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Neuro2A cells were co-transfected with FLAG-tagged SOD1 and HA-tagged CHGB.Arrowheads indicate CHGB accumulation and SOD1 Additional evidence of defective binding of CHGB variants (L413 and R230) with mutant SOD1 or misf SOD1 was obtained by immunofluorescence confocal microscopy of Neuro2a cells after transfection of vectors encoding mouse Cg B or human CHGB variants with SOD1 species. To further investigate the localization of CHGB WT and variants in the context of overexpression of human SOD1 species, Neuro2a cells were co-transfected with HA-CHGB species and FLAG-SOD1 species, and then analyzed by double immunofluorescence using antibodies against trans-Golgi marker (TGN38) or synaptic vesicle marker (synaptophysin).The number of α-motor neurons, which were positive for both Ch AT and Neu N, in SOD13–4) was analyzed.

These transgenic mice also exhibited similar excess CHGB protein levels in the microsomal fraction of the spinal cord when examined at 210 days of age ( showed no deficiency on the rotarod test or on body weight loss until 500 days of age (data not shown).

The disease onset, as defined by 30% loss of maximum recorded time on rotarod (37,38), occurred at mean age of 365.7 ± 3.0 days in SOD1 mice (Fig. Furthermore, we have stratified the tested group of mice according to their sex status and determined whether sex had an effect of disease onset by analyzing rotarod performance. To investigate the degeneration of spinal α-motor neurons, lumbar cord sections at 345 days of age were immunostained with both anti-Ch AT and anti-Neu N antibody.

(D) Enhanced Bip expression due to misf SOD1 accumulation (C4F6 antibody) in motor neurons of SOD1 female mice at 300 days of age. Post-ANOVA Turkey test was used in each sex (same for all figures).

The cytosolic/microsomal fractions of spinal cords were immunoblotted and the immunoprecipitates with anti-SOD1 antibody were immunoblotted. The panels have been spliced for the IP-SOD1 results of male and female samples because results were obtained from two distinct immunoprecipitation experiments for male and female samples. (D) Enhanced Bip expression due to misf SOD1 accumulation (C4F6 antibody) in motor neurons of SOD1 female mice at 300 days of age.

However, the expression of CHGB (WT, L413 and R230) and mouse Cg B did not promote the secretion of endogenous mouse SOD1 in cells (data not shown).


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