Spinal cord homogenates from SOD1 familial amyotrophic lateral sclerosis induce SOD1 aggregation in living cells

<div><p>Mutant Cu/Zn superoxide dismutase (SOD1) can confer its misfolding on wild-type SOD1 in living cells; the propagation of misfolding can also be transmitted between cells <i>in vitro</i>. Recent studies identified fluorescently-tagged SOD1^G85R as a promiscuous substrate that is highly prone to aggregate by a variety of templates, <i>in vitro</i> and <i>in vivo</i>. Here, we utilized several SOD1-GFP reporter proteins with G37R, G85R, or G93A mutations in SOD1. We observed that human spinal cord homogenates prepared from SOD1 familial ALS (FALS) can induce significantly more intracellular reporter protein aggregation than spinal cord homogenates from sporadic ALS, Alzheimer’s disease, multiple system atrophy or healthy control individuals. We also determined that the induction of reporter protein aggregation by SOD1-FALS tissue homogenates can be attenuated by incubating the cells with the SOD1 misfolding-specific antibody 3H1, or the small molecule 5-fluorouridine. Our study further implicates SOD1 as the seeding particle responsible for the spread of SOD1-FALS neurodegeneration from its initial onset site(s), and demonstrates two potential therapeutic strategies for SOD1-mediated disease. This work also comprises a medium-throughput cell-based platform of screening potential therapeutics to attenuate propagated aggregation of SOD1.</p></div

Homogenates prepared from familial ALS spinal cord tissue induce SOD1 aggregation.

<p>A) SOD1 inclusions in 4 SOD1-FALS cases (A4V, D90A, G93S, I113T) were confirmed by immunohistochemistry. B) Homogenates prepared from human spinal cord tissue were incubated with HEK293FT cells pre-transfected with the indicated reporter protein (G37R, G85R or G93A-based). Cells were imaged 48 h post treatment and analyzed for the presence of inclusions using our algorithm. Representative immunocytochemistry micrographs demonstrate induced aggregation of SOD1^G85R-GFP in cells incubated with the indicated homogenate. Arrowheads point towards visible reporter protein inclusions. C) Summary of the effect of FALS, SALS and non-ALS control tissue homogenates on the reporter proteins. Bar graphs represent the percentage of reporter protein in inclusion form out of total reporter protein (area). Statistical significance was established using one way ANOVA followed by Dunnett’s test for multiple comparisons. D) Induced aggregation of the reporter protein using the individual homogenates grouped in (C). Each homogenate was tested 8–16 times with 2 technical repeat per run. *** p < 0.001, * p < 0.05. Scale bar: 40 μm.</p

SOD1-misfolding specific antibodies and 5-fluorouridine reduce induced aggregation of SOD1-GFP by SOD1-FALS homogenates.

<p>Following a 4–6 h transfection of HEK293FT cells using SOD1^G85R-GFP reporter protein, 5-FUr or 3H1 were added to the cells at a final concentration of 5 μM or 20 μg/ml, respectively, shorty prior to incubation with SOD1-FALS tissue homogenates. Cells were then incubated for an additional 48 h period, and analyzed for the presence of induced aggregates (<b>A</b>). We find that 5-FUr and 3H1 are effective at reducing induced reporter protein aggregation by SOD1-D90A, G93S or I113T spinal cord homogenates. The summary bar graph (FALS-SOD1) includes all the repeats from the SOD1-D90A, G93S or I113T. Unpaired t-test was used to demonstrate statistically significant reduction in detectable reporter protein inclusions between untreated and treated cells (<b>B</b>). Arrowheads point towards visible reporter protein inclusions. Five biological repeats were performed for each homogenate. *, p < 0.05; **, p < 0.01. Scale bar: 40 μm.</p

Detection of misfolded SOD1 in various structures and regions by immunohistochemistry.

<p>Relative abundance of DSE antibody (10E11C11) staining in various regions and structures of patient spinal cord sections (N = 5 normal control; N = 5 SOD1-FALS; N = 3 non-SOD1 FALS including R521C-FUS; N = 20 non-SOD1 SALS). Neuronal cytoplasmic inclusions, axonal swellings and axons are sub-structures within the ventral grey matter. Positive staining in corticospinal tract, other tracts and motor roots was in axons. –, no staining; +, some staining; ++, moderate staining; +++, abundant staining.</p

Transfection of mutant FUS is associated with SOD1 misfolding by immunocytochemistry.

<p>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). (<b>A</b>, <b>B</b>) Human wild-type FUS localizes in the nucleus and no misfolded SOD1 is detected. (<b>C</b>–<b>F</b>) Both of the truncated variant, R495x-FUS (<b>B</b>, <b>C</b>), and point mutation variant, P525L-FUS (<b>E</b>, <b>F</b>), 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.</p

Misfolded SOD1 immunohistochemistry in ventral grey matter and corticospinal tracts of cervical spinal cord of ALS patients.

<p>(<b>A</b>) 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>B</b>) Cases of SALS with TDP43 pathology had misfolded SOD1 accumulation only in small numbers of normal sized axons (arrows). (<b>C</b>, <b>D</b>) In the case of FALS with FUS mutation misfolded SOD1 accumulated in some swollen axons in the ventral grey matter (<b>C</b>, arrows) and a moderate number of normal sized axons in corticospinal tract (<b>D</b>, arrows). Scale bar, 60 µm (<b>A</b>, <b>C</b>); 30 µm (<b>B</b>, <b>D</b>).</p

Expression of transfected wild-type and mutant FUS and TDP43 in SH-SY5Y cells.

<p>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.</p

Quantitative immunoprecipitation of misfolded human SOD1 in SH-SY5Y cells transfected with wild-type or mutant FUS and TDP43.

<p>(<b>A</b>) 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>B</b>–<b>E</b>) Show percentage of immunoprecipitable misfolded SOD1 (out of the total precipitable SOD1) using 3H1 (<b>B</b>, <b>D</b>) and 10C12 (<b>C</b>, <b>E</b>) from lysates of transfected SH-SY5Y cell cultures. Appreciable differences are indicated (*, <i>p</i><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.</p

SOD1 misfolding in wild-type and ΔNLS-TDP43-transfected SH-SY5Y cells and human wtSOD1 expressing primary neural cells.

<p>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 (<b>A</b>, <b>B</b>) while the mutant ΔNLS-TDP43 localizes in the cytosol (<b>C</b>, <b>D</b>). 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.</p