Date of Award
Doctor of Philosophy (PhD)
During development of the peripheral nervous system, sensory axons extend to the periphery in excess where they compete for limiting target-derived neurotrophic support. Local neurotrophin insufficiency triggers axon degeneration, resulting in the pruning of over half of all sensory axons during development. Although axon degeneration facilitates the essential sculpting of the developing nervous system, its improper activation may underlie several neurodegenerative disorders. This process can be modeled in vitro by culturing sensory neurons from mouse dorsal root ganglia in the presence of nerve growth factor either as explant cultures or in compartmented chambers that allow independent manipulation of cell bodies and axons. The mitochondrial apoptotic pathway and the effector caspases, Caspase-3 and Caspase-6, mediate axon degeneration in both this in vitro model system and in vivo, clearly indicating a role for classical apoptotic machinery in axon degeneration. However, the full mechanism executing this process has yet to be determined. Previous studies proposed a model whereby the signaling mechanisms that driving axon degeneration in response to local deprivation resides completely within the axon itself. This view has been challenged by results showing that transcriptional inhibition prevents caspase activation in axons, as does physically separating the axon from its cell body. We used the in vitro culture system described above to address this controversy and conclusively show that, although the apoptotic machinery is present in axons, the cell body is required for gating axonal caspase activation and axon degeneration in response to trophic factor withdrawal. Use of selective pharmacological inhibitors and knockdown of candidate genes identifies a pathway whereby local trophic deprivation results in loss of Akt signaling and activation of DLK signaling, leading to activation of the JNK/c-jun signaling and a Foxo3a dependent transcriptional program. The cell body acts as a convergence point for these two pathways resulting in upregulation of the proapoptotic protein Puma, itself identified through a genetic knockout screen. We further show that although Puma is unexpectedly confined to the cell body, rising levels of somatic Puma in response to trophic deprivation overcome inhibition by pro-survival Bcl-xL and Bcl-w to initiate an anterograde, JNK-dependent, prodegenerative program. Taken together, our results identify the cell body as a key arbiter of large-scale axon removal.
Pitts, Jason, "Retrograde Activation of a Somatic Transcriptional Program Regulates Distal Axon Degeneration" (2016). Student Theses and Dissertations. 316.