Control of motoneuron survival by angiogenin

Abstract

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.

Figure 1.

Angiogenin is a neuroprotective factor for cultured motoneurons. A, Direct counts of motoneuron survival in primary ventral horn motoneuron cultures exposed to AMPA (50 μm, 24 h), AMPA and angiogenin (100 ng/ml), angiogenin alone and AMPA and heat denatured angiogenin (*p ≤ 0.05, mean ± SEM, n = 12). B, Direct counts of motoneuron survival in primary motoneuron cultures exposed to tunicamycin (500 nm, 24 h) and/or angiogenin (100 ng/ml, *p ≤ 0.05, mean ± SEM, n = 12). C–E, Western blot analysis of Akt-1 and Ser473-phosphorylated Akt-1 (p-Akt) expression in angiogenin- and IGF-I-treated (C, D), and angiogenin-plus AMPA-treated (E) primary motoneuron cultures. F, Inhibition of the PI-3-K pathway using wortmannin (100 nm, 24 h) reduces the neuroprotective effect of angiogenin in primary motoneuron cultures exposed to AMPA (*p ≤ 0.05, mean ± SEM, n = 3).

Figure 2.

Lack of activity of the ALS-associated ANG mutants and potentiation of stress-induced cell injury by ANG knockdown. A, Western blot (two top panels) and quantitative real-time PCR analysis (two bottom panels) showing human ANG levels in NSC34 cells transiently transfected with pIRES-DsRed2 constructs alone, or containing either ANG, or K40I mutant ANG. B, NSC34 cells transiently transfected with pIRES-DsRed2/ANG are more resistant to tunicamycin-induced ER stress (500 nm, 24 h) than cells transfected with K40I ANG or with empty vector. Apoptotic cell death was assessed after Hoechst 33258 staining. (*p < 0.01 vs treated empty vector, mean ± SEM, n = 7–10). C, Wild-type recombinant angiogenin protein protects NSC34 cells from serum deprivation-induced toxicity (24 h), whereas mutant K40I angiogenin protein shows no protective effect (*p < 0.01, mean ± SEM, n = 7–10). D, Wild-type angiogenin protein, but not K40I mutant angiogenin protein, elevated the levels of pAkt-1 in NSC34 cells during trophic factor withdrawal.

Figure 3.

Knock-down of endogenous angiogenin potentiates stress-induced cell death in motoneurons in vitro. A, Western blot analysis demonstrating efficient knock-down of murine ANG1 in NSC34 cells with siRNAs 48 h after transfection. B, Apoptotic cell death of primary motoneuron cultures transfected with murine ANG1 siRNA and treated with AMPA was assessed after Hoechst 33258 staining. (*p < 0.01 vs nontransfected sister cultures, mean ± SEM, n = 7–10).

Figure 4.

Angiogenin treatment after symptom onset in SOD1^G93A mice delays disease progression and extends lifespan. A, Kaplan–Meyer analysis of probability of survival. Treatment from 50 d, angiogenin-treated SOD1 mice survived for 135 d (±1.5, n = 12), whereas vehicle-treated SOD1 mice survived for only 122.8 d (±2.2, n = 15, p < 0.0001). Treatment from 90 d, angiogenin-treated SOD1 mice survived for 132.8 d (±1.8, n = 12), whereas vehicle-treated SOD1 mice survived for only 122.2 d (±2.1, n = 15, p = 0.0005). B, Stride length analysis in the identical treatment paradigms (*p ≤ 0.05, mean ± SEM). C, Quantitative analysis of sciatic motoneuron survival (*p ≤ 0.05, mean ± SEM, n = 6). D, Western blot analysis of Akt-1, p-Akt, ERK1/2 and p-ERK1/2 in spinal cord homogenate of wild-type and untreated SOD1^G93A mice. E, Western blot analysis of Akt, p-Akt, ERK1/2 and p-ERK1/2 in spinal cord homogenate from wild-type littermates, untreated SOD1^G93A mice, SOD1^G93A mice treated with angiogenin from 50 or 90 d. F, Western blot analysis of ICAM-1 in spinal cord homogenate from wild-type littermates, untreated SOD1^G93A mice, and SOD1^G93A mice treated with angiogenin from 50 d.