angiogenin and Amyotrophic-Lateral-Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that is characterized with progressive muscle atrophy. We have attempted to establish the link between angiogenesis and cellular survival in the pathogenesis of ALS by compiling evidence described in various scientific reports. The phenotypes of human ALS have earlier been captured in the mutant SOD1 mice as well as by targeted deletion of the hypoxia response element (HRE) from the promoter of the mouse gene for vascular endothelial growth factor (VEGF). Indirect evidence shows that angiogenesis can help prevent oxidative stress, and hence, enhance cell survival. VEGF and angiogenin chiefly regulate the process of angiogenesis. Transactive response DNA-binding protein 43 (TDP-43) is usually found inside the nucleus, but in large number of cases of ALS, it accumulates in the cytoplasm (TDP-43 proteinopathy). Interestingly, TDP-43 proteinopathy is found to be aggravated in the presence of the OPTN mutation, which is the genetic factor that is responsible for such accumulation. Interaction of TDP-43 with progranulin can further affect the angiogenesis in ALS patients by regulating activity of VEGF receptors, but conclusive evidence is needed to establish its role in pathogenesis of ALS. Certain mutations in UBQLN2 and UBQLN4 indicate that ubiquitination has a role in ALS pathobiology, but its link to angiogenesis has not been adequately studied. Recent studies have shown that several mutations in RNA-binding proteins (RBPs) can also cause ALS. Conclusively, in this review, we have attempted to argue the role of angiogenesis in enhanced ALS survival rate is probably regulated with the activation of NF-κβ. Additionally, interaction between OPTN and TDP-43 can also impact the transcription of various angiogenic molecules. Whether targeting angiogenic substances or TDP-43 can provide clues about extending ALS survival rate, in combination with current treatments, can only be evaluated after additional studies.

Topics: Amyotrophic Lateral Sclerosis; Carrier Proteins; Cell Cycle Proteins; Cytoplasm; DNA-Binding Proteins; Humans; Membrane Transport Proteins; Mutation; Neovascularization, Pathologic; Nuclear Proteins; Progranulins; Ribonuclease, Pancreatic; Vascular Endothelial Growth Factor A

To perform a meta-analysis to help resolve the controversy of whether the Angiogenin (ANG) rs11701 polymorphism is associated with amyotrophic lateral sclerosis (ALS) risk. A literature search of PubMed, Embase, Web of Science, Chinese National Knowledge Infrastructure, Wanfang and SinoMed was conducted for eligible studies published up to Jun 5, 2015. The strength of the association between the polymorphism and ALS susceptibility was estimated by odds ratio (OR) and associated 95 % confidence interval (CI). The pooled ORs were assessed for the dominant model (TG + GG vs. TT), recessive model (GG vs. TG + TT), heterozygote model (TG vs. TT), homozygote model (GG vs. TT) and allele model (G vs. T). Ten eligible articles were identified, which reported 14 case-control studies and a total of 5807 cases and 3861 controls. Analysis of pooled ORs and 95 % CIs suggested lack of association between the ANG rs11701 polymorphism and risk for ALS, Familial ALS or Sporadic ALS (all p value for z test >0.05). A stratified analysis according to Caucasian or Han Chinese origin further showed that the rs11701 polymorphism was not associated with the disease risk in Caucasians or Han Chinese. There is no difference in the polymorphism frequencies between patients with FALS or SALS. The ANG rs11701 polymorphism was not associated with risk for ALS, FALS or SALS. There is no difference between the polymorphism frequencies in patients with FALS or SALS. Further well-designed studies with larger populations are required to validate these results.

Topics: Amyotrophic Lateral Sclerosis; Genetic Association Studies; Humans; Polymorphism, Single Nucleotide; Ribonuclease, Pancreatic

The study purpose is to perform a meta-analysis to help resolve the debate of whether the Angiogenin (ANG) K17I variant is associated with amyotrophic lateral sclerosis (ALS) risk in Caucasian. Three literature databases were searched for eligible studies published up to January 8, 2015: PubMed, Embase and Web of Science using the following search terms: amyotrophic lateral sclerosis or ALS and Angiogenin or ANG. Five eligible articles were identified, which reported 6 case-control studies and a total of 2326 cases and 3799 controls. The overall results suggested low frequencies of the K17I variant in Caucasian patients (10/2326, 0.43 %) and controls (6/3799, 0.16 %). There is no difference in the variant frequencies between patients with FALS or SALS (p = 0.069). Analysis of pooled odds ratios (ORs) and 95 % confidence intervals (CIs) revealed that the ANG K17I variant increases the risk for ALS (AT vs. AA: OR 2.65, 95 % CI 1.05-6.66, p = 0.038) and familial ALS (FALS) (AT vs. AA: OR 11.81, 95 % CI 2.11-66.15, p = 0.005) but not for sporadic ALS (SALS) (AT vs. AA: OR 1.63, 95 % CI 0.55-4.82, p = 0.378). The ANG K17I variant is rare in Caucasian patients and controls and increases the risk for ALS and FALS but not for SALS in Caucasian populations. Further well-designed studies with larger samples are needed to validate these results.

Topics: Amyotrophic Lateral Sclerosis; Case-Control Studies; Female; Genetic Predisposition to Disease; Humans; Male; Mutation; Polymorphism, Single Nucleotide; Ribonuclease, Pancreatic; Risk; White People

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive muscle weakness that reflects degeneration of motor neurons in the primary motor cortex, corticospinal tracts, brainstem, and spinal cord. Most ALS cases are sporadic, but about 5%-10% are familial. The majority of familial ALS (FALS) cases follow an autosomal dominant inheritance pattern, and include the following mutations: ALS1, Cu/Zn superoxide dismutase (SOD1); ALS3; ALS4, senataxin; ALS6, fused in sarcoma (FUS); ALS7; ALS8, vesicle-associated membrane protein; ALS9, angiogenin; ALS10, TAR DNA-binding protein (TARDBP); and ALS11/FIG4. Some of these gene mutations are rarely seen in sporadic ALS cases. ALS2/alsin and ALS5 show an autosomal recessive inheritance pattern. Recently, mutations in the gene encoding optineurin, earlier reported to be a causative gene for primary open-angle glaucoma, have also been found in patients with ALS. It has also been demonstrated that a mutation in the D-amino acid oxidase gene is associated with classic adult-onset FALS. However, these genetic defects occur in only about 20%-30% FLAS cases, while most genes causing FALS remain unknown.

Topics: Amyotrophic Lateral Sclerosis; Cell Cycle Proteins; D-Amino-Acid Oxidase; DNA Helicases; DNA-Binding Proteins; Flavoproteins; Guanine Nucleotide Exchange Factors; Humans; Membrane Transport Proteins; Multifunctional Enzymes; Mutation; Phosphoric Monoester Hydrolases; R-SNARE Proteins; Ribonuclease, Pancreatic; RNA Helicases; Superoxide Dismutase; Superoxide Dismutase-1; Transcription Factor TFIIIA

Several studies have suggested an increased frequency of variants in the gene encoding angiogenin (ANG) in patients with amyotrophic lateral sclerosis (ALS). Interestingly, a few ALS patients carrying ANG variants also showed signs of Parkinson disease (PD). Furthermore, relatives of ALS patients have an increased risk to develop PD, and the prevalence of concomitant motor neuron disease in PD is higher than expected based on chance occurrence. We therefore investigated whether ANG variants could predispose to both ALS and PD.. We reviewed all previous studies on ANG in ALS and performed sequence experiments on additional samples, which allowed us to analyze data from 6,471 ALS patients and 7,668 controls from 15 centers (13 from Europe and 2 from the USA). We sequenced DNA samples from 3,146 PD patients from 6 centers (5 from Europe and 1 from the USA). Statistical analysis was performed using the variable threshold test, and the Mantel-Haenszel procedure was used to estimate odds ratios.. Analysis of sequence data from 17,258 individuals demonstrated a significantly higher frequency of ANG variants in both ALS and PD patients compared to control subjects (p = 9.3 × 10(-6) for ALS and p = 4.3 × 10(-5) for PD). The odds ratio for any ANG variant in patients versus controls was 9.2 for ALS and 6.7 for PD.. The data from this multicenter study demonstrate that there is a strong association between PD, ALS, and ANG variants. ANG is a genetic link between ALS and PD.

Topics: Amyotrophic Lateral Sclerosis; Databases, Factual; Europe; Female; Genetic Predisposition to Disease; Genetic Variation; Humans; Male; Multicenter Studies as Topic; Parkinson Disease; Ribonuclease, Pancreatic; United States

RNA processing is a tightly regulated, highly complex pathway which includes RNA transcription, pre-mRNA splicing, editing, transportation, translation, and degradation of RNA. Over the past few years, several RNA processing genes have been shown to be mutated or genetically associated with amyotrophic lateral sclerosis (ALS), including the RNA-binding proteins TDP-43 and FUS/TLS. These findings suggest that RNA processing may represent a common pathogenic mechanism involved in development of ALS. In this review, we will discuss six ALS-related, RNA processing genes including their discovery, function, and commonalities.

Topics: Amyotrophic Lateral Sclerosis; Animals; DNA Helicases; DNA-Binding Proteins; Genetic Predisposition to Disease; Histone Acetyltransferases; Humans; Mice; Multifunctional Enzymes; Nerve Tissue Proteins; Ribonuclease, Pancreatic; RNA Helicases; RNA Processing, Post-Transcriptional; RNA Splicing; RNA-Binding Protein FUS; SMN Complex Proteins; Transcription, Genetic

The review is devoted to an analysis of recently published data on the biological functions of angiogenins with a view to their potential use as an active basis for new pharmaceuticals and cosmetics.

Topics: Amyotrophic Lateral Sclerosis; Angiogenesis Inducing Agents; Animals; Anti-Infective Agents; Anti-Inflammatory Agents; Cell Degranulation; Cytokines; Genetic Therapy; Humans; Milk; Neutrophils; Ribonuclease, Pancreatic

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder of the motor neurons in the spinal cord, brainstem, and motor cortex. Ten percent of ALS cases are familial, with both autosomal dominant and recessive modes of inheritance reported. Mutations in the copper/zinc superoxide-dismutase-1 (SOD-1) gene, the first gene linked with ALS, result in the classical ALS phenotype. To date, 135 mutations have been identified in the SOD-1 gene, accounting for approximately 20% of familial ALS cases. Mutations are widely distributed throughout the gene with preponderance for exon 4 and 5. Although mutations result in a toxic gain of function of the SOD-1 enzyme, which normally functions as a free radical scavenger, the mechanisms underlying motor neuron degeneration have not been clearly elucidated. Evidence is emerging of a complex interaction between genetic and molecular factors, with resultant damage of critical target proteins and organelles within the motor neuron. The clinical effectiveness afforded by anti-glutamatergic agents such as riluzole, suggests that glutamate excitotoxicity contributes to neurodegeneration in ALS, with glutamate excitotoxicity mediated via corticomotoneurons that provide a direct link between the motor cortex and the spinal motor neuron. This review provides an overview of the genetics of ALS, and describes recent advances in the understanding of the pathophysiological mechanisms underlying neurodegeneration.

Topics: Amyotrophic Lateral Sclerosis; Animals; Ciliary Neurotrophic Factor; Glutamic Acid; Humans; Intermediate Filament Proteins; Membrane Glycoproteins; Mitochondria; Motor Neurons; Mutation; Nerve Degeneration; Nerve Tissue Proteins; Neurofilament Proteins; Peripherins; Ribonuclease, Pancreatic; Sodium-Potassium-Exchanging ATPase; Superoxide Dismutase; Superoxide Dismutase-1; Survival of Motor Neuron 1 Protein; Vascular Endothelial Growth Factor A

Missense heterozygous mutations in the coding region of angiogenin (ANG) gene, encoding a 14 kDa angiogenic RNase, were recently found in patients of amyotropic lateral sclerosis (ALS). Functional analyses have shown that these are loss-of-function mutations, implying that angiogenin deficiency is associated with ALS pathogenesis and that increasing ANG expression or angiogenin activity could be a novel approach for ALS therapy.. Review the evidence showing the involvement of angiogenin in motor neuron physiology and function, and provide a rationale for targeting angiogenin in ALS therapy.. Review the current understanding of the mechanism of angiogenin action in connection with ALS genetics, pathogenesis and therapy.. ANG is the first gene whose loss-of-function mutations are associated with ALS pathogenesis. Therapeutic modulation of angiogenin level and activity in the spinal cord, either by systemic delivery of angiogenin protein or through retrograde transport of ANG-encoding viral particles, may be beneficial for ALS patients.

Topics: Amyotrophic Lateral Sclerosis; Clinical Trials as Topic; Humans; Ribonuclease, Pancreatic; RNA, Ribosomal; Transcription, Genetic

Motor neuron disorders (MND) form a heterogeneous group of neurodegenerative affections: phenotypic description is based on selective injury to the upper motor neuron or lower motor neuron or both. Phenotypic heterogeneity is also present concerning genetic features: genetic factors involved in MND may be causative or susceptibility factors. Consequences of genetic abnormalities lead to metabolic or functional cellular disturbances that are apparently specific for motor neuron disorder. Genetics greatly contribute to our understanding of the pathophysiological mechanisms of motor neuron degeneration. Genetic studies provide pathological hypotheses considering the function of protein encoded. Moreover, when a gene mutation is identified, animal models can be developed to search for modifications induced by the mutation. We propose to detail causative and susceptibility genetic factors involved in MND and to discuss pathological mechanisms that may explain motor neuron death.

Topics: Adaptor Proteins, Signal Transducing; Amyotrophic Lateral Sclerosis; Guanine Nucleotide Exchange Factors; Humans; Motor Neuron Disease; Motor Neurons; Ribonuclease, Pancreatic; Spinal Cord Diseases; Superoxide Dismutase; Superoxide Dismutase-1

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized clinically by rapidly progressive paralysis leading ultimately to death from respiratory failure. There is substantial evidence suggesting that ALS is a heritable disease, and a number of genes have been identified as being causative in familial ALS. In contrast, the genetics of the much commoner sporadic form of the disease is poorly understood and no single gene has been definitively shown to increase the risk of developing ALS. In this review, we discuss the genetic evidence for each candidate gene that has been putatively associated with increased risk of sporadic ALS. We also review whole genome association studies of ALS and discuss the potential of this methodology for identifying genes relevant to motor neuron degeneration.

Topics: Amyotrophic Lateral Sclerosis; Aryldialkylphosphatase; Cyclic AMP Response Element-Binding Protein; DNA-(Apurinic or Apyrimidinic Site) Lyase; Dynactin Complex; Endosomal Sorting Complexes Required for Transport; Hemochromatosis Protein; Histocompatibility Antigens Class I; Humans; Intercellular Signaling Peptides and Proteins; Intermediate Filament Proteins; Membrane Glycoproteins; Membrane Proteins; Microtubule-Associated Proteins; Nerve Degeneration; Nerve Tissue Proteins; Neurofilament Proteins; Peripherins; Progranulins; Ribonuclease, Pancreatic; RNA-Binding Proteins; SMN Complex Proteins; Superoxide Dismutase; Superoxide Dismutase-1; Vascular Endothelial Growth Factor A

Human Angiogenin (hANG, or ANG, 14.1 kDa) promotes vessel formation and is also called RNase 5 because it is included in the pancreatic-type ribonuclease (pt-RNase) super-family. Although low, its ribonucleolytic activity is crucial for angiogenesis in tumor tissues but also in the physiological development of the Central Nervous System (CNS) neuronal progenitors. Nevertheless, some ANG variants are involved in both neurodegenerative Parkinson disease (PD) and Amyotrophic Lateral Sclerosis (ALS). Notably, some pt-RNases acquire new biological functions upon oligomerization. Considering neurodegenerative diseases correlation with massive protein aggregation, we analyzed the aggregation propensity of ANG and of three of its pathogenic variants, namely H13A, S28N, and R121C. We found no massive aggregation, but wt-ANG, as well as S28N and R121C variants, can form an enzymatically active dimer, which is called ANG-D. By contrast, the enzymatically inactive H13A-ANG does not dimerize. Corroborated by a specific cross-linking analysis and by the behavior of H13A-ANG that in turn lacks one of the two His active site residues necessary for pt-RNases to self-associate through the three-dimensional domain swapping (3D-DS), we demonstrate that ANG actually dimerizes through 3D-DS. Then, we deduce by size exclusion chromatography (SEC) and modeling that ANG-D forms through the swapping of ANG N-termini. In light of these novelties, we can expect future investigations to unveil other ANG determinants possibly related with the onset and/or development of neurodegenerative pathologies.

Topics: Amyotrophic Lateral Sclerosis; Chromatography; Crystallography, X-Ray; Dimerization; Genetic Variation; Humans; Models, Molecular; Mutation; Parkinson Disease; Phosphorylation; Protein Conformation; Protein Domains; Ribonuclease, Pancreatic; Ribonucleases; Sulfones

Protein oligomerization is key to countless physiological processes, but also to abnormal amyloid conformations implicated in over 25 mortal human diseases. Human Angiogenin (h-ANG), a ribonuclease A family member, produces RNA fragments that regulate ribosome formation, the creation of new blood vessels and stress granule function. Too little h-ANG activity leads to abnormal protein oligomerization, resulting in Amyotrophic Lateral Sclerosis (ALS) or Parkinson's disease. While a score of disease linked h-ANG mutants has been studied by X-ray diffraction, some elude crystallization. There is also a debate regarding the structure that RNA fragments adopt after cleavage by h-ANG. Here, to better understand the beginning of the process that leads to aberrant protein oligomerization, the solution secondary structure and residue-level dynamics of WT h-ANG and two mutants i.e., H13A and R121C, are characterized by multidimensional heteronuclear NMR spectroscopy under near-physiological conditions. All three variants are found to adopt well folded and highly rigid structures in the solution, although the elements of secondary structure are somewhat shorter than those observed in crystallography studies. R121C alters the environment of nearby residues only. By contrast, the mutation H13A affects local residues as well as nearby active site residues K40 and H114. The conformation characterization by CD and 1D

Topics: Alanine; Amyotrophic Lateral Sclerosis; Catalytic Domain; Crystallography, X-Ray; G-Quadruplexes; Humans; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Conformation; Mutation; Protein Structure, Secondary; Ribonuclease, Pancreatic; Ribonucleases; RNA; RNA, Transfer; RNA, Transfer, Ala; X-Ray Diffraction

Genetic risk factors are occasionally shared between different neurodegenerative diseases. Previous studies have linked ANG, a gene encoding angiogenin, to both Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). Functional studies suggest ANG plays a neuroprotective role in both PD and amyotrophic lateral sclerosis by reducing cell death. We further explored the genetic association between ANG and PD by analyzing genotype data from the International Parkinson's Disease Genomics Consortium (14,671 cases and 17,667 controls) and whole genome sequencing data from the Accelerating Medicines Partnership - Parkinson's disease initiative (AMP-PD, https://amp-pd.org/) (1,647 cases and 1,050 controls). Our analysis did not replicate the findings of previous studies and identified no significant association between ANG variants and PD risk.

Topics: Amyotrophic Lateral Sclerosis; Cell Death; Genetic Variation; Genome-Wide Association Study; Genotype; Humans; Parkinson Disease; Ribonuclease, Pancreatic; Risk Factors; Whole Genome Sequencing

Amyotrophic Lateral Sclerosis (ALS) is a progressive and incurable neurodegenerative disorder characterized by the degeneration of motor neurons leading to severe muscle atrophy, respiratory failure and death within 3-5 years of disease onset. Missense mutations in Angiogenin (ANG) cause ALS through loss of either ribonucleolytic activity or nuclear translocation activity or both of these functions. Although loss-of-function mechanisms of several rare and ALS-causing ANG variants have been studied before, the structure-function relationship and subsequent functional loss mechanisms of certain novel and uncharacterized rare variants have not been deciphered hitherto. In this study, the structural and dynamic properties of the distantly-located I71V variant, on the functional sites of ANG have been investigated to understand its role in ALS etiology and progression. The I71V variant has a minor allele frequency of <0.06% and thus is classified as a rare variant. Our extensive in silico investigation comprising 1-μs molecular dynamics (MD) simulations, conformational dynamics and related integrated analyses reveal that the I71V variant induces a characteristic conformational switching of catalytic His114 residue resulting in loss of ribonucleolytic activity. Molecular docking and a residue-residue interaction network propagated by an allosteric pathway further support these findings. Moreover, while no conformational alteration of nuclear localization signal governing the nuclear translocation activity was observed, an escalation in mutant plasticity was detected in the structural and essential dynamics simulations. Overall, our study emphasizes that the structure-function relationship of frequently mutating novel ANG variants needs to be established and prioritized in order to advance the pathophysiology and therapeutics of ALS.

Topics: Amyotrophic Lateral Sclerosis; Humans; Molecular Docking Simulation; Molecular Dynamics Simulation; Mutation; Protein Conformation; Ribonuclease, Pancreatic

0.5-1% of ALS (Amyotrophic Lateral Sclerosis) and Parkinson's disease (PD) are associated with mutations in the angiogenin (ANG). These mutations are thought to cause disease through a loss of ANG function, but this hypothesis has not been evaluated statistically. In addition, the potential for ANG to promote disease has not been considered. With the goal of better defining the etiology of ANG-ALS, we assembled all clinical onset and disease duration data and determined if these were correlated with biochemical properties of ANG variants. Loss of ANG stability and ribonuclease activity were found to correlate with early ALS onset, confirming an aspect of the prevailing model of ANG-ALS. Conversely, loss of ANG stability and ribonuclease activity correlated with longer survival following diagnosis, which is inconsistent with the prevailing model. These results indicate that functional ANG appears to decrease the risk of developing ALS but exacerbate ALS once in progress. These findings are rationalized in terms of studies demonstrating that distinct mechanisms contribute to ALS onset and progression and propose that ANG replacement or stabilization would benefit pre-symptomatic ANG-ALS patients. However, this study challenges the prevailing hypothesis that augmenting ANG will benefit symptomatic ANG-ALS patients. Instead, our results suggest that silencing of ANG activity may be beneficial for symptomatic ALS patients. This study will serve as a call-to-arms for neurologists to consistently publish ALS and PD patient's clinical data-if all ANG-ALS patients' data were available our findings could be tested with considerable statistical power.

Topics: Adult; Aged; Aged, 80 and over; Amyotrophic Lateral Sclerosis; Humans; Loss of Function Mutation; Middle Aged; Protein Stability; Ribonuclease, Pancreatic; Survival

Evidence-based information about cerebrospinal fluid (CSF) levels of biomarkers in patients with amyotrophic lateral sclerosis (ALS) is limited.. Vascular endothelial growth factor (VEGF) and its receptor vascular endothelial growth factor receptor 2 (VEGFR2), optineurin (OPTN), monocyte chemoattractant protein-1 (MCP-1), angiogenin (ANG), and TAR DNA-binding protein (TDP-43) were quantified by enzyme-linked immunoassay in the CSF of 54 patients with sporadic ALS and 32 controls in a case-control study design.. CSF levels of VEGF (P = .014) and ANG (P = .009) were decreased, whereas VEGFR2 was higher (P = .002) in patients with ALS than in controls. TDP-43 positively correlated with MCP-1 (P = .003), VEGF (P < .001), and VEGFR2 (P < .001) in patients with ALS.. Our findings suggest possible utility of VEGF, VEGFR2, and ANG as biomarkers for use in ALS treatment trials.

Topics: Adult; Amyotrophic Lateral Sclerosis; Biomarkers; Case-Control Studies; Cell Cycle Proteins; Chemokine CCL2; DNA-Binding Proteins; Female; Humans; India; Male; Membrane Transport Proteins; Middle Aged; Ribonuclease, Pancreatic; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Rare missense variants play a crucial role in amyotrophic lateral sclerosis (ALS) pathophysiology. We report rare/novel missense variants from 154 Indian ALS patients, identified through targeted sequencing of 25 ALS-associated genes. As pathogenic variants could explain only a small percentage of ALS pathophysiology in our cohort, we investigated the frequency of tolerated and benign novel/rare variants, which could be potentially ALS susceptible. These variants were identified in 5.36% (8/149) of sporadic ALS (sALS) cases; with one novel variant each in ERBB4, SETX, DCTN1, and MATR3; four rare variants, one each in PON2 and ANG and two different rare variants in SETX. Identified variants were either absent or present at extremely rare frequencies (MAF < 0.01) in large population databases and were absent in 50 healthy controls sequenced through Sanger method. Furthermore, an oligogenic basis of ALS was observed in three sALS, with co-occurrence of intermediate-length repeat expansions in ATXN2 and a rare/novel variant in DCTN1 and SETX genes. Additionally, molecular dynamics and biochemical functional analysis of an angiogenin variant (R21G) identified from our cohort demonstrated loss of ribonucleolytic and nuclear translocation activities. Our findings suggest that rare variants could be potentially pathogenic and functional studies are warranted to decisively establish the pathogenic mechanisms associated with them.

Topics: Active Transport, Cell Nucleus; Adult; Amyotrophic Lateral Sclerosis; Aryldialkylphosphatase; Computational Biology; Crystallography, X-Ray; DNA Helicases; Dynactin Complex; Female; Genetic Predisposition to Disease; Genetic Variation; HeLa Cells; Heterozygote; Humans; India; Male; Middle Aged; Molecular Dynamics Simulation; Multifunctional Enzymes; Mutation, Missense; Neovascularization, Pathologic; Nuclear Matrix-Associated Proteins; Polymorphism, Genetic; Protein Structure, Secondary; Receptor, ErbB-4; Ribonuclease, Pancreatic; RNA Helicases; RNA-Binding Proteins

Angiogenin (hANG), a member of the Ribonuclease A superfamily has angiogenic, neurotrophic and neuroprotective activities. Mutations in hANG have been found in patients with Amyotrophic lateral sclerosis (ALS). The zebrafish (Danio rerio) rnasel-1, 2 and 3 are orthologues of hANG and of these only Rnasel-1 and Rnasel-2 have been shown to be angiogenic. Herein we show that NCI-65828, a potent and specific small molecule inhibitor of hANG inhibits Rnasel-1 to a similar extent. Treatment of early zebrafish embryos with NCI-65828, or with terrein, a fungal metabolite which prevents the secretion of hANG, resulted in spinal neuron aberrations as well defects in trunk vasculature. Our detailed expression analysis and inhibitor studies suggest that Rnasel-1 plays important roles in neuronal migration and pathfinding as well as in angiogenesis in zebrafish. Our studies suggest the usefulness of the zebrafish as a model to dissect the molecular consequences of the ANG ALS variants.

Topics: Amyotrophic Lateral Sclerosis; Animals; Blood Vessels; Catalysis; Cell Movement; Humans; Motor Neurons; Mutation; Neurogenesis; Neurons, Efferent; Ribonuclease, Pancreatic; Ribonucleases; Zebrafish; Zebrafish Proteins

Mutations in the angiogenin (ANG) gene are known to be associated with both familial and sporadic amyotrophic lateral sclerosis (ALS). The majority of disease-causing mutations of ANG result in loss of either ribonucleolytic activity, nuclear translocation activity or both.. We sequenced ANG gene from a total of 136 sporadic ALS patients and 112 healthy controls of Hungarian origin. To elucidate the role of the R33W mutation in the disease mechanism, computational, and functional analyses were performed.. Mutation screening revealed a mutation located in the signal peptide (M-24I) and two mutations that affect the mature protein (R33W, V103I). The R33W mutation, which has not been previously detected in ALS patients, affects the key amino acid of the nuclear translocation signal of the ANG protein. Molecular dynamics simulations suggested that the R33W mutation results in partial loss of ribonucleolytic activity and reduced nuclear translocation activity. The ribonucleolytic assay and nuclear translocation assay of the R33W ANG protein confirmed the molecular dynamics results.. In the Hungarian ALS population, the observed frequency of ANG mutations was 2.9%, which is higher than previously reported for sporadic cohorts. The evidence from computational and functional analyses support the deleterious effect of the novel R33W variant detected in this study.

Topics: Adult; Aged; Aged, 80 and over; Amyotrophic Lateral Sclerosis; Female; Humans; Hungary; Male; Middle Aged; Molecular Dynamics Simulation; Mutation; Nucleic Acid Conformation; Ribonuclease, Pancreatic; Translocation, Genetic

The RNaseA superfamily member Angiogenin (ANG) is a secreted protein involved in neovascularization, cell proliferation and stress response. Dysregulation of ANG expression is found in many cancers with poor prognosis and mutations in ANG are associated with neurodegenerative diseases. While the uptake and nuclear translocation of ANG is relatively well characterised, little is known about how it reaches the plasma membrane and its mode of secretion. We generated SH-SY5Y neuroblastoma cell lines constitutively expressing wild type (WT) Hemagglutinin (HA) epitope tagged mouse Ang1 (mAng1), and two amyotrophic lateral sclerosis associated ANG variants (C39W and K40I). Herein, we show that these cell lines secrete mAng1 into the culture media. Using small molecule inhibitors we probed the route taken between the endoplasmic reticulum and trans-Golgi network during secretion and have characterised it as COPII and microtubule dependent. In addition, we show that disruption by the PI3-kinase inhibitor wortmannin of the later stages of transit to the plasma membrane leads to mAng1 trafficking to lysosomal compartments. This suggests an autophagy dependent regulation of secretion.

Topics: Amyotrophic Lateral Sclerosis; Animals; Autophagy; Cell Differentiation; Cell Proliferation; COP-Coated Vesicles; Humans; Mice; Microtubules; Motor Neurons; Mutant Proteins; Protein Transport; Ribonuclease, Pancreatic; Secretory Pathway; Tumor Cells, Cultured

Amyotrophic Lateral Sclerosis (ALS), a debilitating neurodegenerative disorder is related to mutations in a number of genes, and certain genes of the Ribonuclease (RNASE) superfamily trigger ALS more frequently. Even though missense mutations in Angiogenin (ANG) and Ribonuclease 4 (RNASE4) have been previously shown to cause ALS through loss-of-function mechanisms, understanding the role of rare variants with a plausible explanation of their functional loss mechanisms is an important mission. The study aims to understand if any of the rare ANG and RNASE4 variants catalogued in Project MinE consortium caused ALS due to loss of ribonucleolytic or nuclear translocation or both these activities. Several in silico analyses in combination with extensive molecular dynamics (MD) simulations were performed on wild-type ANG and RNASE4, along with six rare variants (T11S-ANG, R122H-ANG, D2E-RNASE4, N26K-RNASE4, T79A-RNASE4 and G119S-RNASE4) to study the structural and dynamic changes in the catalytic triad and nuclear localization signal residues responsible for ribonucleolytic and nuclear translocation activities respectively. Our comprehensive analyses comprising 1.2 μs simulations with a focus on physicochemical, structural and dynamic properties reveal that T11S-ANG, N26K-RNASE4 and T79A-RNASE4 variants would result in loss of ribonucleolytic activity due to conformational switching of catalytic His114 and His116 respectively but none of the variants would lose their nuclear translocation activity. Our study not only highlights the importance of rare variants but also demonstrates that elucidating the structure-function relationship of mutant effectors is crucial to gain insights into ALS pathophysiology and in developing effective therapeutics.

Topics: Amyotrophic Lateral Sclerosis; Computer Simulation; Humans; Loss of Function Mutation; Molecular Dynamics Simulation; Mutation, Missense; Protein Conformation; Ribonuclease, Pancreatic; Ribonucleases

Amyotrophic lateral sclerosis (ALS) presents a poorly understood pathogenesis. Evidence from patients and mutant SOD1 mouse models suggests vascular damage may precede or aggravate motor dysfunction in ALS. We have previously shown angiogenin (ANG) treatment enhances motor neuron survival, delays motor dysfunction and prevents vascular regression in the SOD1

Topics: Amyotrophic Lateral Sclerosis; Animals; Blood Vessels; Cell Count; Disease Models, Animal; Humans; Mice, Inbred C57BL; Mice, Transgenic; MicroRNAs; Motor Neurons; Ribonuclease, Pancreatic; RNA-Binding Protein FUS; Sialoglycoproteins; Survival Analysis

Angiogenin (ANG), a member of the RNase superfamily (also known as RNase 5) has neurotrophic, neuroprotective and angiogenic activities. Recently it has also been shown to be important in stem cell homeostasis. Mutations in ANG are associated with neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS) and Fronto-temporal dementia (FTD). ANG is a secreted protein which is taken up by cells and translocated to the nucleus. However, the import pathway/s through which ANG is taken up is/are still largely unclear. We have characterised the uptake of ANG in neuronal, astrocytic and microglial cell lines as well as primary neurons and astrocytes using pharmacological agents as well as dominant negative dynamin and Rab5 to perturb uptake and intracellular trafficking. We find that uptake of ANG is largely clathrin/dynamin independent and microtubule depolymerisation has a marginal effect. Perturbation of membrane ruffling and macropinocytosis significantly inhibited ANG uptake suggesting an uptake mechanism similar to RNase A. Our findings shed light on why mutations which do not overtly affect RNase activity but cause impaired localization are associated with neurodegenerative disease.

Topics: Amyotrophic Lateral Sclerosis; Animals; Astrocytes; Cell Line; Clathrin; Dynamins; Humans; Membrane Microdomains; Mice; Microglia; Neurons; Ribonuclease, Pancreatic

Discovering novel mutations in C9orf72, FUS, ANG, and TDP-43 genes in ALS patients arises necessities for better clinical characterizations of these subjects. The aim is to determine clinical and cognitive profile of genetically positive Serbian ALS patients. 241 ALS patients were included in the study (17 familiar and 224 apparently sporadic). The following genes were analyzed: SOD1, C9orf72, ANG, FUS, and TDP-43. An extensive battery of classic neuropsychological tests was used in 27 ALS patients (22 SOD1 positive and 5 SOD1 negative) and 82 healthy controls (HCs). Overall 37 (15.4%) of 241 ALS patients carried mutations in tested genes-among 17 familiar ALS patients 16 (94.1%) were positive and among 224 apparently sporadic 21 (9.4%) had causative mutation. Mutations in SOD1 gene were the most common, representing 27 (73.0%) of all genetically positive ALS patients. The main clinical characteristics of SOD1 positive patients were: spinal onset in lower extremities, common sphincter and sensitive disturbances, and dysexecutive syndrome. Within SOD1 positive patients, we noticed somewhat earlier onset in patients with A145G, sensory and sphincter disturbances were dominant in patients with L144F, while D90A patients had significant sensory involvement. SOD1 negative group consisted of ten (27.0%) patients (six C9orf72, two ANG, one TDP-43, and one patient baring triple FUS, C9orf72 expansion, and ANG variants). Bulbar involvement and more extensive neuropsychological impairment (including executive, visuospatial, and memory difficulties) were the main features of SOD1 negative cohort. Our results suggest that meaningful clinical suspicion of certain ALS genotype might be made based on thorough clinical evaluation of patients.

Topics: Amyotrophic Lateral Sclerosis; C9orf72 Protein; Cognition Disorders; Cohort Studies; Disability Evaluation; DNA-Binding Proteins; Female; Genetic Association Studies; Genetic Testing; Humans; Male; Mutation; Neuropsychological Tests; Ribonuclease, Pancreatic; RNA-Binding Protein FUS; Serbia; Superoxide Dismutase-1; Tertiary Care Centers

To understand disease mechanisms, a large-scale analysis of human-yeast genetic interactions was performed. Of 1305 human disease genes assayed, 20 genes exhibited strong toxicity in yeast. Human-yeast genetic interactions were identified by en masse transformation of the human disease genes into a pool of 4653 homozygous diploid yeast deletion mutants with unique barcode sequences, followed by multiplexed barcode sequencing to identify yeast toxicity modifiers. Subsequent network analyses focusing on amyotrophic lateral sclerosis (ALS)-associated genes, such as optineurin (

Topics: Amyotrophic Lateral Sclerosis; Animals; Cell Cycle Proteins; Humans; MAP Kinase Kinase 5; Membrane Transport Proteins; Molecular Targeted Therapy; Mutant Proteins; Mutation; Protein Interaction Maps; Ribonuclease, Pancreatic; Saccharomyces cerevisiae; Sequence Deletion; Transcription Factor TFIIIA; Zebrafish

Angiogenin (ANG) is a human ribonuclease that is compromised in patients with amyotrophic lateral sclerosis (ALS). ANG also promotes neovascularization, and can induce hemorrhage and encourage tumor growth. The causal neurodegeneration of ALS is associated with reactive oxygen species, which are also known to elicit the oxidative cleavage of carbon-boron bonds. We have developed a synthetic boronic acid mask that restrains the ribonucleolytic activity of ANG. The masked ANG does not stimulate endothelial cell proliferation but protects astrocytes from oxidative stress. By differentiating between the two dichotomous biological activities of ANG, this strategy could provide a viable pharmacological approach for the treatment of ALS.

Topics: Amyotrophic Lateral Sclerosis; Boronic Acids; Cell Proliferation; Humans; Neovascularization, Pathologic; Neuroprotective Agents; Oxidative Stress; Reactive Oxygen Species; Ribonuclease, Pancreatic

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by loss of motor neurons. We have recently identified SOD1 and FUS mutations as the most common causes in a consecutive series of 111 familial ALS pedigrees in Japan. To reveal possible genetic causes for the remaining 51 patients with familial ALS (45 pedigrees), we performed targeted next-generation sequencing of 35 known ALS/motor neuron diseases-related genes. Known variants in ANG, OPTN, SETX, and TARDBP were identified in 6 patients. A novel likely pathogenic homozygous variant in ALS2 was identified in 1 patient. In addition, 18 patients harbored 1-3 novel variants of uncertain significance, whereas hexanucleotide repeat expansions in C9ORF72 were not detected using repeat-primed polymerase chain reaction. Collectively, in our Japanese cohort, the frequencies of SOD1, FUS, SETX, TARDBP, ANG, and OPTN variants were 32%, 11%, 2%, 2%, 1%, and 1%, respectively. These findings indicate considerable differences in the genetic variations associated with familial ALS across populations. Further genetic analyses and functional studies of novel variants are warranted.

Topics: Amyotrophic Lateral Sclerosis; Asian People; C9orf72 Protein; Cell Cycle Proteins; Cohort Studies; DNA Helicases; DNA-Binding Proteins; Genetic Association Studies; Genetic Predisposition to Disease; Genetic Variation; High-Throughput Nucleotide Sequencing; Humans; Membrane Transport Proteins; Multifunctional Enzymes; Ribonuclease, Pancreatic; RNA Helicases; RNA-Binding Protein FUS; Superoxide Dismutase-1; Transcription Factor TFIIIA

Mutations in Angiogenin (ANG), a member of the Ribonuclease A superfamily (also known as RNase 5) are known to be associated with Amyotrophic Lateral Sclerosis (ALS, motor neurone disease) (sporadic and familial) and Parkinson's Disease (PD). In our previous studies we have shown that ANG is expressed in neurons during neuro-ectodermal differentiation, and that it has both neurotrophic and neuroprotective functions. In addition, in an extensive study on selective ANG-ALS variants we correlated the structural changes to the effects on neuronal survival and the ability to induce stress granules in neuronal cell lines. Furthermore, we have established that ANG-ALS variants which affect the structure of the catalytic site and either decrease or increase the RNase activity affect neuronal survival. Neuronal cell lines expressing the ANG-ALS variants also lack the ability to form stress granules. Here, we report a detailed experimental structural study on eleven new ANG-PD/ALS variants which will have implications in understanding the molecular basis underlying their role in PD and ALS.

Topics: Amyotrophic Lateral Sclerosis; Crystallography, X-Ray; Genetic Variation; Humans; Mutant Proteins; Mutation; Parkinson Disease; Protein Conformation; Ribonuclease, Pancreatic

Juvenile onset ALS is a very rare form of motor neuron disease, with the first symptoms of motor neuron degeneration manifested before 25 years of age. Mutations in the alsin (ALS2), senataxin (SETX), and spatacsin (SPG11) genes have been associated with familial ALS with juvenile onset and slow progression, whereas the genetic architecture of sporadic juvenile ALS remains unclear. We screened mutations in C9orf72, SOD1, FUS, TARDBP, ANG, VCP and PFN1 in 16 juvenile sporadic ALS patients. Four cases (25%) carrying FUS mutations and one individual (6%) harbouring a SOD1 mutation were identified. All cases had an aggressive disease course. Our results suggest that FUS mutations are the most frequent genetic cause in early-onset sporadic ALS patients of Chinese origin. Genetic testing of FUS should be performed in early-onset ALS patients especially those with an aggressive disease course.

Topics: Adenosine Triphosphatases; Adolescent; Adult; Age of Onset; Amyotrophic Lateral Sclerosis; Asian People; Cell Cycle Proteins; DNA-Binding Proteins; Female; Genetic Testing; Humans; Male; Membrane Proteins; Mutation; Profilins; Ribonuclease, Pancreatic; RNA-Binding Protein FUS; Superoxide Dismutase-1; Valosin Containing Protein; Young Adult

To investigate effect of genetic variants in angiogenin (ANG) on the susceptibility of familial amyotrophic lateral sclerosis (FALS) in Han Chinese.. Thirty-one FALS families from 2009 to 2012 were collected and ANG gene was screened in the probands using PCR and direct sequencing.. All 31 ALS families were autosomal dominant inheritance. No mutations and single nucleotide polymorphism were detectable in ANG gene in the 31 probands.. Our study suggests that ANG gene variations may be rare in Chinese Han FALS.

Topics: Amyotrophic Lateral Sclerosis; Asian People; China; Humans; Mutation; Polymorphism, Single Nucleotide; Ribonuclease, Pancreatic

Topics: Adult; Aged; Aged, 80 and over; Amyotrophic Lateral Sclerosis; Case-Control Studies; Female; Humans; Male; Middle Aged; Parkinson Disease; Ribonuclease, Pancreatic; Young Adult

The Angiogenin (ANG) gene is frequently mutated in patients suffering from the neurodegenerative disease--amyotrophic lateral sclerosis (ALS). Most of the ALS-causing mutations in Angiogenin affect either its ribonucleolytic or nuclear translocation activity. Here we report the functional characterization of two previously uncharacterized missense mutations in Angiogenin--D22G and L35P. We predict the nature of loss-of-function(s) in these mutants through our previously established Molecular Dynamics (MD) simulation extended to 100 ns, and show that the predictions are entirely validated through biochemical studies with wild-type and mutated proteins. Based on our studies, we provide a biological explanation for the loss-of-function of D22G-Angiogenin leading to ALS, and suggest that the L35P-Angiogenin mutation would probably cause ALS symptoms in individuals harboring this mutation. Our study thus highlights the strength of MD simulation-based predictions, and suggests that this method can be used for correlating mutations in Angiogenin or other effector proteins with ALS symptoms.

Topics: Amino Acid Substitution; Amyotrophic Lateral Sclerosis; Humans; Models, Molecular; Mutation; Mutation, Missense; Protein Structure, Tertiary; Ribonuclease, Pancreatic

Certain single nucleotide polymorphisms causing missense mutations in angiogenin result in its loss-of-function and onset of amyotrophic lateral sclerosis (ALS). Although several such associations are reported across diverse ethnic groups, no method is available for predicting if a new mutation is deleterious. We present here a fast molecular dynamics based method for determining the mechanisms of functional loss caused by mutations, and attributes to ascertain whether a mutation causes ALS.

Topics: Amyotrophic Lateral Sclerosis; Humans; Molecular Dynamics Simulation; Mutation; Protein Conformation; Ribonuclease, Pancreatic; Solvents; Time Factors

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting lower and upper motoneurons. Recent studies have shown that both motor neurons and non-neuronal neighbouring cells such as astrocytes and microglia contribute to disease pathology. Loss-of-function mutations in the angiogenin (ANG) gene have been identified in ALS patients. Angiogenin is enriched in motor neurons and exerts neuroprotective effects in vitro and in vivo. We have recently shown that motoneurons secrete angiogenin, and that secreted angiogenin is exclusively taken up by astrocytes, suggesting a paracrine mechanism of neuroprotection. To gain insights into astrocyte effectors of angiogenin-induced neuroprotection, we examined alterations in the astrocyte secretome induced by angiogenin treatment using quantitative proteomics based on Stable Isotope Labelling by Amino Acids in Cell Culture (SILAC). We identified 2128 proteins in conditioned media from primary cultured mouse astrocytes, including 1247 putative secreted proteins. Of these, 60 proteins showed significant regulation of secretion in response to angiogenin stimulation. Regulated proteins include chemokines and cytokines, proteases and protease inhibitors as well as proteins involved in reorganising the extracellular matrix. In conclusion, this proteomic analysis increases our knowledge of the astrocyte secretome and reveals potential molecular substrates underlying the paracrine, neuroprotective effects of angiogenin.. This study provides the most extensive list of astrocyte-secreted proteins available and reveals novel potential molecular substrates of astrocyte-neuron communication. It also identifies a set of astrocyte-derived proteins that might slow down ALS disease progression. It should be relevant to a large readership of neuroscientists and clinicians, in particular those with an interest in the physiological and pathological roles of astrocytes and in the molecular and cellular mechanisms underlying neurodegenerative disorders.

Topics: Amyotrophic Lateral Sclerosis; Animals; Animals, Newborn; Astrocytes; Cell Communication; Cell Survival; Culture Media, Conditioned; Disease Progression; Extracellular Matrix; Gene Expression Regulation; Mice; Mice, Inbred C57BL; Motor Neurons; Mutation; Neurons; Proteome; Proteomics; Ribonuclease, Pancreatic

In amyotrophic lateral sclerosis (ALS) cerebrospinal fluid (CSF) analysis is usually performed to exclude inflammatory processes of the central nervous system. Although in a small subset of patients an intrathecal synthesis of IgG is detectable, usually there is no clear explanation for this evidence. This study investigates the occurrence of oligoclonal bands (OCBs) in the CSF of a large series of ALS patients, attempting a correlation with genotype data. CSF was collected from 259 ALS patients. CSF parameters were measured according to standard procedures, and detection of OCBs performed by isoelectric focusing. The patients were screened for mutations in SOD1, FUS, TARDBP, ANG, OPTN, and C9ORF72. We observed the presence of OCBs in the CSF of 9/259 ALS patients (3.5 %), and of disease-associated mutations in 12 cases. OCBs were significantly more frequent in mutation carriers compared to the remaining cohort (3/12 vs 6/247; p < 0.01). Among patients with OCBs, two patients had the TARDBP p.A382T mutation (one of which in homozygous state), and one the ANG p.P-4S variant. Both patients carrying the p.A382T mutation had an atypical phenotype, one of them manifesting signs suggestive of a cerebellar involvement, and the other presenting neuroradiological findings suggestive of an inflammatory disorder of the central nervous system. Our results suggest that ALS patients with OCBs may harbor mutations in disease-causing genes. We speculate that mutations in both TARDBP and ANG genes may disrupt the blood-brain barrier (BBB), promoting local immune responses and neuroinflammation. The role of mutant TARDBP and ANG genes on BBB integrity of ALS patients warrants further investigation.

Topics: Adult; Aged; Amyotrophic Lateral Sclerosis; Blood Cell Count; Blood Sedimentation; Brain; C-Reactive Protein; C9orf72 Protein; Cell Cycle Proteins; Cohort Studies; DNA Mutational Analysis; DNA-Binding Proteins; Female; Humans; Magnetic Resonance Imaging; Male; Membrane Transport Proteins; Middle Aged; Mutation; Oligoclonal Bands; Proteins; Ribonuclease, Pancreatic; Superoxide Dismutase; Superoxide Dismutase-1; Transcription Factor TFIIIA

Five to 10% of cases of amyotrophic lateral sclerosis are familial, with the most common genetic causes being mutations in the C9ORF72, SOD1, TARDBP and FUS genes. Mutations in the angiogenin gene, ANG, have been identified in both familial and sporadic patients in several populations within Europe and North America. The aim of this study was to establish the incidence of ANG mutations in a large cohort of 517 patients from Northern England and establish the neuropathology associated with these cases.. The single exon ANG gene was amplified, sequenced and analysed for mutations. Pathological examination of brain, spinal cord and skeletal muscle included conventional histology and immunohistochemistry.. Mutation screening identified a single sporadic amyotrophic lateral sclerosis case with a p.K54E mutation, which is absent from 278 neurologically normal control samples. The clinical presentation was of limb onset amyotrophic lateral sclerosis, with rapid disease progression and no evidence of cognitive impairment. Neuropathological examination established the presence of characteristic ubiquitinated and TDP-43-positive neuronal and glial inclusions, but no abnormality in the distribution of angiogenin protein.. There is only one previous report describing the neuropathology in a single case with a p.K17I ANG mutation which highlighted the presence of eosinophilic neuronal intranuclear inclusions in the hippocampus. The absence of this feature in the present case indicates that patients with ANG mutations do not always have pathological changes distinguishable from those of sporadic amyotrophic lateral sclerosis.

Topics: Amyotrophic Lateral Sclerosis; Brain; DNA Mutational Analysis; Female; Humans; Male; Middle Aged; Muscle, Skeletal; Mutation; Neuroglia; Ribonuclease, Pancreatic; Spinal Cord

Angiogenin (ANG) is a member of the ribonuclease superfamily which is implicated in angiogenesis. ANG maintains normal vasculature and thereby protects motor neurons from various stress conditions. It is suggested that ANG may play a role in pathomechanism of amyotrophic lateral sclerosis (ALS). However, there have been no studies of ANG in ALS skin. We made a quantitative immunohistochemical study of the expression of ANG in the skin from 20 patients with sporadic ALS, 20 patients with other neurologic or muscular disorders (control group A), and 20 patients without neurologic or muscular disorders (control group B). The nuclei of the epidermal cells showed a weak ANG immunoreactivity in ALS patients. These findings became more marked as ALS progressed. The optical density for ANG immunoreactivity of the nucleus in the epidermal cells in ALS patients was significantly lower (p<0.001) than in control groups A and B. There was a significant negative relationship (r=-0.82, p<0.001) between the optical density for ANG immunoreactivity of the nucleus and duration of illness in ALS patients. These data suggest that changes of ANG in ALS skin are related to the disease process and that metabolic alterations of ANG may take place in the skin of ALS patients.

Topics: Aged; Amyotrophic Lateral Sclerosis; Biomarkers; Female; Humans; Male; Middle Aged; Ribonuclease, Pancreatic; Skin

Several missense mutations in the coding region of angiogenin (ANG) gene have been identified in Amyotrophic Lateral Sclerosis (ALS) patients. These mutations lead to loss of either ribonucleolytic activity or nuclear translocation activity or both of ANG (protein encoded by ANG gene) causing ALS. We present here a cohesive and comprehensive picture of the molecular origins of functional loss of all ALS associated ANG mutants, emerging via extensive molecular dynamics simulations. Our method effectively predicts that conformational change of His114 results in loss of ribonucleolytic activity and that reduction of solvent accessible surface area of nuclear localization signal residues (31)RRR(33) results in loss of nuclear translocation activity. These predictions hold true, without exception, for all ANG mutants studied and can be employed to infer whether a new ANG mutation is causative of ALS or benign ahead of experimental findings.

Topics: Amyotrophic Lateral Sclerosis; Histidine; Humans; Hydrogen Bonding; Molecular Dynamics Simulation; Mutation; Mutation, Missense; Nuclear Localization Signals; Protein Conformation; Ribonuclease, Pancreatic

About 5% of amyotrophic lateral sclerosis (ALS) cases are known to be familial (fALS) and mutations in SOD1 and other genes are found in more than 20% of fALS patients and in 2%-4% of apparently sporadic ALS (sALS) cases. However, there are few reports on the proportion of fALS and the frequency of mutations in Korean patients with ALS. We screened mutations in the SOD1, FUS, TARDBP, ANG, and OPTN genes in 258 consecutively enrolled Korean patients with ALS from October 2006 to November 2010. The frequency of fALS was estimated to be 3.5% (9/258), and mutations were identified in 88.9% (8/9) of fALS patients but only in 2.8% (7/249) of sALS patients. Seven fALS and 3 sALS patients had mutations in SOD1 gene while all the others had FUS gene. The proportion of fALS was lower than that reported in Caucasian populations but the frequency of SOD1 gene mutations in Korean fALS patients (77.8%, 7/9) was much higher than that reported in other ethnic groups. These findings might suggest that there is an ethnic difference in the proportion of fALS and the genetic background of ALS.

Topics: Adult; Aged; Aged, 80 and over; Amyotrophic Lateral Sclerosis; Asian People; Cell Cycle Proteins; DNA-Binding Proteins; Female; Genetic Predisposition to Disease; Genetic Testing; Humans; Male; Membrane Transport Proteins; Middle Aged; Pedigree; Republic of Korea; Ribonuclease, Pancreatic; RNA-Binding Protein FUS; Superoxide Dismutase; Superoxide Dismutase-1; Transcription Factor TFIIIA

Abstract Angiogenin (ANG) gene mutations have been identified in both familial and sporadic amyotrophic lateral sclerosis (ALS) patients from multiple European and North American populations. However, no ANG mutation has yet been reported in Asian ALS populations. Here, we screened for ANG mutations in a Chinese ALS cohort. The entire coding region of the ANG gene was sequenced in 10 familial ALS pedigrees, 202 sporadic ALS patients, and 151 healthy controls. All patients were negative for SOD1, FUS, and TARDBP mutations. We identified a novel missense mutation, c.379G > A (p.V103I), in one sporadic ALS patient, but not in the controls. No mutations were found in the familial ALS patients. A novel missense variant, c.323A > G (p.H84R), was detected in one healthy individual. We identified the presence of the known single nucleotide polymorphism, rs11701 (T/G), in both ALS cases and controls. However, no significant association of the G allele with ALS susceptibility was demonstrated. In conclusion, ANG mutations accounted for 0.5% of our SOD1-, FUS-, TARDBP- mutation-negative ALS cohort. Our findings highlight that the genetic background of ALS differs between different populations, and suggest that ANG mutation may be involved in the aetiology of ALS in the Han Chinese population.

Topics: Adult; Alleles; Amyotrophic Lateral Sclerosis; Asian People; Cohort Studies; DNA Mutational Analysis; Female; Genetic Predisposition to Disease; Genotype; Humans; Male; Middle Aged; Mutation, Missense; Polymorphism, Single Nucleotide; Ribonuclease, Pancreatic

SOD1, ANG, TARDBP and FUS mutations have been associated with amyotrophic lateral sclerosis (ALS). Our goal was to extend molecular genetic analysis to newly identified ALS genetic loci and to determine the frequency of mutations, distribution of disease genes, and variant spectrum of these genes in a large United States ALS-phenotype cohort. We screened 1220 probands with an ALS phenotype, referred originally for SOD1 molecular genetic analysis. 1128 SOD1-negative probands were screened for ANG, and 277 and 223 SOD1- and ANG-negative samples were screened for TARDBP and FUS, respectively. One hundred additional probands were specifically screened only for FUS exon 15. We identified a total of 36 different SOD1 mutations, including three novel mutations, in 92 probands. ANG screening identified three mutations, including two novel mutations, and TARDBP screening identified two previously reported TARDBP mutations. We also identified four mutations in FUS, including the reported FUS in-frame deletion, c.430_447del, p.Gly144_Tyr149del, in a patient with inclusion body myositis, and two known FUS missense mutations. From this study, we estimate frequencies for SOD1, ANG, TARDBP and FUS mutations, in this United States cohort, to be 7.5%, 0.71%, 0.72% and 1.9%, respectively. In conclusion, we identify novel variants in SOD1, ANG, TARDBP and FUS, and expand the FUS-associated clinicopathologic phenotype.

Topics: Amyotrophic Lateral Sclerosis; Clinical Laboratory Techniques; DNA-Binding Proteins; Genetic Predisposition to Disease; Genetic Testing; Humans; Mutation; Phenotype; Ribonuclease, Pancreatic; RNA-Binding Protein FUS; Superoxide Dismutase; Superoxide Dismutase-1; United States

Mutations in the coding region of angiogenin (ANG) gene have been found in patients suffering from Amyotrophic Lateral Sclerosis (ALS). Neurodegeneration results from the loss of angiogenic ability of ANG (protein coded by ANG). In this work, we performed extensive molecular dynamics (MD) simulations of wild-type ANG and disease associated ANG variants to elucidate the mechanism behind the loss of ribonucleolytic activity and nuclear translocation activity, functions needed for angiogenesis.. MD simulations were carried out to study the structural and dynamic differences in the catalytic site and nuclear localization signal residues between WT-ANG (Wild-type ANG) and six mutants. Variants K17I, S28N, P112L and V113I have confirmed association with ALS, while T195C and A238G single nucleotide polymorphisms (SNPs) encoding L35P and K60E mutants respectively, have not been associated with ALS. Our results show that loss of ribonucleolytic activity in K17I is caused by conformational switching of the catalytic residue His114 by 99°. The loss of nuclear translocation activity of S28N and P112L is caused by changes in the folding of the residues (31)RRR(33) that result in the reduction in solvent accessible surface area (SASA). Consequently, we predict that V113I will exhibit loss of angiogenic properties by loss of nuclear translocation activity and L35P by loss of both ribonucleolytic activity and nuclear translocation activity. No functional loss was inferred for K60E. The MD simulation results were supported by hydrogen bond interaction analyses and molecular docking studies.. Conformational switching of catalytic residue His114 seems to be the mechanism causing loss of ribonucleolytic activity and reduction in SASA of nuclear localization signal residues (31)RRR(33) results in loss of nuclear translocation activity in ANG mutants. Therefore, we predict that L35P mutant, would exhibit loss of angiogenic functions, and hence would correlate with ALS while K60E would not show any loss.

Topics: Active Transport, Cell Nucleus; Amyotrophic Lateral Sclerosis; Crystallography, X-Ray; Genetic Variation; Humans; Hydrogen Bonding; Models, Molecular; Molecular Dynamics Simulation; Mutation; Neovascularization, Pathologic; Protein Conformation; Ribonuclease, Pancreatic; Risk Factors; Signal Transduction; Solvents; Surface Properties

To classify familial amyotrophic lateral sclerosis (FALS) on the base of family history, and to determine whether frequency of mutations in major amyotrophic lateral sclerosis (ALS) genes varies in different FALS categories.. Included in the study are 53 FALS families. Patients were classified as definite, probable and possible FALS, according to recently proposed criteria. Seven ALS-associated genes, including SOD1, TARDBP, FUS, ANG, ATXN2, OPTN and C9ORF72, were analysed.. Thirteen patients (24.5%) were included in the definite group. The great majority of our FALS cases (40/53, 75.5%) were families with only two affected relatives; of these, 31 (58.5%) were included in the probable, and 9 (17%) in the possible FALS categories. The percentage of mutations was 61.5% in definite, 41.9% in probable and 11.1% in possible FALS. With respect to probable FALS, if cases with parent-to-child transmission of the disease were considered separately, the mutational load increased to 61.5%, as observed in definite FALS.. Our findings provide evidence that frequency of mutations in currently known ALS genes varies widely among different FALS categories. Families with only two affected relatives have heterogeneous genetic components, the chance to detect mutations being higher in cases with parent-to-child transmission.

Topics: Adult; Aged; Amyotrophic Lateral Sclerosis; Ataxins; C9orf72 Protein; Cell Cycle Proteins; DNA-Binding Proteins; Family; Female; Gene Frequency; Humans; Male; Membrane Transport Proteins; Middle Aged; Mutation; Nerve Tissue Proteins; Proteins; Ribonuclease, Pancreatic; RNA-Binding Protein FUS; Sequence Analysis, DNA; Superoxide Dismutase; Superoxide Dismutase-1; Transcription Factor TFIIIA

Mutations in angiogenin (ANG), a member of the ribonuclease A superfamily, are associated with amyotrophic lateral sclerosis (ALS; sporadic and familial) and Parkinson's disease. We have previously shown that ANG is expressed in neurons during neuro-ectodermal differentiation, and that it has both neurotrophic and neuroprotective functions. Here we report the atomic resolution structure of native ANG and 11 ANG-ALS variants. We correlate the structural changes to the effects on neuronal survival and the ability to induce stress granules in neuronal cell lines. ANG-ALS variants that affect the structure of the catalytic site and either decrease or increase the RNase activity affect neuronal survival. Neuronal cell lines expressing the ANG-ALS variants also lack the ability to form stress granules. Our structure-function studies on these ANG-ALS variants are the first to provide insights into the cellular and molecular mechanisms underlying their role in ALS.

Topics: Amyotrophic Lateral Sclerosis; Animals; Cell Line; Cell Line, Tumor; Crystallography, X-Ray; Cytoplasmic Granules; Humans; Immunohistochemistry; Mice; Mutation; Neurons; Ribonuclease, Pancreatic

To assess the frequency and clinical characteristics of patients with mutations of major amyotrophic lateral sclerosis (ALS) genes in a prospectively ascertained, population-based epidemiologic series of cases.. The study population includes all ALS cases diagnosed in Piemonte, Italy, from January 2007 to June 2011. Mutations of SOD1, TARDBP, ANG, FUS, OPTN, and C9ORF72 have been assessed.. Out of the 475 patients included in the study, 51 (10.7%) carried a mutation of an ALS-related gene (C9ORF72, 32; SOD1, 10; TARDBP, 7; FUS, 1; OPTN, 1; ANG, none). A positive family history for ALS or frontotemporal dementia (FTD) was found in 46 (9.7%) patients. Thirty-one (67.4%) of the 46 familial cases and 20 (4.7%) of the 429 sporadic cases had a genetic mutation. According to logistic regression modeling, besides a positive family history for ALS or FTD, the chance to carry a genetic mutation was related to the presence of comorbid FTD (odds ratio 3.5; p = 0.001), and age at onset ≤54 years (odds ratio 1.79; p = 0.012).. We have found that ∼11% of patients with ALS carry a genetic mutation, with C9ORF72 being the commonest genetic alteration. Comorbid FTD or a young age at onset are strong indicators of a possible genetic origin of the disease.

Topics: Adult; Aged; Amyotrophic Lateral Sclerosis; C9orf72 Protein; Cell Cycle Proteins; Community Health Planning; DNA Mutational Analysis; DNA-Binding Proteins; Family Health; Frontotemporal Dementia; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Italy; Membrane Transport Proteins; Middle Aged; Mutation; Neuropsychological Tests; Proteins; Retrospective Studies; Ribonuclease, Pancreatic; RNA-Binding Protein FUS; Superoxide Dismutase; Superoxide Dismutase-1; Transcription Factor TFIIIA

Progressive muscular atrophy (PMA) and amyotrophic lateral sclerosis (ALS) are devastating motor neuron diseases (MNDs), which result in muscle weakness and/or spasticity. We compared mutation frequencies in genes known to be associated with MNDs between patients with apparently sporadic PMA and ALS. A total of 261 patients with adult-onset sporadic PMA, patients with sporadic ALS, and control subjects of Dutch descent were obtained at national referral centers for neuromuscular diseases in The Netherlands. Sanger sequencing was used to screen these subjects for mutations in the coding regions of superoxide dismutase-1 (SOD1), angiogenin (ANG), fused in sarcoma/translated in liposarcoma (FUS/TLS), TAR DNA-binding protein 43 (TARDBP), and multivesicular body protein 2B (CHMP2B). In our cohort of PMA patients we identified two SOD1 mutations (p.D90A, p.I113T), one ANG mutation (p.K17I), one FUS/TLS mutation (p.R521H), one TARDBP mutation (p.N352S), and one novel CHMP2B mutation (p.R69Q). The mutation frequency of these genes was similar in sporadic PMA (2.7%) and ALS (2.0%) patients, and therefore, our findings demonstrate a genetic overlap between apparently sporadic PMA and ALS.

Topics: Adult; Age of Onset; Aged; Amyotrophic Lateral Sclerosis; DNA-Binding Proteins; Endosomal Sorting Complexes Required for Transport; Female; Genotype; Haplotypes; Humans; Male; Middle Aged; Muscular Atrophy, Spinal; Mutation; Ribonuclease, Pancreatic; RNA-Binding Protein FUS; Superoxide Dismutase; Superoxide Dismutase-1

SOD1 G93D mutation has been described in amyotrophic lateral sclerosis (ALS) patients with slowly progressive disease. We describe an Italian patient affected by sporadic ALS with the SOD1 G93D mutation that disclosed an unusual rapid progression with death occurring after 30 months from the symptom onset. Considering the atypical clinical course further genes associated with ALS or known to be causative were studied including ANG, PGRN, TARDBP, FUS, VCP, CHRNA3, CHRNA4, and CHRNB4. A novel heterozygous ANG missense variant (c.433 C>T, p.R145C) was identified which is neither reported in controls nor in 1000 genomes and single nucleotide polymorphism (SNP) databases. This report confirms that clinical course of SOD1-related ALS may be modulated by other causative or associated genes, including ANG and suggests that extensive screening of ALS-associated genes in patients with an already identified mutation may be helpful for better knowledge of genetic architecture of ALS.

Topics: Aged; Amyotrophic Lateral Sclerosis; Disease Progression; DNA Mutational Analysis; Female; Follow-Up Studies; Genetic Association Studies; Genetic Predisposition to Disease; Humans; Italy; Polymorphism, Single Nucleotide; Ribonuclease, Pancreatic; Superoxide Dismutase

FUS gene mutations were recently identified in familial amyotrophic lateral sclerosis (ALS). The present studies sought to define the clinical, post-mortem and neurophysiological phenotypes in ALS families with FUS mutations and to determine the frequency of FUS mutations in familial and sporadic ALS.. FUS was screened for mutations in familial and sporadic ALS cases. Clinical, post-mortem and neurophysiological features of large families with FUS mutations are described.. FUS mutations were evident in 3.2% (4/124) of familial ALS, representing the second most common gene abnormality to be described in familial ALS after SOD1. No mutations were present in 247 sporadic ALS cases. The clinical presentation in 49 affected patients was consistent with a predominantly lower motor neuron disorder, supported by post-mortem findings. Upper motor neuron involvement varied, with Wallerian degeneration of corticospinal tracts present in one post-mortem case but absent in a second case from the same family. Features of cortical hyperexcitability demonstrated upper motor neuron involvement consistent with other forms of familial and sporadic ALS. One case presented with frontotemporal dementia (FTD) indicating that this may be a rare presenting feature in families with FUS mutation. Ubiquitin-positive cytoplasmic skein-like inclusions were present in lower motor neurons, but in contrast to sporadic ALS, no TDP-43 pathology was evident. Mutation-specific clinical features were identified. Patients with a R521C mutation were significantly more likely to develop disease at a younger age, and dropped-head syndrome was a frequent feature. Reduced disease penetrance was evident among most affected families.

Topics: Adult; Aged; Amyotrophic Lateral Sclerosis; Brain; Cognition Disorders; DNA Mutational Analysis; DNA-Binding Proteins; Dynactin Complex; Endosomal Sorting Complexes Required for Transport; Female; Genetic Testing; Humans; Male; Microtubule-Associated Proteins; Middle Aged; Nerve Tissue Proteins; Neuropsychological Tests; Point Mutation; Ribonuclease, Pancreatic; RNA-Binding Protein FUS; RNA, Messenger; Severity of Illness Index; Superoxide Dismutase; Superoxide Dismutase-1; Vesicular Transport Proteins; Young Adult

Mutations in SOD1, ANG, VAPB, TARDBP and FUS genes have been identified in amyotrophic lateral sclerosis (ALS).. The relative contributions of the different mutations to ALS were estimated by systematically screening a cohort of 162 families enrolled in France and 500 controls (1000 chromosomes) using molecular analysis techniques and performing phenotype-genotype correlations.. 31 pathogenic missense mutations were found in 36 patients (20 SOD1, 1 ANG, 1 VAPB, 7 TARDBP and 7 FUS). Surprisingly two FUS mutation carriers also harboured ANG variants. One family of Japanese origin with the P56S VAPB mutation was identified. Seven novel mutations (three in SOD1, two in TARDBP, two in FUS) were found. None of them was detected in controls. Segregation of detected mutations with the disease was confirmed in 11 families including five pedigrees carrying the novel mutations. Clinical comparison of SOD1, TARDBP, FUS and other familial ALS patients (with no mutation in the screened genes) revealed differences in site of onset (predominantly lower limbs for SOD1 and upper limbs for TARDBP mutations), age of onset (younger with FUS mutations), and in lifespan (shorter for FUS carriers). One third of SOD1 patients survived more than 7 years: these patients had earlier disease onset than those presenting with a more typical course. Differences were also observed among FUS mutations, with the R521H FUS mutation being associated with longer disease duration.. This study identifies new genetic associations with ALS and provides phenotype-genotype correlations with both previously reported and novel mutations.

Topics: Adult; Age Distribution; Age of Onset; Aged; Aged, 80 and over; Amyotrophic Lateral Sclerosis; DNA-Binding Proteins; Family; Female; Genetic Association Studies; Humans; Longevity; Male; Middle Aged; Mutation; Ribonuclease, Pancreatic; RNA-Binding Protein FUS; Superoxide Dismutase; Vesicular Transport Proteins

To determine whether 5 single nucleotide polymorphisms (SNPs) associate with ALS in 3 different populations. We also assessed the contribution of genotype to angiogenin levels in plasma and CSF.. Allelic association statistics were calculated for polymorphisms in the ANG gene in 859 patients and 1047 controls from Sweden, Ireland and Poland. Plasma, serum and CSF angiogenin levels were quantified and stratified according to genotypes across the ANG gene. The contribution of SNP genotypes to variance in circulating angiogenin levels was estimated in patients and controls.. All SNPs showed association with ALS in the Irish group. The SNP rs17114699 replicated in the Swedish cohort. No SNP associated in the Polish cohort. Age- and sex-corrected circulating angiogenin levels were significantly lower in patients than in controls (p<0.001). An allele dose-dependent regulation of angiogenin levels was observed in controls. This regulation was attenuated in the ALS cohort. A significant positive correlation between CSF plasma angiogenin levels was present in controls and abolished in ALS.. ANG variants associate with ALS in the Irish and Swedish populations, but not in the Polish. There is evidence of dysregulation of angiogenin expression in plasma and CSF in sporadic ALS. Angiogenin expression is likely to be important in the pathogenesis of ALS.

Topics: Alleles; Amyotrophic Lateral Sclerosis; Gene Frequency; Genetic Predisposition to Disease; Genotype; Haplotypes; Humans; Ireland; Linkage Disequilibrium; Poland; Polymorphism, Single Nucleotide; Ribonuclease, Pancreatic; Sweden

Topics: Aged; Amino Acid Sequence; Amino Acid Substitution; Amyotrophic Lateral Sclerosis; Base Sequence; Dementia; Female; Heterozygote; Humans; Isoleucine; Lysine; Male; Middle Aged; Mutation; Pedigree; Ribonuclease, Pancreatic

Our objective was to assess the regulation of the hypoxia response of angiogenic and inflammatory factors from 76 cerebrospinal fluids (CSF) of sporadic amyotrophic lateral sclerosis (ALS) patients with different respiratory status. We first analysed the hypoxia response capacity by measuring CSF levels of angiogenin (ANG), VEGF, angiopoietin-2 (ANG-2) and PGE-2 in 40 ALS patients according to their hypoxaemia level and compared it with 40 neurological controls. We then compared the ANG, VEGF, EPO and ANG-2 CSF levels of 36 other ALS patients, divided into three groups with either 1) normoxaemia, 2) intermittent desaturation in the absence of hypoxaemia, or 3) chronic hypoxaemia with or without desaturation. We demonstrated a lack of up-regulation of both ANG and VEGF during hypoxaemia in ALS, compared with hypoxaemic controls. In contrast, PGE-2 and ANG-2 levels were increased in both hypoxaemic ALS patients and controls. ANG and VEGF levels did not increase in patients with long disease durations and with intermittent or chronic hypoxaemia. ANG-2 and EPO levels were up-regulated early in intermittent hypoxaemia and late in chronic hypoxaemia, respectively. Our results suggest alteration of the HIF-1alpha-mediated response to hypoxia during sporadic ALS, whereas the NFK-B pathway seems early activated.

Topics: Aged; Amyotrophic Lateral Sclerosis; Angiopoietin-2; Dinoprostone; Erythropoietin; Humans; Hypoxia; Inflammation; Middle Aged; Oxygen; Respiration; Ribonuclease, Pancreatic; Vascular Endothelial Growth Factor A

Amyotrophic lateral sclerosis (ALS) is a fatal progressive neurodegenerative disease characterized by the selective death of motor neurons in the motor cortex, brain stem and spinal cord. Recently, missense variants in the angiogenin gene (ANG), an angiogenic factor expressed in ventral horn motor neurons that is up-regulated by hypoxia, have been found in ALS patients of Irish/Scottish, North American, Italian, French and Dutch descent. To investigate the role of ANG in the German population, we screened for mutations by sequencing the entire coding region of the ANG gene in a large sample of 581 German ALS cases and 616 sex- and age-matched healthy controls. We identified two heterozygous missense variants, F(-13)L and K54E, in two German sporadic ALS cases but not in controls. Both missense variants are novel and have not been previously found in ALS cases. Our results suggest that missense variants in the ANG gene play a role in ALS in the German population and provide further evidence to support the hypothesis that angiogenic factors up-regulated by hypoxia are involved in the pathophysiology of ALS.

Topics: Amyotrophic Lateral Sclerosis; Central Nervous System; Cohort Studies; DNA Mutational Analysis; Female; Genetic Markers; Genetic Predisposition to Disease; Genetic Testing; Genetic Variation; Genotype; Germany; Heterozygote; Humans; Hypoxia; Male; Middle Aged; Mutation, Missense; Oxidative Stress; Ribonuclease, Pancreatic; White People

Cells can adapt to hypoxia through the activation of hypoxia-inducible factor-1 (HIF-1), which in turn regulates the expression of hypoxia-responsive genes. Defects in hypoxic signaling have been suggested to underlie the degeneration of motoneurons in amyotrophic lateral sclerosis (ALS). We have recently identified mutations in the hypoxia-responsive gene, angiogenin (ANG), in ALS patients, and have shown that ANG is constitutively expressed in motoneurons. Here, we show that HIF-1alpha is sufficient and required to activate ANG in cultured motoneurons exposed to hypoxia, although ANG expression does not change in a transgenic ALS mouse model or in sporadic ALS patients. Administration of recombinant ANG or expression of wild-type ANG protected motoneurons against hypoxic injury, whereas gene silencing of ang1 significantly increased hypoxia-induced cell death. The previously reported ALS-associated ANG mutations (Q12L, K17I, R31K, C39W, K40I, I46V) all showed a reduced neuroprotective activity against hypoxic injury. Our data show that ANG plays an important role in endogenous protective pathways of motoneurons exposed to hypoxia, and suggest that loss of function rather than loss of expression of ANG is associated with ALS.

Topics: Amino Acid Substitution; Amyotrophic Lateral Sclerosis; Animals; Apoptosis; Cell Hypoxia; Cells, Cultured; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, Transgenic; Motor Neurons; Mutation; Recombinant Proteins; Ribonuclease, Pancreatic; RNA, Small Interfering; Signal Transduction; Vascular Endothelial Growth Factors

A K17I mutation in the ANG gene encoding angiogenin has been identified in a case that we previously published as ALS with neuronal intranuclear protein inclusions (Seilhean et al. in Acta Neuropathol 108:81-87, 2004). These inclusions were immunoreactive for smooth muscle alpha-actin but not for angiogenin. Moreover, they were not labeled by anti-TDP-43 antibodies, while numerous cytoplasmic inclusions immunoreactive for ubiquitin, p62 and TDP-43 were detected in both oligodendrocytes and neurons in various regions of the central nervous system. In addition, expression of smooth muscle alpha-actin was increased in the liver where severe steatosis was observed. This is the first neuropathological description of a case with an ANG mutation. Angiogenin is known to interact with actin. Like other proteins involved in ALS pathogenesis, such as senataxin, TDP-43 and FUS/TLS, it plays a role in RNA maturation.

Topics: Actins; Amyotrophic Lateral Sclerosis; Brain; DNA Mutational Analysis; DNA-Binding Proteins; Fatty Liver; Female; Humans; Immunohistochemistry; Inclusion Bodies; Liver; Middle Aged; Motor Neurons; Muscle, Skeletal; Mutation; Neurons; Oligodendroglia; Ribonuclease, Pancreatic; Spinal Cord

Genetic variations in two hypoxia-inducible angiogenic genes, VEGF and ANG, have been linked with sporadic amyotrophic lateral sclerosis (SALS). Common variations in these genes may reduce the levels or functioning of their products. VEGF and ANG belong to a larger group of angiogenic genes that are up-regulated under hypoxic conditions. We hypothesized that common genetic variation across other members of this group may also predispose to sporadic ALS. To screen other hypoxia-inducible angiogenic genes for association with SALS, we selected 112 tagging single nucleotide polymorphisms (tgSNPs) that captured the common genetic variation across 16 VEGF-like and eight ANG-like hypoxia-inducible genes. Screening for association was performed in 270 Irish individuals with typical SALS and 272 ethnically matched unrelated controls. SNPs showing association in the Irish phase were genotyped in a replication sample of 281 Swedish sporadic ALS patients and 286 Swedish controls. Seven markers showed association in the Irish. The one modest replication signal observed in the Swedish replication sample, at rs3801158 in the gene inhibin beta A, was for the opposite allele vs. the Irish cohort. We failed to detect association of common variation across 24 candidate hypoxia-inducible angiogenic genes with SALS.

Topics: Aged; Amyotrophic Lateral Sclerosis; Female; Genetic Predisposition to Disease; Genetic Testing; Genotype; Humans; Hypoxia; Ireland; Male; Middle Aged; Polymorphism, Single Nucleotide; Ribonuclease, Pancreatic; Sequence Analysis, DNA; Sweden; Vascular Endothelial Growth Factor A

Mutations in the angiogenin gene, ANG, have been associated recently with familial and sporadic forms of amyotrophic lateral sclerosis (ALS). However, the cellular and molecular mechanisms that link ANG, a multidomain protein, to ALS are still unknown.. To assess the frequency of ANG gene mutations in 855 French patients with sporadic ALS.. We analyzed by direct sequencing the full coding region of the ANG gene in a cohort of French patients with sporadic ALS. The clinical characteristics of patients carrying ANG mutations are detailed.. French ALS Study Group. Patients A total of 855 patients with sporadic ALS.. Results of genetic analyses.. We observed a previously identified mutation (pI46V) in 2 patients with ALS without a known family link and found a novel mutation (pR121H) in 1 patient who developed ALS with rapid progression. We did not observe an association between patients with ALS and the rs11701 polymorphism, as previously reported in certain ALS populations of other ethnic origins.. Overall, our findings support the implication of ANG gene mutations as a rare but widespread cause of ALS.

Topics: Adult; Aged; Amyotrophic Lateral Sclerosis; Catalytic Domain; Central Nervous System; Cohort Studies; DNA Mutational Analysis; Female; France; Gene Frequency; Genetic Markers; Genetic Predisposition to Disease; Genetic Testing; Genotype; Humans; Male; Middle Aged; Mutation; Polymorphism, Single Nucleotide; Protein Structure, Tertiary; Ribonuclease, Pancreatic

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease. It is suggested that angiogenin (ANG) may play a role in the pathomechanism of this disease. The aim of the study was to measure cerebrospinal fluid (CSF) ANG levels in patients with ALS. Twenty ALS patients and 15 control subjects were included in the study. CSF ANG levels were measured by ELISA. Study results showed that CSF ANG level did not differ between ALS patients and control group (p > 0.05). There was no significant correlation between CSF ANG level and clinical state of ALS patients either (p > 0.05). The present study conducted on CSF of patients with ALS did not confirm previous observation on the possible role of ANG in neurodegeneration in this disease.

Topics: Adult; Aged; Aged, 80 and over; Amyotrophic Lateral Sclerosis; Angiogenesis Inducing Agents; Female; Humans; Male; Middle Aged; Ribonuclease, Pancreatic

Mutations in the hypoxia-inducible factor angiogenin (ANG) have been identified in Amyotrophic Lateral Sclerosis (ALS) patients, but the potential role of ANG in ALS pathogenesis was undetermined. Here we show that angiogenin promotes motoneuron survival both in vitro and in vivo. Angiogenin protected cultured motoneurons against excitotoxic injury in a PI-3-kinase/Akt kinase-dependent manner, whereas knock-down of angiogenin potentiated excitotoxic motoneuron death. Expression of wild-type ANG protected against endoplasmic reticulum (ER) stress-induced and trophic-factor-withdrawal-induced cell death in vitro, whereas the ALS-associated ANG mutant K40I exerted no protective activity and failed to activate Akt-1. In SOD1(G93A) mice angiogenin delivery increased lifespan and motoneuron survival, restored the disease-associated decrease in Akt-1 survival signaling, and reversed a pathophysiological increase in ICAM-1 expression. Our data demonstrate that angiogenin is a key factor in the control of motoneuron survival.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Amyotrophic Lateral Sclerosis; Androstadienes; Animals; Cell Survival; Cells, Cultured; Disease Models, Animal; Embryo, Mammalian; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Female; Gene Expression; Gene Expression Regulation; Insulin-Like Growth Factor I; Intercellular Adhesion Molecule-1; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Motor Neurons; Mutagenesis, Site-Directed; Neuroprotective Agents; Oncogene Protein v-akt; Ribonuclease, Pancreatic; RNA, Small Interfering; Spinal Cord; Superoxide Dismutase; Time Factors; Transfection; Tunicamycin; Wortmannin

To investigate the role of vascular endothelial growth factor (VEGF) and angiogenin (ANG) as genetic determinants in the susceptibility to sporadic ALS in Italian patients. VEGF genotype and haplotype analysis revealed no association between any variants and the risk of ALS. Regarding ANG gene, no mutation was detected and the rs11701 polymorphism, previously described as associated with ALS, was not differently distributed between patients and controls. Overall, our data argue against the hypothesis of both genes as risk factors for motoneuron neurodegeneration, at least in an Italian population.

Topics: Amyotrophic Lateral Sclerosis; Humans; Italy; Mutation; Polymorphism, Genetic; Ribonuclease, Pancreatic; Vascular Endothelial Growth Factor A

Mutations in the Angiogenin gene (ANG) linked to 14q11.2 have been recently discovered to be associated with Amyotrophic Lateral Sclerosis (ALS) in Irish and Scottish populations. In our study we investigated the role of ANG gene in ALS patients from southern Italy. We found a novel mutation in the signal peptide of the ANG gene in a sporadic patient with ALS (SALS). The molecular analysis of the ANG gene also demonstrated an allelic association with the rs11701 single nucleotide polymorphism (SNP) in familial ALS (FALS) but not in SALS patients. Our finding supports the evidence that the ANG gene is involved in ALS.

Topics: Adult; Aged; Amino Acid Substitution; Amyotrophic Lateral Sclerosis; Chromosome Mapping; Chromosomes, Human, Pair 14; Cytoprotection; DNA Mutational Analysis; Female; Genetic Linkage; Genetic Markers; Genetic Predisposition to Disease; Genetic Testing; Humans; Italy; Male; Middle Aged; Motor Neurons; Mutation; Nerve Degeneration; Polymorphism, Single Nucleotide; Ribonuclease, Pancreatic

Amyotrophic lateral sclerosis (ALS) is a late onset neurodegenerative disorder affecting upper and lower motor neurons (MNs). The molecular mechanisms underlying ALS are poorly understood. Mutations in SOD1 is one of the known causes of ALS but occur only in a very small number of cases of ALS. Interestingly, mutations in human angiogenin (hANG), a member of the ribonuclease A (RNase A) superfamily known to be involved in neovascularization, have been recently reported in patients with ALS, but the effects of these mutations on MN differentiation and survival has not been investigated. We have used the well-characterized pluripotent P19 embryonal carcinoma (EC) cell culture model of neuro-ectodermal differentiation to study the effects of hANG-ALS variants on MN differentiation and survival. Here we report that P19 EC cells induced to differentiate in the presence of hANG and hANG-ALS-associated variants internalize the wild-type and variant proteins. The P19 EC cells differentiate to form neurons but the ability of the neurites to extend and make contacts with neighbouring neurites is compromised when treated with the hANG-ALS variants. In addition, hANG-ALS variants also have a cytotoxic effect on MNs leading to their degeneration. hANG was able to protect neurons from hypoxia-induced cell death, but the variants of hANG implicated in ALS lacked the neuroprotective activity. Our findings show that ANG plays an important role in neurite extension/pathfinding and survival providing a causal link between mutations in hANG and ALS.

Topics: Amino Acid Sequence; Amyotrophic Lateral Sclerosis; Animals; Cell Differentiation; Cell Line; Cell Survival; Coculture Techniques; Genetic Variation; Humans; Mice; Models, Molecular; Molecular Sequence Data; Motor Neurons; Mutation; Neurites; Neuroprotective Agents; Recombinant Proteins; Ribonuclease, Pancreatic

Angiogenin (ANG) gene, coding for an angiogenic factor up-regulated by hypoxia and expressed in ventral horn motor neurons, is a novel candidate for the pathogenesis of amyotrophic lateral sclerosis (ALS). ALS is a fatal neurodegenerative disease characterized by the selective loss of cortical and spinal motor neurons. Missense mutations in ANG gene have been identified in two ALS populations from Northern Europe and North America, both in familial (FALS) and sporadic (SALS) patients, but they do not seem to be frequent in the Italian population. We performed a mutational screening in a large cohort of 737 Italian ALS patients, including 605 SALS and 132 FALS cases. We identified seven different mutations, five of which are novel, in nine patients (six SALS and three FALS), but not in 515 healthy controls. Three mutations are located in the signal peptide region, three in the coding sequence, and one in the 3' untranslated region. In our ALS population, the observed mutational frequency of ANG gene accounts for about 1.2%, with an overrepresentation of FALS (2.3%) compared to SALS (1%) cases. We also found the previously described I46V substitution in six patients and four controls, suggesting that this mutation may represent a benign variant, at least in the Italian population. Our results provide further evidence of a tight link between angiogenesis and ALS pathogenesis and suggest that mutations in ANG gene are associated with an increased risk to develop ALS.

Topics: 3' Untranslated Regions; Adult; Aged; Alleles; Amino Acid Substitution; Amyotrophic Lateral Sclerosis; Base Sequence; Case-Control Studies; Cohort Studies; DNA; DNA Mutational Analysis; Female; Gene Frequency; Humans; Italy; Male; Middle Aged; Models, Molecular; Mutation; Open Reading Frames; Pedigree; Polymorphism, Single Nucleotide; Protein Conformation; Protein Sorting Signals; Ribonuclease, Pancreatic

Potentially causative missense variations in the ANG gene and a positive association with the synonymous rs11701-G substitution was detected mainly in Irish and Scottish ALS patients. We screened 262 Italian SOD1 negative ALS patients (250 sporadic) and 415 matched controls for sequence variations in the coding, 3'/5' UTR and 5' flanking (642 bp) regions of the ANG gene. We identified 53 sequence variations of which 46 new, 20 with a minor allele frequency (MAF) >or=0.01 and only three localised in the coding sequence, namely the missense I46V, identified in one patient and two controls, and the synonymous G86G and T97T corresponding to rs11701 and rs2228653. None of the detected SNPs or of their haplotypic combinations was significantly associated with ALS susceptibility or clinical features. In conclusion, we did not detect the association with rs11701-G or with any other newly detected variation in the ANG regulatory region. Furthermore we did not identify potentially causal mutations in the coding region.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Amyotrophic Lateral Sclerosis; Disease Susceptibility; DNA Mutational Analysis; Female; Gene Frequency; Genetic Variation; Humans; Italy; Male; Middle Aged; Promoter Regions, Genetic; Ribonuclease, Pancreatic

Mutations in human angiogenin (hANG), an angiogenic member of the RNase A superfamily, have been recently reported in patients with amyotrophic lateral sclerosis (ALS), a progressive late-onset neurodegenerative disorder. However, very little is known about the expression and subcellular distribution of ANG in the nervous system or its role in differentiation. Here we report that mouse angiogenin-1 (mAng-1) is strongly expressed in the developing nervous system during mouse embryogenesis and neuroectodermal differentiation of pluripotent P19 embryonal carcinoma cells. mAng1 is strongly expressed in motor neurons (MNs) in the spinal cord and dorsal root ganglia as well as in post-mitotic MNs derived from P19 cells. We also show for the first time that ANG expression is in the growth cones and neurites. NCI 65828, an inhibitor of the ribonucleolytic activity of hANG, affected pathfinding by P19-derived neurons but not neuronal differentiation. Our findings clearly show that ANG plays an important role in neurite pathfinding and this has implications for ALS.

Topics: Amyotrophic Lateral Sclerosis; Animals; Carcinoma, Embryonal; Cell Differentiation; Embryo, Mammalian; Female; Fluorescent Antibody Technique; Male; Mice; Mice, Inbred C57BL; Naphthalenesulfonates; Neurites; Ribonuclease, Pancreatic; Tretinoin; Tumor Cells, Cultured

Heterozygous missense mutations in the coding region of angiogenin (ANG), an angiogenic ribonuclease, have been reported in amyotrophic lateral sclerosis (ALS) patients. However, the role of ANG in motor neuron physiology and the functional consequences of these mutations are unknown. We searched for new mutations and sought to define the functional consequences of these mutations.. We sequenced the coding region of ANG in an independent cohort of North American ALS patients. Identified ANG mutations were then characterized using functional assays of angiogenesis, ribonucleolysis, and nuclear translocation. We also examined expression of ANG in normal human fetal and adult spinal cords.. We identified four mutations in the coding region of ANG from 298 ALS patients. Three of these mutations are present in the mature protein. Among the four mutations, P(-4)S, S28N, and P112L are novel, and K17I has been reported previously. Functional assays show that these ANG mutations result in complete loss of function. The mutant ANG proteins are unable to induce angiogenesis because of a deficiency in ribonuclease activity, nuclear translocation, or both. As a correlate, we demonstrate strong ANG expression in both endothelial cells and motor neurons of normal human spinal cords from the developing fetus and adult.. We provide the first evidence that ANG mutations, identified in ALS patients, are associated with functional loss of ANG activity. Moreover, strong ANG expression, in normal human fetal and adult spinal cord neurons and endothelial cells, confirms the plausibility of ANG dysfunction being relevant to the pathogenesis of ALS.

Topics: Active Transport, Cell Nucleus; Adult; Aged, 80 and over; Amyotrophic Lateral Sclerosis; Cell Nucleus; Cohort Studies; Endothelial Cells; Female; Fetus; Heterozygote; Humans; Male; Middle Aged; Motor Neurons; Mutation; Mutation, Missense; Neovascularization, Physiologic; Ribonuclease, Pancreatic; Ribonucleases; Spinal Cord

Human angiogenin (ANG), the first member of the angiogenin family (from the pancreatic ribonuclease A superfamily) to be identified, is an angiogenic factor that induces neovascularization. It has received much attention due to its involvement in the growth of tumors and its elevated expression level in pancreatic and several other cancers. Recently the biological role of ANG has been shown to extend to the nervous system. Mutations in ANG have been linked with familial as well as sporadic forms of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder characterized by selective destruction of motor neurons. Furthermore, mouse angiogenin-1 has been shown to be expressed in the developing nervous system and during the neuronal differentiation of pluripotent stem cells. We have now characterized the seven variants of ANG reported in ALS patients with respect to the known biochemical properties of ANG and further studied the biological properties of three of these variants. Our results show that the ribonucleolytic activity of six of the seven ANG-ALS implicated variants is significantly reduced or lost and some variants also show altered thermal stability. We report a significant reduction in the cell proliferative and angiogenic activities of the three variants that we chose to investigate further. Our studies on the biochemical and structural features of these ANG variants now form the basis for further investigations to determine their role(s) in ALS.

Topics: Amyotrophic Lateral Sclerosis; Cell Line, Tumor; Cell Proliferation; Chromatography, High Pressure Liquid; Circular Dichroism; Enzyme Stability; Formazans; Genetic Variation; Humans; Melanoma; Models, Molecular; Molecular Weight; Mutation; Protein Denaturation; Protein Engineering; Protein Renaturation; Ribonuclease, Pancreatic; Temperature; Tetrazolium Salts

We recently identified angiogenin (ANG) as a candidate susceptibility gene for amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder characterized by adult-onset loss of motor neurons. We now report the finding of seven missense mutations in 15 individuals, of whom four had familial ALS and 11 apparently 'sporadic' ALS. Our findings provide further evidence that variations in hypoxia-inducible genes have an important role in motor neuron degeneration.

Topics: Adult; Aged; Aged, 80 and over; Amyotrophic Lateral Sclerosis; Female; Humans; Male; Middle Aged; Models, Molecular; Mutation, Missense; Pedigree; Protein Conformation; Ribonuclease, Pancreatic

A new study by Greenway and colleagues links mutations in the angiogenin gene to patients with amyotrophic lateral sclerosis (ALS)--a progressive and fatal motoneuron disease. This is an unexpected finding because angiogenin was originally identified as a molecule involved in the formation of blood vessels (angiogenesis). Angiogenin bears striking similarity to vascular endothelial growth factor (VEGF), which is the prototypic angiogenic factor that has recently emerged as a molecule with important neuroprotective activities. Besides VEGF, angiogenin is the second so-called angiogenic factor implicated in ALS, raising the question of whether additional angiogenic factors might have a role in ALS. Overall, these findings identify angiogenin as a novel candidate gene in the pathogenesis of ALS--a discovery that ultimately might lead to the development of new therapeutic strategies.

Topics: Amyotrophic Lateral Sclerosis; Genetic Predisposition to Disease; Humans; Models, Biological; Ribonuclease, Pancreatic; Vascular Endothelial Growth Factor A

The role of hypoxia responsive genes in the pathogenesis of ALS was first suggested when deletions of the hypoxia-responsive element of vascular endothelial growth factor (VEGF) promoter caused a motor neuron disease phenotype in mice. The discovery of ALS-associated mutations in ANG, a hypoxia responsive gene coding for the protein angiogenin, has further supported this pathogenic mechanism in human ALS. In endothelium, angiogenin can regulate expression of VEGF. To date, the patterns of serum angiogenin expression among patients with ALS have not been assessed.. Serum angiogenin and VEGF levels were quantified at diagnosis in 79 patients with definite or probable ALS and 72 healthy controls, using a quantitative sandwich enzyme-linked immunoassay.. Patients with ALS exhibited higher serum angiogenin (p = 0.006) but not VEGF (p = 0.55) levels than matched control subjects. Subgroup analysis showed a greater elevation in angiogenin levels for spinal- (p < 0.001) than bulbar- (p = 0.11) onset ALS vs controls. At 12 months, angiogenin levels remained elevated. No correlation was noted between angiogenin and VEGF levels (r = -0.08, p = 0.49) in ALS patient serum.. These data suggest a modest elevation in serum angiogenin in ALS at diagnosis. Further investigation will be required to assess the utility of serum angiogenin as a biomarker for ALS and as a predictor of disease progression.

Topics: Amyotrophic Lateral Sclerosis; Biomarkers; Female; Humans; Hypoxia; Male; Microcirculation; Neovascularization, Physiologic; Predictive Value of Tests; Ribonuclease, Pancreatic; Spinal Cord; Survival Rate; Up-Regulation; Vascular Endothelial Growth Factor A

Sequence variations with biologic effect in ALS have been identified in the gene for vascular endothelial growth factor (VEGF). The gene for a related protein, angiogenin, lies on chromosome 14q11.2. Analysis of the angiogenin (ANG) gene in the authors' population has demonstrated a significant allelic association with the rs11701 single nucleotide polymorphism (SNP) and identified a novel mutation in two individuals with sporadic ALS that potentially inhibits angiogenin function. These observations propose a candidate region for ALS on chromosome 14q11.2 and suggest that other genes with similar function to VEGF may be important in the pathogenesis of ALS.

Topics: Aged; Alleles; Amino Acid Substitution; Amyotrophic Lateral Sclerosis; Chromosome Mapping; Chromosomes, Human, Pair 14; Codon; Cohort Studies; DNA-(Apurinic or Apyrimidinic Site) Lyase; Female; Gene Frequency; Genetic Predisposition to Disease; Humans; Ireland; Male; Middle Aged; Mutation, Missense; Point Mutation; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Ribonuclease, Pancreatic; Vascular Endothelial Growth Factor A