p75NTR介导神经细胞凋亡研究进展

在神经发育过程中,神经营养因子通过其低亲和力受体p75NTR与高亲和力受体Trk的介导,在神经元的存活、分化与髓鞘形成中发挥着重要的作用;与存活、生长这些“正性”信号相反,p75NTR也介导受损后及新生神经元凋亡这一“负性”信号。一直以来,p75NTR是如何介导这些截然相反的功能仍不清楚,尤其是何时引起凋亡,其机制如何更是所知甚少。近来,随着s-p75NTR、proNGF、sortilin复合受体等的发现,其中的一些机制开始有所明朗。本文就p75NTR介导神经元凋亡的研究进展做一综述。     

Nogo-66 and myelin-associated glycoprotein (MAG) inhibit the adhesion and migration of Nogo-66 receptor expressing human glioma cells

Malignant gliomas are common and aggressive brain tumours associated with significant morbidity and mortality. We showed in this report that substratum adherence and migration by human U87MG glioma cells in culture were significantly attenuated by the extracellular domains of Nogo-A (Nogo-66) and the myelin-associated glycoprotein (MAG). U87MG cells contained significant amounts of endogenous Nogo-66 receptor (NgR), and treatment of the cells with phosphatidylinositol-specific phospholipase C (PI-PLC) or NgR antibodies resulted in an increase in their ability to adhere to, or migrate through, Nogo-66- and MAG-coated substrates. Nogo-66 and MAG may therefore modulate glioma growth and migration by acting through the NgR, a phenomenon that has potential therapeutic implications.     

Binding of soluble myelin-associated glycoprotein to specific gangliosides induces the association of p75NTR to lipid rafts and signal transduction

Myelin-associated glycoprotein (MAG) is a potent inhibitor of neurite outgrowth from a variety of neurons. Here we show that gangliosides, GT1b and GD1a, as well as the Nogo receptor, are functional binding partners for soluble MAG-Fc. Postnatal cerebellar neurons from mice deficient in the GalNAcT gene are insensitive to MAG with regard to neurite outgrowth and lack in the activation of RhoA. MAG-Fc or the antibody to GT1b and GD1a elicits recruitment of p75(NTR.) to lipid rafts, specialized microdomain for signal transduction. Disruption of lipid rafts results in abolishment of inhibitory effects of MAG-Fc and the Nogo peptide. These findings establish gangliosides as functional binding partners for soluble MAG. Gangliosides may play a role in translocation of p75(NTR.) to lipid rafts for initiation of the signal transduction.     

Astrocytic production of nerve growth factor in motor neuron apoptosis: implications for amyotrophic lateral sclerosis

Reactive astrocytes frequently surround degenerating motor neurons in patients and transgenic animal models of amyotrophic lateral sclerosis (ALS). We report here that reactive astrocytes in the ventral spinal cord of transgenic ALS-mutant G93A superoxide dismutase (SOD) mice expressed nerve growth factor (NGF) in regions where degenerating motor neurons expressed p75 neurotrophin receptor (p75(NTR)) and were immunoreactive for nitrotyrosine. Cultured spinal cord astrocytes incubated with lipopolysaccharide (LPS) or peroxynitrite became reactive and accumulated NGF in the culture medium. Reactive astrocytes caused apoptosis of embryonic rat motor neurons plated on the top of the monolayer. Such motor neuron apoptosis could be prevented when either NGF or p75(NTR) was inhibited with blocking antibodies. In addition, nitric oxide synthase inhibitors were also protective. Exogenous NGF stimulated motor neuron apoptosis only in the presence of a low steady state concentration of nitric oxide. NGF induced apoptosis in motor neurons from p75(NTR +/+) mouse embryos but had no effect in p75(NTR -/-) knockout embryos. Culture media from reactive astrocytes as well as spinal cord lysates from symptomatic G93A SOD mice-stimulated motor neuron apoptosis, but only when incubated with exogenous nitric oxide. This effect was prevented by either NGF or p75(NTR) blocking-antibodies suggesting that it might be mediated by NGF and/or its precursor forms. Our findings show that NGF secreted by reactive astrocytes induce the death of p75-expressing motor neurons by a mechanism involving nitric oxide and peroxynitrite formation. Thus, reactive astrocytes might contribute to the progressive motor neuron degeneration characterizing ALS.     

神经营养因子前体蛋白功能的研究进展

神经营养因子是一组在结构与功能上具有相关性的多肽性因子,它们通过前体蛋白的切割成为具有特定功能的成熟蛋白,为不同的神经细胞亚群提供营养支持,在中枢神经系统和周围神经系统的发育分化及病理生理中起着重要的作用.以前认为神经营养因子的前体不具有生理功能,最近的研究则表明,神经营养因子前体蛋白具有不同于神经营养因子的功能.研究发现,神经营养因子前体,至少神经生长因子和脑源性神经营养因子的前体大量存在于细胞外,它们通过与p75NTR和sortilin受体组成三聚体诱导神经细胞的凋亡.这一机制可能与神经发育时调节神经细胞的比例,神经损伤后神经细胞的死亡以及某些人类疾病的发生有密切联系.此外,神经营养因子前体还可能具有其他未知的新功能,对神经营养因子前体功能的深入研究将使人们对神经系统的发生、发育及神经系统疾病的发病机制有更加深入的了解,并有助于神经系统疾病新药物、新疗法的开发与研究.     

Nogo-P4对神经干细胞分化成神经元样细胞的影响

目的观察Nogo-P4对神经干细胞分化成神经元样细胞是否存在抑制作用。方法体外培养大鼠脊髓来源神经干细胞,分为A、B、C和D组,在神经干细胞分化过程中分别加入0μmol/L、2μmol/L、4μmol/L和6μmol/L的Nogo-P4,免疫荧光标记神经元样细胞,计数神经元样细胞分化比率,使用Image-ProPlus5.0软件测量神经元样细胞神经突长度。结果神经干细胞分化第3d,A-D各组神经元样细胞平均神经突长度分别为97.80±6.97μm、88.25±5.83μm、80.54±6.75μm和79.31±6.57μm;神经元样细胞比率分别为(35.82±6.53)%、(35.31±6.11)%、(38.97±5.79)%和(34.75±5.61)%。A-C组神经突长度随着Nogo-P4浓度的升高而下降,P<0.05;C、D组神经元样细胞神经突长度较A组缩短约17%;各组神经元样细胞分化比率无显著差异。结论Nogo-P4对神经干细胞分化过程中形成的神经元样细胞具有神经突生长抑制作用,对神经元样细胞分化比率无影响。     

A novel Rieske-type protein derived from an apoptosis-inducing factor-like (AIFL) transcript with a retained intron 4 induces change in mitochondrial morphology and growth arrest

Upon spinal cord injury, the myelin inhibitors, including the myelin-associated glycoprotein (MAG), Nogo-A and the oligodendrocyte myelin glycoprotein (OMgp), bind to and signal via a single neuronal receptor/co-receptor complex comprising of Nogo receptor 1(NgR1)/LINGO-1 and p75 or TROY, impeding regeneration of injured axons. We employed a cell-free system to study the binding of NgR1 to its co-receptors and the myelin inhibitor Nogo-A, and show that gangliosides mediate the interaction of NgR1 with LINGO-1. Solid phase binding assays demonstrate that the sialic acid moieties of gangliosides and the stalk of NgR1 are the principal determinants of these molecular interactions. Moreover, the tripartite complex comprising of NgR1, LINGO-1 and ganglioside exhibits stronger binding to Nogo-A (Nogo-54) in the presence of p75, suggesting the gangliosides modulate the myelin inhibitor-receptor signaling.     

Why do Nogo/Nogo‐66 receptor gene knockouts result in inferior regeneration compared to treatment with neutralizing agents?

IN-1, the monoclonal antibody against the exon 3-encoded N-terminal domain of Nogo-A, and the Nogo-66 receptor (NgR) antagonist NEP1-40 have both shown efficacy in promoting regeneration in animal spinal cord injury models, the latter even when administered subcutaneously 1 week after injury. These results are supportive of the hypothesis that the Nogo-NgR axis is a major path for inhibition of spinal cord axonal regeneration and uphold the promises of these neutralizing agents in clinical applications. However, mice with targeted disruption of Nogo and NgR have, surprisingly, only modest regenerative capacity (if any) compared with treatment with IN-1 or NEP1-40. Disruption of the Nogo gene by various groups yielded results ranging from significant regenerative improvement in young mice to no improvement. Likewise, knockout of NgR produced some improvement in raphespinal and rubrospinal axonal regeneration, but not that of corticospinal neurons. Other than invoking possible differences in genetic background, we suggest here some possible and testable explanations for the above phenomena. These possibilities include effects of IN-1 and NEP1-40 on the CNS beyond neutralization of Nogo and NgR functions, and the latter's possible role in the CNS beyond that of neuronal growth inhibition.     

Myelin-associated glycoprotein-mediated signaling in central nervous system pathophysiology

The myelin-associated glycoprotein (MAG) is a type I membrane-spanning protein expressed exclusively in oligoden drocytes and Schwann cells. It has two generally known pathophysiological roles in the central nervous system (CNS): maintenance of myelin integrity and inhibition of CNS axonal regeneration. The subtle CNS phenotype resulting from genetic ablation of MAG expression has made mechanistic analysis of its functional role in these difficult. However, the past few years have brought some major revelations, particularly in terms of mechanisms of MAG signaling through the Nogo-66 receptor (NgR) complex. Although apparently converging through NgR, a readily noticeable fact is that the neuronal growth inhibitory effect of MAG differs from that of Nogo-66. This may result from the influence of coreceptors in the form of gangliosides or from MAG-specific neuronal receptors such as NgR2. MAG has several other neuronal binding partners, and some of these may modulate its interaction with the NgR complex or downstream signaling. This article discusses new findings in MAG-forward and-reverse signaling and its role in CNS pathophysiology.     

RanBPM is a novel binding protein for p75NTR

The neurotrophin receptor p75NTR can induce signal transduction both in vivo and in vitro. The mechanisms by which p75NTR transduces signals have remained mostly unknown. Using yeast two-hybrid system, we identified the Ran-binding protein (RanBPM) as an interactor with the intracytoplasmic domain of p75NTR (p75ICD). The interaction was then validated by immunoprecipitation in mammalian cells and immunoblotting analysis. The domain in p75ICD interacting with RanBPM was mapped to the death domain.     

Inter- and intracellular interactions of Nogo: new findings and hypothesis

The molecule Nogo has captured the imagination of many as a possible key player, and therefore therapeutic target, in the pathological settings of central nervous system (CNS) injury and degenerative pathology. Found in both glial cells and neurons, the endogenous, physiological role of Nogo is as yet unknown. Recently reported targeted disruption of the Nogo gene did not result in any obvious neuro‐anatomical or neurological phenotype. Compared with wild‐type mice, Nogo‐deficient mice also did not exhibit a truly convincing enhancement in their ability to regenerate CNS neurons upon injury. Does the molecule have any important physiological function at all? Other recent discoveries of new interacting partners of Nogo at the mitochondria and the CNS paranode suggest intriguing links to the modulation of apoptosis and developmental organization or signalling at the axoglial junction.     

Rational design, solution conformation and identification of functional residues of the soluble and structured Nogo-54, which mimics Nogo-66 in inhibiting the CNS neurite outgrowth

The interaction between Nogo-66 and its receptor NgR represents a promising target for designing drugs to treat CNS axonal injury which often leads to permanent disability. Unfortunately, the isolated Nogo-66 is highly insoluble while its truncated form Nogo-40 is soluble but unstructured, thus retarding further characterization and application. Here, we rationally design another soluble form Nogo-54. CD and NMR characterization reveals that Nogo-54 is structured, and importantly, is able to mimic Nogo-66 in inhibiting neurite outgrowth. Strikingly, mutating its C-terminal four residues (Lys50, Glu51, Arg53, and Arg54) leads to a mutant Nogo-54m which has no dramatic structural change but whose inhibitory activity is completely abolished. This strongly suggests that the four charged residues contribute significantly to the inhibitory action of Nogo-66. Furthermore, our study also provides a soluble and structured mimic as well as a possible antagonist for Nogo-66 which may hold promising potential for various medical applications.     

神经营养因子对p75NTR诱导哺乳动物神经细胞凋亡的影响

目的：研究R2L1细胞的凋亡以及神经营养因子对凋亡的影响,方法：形态学观察,细胞活性测定和流式细胞仪分析。结果：R2L1细胞去和因清培养12小时即发生凋亡。24小时后大部分细胞凋亡。48小时后细胞基本上都死亡。结论：NGF、NT－3和抗p75NTR抗体能抑制R2L1细胞在去血清培养后发生的凋亡,而BDNF却无此报制功能。     

p75 neurotrophin receptor mediates apoptosis in transit-amplifying cells and its overexpression restores cell death in psoriatic keratinocytes

p75 neurotrophin receptor (p75NTR) belongs to the TNF-receptor superfamily and signals apoptosis in many cell settings. In human epidermis, p75NTR is mostly confined to the transit-amplifying (TA) sub-population of basal keratinocytes. Brain-derived neurotrophic factor (BDNF) or neurotrophin-4 (NT-4), which signals through p75NTR, induces keratinocyte apoptosis, whereas β-amyloid, a ligand for p75NTR, triggers caspase-3 activation to a greater extent in p75NTR transfected cells. Moreover, p75NTR co-immunoprecipitates with NRAGE, induces the phosphorylation of c-Jun N-terminal kinase (JNK) and reduces nuclear factor kappa B (NF-κB) DNA-binding activity. p75NTR also mediates pro-NGF-induced keratinocyte apoptosis through its co-receptor sortilin. Furthermore, BDNF or β-amyloid cause cell death in TA, but not in keratinocyte stem cells (KSCs) or in p75NTR silenced TA cells. p75NTR is absent in lesional psoriatic skin and p75NTR levels are significantly lower in psoriatic than in normal TA keratinocytes. The rate of apoptosis in psoriatic TA cells is significantly lower than in normal TA cells. BDNF or β-amyloid fail to induce apoptosis in psoriatic TA cells, and p75NTR retroviral infection restores BDNF- or β-amyloid-induced apoptosis in psoriatic keratinocytes. These results demonstrate that p75NTR has a pro-apoptotic role in keratinocytes and is involved in the maintenance of epidermal homeostasis.