ABSTRACT
Title
Adult Mouse Post Mortem Neural Precursors differentiation depends from EPO and require mTOR
Authors
F. Messaggio1
Dottorato di Ricerca in Fisiopatologia, Farmacologia e Malattie Metaboliche
1 Laboratories of Pharmacology, Dept. Medicine, Surgery and Dentistry, University of Milan; Milan Italy.
Dottorato di Ricerca in Fisiopatologia, Farmacologia e Malattie Metaboliche
1 Laboratories of Pharmacology, Dept. Medicine, Surgery and Dentistry, University of Milan; Milan Italy.
Abstract
Neural stem cells from the subventricular zone of the forebrain, because of their proliferation and differentiation characteristics, are a good tool for tissue replacement therapies. We recently isolated a subclass of neural progenitors, capable of surviving a powerful ischemia insult: these cells were named Post Mortem Neural Precursor Cells (PM-NPCs). Differentiation yield mostly neurons (about 30-40%) compared to regular NPCs. Also the cholinergic yield is higher.
The involvement of MAP Kinases and mTOR in EPO-dependent PM-NPCs differentiation was investigated. T0 NPCs and T6 PM-NPCs were cultured for 7 days in presence of PD98059 at 3 and 30 mM or rapamycin 0.05 ng/ml, that were respectively added to the differentiation medium. PD98059 affected mildly PM-NPCs differentiation at the concentration of 3 mM, but the exposure to 30 mM reduced very significantly the number of b tubulin III-positive PM-NPCs. T0 were unaffected by the treatment. PM-NPCs neuronal differentiation is, however, extremely dependent upon mTOR function, since rapamycin at dose as low as 0.05 ng/ml obliterated the higher neuronal-differentiation typical of PM-NPCs. This low dose of rapamycin did not affect differentiation of T0 NPCs. The addition of exogenous EPO to the medium promotes NPCs differentiation into neurons, while PM-NPCs neuronal differentiation is dependent upon endogenous EPO production and EPO dependent activation of EPO-R. To this point we added rh-EPO to the differentiation medium at different concentrations (1U/ml, 10U/ml, 50U/ml). rh-EPO addition significantly (p<0.001) increased the percentage of differentiated T0 NPCs that expressed b tubulin III.
Differently the percentage of PM-NPCs neuronal differentiation was not affected by the addition of rh-EPO to the medium. To verify the role of endogenous EPO on the neuronal differentiation of PM-NPCs, both T0 NPCs and PM-NPCs were exposed to EPO neutralizing antibody during the differentiation period. This resulted in the almost total abolition of b tubulin III expression (p<0.001) in PM-NPC and a 50% reduction in T0. The effect of anti- EPO antibody in T0 NPCs differentiation may suggest that EPO-dependent differentiation may also be present in T0. Also the blockade of EPO-R by means of specific antibody (Verdier et al 2006) inhibits the neuronal differentiation of PM-NPCs and reduces by 50% the T0 differentiation. The addition of unspecific IgG does not affect such a process. Thus it appears that anti-EPO and anti EPO-R exposure blocks effectively the action of endogenous EPO likely released by PM-NPCs and impairs the differentiation towards neuronal phenotype. Differently, rapamycin reduced T6 Epo-dependent neuronal differentiation. In conclusion, our data strongly suggest that EPO-mediated neuronal differentiation of neural progenitor requires mTOR functionality.
The involvement of MAP Kinases and mTOR in EPO-dependent PM-NPCs differentiation was investigated. T0 NPCs and T6 PM-NPCs were cultured for 7 days in presence of PD98059 at 3 and 30 mM or rapamycin 0.05 ng/ml, that were respectively added to the differentiation medium. PD98059 affected mildly PM-NPCs differentiation at the concentration of 3 mM, but the exposure to 30 mM reduced very significantly the number of b tubulin III-positive PM-NPCs. T0 were unaffected by the treatment. PM-NPCs neuronal differentiation is, however, extremely dependent upon mTOR function, since rapamycin at dose as low as 0.05 ng/ml obliterated the higher neuronal-differentiation typical of PM-NPCs. This low dose of rapamycin did not affect differentiation of T0 NPCs. The addition of exogenous EPO to the medium promotes NPCs differentiation into neurons, while PM-NPCs neuronal differentiation is dependent upon endogenous EPO production and EPO dependent activation of EPO-R. To this point we added rh-EPO to the differentiation medium at different concentrations (1U/ml, 10U/ml, 50U/ml). rh-EPO addition significantly (p<0.001) increased the percentage of differentiated T0 NPCs that expressed b tubulin III.
Differently the percentage of PM-NPCs neuronal differentiation was not affected by the addition of rh-EPO to the medium. To verify the role of endogenous EPO on the neuronal differentiation of PM-NPCs, both T0 NPCs and PM-NPCs were exposed to EPO neutralizing antibody during the differentiation period. This resulted in the almost total abolition of b tubulin III expression (p<0.001) in PM-NPC and a 50% reduction in T0. The effect of anti- EPO antibody in T0 NPCs differentiation may suggest that EPO-dependent differentiation may also be present in T0. Also the blockade of EPO-R by means of specific antibody (Verdier et al 2006) inhibits the neuronal differentiation of PM-NPCs and reduces by 50% the T0 differentiation. The addition of unspecific IgG does not affect such a process. Thus it appears that anti-EPO and anti EPO-R exposure blocks effectively the action of endogenous EPO likely released by PM-NPCs and impairs the differentiation towards neuronal phenotype. Differently, rapamycin reduced T6 Epo-dependent neuronal differentiation. In conclusion, our data strongly suggest that EPO-mediated neuronal differentiation of neural progenitor requires mTOR functionality.