FKBP12 mRNA expression is upregulated by intrinsic CNS neurons regenerating axons into peripheral nerve grafts in the brain.

We have examined the expression of the immunophilin FKBP12 in adult rat intrinsic CNS neurons stimulated to regenerate axons by the implantation of segments of autologous tibial nerve into the thalamus or cerebellum. After survival times of 3 days to 6 weeks, the brains were fresh-frozen. In some animals the regenerating neurons were retrogradely labelled with cholera toxin subunit B 1 day before they were killed. Sections through the thalamus or cerebellum were used for in situ hybridization with digoxygenin-labelled riboprobes for FKBP12 or immunohistochemistry to detect cholera toxin subunit B-labelled neurons. FKBP12 was constitutively expressed by many neurons, and was very strongly expressed in the hippocampus and by Purkinje cells. Regenerating neurons were found in the thalamic reticular nucleus and deep cerebellar nuclei of animals that received living grafts. Neurons in these nuclei upregulated FKBP12 mRNA; such neurons were most numerous at 3 days post grafting but were most strongly labelled at 2 weeks post grafting. Regenerating neurons identified by retrograde labelling were found to have upregulated FKBP12 mRNA. No upregulation was seen in neurons in animals that received freeze-killed grafts, which do not support axonal regeneration. We conclude that FKBP12 is a regeneration-associated gene in intrinsic CNS neurons.

Transcriptional upregulation of SCG10 and CAP-23 is correlated with regeneration of the axons of peripheral and central neurons in vivo.

We have compared SCG10 and CAP-23 expression with that of GAP-43 during axonal regeneration in the peripheral and central nervous systems (PNS, CNS) of adult rats. SCG10, CAP-23, and GAP-43 mRNAs were strongly upregulated by motor and dorsal root ganglion (DRG) neurons following sciatic nerve crush, but not after dorsal rhizotomy. When the sciatic nerve was cut and ligated to prevent reinnervation of targets, expression of all three mRNAs was prolonged. Neurons in the thalamic reticular nucleus and deep cerebellar nuclei transiently upregulated these mRNAs after axotomy, and showed prolonged upregulation of all three molecules when regenerating axons into peripheral nerve grafts inserted into the thalamus of cerebellum. Neurons in the dorsal thalamus and cerebellar cortex showed poor regenerative capacity and most did not upregulate any of these mRNAs. Thus, in both PNS and CNS neurons, the transcription of SCG10, CAP-23, and GAP-43 mRNAs is coregulated following axotomy and during regeneration. Signals from living peripheral nerve appear to maintain expression of all three mRNAs in regenerating neurons, and in PNS neurons downregulation correlates with target reinnervation. Thus, SCG10 and CAP-23, as well as GAP-43, are likely to be important neuronal determinants of regenerative ability.

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.

Overexpression of GAP-43 in thalamic projection neurons of transgenic mice does not enable them to regenerate axons through peripheral nerve grafts.

It is well established that some populations of neurons of the adult rat central nervous system (CNS) will regenerate axons into a peripheral nerve implant, but others, including most thalamocortical projection neurons, will not. The ability to regenerate axons may depend on whether neurons can express growth-related genes such as GAP-43, whose expression correlates with axon growth during development and with competence to regenerate. Thalamic projection neurons which fail to regenerate into a graft also fail to upregulate GAP-43. We have tested the hypothesis that the absence of strong GAP-43 expression by the thalamic projection neurons prevents them from regenerating their axons, using transgenic mice which overexpress GAP-43. Transgene expression was mapped by in situ hybridization with a digoxigenin-labeled RNA probe and by immunohistochemistry with a monoclonal antibody against the GAP-43 protein produced by the transgene. Many CNS neurons were found to express the mRNA and protein, including neurons of the mediodorsal and ventromedial thalamic nuclei, which rarely regenerate axons into peripheral nerve grafts. Grafts were implanted into the region of these nuclei in the brains of transgenic animals. Although these neurons strongly expressed the transgene mRNA and protein and transported the protein to their axon terminals, they did not regenerate axons into the graft, suggesting that lack of GAP-43 expression is not the only factor preventing thalamocortical neurons regenerating their axons.

The effects of a lesion or a peripheral nerve graft on GAP-43 upregulation in the adult rat brain: an in situ hybridization and immunocytochemical study.

We have sought to determine (1) if thalamic neurons upregulate the growth associated protein GAP-43 as a response to injury, or if a peripheral nerve graft is required to induce, enhance or sustain such a response, and (2) if thalamic neurons with different regenerative potentials also display different GAP-43 responses. Levels of GAP-43 protein (detected by LM and EM immunohistochemistry) and of GAP-43 mRNA (detected by in situ hybridization) were compared in the thalamus of adult rats between 1 d and 180 d after making a stab lesion or after implanting a peripheral nerve autograft. Stab injury is a sufficient stimulus to cause a transient upregulation in GAP-43 expression by neurons in the thalamus (both around the graft tip and in particular in the thalamic reticular nucleus) in the first week after injury but this response is both prolonged, and enhanced in the presence of a peripheral nerve graft. In addition, we demonstrate directly, by double labelling, that neurons of the thalamic reticular nucleus displaying high levels of the mRNA for GAP-43, have axons regenerating in the distal portion of the graft. These findings lend direct support to the hypothesis that upregulation of the GAP-43 gene is essential for prolonged regenerative axonal growth. We also demonstrate GAP-43 protein in graft Schwann cells and in brain astrocytes close to the site of graft implantation.

Differential regenerative growth of CNS axons into tibial and peroneal nerve grafts in the thalamus of adult rats.

Segments of peripheral nerve were autografted into the thalamus of adult rats. The peroneal nerve was used in one group, the tibial nerve (which has approximately twice the cross-sectional area of the peroneal nerve) in a second group, and two lengths of peroneal nerve side by side in a third group. Between 1 and 4 months later HRP was applied to the distal end of each graft to label neurons which had regenerated their axons into the graft. Serial coronal sections of each brain were reacted to reveal retrogradely transported HRP, and the positions of all labeled neurons were recorded in camera lucida drawings. In all three groups a few labeled neurons resembling thalamocortical projection cells were found in the dorsal thalamus close to the graft tip (mean number, 29 in the single peroneal group; 22 in the tibial group; and 14 in the double-peroneal group). However, neurons in the thalamic reticular nucleus (TRN) regenerated much more successfully into the larger nerve grafts; many more retrogradely labeled cells were found in animals with tibial or double-peroneal nerve grafts (mean number, 1.1 in the single-peroneal group; 272 in the tibial group; and 163 in the double-peroneal group). These neurons were concentrated in the sector of TRN known to project to the part of the dorsal thalamus containing the graft tip. The largest numbers of labeled neurons were found when the graft tip encroached upon the TRN. These results suggest that both graft size and graft position are critical determinants of the extent of axonal regeneration from the TRN. Larger grafts may be more copiously invaded by regenerating axons because such grafts damage larger numbers of TRN axons when implanted and/or because they stimulate regeneration by releasing critical quantities of neurotrophic factors.