Astroglial Differentiation Research Articles

Prolactin Induces MAPK Signaling in Neural Progenitors without Alleviating Glucocorticoid-Induced Inhibition of in vitro Neurogenesis

We recently demonstrated that prolactin (PRL) prevents chronic stress-induced inhibition of adult hippocampal neurogenesis. It remained unsettled, however, whether PRL is acting directly on neural stem and progenitors cells (NPCs) or if neurogenesis is affected by an indirect mechanism, for example through the extensively described effects of PRL on the HPA axis. To address this point, we used neurosphere cultures derived from the adult rat hippocampus as an in vitro model for NPCs. Dexamethasone (DEX) was applied to stress the NPCs, and proliferation, survival and differentiation of cells were examined. DEX markedly inhibited proliferation of NPCs and cells entered the G₀ phase of cell cycle. Moreover, DEX reduced NPC survival and repressed astroglial differentiation, which is normally induced by serum or bone morphogenetic protein application. Even though we could demonstrate that NPCs express the PRL receptor and ERK1/2 signaling is induced by PRL, we did not observe any effect of PRL on NPCs proliferation, differentiation or survival, neither in the presence nor during absence of DEX. In summary, our results indicate that PRL action on NPCs and neurogenesis in vivo occurs via an indirect mechanism.

Id2 Is Required for Specification of Dopaminergic Neurons during Adult Olfactory Neurogenesis

Understanding the biology of adult neural stem cells has important implications for nervous system development and may contribute to our understanding of neurodegenerative disorders and their treatment. We have characterized the process of olfactory neurogenesis in adult mice lacking inhibitor of DNA binding 2(-/-) (Id2(-/-)). We found a diminished olfactory bulb containing reduced numbers of granular and periglomerular neurons with a distinct paucity of dopaminergic periglomerular neurons. While no deficiency of the stem cell compartment was detectable, migrating neuroblasts in Id2(-/-) mutant mice prematurely undergo astroglial differentiation within a disorganized rostral migratory stream. Further, when evaluated in vitro loss of Id2 results in decreased proliferation of neural progenitors and decreased expression of the Hes1 and Ascl1 (Mash1) transcription factors, known mediators of neuronal differentiation. These data support a novel role for sustained Id2 expression in migrating neural progenitors mediating olfactory dopaminergic neuronal differentiation in adult animals.

Expression of Disabled 1 suppresses astroglial differentiation in neural stem cells

Disabled 1 (Dab1), a cytoplasmic adaptor protein expressed predominantly in the CNS, transduces a Reelin-initiated signaling that controls neuronal migration and positioning during brain development. To determine the role of Dab1 in neural stem cell (NSC) differentiation, we established a culture of neurospheres derived from the embryonic forebrain of the Dab1 −/− mice, yotari. Differentiating Dab1 −/− neurospheres exhibited a higher expression of GFAP, an astrocytic marker, at the expense of neuronal markers. Under Dab1-deficient condition, the expression of NeuroD, a transcription factor for neuronal differentiation, was decreased and the JAK-STAT pathway was evidently increased during differentiation of NSC, suggesting the possible involvement of Dab1 in astrocyte differentiation via JAK-STAT pathway. Notably, expression of neural and glial markers and the level of JAK-STAT signaling molecules were not changed in differentiating NSC by Reelin treatment, indicating that differentiation of NSC is Reelin-independent. Immunohistochemical analyses showed a decrease in the number of neurons and an increase in the number of GFAP-positive cells in developing yotari brains. Our results suggest that Dab1 participates in the differentiation of NSCs into a specific cell lineage, thereby maintaining a balance between neurogenesis and gliogenesis.

AICAR Induces Astroglial Differentiation of Neural Stem Cells via Activating the JAK/STAT3 Pathway Independently of AMP-activated Protein Kinase

Neural stem cell differentiation and the determination of lineage decision between neuronal and glial fates have important implications in the study of developmental, pathological, and regenerative processes. Although small molecule chemicals with the ability to control neural stem cell fate are considered extremely useful tools in this field, few were reported. AICAR is an adenosine analog and extensively used to activate AMP-activated protein kinase (AMPK), a metabolic "fuel gauge" of the biological system. In the present study, we found an unrecognized astrogliogenic activity of AICAR on not only immortalized neural stem cell line C17.2 (C17.2-NSC), but also primary neural stem cells (NSCs) derived from post-natal (P0) rat hippocampus (P0-NSC) and embryonic day 14 (E14) rat embryonic cortex (E14-NSC). However, another AMPK activator, Metformin, did not alter either the C17.2-NSC or E14-NSC undifferentiated state although both Metformin and AICAR can activate the AMPK pathway in NSC. Furthermore, overexpression of dominant-negative mutants of AMPK in C17.2-NSC was unable to block the gliogenic effects of AICAR. We also found AICAR could activate the Janus kinase (JAK) STAT3 pathway in both C17.2-NSC and E14-NSC but Metformin fails. JAK inhibitor I abolished the gliogenic effects of AICAR. Taken together, these results suggest that the astroglial differentiation effect of AICAR on neural stem cells was acting independently of AMPK and that the JAK-STAT3 pathway is essential for the gliogenic effect of AICAR.

Epigenetic-Mediated Dysfunction of the Bone Morphogenetic Protein Pathway Inhibits Differentiation of Glioblastoma-Initiating Cells

Despite similarities between tumor-initiating cells with stem-like properties (TICs) and normal neural stem cells, we hypothesized that there may be differences in their differentiation potentials. We now demonstrate that both bone morphogenetic protein (BMP)-mediated and ciliary neurotrophic factor (CNTF)-mediated Jak/STAT-dependent astroglial differentiation is impaired due to EZH2-dependent epigenetic silencing of BMP receptor 1B (BMPR1B) in a subset of glioblastoma TICs. Forced expression of BMPR1B either by transgene expression or demethylation of the promoter restores their differentiation capabilities and induces loss of their tumorigenicity. We propose that deregulation of the BMP developmental pathway in a subset of glioblastoma TICs contributes to their tumorigenicity both by desensitizing TICs to normal differentiation cues and by converting otherwise cytostatic signals to proproliferative signals.

Effect of Choline-Containing Phospholipids on Transglutaminase Activity in Primary Astroglial Cell Cultures

The aim of the present investigation was to study the effects of choline and choline-containing phospholipids CDP-choline (CDPC) and L-alpha-glyceryl-phosphorylcholine (AGPC) on transglutaminase (TG) activity and expression in primary astrocyte cultures. TG is an important Ca2+-dependent protein that represents a normal constituent of nervous systems during fetal stages of development, playing a role in cell signal transduction, differentiation, and apoptosis. Confocal laser scanning microscopy (CLSM) analysis showed an increase of TG activity in astrocyte cultures treated with choline, CDPC, or AGPC at 0.1 μM or 1 μM concentrations. Comparatively, AGPC induced the most conspicuous effects enhancing monodansyl-cadaverine fluorescence both in cytosol and in nuclei, supporting the evidence of the important role played by AGPC throughout differentiation processes tightly correlated to nucleus-cytosol cross- talk during astroglial cells proliferation and development. Western blot analysis showed that in 24h 1 μM AGPC and choline-treated astrocytes increased TG-2, whereas no effect was observed in 24h 1 μM CDP-choline treated astrocytes. Our data suggest a crucial role of choline precursors during different stages of astroglial cell proliferation and differentiation in cultures.

Effect of growth factors and steroids on transglutaminase activity and expression in primary astroglial cell cultures

Type-2 transglutaminase (TG-2) is a multifunctional enzyme involved in the regulation of cell differentiation and survival that recently has been shown to play an emerging role in astrocytes, where it is involved in both proliferation and differentiation processes. Growth factors (GFs) such as EGF, basic fibroblast growth factor, insulin-like growth factor-I (IGF-I), and insulin (INS) are trophic and mitogenic peptides that participate in neuron-glia interactions and stimulate neuronal and astroglial proliferation and differentiation. Steroid hormones such as glucocorticoids and estrogens also play a pivotal role in neuronal and astroglial proliferation and differentiation and are key hormones in neurodegenerative and neuroprotective processes. We investigated the effects of the interaction of GFs with dexamethasone (DEX) or 17beta-estradiol (E(2)) on TG-2 activity and their expression in cultured astrocytes. We observed a significant increase in TG-2 activity and expression in astroglial cells treated for 24 hr with IGF-I, EGF, or INS. Priming of the cells with DEX or E(2), for 48 hr also led to an increase in TG-2 levels. When growth factors were present in the last 24 hr of the steroid treatment, a reduction in TG-2 expression and activity and a different subcellular TG-2 distribution were found. Our data indicate that steroid hormone-GF interaction may play an important role in astroglial function. The effect on TG-2 could be part of the regulation of intracellular pathways associated with the astrocyte response observed in physiological conditions and, possibly, also in neuropathological diseases.

Neurons or Glia? Can SHP2 Know It All?

Normal development of the nervous system relies on the spatially and temporally well-controlled differentiation of neurons and glia. Here, we discuss the intra- and extracellular molecular mechanisms that underlie the sequential genesis of neurons and glia, emphasizing recent studies describing the role of a signaling molecule, the tyrosine phosphatase SHP2, in normal brain development. Activation of SHP2 simultaneously enhances downstream activation of the MEK-ERK pathway, which subsequently promotes neurogenesis, while inhibiting the JAK-STAT pathway, which is critical for astroglial differentiation. Mutations in SHP2 that increase its tyrosine phosphatase activity cause a mental retardation-related disorder, Noonan syndrome. An imbalance in neurogenesis versus gliogenesis due to SHP2 mutations may contribute to Noonan syndrome.

HIV/gp120 Decreases Adult Neural Progenitor Cell Proliferation via Checkpoint Kinase-Mediated Cell-Cycle Withdrawal and G1 Arrest

Impaired adult neurogenesis has been observed in several neurodegenerative diseases, including human immunodeficiency virus (HIV-1)-associated dementia (HAD). Here we report that the HIV-envelope glycoprotein gp120, which is associated with HAD pathogenesis, inhibits proliferation of adult neural progenitor cells (aNPCs) in vitro and in vivo in the dentate gyrus of the hippocampus of HIV/gp120-transgenic mice. We demonstrate that HIV/gp120 arrests cell-cycle progression of aNPCs at the G1 phase via a cascade consisting of p38 mitogen-activated protein kinase (MAPK) --> MAPK-activated protein kinase 2 (a cell-cycle checkpoint kinase) --> Cdc25B/C. Our findings define a molecular mechanism that compromises adult neurogenesis in this neurodegenerative disorder.

Melatonin influences the proliferative and differentiative activity of neural stem cells

Though melatonin has a wide variety of biological functions, its effects on the neural stem cells (NSCs) is still unknown. In this study, we examined the effects of melatonin at either physiological (0.01-10 nm) or pharmacological concentrations (1-100 microM) on the proliferation and neural and astroglial differentiation of NSCs derived from the mouse embryo striatum using an in vitro culture system. We found that melatonin at pharmacological concentrations, but not at physiological concentrations, suppressed epidermal growth factor (EGF)-stimulated NSC proliferation (increment of viable cells, DNA synthesis and neurosphere formation) in a concentration-dependent manner. Furthermore, treatment with melatonin at a pharmacological concentration during the proliferation period facilitated 1% FBS-induced neural differentiation of NSCs without affecting the astroglial differentiation. In contrast, the treatment with melatonin at pharmacological concentrations during the differentiation period decreased the neural differentiation of the NSCs. As with melatonin, MCI-186, an antioxidant, suppressed EGF-stimulated NSC proliferation and facilitated the subsequent neural differentiation of NSCs. These results suggest that melatonin exerts potent modulatory effects on NSC functions including the suppression of the proliferation and facilitation of neuronal differentiation, likely via its antioxidant activity. As neurogenesis is thought to play an important role in ameliorating the deficit in neurodegenerative diseases, melatonin might be beneficially used for the treatment diseases such as cerebral infarction.

N-CoR Pathway Targeting Induces Glioblastoma Derived Cancer Stem Cell Differentiation

Nuclear receptor co-repressor (N-CoR) is a critical regulator of neural stem cell differentiation. Nuclear localization of N-CoR is a feature of undifferentiated neural stem cells and cytoplasmic translocation of N-CoR leads to astrocytic differentiation. Comparative proteomic analysis of microdissected glioblastoma multiforme (GBM) specimens and matched normal glial tissue reveals increased expression of N-CoR in GBM. In GBM primary cell cultures, tumor cells with nuclear localization of N-CoR demonstrate an undifferentiated phenotype, but are subject to astroglial differentiation upon exposure to agents promoting phosphorylation of N-CoR and its subsequent translocation to the cytoplasm. Treatment of glioma cell lines with a combination of retinoic acid and low-dose okadaic acid decreases the co-repressor effect of N-CoR and has a striking synergistic effect on growth inhibition. The identification of N-CoR in GBM provides insights into the tumorigenesis process and supports the development of differentiation-based therapeutic strategies.

PPARs, RXRs, and Stem Cells

Welcome to this special issue of PPAR Research: PPARs, RXRs, and Stem Cells. Within the past decade, there has been a burgeoning interest regarding the mechanisms regulating stem cell regeneration and differentiation in embryonic and adult tissues. Recent studies have identified stem or progenitor cells within most, if not all, somatic tissues. Cell biologists have explored a number of transcriptional regulatory pathways in the context of stem cell self-renewal and lineage commitment. While there has been a wealth of attention given to the Wnt pathway, Oct4, nanog, and STAT transcription factors in this context, the role of PPARs and related nuclear hormone receptors in regulating stem cells remains relatively unexplored. The current issue of PPAR Research has called for manuscripts that will spotlight the PPAR-Stem Cell relationship. We are fortunate to have received a mixture of excellent primary research manuscripts and comprehensive review articles from experts in the field. Mullen, Gu, and Cooney (Houston, Tex) have explored the role of nuclear hormone receptors in murine embryonic stem cell differentiation and function. Purton (Boston, Mass) has comprehensively reviewed the literature concerning the role of retinoid receptors in hematopoietic stem cells. Casteilla, Cousin, and Carmona (Toulouse, Fla) evaluate the classical role of PPARγ as an adipogenic regulator in adipose tissue-derived stromal/stem cells (ASCs). Three investigators use bone marrow-derived mesenchymal stem cell (MSC) models. Isales et al. (Augusta, Ga) provide novel findings relating to the role of mystatin and GILZ on adipogenesis in response to PPAR ligands. Shockley et al. (Bar Harbor, Me & Little Rock, Ark) report the transcriptomic response of MSCs to PPARγ agonists. Duque, Rivas, and Akter (Montreal, Canada) describe a role for farnesylation in modulating MSC adipogenesis. Finally, Cimini et al. (L’Aquila, IT) provide novel insights into the effect of PPARγ during neural stem cell (NSC) astroglial differentiation. We hope that this issue will stimulate other investigators to pursue novel avenues related to the converging themes of PPARs, nuclear hormone receptors, and stem cell biology. The outcomes of such investigations will have far reaching implications regarding fundamental questions relating to normal development, tumor biology, and tissue engineering and regenerative medicine. Z. Elizabeth Floyd Jeffrey M. Gimble

Activation of GABAA receptors facilitates astroglial differentiation induced by ciliary neurotrophic factor in neural progenitors isolated from fetal rat brain

Immunocytochemical analysis confirmed the validity of isolation procedures of neural progenitors capable of self-replication and differentiation from discrete fetal rat brain structures. A reverse transcription-polymerase chain reaction analysis revealed the expression of particular GABA(A) receptor (GABA(A)R), GABA(B)R-1 and GABA(C)R, but not GABA(B)R-2, subunits in neocortical cells before commitment. Sustained exposure to the GABA(A)R agonist muscimol at 100 mumol/L led to significant increases in the mitochondrial activity and the total areas of neocortical neurospheres formed during the cultivation for 12 days in a manner sensitive to a GABA(A)R antagonist, with lactate dehydrogenase release being unchanged. Moreover, prior sustained exposure to muscimol significantly facilitated the subsequent expression of an astroglial marker protein in cells differentiated by ciliary neurotrophic factor (CNTF) toward an astroglial lineage, with a concomitant decrease in the neuronal marker protein expression, in an antagonist-sensitive manner on Western blotting analysis. However, muscimol failed to significantly affect the expression of both marker proteins in cells differentiated in either the presence or absence of all-trans-retinoic acid. These results suggest that prior activation of GABA(A)R may preferentially facilitate the commitment by CNTF of neural progenitor cells toward an astroglial lineage after simulation of the self-replication activity in the developing rat brain.

Nitric Oxide Exposure Diverts Neural Stem Cell Fate from Neurogenesis Towards Astrogliogenesis

Regeneration of cells in the central nervous system is a process that might be affected during neurological disease and trauma. Because nitric oxide (NO) and its derivatives are powerful mediators in the inflammatory cascade, we have investigated the effects of pathophysiological concentrations of NO on neurogenesis, gliogenesis, and the expression of proneural genes in primary adult neural stem cell cultures. After exposure to NO, neurogenesis was downregulated, and this corresponded to decreased expression of the proneural gene neurogenin-2 and beta-III-tubulin. The decreased ability to generate neurons was also found to be transmitted to the progeny of the cells. NO exposure was instead beneficial for astroglial differentiation, which was confirmed by increased activation of the Janus tyrosine kinase/signal transducer and activator of transcription transduction pathway. Our findings reveal a new role for NO during neuroinflammatory conditions, whereby its proastroglial fate-determining effect on neural stem cells might directly influence the neuroregenerative process.

Neuronal and glial apoptosis in human traumatic brain injury

To establish reliable methods to aid the timing of brain damage after traumatic brain injury (TBI), brain tissue from 56 autopsy cases with TBI and known survival times, ranging from a few minutes to 126 days, were tested for apoptotic changes to the neuronal and glial cells. Apoptosis was established using the TdT-mediated dUTP nick end labelling (TUNEL) method of in-situ labelling and immunohistochemical reaction of caspase 3. In addition, cellular reaction and astroglial cell differentiation were investigated using histological and immunohistochemical markers. From a survival time of 120 min up to 12 days, TUNEL-positive apoptotic neuronal cells were frequently detected in the contusion zone. The earliest positive caspase 3 reaction in cortical neurons was evident after a posttraumatic interval of 80 min. Detection of apoptotic glial cells using the TUNEL technique showed that as in the case of neuronal cells, the earliest positive TUNEL reaction was obtained after 110 min. In cases of survival times of 120 min up to 4 days, apoptotic glial cells could frequently be detected. However, the first caspase 3-positive glial cells appeared 5 h after injury. Cerebral apoptosis was significantly associated with TBI cases as compared to control cases (P<0.001). The reference histological findings of neutrophilic granulocytes, CD3-positive T-lymphocytes, CD68-positive activated microglial cells/macrophages and TUNEL-positive neuronal cells increases the degree of certainty in determining the probable age of traumatic brain injury to 87.5%.

Pax2 regulates neuronal–glial cell fate choice in the embryonic optic nerve

During development, neural cell fate in the vertebrate optic nerve is restricted to the astroglial lineage. However, when isolated from the embryo and explanted in vitro, optic nerve progenitors generate neurons instead of astrocytes, suggesting that neuronal potentialities exist and are repressed in progenitors in vivo. Here we have investigated the mechanisms controlling cell fate in the optic nerve. The optic nerve is characterized by expression of the homeodomain transcription factor Pax2 which is maintained in differentiated astrocytes. We have observed that Pax2 is rapidly down-regulated in explanted optic nerves that generate neurons, and that its overexpression by electroporation in the optic nerve, or ectopically in the neural tube, is sufficient to block neuronal differentiation and allow glial development, showing that Pax2 plays a major role in controlling cell fate in the optic nerve. In vitro and ex vivo experiments further show that a signaling cascade that involves successively Sonic hedgehog and FGF is required to maintain Pax2 expression in optic nerve precursors whereby inhibiting the neuronal fate and promoting astroglial differentiation.

Dexamethasone blocks astroglial differentiation from neural precursor cells

In previous studies, we demonstrated functional neuronal and dopaminergic differentiation of fetal mesencephalic neural precursor cells. The major factors for orienting their progeny towards a dopaminergic phenotype are forskolin and interleukin-1beta. Here, we investigated the effects of dexamethasone (10 microM) on neuronal and glial differentiation. Exposure of mesencephalic neural precursor cells to dexamethasone significantly reduces the amount of glial fibrillary acidic protein astroglia, but not of galactocerebrosidase C oligodendroglia, MAP2ab neurons and tyrosine hydroxylase dopaminergic cells. Presuming a possible involvement of the nuclear factor-kappaB pathway, we examined the effects of wortmannin (phosphatidylinositol 3'-kinase inhibitor) and SN50 (nuclear factor-kappaB inhibitor) on gliogenesis. Both wortmannin and SN50 mimicked the effects of dexamethasone suggesting that dexamethasone specifically blocks astroglial differentiation from mesencephalic neural precursor cells most likely via inhibition of the nuclear factor-kappaB pathway.

Combinatorial protein display for the cell-based screening of biomaterials that direct neural stem cell differentiation

Neural stem cell (NSC) has emerged as a potential source for cell replacement therapy following traumatic injuries and degenerative diseases of the central nervous system. However, clinical applications of NSC further require technological advances especially for controlling differentiation of NSC. This study aimed at developing biomaterials that serve to expand undifferentiated NSC or to induce cells with specific phenotypes. Our approach is to construct composite biomaterials that consist of extracellular matrix components and growth factors. In order to optimize matrix–growth factor combinations, we conducted the parallel and rapid screening of composite biomaterials through assays using cell-based arrays. The photo-assisted patterning of an alkanethiol self-assembled monolayer was employed to achieve site-addressable combinatorial immobilization of natural and synthetic matrices incorporated with growth factors including epidermal growth factor (EGF), ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), and neurotrophin-3 (NT-3). NSC obtained from the rat embryonic striatum was cultured directly on the array to screen for cell adhesion, proliferation, and promotion of neuronal and glial specification. The results showed that the significant number of cells adhered to laminin-1, fibronectin, ProNectin™, and poly(ethyleneimine). It was found that cells proliferated most extensively on a spot with immobilized EGF among the spots with different matrix–growth factor combinations. The results also showed that neuronal differentiation was promoted on the spots with immobilized NGF or NT-3, and astroglial differentiation with CNTF. Importantly, observed effects of growth factors were frequently altered depending on the type of co-immobilized matrices, suggesting synergic effects of adhesion and growth factor signals.

Reactive astrogliosis induces astrocytic differentiation of adult neural stem/progenitor cells in vitro

Neural stem cells reside in defined areas of the adult mammalian brain, including the dentate gyrus of the hippocampus. Rat neural stem/progenitor cells (NSPCs) isolated from this region retain their multipotency in vitro and in vivo after grafting into the adult brain. Recent studies have shown that endogenous or grafted NSPCs are activated after an injury and migrate toward lesioned areas. In these areas, reactive astrocytes are present and secrete numerous molecules and growth factors; however, it is not currently known whether reactive astrocytes can influence the lineage selection of NSPCs. We investigated whether reactive astrocytes could affect the differentiation, proliferation, and survival of adult NSPCs by modelling astrogliosis in vitro, using mechanical lesion of primary astrocytes. Initially, it was found that conditioned medium from lesioned astrocytes induced astrocytic differentiation of NSPCs without affecting neuronal or oligodendrocytic differentiation. In addition, NSPCs in coculture with lesioned astrocytes also displayed increased astrocytic differentiation and some of these NSPC-derived astrocytes participated in glial scar formation in vitro. When proliferation and survival of NSPCs were analyzed, no differential effects were observed between lesioned and nonlesioned astrocytes. To investigate the molecular mechanisms of the astrocyte-inducing activity, the expression of two potent inducers of astroglial differentiation, ciliary neurotrophic factor and leukemia inhibitory factor, was analyzed by Western blot and shown to be up-regulated in conditioned medium from lesioned astrocytes. These results demonstrate that lesioned astrocytes can induce astroglial differentiation of NSPCs and provide a mechanism for astroglial differentiation of these cells following brain injury.

Involvement of the Mouse Prp19 Gene in Neuronal/Astroglial Cell Fate Decisions

The molecular mechanisms involved in neuronal/astroglial cell fate decisions during the development of the mammalian central nervous system are poorly understood. Here, we report that PRP19beta, a splice variant of mouse PRP19alpha corresponding to the yeast PRP19 protein, can function as a neuron-astroglial switch during the retinoic acid-primed neural differentiation of P19 cells. The beta-variant possesses an additional 19 amino acid residues in-frame in the N-terminal region of the alpha-variant. The forced expression of the alpha-variant RNA caused the down-regulation of oct-3/4 and nanog mRNA expression during the 12-48 h of the late-early stages of neural differentiation and was sufficient to convert P19 cells into neurons (but not glial cells) when the cells were cultured in aggregated form without retinoic acid. In contrast, the forced expression of the beta-variant RNA suppressed neuronal differentiation and conversely stimulated astroglial cell differentiation in retinoic acid-primed P19 cells. Based on yeast two-hybrid screening, cyclophilin A was identified as a specific binding partner of the beta-variant. Luciferase reporter assay mediated by the oct-3/4 promoter revealed that cyclophilin A could act as a transcriptional activator and that its activity was suppressed by the beta-variant, suggesting that cyclophilin A takes part in the induction of oct-3/4 gene expression, which might lead to neuroectodermal otx2 expression within 12 h of the immediate-early stages of retinoic acid-primed neural differentiation. These results show that the alpha-variant gene plays a pivotal role in neural differentiation and that the beta-variant participates in neuronal/astroglial cell fate decisions.