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PTP? Knockdown in Lampreys Impairs Reticulospinal Axon Regeneration and Neuronal Survival After Spinal Cord Injury.

ABSTRACT: Traumatic spinal cord injury (SCI) results in persistent functional deficits due to the lack of axon regeneration within the mammalian CNS. After SCI, chondroitin sulfate proteoglycans (CSPGs) inhibit axon regrowth via putative interactions with the LAR-family protein tyrosine phosphatases, PTP? and LAR, localized on the injured axon tips. Unlike mammals, the sea lamprey, Petromyzon marinus, robustly recovers locomotion after complete spinal cord transection (TX). Behavioral recovery is accompanied by heterogeneous yet predictable anatomical regeneration of the lamprey's reticulospinal (RS) system. The identified RS neurons can be categorized as "good" or "bad" regenerators based on the likelihood that their axons will regenerate. Those neurons that fail to regenerate their axons undergo a delayed form of caspase-mediated cell death. Previously, this lab reported that lamprey PTP? mRNA is selectively expressed in "bad regenerator" RS neurons, preceding SCI-induced caspase activation. Consequently, we hypothesized that PTP? deletion would reduce retrograde cell death and promote axon regeneration. Using antisense morpholino oligomers (MOs), we knocked down PTP? expression after TX and assessed the effects on axon regeneration, caspase activation, intracellular signaling, and behavioral recovery. Unexpectedly, PTP? knockdown significantly impaired RS axon regeneration at 10 weeks post-TX, primarily due to reduced long-term neuron survival. Interestingly, cell loss was not preceded by an increase in caspase or p53 activation. Behavioral recovery was largely unaffected, although PTP? knockdowns showed mild deficits in the recovery of swimming distance and latency to immobility during open field swim assays. Although the mechanism underlying the cell death following TX and PTP? knockdown remains unknown, this study suggests that PTP? is not a net negative regulator of long tract axon regeneration in lampreys.

SUBMITTER: Rodemer W

PROVIDER: S-EPMC7096546 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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PTPσ Knockdown in Lampreys Impairs Reticulospinal Axon Regeneration and Neuronal Survival After Spinal Cord Injury.

Rodemer William W Zhang Guixin G Sinitsa Isabelle I Hu Jianli J Jin Li-Qing LQ Li Shuxin S Selzer Michael E ME

Frontiers in cellular neuroscience 20200319

Traumatic spinal cord injury (SCI) results in persistent functional deficits due to the lack of axon regeneration within the mammalian CNS. After SCI, chondroitin sulfate proteoglycans (CSPGs) inhibit axon regrowth <i>via</i> putative interactions with the LAR-family protein tyrosine phosphatases, PTPσ and LAR, localized on the injured axon tips. Unlike mammals, the sea lamprey, <i>Petromyzon marinus</i>, robustly recovers locomotion after complete spinal cord transection (TX). Behavioral recove ...[more]

PMID: 32265663

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Project description:Paralysis following spinal cord injury (SCI) is due to interruption of axons and their failure to regenerate. It has been suggested that the small GTPase RhoA may be an intracellular signaling convergence point for several types of growth-inhibiting extracellular molecules. Even if this is true in vitro, it is not clear from studies in mammalian SCI, whether the effects of RhoA manipulations on axon growth in vivo are due to a RhoA-mediated inhibition of true regeneration or only of collateral sprouting from spared axons, since work on SCI generally is performed with partial injury models. RhoA also has been implicated in local neuronal apoptosis after SCI, but whether this reflects an effect on axotomy-induced cell death or an effect on other pathological mechanisms is not known. In order to resolve these ambiguities, we studied the effects of RhoA knockdown in the sea lamprey central nervous system (CNS), where after complete spinal cord transection (TX), robust but incomplete regeneration of large axons belonging to individually identified reticulospinal (RS) neurons occurs, and where some RS neurons show unambiguous delayed retrograde apoptosis after axotomy. RhoA protein was detected in neurons and axons of the lamprey brain and spinal cord, and its expression was increased post-TX. Knockdown of RhoA in vivo by retrogradely-delivered morpholino antisense oligonucleotides (MOs) to the RS neurons significantly reduced retrograde apoptosis signaling in identified RS neurons post-SCI, as indicated by Fluorochrome Labeled Inhibitor of Caspases (FLICA) in brain wholemounts. In individual RS neurons, the reduction of caspase activation by RhoA knockdown began at 2weeks post-TX and was still seen at 8weeks. RhoA knockdown slowed axon retraction and possibly increased early axon regeneration in the proximal stump. The number of axons regenerating beyond the lesion more than 5mm at 10weeks post-TX also was increased. Thus RhoA knockdown both enhanced true axon regeneration and inhibited retrograde apoptosis signaling after SCI.

Project description:Promoting the combination of robust regeneration of damaged axons and synaptic reconnection of these growing axon populations with appropriate neuronal targets represents a major therapeutic goal following spinal cord injury (SCI). A key impediment to achieving this important aim includes an intrinsic inability of neurons to extend axons in adult CNS, particularly in the context of the chronically-injured spinal cord. We tested whether an inhibitory peptide directed against phosphatase and tensin homolog (PTEN: a central inhibitor of neuron-intrinsic axon growth potential) could restore inspiratory diaphragm function by reconnecting critical respiratory neural circuitry in a rat model of chronic cervical level 2 (C2) hemisection SCI. We found that systemic delivery of PTEN antagonist peptide 4 (PAP4) starting at 8 weeks after C2 hemisection promoted substantial, long-distance regeneration of injured bulbospinal rostral Ventral Respiratory Group (rVRG) axons into and through the lesion and back toward phrenic motor neurons (PhMNs) located in intact caudal C3-C5 spinal cord. Despite this robust rVRG axon regeneration, PAP4 stimulated only minimal recovery of diaphragm function. Furthermore, re-lesion through the hemisection site completely removed PAP4-induced functional improvement, demonstrating that axon regeneration through the lesion was responsible for this partial functional recovery. Interestingly, there was minimal formation of putative excitatory monosynaptic connections between regrowing rVRG axons and PhMN targets, suggesting that (1) limited rVRG-PhMN synaptic reconnectivity was responsible at least in part for the lack of a significant functional effect, (2) chronically-injured spinal cord presents an obstacle to achieving synaptogenesis between regenerating axons and post-synaptic targets, and (3) addressing this challenge is a potentially-powerful strategy to enhance therapeutic efficacy in the chronic SCI setting. In conclusion, our study demonstrates a non-invasive and transient pharmacological approach in chronic SCI to repair the critically-important neural circuitry controlling diaphragmatic respiratory function, but also sheds light on obstacles to circuit plasticity presented by the chronically-injured spinal cord.

Project description:Transected axons fail to regrow across anatomically complete spinal cord injuries (SCI) in adults. Diverse molecules can partially facilitate or attenuate axon growth during development or after injury1-3, but efficient reversal of this regrowth failure remains elusive4. Here we show that three factors that are essential for axon growth during development but are attenuated or lacking in adults-(i) neuron intrinsic growth capacity2,5-9, (ii) growth-supportive substrate10,11 and (iii) chemoattraction12,13-are all individually required and, in combination, are sufficient to stimulate robust axon regrowth across anatomically complete SCI lesions in adult rodents. We reactivated the growth capacity of mature descending propriospinal neurons with osteopontin, insulin-like growth factor 1 and ciliary-derived neurotrophic factor before SCI14,15; induced growth-supportive substrates with fibroblast growth factor 2 and epidermal growth factor; and chemoattracted propriospinal axons with glial-derived neurotrophic factor16,17 delivered via spatially and temporally controlled release from biomaterial depots18,19, placed sequentially after SCI. We show in both mice and rats that providing these three mechanisms in combination, but not individually, stimulated robust propriospinal axon regrowth through astrocyte scar borders and across lesion cores of non-neural tissue that was over 100-fold greater than controls. Stimulated, supported and chemoattracted propriospinal axons regrew a full spinal segment beyond lesion centres, passed well into spared neural tissue, formed terminal-like contacts exhibiting synaptic markers and conveyed a significant return of electrophysiological conduction capacity across lesions. Thus, overcoming the failure of axon regrowth across anatomically complete SCI lesions after maturity required the combined sequential reinstatement of several developmentally essential mechanisms that facilitate axon growth. These findings identify a mechanism-based biological repair strategy for complete SCI lesions that could be suitable to use with rehabilitation models designed to augment the functional recovery of remodelling circuits.

Dataset Information

PTP? Knockdown in Lampreys Impairs Reticulospinal Axon Regeneration and Neuronal Survival After Spinal Cord Injury.

Publications

PTPσ Knockdown in Lampreys Impairs Reticulospinal Axon Regeneration and Neuronal Survival After Spinal Cord Injury.

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