The Cytoskeletal Stress Response Pathway: a homeostatic system driven by Dual Leucine Zipper Kinase (DLK)

Injury to peripheral axons initiates a complex cascade of cellular responses, including cytoskeletal disassembly, axon transport disruption, and ultimately axon regeneration. Central to this process is the MAP triple kinase Dual-Leucine Zipper Kinase (DLK), activated by injury and other neuronal stressors. Here, we propose the existence of a homeostatic mechanism termed the Cytoskeletal Perturbation Response (CPR). We investigate this hypothesis by examining the response of cultured dorsal root ganglion (DRG) neurons to low dose nocodazole treatment, a cytoskeletal perturbing agent. To gain insights into DLK-dependent transcriptional changes following cytoskeletal insult, we performed bulk RNA sequencing on cultured neurons treated for 16 hours. Using a fully crossed two-factor design, we determined the interactive effect of DLK on nocodazole- dependent transcriptional changes. Our study demonstrates that cytoskeletal perturbation triggers DLK-dependent signaling cascades, leading to significant transcriptional changes. These changes involve transcription factors (Jun, Egr1, Atf3) and MAP kinase regulators (DUSPs), pointing to a regulatory network that attenuates DLK signaling. Taken together, our findings suggest that cytoskeletal perturbation activates a DLK-dependent homeostatic mechanism, the CPR, which orchestrates transcriptional changes and morphological adaptations to repair neuronal damage. The CPR bears similarities to established homeostatic responses, offering insights into the intricate processes that underlie axon regeneration and cellular repair. Primary DRGs were cultured from CD1 mouse embryos. At 6 days in vitro (DIV), neurons were pretreated with DMSO or 500nM DLK inhibitor (GNE3511). After 15 minutes, neurons were then treated with DMSO, or 200nM nocodazole. This produced 4 groups: ((1)DMSO, DMSO; (2) DMSO, Noc; (3) DLKi, DMSO and (4) DLKi, Noc). We collected 4 replicates per group. We performed differential gene expression profiliing using data obtained from RNA seq of these 4 group using 4 replicates.