Investigating Mediation of Axonal Khsrp Translation and Implications for Nerve Regeneration

Axons can reach long distances throughout our bodies, providing pathways for sensory and motor signaling. However, their considerable lengths make the system vulnerable to disruption by injury and disease. Although PNS axons have the ability to regenerate, the time required for axons to regrow and reinnervate their targets often causes regeneration to fail in humans. Therefore, it is of critical need to develop strategies to enhance neuronal regeneration capacity. Our lab has shown that the RNA binding protein KHSRP slows axon regeneration, as regeneration is accelerated in mice lacking neuronal KHSRP. KHSRP is upregulated injury via Ca2+ → PERK → eIF2aPS51 pathway, and axonal KHSRP remains elevated out to 28 days after axotomy (Patel et al., 2022). All available evidence indicated that the increase in axonal Ca2+ signaling subsides a few hours after initial injury, so it was not clear how KHSRP could remain elevated in regenerating axons. Thus, we looked for potential modulators of KHSRP synthesis and found that REG3A, a secreted calcium-dependent lectin protein that is upregulated in regenerating axons (Kalinski et al., 2015), decreases axon growth, elevates axonal Ca2+, and increases translation of axonal Khsrp mRNA. My project aimed to identify the source of axonal Ca2+ elevation driven by REG3A. We see that primary mouse dorsal root ganglion (DRG) cultures treated with recombinant REG3A (recREG3A) have decreased axon length, increased branching, and increased axonal KHSRP. Using Ca2+ chelators, we find that recREG3A treatment decreases axon growth by inhibiting release of intracellular Ca2+ stores rather than entry into the axons from extracellular sources. Further, inhibitors of ER receptors that are known to mitigate Ca2+ release from the ER partially attenuate effects of recREG3A on both axon growth and elevation of axonal KHSRP. recREG3A increases eIF2a phosphorylation in axons, and this increase as well as the effects on axon growth are blocked by inhibition of PERK. Together, these findings indicate that REG3A slows axon growth by release of axonal ER Ca2+ stores to activate PERK, phosphorylate eIF2a, and increase translation of axonal Khsrp mRNA.