Sensory neurons that innervate the skin are easily damaged. When damage exceeds the capacity of these cells for repair, peripheral neuropathy (PN) manifests. Cancer patients who survive chemotherapy, or diabetics who struggle to control blood sugars, may be left with months to a lifetime of numbness or shooting pain. Free nerve endings in the skin mediate pain and temperature sensation, both of which are compromised early in most forms of PN. The repair mechanism of these sensory endings has not been elucidated, although it is the key to understanding the onset of PN and may also be critical for developing strategies to delay or cure PN. Axon regeneration uses a common set of machinery in all organisms and neurons in which it has been studied. This core machinery includes a MAP kinase cascade initiated by DLK and the activation of AP-1 transcription factors. This core pathway mediates regeneration when axons of sensory neurons in Drosophila are injured, but regeneration of peripheral sensory endings in the same cells is completely independent of this machinery. In Drosophila, sensory endings have been defined as dendrites by cell biology studies, suggesting that the DLK pathway may be specific to axon regeneration and not used for dendrite regeneration. Determining whether sensory endings in vertebrates are repaired using the conserved axon regeneration pathway or a completely unexplored dendrite regeneration pathway will be a critical foundation for understanding PN. In this exploratory work, two major lines of experimentation will be used to determine the repair pathway used to regenerate vertebrate sensory endings in the skin. The sensory neurons that innervate the skin in zebrafish will be used as a model for all of these experiments. Zebrafish larval sensory neurons known as Rohon-Beard (RB) neurons are accessible for imaging and manipulation with current methodology, so these cells will be used as a first model in which to study sensory ending cell biology and injury responses. Analogous methods will then be developed for dorsal root ganglion neurons (DRGs), as these are the most relevant for understanding PN. To determine whether vertebrate free sensory endings are dendrites as in Drosophila, or axons as they are historically classified in vertebrates, we will determine the polarity of their microtubules. All known dendrites contain at least 50% minus-end-out microtubules while axons are close to 100% plus-end-out. Microtubule polarity has never been assayed in vertebrate sensory endings, so this will be done in both RB and DRG cells. To determine whether injury to sensory endings triggers the identified axon regeneration pathway or a novel dendrite regeneration pathway, we will develop markers for DLK signaling and compare responses to central axon injury and peripheral sensory ending injury in zebrafish sensory neurons. These two lines of experimentation will form a foundation for understanding how sensory endings are repaired, and how this process fails in patients with PN.
The NIH and medical foundations counsel diabetics and cancer patients living with peripheral neuropathy that, while this condition may result in amputation or discontinuation of chemotherapy, its cause is not well understood. To form a framework for understanding peripheral neuropathy, the fragile free sensory endings in the skin must be characterized. We will determine whether these easily damaged structures are axons or dendrites, and whether they respond to injury using the identified axon regeneration machinery or a still uncharacterized dendrite regeneration pathway.
