Transriptional effects of blocking mitochondrial fission-fusion in aging Drosophila clock neurons
Mitochondria are dynamic organelles and undergo fission-fusion in response to various environemtal cues. But the roles of these two very different mitochondrial forms – predominantly spherical and tubular - are not well-characterized in neurons of animals and especially in aging neurons. This is important because neurons are long-lived and mitochondrial dynamics is associated with neurodegenerative diseases. We used here an efficient cell type-specific CRISPR approach to knockout key fission-fusion genes and disrupt mitochondrial dynamics within the inessential clock neurons of Drosophila. In order to understand the cellular effects of these perturbations, transcriptomic profiling was performed from young and old clock neurons with and without disrupted mitochondrial dynamics. Overall design: To perturb mitochondrial dynamics in Drosophila clock neurons we used a cell type-sepcific CRISPR approach. We used CLK856-Gal4 driver to label clock neurons and under UAS-control, expressed Cas9 and 3X-gRNA against gene of interest along with eGFP to label the neurons for sorting. Following genes were targeted: Drp1 (involved in fission), Marf (involved in fusion of the outer mitochondrial membrane) and Opa1 (involved in fusion of inner mitochondrial membrane). Cas9 alone was used as a control. Bulk RNAseq was performed from FACS-purified clock neurons of the 4 genotypes (Cas9-control, Drp1-mut, Marf-mut and Opa1-mut) at two ages - Young (~15days old) and Old (~35 days old). Differential expression analysis was performed for each of the mutants from the Cas9 control at both ages independently