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Activated Erk Is an Early Retrograde Signal After Spinal Cord Injury in the Lamprey.

ABSTRACT: We previously reported that spinal cord transection (TX) in the lamprey causes mRNA to accumulate in the injured tips of large reticulospinal (RS) axons. We sought to determine whether this mRNA accumulation results from phosphorylation and transport of retrograde signals, similar to what has been reported in mammalian peripheral nerve. Extracellular signal-regulated protein kinase (Erk), mediates the neurite outgrowth-promoting effects of many neurotrophic factors. To assess the role of Erk in retrograde signaling of RS axon injury, we used immunoblot and immunohistochemistry to determine the changes in phosphorylated Erk (p-Erk) in the spinal cord after spinal cord TX. Immunostaining for p-Erk increased within axons and local cell bodies, most heavily within the 1-2 mm closest to the TX site, at between 3 and 6 h post-TX. In axons, p-Erk was concentrated in 3-5 ?m granules that became less numerous with distance from the TX. The retrograde molecular motor dynein colocalized with p-Erk, but vimentin, which in peripheral nerve was reported to participate with p-Erk as part of a retrograde signal complex, did not colocalize with p-Erk, even though vimentin levels were elevated post-TX. The results suggest that p-Erk, but not vimentin, may function as a retrograde axotomy signal in lamprey central nervous system neurons, and that this signal may induce transcription of mRNA, which is then transported down the axon to its injured tip.

SUBMITTER: Jin LQ

PROVIDER: S-EPMC7674770 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Activated Erk Is an Early Retrograde Signal After Spinal Cord Injury in the Lamprey.

Jin Li-Qing LQ John Brittany H BH Hu Jianli J Selzer Michael E ME

Frontiers in neuroscience 20201105

We previously reported that spinal cord transection (TX) in the lamprey causes mRNA to accumulate in the injured tips of large reticulospinal (RS) axons. We sought to determine whether this mRNA accumulation results from phosphorylation and transport of retrograde signals, similar to what has been reported in mammalian peripheral nerve. Extracellular signal-regulated protein kinase (Erk), mediates the neurite outgrowth-promoting effects of many neurotrophic factors. To assess the role of Erk in ...[more]

PMID: 33250705

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Project description:Previously, we reported that RhoA knockdown by morpholino antisense oligonucleotides (MOs), and enzymatic digestion of chondroitin sulfate proteoglycans (CSPGs) at the site of injury with chondroitinase ABC (ChABC), each can reduce retrograde neuronal apoptosis after spinal cord transection in the lamprey. To elucidate the mechanisms in neuronal survival and axon regeneration, we have investigated whether these two effects are additive in vivo. We used lampreys as a spinal cord injury model. MOs were used to knockdown RhoA and Chondroitinase ABC (ChABC) was used to digest CSPGs in vivo. Retrograde labeling, fluorochrome-labeled inhibitor of caspase activity (FLICA), immunohistochemistry, and western blots were performed to assess axonal regeneration, neuronal apoptotic signaling and Akt activation. Four treatment combinations were evaluated at 2-, 4-, and 10-weeks post-transection: (1) Control MO plus enzyme buffer (Ctrl); (2) control MO plus ChABC; (3) RhoA MO plus enzyme buffer (RhoA MO); and (4) RhoA MO plus ChABC (RhoA MO + ChABC). Consistent with our previous findings, at 4-weeks post-transection, there was less caspase activation in the ChABC and RhoA MO groups than in the Ctrl group. Moreover, the RhoA MO plus ChABC group had the best protective effect on identified reticulospinal (RS) neurons among the four treatment combinations. At 2 weeks post-transection, when axons have retracted maximally in the rostral stump and are beginning to regenerate back toward the lesion, the axon tips in the three treatment groups each were closer to the transection than those in the Ctr MO plus enzyme buffer group. Long-term axon regeneration also was evaluated for the large, individually identified RS neurons at 10 weeks post-transection by retrograde labeling. The percent regenerated axons in the RhoA MO plus ChABC group was greater than that in any of the other groups. Akt phosphorylation levels at threonine 308 was quantified in the identified RS neurons by western blots and immunofluorescence. The RhoA MO plus ChABC treatment enhanced pAkt-308 phosphorylation more than any of the other treatment groups. Although some of the effects of CSPGs are mediated through RhoA activation, some growth-inhibiting mechanisms of RhoA and CSPGs are independent of each other, so combinatorial therapies may be warranted.

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:Axon regrowth after spinal cord injury (SCI) is inhibited by several types of inhibitory extracellular molecules in the central nervous system (CNS), including chondroitin sulfate proteoglycans (CSPGs), which also are components of perineuronal nets (PNNs). The axons of lampreys regenerate following SCI, even though their spinal cords contain CSPGs, and their neurons are enwrapped by PNNs. Previously, we showed that by 2 weeks after spinal cord transection in the lamprey, expression of CSPGs increased in the lesion site, and thereafter, decreased to pre-injury levels by 10 weeks. Enzymatic digestion of CSPGs in the lesion site with chondroitinase ABC (ChABC) enhanced axonal regeneration after SCI and reduced retrograde neuronal death. Lecticans (aggrecan, versican, neurocan, and brevican) are the major CSPG family in the CNS. Previously, we cloned a cDNA fragment that lies in the most conserved link-domain of the lamprey lecticans and found that lectican mRNAs are expressed widely in lamprey glia and neurons. Because of the lack of strict one-to-one orthology with the jawed vertebrate lecticans, the four lamprey lecticans were named simply A, B, C, and D. Using probes that distinguish these four lecticans, we now show that they all are expressed in glia and neurons but at different levels. Expression levels are relatively high in embryonic and early larval stages, gradually decrease, and are upregulated again in adults. Reductions of lecticans B and D are greater than those of A and C. Levels of mRNAs for lecticans B and D increased dramatically after SCI. Lectican D remained upregulated for at least 10 weeks. Multiple cells, including glia, neurons, ependymal cells and microglia/macrophages, expressed lectican mRNAs in the peripheral zone and lesion center after SCI. Thus, as in mammals, lamprey lecticans may be involved in axon guidance and neuroplasticity early in development. Moreover, neurons, glia, ependymal cells, and microglia/macrophages, are responsible for the increase in CSPGs during the formation of the glial scar after SCI.

Project description:The number of elderly patients with spinal cord injury without radiographic abnormalities (SCIWORA) has been increasing in recent years and common of most cervical spinal cord injuries. Basic research has shown the effectiveness of early decompression after spinal cord injury on the spinal cord without stenosis; no studies have reported the efficacy of decompression in models with spinal cord compressive lesions. The purpose of this study was to evaluate the effects of decompression surgery after acute spinal cord injury in rats with chronic spinal cord compressive lesions, mimicking SCIWORA. A water-absorbent polymer sheet (Aquaprene DX, Sanyo Chemical Industries) was inserted dorsally into the 4-5th cervical sublaminar space in 8-week-old Sprague Dawley rats to create a rat model with a chronic spinal compressive lesion. At the age of 16 weeks, 30 mildly myelopathic or asymptomatic rats with a Basso, Beattie, and Bresnahan score (BBB score) of 19 or higher were subjected to spinal cord compression injuries. The rats were divided into three groups: an immediate decompression group (decompress immediately after injury), a sub-acute decompression group (decompress 1 week after injury), and a non-decompression group. Behavioral and histological evaluations were performed 4 weeks after the injury. At 20 weeks of age, the BBB score and FLS (Forelimb Locomotor Scale) of both the immediate and the sub-acute decompression groups were significantly higher than those of the non-decompression group. There was no significant difference between the immediate decompression group and the sub-acute decompression group. TUNEL (transferase-mediated dUTP nick end labeling) staining showed significantly fewer positive cells in both decompression groups compared to the non-decompression group. LFB (Luxol fast blue) staining showed significantly more demyelination, and GAP-43 (growth associated protein-43) staining tended to show fewer positive cells in the non-decompression group. Decompression surgery in the acute or sub-acute phase of injury is effective after mild spinal cord injury in rats with chronic compressive lesions. There was no significant difference between the immediate decompression and sub-acute decompression groups.

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Activated Erk Is an Early Retrograde Signal After Spinal Cord Injury in the Lamprey.

Publications

Activated Erk Is an Early Retrograde Signal After Spinal Cord Injury in the Lamprey.

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