Additive amelioration of ALS by co-targeting independent pathogenic mechanisms.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which glia are central mediators of motor neuron (MN) death. Since multiple cell types are involved in disease pathogenesis, the objective of this study was to determine the benefit of co-targeting independent pathogenic mechanisms in a familial ALS mouse model. METHODS: Recently, our laboratory identified that ALS microglia induce MN death in an NF-κB-dependent mechanism. We also demonstrated that a single, post-natal, intravenous injection of adeno-associated viral vector serotype 9 encoding a shRNA against mutant SOD1 is able to traverse the blood-brain barrier of ALS mice and reduce SOD1-expression in astrocytes and MNs. Reducing mutant SOD1 in MNs and astrocytes led to a robust increase in survival. To evaluate the benefit of co-targeting multiple cell types in ALS, we combined microglial NF-κB suppression with SOD1 reduction in astrocytes and MNs. RESULTS: Targeting both astrocytes and microglia resulted in an additive increase in survival and motor function by delaying both onset and progression. Strikingly, targeting all three cell types (astrocytes, motor neurons [MNs], and microglia) resulted in an additive increase in lifespan and motor function, with maximum survival reaching 204 days, 67 days longer than the mean survival of untreated control animals. INTERPRETATION: Our data suggest that a combinatorial approach co-targeting different pathogenic mechanisms in independent cell types is a beneficial therapeutic strategy for ALS

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

Characterization of FUS Mutations in Amyotrophic Lateral Sclerosis Using RNA-Seq

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease resulting in severe muscle weakness and eventual death by respiratory failure. Although little is known about its pathogenesis, mutations in fused in sarcoma/translated in liposarcoma (FUS) are causative for familial ALS. FUS is a multifunctional protein that is involved in many aspects of RNA processing. To elucidate the role of FUS in ALS, we overexpressed wild-type and two mutant forms of FUS in HEK-293T cells, as well as knocked-down FUS expression. This was followed by RNA-Seq to identify genes which displayed differential expression or altered splicing patterns. Pathway analysis revealed that overexpression of wild-type FUS regulates ribosomal genes, whereas knock-down of FUS additionally affects expression of spliceosome related genes. Furthermore, cells expressing mutant FUS displayed global transcription patterns more similar to cells overexpressing wild-type FUS than to the knock-down condition. This observation suggests that FUS mutants do not contribute to the pathogenesis of ALS through a loss-of-function. Finally, our results demonstrate that the R521G and R522G mutations display differences in their influence on transcription and splicing. Taken together, these results provide additional insights into the function of FUS and how mutations contribute to the development of ALS.ALS Foundation NetherlandsAdessium FoundationSeventh Framework Programme (European Commission) (grant number 259867)Thierry Latran FoundationNational Institutes of Health (U.S.) (NIH/NINDS grant R01NS073873)National Institute of Neurological Disorders and Stroke (U.S.) (NIH/NINDS grant numbers 1R01NS065847

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