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.     

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.     

Nogo receptor 3, a paralog of Nogo-66 receptor 1 （NgR1）, may function as a NgR1 co-receptor for Nogo-66

Nogo-A is a major myelin associated inhibitor that blocks regeneration of injured axons in the central nervous system (CNS).Nogo-66 (a 66-residue domain of Nogo-A) expressed on the surface of oligodendrocytes has been shown to directly interact with Nogo-66 receptor 1 (NgR1).A number of additional components of NgR1 receptor complex essential for its signaling have been uncovered.However,detailed composition of the complex and its signaling mechanisms remain to be fully elucidated.In this study,we show that Nogo receptor 3 (NgR3),a paralog of NgR1,is a binding protein for NgR1.The interaction is highly specific because other members of the reticulin family,to which Nogo-A belongs,do not bind to NgR3.Neither does NgR3 show any binding activity with Nogo receptor 2 (NgR2),another NgR1 paralog.Majority of NgR3 domains are required for its binding to NgR1.Moreover,a truncated NgR3 with the membrane anchoring domain deleted can function as a decoy receptor to reverse neurite outgrowth inhibition caused by Nogo-66 in culture.These in vitro results,together with previously reported overlapping expression profile between NgR1 and NgR3,suggest that NgR3 may be associated with NgR1 in vivo and that their binding interface may be targeted for treating neuronal injuries.     

Ectodomain shedding of human Nogo-66 receptor homologue-1 by zinc metalloproteinases

The Nogo-66 receptor (NgR) plays a pivotal role in the inhibition of neuroregeneration as the receptor for multiple neurite outgrowth inhibitors such as Nogo-A. We have previously shown that NgR undergoes zinc metalloproteinase-mediated ectodomain shedding in neuroblastoma cells. Here, we demonstrate that the NgR-related protein NgR homologue-1 is released from neuroblastoma cells as a full-length ectodomain (NgRH1-ecto) and an N-terminal fragment (NTF-NgRH1) containing the leucine-rich repeat region of the protein. Inhibitors of the major protease classes failed to block the release of NgRH1-ecto, suggesting that this occurs via a protease-independent mechanism, presumably by a phospholipase-like enzyme. The release of NTF-NgRH1 was blocked by a hydroxamate-based zinc metalloproteinase inhibitor and tissue inhibitor of metalloproteinases-2 and -3, but not -1, implicating the involvement of membrane-type matrix metalloproteinases in this process. Our findings thus highlight the parallels between the ectodomain shedding of NgRH1 and that previously described for NgR.     

Chitotriosidase - a putative biomarker for sporadic amyotrophic lateral sclerosis

Background

Potential biomarkers to aid diagnosis and therapy need to be identified for Amyotrophic Lateral Sclerosis, a progressive motor neuronal degenerative disorder. The present study was designed to identify the factor(s) which are differentially expressed in the cerebrospinal fluid (CSF) of patients with sporadic amyotrophic lateral sclerosis (SALS; ALS-CSF), and could be associated with the pathogenesis of this disease.

Results

Quantitative mass spectrometry of ALS-CSF and control-CSF (from orthopaedic surgical patients undergoing spinal anaesthesia) samples showed upregulation of 31 proteins in the ALS-CSF, amongst which a ten-fold increase in the levels of chitotriosidase-1 (CHIT-1) was seen compared to the controls. A seventeen-fold increase in the CHIT-1 levels was detected by ELISA, while a ten-fold elevated enzyme activity was also observed. Both these results confirmed the finding of LC-MS/MS. CHIT-1 was found to be expressed by the Iba-1 immunopositive microglia.

Conclusion

Elevated CHIT-1 levels in the ALS-CSF suggest a definitive role for the enzyme in the disease pathogenesis. Its synthesis and release from microglia into the CSF may be an aligned event of neurodegeneration. Thus, high levels of CHIT-1 signify enhanced microglial activity which may exacerbate the process of neurodegeneration. In view of the multifold increase observed in ALS-CSF, it can serve as a potential CSF biomarker for the diagnosis of SALS.     

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.     

Mutant SOD1 inhibits ER‐Golgi transport in amyotrophic lateral sclerosis

Cu/Zn‐superoxide dismutase is misfolded in familial and sporadic amyotrophic lateral sclerosis, but it is not clear how this triggers endoplasmic reticulum (ER) stress or other pathogenic processes. Here, we demonstrate that mutant SOD1 (mSOD1) is predominantly found in the cytoplasm in neuronal cells. Furthermore, we show that mSOD1 inhibits secretory protein transport from the ER to Golgi apparatus. ER‐Golgi transport is linked to ER stress, Golgi fragmentation and axonal transport and we also show that inhibition of ER‐Golgi trafficking preceded ER stress, Golgi fragmentation, protein aggregation and apoptosis in cells expressing mSOD1. Restoration of ER‐Golgi transport by over‐expression of coatomer coat protein II subunit Sar1 protected against inclusion formation and apoptosis, thus linking dysfunction in ER‐Golgi transport to cellular pathology. These findings thus link several cellular events in amyotrophic lateral sclerosis into a single mechanism occurring early in mSOD1 expressing cells.

     

Proteomic profiling of cerebrospinal fluid identifies biomarkers for amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motor neurons. We tested the hypothesis that proteomic analysis will identify protein biomarkers that provide insight into disease pathogenesis and are diagnostically useful. To identify ALS specific biomarkers, we compared the proteomic profile of cerebrospinal fluid (CSF) from ALS and control subjects using surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS). We identified 30 mass ion peaks with statistically significant (p < 0.01) differences between control and ALS subjects. Initial analysis with a rule-learning algorithm yielded biomarker panels with diagnostic predictive value as subsequently assessed using an independent set of coded test subjects. Three biomarkers were identified that are either decreased (transthyretin, cystatin C) or increased (carboxy-terminal fragment of neuroendocrine protein 7B2) in ALS CSF. We validated the SELDI-TOF-MS results for transthyretin and cystatin C by immunoblot and immunohistochemistry using commercially available antibodies. These findings identify a panel of CSF protein biomarkers for ALS.     

Characterization of two novel proteins,NgRH1 and NgRH2, structurally and biochemically homologous to the Nogo-66 receptor

Nogo-66 receptor (NgR) has recently been identified as the neuronal receptor of the myelin-associated proteins Nogo-A, oligodendrocyte protein (OMgp) and myelin-associated glycoprotein (MAG), and mediates inhibition of axonal regeneration both in vitro and in vivo. Through database searches, we have identified two novel proteins (NgRH1 and NgRH2) that turned out to be homologous in their primary structures, biochemical properties and expression patterns to NgR. Like NgR, the homologues contain eight leucine-rich repeats (LRR) flanked by a leucine-rich repeat C-terminus (LRRCT) and a leucine-rich repeat N-terminus (LRRNT), and also have a C-terminal GPI signal sequence. Northern blot analysis showed predominant expression of NgRH1 and NgRH2 mRNA in the brain. In situ hybridization and immunohistochemistry on rat brain slices revealed neuronal expression of the genes. NgRH1 and NgRH2 were detected on the cell surface of recombinant cell lines as N-glycosylated GPI anchored proteins and, consistent with other GPI anchored proteins, were localized within the lipid rafts of cellular membranes. In addition, an N-terminal proteolytic fragment of NgR comprising the majority of the ectodomain was found to be constitutively secreted from cells. Our data indicate that NgR, NgRH1 and NgRH2 constitute a novel receptor protein family, which may play related roles within the CNS.     

MicroRNAs as regulators of cell death mechanisms in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting upper and lower motor neurons (MNs), resulting in paralysis and precocious death from respiratory failure. Although the causes of ALS are incompletely understood, the role of alterations in RNA metabolism seems central. MicroRNAs (miRNAs) are noncoding RNAs implicated in the regulation of gene expression of many relevant physiological processes, including cell death. The recent model of programmed cell death (PCD) encompasses different mechanisms, from apoptosis to regulated necrosis (RN), in particular necroptosis. Both apoptosis and necroptosis play a significant role in the progressive death of MNs in ALS. In this review, we present key research related to miRNAs that modulate apoptosis and RN pathways in ALS. We also discuss whether these miRNAs represent potential targets for therapeutic development in patients.     

肌萎缩侧索硬化患者肌电图F波和束颤波的特点及其相关性*

目的:研究肌萎缩侧索硬化(ALS)患者肌电图检查中异常F波与束颤波的特点及其相关性。方法:收集2016年9月至2018年2月就诊于浙江大学医学院附属第二医院神经内科的54例ALS患者,进行常规肌电图检查和F波测定,记录216条正中神经、胫后神经的F波以及324块肌肉的束颤波相关参数,计算F波出现率、巨大F波、束颤波的出现率及其异常率,分析巨大F波、束颤波与病程的关系以及巨大F波和束颤波之间的关联性。结果:F波出现率的异常率88.89%,巨大F波出现率55.56%,束颤波出现率48.15%;有束颤波的病程与没有束颤波病程比较具有明显差异(P＜0.01),有巨大F波的病程与未出现巨大F波病程比较无明显差异(P＞0.05);上肢正中神经巨大F波出现与束颤波的出现无关联(P＞0.05),下肢胫后神经巨大F波出现与束颤波的出现比较具有相关性(P=0.05)。结论:以上肢为主的F波出现率异常、以下肢为主的巨大F波和束颤波的出现可作为ALS电生理诊断阳性指标。有束颤波或巨大F波时可考虑疾病相对较早,且有较好的神经再支配及代偿,进展相对较慢。     

Trace elements, age, and sex in amyotrophic lateral sclerosis disease

The statistical tests analysis of variance, analysis of covariance, correlation coefficient, Kolmogorov-Smirnov test,t-test, and Tukey test were applied to copper, magnesium, managenese, and zinc content in serum (S) and in cerebrospinal fluid (CSF) of controls and of a sporadic form of Amyotrophic Lateral Sclerosis (ALS) disease. This is carried out in order to evaluate statistically the possible relationships among the trace elements when ALS patients and controls are considered as independent groups, within sex groups and within age decades of both patients and control classes. A statistically significant difference between older controls (age >40) and ALS patients (age>40) for copper in CSF, copper in S, manganese in S, and zinc in CSF was found. Statistically significant correlation coefficients within the different classes formed for this study were observed. Within this pool, a correlation of patient group can differ statistically from the corresponding one of controls and vice versa. Thus, this correlation could be characteristic of the group from which is extracted, e.g., the correlation between copper in S and zinc in S, which is characteristic of ALS patients when considered as an independent group as well as members of the male patient class.     

Protein kinase and protein phosphatase expression in amyotrophic lateral sclerosis spinal cord

The Kinetworks trade mark multi-immunoblotting technique was used to evaluate the expressions of 78 protein kinases, 24 protein phosphatases and phosphorylation states of 31 phosphoproteins in thoracic spinal cord tissue from control subjects and patients having the sporadic form of amyotrophic lateral sclerosis (ALS). In both the cytosolic (C) and particulate (P) fractions of spinal cord from ALS patients as compared with controls, there were increased levels of calcium/calmodulin-dependent protein kinase kinase (CaMKK; C = 120% increase/P = 580% increase;% change, compared with control), extracellular regulated kinase 2 (ERK2; C = 120% increase/P = 170% increase), G protein-coupled receptor kinase 2 (GRK2; C = 140% increase/P = 140% increase), phospho-Y279/216 glycogen synthase kinase 3 alpha/beta (GSK3alpha/beta; C = 90% increase/P = 220% increase), protein kinase B alpha (PKBalpha; C = 360% increase/P = 200% increase), phospho-T638 PKCalpha/beta (C = 630% increase/P = 170% increase), cGMP-dependent protein kinase (PKG; C = 100% increase/P = 75% increase), phospho-T451 dsRNA-dependent protein kinase (PKR; C = 2600% increase/P = 3330% increase), ribosomal S6 kinase 1 (RSK1; C = 750% increase/P = 630% increase), phospho-T389 p70 S6 kinase (S6K; C = 1000% increase/P = 460% increase), and protein-tyrosine phosphatase 1 delta (PTP1delta; C = 43% increase/P = 70% increase). Cytosolic increases in phospho-alpha-S724/gamma-S662 adducin (C = 15650% increase), PKCalpha (C = 100% increase) and PKCzeta (C = 190% increase) were found in ALS patients as compared with controls, while particulate increases in cAMP-dependent protein kinase (PKA; 43% increase), protein kinase C beta (PKCbeta; 330% increase), and stress-activated protein kinase beta (SAPKbeta; 34% increase) were also observed. Cyclin-dependent kinase-associated phosphatase (KAP) was apparently translocated, as it was reduced (31% decrease) in cytosolic fractions but elevated (100% increase) in particulate fractions of ALS spinal cord tissue. Our observations indicate that ALS is associated with the elevated expression and/or activation of many protein kinases, including PKCalpha, PKCbeta, PKCzeta and GSK3alpha/beta, which may augment neural death in ALS, and CaMKK, PKBalpha, Rsk1, S6K, and SAPK, which may be a response to neuronal injury that potentially can mitigate cell death.     

Misfolded superoxide dismutase-1 in CSF from amyotrophic lateral sclerosis patients

Several of the superoxide dismutase-1 (SOD1) mutations linked to amyotrophic lateral sclerosis (ALS) lead to synthesis of structurally defective molecules, suggesting that any cytotoxic conformational species common for all mutations should be misfolded. SOD1 can be secreted and evidence from ALS model systems suggests that extracellular SOD1 may be involved in cytotoxicity. Three ELISAs specifically reacting with different sequence segments in misfolded SOD1 species were used for analysis of CSF from 38 neurological controls and from 96 ALS patients, 57 of whom were sporadic cases and 39 familial, including 22 patients carrying SOD1 mutations. Misfolded SOD1 was found in all samples. There were, however, no significant differences between patients with and without mutations, and between all the ALS patients and the controls. The estimated concentration of misfolded SOD1 in the interstitium of the CNS is a 1000 times lower than that required for appreciable cytotoxicity in model systems. The results argue against a direct cytotoxic role of extracellular misfolded SOD1 in ALS. Misfolded SOD1 in CSF cannot be used as a biomarker of ALS in patients with and without mutations in the enzyme.     

Oncomodulin/Truncated Protamine-Mediated Nogo-66 Receptor Small Interference RNA Delivery Promotes Axon Regeneration in Retinal Ganglion Cells

The optic nerve often suffers regenerative failure after injury, leading to serious visual impairment such as glaucoma. The main inhibitory factors, including Nogo-A, oligodendrocyte myelin glycoprotein, and myelin-associated glycoprotein, exert their inhibitory effects on axonal growth through the same receptor, the Nogo-66 receptor (NgR). Oncomodulin (OM), a calcium-binding protein with a molecular weight of an ∼12 kDa, which is secreted from activated macrophages, has been demonstrated to have high and specific affinity for retinal ganglion cells (RGC) and promote greater axonal regeneration than other known polypeptide growth factors. Protamine has been reported to effectively deliver small interference RNA (siRNA) into cells. Accordingly, a fusion protein of OM and truncated protamine (tp) may be used as a vehicle for the delivery of NgR siRNA into RGC for gene therapy. To test this hypothesis, we constructed OM and tp fusion protein (OM/tp) expression vectors. Using the indirect immunofluorescence labeling method, OM/tp fusion proteins were found to have a high affinity for RGC. The gel shift assay showed that the OM/tp fusion proteins retained the capacity to bind to DNA. Using OM/tp fusion proteins as a delivery tool, the siRNA of NgR was effectively transfected into cells and significantly down-regulated NgR expression levels. More importantly, OM/tp-NgR siRNA dramatically promoted axonal growth of RGC compared with the application of OM/tp recombinant protein or NgR siRNA alone in vitro. In addition, OM/tp-NgR siRNA highly elevated intracellular cyclic adenosine monophosphate (cAMP) levels and inhibited activation of the Ras homolog gene family, member A (RhoA). Taken together, our data demonstrated that the recombinant OM/tp fusion proteins retained the functions of both OM and tp, and that OM/tp-NgR siRNA might potentially be used for the treatment of optic nerve injury.     

肌萎缩侧索硬化患者SOD1基因突变检测及突变与临床表型的关系

应用PCR技术结合DNA直接测序方法对8例临床确诊为家族性肌萎缩侧索硬化(Familiar amyotrophic lateral sclerosis,FALS)家系的先证者进行铜锌超氧化物歧化酶基因(SOD1)的突变筛查,在3例先证者中检出2种SOD1基因突变,其中,2例携带了位于4号外显子的错义突变Cys111Tyr(c.332G>A),另1例携带了位于5号外显子的错义突变Gly147Asp(c.440G>A),这2种突变在中国ALS患者中属首次报道。该结果扩大了中国FALS患者的SOD1基因突变谱,对研究中国FALS患者SOD1基因突变特点和分布规律有一定帮助。分析携带这2个突变患者的临床特点,提示Cys111Tyr突变导致的临床表型相对温和,而Gly147Asp突变可导致病情进展较快。该结果有待在更多的病例中进行证实。     

Harnessing cellular aging in human stem cell models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive neurodegenerative condition that is invariably fatal, usually within 3 to 5 years of diagnosis. The etiology of ALS remains unresolved and no effective treatments exist. There is therefore a desperate and unmet need for discovery of disease mechanisms to guide novel therapeutic strategies. The single major risk factor for ALS is aging, yet the molecular consequences of cell type‐specific aging remain understudied in this context. Induced pluripotent stem cells (iPSCs) have transformed the standard approach of examining human disease, generating unlimited numbers of disease‐relevant cells from patients, enabling analysis of disease mechanisms and drug screening. However, reprogramming patient cells to iPSCs reverses key hallmarks of cellular age. Therefore, although iPSC models recapitulate some disease hallmarks, a crucial challenge is to address the disparity between the advanced age of onset of neurodegenerative diseases and the fetal‐equivalent maturational state of iPSC‐derivatives. Increasing recognition of cell type‐specific aging paradigms underscores the importance of heterogeneity in ultimately tipping the balance from a state of compensated dysfunction (clinically pre‐symptomatic) to decompensation and progression (irreversible loss of neurological functions). In order to realize the true promise of iPSC technology in ALS, efforts need to prioritize faithfully recapitulating the clinical pathophysiological state, with proportionate emphasis on capturing the molecular sequelae of both cellular age and non‐cell‐autonomous disease mechanisms within this context.     

Nogo goes in the pure water: solution structure of Nogo-60 and design of the structured and buffer-soluble Nogo-54 for enhancing CNS regeneration

The inability to determine the structure of the buffer-insoluble Nogo extracellular domain retarded further design of Nogo receptor (NgR) antagonists to treat CNS axonal injuries. Very surprisingly, we recently discovered that Nogo-60 was soluble and structured in salt-free water, thus allowing the determination of the first Nogo structure by heteronuclear NMR spectroscopy. Nogo-60 adopts an unusual helical structure with the N- and C-terminal helices connected by a long middle helix. While the N-helix has no contact with the rest of the molecule, the C-helix flips back to pack against the 20-residue middle helix. This packing appears to trigger the formation of the stable Nogo-60 structure because Nogo-40 with the last helix truncated is unstructured. The Nogo-60 structure offered us rationales for further design of the structured and buffer-soluble Nogo-54, which may be used as a novel NgR antagonist. Furthermore, our discovery may imply a general solution to solubilizing a category of buffer-insoluble proteins for urgent structural investigations.     

不同行为学测试方法在家族性肌萎缩侧索硬化小鼠模型的比较

目的比较目前常用的5种行为学检测方法在家族性肌萎缩侧索硬化鼠模型研究中的作用。方法分为模型组(SOD1-G93A转基因鼠)和阴性对照组(同窝阴性对照)。使用5种行为学评价方法(一般状况评分、体重测定、转棒试验、抓力测定和步长分析)评价其行为学变化。结果 (1)一般状况评分:在第89天,模型组的一般状况评分开始下降。在第101天时,与对照组相比开始有统计学差异(P=0.000)。(2)体重测定:15周(第105天)时,模型组的体重开始下降,且与阴性对照组相比(P=0.026),开始有统计学差异。(3)转棒试验:11周(第77天)时,模型组的转棒时间开始下降。第13周(第91天)时,与阴性对照组相比开始有统计学差异(P=0.047)。(4)抓力测定:10周(第70天)时,模型组的后肢抓力开始下降。第13周(第91天)时,与阴性对照组相比开始有统计学差异(P=0.000)。(5)步长分析:第14周(第98天)后模型组的步长开始变短。15周(第105天)时与阴性对照组相比开始有统计学意义(P=0.000)。结论抓力测定优于其他行为学检测方法。