEK cells grown in high glucose medium, while mitochondrial and ER biogenesis are transcriptionally repressed in CLN3 KO HEK cells starved in no glucose medium. Moreover, we observed a substantial reduction in levels of proteins that belong to respiratory chain subunits in whole cell extracts, apart from complex V. On the contrary, the level of native OxPhos complexes in mitochondria isolated from HEK cells grown in a high glucose medium remains unaltered. Importantly, we report compromised mitochondrial respiration, increased mitochondrial superoxide levels, a decline in mitochondrial membrane potential and reduced cell viability in CLN3 KO HEK cells in both, high glucose and no glucose condition. Overall, we introduce extensive evidence of mitochondrial dysfunction in CLN3 KO HEK cells. In addition, pathway analysis completed on transcriptome data revealed multiple signaling pathways that are significantly affected by CLN3 KO and may play an important role in Batten disease progression. Particular attention should be given to cell proliferation signaling, including Hippo, mTOR, p70 S6K1 signalling pathways, as well as stress response mechanisms, such as unfolded protein response and eIF2 signaling. Additionally, transcription factor analysis performed on the transcriptome data suggested that YAP, which is regulated by Hippo and mTOR signaling, is probably a key regulator of the cellular response to CLN3-loss-of-function. Indeed, our immunoblotting results indicate increased YAP activity in CLN3 KO HEK under both normal and starvation conditions. Likewise, we observed higher activity of S6K1 and mTORC1 in CLN3 KO HEK cells under starvation. Moreover, integrated stress response and unfolded protein response are upregulated in starved CLN3 KO HEK cells. In conclusion, our findings indicate several potential therapeutic targets and offer insight into the Batten disease mechanism.2022-09-1