Soluble misfolded subfractions of mutant superoxide dismutase-1s are enriched in spinal cords throughout life in murine ALS models

Abstract

Mutants of superoxide dismutase-1 (SOD1) cause ALS by an unidentified cytotoxic mechanism. We have previously shown that the stable SOD1 mutants D90A and G93A are abundant and show the highest levels in liver and kidney in transgenic murine ALS models, whereas the unstable G85R and G127X mutants are scarce but enriched in the CNS. These data indicated that minute amounts of misfolded SOD1 enriched in the motor areas might exert the ALS-causing cytotoxicity. A hydrophobic interaction chromatography (HIC) protocol was developed with the aim to determine the abundance of soluble misfolded SOD1 in tissues in vivo. Most G85R and G127X mutant SOD1s bound in the assay, but only minute subfractions of the D90A and G93A mutants. The absolute levels of HIC-binding SOD1 were, however, similar and broadly inversely related to lifespans in the models. They were generally enriched in the susceptible spinal cord. The HIC-binding SOD1 was composed of disulfide-reduced subunits lacking metal ions and also subunits that apparently carried nonnative intrasubunit disulfide bonds. The levels were high from birth until death and were comparable to the amounts of SOD1 that become sequestered in aggregates in the terminal stage. The HIC-binding SOD1 species ranged from monomeric to trimeric in size. These species form a least common denominator amongst SOD1 mutants with widely different molecular characteristics and might be involved in the cytotoxicity that causes ALS.

Fig. 1.

Reduced immunoblots of soluble HIC-binding hSOD1 fractions in transgenic mice. (A) Total hSOD1 and the HIC-binding subfractions in tissue extracts from a 34-day-old G93A transgenic mouse. The figures indicate the fractions (percentages) of total hSOD1 that bound. (B) HIC-binding hSOD1 fractions of tissue extracts from 100-day-old G127, G85R, and wild-type hSOD1 mice. In addition to the monomer band at ≈17 kDa, tissues form G127X mice contain a prominent band at 33 kDa (7). In quantifications of G127X hSOD1, both bands were included. (C) HIC-binding hSOD1 subfractions in tissues of G93A mice of different ages (in days). Two spinal cords were pooled from 1-day-old and 10-day-old mice; the other data are from individual mice. The percentages and absolute amounts bound (μg/g of wet weight) in the spinal cords were, respectively, 5.3% and 11 at day 1, 3.5% and 19 at day 10, 3.2% and 44 at day 19, 4.8% and 50 at day 34, 3.8% and 43 at day 50, 2.8% and 42 at day 100, and 4.0% and 45 in the terminal mouse. (D) HIC-binding hSOD1 in tissues from D90A mice of different ages. The corresponding data for the D90A mice were, respectively, 0.49% and 4.1 at day 100, 0.33% and 4.8 at day 200, 0.43% and 6.2 at day 300, and 2.6% and 25 in the terminal mouse. Sp.c., spinal cord; Kidn., kidney.

Fig. 2.

Gel chromatography of spinal cord extracts from 100-day-old mice. (A) The distributions of the hSOD1s analyzed by immunoblots from the void volume to the end of the monomer peaks. Tubes 26–35 were virtually devoid of hSOD1 in the D90A, G85R, and wild-type hSOD1 chromatograms, and the data points were omitted. The G93A chromatogram contained minute amounts of hSOD1 in some of these fractions, and the data were multiplied by 10 to increase visibility. (B) Analysis of disulfide oxidation by nonreduced immunoblotting. Subunits lacking disulfide bond are more retarded in the gel than subunits restricted by the C57-C146 disulfide bond (8). Note that D90A mutant SOD1 has a higher mobility than wild-type and G93A mutant human SOD1s (17). The extracts were electrophoresed together with the dimer (tube 39) and monomer (tube 43) peak fractions.

Fig. 3.

Nonreduced immunoblots of soluble HIC-binding hSOD1 subfractions in spinal cords from transgenic mice. (A) Dilutions of the extract and the HIC-binding subfraction of a 34-day-old G93A mouse. The SDS/PAGE gel was cut in half, and the right side was subjected to in-gel reduction. The two halves were then mounted together for the electroblotting and immunostaining. (B) Similar analysis of the other transgenic models. (C) Comparison of a G85R spinal cord extract (Extr.) with a recombinant G85R/C6A/C111A hSOD1 preparation. The pair to the left shows the pattern in a regular reduced immunoblot, the intermediate a nonreduced immunoblot, and the pair to the right a nonreduced immunoblot subjected to in-gel reduction. The samples were electrophoresed on one gel, and the pieces were mounted together for electroblotting and immunostaining. (D) Comparison of a G93A spinal cord extract and the HIC-binding subfraction with various recombinant mutants harboring combinations of cysteine mutations. The spinal cord fractions were treated with iodoacetamide. The recombinant variants were denatured, reduced, deprived of metals, and allowed to form disulfide bonds spontaneously. The lower row shows nonreduced immunoblots subjected to in-gel reduction, and the upper row shows reduced immunoblots. Sp.c., spinal cord; extr., extract; bind., binding.