Enhanced expression of cellular prion protein gene by insulin or nerve growth factor in immortalized mouse neuronal precursor cell lines

In order to understand the fundamental and putative roles of PrP(c) in the central nervous system, neuronal cell lines were established. Cells were immortalized by recombinant retrovirus vector-mediated transduction of SV40 T-antigen gene. Among these, two cell lines were selected based on their RT-PCR expressions of neuron-specific neurofilament (NF-H, NF-M) and cell morphology. These cell lines showed the properties of neuronal progenitor cells in antigenicity, morphology and responses to differentiating agents. Expression of PrP(c) was detected by immunocytochemical analysis. These cell lines responded to differentiating agents such as dibutyl cyclic AMP (dcAMP) and phorbol 12-myristate 13-acetate (PMA) before developing into neuronal-like cells. Neurite extensions were observed 20 min after incubation with the differentiating agents. Treatment with nerve growth factor (NGF) and insulin induced cell differentiation and enhanced expression of PrP gene (Prnp) mRNA and protein. The latter phenomenon was not inhibited by wortmannin, which is a specific inhibitor of phosphatidylinositol 3-kinase. These results suggest that PrP(c) plays an important role in the differentiation-mediated classic signaling pathway of neuronal cell.     

Purkinje-cell-derived Sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum

Purkinje cells (PCs) are the projection neurons of the cerebellar cortex. They receive two major types of synaptic input - that from the inferior olive via climbing fibres and that from the granule neurons via parallel fibres. The precursors of granule neurons proliferate at the surface of the developing cerebellumin the external granule layer (EGL), which persists until postnatal day 14 in the mouse [1]. PCs are thought to provide trophic support for granule neurons [2][3] and to stimulate the proliferation of cells in the EGL [4], but the signalling molecules that mediate these cell-cell interactions have not been identified. I show here that PCs in the developing mouse cerebellum express the gene encoding the morphogen Sonic hedgehog (Shh) and that dividing cells in the EGL express Patched (Ptc) and Gli1, two target genes of which expression is upregulated in response to Hedgehog signalling (see [5] and references therein). Treatment of developing mice with hybridoma cells that secrete neutralizing anti-Shh antibodies [6] disrupted cerebellar development and reduced bromodeoxyuridine (BrdU) incorporation in the EGL of neonatal mice, whereas treatment of dissociated granule neuron cultures with recombinant Shh stimulated BrdU incorporation. These results suggest that PC-derived Shh normally promotes the proliferation of granule neuron precursors in the EGL.     

Astrocytes support incomplete differentiation of an oligodendrocyte precursor cell.

Glial fibrillary acidic protein-positive astrocytes, but not neurons or fibroblasts, support the differentiation of an oligodendroglial precursor cell expressing O4 antigen and vimentin into an O4 antigen-positive, but vimentin-negative oligodendrocyte. Further maturation into galactocerebroside (O1)-positive oligodendrocytes is, however, not achieved under the culture conditions used, neither in the presence of astrocytes nor neurons.     

Differential proliferation rhythm of neural progenitor and oligodendrocyte precursor cells in the young adult hippocampus

Oligodendrocyte precursor cells (OPCs) are a unique type of glial cells that function as oligodendrocyte progenitors while constantly proliferating in the normal condition from rodents to humans. However, the functional roles they play in the adult brain are largely unknown. In this study, we focus on the manner of OPC proliferation in the hippocampus of the young adult mice. Here we report that there are oscillatory dynamics in OPC proliferation that differ from neurogenesis in the subgranular zone (SGZ); the former showed S-phase and M-phase peaks in the resting and active periods, respectively, while the latter only exhibited M-phase peak in the active period. There is coincidence between different modes of proliferation and expression of cyclin proteins that are crucial for cell cycle; cyclin D1 is expressed in OPCs, while cyclin D2 is observed in neural stem cells. Similar to neurogenesis, the proliferation of hippocampal OPCs was enhanced by voluntary exercise that leads to an increase in neuronal activity in the hippocampus. These data suggest an intriguing control of OPC proliferation in the hippocampus.     

Pelizaeus-Merzbacher disease-associated proteolipid protein 1 inhibits oligodendrocyte precursor cell differentiation via extracellular-signal regulated kinase signaling

Oligodendrocytes (OLs) are myelin-forming glial cells in the central nervous system (CNS) and their dysfunction causes neuropathies such as demyelinating diseases. Proteolipid protein 1 (PLP1) is an oligodendrocyte myelin-rich tetraspan membrane protein and aberration of the plp1 gene is known to be responsible for dysmyelinating Pelizaeus-Merzbacher disease (PMD). Among previously identified gene alternations, multiplication of the plp1 gene causes increased expression of PLP1, resulting in a phenotype with severe dysmyelination in human and also rodent models. Yet little is known about the relationship between increased PLP1 expression and oligodendrocyte precursor cell (OPC) differentiation and the intracellular molecular mechanism. Here we show that expression of PLP1 in OPCs markedly inhibits their differentiation, and that this inhibitory effect is effectively improved by inhibition of extracellular signal-regulated kinase (ERK) activity. Furthermore, in cocultures with dorsal root ganglion (DRG) neurons, ERK inhibition also improves PLP1-induced dysmyelination. Thus, ERK inhibition helps to improve defective OPC differentiation induced by PLP1 expression, suggesting that molecules belonging to ERK signaling cascade may be new PMD therapeutic targets.     

Laminin-induced PC-12 cell differentiation is inhibited following laser inactivation of cellular prion protein

Prions, the etiological agents for infectious degenerative encephalopathies, act by inducing structural modifications in the cellular prion protein (PrPc). Recently, we demonstrated that PrPc binds laminin (LN) and that this interaction is important for the neuritogenesis of cultured hippocampal neurons. Here we have used the PC-12 cell model to explore the biological role of LN-PrPc interaction. Antibodies against PrPc inhibit cell adhesion to LN-coated culture plaques. Furthermore, chromophore-assisted laser inactivation of cell surface PrPc perturbs LN-induced differentiation and promotes retraction of mature neurites. These results point out to the importance of PrPc as a cell surface ligand for LN.     

Dystroglycan regulates structure, proliferation and differentiation of neuroepithelial cells in the developing vertebrate CNS

In the developing CNS alpha- and beta-dystroglycan are highly concentrated in the endfeet of radial neuroepithelial cells at the contact site to the basal lamina. We show that injection of anti-dystroglycan Fab fragments, knockdown of dystroglycan using RNAi, and overexpression of a dominant-negative dystroglycan protein by microelectroporation in neuroepithelial cells of the chick retina and optic tectum in vivo leads to the loss of their radial morphology, to hyperproliferation, to an increased number of postmitotic neurons, and to an altered distribution of several basally concentrated proteins. Moreover, these treatments also altered the oriented growth of axons from retinal ganglion cells and from tectal projection neurons. In contrast, expression of non-cleavable dystroglycan protein in neuroepithelial cells reduced their proliferation and their differentiation to postmitotic neurons. These results demonstrate that dystroglycan plays a key role in maintaining neuroepithelial cell morphology, and that interfering with dystroglycan function influences proliferation and differentiation of neuroepithelial cells. These data also suggest that an impaired dystroglycan function in neuroepithelial cells might be responsible for some of the severe brain abnormalities observed in certain forms of congenital muscular dystrophy.     

Toxic effect of blood components on perinatal rat subventricular zone cells and oligodendrocyte precursor cell proliferation, differentiation and migration in culture

The germinal matrix of human brain gives rise to oligodendrocytes and astrocytes after mid-gestation. Hemorrhage in the germinal matrix of premature infants is associated with suppressed cell proliferation. We hypothesize that soluble blood constituents have an adverse effect on the proliferation of cultured rat subventricular zone (SVZ) cells and the proliferation, migration, and differentiation of oligodendrocyte progenitor cells (OPC). Using caspase 3 activation and lactate dehydrogenase release assays, rat plasma, serum, thrombin, and kallikrein killed SVZ cells when grown in the presence (but not absence) of platelet derived growth factor. Plasma and serum killed OPC at 1 : 1 to 1 : 100 dilutions. Using a bromodeoxyuridine incorporation assay OPC proliferation was reduced by plasma, serum, thrombin and plasmin. Blood proteins also suppressed OPC migration in a concentration dependent manner. However, differentiation of OPC into myelin basic protein expressing cells was suppressed only by thrombin. We conclude that soluble blood components, particularly thrombin, have an adverse effect on maturing SVZ cells and OPC derived from newborn rat brain.     

Efficient CPP-mediated Cre protein delivery to developing and adult CNS tissues

Background  

Understanding and manipulating gene function in physiological conditions is a major objective for both fundamental and applied research. In contrast to other experimental settings, which use either purely genetic or gene delivery (viral or non-viral) strategies, we report here a strategy based on direct protein delivery to central nervous system (CNS) tissues. We fused Cre recombinase with cell-penetrating peptides and analyzed the intracellular biological activity of the resulting chimerical proteins when delivered into cells endowed with Cre-mediated reporter gene expression.     

Role of the retinoblastoma tumor suppressor protein in cellular differentiation

The retinoblastoma protein (pRb105) is a true tumor suppressor as deregulation of the Rb pathway by either mutation of pRb105 itself or other proteins in the pathway, such as p16INK4a, occur in most cancers. This prototypical family member, along with the related p107 and p130, are involved in the control of cell cycle regulation, but pRb105 has also been shown to be involved in tissue development and differentiation. This prospective will discuss the increasing evidence for a role of pRb105 in cellular differentiation and the fact that various cancers, which contain mutant pRb105, or mutations in proteins in the pRb105 pathway, are perhaps a result of deregulation of differentiation.     

Chemokines and inflammatory mediators interact to regulate adult murine neural precursor cell proliferation, survival and differentiation

Adult neural precursor cells (NPCs) respond to injury or disease of the CNS by migrating to the site of damage or differentiating locally to replace lost cells. Factors that mediate this injury induced NPC response include chemokines and pro-inflammatory cytokines, such as tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ), which we have shown previously promotes neuronal differentiation. RT-PCR was used to compare expression of chemokines and their receptors in normal adult mouse brain and in cultured NPCs in response to IFNγ and TNFα. Basal expression of many chemokines and their receptors was found in adult brain, predominantly in neurogenic regions, with OB?SVZ>hippocampus and little or no expression in non-neurogenic regions, such as cortex. Treatment of SVZ-derived NPCs with IFNγ and TNFα (alone and in combination) resulted in significant upregulation of expression of specific chemokines, with CXCL1, CXCL9 and CCL2 most highly upregulated and CCL19 downregulated. Unlike IFNγ, chemokine treatment of NPCs in vitro had little or no effect on survival, proliferation or migration. Neuronal differentiation was promoted by CXCL9, CCL2 and CCL21, while astrocyte and total oligodendrocyte differentiation was not affected. However, IFNγ, CXCL1, CXCL9 and CCL2 promoted oligodendrocyte maturation. Therefore, not only do NPCs express chemokine receptors, they also produce several chemokines, particularly in response to inflammatory mediators. This suggests that autocrine or paracrine production of specific chemokines by NPCs in response to inflammatory mediators may regulate differentiation into mature neural cell types and may alter NPC responsiveness to CNS injury or disease.     

Monoacylated cellular prion protein modifies cell membranes, inhibits cell signaling, and reduces prion formation

Prion diseases occur following the conversion of the cellular prion protein (PrP(C)) into a disease related, protease-resistant isoform (PrP(Sc)). In these studies, a cell painting technique was used to introduce PrP(C) to prion-infected neuronal cell lines (ScGT1, ScN2a, or SMB cells). The addition of PrP(C) resulted in increased PrP(Sc) formation that was preceded by an increase in the cholesterol content of cell membranes and increased activation of cytoplasmic phospholipase A(2) (cPLA(2)). In contrast, although PrP(C) lacking one of the two acyl chains from its glycosylphosphatidylinositol (GPI) anchor (PrP(C)-G-lyso-PI) bound readily to cells, it did not alter the amount of cholesterol in cell membranes, was not found within detergent-resistant membranes (lipid rafts), and did not activate cPLA(2). It remained within cells for longer than PrP(C) with a conventional GPI anchor and was not converted to PrP(Sc). Moreover, the addition of high amounts of PrP(C)-G-lyso-PI displaced cPLA(2) from PrP(Sc)-containing lipid rafts, reduced the activation of cPLA(2), and reduced PrP(Sc) formation in all three cell lines. In addition, ScGT1 cells treated with PrP(C)-G-lyso-PI did not transmit infection following intracerebral injection to mice. We propose that that the chemical composition of the GPI anchor attached to PrP(C) modified the local membrane microenvironments that control cell signaling, the fate of PrP(C), and hence PrP(Sc) formation. In addition, our observations raise the possibility that pharmacological modification of GPI anchors might constitute a novel therapeutic approach to prion diseases.     

Differential requirement of cyclin-dependent kinase 2 for oligodendrocyte progenitor cell proliferation and differentiation

ABSTRACT: Cyclin-dependent kinases (Cdks) and their cyclin regulatory subunits control cell growth and division. Cdk2-cyclin E complexes, phosphorylating the retinoblastoma protein, drive cells through the G1/S transition into the S phase of the cell cycle. Despite its fundamental role, Cdk2 was found to be indispensable only in specific cell types due to molecular redundancies in its function. Converging studies highlight involvement of Cdk2 and associated cell cycle regulatory proteins in oligodendrocyte progenitor cell proliferation and differentiation. Giving the contribution of this immature cell type to brain plasticity and repair in the adult, this review will explore the requirement of Cdk2 for oligodendrogenesis, oligodendrocyte progenitor cells proliferation and differentiation during physiological and pathological conditions.     

Cytosolic prion protein toxicity is independent of cellular prion protein expression and prion propagation

Prion diseases are transmissible neurodegenerative diseases caused by a conformational isoform of the prion protein (PrP), a host-encoded cell surface sialoglycoprotein. Recent evidence suggests a cytosolic fraction of PrP (cyPrP) functions either as an initiating factor or toxic element of prion disease. When expressed in cultured cells, cyPrP acquires properties of the infectious conformation of PrP (PrP(Sc)), including insolubility, protease resistance, aggregation, and toxicity. Transgenic mice (2D1 and 1D4 lines) that coexpress cyPrP and PrP(C) exhibit focal cerebellar atrophy, scratching behavior, and gait abnormalities suggestive of prion disease, although they lack protease-resistant PrP. To determine if the coexpression of PrP(C) is necessary or inhibitory to the phenotype of these mice, we crossed Tg1D4(Prnp(+/+)) mice with PrP-ablated mice (TgPrnp(o/o)) to generate Tg1D4(Prnp(o/o)) mice and followed the development of disease and pathological phenotype. We found no difference in the onset of symptoms or the clinical or pathological phenotype of disease between Tg1D4(Prnp(+/+)) and Tg1D4(Prnp(o/o)) mice, suggesting that cyPrP and PrP(C) function independently in the disease state. Additionally, Tg1D4(Prnp(o/o)) mice were resistant to challenge with mouse-adapted scrapie (RML), suggesting cyPrP is inaccessible to PrP(Sc). We conclude that disease phenotype and cellular toxicity associated with the expression of cyPrP are independent of PrP(C) and the generation of typical prion disease.     

Isolation and culture of rat and mouse oligodendrocyte precursor cells

The ability to isolate oligodendroglial precursor cells (OPCs) provides a powerful means to characterize their differentiation, properties and potential for myelin repair. Although much knowledge is available for isolation of OPCs from the rat central nervous system, preparation and maintenance of mouse OPCs has been until recently a challenge owing to difficulties in obtaining a sufficient quantity of purified OPCs. Here, we describe protocols to prepare highly enriched rat OPCs and nearly homogenous mouse OPCs. The mouse method generates predominantly OPCs from cortical neural progenitor cells as clonal aggregates called "oligospheres" by taking advantage of molecular genetic tools. Isolated OPCs can be further differentiated into oligodendrocytes. Collectively, we describe simple and efficient methods for the preparation and in vitro maintenance of enriched OPCs from rats and mice. Isolation and culture of a large, homogenous population of rodent OPCs should significantly facilitate studies on OPC lineage progression and their utility in myelin repair after injury.     

Prion protein scrapie and the normal cellular prion protein

Prions are infectious proteins and over the past few decades, some prions have become renowned for their causative role in several neurodegenerative diseases in animals and humans. Since their discovery, the mechanisms and mode of transmission and molecular structure of prions have begun to be established. There is, however, still much to be elucidated about prion diseases, including the development of potential therapeutic strategies for treatment. The significance of prion disease is discussed here, including the categories of human and animal prion diseases, disease transmission, disease progression and the development of symptoms and potential future strategies for treatment. Furthermore, the structure and function of the normal cellular prion protein (PrP^C) and its importance in not only in prion disease development, but also in diseases such as cancer and Alzheimer's disease will also be discussed.