Patodia, S; (2013) Transcriptional regulation and downstream effectors of peripheral nerve regeneration. Doctoral thesis (PhD), UCL (University College London).

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Abstract

Unlike the central nervous system, robust axon regeneration after peripheral nerve injury is driven by transcriptional activation of a regeneration programme. Transcription factors form a vital link in the chain of regeneration, converting injuryinduced stress signals into increased expression of a wide range of downstream effector molecules like neuropeptides, neurotrophic factors, adhesion molecules and cytoskeletal adaptor proteins. To gain insight into the in vivo function of some of these regeneration associated molecules, we examined the effects of global or celltype specific, and single and combined deletions of transcription factors - STAT3, c- Jun and C/EBPδ, and effector molecules - CAP23 and integrin β1, on axonal regeneration after facial nerve axotomy. The facial nerve axotomy model is a wellestablished experimental paradigm, providing insights into molecular signals that determine axonal regeneration, target re-innervation and neuronal cell death. Neuronal STAT3 deletion (STAT3ΔS) caused profound and persistent defects in regeneration and functional recovery after mild (crush) and severe (cut) nerve injuries. Axotomised STAT3-deficient motoneurons appeared shrunk by 50-60% in size but displayed no cell death. There was also a severe reduction in microglial activation and recruitment of lymphocytes, in the expression of regenerationassociated molecules CD44, α7β1 integrin, in the nuclear translocation of ATF3, and in perineuronal sprouting of the CGRP+ and galanin+ facial motoneurons. Deletion of STAT3 in Schwann cells produced no apparent deficiencies in regeneration. As with neuronal STAT3 deletion, neuronal deletion of c-Jun (c-JunΔS) (Raivich et al, 2004; Ruff et al, 2012) abolishes most of the cell body response, neuronal cell death, and re-innervation after axotomy. Deletion of c-Jun in peripheral nerve Schwann cells (c-JunΔP0) had a very different effect. Neuronal cell death was increased by 2- 3 fold, even though most of the cell body response was not affected. Axonal regeneration was reduced, but most of the defect in target re-innervation and functional recovery appeared to be due to excessive neuronal cell death. Mutants lacking both neuronal c-jun and STAT3 did not show more exacerbated regeneration defects than the single deletions, suggesting that deletion of either 4 transcription factor – c-Jun or STAT3 – will produce a saturating effect on the regeneration-deficient phenotype. Expression of c-jun itself was not affected in the neuronal STAT3 null mice, and vice versa, confirming the above hypothesis. Double deletion of c-jun in both neurons and in Schwann cells, blocked neuronal cell death seen in Schwann cell c-Jun deficient mutants, and completely obliterated target re-innervation after facial axotomy, suppressing even the mild 20% regeneration seen in the neuronal c-jun null mutants. Since both neuronal and Schwann cell c-Jun are phosphorylated at its N-terminus following nerve injury, we also explored the effects of c-Jun phosphorylation. Global replacement of all 4 Nterminal c-Jun phosphorylation sites (Ser63&73, Thr91&93) with alanines (c-jun4A) produced a significant increase (1.8x) in neuronal cell death, an approximate 40% reduction in target re-innervation and delayed functional recovery. This phenotype was more alike that observed with Schwann cell rather than neuronal c-Jun deletion. Global C/EBPδ deletion reduced axotomy induced neuronal cell death, and had moderate effects on microglial activation and axonal sprouting. Combined deletion of C/EBPδ and STAT3 did not exacerbate the defect in regeneration seen with STAT3 deletion alone, but seemed to speed up functional recovery. Deletion of neuronal CAP23, a downstream effector gene, led to impaired target reinnervation, reduced galanin+ perineuronal sprouting and early microglial function. Neuron-specific deletion of beta 1 integrin, another effector molecule, resulted in a 2.5-fold increase in neuronal cell death, a commensurate 60% reduction in target reinnervation and transiently delayed functional recovery. This phenotype was similar to c-junΔP0 and c-jun4A mutants. In summary, there appears to be two neuronal sub-populations which are controlled by different aspects of the regenerative programme A) one dependent on ‘intrinsic’ activation of transcriptional master switches like c-jun and STAT3 in neurons and B) the other on ‘extrinsic’ post-traumatic trophic signalling elicited by the Jun-expressing and N-terminal phosphorylation-dependent Schwann cells. c-Jun phosphorylation and neuronal β1 integrin appear to be critical co-factors in the signalling response elicited by the Schwann cells.

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