Intracelluar mediators of axonal sprouting in vivo

Generation of new axonal sprouts and the process of axonal elongation play a vital role in neural regeneration and repair. The facial nerve axotomy model is a well-established, prototypical experimental paradigm for the systematic study of nerve regeneration and degeneration, providing insights into molecular signals that determine axonal regeneration and neuronal cell death. Interestingly, this model of peripheral injury induces a delayed appearance of galanin+ and calcitonin gene-related peptide+ (CGRP) neuropeptide-immunoreactive growth cones in the facial nucleus which peak at 14 days following axotomy and surprisingly, increase in number if recut within this time window. Furthermore, application of the retrograde tracer mini-ruby to the distal portion of the cut nerve demonstrates the motoneuron origin of these sprouting neurites. To gain an insight into the molecular mechanisms inducing the sprouting response we examined how neuronal cell death and the inflammatory response of various transgenics affected sprouting and regeneration. Deletion of the a7 integrin, which demonstrated a moderate reduction in regeneration, showed enhanced sprouting; neural c-jun blocked regeneration, abolished regenerationassociated neuronal proteins and neuronal cell death, also completely eliminated central axonal sprouting. Absence of TNFR1&2 which displayed reduced neuronal cell death and inflammation, showed a tendency toward enhanced sprouting; TGF(31 deletion, which showed an elevated inflammatory response and a 4-fold increase in neuronal cell death, resulted in decreased central sprouting. Similarly, enhanced neural inflammation following systemic injection of E.coli lipopolysaccharide (LPS) also reduced central sprouting. Finally, neuronally expressed constitutively active Ras (Ras+), dominantnegative MEK1 (MEKIdn) and Ras+xMEK1dn double mutant (DM) all demonstrated reduced neuronal cell death as well as substantially enhanced central sprouting, particularly in the MEKIdn mutant, suggesting that the sprouting response in these mutants may be beneficial for improving regeneration in the CNS. Sprouting and regeneration studies in Ras+ and MEKIdn mutants were therefore extended to the injured corticospinal tract (CST) and rubrospinal tract. These mutants showed extensive collateral sprouting of corticospinal tract (CST) axons, in the grey and white matter on the ipsilateral side in Ras+,