STAT3 but Not STAT1 Is Required for Astrocyte Differentiation

The JAK-STAT signaling pathway has been implicated in astrocyte differentiation. Both STAT1 and STAT3 are expressed in the central nervous system and are thought to be important for glial differentiation, as mainly demonstrated in vitro; however direct in vivo evidence is missing. We investigated whether STAT1 and STAT3 are essential for astrocyte development by testing the STAT responsiveness of astrocyte progenitors. STAT3 was absent in the ventricular zone where glial progenitors are born but begins to appear at the marginal zone at E16.5. At E18.5, both phospho-STAT1 and phospho-STAT3 were present in glial fibrillary acidic protein (GFAP)-expressing white matter astrocytes. Overexpression of STAT3 by electroporation of chicks in ovo induced increased numbers of astrocyte progenitors in the spinal cord. Likewise, elimination of STAT3 in Stat3 conditional knockout (cKO) mice resulted in depletion of white matter astrocytes. Interestingly, elimination of STAT1 in Stat1 null mice did not inhibit astrocyte differentiation and deletion of Stat1 failed to aggravate the glial defects in Stat3 cKO mice. Measuring the activity of STAT binding elements and the gfap promoter in the presence of various STAT mutants revealed that transactivation depended on the activity of STAT3 not STAT1. No synergistic interaction between STAT1 and STAT3 was observed. Cortical progenitors of Stat1 null; Stat3 cKO mice generated astrocytes when STAT3 or the splice variant Stat3β was supplied, but not when STAT1 was introduced. Together, our results suggest that STAT3 is necessary and sufficient for astrocyte differentiation whereas STAT1 is dispensable.     

MicroRNA-223 Regulates Granulopoiesis but Is Not Required for HSC Maintenance in Mice

MIR233 is genetically or epigenetically silenced in a subset of acute myeloid leukemia (AML). MIR223 is normally expressed throughout myeloid differentiation and highly expressed in hematopoietic stem cells (HSCs). However, the contribution of MIR223 loss to leukemic transformation and HSC function is largely unknown. Herein, we characterize HSC function and myeloid differentiation in Mir223 deficient mice. We show that Mir223 loss results in a modest expansion of myeloid progenitors, but is not sufficient to induce a myeloproliferative disorder. Loss of Mir223 had no discernible effect on HSC quiescence, long-term repopulating activity, or self-renewal capacity. These results suggest that MIR223 loss is likely not an initiating event in AML but may cooperate with other AML associated oncogenes to induce leukemogenesis.     

Lymphotoxin, but Not TNF, Is Required for Prion Invasion of Lymph Nodes

Neuroinvasion and subsequent destruction of the central nervous system by prions are typically preceded by a colonization phase in lymphoid organs. An important compartment harboring prions in lymphoid tissue is the follicular dendritic cell (FDC), which requires both tumor necrosis factor receptor 1 (TNFR1) and lymphotoxin β receptor (LTβR) signaling for maintenance. However, prions are still detected in TNFR1^−/− lymph nodes despite the absence of mature FDCs. Here we show that TNFR1-independent prion accumulation in lymph nodes depends on LTβR signaling. Loss of LTβR signaling, but not of TNFR1, was concurrent with the dedifferentiation of high endothelial venules (HEVs) required for lymphocyte entry into lymph nodes. Using luminescent conjugated polymers for histochemical PrP^Sc detection, we identified PrP^Sc deposits associated with HEVs in TNFR1^−/− lymph nodes. Hence, prions may enter lymph nodes by HEVs and accumulate or replicate in the absence of mature FDCs.     

Fibroblast Growth Factor 4 Is Required but not Sufficient for the Astrocyte Dedifferentiation

Our recent studies demonstrated that mature astrocytes from spinal cord can be reprogrammed in vitro and in vivo to generate neural stem/progenitor cells (NSPCs) following treatment with conditioned medium collected from mechanically injured astrocytes. However, little is known regarding the molecular mechanisms underlying the reprogramming of astrocytes. Here, we show that fibroblast growth factor 4 (FGF4) exerts a critical role in synergistically converting astrocytes into NSPCs that can express multiple neural stem cell markers (nestin and CD133) and are capable of both self-renewal and differentiation into neurons and glia. Lack of FGF4 signals fails to elicit the dedifferentiation of astrocytes towards NSPCs, displaying a substantially lower efficiency in the reprogramming of astrocytes and a slower transition through fate-determined state. These astrocyte-derived NSPCs displayed relatively poor self-renewal and multipotency. More importantly, further investigation suggested that FGF4 is a key molecule necessary for activating PI3K/Akt/p21 signaling cascades, as well as their downstream effectors responsible for directing cell reprogramming towards NSPCs. Collectively, these findings provide a molecular basis for astrocyte dedifferentiation into NSPCs after central nervous system (CNS) injury and imply that FGF4 may be a clinically applicable molecule for in situ neural repair in the CNS disorders.     

Nucleotide Excision Repair Is Not Required for the Antiapoptotic Function of Insulin-like Growth Factor 1

The expression of ERCC1, a member of the nucleotide excision repair (NER) family, is enhanced in cells transfected with insulin-like growth factor 1 (IGF-1) receptors. Of interest, an excellent concordance between ERCC1 expression and NER-mediated cell survival has been demonstrated. The two aims of the present study were to determine the signaling pathways used by IGF-1 to confer protection against apoptotic cell death in Chinese hamster ovary (CHO) cells and to assess the role of NER in this IGF-1 action. Experiments with pharmacological inhibitors indicated that phosphatidylinositol 3-kinase (PI 3-kinase) but not mitogen-activated protein kinase (ERK1/ERK2) mediates IGF-1 antiapoptotic activity. Using two series of CHO cells that have altered expression of ERCC1 or XPB/ERCC3, we examined IGF-1's ability to delay apoptotic death and reduction of mitochondrial oxidative function mediated by growth factor withdrawal. IGF-1 effectively blocked apoptosis, concomitant with increased MTT activity, in a pair of CHO cell lines expressing inactive ERCC1 (43-3B cells) and the transfected line of the mutant carrying the expressed human ERCC1 gene (83-G5 cells). Similarly, repair-deficient UV24 cells, which lack XPB/ERCC3, and their parental line AA8 were also responsive to the IGF-1's antiapoptotic capacity. In the presence of IGF-1, these cell lines became resistant to the cleavage of poly(ADP-ribose) polymerase, a key player in DNA damage recognition and DNA repair. These results suggest that PI 3-kinase activation plays a determinant role in the antiapoptotic function of IGF-1, but that functional NER does not play a critical part in mediating this IGF-1 response.