Cellular prion protein coupling to TACE-dependent TNF-α shedding controls neurotransmitter catabolism in neuronal cells

Despite considerable efforts to unravel the role of cellular prion protein (PrP^C) in neuronal functions, the mechanisms by which PrP^C takes part in the homeostasis of a defined neuronal phenotype remain poorly characterized. By taking advantage of a neuroectodermal cell line (1C11) endowed with the capacity to differentiate into serotonergic (1C11^5-HT) or noradrenergic (1C11^NE) neurons, we assessed the contribution of PrP^C to bioaminergic cell functions. We established that in 1C11-derived neuronal cells antibody-mediated PrP^C ligation triggered tumor necrosis factor (TNF)-α release, through recruitement of the metalloproteinase TNF-α converting enzyme (TACE). TNF-α shed in response to PrP^C acts as a second message signal, eliciting serotonin (5-HT) or norepinephrine (NE) degradation in 1C11^5-HT or 1C11^NE cells, respectively. Our data thus introduced TNF-α as a PrP^C-dependent modulator of neuronal metabolism. Of note, we previously reported on a control of neurotransmitter catabolism by 5-HT_2B or α_1D autoreceptors in 1C11 bioaminergic neurons, via the same TACE/TNF-α pathway (Ann. N Y Acad. Sci. 1091, 123). Here, we show that combined stimulation of PrP^C and these two bioaminergic receptors add their effects on neurotransmitter degradation. Overall, these observations unveil a novel contribution of PrP^C to the control of neuronal functions and may have implications regarding dysfunction of the bioaminergic systems in prion diseases.     

Regulation by neurotransmitter receptors of serotonergic or catecholaminergic neuronal cell differentiation

The murine F9-derived 1C11 clone exhibits a stable epithelial morphology, expresses nestin, an early neuroectodermal marker, and expresses genes involved in neuroectodermal cell fate. Upon appropriate induction, 100% of 1C11 precursor cells develop neurite extensions and acquire neuronal markers (N-CAM, synaptophysin, gammagamma-enolase, and neurofilament) as well as the general functions of either serotonergic (1C11*(/5HT)) (5HT, 5-hydroxytryptamine) or noradrenergic (1C11**(/NE)) (NE, norepinephrine) neurons. The two programs are shown to be mutually exclusive. 1C11 thus behaves as a neuroepithelial cell line with a dual bioaminergic fate. 1C11*(/5HT) cells implement a functional 5-HT transporter and thereby a complete serotonergic phenotype within 4 days, whereas 5-HT(1B/D), 5-HT(2B), and 5-HT(2A) receptors are sequentially induced. The accurate time schedule of catecholaminergic differentiation was defined. Catecholamine synthesis, storage, and catabolism are acquired within 4 days; the noradrenergic phenotype is complete at day 12 and includes a functional norepinephrine transporter and an alpha(1D)-adrenoreceptor (day 8). The time-dependent onset of neurotransmitter-associated functions proper to either program is similar to in vivo observations. Along each pathway, the selective induction of serotonergic or adrenergic receptors is shown to be an essential part of the differentiation program, since they promote an autoregulation of the corresponding phenotype.     

Overstimulation of PrPC signaling pathways by prion peptide 106-126 causes oxidative injury of bioaminergic neuronal cells

Transmissible spongiform encephalopathies, also called prion diseases, are characterized by neuronal loss linked to the accumulation of PrP(Sc), a pathologic variant of the cellular prion protein (PrP(C)). Although the molecular and cellular bases of PrP(Sc)-induced neuropathogenesis are not yet fully understood, increasing evidence supports the view that PrP(Sc) accumulation interferes with PrP(C) normal function(s) in neurons. In the present work, we exploit the properties of PrP-(106-126), a synthetic peptide encompassing residues 106-126 of PrP, to investigate into the mechanisms sustaining prion-associated neuronal damage. This peptide shares many physicochemical properties with PrP(Sc) and is neurotoxic in vitro and in vivo. We examined the impact of PrP-(106-126) exposure on 1C11 neuroepithelial cells, their neuronal progenies, and GT1-7 hypothalamic cells. This peptide triggers reactive oxygen species overflow, mitogen-activated protein kinase (ERK1/2), and SAPK (p38 and JNK1/2) sustained activation, and apoptotic signals in 1C11-derived serotonergic and noradrenergic neuronal cells, while having no effect on 1C11 precursor and GT1-7 cells. The neurotoxic action of PrP-(106-126) relies on cell surface expression of PrP(C), recruitment of a PrP(C)-Caveolin-Fyn signaling platform, and overstimulation of NADPH-oxidase activity. Altogether, these findings provide actual evidence that PrP-(106-126)-induced neuronal injury is caused by an amplification of PrP(C)-associated signaling responses, which notably promotes oxidative stress conditions. Distorsion of PrP(C) signaling in neuronal cells could hence represent a causal event in transmissible spongiform encephalopathy pathogenesis.     

PrPc介导的细胞信号转导

朊病毒病的发生是由于细胞正常朊蛋白PrPc转变成了异常构象的PrPc形式。PrPc的生理学功能目前尚不完全明确,可能与铜离子代谢、脂质摄取以及细胞信号传递有关。PrPc可以与小窝蛋白相互作用而活化Fyn非受体酪氨酸激酶从而引起下游信号通路的转导;可以作为受体与PrPc键合多肽结合后激活cAMP／PKA信号通路;以及引起细胞内钙离子浓度变化而活化信号通路。     

The cellular prion protein: a new partner of the lectin CBP70 in the nucleus of NB4 human promyelocytic leukemia cells.

Prion diseases are characterized by the presence of an abnormal isoform of the cellular prion protein (PrPc) whose physiological role still remains elusive. To better understand the function of PrPc, it is important to identify the different subcellular localization(s) of the protein and the different partners with which it might be associated. In this context, the PrPc-lectins interactions are investigated because PrPc is a sialoglycoprotein which can react with lectins which are carbohydrate-binding proteins. We have previously characterized a nuclear lectin CBP70 able to recognize N-acetyl-beta-D-glucosamine residues in HL60 cells. Using confocal immunofluorescence, flow-cytofluorometry, and Western-blotting, we have found that PrPc is expressed in the nucleus of the NB4 human promyelocytic leukemia cell line. It was also found that the lectin CBP70 is localized in NB4 cell nuclei. Moreover, several approaches revealed that PrPc and CBP70 are colocalized in the nucleus. Immunoprecipitation experiments showed that these proteins are coprecipitated and interact via a sugar-dependent binding moiety. In conclusion, PrPc and CBP70 are colocalized in the nuclear compartment of NB4 cells and this interaction may be important to better understand the biological function and possibly the conversion process of PrPc into its pathological form (PrPsc).     

The Cellular Prion Protein Interacts with the Tissue Non-Specific Alkaline Phosphatase in Membrane Microdomains of Bioaminergic Neuronal Cells

Background

The cellular prion protein, PrP^C, is GPI anchored and abundant in lipid rafts. The absolute requirement of PrP^C in neurodegeneration associated to prion diseases is well established. However, the function of this ubiquitous protein is still puzzling. Our previous work using the 1C11 neuronal model, provided evidence that PrP^C acts as a cell surface receptor. Besides a ubiquitous signaling function of PrP^C, we have described a neuronal specificity pointing to a role of PrP^C in neuronal homeostasis. 1C11 cells, upon appropriate induction, engage into neuronal differentiation programs, giving rise either to serotonergic (1C11^5-HT) or noradrenergic (1C11^NE) derivatives.

Methodology/Principal Findings

The neuronal specificity of PrP^C signaling prompted us to search for PrP^C partners in 1C11-derived bioaminergic neuronal cells. We show here by immunoprecipitation an association of PrP^C with an 80 kDa protein identified by mass spectrometry as the tissue non-specific alkaline phosphatase (TNAP). This interaction occurs in lipid rafts and is restricted to 1C11-derived neuronal progenies. Our data indicate that TNAP is implemented during the differentiation programs of 1C11^5-HT and 1C11^NE cells and is active at their cell surface. Noteworthy, TNAP may contribute to the regulation of serotonin or catecholamine synthesis in 1C11^5-HT and 1C11^NE bioaminergic cells by controlling pyridoxal phosphate levels. Finally, TNAP activity is shown to modulate the phosphorylation status of laminin and thereby its interaction with PrP.

Conclusion/Significance

The identification of a novel PrP^C partner in lipid rafts of neuronal cells favors the idea of a role of PrP in multiple functions. Because PrP^C and laminin functionally interact to support neuronal differentiation and memory consolidation, our findings introduce TNAP as a functional protagonist in the PrP^C-laminin interplay. The partnership between TNAP and PrP^C in neuronal cells may provide new clues as to the neurospecificity of PrP^C function.     

Depletion of Janus kinase-2 promotes neuronal differentiation of mouse embryonic stem cells

Janus kinase 2 (JAK2), a non-receptor tyrosine kinase, is a critical component of cytokine and growth factor signaling pathways regulating hematopoietic cell proliferation. JAK2 mutations are associated with multiple myeloproliferative neoplasms. Although physiological and pathological functions of JAK2 in hematopoietic tissues are well-known, such functions of JAK2 in the nervous system are not well studied yet. The present study demonstrated that JAK2 could negatively regulate neuronal differentiation of mouse embryonic stem cells (ESCs). Depletion of JAK2 stimulated neuronal differentiation of mouse ESCs and activated glycogen synthase kinase 3β, Fyn, and cyclin-dependent kinase 5. Knockdown of JAK2 resulted in accumulation of GTP-bound Rac1, a Rho GTPase implicated in the regulation of cytoskeletal dynamics. These findings suggest that JAK2 might negatively regulate neuronal differentiation by suppressing the GSK-3β/Fyn/CDK5 signaling pathway responsible for morphological maturation.     

Glycosylation-related genes are variably expressed depending on the differentiation state of a bioaminergic neuronal cell line: implication for the cellular prion protein

A striking feature of the cellular prion protein (PrP^C) is the heterogeneity of its glycoforms, whose contribution to PrP^C function has yet to be defined. Using the 1C11 neuronal bioaminergic differentiation model and a glycomics approach, we show here a correlation between differential PrP^C N-glycosylations in 1C11^5-HT serotonergic and 1C11^NE noradrenergic cells compared to their 1C11 precursor cells and a variation of the glycogenome expression status in these cells. In particular, expression of genes involved in N-glycan synthesis or in the modeling of chondroitin and heparan sulfate proteoglycans appeared to be modulated. Our results highlight that, the expression of glycosylation-related genes is regulated during bioaminergic neuronal differentiation, consistent with a participation of glycoconjugates in neuronal development and plasticity. A neuronal regulation of glycosylation processes may have direct implications on some neurospecific functions of PrP^C and may participate in specific brain targeting of prion strains. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

朊病毒研究的现状

朊病毒疾病是人和动物中的一种传染性,散发性和遗传性的神经退行性脑病,到目前为止,关于此病的致病机制并不十分清楚,但有研究表明该病是由一种正常蛋白PrP的不正常折叠形式在大脑中积聚所致,许多哺乳动物和鸟类的朊病毒基因和氨基酸序列都已被分析,而且传染源的部分性质已经被阐明,朊病毒疾病的传染和潜伏期在人和动物中有很大差异,一些变异与疾病的自发性,易感性和抵抗性有关,为得到关于现毒更多的信息,已对其基因两端进行了大范围的DNA测序分析,至于正常细胞蛋白PrP的生理功能,现在也并不确定。     

Derivation of high purity neuronal progenitors from human embryonic stem cells

The availability of human neuronal progenitors (hNPs) in high purity would greatly facilitate neuronal drug discovery and developmental studies, as well as cell replacement strategies for neurodegenerative diseases and conditions, such as spinal cord injury, stroke, Parkinson's disease, Alzheimer's disease, and Huntington's disease. Here we describe for the first time a method for producing hNPs in large quantity and high purity from human embryonic stem cells (hESCs) in feeder-free conditions, without the use of exogenous noggin, sonic hedgehog or analogs, rendering the process clinically compliant. The resulting population displays characteristic neuronal-specific markers. When allowed to spontaneously differentiate into neuronal subtypes in vitro, cholinergic, serotonergic, dopaminergic and/or noradrenergic, and medium spiny striatal neurons were observed. When transplanted into the injured spinal cord the hNPs survived, integrated into host tissue, and matured into a variety of neuronal subtypes. Our method of deriving neuronal progenitors from hESCs renders the process amenable to therapeutic and commercial use.     

Human Doppel and prion protein share common membrane microdomains and internalization pathways

Doppel is the first identified homologue of the prion protein (PrPc) implicated in prion disease. Doppel is considered an N-truncated form of PrPc, and shares with PrPc several structural and biochemical features. When over expressed in the brain of some PrP knockout animals, it provokes cerebellar ataxia. As this phenotype is rescued by reintroducing the PrP gene, it has been suggested that Doppel and PrPc have antagonistic functions and may compete for a common ligand. However, a direct interaction between the two proteins has recently been observed. To investigate whether the neuronal environment is suitable for such possibility, human Doppel and PrPc were expressed separately, or together, in neuroblastoma cells, and then studied by biochemical and immunomicroscopic tools, as well as in intact cells expressing fluorescent fusion constructs. The results demonstrate that Doppel and PrPc co-patch extensively at the plasma membrane, and get internalized together after ganglioside cross-linking by cholera toxin or addition of an antibody against only one of the proteins. These processes no longer occur if the integrity of rafts is disrupted. We also show that, whereas each protein expressed alone occupies Triton X-100-insoluble membrane microdomains, co-transfected Doppel and PrPc redistribute together into a less ordered lipidic environment. All these features are consistent with interactions occurring between Doppel and PrPc in our neuronal cell model.     

Functions of prion protein PrPc

It is now well established that both normal and pathological (or scrapie) isoforms of prion protein, PrPc and PrPsc respectively, are involved in the development and progression of various forms of neurodegenerative diseases, including scrapie in sheep, bovine spongiform encephalopathy (or "mad cow disease") and Creutzfeldt-Jakob disease in human, collectively known as prion diseases. The protein PrPc is highly expressed in the central nervous system in neurons and glial cells, and also present in non-brain cells, such as immune cells or epithelial and endothelial cells. Identification of the physiological functions of PrPc in these different cell types thus appears crucial for understanding the progression of prion diseases. Recent studies highlighted several major roles for PrPc that may be considered in two major domains : (1) cell survival (protection against oxidative stress and apoptosis) and (2) cell adhesion. In association with cell adhesion, distinct functions of PrPc were observed, depending on cell types : neuronal differentiation, epithelial and endothelial barrier integrity, transendothelial migration of monocytes, T cell activation. These observations suggest that PrPc functions may be particularly relevant to cellular stress, as well as inflammatory or infectious situations.     

Selective prion protein binding to synaptic components is modulated by oxidative and nitrosative changes induced by copper(II) and peroxynitrite in cholinergic synaptosomes, unveiling a role for calcineurin B and thioredoxin

Choline acetyltransferase (ChAT) and choline transport are decreased after nitrosative stress. ChAT activity is altered in scrapie-infected neurons, where oxidative stress develops. Cellular prion protein (PrPc) may play a neuroprotective function in participating in the redox control of neuronal environment and regulation of copper metabolism, a role impaired when PrPc is transformed into PrPSc in prion pathologies. The complex cross-talk between PrPc and cholinergic neurons was analyzed in vitro using peroxynitrite and Cu2+ treatments on nerve endings isolated from Torpedo marmorata, a model of the motoneuron pre-synaptic element. Specific interactions between solubilized synaptic components and recombinant ovine prion protein (PrPrec) could be demonstrated by Biacore technology. Peroxynitrite abolished this interaction in a concentration-dependent way and induced significant alterations of neuronal targets. Interaction was restored by prior addition of peroxynitrite trapping agents. Cu2+ (in the form of CuSO4) treatment of synaptosomes triggered a milder oxidative effect leading to a bell-shaped increase of PrPrec binding to synaptosomal components, counteracted by the natural thiol agents, glutathione and thioredoxin. Copper(II)-induced modifications of thiols in several neuronal proteins. A positive correlation was observed between PrPrec binding and immunoreactive changes for calcineurin B and its partners, suggesting a synergy between calcineurin complex and PrP for copper regulation.     

Axonal transport of the cellular prion protein is increased during axon regeneration

The cellular prion protein, PrPc, is a glycosylphosphatidylinositol-anchored cell surface glycoprotein and a protease-resistant conformer of the protein may be the infectious agent in transmissible spongiform encephalopathies. PrPc is localized on growing axons in vitro and along fibre bundles that contain elongating axons in developing and adult brain. To determine whether the growth state of axons influenced the expression and axonal transport of PrPc, we examined changes in the protein following post-traumatic regeneration in the hamster sciatic nerve. Our results show (1) that PrPc in nerve is significantly increased during nerve regeneration; (2) that this increase involves an increase in axonally transported PrPc; and (3) that the PrPc preferentially targeted for the newly formed portions of the regenerating axons consists of higher molecular weight glycoforms. These results raise the possibility that PrPc may play a role in the growth of axons in vivo, perhaps as an adhesion molecule interacting with the extracellular environment through specialized glycosylation.     

Prions impair bioaminergic functions through serotonin- or catecholamine-derived neurotoxins in neuronal cells

The conversion of the cellular prion protein, PrP(C), to an abnormal isoform, PrP(Sc), is a central event leading to neurodegeneration in prion diseases. Deciphering the molecular and cellular changes imparted by PrP(Sc) accumulation remains an arduous task due to the small number of cell lines supporting prion replication. Here we introduce the 1C11 cell line as a new in vitro model to investigate prion pathogenesis. This cell line is a committed neuroectodermal progenitor able to differentiate into fully functional serotonergic or catecholaminergic neurons. 1C11 cells, which naturally express PrP(C) from the undifferentiated state, can be chronically infected with various prion strains. Prion infection does not promote any noticeable phenotypic change in the progenitor cells nor prevent the onset of the serotonergic and catecholaminergic differentiation programs. Pathogenic prions, however, deviate the overall neurotransmitter-metabolism in both pathways by decreasing bioamine synthesis, storage, and transport, and enhancing catabolism. Noteworthy, oxidized derivatives of both serotonin and catecholamines are selectively detected in the differentiated progenies of infected cells and contribute to irreversible impairment in bioamine synthesis. Finally, the level of PrP(Sc) accumulation, that of infectivity, and the extent of all prion-induced changes in infected cells appear to be correlated. The report of such specific effects of infection on neuronal functions provides a foundation for dissecting the events underlying loss of neuronal homeostasis in prion diseases.     

Role of Src kinases in the ADAM-mediated release of L1 adhesion molecule from human tumor cells

The ectodomain of certain transmembrane molecules can be released by proteolysis, and the solubilized antigens often exert important biological functions. We demonstrated before that the L1 adhesion molecule is shed from the cell surface. Here we show that L1 release in AR breast carcinoma cells is mediated by a member of the disintegrin metalloproteinase (ADAM) family of proteinases. Up-regulation of L1 shedding by phorbol ester or pervanadate involved distinct mechanisms. Pervanadate induced shedding and rounding-up of cells from the substrate, which was blocked by the Src kinase inhibitor PP2. Tyr phosphorylation of the L1 cytoplasmic tail and the Src kinase Fyn was observed following pervanadate treatment. Up-regulation of L1 release and activation of Fyn occurred also when cells were detached by EDTA suggesting that the regulation of L1 shedding by this pathway was linked to cell morphology and adhesion. The phorbol 12-myristate 13-acetate-induced shedding was inhibited by the protein kinase C inhibitor bisindolylmaleimide I and by PD98059, a specific inhibitor of the mitogen-activated protein kinase pathway. Soluble L1 binds to the proteoglycan neurocan and in bound form could support integrin-mediated cell adhesion and migration. We propose that the release of cell-associated adhesion molecules such as L1 may be relevant to promote cell migration.