Molecular dissection of ALS-associated toxicity of SOD1 in transgenic mice using an exon-fusion approach

Han-Xiang Deng, Hujun Jiang, Ronggen Fu, Hong Zhai, Yong Shi, Erdong Liu, Makito Hirano, Mauro C Dal Canto, Teepu Siddique, Han-Xiang Deng, Hujun Jiang, Ronggen Fu, Hong Zhai, Yong Shi, Erdong Liu, Makito Hirano, Mauro C Dal Canto, Teepu Siddique

Abstract

Mutations in Cu,Zn superoxide dismutase (SOD1) are associated with amyotrophic lateral sclerosis (ALS). Among more than 100 ALS-associated SOD1 mutations, premature termination codon (PTC) mutations exclusively occur in exon 5, the last exon of SOD1. The molecular basis of ALS-associated toxicity of the mutant SOD1 is not fully understood. Here, we show that nonsense-mediated mRNA decay (NMD) underlies clearance of mutant mRNA with a PTC in the non-terminal exons. To further define the crucial ALS-associated SOD1 fragments, we designed and tested an exon-fusion approach using an artificial transgene SOD1(T116X) that harbors a PTC in exon 4. We found that the SOD1(T116X) transgene with a fused exon could escape NMD in cellular models. We generated a transgenic mouse model that overexpresses SOD1(T116X). This mouse model developed ALS-like phenotype and pathology. Thus, our data have demonstrated that a 'mini-SOD1' of only 115 amino acids is sufficient to cause ALS. This is the smallest ALS-causing SOD1 molecule currently defined. This proof of principle result suggests that the exon-fusion approach may have potential not only to further define a shorter ALS-associated SOD1 fragment, thus providing a molecular target for designing rational therapy, but also to dissect toxicities of other proteins encoded by genes of multiple exons through a 'gain of function' mechanism.

Figures

Figure 1.
Figure 1.
NMD underlying removal of mutant SOD1 mRNA with PTCs in exons 3 and 4. (A) Southern blot showing that multiple copies of hSOD1E77X or hSOD1K91X transgene were integrated into mouse genome in the transgenic mice. Lane 2, human genomic DNA with two copies of SOD1 gene as a control; lanes 1 and 3–7, DNA from transgenic mice harboring transgene hSOD1E77X; lanes 8–10, DNA from transgenic mice harboring transgene hSOD1K91X. (B) The northern blot analysis of SOD1E77X and SOD1K91X transgenic mouse lines compared with established ALS mouse models of mutant SOD1. (Top panel) mRNA expression of human SOD1 transgenes detected by human SOD1-specific probe. (Low panel) mRNA expression of mouse endogenous SOD1 detected by mouse SOD1-specific probe as an internal control. Lane 1, SOD1G93A transgenic mice that develop disease around 100 days. Lane 2, wild-type mouse (non-transgenic) as a control. Lane 3, SOD1L126Z transgenic mice that develop disease around 350 days. Lanes 4–7, SOD1E77X transgenic mice. Lane 8, SOD1K91X transgenic mice. (C) Representative sequence electropherograms showing NMD of SOD1E77X. Plasmid DNA containing cDNA of SOD1 with SOD1E77X mutation was mixed with plasmid containing wild-type cDNA of SOD1 at 10 different ratios by amount, i.e. from 0.1 to 1.0 (mutant/wild-type). Mixed DNA samples were used as templates for PCR-sequencing. The relative ratio of the peak height (T/G) was established by measuring the individual peak heights representing SOD1E77X (T) and wtSOD1 (G). The known molecular ratios (0.1–1.0) of the mutant/wild-type are labeled on the top of the sequence electropherograms of the sub-panels (A–j). Sub-panel (k) shows a sequence electropherogram of a stop codon (TAA) at codon 77, and (l) shows wtSOD1 at codon 77 when a single type of DNA was used. Sub-panels (m and n) show the sequence electropherograms of RT-PCR product of human SOD1 from NIH/3T3 cells transfected with equal molar ratio of SOD1E77X/wtSOD1 plasmids; CT, cycloheximide-treated; NT, not treated with cycloheximide. Sub-panels (o) and (p) show the sequence electropherograms of RT–PCR product of human SOD1 from another cell line (NSC34) transfected with equal molar ratio of SOD1E77X/wtSOD1 plasmids. (D) A standard curve showing ratios of peak height (T/G) against known molecular ratios (SOD1E77X/wtSOD1). Cyc- (+), cycloheximide-treated; Cyc- (−), not treated with cycloheximide. (E) Relative steady-state mRNA levels of SOD1E77X in cell lines of NIH/3T3 and NSC34 compared with wtSOD1. Approximately 80% of mRNA transcribed from human SOD1E77X was eliminated, and inhibition of translation by cycloheximide significantly suppressed such elimination in both NIH/3T3 and NSC34 cell lines (Student's t-test, P < 0.001).
Figure 2.
Figure 2.
Escape of NMD via exon-fusion approach. (A) A standard curve showing ratios of peak height (T/A) against known molecular ratios (hSOD1T116X/hwtSOD1). (B) Relative steady-state mRNA levels of hSOD1T116X in cell lines of NIH/3T3 and NSC34 represent approximately half of hwtSOD1 mRNA. However, inhibition of translation by cycloheximide does not affect the steady-state mRNA level of hSOD1T116X in either NIH/3T3 or NSC34 cell line (Student's t-test, P > 0.5). (C) The northern blot analysis of hSOD1T116X transgenic mouse lines compared to established ALS mouse models of mutant SOD1. Lane 1, SOD1G93A mice that develop disease by 100 days. Lane 2, SOD1A4V low expressor line that never develops disease, even when crossed with hwtSOD1 transgenic mice. Lane 3, SOD1A4V high expressor line that do not develop disease alone. However, they will develop disease by 8 months when crossed with the hwtSOD1 transgenic mice (6). Lane 4, hwtSOD1 transgenic mice that do not develop disease (3). Lane 5, SOD1L126Z high expressor line that develops disease by 12 months. They will develop disease by 6 months when crossed with hwtSOD1 transgenic mice (5). Lane 6, SOD1L126Z low expressor line that never develops disease even when crossed with hwtSOD1 transgenic mice. Lane 7, SOD1T1116X high expressor line that develops disease by 10 months in the homozygous state (double dose). They will develop disease by 13–18 months when crossed with hwtSOD1 transgenic mice. Lanes 8 and 9, SOD1T1116X low expressor lines that do not develop disease in their life time.
Figure 3.
Figure 3.
Development of ALS-like phenotype and pathology in the hSOD1T116X transgenic mice. (A) hSOD1T116X/hwtSOD1 double transgenic mice showing ALS-like phenotype, including motor impairment, paralysis, muscle atrophy (especially in the hind legs) and loss ∼30% of body weight by the end-stage. (B) A representative section showing severe loss of motor neurons in the anterior horn of lumbar spinal cord. Immunohistochemistry using an antibody against ChAT shows a ChAT-positive motor neuron (arrow). An average number of motor neurons in the L1 to L3 segments in an affected mouse that we analyzed in detail was 6.4 ± 2.6 per anterior horn, compared to 14.6 ± 4.3 per anterior horn in a control (Student's t-test, P < 0.001); bar, 100 µm. (C–E) Confocal microscopy showing SOD1 aggregates (green in C) and astrocytosis (D) in spinal cord sections of affected mice. Astrocytes are shown as glial fibrillary acidic protein-positive cells in red (D). No apparent SOD1 aggregates have been observed in astrocytes (overlay in E).
Figure 4.
Figure 4.
Recruitment of wtSOD1 into aggregates in spinal cord and anterior root axons of hSOD1T116X/hwtSOD1 double transgenic mice. (A and B) Immunohistochemical staining with an antibody against the last 28 amino acid of SOD1 (c-SOD1) (5) showed a large number of wtSOD1 aggregates in the spinal cord sections of the affected mice (A and B). These aggregates were predominantly present in neuritic processes (arrows). Some wtSOD1 aggregates were found in surviving large neurons (arrowhead in B). Some neuritic processes with wtSOD1 aggregates were apparently swollen (large arrows in A and B). (C–E) Immuno-reactive SOD1 aggregates containing ubiquitin. Confocal microscopy showing c-SOD1-positive aggregates (green in C) and ubiquitin (red in D) in spinal cord sections of affected mice. Signals from SOD1 and ubiquitin largely overlapped (overlay in E). (F–H) Prominent SOD1 aggregates in anterior root axons. Confocal microscopy showing c-SOD1-positive aggregates (green in F) in anterior root axons with neurofilament medium chain staining (red in G) in the spinal cord sections of affected mice. Compared with axons in the neighboring spinal cord, the anterior root axons had more prominent SOD1 aggregates. A representative anterior root axon without apparent SOD1 aggregates is shown by a large arrow, and a representative anterior root axon with SOD1 aggregates is shown by a small arrow. Most of the anterior root axons showed prominent SOD1 aggregates (overlay in E).

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