Differentiation and neural integration of hippocampal neuronal progenitors: signaling pathways sequentially involved.
In the context of their potential implication in regenerative strategies, we characterized cell mechanisms underlying the fate of embryonic rat hippocampal H19-7 progenitors in culture upon induction of their differentiation, and tested their capacities to integrate into a neuronal network in vitro. Without addition of growth factors, nearly 100% of cells expressed various neuronal markers, with a progressive rise of the expression of Synapsin I and II, suggesting that cells developed as mature neurons with synaptogenic capacities. Fully differentiated neurons were identified as glutamatergic and expressed the receptor-associated protein PSD-95. Quantification of ATP showed that 60% of cells died within 24 h after differentiation. Cell death was shown to imply Erk1/2-dependent intrinsic mitochondrial apoptosis signaling pathway, with activation of caspase-9 and -3, finally leading to single-strand DNA. Surviving neurons displayed high levels of Akt, phospho-Akt, and antiapoptotic proteins such as Bcl-2 and Bcl-XL, with decreased caspase activation. In the absence of trophic support, the proapoptotic death-associated protein (DAP) kinase was dramatically stimulated by 24 h postdifferentiation, along with increased levels of p38 and phospho-p38, and caspase reactivation. These findings show that different signaling pathways are sequentially triggered by differentiation, and highlight that ultimate cell death would involve p38 and DAP kinase activation. This was supported by the improvement of cell survival at 24-h postdifferentiation when cells were treated by PD169316, a specific inhibitor of p38. Finally, when seeded on rat hippocampal primary cultured neurons, a significant number of differentiated H19-7 cells were able to survive and to develop cell-cell communication.
Akchiche
Nassila
N
Inserm U954, Vandoeuvre-lès-Nancy, F-54500, France.
Bossenmeyer-Pourié
Carine
C
Pourié
Grégory
G
Koziel
Violette
V
Nédélec
Emmanuelle
E
Guéant
Jean-Louis
JL
Daval
Jean-Luc
JL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001973:Bromodeoxyuridine / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods; D000076122:Disks Large Homolog 4 Protein; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006624:Hippocampus / Q000166:cytology*; D007093:Imidazoles / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000502:physiology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013234:Stem Cells / Q000187:drug effects / Q000502:physiology*; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D050599:Vesicular Glutamate Transport Protein 2 / Q000235:genetics / Q000378:metabolism; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism
C494813:Bcl2l1 protein, rat; D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D004791:Enzyme Inhibitors; D007093:Imidazoles; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D050599:Vesicular Glutamate Transport Protein 2; D051020:bcl-X Protein; D000255:Adenosine Triphosphate; D020929:Mitogen-Activated Protein Kinase Kinases; D020169:Caspases; D001973:Bromodeoxyuridine; C408604:2-(4-nitrophenyl)-4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazole