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Importantly, drugs are present only during induction of the regeneration program, not during axon sprouting or outgrowth. The major advantage that this assay has over the in vivo counterpart is that injury signaling is induced in culture and therefore is amenable to pharmacological perturbations. Preconditioned neurons grow extensive neurites in a short time compared with uninjured neurons. Twenty-four hours later, the regeneration program is active, and we administer the testing injury via replating of the neurons. Traditionally, this paradigm is performed in vivo, but we and others have recently described an in vitro version of this assay in which dissection of mouse dorsal root ganglia (DRG) neurons serves as the preconditioning lesion ( 17– 19). We took advantage of the preconditioning phenomenon, in which a conditioning injury activates the regeneration program and a second test injury assays its state ( 16). To accomplish this, we developed an in vitro screen to identify injury signals required for induction of the proregenerative program. We sought to identify additional components of the axon injury response, including previously unidentified pathways or undescribed regulators of known signals, such as DLK. Along with DLK, a handful of other kinases, transcription factors, and histone modifiers drive regenerative axon signaling, and other factors are likely yet undiscovered ( 13– 15). DLK promotes retrograde transport of injury signals and is required for axon regeneration in mice, Drosophila, and Caenorhabditis elegans ( 9– 12). Our findings support the hypothesis that HSP90 chaperones DLK and is required for DLK functions, including proregenerative axon injury signaling.ĭual leucine zipper kinase (DLK) is an essential axon injury sensor and MAP triple kinase that activates the JNK and p38 families ( 6– 8). Genetic knockdown of Drosophila HSP90, Hsp83, decreases levels of Drosophila DLK, Wallenda, and blocks Wallenda-dependent synaptic terminal overgrowth and injury signaling. This phenomenon is evolutionarily conserved in Drosophila. Moreover, HSP90 is required for DLK stability in vivo, where HSP90 inhibitor reduces DLK protein in the sciatic nerve. 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. HSP90 is an atypical chaperone that promotes the stability of signaling molecules. 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 inhibition blocks injury-induced activation of the proregenerative transcription factor cJun and several regeneration-associated genes. The top hits were inhibitors to heat shock protein 90 (HSP90), a chaperone with no known role in axon injury. Of 480 compounds, 35 prevented injury-induced neurite regrowth. 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. 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. While many regeneration-associated genes are known, the mechanisms by which injury activates them are less well-understood. Peripheral nerve injury induces a robust proregenerative program that drives axon regeneration.