Deletion of the BH3-only protein puma protects motoneurons from ER stress-induced apoptosis and delays motoneuron loss in ALS mice

Abstract

BH3-only proteins couple diverse stress signals to the evolutionarily conserved mitochondrial apoptosis pathway. Previously, we reported that the activation of the BH3-only protein p53-up-regulated mediator of apoptosis (Puma) was necessary and sufficient for endoplasmic reticulum (ER) stress- and proteasome inhibition-induced apoptosis in neuroblastoma and other cancer cells. Defects in protein quality control have also been suggested to be a key event in ALS, a fatal neurodegenerative condition characterized by motoneuron degeneration. Using the SOD1(G93A) mouse model as well as human post mortem samples from ALS patients, we show evidence for increased ER stress and defects in protein degradation in motoneurons during disease progression. Before symptom onset, we detected a significant up-regulation of Puma in motoneurons of SOD1(G93A) mice. Genetic deletion of puma significantly improved motoneuron survival and delayed disease onset and motor dysfunction in SOD1(G93A) mice. However, it had no significant effect on lifespan, suggesting that other ER stress-related cell-death proteins or other factors, such as excitotoxicity, necrosis, or inflammatory injury, may contribute at later disease stages. Indeed, further experiments using cultured motoneurons revealed that genetic deletion of puma protected motoneurons against ER stress-induced apoptosis but showed no effect against excitotoxic injury. These findings demonstrate that a single BH3-only protein, the ER stress-associated protein Puma, plays an important role during the early stages of chronic neurodegeneration in vivo.

Fig. 1.

Protein degradation defects in SOD1^G93A mice and ALS patients. (A) Immunocytochemistry in spinal cord sections for KDEL, which recognizes both Grp78 and Grp94. (Scale bar: 75 μm.) (B) Western blot for Grp78 expression. (C) Immunocytochemistry in spinal cord cross-sections for ubiquitin. (Scale bar: 100 μm.) (D) Western blot for ubiquitin expression with molecular mass marker. (E and F) Post mortem spinal cord cross-sections showing anterior horn motoneurons immunostained with antibodies to KDEL (brown) (E) or ubiquitin (brown) (F) both counterstained with haematoxylin (blue). (Scale bars: E, 0.5 mm; F, 0.3 mm)

Fig. 2.

ER-stress in SOD1^G93A mice. (A) Western blot analysis of CHOP, Puma, and Bim expression. (B–D) Real-time quantitative PCR analysis of CHOP mRNA (B), Bim mRNA (C), and Puma mRNA (D). n = 3. Error bars are ± SEM. (E) Immunocytochemistry in spinal cord sections at 70 days for Puma (green) and the nuclear stain DAPI (blue). (Scale bar: 50 μm.)

Fig. 3.

Lifespan analysis in cross-bred mice. (A) Immunocytochemistry on spinal cord sections for human SOD1 (green) and the nuclear stain DAPI (blue). (Scale bar: 100 μm.) Western blot analysis of human SOD1 expression in cross-bred mice. (B) Western blot analysis of Bim and Bid expression in cross-bred mice. n.s. indicates a nonspecific band showing equal sample loading. (C) Kaplan–Meyer analysis of survival.

Fig. 4.

Assessment of disease progression. (A) Body weight of mice during disease progression. (B) Analysis of PaGE test performance during disease progression (expressed as a percentage of maximum performance at 10 weeks). (C) Stride-length analysis. (All error bars are ± SEM. n = 10–15 in each group. *, P ≤ 0.05).

Fig. 5.

Motoneuron survival and histology. (A) Motoneuron survival in the sciatic motor pool. n = 7–9. *, P ≤ 0.05. (B and C) Immunocytochemistry in lumbar spinal cord cross-sections for the astrocyte marker GFAP (green) and the nuclar stain DAPI (blue) (B) and the microglial marker CD11b (green) and the nuclar stain DAPI (blue) (C). (Scale bars, 150 μm.)

Fig. 6.

Puma^−/− motoneurons in vitro. (A) MTT reduction after tunicamycin (tuni) treatment. n = 3. (B) MTT reduction after AMPA treatment (50 μM). n = 3. (C) Photomicrograhps of SMI-32- (green) and DAPI- (blue) immunostained primary motoneuron cultures after AMPA treatment. (Scale bar: 75 μm.) (D and E) Direct counts of motoneuron survival after tunicamycin (D) and AMPA (E) treatment. n = 3. (F) Photomicrograhps of SMI-32- (green) and DAPI- (blue) immunostained primary motoneuron cultures after tunicamycin treatment. (Scale bar: 100 μm.) (G and H) Direct counts of caspase 9-positive motoneurons in cultures after tunicamycin (G) and AMPA (H) treatment. All Error bars are ± SEM. *, P ≤ 0.05.