The expression of chemorepulsive guidance receptors and the regenerative abilities of spinal-projecting neurons after spinal cord injury.

Spinal cord injury (SCI) in mammals leads to permanent loss of function because axons do not regenerate in the central nervous system (CNS). To date, treatments based on neutralizing inhibitory environmental cues, such as the myelin-associated growth inhibitors and chondroitin sulfate proteoglycans, or on adding neurotrophic factors, have had limited success in enhancing regeneration. Published studies suggested that multiple axon guidance cues (repulsive guidance molecule (RGM) family, semaphorins, ephrins, and netrins) persist in adult animals, and that their expression is upregulated after CNS injury. Moreover, many adult CNS neurons continue to express axon guidance receptors. We used the advantages of the lamprey CNS to test the hypotheses that the regenerative abilities of spinal-projecting neurons depend upon their expression of chemorepulsive guidance receptors. After complete spinal transection, lampreys recover behaviorally, and injured axons grow selectively in their correct paths. However, the large identified reticulospinal (RS) neurons in the lamprey brain are heterogeneous in their regenerative abilities - some are high regeneration capacity neurons (probability of axon regeneration >50%), others are low regeneration capacity neurons (<30%). Here we report that the RGM receptor Neogenin is expressed preferentially in the low regeneration capacity RS neurons that regenerate poorly, and that downregulation of Neogenin by morpholino antisense oligonucleotides enhances regeneration of RS axons after SCI. Moreover, lamprey CNS neurons co-express multiple guidance receptors (Neogenin, UNC5 and PlexinA), suggesting that the regenerative abilities of spinal-projecting neurons might reflect the summed influences of the chemorepulsive guidance receptors that they express.

Differential expression of HDACs and KATs in high and low regeneration capacity neurons during spinal cord regeneration.

After spinal cord injury (SCI) in mammals, injured axons fail to regenerate. By contrast, lampreys recover from complete spinal transection and axons regenerate selectively in their correct paths. Yet the large, identified reticulospinal neurons in the lamprey brain vary greatly in their regenerative abilities - some have high regeneration capacity (probability of regeneration >50%) and others have low regeneration capacity (<30%) - even though they have similar projection paths. The presence of both regenerating and non-regenerating neurons located in the same brain region and projecting to the same axon tracts suggests that differences in their regenerating abilities depend upon factors intrinsic to the neurons. Previous work has suggested that axon regeneration, especially in PNS, could depend on epigenetic mechanisms of histone modifications, such as the acetylation of histone tails. Our data indicated that expression of the enzymes responsible for regulating the acetylation of histone (KATs and HDACs) - KAT2A, KAT5 and P300 and HDAC3 did not change after SCI in either high regeneration capacity or low regeneration capacity neurons. In the present report, we show a novel and unexpected relationship between neuron regeneration abilities and expression of HDAC1. While HDAC1 expression was downregulated in both high and low regeneration capacity neurons 2 and 4weeks after SCI, it was upregulated at 7weeks at almost all RS neurons. However, at 10weeks post-transection only high regeneration capacity neurons displayed elevated HDAC1 mRNA expression and HDAC1 expression was again downregulated in low regeneration capacity neurons. Moreover, we show that HDAC1 is preferentially expressed in regenerated neurons, but not in non-regenerating neurons. Together, these results suggest that SCI causes significant changes in HDAC1 expression and that HDAC1 expression in regenerating neurons may modulates a survival or regeneration programs.

The sea lamprey UNC5 receptors: cDNA cloning, phylogenetic analysis and expression in reticulospinal neurons at larval and adult stages of development.

UNC5 receptors mediate repulsive signaling of netrin on neurons. Although only one UNC5 receptor has been identified in invertebrates, four members of the UNC5 family have been identified in gnathostomes. Lampreys, together with mixynes, belong to the oldest branch of extant vertebrates, and their phylogenetic position near to the vertebrate root makes them an interesting model for understanding molecular evolution. Here, we cloned three sea lamprey UNC5 (UNC5L) receptors, and phylogenetic analyses indicated that the first two duplications of the ancestral UNC5 gene occurred before the separation of jawless and jawed vertebrates. UNC5 receptors play important roles during early development, but expression studies have also suggested that UNC5 receptors play roles in the mature nervous system. Here, we report the expression of the different UNC5L receptor transcripts in identified reticulospinal neurons of mature larval or adult sea lampreys detected by in situ hybridization in wholemounted brain preparations. In addition, an extensive expression of the UNC5 receptors was also observed in most brain regions of the adult lamprey. An increase in the types of identifiable reticulospinal neurons expressing the UNC5L receptors was observed in adults compared with larvae. Expression of UNC5 receptors at late developmental stages appears to be a shared characteristic of lampreys and mammals. In larvae, expression of UNC5L receptors was observed in reticulospinal neurons that when axotomized are known to be "bad regenerators." Results in lampreys and mammals suggest that the UNC5-Netrin axonal guidance system may play a role in limiting axonal regeneration after spinal cord injury.

Multiple neurofilament subunits are present in lamprey CNS.

In mammals, there are three neurofilament (NF) subunits (NF-L, NF-M, and NF-H), but it was thought that only a single NF, NF180, exists in lamprey. However, NF180 lacked the ability to self-assemble, suggesting that like mammalian NFs, lamprey NFs are heteropolymers, and that additional NF subunits may exist. The present study provides evidence for the existence of a lamprey NF-L homolog (L-NFL). Genes encoding two new NF-M isoforms (NF132 and NF95) also have been isolated and characterized. With NF180, this makes three NF-M-like isoforms. In situ hybridization showed that all three newly cloned NFs are expressed in spinal cord neurons and in spinal-projecting neurons of the brainstem. Like NF180, there were no KSP multiphosphorylation repeat motifs in the tail regions of NF132 or NF95. NF95 was highly identical to homologous parts of NF180, sharing 2 common pieces of DNA with it. Northern blots suggested that NF95 may be expressed at very low levels in older larvae. The presence of L-NFL in lamprey CNS may support the hypothesis that as in mammals, NFs in lamprey are obligate heteropolymers, in which NF-L is a required subunit.

Expression of the repulsive guidance molecule RGM and its receptor neogenin after spinal cord injury in sea lamprey.

The sea lamprey recovers normal-appearing locomotion after spinal cord transection and its spinal axons regenerate selectively in their correct paths. However, among identified reticulospinal neurons some are consistently bad regenerators and only about 50% of severed reticulospinal axons regenerate through the site of injury. We previously suggested (Shifman, M. I., and Selzer, M. E., 2000a. Expression of netrin receptor UNC-5 in lamprey brain; modulation by spinal cord transection. Neurorehabilitation and Neural Repair 14, 49-58; Shifman, M. I., and Selzer, M. E., 2000b. In situ hybridization in wholemounted lamprey spinal cord: localization of netrin mRNA expression. Journal of Neuroscience Methods 104, 19-25) that selective chemorepulsion might explain why some neurons are bad regenerators and others not. To explore the role of additional chemorepulsive axonal guidance molecules during regeneration, we examined the expression of the repulsive guidance molecule (RGM) and its receptor neogenin by in situ hybridization and quantitative PCR. RGM mRNA was expressed in the spinal cord, primarily in neurons of the lateral gray matter and in dorsal cells. Following spinal cord transection, RGM message was downregulated in neurons close (within 10 mm) to the transection at 2 and 4 weeks, although it was upregulated in reactive microglia at 2 weeks post-transection. Neogenin mRNA expression was unchanged in the brainstem after spinal cord transection, and among the identified reticulospinal neurons, was detected only in "bad regenerators", neurons that are known to regenerate well never expressed neogenin. The downregulation of RGM expression in neurons near the transection may increase the probability that regenerating axons will regenerate through the site of injury and entered caudal spinal cord.