Exploring mechanisms of axon growth and circuit connectivity for promoting respiratory function recovery following cervical spinal cord injury

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Project Summary / Abstract (30-line maximum). A majority of traumatic spinal cord injury (SCI) cases occur in the cervical spinal cord, resulting in persistent diaphragmatic respiratory dysfunction that is associated with mortality, a host of morbidities such as respiratory infections, and greatly reduced quality of life. Diaphragm is directly controlled by phrenic motor neurons (PMNs) located at levels C3-5. PMNs are mono-synaptically activated by supraspinal brainstem neurons located in the rostral Ventral Respiratory Group (rVRG). Cervical SCI results in axotomy of descending rVRG fibers, denervation and silencing of spared PMNs, and partial-to-complete hemi-diaphragm paralysis. In this Competing Continuation (“Renewal”) application, we aim to promote reconnection of rVRG-PMN- diaphragm circuitry in a rat model of cervical SCI, a critically important therapeutic goal for individuals with SCI. We developed inhibitory peptides against PTEN (phosphatase and tensin homolog: a central inhibitor of neuron-intrinsic axon growth potential) and PTPσ (protein tyrosine phosphatase-sigma: an axonally-expressed receptor that mediates the neuron-extrinsic axon growth inhibitory effects of chondroitin sulfate proteoglycans). Our exciting preliminary findings show that systemic delivery of these peptides each result in robust – but partial – recovery of diaphragm function in the C2 hemisection model of SCI. These initial studies also provide compelling data suggesting that PTEN and PTPσ inhibition may promote recovery via different modes of rVRG axon growth: (1) robust regeneration of injured ipsilateral rVRG axons with PTEN inhibition; (2) extensive sprouting of spared contralateral rVRG axons into the PMN pool (ipsilateral to the lesion) with PTPσ inhibition. Importantly, we do not understand which modes of axon growth can promote recovery of diaphragm function after SCI, which significantly limits ability to develop targeted therapies. To address this critical issue, we will use chemogenetic DREAAD manipulations to selectively-silence defined neuronal populations involved in respiratory control in order to determine the mode(s) of circuit re-connectivity that causally drive recovery in response to PTEN and PTPσ manipulation. We will target PTEN and PTPσ with systemic delivery of inhibitory peptides and rVRG neuron-specific transduction with AAV-shRNA. We will compliment this approach using an array of cutting-edge functional and axonal/synaptic tracing methods to assess rVRG-PMN circuit plasticity. We hypothesize that stimulating (1) regeneration of injured rVRG axons, (2) sprouting of spared fibers originating in contralateral rVRG, and (3) synaptic connectivity of these growing rVRG axons with PMNs (located caudal to the lesion) will causally promote recovery of diaphragmatic respiratory function following cervical SCI. We also hypothesize that the combination of rVRG axon regeneration and sprouting of spared rVRG fibers will promote robust diaphragm recovery in the clinically-associated cervical contusion SCI model. We will acquire an in-depth understanding of how modulating axon growth inhibition can induce rVRG- PMN circuit plasticity and, importantly, which modes of connectivity promote diaphragm recovery after SCI.