Neuron-specific expression of mutant superoxide dismutase 1 in transgenic mice does not lead to motor impairment

Abstract

Mutations were identified in the Cu/Zn superoxide dismutase gene (SOD1) in approximately 15% of patients with familial amyotrophic lateral sclerosis. Transgenic animals expressing mutant SOD1 in all tissues develop an ALS-like phenotype. To determine whether neuron-specific expression of mutant SOD1 is sufficient to produce such a phenotype, we generated transgenic animals carrying the G37R mutation that is associated with the familial form of ALS (FALS), which is driven by the neurofilament light chain promoter. The transgenic animals express high levels of the human SOD1 protein in neuronal tissues, especially in the large motor neurons of the spinal cord, but they show no apparent motor deficit at up to 1.5 years of age. Our animal model suggests that neuron-specific expression of ALS-associated mutant human SOD1 may not be sufficient for the development of the disease in mice.

Fig. 1.

1, Diagram of the construct used to generate the transgenic animals. The pGCHNFL vector contains the promoter region, some enhancing intronic sequences, and the 3′-untranslated region (3′-UTR) of the neurofilament light chain gene. 2, RT-PCR and Western blots show expression of the human transgene in nervous tissue both at the mRNA and protein levels. 2A, Multiple tissue RT-PCR showing expression of the human SOD1 cDNA. 2B, Same as2A but using primers amplifying the GAPDH cDNA.2C, Western blots probed with an anti-SOD1 antibody recognizing both human and mouse proteins, showing expression of the hSOD1 in nervous tissue exclusively. mSOD1, Mouse SOD1;hSOD1, human SOD1.

Fig. 2.

Characterization of the human SOD1 antibody.A, Western blot probed with preimmune serum.B, Western blot probed with the human SOD1 antibody, which specifically recognizes the hSOD1, but not the mSOD1, protein.C, Western blot probed with peptide-preabsorbed human SOD1 antibody. h/mSOD1, Protein extract from spinal cord of transgenic animal overexpressing hSOD1; mSOD1, protein extract from spinal cord of nontransgenic animal;hSOD1, human SOD1 (Sigma).

Fig. 3.

Human SOD1 is expressed in large neurons in the ventral horn of the spinal cord in transgenic animals. A, B, Normal nontransgenic animal. C, D, Transgenic line G37R-3156. E, F, Transgenic line G37R-4012.G, H, Transgenic line SOD1-4305. Scale bars: A, C, E, G, 200 μm; B, 100 μm; D, F, H, 50 μm.

Fig. 4.

Limb grip strength measurement. There is no significant difference in the performance of transgenic versus nontransgenic animals.

Fig. 5.

Size of motor neurons in L4 and L5 ventral roots.1, Photographs of ventral root axons of 1-year-old normal and transgenic animals. 1A, Nontransgenic animal.1B, Line SOD1-4305. 1C, Line G37R-3156.1D, Line G37R-4012. Scale bar, 100 μm.2, Axon counts in L4 and L5 ventral roots of 1-year-old normal and transgenic animals. 1, 2, No significant differences are seen between the normal and transgenic animals.