The unfolded protein response in familial amyotrophic lateral sclerosis

Abstract

Mutant superoxide dismutase type 1 (MTSOD1) is thought to cause ∼20% of cases of familial amyotrophic lateral sclerosis (FALS) because it misfolds and aggregates. Previous studies have shown that MTSOD1 accumulates inside the endoplasmic reticulum (ER) and activates the unfolded protein response (UPR), suggesting that ER stress is involved in the pathogenesis of FALS. We used a genetic approach to investigate the role of the UPR in FALS. We crossed G85RSOD1 transgenic mice with pancreatic ER kinase haploinsufficient (PERK(+/-)) mice to obtain G85R/PERK(+/-) mice. PERK(+/-) mice carry a loss of function mutation of PERK, which is the most rapidly activated UPR pathway, but have no abnormal phenotype. Compared with G85R transgenic mice, G85R/PERK(+/-) mice had a dramatically accelerated disease onset as well as shortened disease duration and lifespan. There was also acceleration of the pathology and earlier MTSOD1 aggregation. A diminished PERK response accelerated disease and pathology in G85R transgenic mice presumably because the mice had a reduced capacity to turn down synthesis of misfolded SOD1, leading to an early overloading of the UPR. The results indicate that the UPR has a significant influence on FALS, and suggest that enhancing the UPR may be effective in treating ALS.

Figure 1.

Plots of disease onset (A), the end of early disease (B) and survival (C) as well as diagrams of the duration (with standard deviation) of the early phase (D) and the late phase (E) of disease in G85R/Perk^+/− versus G85R transgenic mice. In the Y-axis of (A)–(C), 1 refers to the total number of animals, which is detailed in the upper right. The asterisk indicates P < 0.05.

Figure 2.

Neuropathological and immunohistochemical studies of the anterior horn of the lumbar spinal cord of G85R/Perk^+/−, G85R and Perk^+/− transgenic mice of varying ages. Sections of the anterior horn of the spinal cord were stained with (A) Nissl for MNs, (B) anti-GFAP antibody for astrocytes, (C) anti-Iba1 antibody for microglia. In this figure and others: Pre, presymptomatic; End, end stage. Scale bar: 50 µm. Bar diagrams showing the mean ± standard deviation from quantitation of cell types in the lumbar anterior horn of G85R/Perk^+/− versus G85R mice: (D) MNs; (E) astrocytes; (F) microglia; *P < 0.05, **P < 0.01.

Figure 3.

Studies of SOD1 aggregation and accumulation. (A) Immunohistochemical studies of the lumbar anterior horn of G85R/Perk^+/−, G85R and Perk^+/− transgenic mice of varying ages. (B) Representative western blot of lumbar spinal cord homogenates from varied aged G85R/Perk^+/−, G85R and control mice (Perk^+/−, WTSOD1/Perk^+/− transgenic mice and non-transgenic littermates) subjected to SDS–PAGE under non-reducing conditions. The spinal cord homogenates were immunostained with anti-human SOD1 antibody. The monomeric and dimeric forms of SOD1 are noted with arrows. Note that the monomeric form of G85R MTSOD1 has a different electrophoretic mobility than WTSOD1, and that high molecular weight forms above the dimeric species are present in both G85R/Perk^+/− and G85R mice, but they are present at an earlier time in the G85R/Perk^+/− mice. Anti-β-tubulin antibody was used as a loading control. In this and subsequent figures: Pre, presymptomatic; Wk, weak; End, end stage. (C) Representative western blot of lumbar spinal cord homogenates from varied aged G85R/Perk^+/− and G85R mice subjected to SDS–PAGE under reducing conditions. (D) Quantitation of total MTSOD1 in the spinal cord homogenates from varied aged G85R/Perk^+/− and G85R mice as detected in western blots of samples electrophoresed under reducing conditions. The mean amount of MTSOD1 in the spinal cord of G85R mice at ∼220 days was arbitrarily given a value of 100. Note that there is more total MTSOD1 in G85R/Perk^+/− compared with G85R mice at 260 and 280 days; no values for G85R/Perk^+/− mice are provided after 280 days because they are dead. **P < 0.01.

Figure 4.

Quantitation and detection of markers of the UPR and apoptosis in varied aged G85R/Perk^+/−, G85R and control mice (Perk^+/−, WTSOD1/Perk^+/− transgenic mice and non-transgenic littermates) by western blots of lumbar spinal cord homogenates. (A) Representative western blots immunostained with antibodies to the following: PERK, BiP, phosphorylated(P)-eIF2α, ATF4, CHOP, ATF6, XBP-1, activated caspase-12. Anti-β-tubulin antibody was used as a loading control. The level of the individual immunostained protein at the end stage in the case of G85R was arbitrarily given a value of 100. (B) Quantitation of UPR markers on western blots. *P < 0.05, **P < 0.01.