Mutant SOD1 inclusions resemble aggresomes. A, 48 h after transfection with SOD1-GFP, HEK293 cells were immunostained for γ-tubulin, a MTOC marker, and analyzed by confocal microscopy. Cells expressing mutant SOD1 developed large inclusions close or around the MTOC. Scale bars are 10 μm. B, quantitative analysis of co-localization of mutant SOD1 inclusions and MTOC. Approximately 100 cells with inclusions were counted for each mutant per experiment, and data from three independent experiments arepresented.

Overexpression of p50 prevents inclusion formation by mutant SOD1. A, HEK293 cells were transfected with 0.5 μg of various SOD1-GFP constructs and different amounts of p50-myc plasmid. The total amount of plasmid transfected in each well was kept constant at 1 μg by adding empty vector control for p50-myc. The percentage of transfected cells with inclusions was measured 48 h after transfection. B, Western blotting of protein extracts prepared from cells in A after counting, showing similar levels of SOD1 and increased levels of p50 with increased amounts of transfected p50 plasmid. C, inclusion analysis in NSC34 cells. Experiments were carried out in the same manner as in A, except NSC34 cells were used. D, Western blotting of protein extracts prepared from NSC34 cells in the same manner as in C. E, amount of SOD1 in the whole extract, supernatant, and detergent-resistant pellet fractions in the presence and absence of p50 overexpression. The amounts of mutant A4V and G85R in the pellet fraction was reduced when p50 was overexpressed. WT SOD1 was not or very weakly detected in the pellet fraction under both conditions. Data presented in A and C were obtained from three independent experiments. ^*, p < 0.04; ^**, p < 0.004; and ^***, p < 0.0004.

H46R and H48Q show little affinity for dynein. GST pulldowns were performed from HEK293 cells transfected with GST-DIC and various FLAG-tagged SOD1 constructs. Western blotting for GST and FLAG revealed that WT, H46R, or H48Q SOD1 was not or to very little extent co-precipitated by DIC-GST. However, the inclusion-forming mutants A4V, G85R, and G93A were all readily pulled down together with DIC-GST. Empty GST vector and A4V SOD1 were co-transfected as negative controls showing no co-precipitation of empty GST and A4V SOD1.

Disruption of the interaction of mutant SOD1 with dynein affects cell viability. Cell viability in the absence and presence of p50 overexpression was determined by the percentage of PI-positive transfected cells. A, representative images show PI-positive HEK293 cells expressing WT or A4V SOD1 in the absence of p50 overexpression. B, quantification of cell viability obtained from three independent experiments. The black bars are the percentage of PI-positive cells without inclusions, and the gray bars are the percentage of PI-positive inclusion-containing cells. Standard deviations shown are for the total number of PI-positive cells. ^*, p < 0.05 and ^**, p < 0.03.

Disruption of the dynein-dynactin complex by p50 overexpression blocks the mutant SOD1-dynein interaction. A, schematic drawing of the dynein-dynactin complex under normal and p50 overexpression conditions. Dynein consists of several subunits including two DHC and two DIC. Dynactin also comprises multiple subunits including eight Arp1, four p50, and two p150^Glued. Overexpressed exogenous p50 has been reported to dissociate the cargo binding region from the remaining dynactin complex (42). B, GST pulldowns were performed from HEK293 cells transfected with SOD1-FLAG (WT or A4V), GST-DIC and p50-myc or vector control. Western blotting showed that co-precipitation of A4V mutant SOD1 with DIC-GST was abolished in the presence of p50-Myc overexpression. WT-FLAG did not interact with DIC-GST either in the presence or absence of p50-myc overexpression. DHC and p150^Glued were co-precipitated with DIC under all conditions. Overexpression of p50-myc increased the amount of p50 co-precipitated with DIC. C, quantitative analysis from three independent experiments showed an approximately eight times reduction in the amount of A4V SOD1 co-precipitated with DIC in the presence of p50-myc. The band intensity of co-precipitated SOD1 was normalized against that of DIC precipitated. ^*, p < 0.02.

H46R and H48Q form none or few HMW complexes compared with other ALS-linked SOD1 mutants. Fresh NSC34 cell lysates containing WT or various SOD1-FLAG mutants were incubated with the cross-linker DSG for 60 min before the reaction was quenched. The reaction mixtures were resolved in 10% SDS-PAGE and blotted with a FLAG antibody. After cross-linking, WT SOD1 migrated as both monomer and dimer; however, all mutants with the exception of H46R and H48Q showed formation of HMW complexes in addition to monomeric and dimeric forms. As the control in SDS-PAGE, all SOD1-FLAG ran as monomers in the absence of DSG (second panel), and actin blotting showed equal loading of all samples (third panel).

H46R and H48Q mutants behave as WT-SOD1 and do not form inclusions in transfected cells. A, H46R and H48Q do not form inclusions like the A4V, G85R, and G93A mutant SOD1s. HEK293 cells were transfected with SOD1-GFP plasmids and photographed 48 h later using a Nikon Eclipse epifluorescence microscope with identical settings for WT and mutant SOD1s. B, quantification of mutant SOD1 inclusion formation in HEK293 cells. A4V, G85R, and G93A showed a statistically significant increase in inclusion formation versus WT SOD1, whereas H46R and H48Q behaved like WT. ^**, p < 0.0003. C, fractionation of HEK293 cell lysate 48 h after cells were transfected with SOD1-GFP. A4V, G85R, and G93A were all detected in the pellet fraction, whereas H46R and H48Q remained soluble, similar to WT SOD1. D, fractionation of HEK293 cell lysate 48 h after cells were transfected with untagged SOD1. Increased levels of untagged A4V, G85R, and G93A were observed in pellet fractions compared with WT, H46R, and H48Q. E, quantification of mutant SOD1 inclusion formation in NSC34 cells. A4V, G85R, and G93A, but neither H46R nor H48Q, showed a statistically significant increase in inclusion formation versus WT SOD1. ^*, p < 0.003. F, fractionation of NSC34 cell lysate as described in C showing increased amounts of A4V, G85R, and G93A in the insoluble pellet fraction. G, quantification of mutant SOD1 inclusion formation in the presence and absence of p62 overexpression. HEK293 cells were transfected with various SOD1-GFP constructs and DsRed-p62 or the vector control. The percentage of transfected cells with inclusions were measured 24 h after transfection. Inclusions formed by H46R or H48Q remained unchanged even with p62 overexpression, whereas inclusions by A4V, G85R, and G93A increased in the presence of p62 overexpression. ^*, p < 0.05 and ^**, p < 0.01. Quantitative data in B, E, and G were obtained from three independent experiments.

Potential role(s) of the dynein-dynactin motor complex in mutant SOD1-mediated ALS. Dynein-dynactin can sequester and transport mutant SOD1 to form large aggresome-like inclusions that require autophagic degradation. Dynein is also involved in the autophagosome-lysosome fusion that is critical to the degradation of misfolded protein inclusions in autophagosomes. Interplay of the two dynein-dependent events, the formation and degradation of inclusions, determines the eventual inclusion burden in neurons. Increased levels of mutant SOD1 associated with dynein as the disease progresses could contribute to decreased axonal transport in ALS by multiple mechanisms. These potential mechanisms illustrated here include competitive occupancy of the dynein-mediated transport capacity, disruption of dynein motor activity, destabilization of microtubules, physical blockage of transport by mutant SOD1 aggregates in the axon. Decreased retrograde transport of cargos such as mitochondria and neurotrophic factors as well as increased aggregation burden may determine the toxicity of mutant SOD1 and ALS disease manifestation.