Spinal motoneurons and molecules related to neurotrophic function after axon injury

The capacity of motoneurons to survive and regenerate new axons after injuries such as axotomy depends on a great variety of parameters. After sciatic nerve transection (SNT) in adult rats and to some extent after a ventral funiculus lesion (VFL), many cells survive and regeneration is highly prioritized. After SNT in neonatal rats or ventral root avulsion (VRA) in adult rats, on the other hand, a large proportion of the lesioned cells die and overall chances of regeneration are small. Differences in protein expression patterns of trophic factors, their receptors and receptors to extracellular matrix molecules, integrins, were studied in motoneurons and scar tissue after the mentioned types of injury. The effect of infiltrating inflammatory cells and substances produced by such cells was studied on rat motoneurons in vivo and in cell cultures, with the aim to determine some of the factors that have an influence on the outcome of the injured motoneuron.

In all lesion models the expression of mRNA encoding growth-associated protein (GAP-43) was highly upregulated. Alpha-CGRP was downregulated in response to a SNT in newborn rats, but was upregulated in two-week old and in adult rats. GAP-43 and CGRP mRNA expressions in spinal motoneurons are regulated, at least in part, by target-derived factors like CNTF and bFGF and may be nerve cell-derived substances that are involved in sprouting and regulation of neuromuscular junctions during development and regeneration.

The trophic factors glial cell-line derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF) and insulin-like growth factors (IGF-1 and -2) have been shown to be crucial for survival and regeneration of spinal motoneurons after various kinds of axotomy and in vitro. The mRNA expression in transected sciatic nerve was correlated to the regulation of their receptor subunits in spinal motoneurons and compared in the different lesion models. GDNF receptor alpha (GFR[alpha]1), c-RET and LIF receptor (LIFR) mRNAs were extensively upregulated after axotomy, with an earlier response after avulsion and highly prioritized expression of GFR[alpha]1 in surviving motoneurons. Levels of CNTF receptor alpha (CNTFR[alpha]) and the BDNF high affinity receptor trkB were rather unaffected. The largest difference in response between SNT and VRA in the adult rat was found for the NT-3 receptor trkC and membrane glycoprotein 130 (gpl30) mRNAs. Thus, after SNT there were only minor changes, while VRA induced a profound down-regulation.

IGF-1 is available for lesioned motoneurons both after peripheral and central axonal lesions, while significant IGF-2 expression was restricted to denervated nerve. Also, the regulation of IGF binding proteins (IGFBPs) are important. Thus, IGFBP-4 and IGFBP-5 were dominant in peripheral nerve, while IGFBP-2 and IGFBP-5 were preferentially found in the CNS. IGFBP-6 mRNA was strongly upregulated in spinal motoneurons and may be of special relevance for the severed cells, probably in relation to the IGF-2 expression in denervated nerve. Both integrin subunits [alpha]7 and ß1 were highly upregulated in spinal motoneurons after SNT, VRA and VFL and, in addition, integrin [alpha]6, which is not normally detectable. The levels of integrin 0 remained elevated longer after SNT than after VRA and VFL whereas integrin [alpha]6 displayed the opposite pattern. Interestingly, at 21 and 42 days after VFL, some motoneurons once again upregulated [alpha]7, possibly indicating a regeneration through the scar and contact with ventral roots.

Also taken in consideration was the lesion-accompanied local inflammatory response. The traditional view has been that the relative immune privilege of the CNS serves to protect delicate neuronal networks from damage by immune reactions, but herein is demonstrated that axotomy induces an upregulation of MHC I and ß2-microglobulin mRNA in motoneurons, indicating an active participation in the immune response after injury.

Experimental autoimmune encephalomyelitis (EAE) induced by active immunization with an encephalitogenic MBP peptide led to a robust survival promoting effect on avulsed motoneurons in spite of a very intense inflammatory reaction with high levels of pro-inflammatory cytokines in the lesioned segments. Furthermore, the expression of the neurotrophic factors BDNF, NT-3 and GDNF by encephalitogenic T and NK cells, may constitute an important mechanism for neuronal protection in CNS inflammation and is supported by the fact that interferon-[gamma] (IFN-[gamma]) or tumor necrosis factor-[alpha] (TNF-[alpha]) treatment increased death of motoneurons only in cultures deprived of neurotrophic support.

In conclusion, this thesis demonstrates a complex regulation of a network of substances that are decisive for the regenerative potential of motoneurons after mechanical nerve trauma.

List of scientific papers

I. Hammarberg H, Piehl F, Cullheim S, Fjell J, Hokfelt T, Fried K (1996). GDNF mRNA in Schwann cells and DRG satellite cells after chronic sciatic nerve injury. Neuroreport. 7(4): 857-860.
https://pubmed.ncbi.nlm.nih.gov/96332944

II. Hammarberg H, Risling M, Hokfelt T, Cullheim S, Piehl F (1998). Expression of insulin-like growth factors and corresponding binding proteins (IGFBP 1-6) in rat spinal cord and peripheral nerve after axonal injuries. J Comp Neurol. 400(1): 57-72.
https://pubmed.ncbi.nlm.nih.gov/98433830

III. Piehl F, Hammarberg H, Hokfelt T, Cullheim S (1998). Regulatory effects of trophic factors on expression and distribution of CGRP and GAP-43 in rat motoneurons. J Neurosci Res. 51(1): 1-14.
https://pubmed.ncbi.nlm.nih.gov/98112683

IV. Hammarberg H, Piehl F, Risling M, Cullheim S. (1970). Differential regulation of trophic factor and receptor mRNAs after peripheral and central axotomy of rat spinal motoneurons. [Submitted]

V. Hammarberg H, Wallquist W, Piehl F, Risling M, Cullheim S (1970). Regulation of laminin associated integrin subunit alfa6, alfa7 and beta1 in rat spinal motoneurons during postnatal development and after axonal injury. [Submitted]

VI. Lindå H, Hammarberg H, Cullheim S, Levinovitz A, Khademi M, Olsson T (1998). Expression of MHC class I and beta2-microglobulin in rat spinal motoneurons: regulatory influences by IFN-gamma and axotomy. Exp Neurol. 150(2): 282-295.

VII. Hammarberg H, Lidman O, Lundberg C , Eltayeb SY, Muhallab S, Svenningsson A, Lindå H, van Der Meide PH, Cullheim S, Olsson T, Piehl F. (1970). Experimental autoimmune encephalomyelitis protects mechanically injured neurons; Evidence for the production of neurotrophins by activated T and NK cells in the CNS. [Submitted]