Figure 1.
Misfolded SOD1 immunohistochemistry in ventral grey matter and corticospinal tracts of cervical spinal cord of ALS patients.
(A) Cases of FALS with SOD1 mutations show extensive accumulation of misfolded SOD1 in swollen and normal sized axons as well as in the perikarya of some lower motor neurons (inset). (B) Cases of SALS with TDP43 pathology had misfolded SOD1 accumulation only in small numbers of normal sized axons (arrows). (C, D) In the case of FALS with FUS mutation misfolded SOD1 accumulated in some swollen axons in the ventral grey matter (C, arrows) and a moderate number of normal sized axons in corticospinal tract (D, arrows). Scale bar, 60 µm (A, C); 30 µm (B, D).
Table 1.
Detection of misfolded SOD1 in various structures and regions by immunohistochemistry.
Figure 2.
Expression of transfected wild-type and mutant FUS and TDP43 in SH-SY5Y cells.
The immunoblot on the left was probed with antibody specific to HA-tag, detecting only the exogenously expressed proteins in these samples. The empty vector (ev; pCINeo) control does not display detectable immunoreactivity with the HA-tag antibody, while both exogenous FUS and TDP43 are detected as 43 kDa and 72 kDa bands, respectively. Top and bottom portions of the immunoblot on the right were probed using FUS and TDP43 specific antibodies, respectively, detecting both the endogenous and exogenous proteins. Endogenous FUS and TDP43 are detected in all samples; however, stronger signals, corresponding to the over-expressed protein, are detected in the transfected samples.
Figure 3.
Transfection of mutant FUS is associated with SOD1 misfolding by immunocytochemistry.
Human neuroblastoma SH-SY5Y cells and primary neural cultures expressing human wtSOD1 were stained for HA-Tag (red), misfolded SOD1 (green), and Hoechst33342 nuclear counterstain (blue). (A, B) Human wild-type FUS localizes in the nucleus and no misfolded SOD1 is detected. (C–F) Both of the truncated variant, R495x-FUS (B, C), and point mutation variant, P525L-FUS (E, F), localize in the cytosol and are associated with misfolding of SOD1 in the same cells, as detected by the immunocytochemistry with the 3H1 SOD1 misfolding-specific mAb. Exogenous FUS was detected using the N-terminal HA-tag. Arrows point to transfected cells. Scale bar, 20µm.
Figure 4.
SOD1 misfolding in wild-type and ΔNLS-TDP43-transfected SH-SY5Y cells and human wtSOD1 expressing primary neural cells.
SH-SY5Y cells and primary neural cells stained against HA-tag (transfected TDP43; red), misfolded SOD1 (green), and Hoechst33342 (blue) nuclear counterstain. Human wtTDP43 predominantly localizes in the nucleus (A, B) while the mutant ΔNLS-TDP43 localizes in the cytosol (C, D). Both variants of TDP43 are associated with misfolding of the endogenous SOD1 in the same cells, as detected by 3H1 immunoreactivity. Arrows point to transfected cells. Scale bar, 20µm.
Figure 5.
Quantitative immunoprecipitation of misfolded human SOD1 in SH-SY5Y cells transfected with wild-type or mutant FUS and TDP43.
(A) Representative immunoblots of immunoprecipitations. SOD1 proteins from transfected (48 h) and untransfected SH-SY5Y cell lysates were precipitated using pan-SOD1 antibody, SOD100 (rabbit polyclonal), and SOD1 misfolding-specific mouse monoclonal antibodies, 3H1 and 10C12. rIgG was used as isotype control for SOD100, and mIgG2a was used as isotype control for the DSE antibodies. Blots were probed with pan-SOD1 antibody. Framed bands show FUS, and TDP43 transfection efficiency of the indicated construct, as was detected by probing with antibody to HA-tag. (B–E) Show percentage of immunoprecipitable misfolded SOD1 (out of the total precipitable SOD1) using 3H1 (B, D) and 10C12 (C, E) from lysates of transfected SH-SY5Y cell cultures. Appreciable differences are indicated (*, p<0.01). N = 5 for each of wtFUS, R495x- and P525L-FUS. N = 6 for each of ev, wtTDP43 and ΔNLS-TDP43. Error bars represent s.e.m.