Aberrant Localization of FUS and TDP43 Is Associated with Misfolding of SOD1 in Amyotrophic Lateral Sclerosis

Background: Amyotrophic lateral sclerosis (ALS) is incurable and characterized by progressive paralysis of the muscles of the limbs, speech and swallowing, and respiration due to the progressive degeneration of voluntary motor neurons. Clinically indistinguishable ALS can be caused by genetic mutations of Cu/Zn superoxide dismutase (SOD1), TAR-DNA binding protein 43 (TDP43), or fused in sarcoma/translocated in liposarcoma (FUS/TLS), or can occur in the absence of known mutation as sporadic disease. In this study, we tested the hypothesis that FUS/TLS and TDP43 gain new pathogenic functions upon aberrant accumulation in the cytosol that directly or indirectly include misfolding of SOD1. Methodology/Principal Findings: Patient spinal cord necropsy immunohistochemistry with SOD1 misfolding-specific antibodies revealed misfolded SOD1 in perikarya and motor axons of SOD1-familial ALS (SOD1-FALS), and in motor axons of R521C-FUS FALS and sporadic ALS (SALS) with cytoplasmic TDP43 inclusions. SOD1 misfolding and oxidation was also detected using immunocytochemistry and quantitative immunoprecipitation of human neuroblastoma SH-SY5Y cells as well as cultured murine spinal neural cells transgenic for human wtSOD1, which were transiently transfected with human cytosolic mutant FUS or TDP43, or wtTDP43. Conclusion/Significance: We conclude that cytosolic mislocalization of FUS or TDP43 in vitro and ALS in vivo may kindle wtSOD1 misfolding in non-SOD1 FALS and SALS. The lack of immunohistochemical compartmental co-localization o

Induction of wild-type SOD1 misfolding, aggregation and its cell-to-cell propagation

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive degeneration and loss of motor neurons that appears to spread through the neuroaxis in a spatiotemporally restricted manner. Misfolded Cu/Zn superoxide dismutase (SOD1) has been detected in all ALS patients, despite SOD1 mutations accounting for only 2% of total cases, while the presence of inclusions containing pathological TAR-DNA binding protein-43 (TDP-43) represent a hallmark of all non-SOD1/FUS familial ALS. We previously reported that TDP-43 and FUS can trigger misfolding of human wild-type SOD1 (HuWtSOD1) in living cells, however the mechanisms and consequences are unknown. Here, we used immunocytochemistry, immunoprecipitation and cell viability studies to demonstrate that TDP-43 or FUS-induced misfolded HuWtSOD1 can propagate from cell-to-cell via conditioned media, and seed cytotoxic misfolding of endogenous HuWtSOD1 in the recipient cells in a prion-like fashion. Knockdown of SOD1 using siRNA in recipient cells, or incubation of conditioned media with misfolded SOD1-specific antibodies, inhibits intercellular transmission, indicating that HuWtSOD1 is an obligate seed and substrate of propagated misfolding. Furthermore, we developed several chimeric SOD1-GFP proteins that we validated to aggregate in the presence of pathological SOD1 or TDP-43 seed. We used this assay, along with immunofluorescence, live-cell microscopy and flow cytometry studies, to show that intermolecular conversion of SOD1 by pathological TDP-43 is mediated by tryptophan residues in both proteins. Furthermore, we used the reporter proteins to show that human spinal cord extracts prepared from familial, but not sporadic, ALS patients can trigger SOD1 aggregation in cultured cells. Finally, we used this system to show that small molecules, akin to 5-fluorouridine, can block this intermolecular kindling of SOD1 aggregation, and demonstrated that our assay can be used as a high-throughput tool for screening drugs against induced SOD1 aggregation. Altogether, our studies indicate that pathological TDP-43 and FUS may exert motor neuron pathology in ALS through the initiation of tryptophan-dependent propagated SOD1 misfolding. Furthermore, it is key to recognize that elucidation of the pathogenic role of a simple structural motif in ALS may provide a framework for understanding other neurodegenerative diseases in which propagated protein misfolding is shown to occur.Medicine, Faculty ofExperimental Medicine, Division ofMedicine, Department ofGraduat

Aberrant localization of fused in sarcoma (FUS) and TAR DNA binding protein (TDP)-43 triggers misfolding of human Cu/Zn superoxide dismutase (SOD1)

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat

Tryptophan 32-mediated SOD1 aggregation is attenuated by pyrimidine-like compounds in living cells

Over 160 mutations in superoxide dismutase 1 (SOD1) are associated with familial amyotrophic lateral sclerosis (fALS), where the main pathological feature is deposition of SOD1 into proteinaceous cytoplasmic inclusions. We previously showed that the tryptophan residue at position 32 (W32) mediates the prion-like propagation of SOD1 misfolding in cells, and that a W32S substitution blocks this phenomenon. Here, we used in vitro protein assays to demonstrate that a W32S substitution in SOD1-fALS mutants significantly diminishes their propensity to aggregate whilst paradoxically decreasing protein stability. We also show SOD1-W32S to be resistant to seeded aggregation, despite its high abundance of unfolded protein. A cell-based aggregation assay demonstrates that W32S substitution significantly mitigates inclusion formation. Furthermore, this assay reveals that W32 in SOD1 is necessary for the formation of a competent seed for aggregation under these experimental conditions. Following the observed importance of W32 for aggregation, we established that treatment of living cells with the W32-interacting 5-Fluorouridine (5-FUrd), and its FDA approved analogue 5-Fluorouracil (5-FU), substantially attenuate inclusion formation similarly to W32S substitution. Altogether, we highlight W32 as a significant contributor to SOD1 aggregation, and propose that 5-FUrd and 5-FU present promising lead drug candidates for the treatment of SOD1-associated ALS

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

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

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

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