ADCY9 Regulates Neural Stem Cells Via Mitofusin-1 to Maintain Planarian (Dugesia japonica) Cephalic Ganglia Regeneration.

ADCY9, a crucial member of the adenylate cyclase family, exerts neuroprotective and analgesic effects in the nervous system by modulating the activity of the cAMP/AMPK signaling pathway. However, the role of the ADCY9 gene in neural regeneration remains unreported. In this study, we utilized Dugesia japonica, a highly regenerative planarian species, as a model to systematically examine the spatiotemporal expression pattern of the ADCY9 gene during planarian brain regeneration and investigate its regulatory function in this process. The results demonstrated that the downregulation of ADCY9 resulted in abnormal brain regeneration in planarians, characterized by partial loss of the nerve cord, reduced numbers of collateral branches, and significant inhibition of the regeneration and differentiation of multiple neuron types. RNA sequencing revealed that the downregulation of ADCY9 led to 499 differentially expressed genes, with KEGG enrichment pathway analysis indicating significant associations with neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Double RNAi experiments demonstrated that simultaneous knockdown of ADCY9 and Mitofusin-1 significantly restored neural regeneration. Collectively, ADCY9 might promote the comprehensive reconstruction of neural structure by hierarchically regulating the regeneration intensity through negative regulation of the downstream inhibitory factor Mitofusin-1. This study discloses the function of ADCY9 in planarian neural regeneration, providing a theoretical foundation for its application in investigating neural regeneration mechanisms and neurodegenerative disease pathogenesis in higher vertebrates.