Regulation and function of neuronal GTP-Ras in facial motor nerve regeneration.
Activation of Ras into the GTP-binding, 'ON' state is a key switch in the neurotrophin-mediated neuronal survival and neurite outgrowth, in vitro as well as in vivo. In the current study we explored changes in GTP-Ras levels following facial nerve injury and the ensuing regeneration and the effects of perturbing these changes in vivo using synapsin-promoter mediated neuronal expression of constitutively active Val12H-Ras (synRas). Quantification of GTP-Ras and total Ras revealed a precipitous drop in the relative GTP-Ras levels in the axotomized facial motor nucleus, to 40% of normal levels at 2 days after cut, followed by a partial recovery to 50-65% at 4-28 days. On western blots, control and axotomized nuclei from synRas mutants showed a 2.2- and 2.5-fold elevation in GTP-Ras, respectively, compared with their wild type littermate controls (p < 5%, anova, TUKEY post-hoc), with the levels in the axotomized synRas nucleus slightly but not significantly above that in the uninjured littermate control (p = 9.9%). Similar increase was also observed in the pERK but not pAKT targets of the Ras cascade. This moderate elevation of GTP-Ras strongly curtailed post-traumatic neuronal cell death (-65%), the influx of T-cells (-48%) as well as other parameters of neuroinflammatory response. Although synRas did not affect the speed of axonal regeneration or functional recovery it caused a very pronounced increase in central axonal sprouting. These current data emphasize the role of reduced active Ras, and by extension, the reduced overall level of retrograde neurotrophin signalling after axotomy, in mediating post-traumatic cell death and inflammation and in restricting the sprouting response. Moreover, the neuroprotective and central sprouting-enhancing effects of neuronal Val12H-Ras could help promote recovery in CNS injury.
Makwana
Milan
M
Perinatal Brain Repair Group, Department of Obstetrics & Gynaecology, EGA Institute of Women's Health, University College London, London, UK.
Serchov
Tsvetan
T
Hristova
Mariya
M
Bohatschek
Marion
M
Gschwendtner
Andreas
A
Kalla
Roger
R
Liu
Zhiqiang
Z
Heumann
Rolf
R
Raivich
Genndij
G
D000704:Analysis of Variance; D000818:Animals; D019771:Axotomy / Q000379:methods; D017252:CD3 Complex / Q000378:metabolism; D015740:Calcitonin Gene-Related Peptide / Q000378:metabolism; D004195:Disease Models, Animal; D004789:Enzyme Activation / Q000502:physiology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D020220:Facial Nerve Injuries / Q000235:genetics / Q000503:physiopathology*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D019004:Galanin / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009154:Mutation; D009416:Nerve Regeneration / Q000235:genetics / Q000502:physiology*; D009474:Neurons / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D020127:Recovery of Function / Q000235:genetics / Q000502:physiology*; D013997:Time Factors; D014633:Valine / Q000235:genetics; D018631:ras Proteins / Q000235:genetics / Q000502:physiology
D017252:CD3 Complex; D006639:Histidine; D019004:Galanin; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D019204:GTP-Binding Proteins; D018631:ras Proteins; D014633:Valine; D015740:Calcitonin Gene-Related Peptide