Tryptophan residues in TDP-43 and SOD1 modulate the cross-seeding and toxicity of SOD1.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons. Neuronal superoxide dismutase-1 (SOD1) inclusion bodies are characteristic of familial ALS with SOD1 mutations, while a hallmark of sporadic ALS is inclusions containing aggregated WT TAR DNA-binding protein 43 (TDP-43). We show here that co-expression of mutant or WT TDP-43 with SOD1 leads to misfolding of endogenous SOD1 and aggregation of SOD1 reporter protein SOD1-GFP in human cell cultures and promotes synergistic axonopathy in zebrafish.

Tryptophan 32 mediates SOD1 toxicity in a in vivo motor neuron model of ALS and is a promising target for small molecule therapeutics.

SOD1 misfolding, toxic gain of function, and spread are proposed as a pathological basis of amyotrophic lateral sclerosis (ALS), but the nature of SOD1 toxicity has been difficult to elucidate. Uniquely in SOD1 proteins from humans and other primates, and rarely in other species, a tryptophan residue at position 32 (W32) is predicted to be solvent exposed and to participate in SOD1 misfolding. We hypothesized that W32 is influential in SOD1 acquiring toxicity, as it is known to be important in template-directed misfolding.

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.

Induction of Cu/Zn Superoxide Dismutase (SOD1) Aggregation in Living Cells.

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. In the familial form of ALS, the presence of any one of over 180 inherited mutations in the gene that encodes Cu/Zn superoxide dismutase (SOD1) leads to its eventual misfolding and aggregation. Once the pathological SOD1 seed is formed, it can continue growing into a larger aggregate through nucleation of other SOD1 substrate molecules.

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

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 in vitro. Recent studies identified fluorescently-tagged SOD1G85R as a promiscuous substrate that is highly prone to aggregate by a variety of templates, in vitro and in vivo. Here, we utilized several SOD1-GFP reporter proteins with G37R, G85R, or G93A mutations in SOD1.

Intercellular Prion-Like Conversion and Transmission of Cu/Zn Superoxide Dismutase (SOD1) in Cell Culture.

The prion hypothesis has extended to the fatal motor neuron disease, amyotrophic lateral sclerosis (ALS), as a means to explain the spatiotemporal spread of pathology from one or more focal points through the neuroaxis. About 20% of inheritable cases of ALS are due to mutation in the gene encoding the Cu/Zn superoxide dismutase (SOD1), causing the protein to misfold and form neurotoxic aggregates. Mutant SOD1 has been shown to impart its misfold onto natively folded wild-type SOD1 in living cells.

TDP-43 or FUS-induced misfolded human wild-type SOD1 can propagate intercellularly in a prion-like fashion.

Amyotrophic lateral sclerosis (ALS), which appears to spread through the neuroaxis in a spatiotemporally restricted manner, is linked to heritable mutations in genes encoding SOD1, TDP-43, FUS, C9ORF72, or can occur sporadically without recognized genetic mutations. Misfolded human wild-type (HuWt) SOD1 has been detected in both familial and sporadic ALS patients, despite mutations in SOD1 accounting for only 2% of total cases. We previously showed that accumulation of pathological TDP-43 or FUS coexist with misfolded HuWtSOD1 in patient motor neurons, and can trigger its misfolding in cultured cells.

Exosome-dependent and independent mechanisms are involved in prion-like transmission of propagated Cu/Zn superoxide dismutase misfolding.

Amyotrophic lateral sclerosis (ALS), a fatal adult-onset degenerative neuromuscular disorder with a poorly defined etiology, progresses in an orderly spatiotemporal manner from one or more foci within the nervous system, reminiscent of prion disease pathology. We have previously shown that misfolded mutant Cu/Zn superoxide dismutase (SOD1), mutation of which is associated with a subset of ALS cases, can induce endogenous wild-type SOD1 misfolding in the intracellular environment in a templating fashion similar to that of misfolded prion protein. Our recent observations further extend the prion paradigm of pathological SOD1 to help explain the intercellular transmission of disease along the neuroaxis.

Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms.

Amyotrophic lateral sclerosis (ALS) is predominantly sporadic, but associated with heritable genetic mutations in 5-10% of cases, including those in Cu/Zn superoxide dismutase (SOD1). We previously showed that misfolding of SOD1 can be transmitted to endogenous human wild-type SOD1 (HuWtSOD1) in an intracellular compartment. Using NSC-34 motor neuron-like cells, we now demonstrate that misfolded mutant and HuWtSOD1 can traverse between cells via two nonexclusive mechanisms: protein aggregates released from dying cells and taken up by macropinocytosis, and exosomes secreted from living cells.

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.

Generalization of the prion hypothesis to other neurodegenerative diseases: an imperfect fit.

Protein misfolding diseases have been classically understood as diffuse errors in protein folding, with misfolded protein arising autonomously throughout a tissue due to a pathologic stressor. The field of prion science has provided an alternative mechanism whereby a seed of pathologically misfolded protein, arising exogenously or through a rare endogenous structural fluctuation, yields a template to catalyze misfolding of the native protein. The misfolded protein may then spread intercellularly to communicate the misfold to adjacent areas and ultimately infect a whole tissue.

Intermolecular transmission of superoxide dismutase 1 misfolding in living cells.

Human wild-type superoxide dismutase-1 (wtSOD1) is known to coaggregate with mutant SOD1 in familial amyotrophic lateral sclerosis (FALS), in double transgenic models of FALS, and in cell culture systems, but the structural determinants of this process are unclear. Here we molecularly dissect the effects of intracellular and cell-free obligately misfolded SOD1 mutant proteins on natively structured wild-type SOD1. Expression of the enzymatically inactive, natural familial ALS SOD1 mutations G127X and G85R in human mesenchymal and neural cell lines induces misfolding of wild-type natively structured SOD1, as indicated by: acquisition of immunoreactivity with SOD1 misfolding-specific monoclonal antibodies; markedly enhanced protease sensitivity suggestive of structural loosening; and nonnative disulfide-linked oligomer and multimer formation.