How does the nervous system respond to axonal injury? The answer is: it depends. In the environment of the central nervous system (CNS), neurons possess extremely low regenerative capacity. In contrast, the peripheral nervous system’s (PNS) neurons exhibit the ability to regrow following injury. While the mechanisms underlying this regrowth is not completely understood, there is significant motivation to elucidate this field for the purpose of developing therapies to enhance axonal regeneration even in the inhibitory environment of the CNS.
Dr. Valeria Cavalli’s group at Washington University decided to study axonal regeneration in the context of the dorsal root ganglion (DRG). What makes the DRG neuron an interesting model system is its structure as a pseudounipolar neuron: from the cell bodies located in the dorsal root ganglion located adjacent to the spinal cord arises a single extension which splits into two branches: a central branch that navigates back to the spinal cord, and a peripheral branch, which as the name suggests projects peripherally. Injury to this peripheral branch of the DRG results in activation of a pro-regenerative program – that is, a series of molecular events that promote the regrowth of the damaged axon. The central branch, keeping in theme with the CNS, does not exhibit such regenerative capacities. What, then, allows a single DRG neuron to exhibit repair on one branch but not the other?
Transcriptional profiling studies in DRG neurons identified a handful of interesting genes upregulated following injury; among these is the gene known as Hypoxia Inducible Factor (HIF), a heterodimeric protein made of HIF-α and HIF-β subunits. HIF-α has a particularly interesting expression profile: at physiologic O2 concentrations, HIF-α is targeted for ubiquitination/degradation pathways. As O2 concentrations drop, this suppression of HIF-α expression is released.
Cho et al., 2015, showed that following peripheral DRG axotomy, a large percentage of HIF-α targeted genes are upregulated over a 1.2 fold threshold; furthermore, this upregulation of gene expression is lost in HIF-α knockdown DRGs. In uninjured DRG neurons, constitutive overexpression of HIF-α, but not knockdown of HIF-α, lead to significant changes in gene expression compared to wildtype uninjured DRG neurons.
To study the regenerative capacity of DRG neurons, Cho et al. infected DRG neurons either with control shRNA, two HIF-α targeting shRNAs, or a lentivirus virally overexpresses HIF-α. These neurons were axotomized and immunostained for SCG10, a marker of axon regeneration. Interestingly, both shRNA-mediated knockdown and lentiviral overexpression of constitutively active HIF-α resulted in reduced SCG10 fluorescence, suggesting that regulated expression of HIF-α (as opposed to constitutive overexpression) is necessary for axonal regrowth. Further downstream, Cho et al. conducted knockdowns of genes targeted by HIF-α, and showed that while knockdowns contributed to reduced axon regeneration for some genes, there were still others genes that, when knocked down, instead exhibited slight increases in axon regeneration. This would suggest that HIF-α targeted genes could either promote or inhibit axon repair.
In order to show how HIF-α regulates axon regrowth in vivo, Cho et al. generated mice lacking HIF-α in sensory neurons by crossing HIFAflox/flox with mice expressing Cre under the Advillin promoter (HIF1AcKO), which is specific to the peripheral sensory nervous system. After confirming HIF-α knockout, the experimenters crushed the sciatic nerve and quantified regeneration of the nerve axons 3 days later by SCG10 staining. Using SCG10 intensity and distance from crush site, the researchers calculated a regeneration index; in the HIF1AcKO, regeneration was limited past the crush site (Fig 4A-C). Injury to the sciatic nerve, but not the dorsal root itself, results in elevated HIF-α expression (Fig 4D,E).
While hypoxia does increase HIF-α levels in vitro, Cho et. al specifically showed that acute intermittent hypoxia (AIH) enhances axon regeneration in vivo via elevating HIF-α and subsequent downstream target genes such as vascular endothelial growth factor (VEGFA). They demonstrated axon regeneration both in the sensory sciatic nerve fibers as well as in sciatic motor neuron: sciatic injury in YFP-16 mice, which express yellow fluorescent protein (YFP) in motor neurons, showed increased colocalization of YFP and neuromuscular junction boutons following AIH treatment for sciatic nerve injury. Taken together, these results suggests that AIH may drive axon regeneration in the periphery via HIF-α-dependent mechanisms.
To learn more about the work conducted in Dr. Cavalli’s lab, attend her talk this Tuesday, March 21th, at 4:00pm in the Center for Neural Circuits and Behavior.
Junmi Saikia is a third year MD/PhD student in the Malinow Lab at UCSD. Her research interests revolve around neurodegenerative diseases. She apologizes in advance for the punishably punfunny title.