WCNR 2012 Poster Abstracts

Poster 377

Abstract 640

Does Ndel1 Promote Axon Regeneration?

Zhang KG¹, Li XM¹, Selzer ME^1,2

¹Shriners Hospitals Pediatric Research Center (Center for Neural Repair and Rehabilitation), Temple University School of Medicine, Philadelphia, USA

²Dept. of Neurology, Temple University School of Medicine, Philadelphia, USA

Background and Aims: In lampreys, unlike mammals, severed spinal axons regenerate. Their tips lack conventional growth cones and are densely packed with neurofilaments (NFs). Because the regenerative abilities of identified lamprey spinal-projecting neurons correlate with their expression of the NFs, synthesis, assembly and/or transport of NFs into the axon tip might generate a protrusive force that contributes to regeneration. How might this be coordinated? The mammalian nuclear distribution protein E-like 1 (Ndel1) has been viewed as an integrator of the cytoskeleton in axon regeneration. It was reported that Ndel1 facilitates the polymerization of NFs through a direct interaction with the NF light subunit (NFL). Thus Ndel1 could be important for NF assembly, transport and neuronal integrity. In mice, local silencing of Ndel1 by siRNA following sciatic nerve sectioning severely reduced the extent of regeneration. In order to investigate the role of Ndel1 in NFs assembly and axonal regeneration in lamprey, we have cloned lamprey Ndel1.

Methods: Genebank screening was used to identify lamprey homologs of Ndel1. A cDNA library was probed with RNA probes constructed from the discovered sequences. Spinal cords (SCs) were transected and at various recovery times, in situ hybridization (ISH) was performed, using these RNA probes.

Results: Two isoforms of Ndel1 were found. In one of them, 100 amino acids were deleted from the middle of the gene. Ndel1 was expressed in both neurons and glial cells. Similar to lamprey NFL, expression of Ndel1 protein and mRNA was decreased following SC transection, and recovered after 7 weeks selectively in neurons previously identified as “good regenerators.” Future experiments will determine whether the two Ndel1 isoforms are expressed differently in lamprey CNS, and if they are associated with NFL.

Conclusions: Ndel1 may be involved in the mechanism of axon regeneration in the lamprey SC.

Poster 393

Abstract 642

Retrograde Cell Death After Spinal Cord Injury is by the Extrinsic Apoptotic Pathway

Barreiro-Iglesias A¹, Selzer ME^1,2, Shifman MI

¹Shriners Hospitals Pediatric Research Center (Center for Neural Repair and Rehabilitation), Temple University School of Medicine, Philadelphia, USA

²Dept. of Neurology, Temple University School of Medicine, Philadelphia, USA

Background and Aims: Spinal cord injury (SCI) leads to permanent disability because in mammals, axons do not regenerate and thus cannot re-connect with appropriate targets. Moreover, axotomy often induces retrograde neuronal death or severe atrophy, which also limits recovery. We have used the lamprey reticulospinal system to determine the mechanisms of this as yet poorly understood retrograde cell death. Unlike in mammals, spinal transection in lampreys is followed by functional regeneration. Approximately 50% of severed reticulospinal axons regenerate through the site of injury. A subset of large reticulospinal neurons can be identified in whole-mount brain preparations. Some of these identified neurons are consistently “bad regenerators” and others “good regenerators.” Previously, we showed that after axotomy, many of the “bad regenerating” neurons undergo apoptotic cell death. The presence of active caspases is a widely accepted marker of apoptosis. The present study was aimed at identifying the specific apoptotic pathway activated in spinal projecting neurons in the lamprey brainstem after SCI.

Methods: We used specific fluorochrome labelled inhibitors of caspases (FLICA) to identify the caspases that are activated after spinal cord transection.

Results: Caspase-8, but not caspase-9, was activated in spinal-projecting neurons at 1 to 2 weeks post-transection. The caspase-8 activation began at the cut axon tip and moved centripetally to the cell body. Experiments using the microtubule stabilizer Taxol showed that the retrograde activation of caspase-8 depends on microtubule retrograde transport. Activated caspase-3/7 was observed in these neurons 10 weeks after axotomy.

Conclusions: These results show that SCI results in slow activation of the extrinsic or death receptor pathway of apoptosis in lamprey spinal-projecting neurons. Future studies will investigate this process in mammalian models of chronic SCI.

Poster 394

Abstract 643

Role of Chondroitin Sulfate Proteoglycans in Spinal Cord Regeneration

Zhang KG¹, Li XM1, Selzer ME^1,2

¹Shriners Hospitals Pediatric Research Center (Center for Neural Repair and Rehabilitation), Temple University School of Medicine, Philadelphia, USA

²Dept. of Neurology, Temple University School of Medicine, Philadelphia, USA

Background and Aims: Disability following spinal cord injury (SCI) is due to failure of axonal regeneration. Axon growth is inhibited by the presence in CNS of several inhibitory molecules, including chondroitin sulfate proteoglycans (CSPG). Digestion of CSPG with chondroitinase ABC (ChABC) enhanced axon growth and functional recovery after SCI in mammals. However, it is not known whether this involved true regeneration of injured axons or collateral sprouting by spared axons. Although sprouting may have beneficial effects in partial lesions, true regeneration might be needed in complete injury. The unique advantages of the lamprey were used to determine whether digesting CSPG can enhance true regeneration of severed spinal axons or only induce compensatory sprouting by uninjured fibers, and if so, whether expression CSPG receptors contributes to determining which neurons are “bad regenerators.”

Methods: Genebank screening was used to identify lamprey homologs of CSPGs and their receptors, protein tyrosine phosphatase sigma (PTPσ) and LAR. Spinal cords (SCs) were hemisected and ChABC applied to the transection site, together with Rhodamine-dextran to label cut reticulospinal axons. Three weeks later a complete transection was performed 1 cm caudal to the hemisection to label the previously spared axons with Alexa 488-dextran. CSPGs and their digestion products were labelled by immunohistochemistry and their mRNA expressions determined by in situ hybridization.

Results: CSPGs were expressed in a distribution similar to the perineuronal nets of mammalian SC. CSPG expression initially increased at the TX site, then decreased by 8 weeks. PTPσ and LAR were expressed in spinal-projecting neurons, and this expression increased with age and specific to “bad-regenerators.” No collateral spouting was seen from spared axons caudal to the hemisection. However, regeneration of cut axons appeared to be enhanced by ChABC.

Conclusions: Bad-regenerating axons selectively express receptors for CSPG and digestion of CSPGs enhances axon regeneration.