HSP90 is a chaperone for DLK and is required for axon injury signaling

Significance Defining mechanisms of axon injury signaling is critical to understand axon regeneration. This knowledge can be used to develop strategies of axonal repair. Identification of such injury signals has been limited by traditional in vivo assays of proregenerative injury signaling. Here, we describe an in vitro screening platform that specifically identifies proregenerative axon injury signals in mouse neurons. We show that HSP90 is required for injury signaling and detail a mechanism by which HSP90 chaperones the essential proregenerative kinase, dual leucine zipper kinase (DLK). Thus, this work also describes HSP90 as a previously unidentified regulator of DLK, a critical neuronal stress sensor that drives axon regeneration, degeneration, and neurological disease. Peripheral nerve injury induces a robust proregenerative program that drives axon regeneration. While many regeneration-associated genes are known, the mechanisms by which injury activates them are less well-understood. To identify such mechanisms, we performed a loss-of-function pharmacological screen in cultured adult mouse sensory neurons for proteins required to activate this program. Well-characterized inhibitors were present as injury signaling was induced but were removed before axon outgrowth to identify molecules that block induction of the program. Of 480 compounds, 35 prevented injury-induced neurite regrowth. The top hits were inhibitors to heat shock protein 90 (HSP90), a chaperone with no known role in axon injury. HSP90 inhibition blocks injury-induced activation of the proregenerative transcription factor cJun and several regeneration-associated genes. These phenotypes mimic loss of the proregenerative kinase, dual leucine zipper kinase (DLK), a critical neuronal stress sensor that drives axon degeneration, axon regeneration, and cell death. HSP90 is an atypical chaperone that promotes the stability of signaling molecules. HSP90 and DLK show two hallmarks of HSP90–client relationships: (i) HSP90 binds DLK, and (ii) HSP90 inhibition leads to rapid degradation of existing DLK protein. Moreover, HSP90 is required for DLK stability in vivo, where HSP90 inhibitor reduces DLK protein in the sciatic nerve. This phenomenon is evolutionarily conserved in Drosophila. Genetic knockdown of Drosophila HSP90, Hsp83, decreases levels of Drosophila DLK, Wallenda, and blocks Wallenda-dependent synaptic terminal overgrowth and injury signaling. Our findings support the hypothesis that HSP90 chaperones DLK and is required for DLK functions, including proregenerative axon injury signaling.