Expression of regeneration-related molecules in injured and regenerating striatal and nigral neurons.

Peripheral nerve grafts in the neostriatum promote axonal regeneration from restricted classes of CNS neuron, principally cells in the substantia nigra pars compacta (SNpc) and striatal cholinergic interneurons. We have examined the molecular responses of CNS neurons induced to regenerate axons by tibial nerve grafting to the neostriatum of adult rats. Brain sections were probed for mRNAs for the transcription factor c-jun, and the cell recognition molecule CHL1, or immunoreacted for TrkA or p75, 1 day to 29 weeks after grafting (dpo; wpo). In unoperated rats, scattered neurons throughout the neostriatum showed weak signals for CHL1 mRNA and slightly stronger signals for c-jun mRNA. Cells of similar appearance strongly expressed TrkA but possessed little p75. By 1 dpo, many neostriatal neurons of various sizes and GFAP + glial cells near the host/graft interface had upregulated CHL1 mRNA, c-jun mRNA and p75. Most of the larger (20-25 microm diameter) CHL1 mRNA+ cells were also TrkA+, indicating that they were NGF-sensitive cholinergic interneurons. From two weeks postgrafting, high levels of CHL1 and c-jun mRNAs and p75 in the neostriatum were confined to a few presumptive cholinergic interneurons; p75+ cells were also TrkA+ and were larger than TrkA+ neurons on the contralateral side. Retrograde labelling showed that most p75+ and some TrkA+ neurons regenerated axons through the graft. Neurons in the SNpc showed a moderate to strong signal for CHL1 mRNA, weaker signal for c-jun mRNA, and no p75 or TrkA. Some SNpc cells upregulated c-jun mRNA after graft implantation, although they did not upregulate CHL1 mRNA, p75 or TrkA. Since neostriatal neurons which regenerate axons into grafts express receptors for NGF, and grafts mimic the effects of NGF treatment on these cells, sensitivity to graft-derived NGF may be a determinant of their high regenerative capacity. The finding that c-jun and CHL1 are consistently expressed by CNS neurons induced to regenerate their axons strongly supports the idea that these molecules are directly involved in axonal regeneration.

Axonal regeneration from CNS neurons in the cerebellum and brainstem of adult rats: correlation with the patterns of expression and distribution of messenger RNAs for L1, CHL1, c-jun and growth-associated protein-43.

Some neurons in the brain and spinal cord will regenerate axons into a living peripheral nerve graft inserted at the site of injury, others will not. We have examined the patterns of expression of four molecules thought to be involved in developmental and regenerative axonal growth, in the cerebellum and brainstem of adult rats, following the implantation into the cerebellum of peripheral nerve grafts. We also determined how the expression patterns observed correlate with the abilities of neurons in these regions to regenerate axons. Three days to 16 weeks after insertion of living tibial nerve autografts, neurons which had regenerated axons into the graft were retrogradely labelled from the distal extremity of the graft with cholera toxin conjugated to horseradish peroxidase, and sections through the cerebellum and brainstem were processed for visualization of transported tracer and/or hybridized with riboprobes to detect messenger RNAs for the cell recognition molecules L1 and CHL1 (close homologue of L1), growth-associated protein-43 and the cellular oncogene c-jun. Retrogradely labelled neurons were present in cerebellar deep nuclei close to the graft and in brainstem nuclei known to project to the cerebellum. Neurons in these same nuclei were found to have up-regulated expression of all four messenger RNAs. Individual retrogradely labelled neurons also expressed high levels of L1, CHL1, c-jun or growth-associated protein-43 messenger RNAs (and vice versa), and every messenger RNA investigated was co-localized with at least one other messenger RNA. Purkinje cells did not regenerate axons into the graft or up-regulate L1, CHL1 or growth-associated protein-43 messenger RNAs, but there was increased expression of c-jun messenger RNA in some Purkinje cells close to the graft. Freeze-killed grafts produced no retrograde labelling of neurons, and resulted in only transient and low levels of up-regulation of the tested molecules, mainly L1 and CHL1. These findings show that cerebellar deep nucleus neurons and precerebellar brainstem neurons, but not Purkinje cells, have a high propensity for axon regeneration, and that axonal regeneration by these neurons is accompanied by increased expression of L1, CHL1, c-jun and growth-associated protein-43. Furthermore, although the patterns of expression of the four molecules investigated are not identical in regenerating neuronal populations, it is probable that all four are up-regulated in all neurons whose axons regenerate into the grafts and that their up-regulation may be required for axon regeneration to occur. Finally, because c-jun up-regulation is seen in Purkinje cells close to the graft, unaccompanied by up-regulation of the other molecules investigated, c-jun up-regulation alone cannot be taken to reliably signify a regenerative response to axotomy.

The cell recognition molecule CHL1 is strongly upregulated by injured and regenerating thalamic neurons.

Close homologue of L1 (CHL1) is a cell recognition molecule known to promote axonal growth in vitro. We have investigated the expression of CHL1 mRNA by regenerating central nervous system (CNS) neurons, by using in situ hybridisation 3 days to 10 weeks following the implantation of living and freeze-killed peripheral nerve autografts into the thalamus of adult rats. At all survival times after implantation of living grafts, neurons of the thalamic reticular nucleus (TRN), close to the graft tip and up to 1 mm away from it, displayed strong signal for CHL1 mRNA, even though TRN neurons show very low levels of CHL1 mRNA expression in unoperated animals. When the cell bodies of regenerating neurons were identified by retrograde labelling from the distal portion of the grafts, 4-6 weeks after operation, most of the labelled cells were found in the TRN and could be shown to haveupregulated CHL1 mRNA. In addition, some neurons in dorsal thalamic nuclei near the graft tip transiently upregulated CHL1 mRNA during the first 3 weeks after graft implantation, and glial cells showing CHL1 mRNA expression were present at the brain/graft interface 3 days to 2 weeks after operation. Freeze-killed grafts, into which axons do not regenerate, caused a transient upregulation of CHL1 in very few TRN neurons near the graft tip and in glial cells at the brain/graft interface but did not produce prolonged CHL1 mRNA expression. CHL1 can therefore be added to the list of molecules (including GAP-43, L1, and c-jun) strongly expressed by CNS neurons that regenerate their axons into nerve grafts, but not by those neurons that fail to regenerate their axons.