Human angiogenin is a neuroprotective factor and amyotrophic lateral sclerosis associated angiogenin variants affect neurite extension/pathfinding and survival of motor neurons.
ABSTRACT 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.
Article: Mechanisms of loss of functions of human angiogenin variants implicated in amyotrophic lateral sclerosis.[show abstract] [hide abstract]
ABSTRACT: 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.PLoS ONE 01/2012; 7(2):e32479. · 4.09 Impact Factor
Article: Mechanisms of loss of functions of human angiogenin variants implicated in amyotrophic lateral sclerosisPLoS ONE 02/2012; · 4.09 Impact Factor
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ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with a substantial heritable component. In pedigrees affected by its familial form, incomplete penetrance is often observed. We hypothesized that this could be caused by a complex inheritance of risk variants in multiple genes. Therefore, we screened 111 familial ALS (FALS) patients from 97 families, and large cohorts of sporadic ALS (SALS) patients and control subjects for mutations in TAR DNA-binding protein (TARDBP), fused in sarcoma/translated in liposarcoma (FUS/TLS), superoxide dismutase-1 (SOD1), angiogenin (ANG) and chromosome 9 open reading frame 72 (C9orf72). Mutations were identified in 48% of FALS families, 8% of SALS patients and 0.5% of control subjects. In five of the FALS families, we identified multiple mutations in ALS-associated genes. We detected FUS/TLS and TARDBP mutations in combination with ANG mutations, and C9orf72 repeat expansions with TARDBP, SOD1 and FUS/TLS mutations. Statistical analysis demonstrated that the presence of multiple mutations in FALS is in excess of what is to be expected by chance (P = 1.57 × 10(-7)). The most compelling evidence for an oligogenic basis was found in individuals with a p.N352S mutation in TARDBP, detected in five FALS families and three apparently SALS patients. Genealogical and haplotype analyses revealed that these individuals shared a common ancestor. We obtained DNA of 14 patients with this TARDBP mutation, 50% of whom had an additional mutation (ANG, C9orf72 or homozygous TARDBP). Hereby, we provide evidence for an oligogenic aetiology of ALS. This may have important implications for the interpretation of whole exome/genome experiments designed to identify new ALS-associated genes and for genetic counselling, especially of unaffected family members.Human Molecular Genetics 05/2012; 21(17):3776-84. · 7.64 Impact Factor