Caspase-2 is upregulated after sciatic nerve transection and its inhibition protects dorsal root ganglion neurons from Apoptosis after serum withdrawal

Sciatic nerve (SN) transection-induced apoptosis of dorsal root ganglion neurons (DRGN) is one factor determining the efficacy of peripheral axonal regeneration and the return of sensation. Here, we tested the hypothesis that caspase-2(CASP2) orchestrates apoptosis of axotomised DRGN both in vivo and in vitro by disrupting the local neurotrophic supply to DRGN. We observed significantly elevated levels of cleaved CASP2 (C-CASP2), compared to cleaved caspase-3 (C-CASP3), within TUNEL+DRGN and DRG glia (satellite and Schwann cells) after SN transection. A serum withdrawal cell culture model, which induced 40% apoptotic death in DRGN and 60% in glia, was used to model DRGN loss after neurotrophic factor withdrawal. Elevated C-CASP2 and TUNEL were observed in both DRGN and DRG glia, with C-CASP2 localisation shifting from the cytosol to the nucleus, a required step for induction of direct CASP2-mediated apoptosis. Furthermore, siRNAmediated downregulation of CASP2 protected 50% of DRGN from apoptosis after serum withdrawal, while downregulation of CASP3 had no effect on DRGN or DRG glia survival. We conclude that CASP2 orchestrates the death of SN-axotomised DRGN directly and also indirectly through loss of DRG glia and their local neurotrophic factor support. Accordingly, inhibiting CASP2 expression is a potential therapy for improving both the SN regeneration response and peripheral sensory recovery

In vitro and in vivo effects on neural crest stem cell differentiation by conditional activation of Runx1 short isoform and its effect on neuropathic pain behavior

INTRODUCTION: Runx1, a Runt domain transcription factor, controls the differentiation of nociceptors that express the neurotrophin receptor Ret, regulates the expression of many ion channels and receptors, and controls the lamina-specific innervation pattern of nociceptive afferents in the spinal cord. Moreover, mice lacking Runx1 exhibit specific defects in thermal and neuropathic pain. We investigated whether conditional activation of Runx1 short isoform (Runx1a), which lacks a transcription activation domain, influences differentiation of neural crest stem cells (NCSCs) in vitro and in vivo during development and whether postnatal Runx1a activation affects the sensitivity to neuropathic pain. METHODS: We activated ectopic expression of Runx1a in cultured NCSCs using the Tet-ON gene regulatory system during the formation of neurospheres and analyzed the proportion of neurons and glial cells originating from NCSCs. In in vivo experiments we applied doxycycline (DOX) to pregnant mice (days 8-11), i.e. when NCSCs actively migrate, and examined the phenotype of offsprings. We also examined whether DOX-induced activation of Runx1a in adult mice affects their sensitivity to mechanical stimulation following a constriction injury of the sciatic nerve. RESULTS: Ectopic Runx1a expression in cultured NCSCs resulted in predominantly glial differentiation. Offsprings in which Runx1a had been activated showed retarded growth and displayed megacolon, pigment defects, and dystrophic dorsal root ganglia. In the neuropathic pain model, the threshold for mechanical sensitivity was markedly increased following activation of Runx1a. CONCLUSION: These data suggest that Runx1a has a specific role in NCSC development and that modulation of Runx1a activity may reduce mechanical hypersensitivity associated with neuropathic pain

Astrocytic Ca2+ Waves Guide CNS Growth Cones to Remote Regions of Neuronal Activity

Activity plays a critical role in network formation during developmental, experience-dependent, and injury related remodeling. Here we report a mechanism by which axon trajectory can be altered in response to remote neuronal activity. Using photoconductive stimulation to trigger high frequency action potentials in rat hippocampal neurons in vitro, we find that activity functions as an attractive cue for growth cones in the local environment. The underlying guidance mechanism involves astrocyte Ca2+ waves, as the connexin-43 antagonist carbenoxolone abolishes the attraction when activity is initiated at a distance greater than 120 µm. The asymmetric growth cone filopodia extension that precedes turning can be blocked with CNQX (10 µM), but not with the ATP and adenosine receptor antagonists suramin (100 µM) and alloxazine (4 µM), suggesting non-NMDA glutamate receptors on the growth cone mediate the interaction with astrocytes. These results define a potential long-range signalling pathway for activity-dependent axon guidance in which growth cones turn towards directional, temporally coordinated astrocyte Ca2+ waves that are triggered by neuronal activity. To assess the viability of the guidance effect in an injury paradigm, we performed the assay in the presence of conditioned media from lipopolysaccharide (LPS) activated purified microglial cultures, as well as directly activating the glia present in our co-cultures. Growth cone attraction was not inhibited under these conditions, suggesting this mechanism could be used to guide regeneration following axonal injury