Basic fibroblast growth factor promotes the generation of microtubule-associated protein 2-positive cells from microglia

We recently demonstrated that microglia as multipotential stem cells give rise to microtubule-associated protein 2 (MAP2)-positive and glial fibrillary acidic protein (GFAP)-positive cells and that microglia-derived MAP2-positive cells possess properties of functional neurons. In this study, we investigated the role of fibroblast growth factor (FGF) signaling in the molecular mechanism underlying the generation of microglia-derived MAP2-positive and GFAP-positive cells. Real-time quantitative PCR analyses demonstrated that mRNA levels of a family of three FGF receptors, Fgfr1-3, were upregulated in microglia treated with 70% fetal bovine serum (FBS). Immunocytochemical analyses demonstrated that basic FGF (bFGF) promoted the generation of microglia-derived MAP2-positive and GFAP-positive cells, and the FGF receptor tyrosine kinase inhibitor SU5402 and the MEK inhibitor PD98059 both inhibited this process. Western blot analyses demonstrated that bFGF increased phosphorylated ERK1/2 levels without altering total ERK1/2 levels. These results suggest that bFGF promotes the generation of microglia-derived MAP2-positive and GFAP-positive cells via FGF receptors and the ERK-MAP kinase pathway

Abeta-induced BACE-1 cleaves N-terminal sequence of mPGES-2.

We previously indicated that amyloid beta (Abeta) augments protein levels of beta-site amyloid precursor protein cleaving enzyme-1 (BACE-1) through oxidative stress. In this study, we revealed that BACE-1 is involved in the cleavage of membrane-bound prostaglandin E2 synthase-2 (mPGES-2) in its N-terminal portion, which, in turn, enhanced the generation of prostaglandin E2 (PGE2). PGE2 results in increased Abeta production, initiating a cell-injuring cycle. Using rat primary cortical neurons, a 48 h treatment with Abeta 1-42 (5 microM) resulted in the enhanced extracellular PGE2 levels up to about 1 ng/mL, which was attenuated by treatment with a BACE-1 inhibitor (200 nM). A synthetic peptide sequence of 20-amino acids that included the cleavage site of mPGES-2 (HTARWHL RAQDLHERS AAQLSLSS) was cleaved by recombinant BACE-1, confirmed using reverse-phase high-performance liquid chromatography. Cleaved or activated mPGES-2 augments the generation of PGE2. In addition, mPGES-2 was determined to be colocalized with BACE-1 and cyclooxygenase-2 in the perinuclear region in cells after exposure to Abeta. Exposure of neurons to PGE2 led to cell death, and Abeta production was enhanced by PGE2 (1 ng/mL, 48 h). Collectively, these results suggest that Abeta might cause neuroinflammation that aggravates Alzheimer's disease pathogenesis

PI3K/Akt/mTOR signaling regulates glutamate transporter 1 in astrocytes.

Reduction in or dysfunction of glutamate transporter 1 (GLT1) is linked to several neuronal disorders such as stroke, Alzheimer's disease, and amyotrophic lateral sclerosis. However, the detailed mechanism underlying GLT1 regulation has not been fully elucidated. In the present study, we first demonstrated the effects of mammalian target of rapamycin (mTOR) signaling on GLT1 regulation. We prepared astrocytes cultured in astrocyte-defined medium (ADM), which contains several growth factors including epidermal growth factor (EGF) and insulin. The levels of phosphorylated Akt (Ser473) and mTOR (Ser2448) increased, and GLT1 levels were increased in ADM-cultured astrocytes. Treatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor or an Akt inhibitor suppressed the phosphorylation of Akt (Ser473) and mTOR (Ser2448) as well as decreased ADM-induced GLT1 upregulation. Treatment with the mTOR inhibitor rapamycin decreased GLT1 protein and mRNA levels. In contrast, rapamycin did not affect Akt (Ser473) phosphorylation. Our results suggest that mTOR is a downstream target of the PI3K/Akt pathway regulating GLT1 expression

Integrin-associated protein promotes neuronal differentiation of neural stem/progenitor cells.

Neural stem/progenitor cells (NSPCs) proliferate and differentiate depending on their intrinsic properties and local environment. During the development of the mammalian nervous system, NSPCs generate neurons and glia sequentially. However, little is known about the mechanism that determines the timing of switch from neurogenesis to gliogenesis. In this study, we established a culture system in which the neurogenic potential of NSPCs is decreased in a time-dependent manner, so that short-term-cultured NSPCs differentiate into more neurons compared with long-term-cultured NSPCs. We found that short-term-cultured NSPCs express high levels of integrin-associated protein form 2 (IAP2; so-called CD47) mRNA using differential display analysis. Moreover, IAP2 overexpression in NSPCs induced neuronal differentiation of NSPCs. These findings reveal a novel mechanism by which IAP2 induces neuronal differentiation of NSPCs

Expression of IAP2 mRNA in NSPCs.

<p>(A) Differential display analysis of 13 DIV neurosphere (left lane) and 20 DIV neurosphere (right lane) RNA using No. 22 upstream primer and 5′-T_nAC-3′ downstream primer sets. Arrowhead indicates the differentially expressed IAP2 cDNA fragment between 13 DIV neurospheres and 20 DIV neurospheres. (B) Schematic representation of the alternative splicing possibilities for generation of IAP forms. Alternative splice forms of IAP were identified by RT-PCR analysis using primer pairs designed in common exon sequences (arrows). Right arrow indicates the position of the forward primer and the left arrow indicates the position of the reverse primer for RT-PCR as shown in (C). (C) Identification of splicing forms of IAP expressed in NSPCs using RT-PCR analysis. Total RNA was isolated from 13 DIV neurospheres and 20 DIV neurospheres. Alternative splice forms were distinguished by the size of PCR products using primer sets shown in (B). (D) Quantification of IAP2 mRNA expression levels in 13 DIV neurospheres and 20 DIV neurospheres. Total RNA was isolated from 13 and 20 DIV neurospheres. The expression levels of IAP2 and Ppia (internal standard) mRNA were determined by real-time quantitative PCR analysis. (E) Western blot analysis of IAP protein expression at 13 DIV (left lane) and 20 DIV (right lane). Each value represents the mean ± SEM from three independent experiments and is expressed in reference to 13 DIV neurospheres. **<i>p</i> < 0.01 compared with 13 DIV neurospheres.</p

<i>In vitro</i> time-dependent decline in neurogenic competency of NSPCs.

<p><b>(A)</b> Strategy for the functional screening of candidate genes involved in the temporal specification of NSPCs using the neurosphere method. (B) Representative immunofluorescent images of NSPCs differentiated from 13 DIV neurospheres (left) and 20 DIV neurospheres (right). Secondary neurospheres were dissociated in NSPC proliferation medium and plated on poly l-lysine-coated cover glass. After 24 hours, NSPCs were exposed to NSPC differentiation medium, fixed at 10 days after differentiation, stained for a neuron marker (Tuj1; green) and an astrocyte marker (GFAP; red), and counterstained with DAPI (blue). Scale bar: 50 μm. Quantification of Tuj1-positive cells (C) or GFAP-positive cells (D) 10 days after differentiation. Data are shown as the mean ± SEM of four independent experiments. *<i>p</i> < 0.05.</p