Angiogenin loss-of-function mutations in amyotrophic lateral sclerosis

Abstract

Objective: 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.

Methods: 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.

Results: 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.

Interpretation: 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.

Fig 1

Angiogenin (ANG) mutations identified in Northern American amyotrophic lateral sclerosis (ALS) patients. (A) DNA sequence traces of mutations identified by bidirectional sequencing. Mutations are indicated using single-letter amino acid code. (B) Amino acid sequence of ANG with the signal peptide underlined. Mutations identified in this study, P(-4)S, K17I, S28N, and P112L, are shown in purple. The RNase catalytic residues (H13, K40, and H114) are shown in red. The nuclear localization signal ( RRRGL35) is shown in green. (C) Crystal structure of ANG (from www.rcsb.org; 1ANG) showing the positions of mutated residues (purple). The catalytic triad is shown in red.

Fig 2

Angiogenic activity of wild-type (WT) and mutant angiogenin (ANG) proteins. Recombinant WT and mutant ANG proteins were expressed and purified. (A) Sodium dode-cyl sulfate polyacrylamide gel electrophoresis and Coomassie blue staining. Five micrograms of the proteins were loaded in each lane. (B–E) Endothelial cell tube formation assay. Human umbilical vein endothelial cells (HUVECs) were cultured on fibrin gels in the presence of (B) WT, (C) K17I, (D) S28N, and (E) P112L. Formation of tubular structures was evaluated using a phase-contrast microscope. Pictures shown are from a representative experiment of five independent repeats. Original magnification ×40.

Fig 3

Ribonucleolytic activity of wild-type (WT) and mutant angiogenin (ANG) proteins. RNase activity was measured with yeast transfer RNA (tRNA) as the substrate. Increasing concentration of WT and mutant ANG proteins were incubated with yeast tRNA (2mg/ml) at 37°C for 2 hours. Undigested tRNA was precipitated by perchloric acid. (A) Absorbance of the supernatants at 260nm. (B) Relative RNase activity of mutant ANG as compared with that of WT ANG (100%). The amount of enzyme required to generate 0.1 optical density (OD) is compared with that of WT ANG to generate the same OD unit. Student’s t test of four independent experiments shows that the difference between WT and each of the three mutant proteins is significant (n = 4; p < 0.001).

Fig 4

Nuclear translocation of wild-type (WT) and mutant angiogenin (ANG) proteins. Human umbilical vein endothelial cells (HUVECs) were incubated with 1μg/ml of (A, E, I) WT, (B, F, J) K17I, (C, G, K) S28N, and (D, H, L) P112L ANG at 37°C for 30 minutes and fixed with −20°C methanol. ANG was visualized by immunofluorescence with the anti-ANG monoclonal antibody 26-2F and Alexa 488 –labeled goat F(ab′)₂ anti–mouse IgG (A–D). (E–H) 46′-diamidino-2-phenylindole-2 HCl staining of the cell nuclei. (I–L) Merge of the green and blue fluorescence. Images are from a representative experiment of five independent repeats. Original magnification ×600. BSA = bovine serum albumin.

Fig 5

Immunohistochemical staining of angiogenin (ANG) in fetal and adult human spinal cords. Spinal cords of (A) 15-, (B) 18-, (C) 21-, (D) 25-, and (E) 30-week-old fetuses and of an (F) adult were collected, fixed in formalin, and embedded in paraffin. Sections of 4μM were cut and stained immunohistochemically for ANG with 26-2F. Images are from the ventral horn area of the spinal cords where motor neurons are located. Arrows denote ANG staining in motor neurons. Original magnification ×100. Insets are the high-magnification (A–E, ×400; F, ×200) images of the motor neuron and its surroundings.

Fig 6

Angiogenin (ANG) expression in motor neurons and blood vessels of fetal and adult human spinal cords. (A–F) Green immunofluorescence for ANG with 26-2F and Alexa 488 –labeled goat anti–mouse IgG. Arrows indicate representative ANG staining in motor neurons. (G–L) Red immunofluorescence for blood vessels with anti–von Willebrand factor polyclonal antibody and Alexa 555–labeled goat anti–rabbit IgG. Arrowheads indicate representative blood vessels. (M–R) Merge of green and red fluorescence. Arrowheads indicate colocalization of ANG and von Willebrand factor. Images are from a representative area of the ventral horns of spinal cords of (A, G, M) 15-, (B, H, N) 18-, (C, I, O) 21-, (D, J, P) 25-, and (E, K, Q) 30-week-old fetuses and an (F, L, R) adult. Original magnification ×100.