Article Figures & Data
Figures
- 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 SOD1G93A 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 SOD1G93A mice. E, Western blot analysis of Akt, p-Akt, ERK1/2 and p-ERK1/2 in spinal cord homogenate from wild-type littermates, untreated SOD1G93A mice, SOD1G93A 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 SOD1G93A mice, and SOD1G93A mice treated with angiogenin from 50 d.
Additional Files
Supplemental Data
Files in this Data Supplement:
- supplemental material - Supplemental Material
- supplemental material - Supplemental Table
- supplemental material - Supplemental Figure 1
- supplemental material - Supplemental Figure 2
- supplemental material - Supplemental Figure 3
- supplemental material - Supplemental Figure 4
