Drosophila with-no-lysine and frayed Act in Parallel to dSarm to Drive Neurodegeneration Following dNmnat Depletion

The maintenance of neuronal circuits is vital for sustaining nervous system function throughout life. Neurons are particularly susceptible to damage from metabolic and environmental stressors, which can lead to various neurodegenerative diseases. Over the past three decades, research has identified critical neuronal survival factors like Nmnat2 and key drivers of axon destruction, such as Sarm1, following neuronal injury. Further research has highlighted Sarm1’s essential role in neurodegeneration; however, much remains unknown about its activation, mechanisms of action, and potential interacting pathways that mediate neurodegeneration.

To explore these questions, we used Drosophila , a model system that previously enabled us to identify mutations in pro-degenerative genes such as dSarm following injury. We developed a novel method to trigger degeneration via neuron-intrinsic dNmnat depletion—a conserved approach for inducing dSarm-dependent degeneration. Conducting a forward genetic screen of nearly 5,000 chromosomes, we identified several protective mutations, including two loss-of-function alleles of the Drosophila with-no-lysine ( dWnk ) gene, which encodes a conserved serine/threonine kinase uniquely sensitive to intracellular chloride and potassium levels. Our findings reveal that dWnk, along with its downstream kinase Frayed (Fray; ortholog of mammalian OSR1/SPAK), promotes neurodegeneration caused by dNmnat depletion and that kinase activities of both proteins are essential to this signaling event. We found that dWnk is activated in parallel with dSarm to drive neurodegeneration following dNmnat depletion. Furthermore, simultaneous inhibition of both dSarm- and dWnk-mediated pathways provides neurons with near-complete protection from dNmnat depletion. Interestingly, activation of the dWnk pathway reduces the neuroprotection afforded by a dSarm null allele following dNmnat depletion but not axonal injury. This distinction highlights mechanistic differences between dNmnat depletion and other injury models and suggests that dWnk/Fray signaling may rely on the presence of the neuronal cell body. Overall, our innovative screening approach revealed pathways driving neurodegeneration in parallel to Sarm1 and indicates that targeting these pathways alongside Sarm1 inhibition could enhance therapeutic strategies against neurodegeneration.