Nogo-A and Nogo-66 receptor in amyotrophic lateral sclerosis

Nogo/reticulon (RTN)-4 has been strongly implicated as a disease marker for the motor neuron disease amyotrophic lateral sclerosis (ALS). Nogo isoforms, including Nogo-A, are ectopically expressed in the skeletal muscle of ALS mouse models and patients and their levels correlate with the disease severity. The notion of a direct involvement of Nogo-A in ALS aetiology is supported by the findings that Nogo-A deletion in mice reduces muscle denervation and prolongs survival, whereas overexpression of Nogo-A destabilizes motor nerve terminals and promotes denervation. Another intriguing, and somewhat paradoxical, recent finding revealed that binding of the Nogo-66 receptor (NgR) by either agonistic or antagonistic Nogo-66-derived peptides protects against p75 neurotrophin receptor (p75(NTR))-dependent motor neuron death. Ligand binding by NgR could result in subsequent engagement of p75(NTR), and this association could preclude pro-apoptotic signalling by the latter. Understanding the intricate interplay among Nogo isoforms, NgR and p75(NTR) in ALS disease progression may provide important, therapeutically exploitable information.     

Getting RIP'd Stunts Your Growth

The p75 neurotrophin receptor (p75NTR) collaborates with the Nogo receptor (NgR) and LINGO-1 to activate RhoA in response to myelin-based growth inhibitors such as myelin-associated glycoprotein (MAG). In this issue of Neuron, Domeniconi et al., in a surprising turn, show that MAG induces intramembrane proteolysis (RIP) of p75NTR and find that p75NTR cleavage is required for MAG-induced RhoA activation and growth inhibition.     

TAJ/TROY, an orphan TNF receptor family member, binds Nogo-66 receptor 1 and regulates axonal regeneration

Myelin-associated inhibitory factors (MAIFs) are inhibitors of CNS axonal regeneration following injury. The Nogo receptor complex, composed of the Nogo-66 receptor 1 (NgR1), neurotrophin p75 receptor (p75), and LINGO-1, represses axon regeneration upon binding to these myelin components. The limited expression of p75 to certain types of neurons and its temporal expression during development prompted speculation that other receptors are involved in the NgR1 complex. Here, we show that an orphan receptor in the TNF family called TAJ, broadly expressed in postnatal and adult neurons, binds to NgR1 and can replace p75 in the p75/NgR1/LINGO-1 complex to activate RhoA in the presence of myelin inhibitors. In vitro exogenously added TAJ reversed neurite outgrowth caused by MAIFs. Neurons from Taj-deficient mice were more resistant to the suppressive action of the myelin inhibitors. Given the limited expression of p75, the discovery of TAJ function is an important step for understanding the regulation of axonal regeneration.     

Rho activation patterns after spinal cord injury and the role of activated Rho in apoptosis in the central nervous system

Growth inhibitory proteins in the central nervous system (CNS) block axon growth and regeneration by signaling to Rho, an intracellular GTPase. It is not known how CNS trauma affects the expression and activation of RhoA. Here we detect GTP-bound RhoA in spinal cord homogenates and report that spinal cord injury (SCI) in both rats and mice activates RhoA over 10-fold in the absence of changes in RhoA expression. In situ Rho-GTP detection revealed that both neurons and glial cells showed Rho activation at SCI lesion sites. Application of a Rho antagonist (C3-05) reversed Rho activation and reduced the number of TUNEL-labeled cells by approximately 50% in both injured mouse and rat, showing a role for activated Rho in cell death after CNS injury. Next, we examined the role of the p75 neurotrophin receptor (p75NTR) in Rho signaling. After SCI, an up-regulation of p75NTR was detected by Western blot and observed in both neurons and glia. Treatment with C3-05 blocked the increase in p75NTR expression. Experiments with p75NTR-null mutant mice showed that immediate Rho activation after SCI is p75NTR dependent. Our results indicate that blocking overactivation of Rho after SCI protects cells from p75NTR-dependent apoptosis.     

TRAF family proteins interact with the common neurotrophin receptor and modulate apoptosis induction.

The common neurotrophin receptor, p75(NTR), has been shown to signal in the absence of Trk tyrosine kinase receptors, including induction of neural apoptosis and activation of NF-kappaB. However, the mechanisms by which p75(NTR) initiates these intracellular signal transduction pathways are unknown. Here we report interactions between p75(NTR) and the six members of TRAF (tumor necrosis factor receptor-associated factors) family proteins. The binding of different TRAF proteins to p75(NTR) was mapped to distinct regions in p75(NTR). Furthermore, TRAF4 interacted with dimeric p75(NTR), whereas TRAF2 interacted preferentially with monomeric p75(NTR). TRAF2-p75(NTR), TRAF4-p75(NTR), and TRAF6-p75(NTR) interactions modulated p75(NTR)-induced cell death and NF-kappaB activation with contrasting effects. Coexpression of TRAF2 with p75(NTR) enhanced cell death, whereas coexpression of TRAF6 was cytoprotective. Furthermore, overexpression of TRAF4 abrogated the ability of dimerization to prevent the induction of apoptosis normally mediated by monomeric p75(NTR). TRAF4 also inhibited the NF-kappaB response, whereas TRAF2 and TRAF6 enhanced p75(NTR)-induced NF-kappaB activation. These results demonstrate that TRAF family proteins interact with p75(NTR) and differentially modulate its NF-kappaB activation and cell death induction.     

Neurotrophin binding to the p75 receptor modulates Rho activity and axonal outgrowth

While the neurotrophin receptor p75NTR is expressed by many developing neurons, its function in cells escaping elimination by programmed cell death remains unclear. The lack of intrinsic enzymatic activity of p75NTR prompted a search for protein interactors expressed in the developing retina, which resulted in the identification of the GTPase RhoA. In transfected cells, p75NTR activated RhoA, and neurotrophin binding abolished RhoA activation. In cultured neurons, inactivation of Rho proteins mimicked the effect of neurotrophins by increasing the rate of neurite elongation. In vivo, axonal outgrowth was retarded in mice carrying a mutation in the p75NTR gene. These results indicate that p75NTR modulates in a ligand-dependent fashion the activity of intracellular proteins known to regulate actin assembly.     

中枢神经再生抑制因子及免疫治疗研究

成体哺乳动物中枢神经损伤后早期轴突再生失败的一个主要原因是由于髓磷脂抑制分子的存在。Nogo、髓磷脂相关糖蛋白以及少突胶质细胞髓磷脂糖蛋白等神经再生抑制因子的发现,大大促进了中枢神经再生分子机制的研究。它们均能独立通过Nogo-66受体产生对轴突再生的抑制效应,髓磷脂抑制分子及其信号转导机制的研究日益成为中枢神经再生的研究热点,髓磷脂及其信号转导分子特别是Nogo-66受体、p75神经营养素受体成为损伤后促进轴突再生、抑制生长锥塌陷的主要治疗靶点。抑制上述抑制因子及相关受体NgR或p75NTR可能有助于中枢神经损伤的修复,围绕这些抑制因子及其相关受体介导的信号转导途径,人们提出了多种治疗中枢神经损伤的新思路,其中免疫学方法尤其受到关注。     

Troy/Taj and its role in CNS axon regeneration

Binding of myelin inhibitors to the NgR1/p75/LINGO-1 signaling complex activates RhoA to mediate the inhibition of axonal outgrowth. The nerve growth factor receptor p75, a TNF family receptor, is absent or poorly expressed in certain types of neurons that respond to myelin inhibitors, thereby prompting speculation that other TNF family receptors are involved in the NgR1 complex. Troy/Taj is an orphan TNF family receptor that is broadly expressed in postnatal and adult neurons. Troy binds to NgR1 and can functionally replace p75 in the p75/NgR1/LINGO-1 complex to activate RhoA and block neurite outgrowth in the presence of myelin inhibitors. Neurons from Troy-deficient mice are more resistant to the suppressive action of the myelin inhibitors. The discovery of TROY function in axon growth is an important step for understanding the complex regulation of axonal regeneration by diverse members of the TNF receptor family.     

Polyubiquitination of the neurotrophin receptor p75 directs neuronal cell survival

Specific binding of nerve growth factor (NGF) to p75 neurotrophin receptor (p75(NTR)) leads to p75(NTR) polyubiquitination and its subsequent interaction with TRAF6 resulting in neuronal cell survival. However, when the binding of NGF to p75(NTR) was blocked with p75 antiserum, p75(NTR) polyubiquitination and neuronal cell survival were impaired. Results showed that tyrosine phosphorylation of p75(NTR) increased the polyubiquitination of p75(NTR) and contributed to the observed apparent neuroprotective effects. Similar to p75(NTR) polyubiquitination, interaction of TRAF6 with p75(NTR) was NGF/tyrosine phosphorylation dependent suggesting that TRAF6 might function as an E3 ubiquitin ligase. In sum, the results show that specific binding of NGF to p75(NTR) mediates neuronal cell survival.     

AMIGO3 Is an NgR1/p75 Co-Receptor Signalling Axon Growth Inhibition in the Acute Phase of Adult Central Nervous System Injury

Axon regeneration in the injured adult CNS is reportedly inhibited by myelin-derived inhibitory molecules, after binding to a receptor complex comprised of the Nogo-66 receptor (NgR1) and two transmembrane co-receptors p75/TROY and LINGO-1. However, the post-injury expression pattern for LINGO-1 is inconsistent with its proposed function. We demonstrated that AMIGO3 levels were significantly higher acutely than those of LINGO-1 in dorsal column lesions and reduced in models of dorsal root ganglion neuron (DRGN) axon regeneration. Similarly, AMIGO3 levels were raised in the retina immediately after optic nerve crush, whilst levels were suppressed in regenerating optic nerves, induced by intravitreal peripheral nerve implantation. AMIGO3 interacted functionally with NgR1-p75/TROY in non-neuronal cells and in brain lysates, mediating RhoA activation in response to CNS myelin. Knockdown of AMIGO3 in myelin-inhibited adult primary DRG and retinal cultures promoted disinhibited neurite growth when cells were stimulated with appropriate neurotrophic factors. These findings demonstrate that AMIGO3 substitutes for LINGO-1 in the NgR1-p75/TROY inhibitory signalling complex and suggests that the NgR1-p75/TROY-AMIGO3 receptor complex mediates myelin-induced inhibition of axon growth acutely in the CNS. Thus, antagonizing AMIGO3 rather than LINGO-1 immediately after CNS injury is likely to be a more effective therapeutic strategy for promoting CNS axon regeneration when combined with neurotrophic factor administration.     

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.     

The p75 receptor transduces the signal from myelin-associated glycoprotein to Rho

Myelin-associated glycoprotein (MAG) is a potent inhibitor of neurite outgrowth from a variety of neurons. The receptor for MAG or signals that elicit morphological changes in neurons remained to be established. Here we show that the neurotrophin receptor p75 (p75(NTR)) is the signal transducing element for MAG. Adult dorsal root ganglion neurons or postnatal cerebellar neurons from mice carrying a mutation in the p75(NTR) gene are insensitive to MAG with regard to neurite outgrowth. MAG activates small GTPase RhoA, leading to retarded outgrowth when p75(NTR)) is present. Colocalization of p75(NTR) and MAG binding is seen in neurons. Ganglioside GT1b, which is one of the binding partners of MAG, specifically associates with p75(NTR). Thus, p75(NTR) and GT1b may form a receptor complex for MAG to transmit the inhibitory signals in neurons.     

Suppression of the p75 receptor signal attenuates the effect of ephrin-B3 and promotes axonal regeneration of the injured optic nerve

The p75 neurotrophin receptor (p75NTR) is known to transduce the signal from some myelin-associated axon growth inhibitors, including Nogo and myelin-associated glycoprotein. As ephrin-B3, a member of the ephrin family, is also expressed in myelin and inhibits axon growth, the purpose of this study was to assess the possible involvement of p75NTR in ephrin-B3 signaling. Here, we report that p75NTR is required for the inhibitory effect of ephrin-B3 on neurite growth in vitro. While ephrin-B3 inhibited neurite elongation of embryonic cortical neurons, the neurons with p75NTR knockdown or with EphA4 knockdown were less sensitive to ephrin-B3. Although no direct interaction of p75NTR with ephrin-B3 was observed, Pep5, a peptide that specifically inhibits RhoA activation mediated by p75NTR, reduced the effect of ephrin-B3. Therefore, p75NTR functions as a signal transducer for ephrin-B3. Moreover, axonal regeneration in vivo was induced by Pep5 application after optic nerve crush injury in mice. Thus, Pep5 is a promising agent that contributes to axonal regeneration in the central nervous system.     

Nogo在中枢神经损伤再生中的作用机制

Nogo是中枢神经系统（CNS）少突胶质细胞分泌的一种髓磷脂蛋白,它的主要功能是抑制损伤后轴突的再生,它含有两个完全独立的具有抑制活性的结构域：位于细胞内的amino—Nogo和位于细胞表面的Nogo-66。Nogo-66是通过与受体复合体NgR／p75／Lingo—1结合,触发Rho信号通路来发挥作用。Nogo及其信号转导机制日益成为CNS损伤再生的研究热点,就Nogo在CNS损伤再生中的作用机制作一综述。     

ProBDNF collapses neurite outgrowth of primary neurons by activating RhoA

Background

Neurons extend their dendrites and axons to build functional neural circuits, which are regulated by both positive and negative signals during development. Brain-derived neurotrophic factor (BDNF) is a positive regulator for neurite outgrowth and neuronal survival but the functions of its precursor (proBDNF) are less characterized.

Methodology/Principal Findings

Here we show that proBDNF collapses neurite outgrowth in murine dorsal root ganglion (DRG) neurons and cortical neurons by activating RhoA via the p75 neurotrophin receptor (p75NTR). We demonstrated that the receptor proteins for proBDNF, p75NTR and sortilin, were highly expressed in cultured DRG or cortical neurons. ProBDNF caused a dramatic neurite collapse in a dose-dependent manner and this effect was about 500 fold more potent than myelin-associated glycoprotein. Neutralization of endogenous proBDNF by using antibodies enhanced neurite outgrowth in vitro and in vivo, but this effect was lost in p75NTR^−/− mice. The neurite outgrowth of cortical neurons from p75NTR deficient (p75NTR^−/−) mice was insensitive to proBDNF. There was a time-dependent reduction of length and number of filopodia in response to proBDNF which was accompanied with a polarized RhoA activation in growth cones. Moreover, proBDNF treatment of cortical neurons resulted in a time-dependent activation of RhoA but not Cdc42 and the effect was absent in p75NTR^−/− neurons. Rho kinase (ROCK) and the collapsin response mediator protein-2 (CRMP-2) were also involved in the proBDNF action.

Conclusions

proBDNF has an opposing role in neurite outgrowth to that of mature BDNF. Our observations suggest that proBDNF collapses neurites outgrowth and filopodial growth cones by activating RhoA through the p75NTR signaling pathway.     

Molecular dissection of the myelin-associated glycoprotein receptor complex reveals cell type-specific mechanisms for neurite outgrowth inhibition

Neuronal Nogo66 receptor-1 (NgR1) binds the myelin inhibitors NogoA, OMgp, and myelin-associated glycoprotein (MAG) and has been proposed to function as the ligand-binding component of a receptor complex that also includes Lingo-1, p75(NTR), or TROY. In this study, we use Vibrio cholerae neuraminidase (VCN) and mouse genetics to probe the molecular composition of the MAG receptor complex in postnatal retinal ganglion cells (RGCs). We find that VCN treatment is not sufficient to release MAG inhibition of RGCs; however, it does attenuate MAG inhibition of cerebellar granule neurons. Furthermore, the loss of p75(NTR) is not sufficient to release MAG inhibition of RGCs, but p75(NTR-/-) dorsal root ganglion neurons show enhanced growth on MAG compared to wild-type controls. Interestingly, TROY is not a functional substitute for p75(NTR) in RGCs. Finally, NgR1(-/-) RGCs are strongly inhibited by MAG. In the presence of VCN, however, NgR1(-/-) RGCs exhibit enhanced neurite growth. Collectively, our experiments reveal distinct and cell type-specific mechanisms for MAG-elicited growth inhibition.     

Functional interaction of Fas-associated phosphatase-1 (FAP-1) with p75(NTR) and their effect on NF-kappaB activation.

The common neurotrophin receptor p75(NTR), a member of the tumor necrosis factor (TNF) receptor superfamily, plays an important role in several cellular signaling cascades, including that leading to apoptosis. FAP-1 (Fas-associated phosphatase-1), which binds to the cytoplasmic tail of Fas, was originally identified as a negative regulator of Fas-mediated apoptosis. Here we have shown by co-immunoprecipitation that FAP-1 also binds to the p75(NTR) cytoplasmic domain in vivo through the interaction between the third PDZ domain of FAP-1 and C-terminal Ser-Pro-Val residues of p75(NTR). Furthermore, cells expressing a FAP-1/green fluorescent protein showed intracellular co-localization of FAP-1 and p75(NTR) at the plasma membrane. To elucidate the functional role of this physical interaction, we examined TRAF6 (TNF receptor-associated factor 6)-mediated NF-kappaB activation and tamoxifen-induced apoptosis in 293T cells expressing p75(NTR). The results revealed that TRAF6-mediated NF-kappaB activation was suppressed by p75(NTR) and that the p75(NTR)-mediated NF-kappaB suppression was reduced by FAP-1 expression. Interestingly, a mutant of the p75(NTR) intracellular domain with a single substitution of a Met for Val in its C-terminus, which cannot interact with FAP-1, displayed enhanced pro-apoptotic activity in 293T transfected cells. Thus, similar to Fas, FAP-1 may be involved in suppressing p75(NTR)-mediated pro-apoptotic signaling through its interaction with three C-terminal amino acids (tSPV). Thus, FAP-1 may regulate p75(NTR)-mediated signal transduction by physiological interaction through its third PDZ domain.     

A role for p75 neurotrophin receptor in the control of hair follicle morphogenesis

During hair follicle (HF) morphogenesis, p75 neurotrophin receptor (p75NTR) reportedly is the first growth factor receptor found to be expressed by those fibroblasts that later develop into the dermal papilla (DP) of the HF. However, the functional role of p75NTR in HF morphogenesis is still unknown. Studying HF development in fetal and neonatal C57BL/6 murine back skin, we show that p75NTR-immunoreactivity (IR) is prominently expressed by DP fibroblasts as well as by skin nerves during the early steps of HF development. In contrast, p75NTR-IR disappears from the DP in the fully developed HF and it is expressed only in the epithelial outer root sheath of the HF. Compared to age-matched wild-type animals, p75NTR knockout (-/-) mice show significant acceleration of HF morphogenesis, and DP fibroblasts of p75NTR knockout mice show reduced proliferative activity in situ, indicating alterations in their transition from proliferation to differentiation. Although no significant differences in the expression of adhesion molecules (NCAM), selected morphogens (TGFbeta-2, HGF/SF, FGF-2, KGF), or their receptors (TGFbetaR-II, m-met, FGFR-1) were seen between DP of p75NTR knockout and wild-type mice, p75NTR mutants showed a prominent upregulation of FGFR-2, a high-affinity receptor for KGF, in both follicular DP and epithelium. Furthermore, the administration of anti-KGF neutralizing antibody significantly inhibited acceleration of HF morphogenesis in p75NTR knockout mice in vivo. These observations suggest that p75NTR plays an important role during HF morphogenesis, functioning as a receptor that negatively controls HF development, most likely via alterations in DP fibroblast proliferation/differentiation and via downregulation of KGF/FGFR-2 signaling in the HF.     

A role for p75 neurotrophin receptor in the control of apoptosis-driven hair follicle regression.

To examine the mechanisms that underlie the neurotrophin-induced, apoptosis-driven hair follicle involution (catagen), the expression and function of p75 neurotrophin receptor (p75NTR), which is implicated in apoptosis control, were studied during spontaneous catagen development in murine skin. By RT-PCR, high steady-state p75NTR mRNA skin levels were found during the anagen-catagen transition of the hair follicle. By immunohistochemistry, p75NTR alone was strongly expressed in TUNEL+/Bcl2- keratinocytes of the regressing outer root sheath, but both p75NTR and TrkB and/or TrkC were expressed by the nonregressing TUNEL-/Bcl2+ secondary hair germ keratinocytes. To determine whether p75NTR is functionally involved in catagen control, spontaneous catagen development was compared in vivo between p75NTR knockout (-/-) and wild-type mice. There was significant catagen retardation in p75NTR knockout mice as compared to wild-type controls (P<0.05). Instead, transgenic mice-overexpressing NGF (promoter: K14) showed substantial acceleration of catagen (P<0.001). Although NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3) accelerated catagen in the organ-cultured skin of C57BL/6 mice, these neurotrophins failed to promote catagen development in the organ-cultured p75NTR null skin. These findings suggest that p75NTR signaling is involved in the control of kerotinocyte apoptosis during catagen and that pharmacological manipulation of p75NTR signaling may prove useful for the treatment of hair disorders that display premature entry into catagen.     

The p75 Neurotrophin Receptor Mediates Neuronal Apoptosis and Is Essential for Naturally Occurring Sympathetic Neuron Death

Abstract. To determine whether the p75 neurotrophin receptor (p75NTR) plays a role in naturally occurring neuronal death, we examined neonatal sympathetic neurons that express both the TrkA tyrosine kinase receptor and p75NTR. When sympathetic neuron survival is maintained with low quantities of NGF or KCl, the neurotrophin brain-derived neurotrophic factor (BDNF), which does not activate Trk receptors on sympathetic neurons, causes neuronal apoptosis and increased phosphorylation of c-jun. Function-blocking antibody studies indicate that this apoptosis is due to BDNF-mediated activation of p75NTR. To determine the physiological relevance of these culture findings, we examined sympathetic neurons in BDNF−/− and p75NTR−/− mice. In BDNF−/− mice, sympathetic neuron number is increased relative to BDNF+/+ littermates, and in p75NTR−/− mice, the normal period of sympathetic neuron death does not occur, with neuronal attrition occurring later in life. This deficit in apoptosis is intrinsic to sympathetic neurons, since cultured p75NTR−/− neurons die more slowly than do their wild-type counterparts. Together, these data indicate that p75NTR can signal to mediate apoptosis, and that this mechanism is essential for naturally occurring sympathetic neuron death.