[Effects of gamma-secretase inhibitor N-(3, 5-difluorophenacetyl-L-alanyl)-S-phenylglycine t-butyl ester on proliferation and differentiation of neural stem cells].
OBJECTIVE
To investigate the role of N-[N-(3,5-Difluorophenacetyl-L-alanyl)-S-phenylglycine t-butyl ester (DAPT), a gamma-secretases inhibitor, on the proliferation and differentiation of neural stem cells (NSCs).


METHODS
NSCs were isolated from Sprague-Dawley rat brain, cultured, and treated with DAPT for 6 weeks. Cell counting was conducted every 24 h. CCK8 assay was used to draw the growth curve. Immunofluorescence staining was performed to observe the proportions of beta-tubulin III positive cells (neurons), glial fibrillary acidic protein (GFAP) positive cells (astrocytes), and 2',3'-cyclic nucleotide3' phosphohydrolase (CNPase) positive cells (oligodendrocytes). RT-PCR was employed to assay the mRNA expression of RBP-Jk and Hes1 genes, downstream genes of the Notch pathway.


RESULTS
Cell counting and CCK-8 assay showed that DAPT reduced the rate of NSC proliferation. Addition of DAPT altered NSC differentiation in vitro, percentage of The proportions neurons of the DAPT group was (13.84 +/- 1.22)%, significantly higher than that of the control group [(3.7 +/- 1.04)% , P <0.01], the proportion of the oligodendrocytes of the DAPT group was (14.75 +/- 1.58)%, significantly higher than that pf the control group [(4.8 +/- 1.22)%, P < 0.01]. However, the proportion of astrocytes of the DAPT group was (63.41 +/- 1.20)%, significantly lower than that of the control group [ (82.84 +/- 3.68)%, P <0.01]. The expression levels of RBP-Jk and Hes1 mRNA (RBP-Jk/GAPDH and Hesl/GAPDH) in the NSC treated with DAPT were 0.52 +/- 0.13 and 0.66 +/- 0.18 respectively, both significantly lower than those of the control group (0.28 +/- 0.06 and 0.16 +/- 0.08 respectively, both P <0.05).


CONCLUSIONS
DAPT inhibits the NSC proliferation and alters the NSC committed differentiation. These effects are mediated via Notch signaling down regulation as a result of the inhibition of gamma-secretase.
Cai
Liang-Zhi
LZ
Research Center of Molecular Medicine, Division of Biochemistry, Fujian Medical University, Fuzhou 350004, China.
Lin
Ling
L
Hu
Jian-Shi
JS
Zheng
Zhi-Hong
ZH
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000378:metabolism; D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D001921:Brain / Q000166:cytology; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D004151:Dipeptides / Q000494:pharmacology*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000187:drug effects; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D013997:Time Factors; D000072056:Transcription Factor HES-1; D014404:Tubulin / Q000378:metabolism
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D053829:Amyloid Precursor Protein Secretases; D051792:Basic Helix-Loop-Helix Proteins; D004151:Dipeptides; D005904:Glial Fibrillary Acidic Protein; D018398:Homeodomain Proteins; D012333:RNA, Messenger; D000072056:Transcription Factor HES-1; D014404:Tubulin; C489735:Hes1 protein, rat; C419410:N-(N-(3,5-difluorophenacetyl)alanyl)phenylglycine tert-butyl ester