Inhibition of stat/smad co-transcription factor P300 regulates differentiation of adult spinal cord neural progenitors and ameliorates functional recovery in chronic spinal cord

Abstract

Background: Endogenous neural progenitor cells (NPCs) are activated in the injured adult spinal cord (SC) and may contribute to neuronal and glial regeneration and prevention of astroglial scar formation. Extracellular signaling molecules secreted by reactive astrocytes such as BMPs and its antagonist noggin play critical role in the neurogenic process that follows spinal cord injury (SCI). JAK/STAT is one of the intracellular signaling pathways, in addition to BMP/Smad pathway, that is activated in reactive astoryctes after SCI. GSK-3β is a crucial activator of STAT3 and can be inhibited by lithium (LiCl). Inhibition of JAK/STAT and BMP/Smad pathways by LiCl and noggin respectively suppresses astrogliosis and promotes neurogenesis. p300 is found to bridge the STAT-Smad complex signaling. It is possible that the combinatory LiCl and noggin treatment enables concomitant suppression of glial scarring and reactivation of neurogensis and remylination, leading to functional recovery after chronic SCI. Objectives: To examine whether LiCl and/or noggin can modulate the proliferation and differentiation of NPCs in the injured adult SC, and to examine the effect of the combined treatment on astroglial scar formation, promotion of axonal regeneration and functional recovery in the chronically injured adult SC. Methods: Thoracic T8-T10 SC segments were dissected from 4-6-week-old Nestin-GFP transgenic mice 24 h after contusion injury. Second-passaged neurosphere cells were grown in differentiation medium with the addition of LiCl (0.5mM-2mM) or noggin (7.5ng/ml-30ng/ml) alone, or a combination of LiCl (1mM) and noggin (15ng/ml) for 7 days. Neurosphere cells were treated with LiCl ± noggin for 4 days before BrdU labeling. Contusiive spinal cord injury at T8-T10, with a force of 375 kdyne, was performed on 6-week-old Nestin-GFP transgenic mice. Four weeks later, osmotic minipumps delivering treatments (LiCl ± noggin) and vehicle were implanted in the injured mice for 2 weeks. Recovery of locomotor movements was assessed by BMS test 2 hours before the mice were sacrificed. Spinal cord tissue 5mm rostral and caudal from the injury epicenter was collected for immunostaining and immunoprecipitation. Result: The combined treatment increased the number of immature neurons, mature neurons, oligodendrocyte precursors and oligodendrocytes while decreased the number of astrocytes and microglial cells after chronic SCI. Astroglial scar formation was significantly attenuated and the animals demonstrated significantly better locomotor recovery. The expression of p300 together with Smad and STAT3 decreased significantly in differentiated NPCs treated by LiCl plus noggin. Conclusion: LiCl and noggin synergistically promoted neuronal and oligodendroglial differentiation of adult SC-derived NPCs and at the same time inhibited astroglia differentiation. Moreover, the synergistic effect is mediated through the downregulation of p300, the co-transcription factor for both the STAT/Smad signaling cascades, suggesting that p300 may be a potential therapeutic target for SCI.