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

Abstract

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. Furthermore, once HuWt-SOD1 propagation has been established, misfolding of HuWt-SOD1 can be efficiently and repeatedly propagated between HEK293 cell cultures via conditioned media over multiple passages, and to cultured mouse primary spinal cord cells transgenically expressing HuWtSOD1, but not to cells derived from nontransgenic littermates. Conditioned media transmission of HuWtSOD1 misfolding in HEK293 cells is blocked by HuWtSOD1 siRNA knockdown, consistent with human SOD1 being a substrate for conversion, and attenuated by ultracentrifugation or incubation with SOD1 misfolding-specific antibodies, indicating a relatively massive transmission particle which possesses antibody-accessible SOD1. Finally, misfolded and protease-sensitive HuWtSOD1 comprises up to 4% of total SOD1 in spinal cords of patients with sporadic ALS (SALS). Propagation of HuWtSOD1 misfolding, and its subsequent cell-tocell transmission, is thus a candidate process for the molecular pathogenesis of SALS, which may provide novel treatment and biomarker targets for this devastating disease.A myotrophic lateral sclerosis (ALS) is a fatal neuromuscular condition that afflicts as many as 1 of 350 males and 420 females over the age of 18 (1). In ALS, degeneration of upper and lower motor neurons causes progressive muscle paralysis and spasticity, affecting mobility, speech, swallowing, and respiration (2). Half of affected individuals die within 3 y, and less than 20% survive for more than 5 y (3); 90-95% of ALS cases are sporadic (SALS) in which some apparently facilitating gene mutations, such as repeat expansions in the gene that encodes ataxin-2 (4), have been identified. The remaining 5-10% of ALS cases are familial (FALS) and predominantly associated with Mendelian-inherited mutations in the genes encoding Cu/Zn superoxide dismutase (SOD1), TAR-DNA-binding protein 43 (TDP-43), fused in sarcoma/translocated in liposarcoma (FUS/ TLS), C9ORF72, and other genes (reviewed in ref. 3).Despite the profusion of functionally diverse genes implicated in FALS and SALS, clinical and pathological similarities between all forms of ALS suggest the existence of a common pathogenic pathway that could be united by a single gene/protein (5). One of the mechanisms by which a mutant or wild-type (WT) protein can dominate pathogenesis of phenotypically diverse diseases is by propagated protein misfolding, such as that underpinning the prion diseases, which has been increa...

Endogenous macrophage migration inhibitory factor reduces the accumulation and toxicity of misfolded SOD1 in a mouse model of ALS

“…Finally, accumulation of misfolded SOD1 has been reported by several groups also in sporadic ALS (27,(41)(42)(43)(44)(45)(46)(47), although other groups have reached the opposite conclusion (48)(49)(50)(51). The identification of MIF as a cytosolic chaperone that stimulates the folding or refolding of misfolded SOD1 and inhibits the aggregation of mutant SOD1 in vivo suggests new avenues for therapy in ALS, mediated by increasing intracellular MIF levels in the nervous system.…”

Spinal motor neuron protein supersaturation patterns are associated with inclusion body formation in ALS

“…Thus, although SOD1 is very stable once native (72), its high expression level leaves it particularly prone to misfolding and aggregation if protein homeostasis is disrupted specifically in motor neurons. Indeed, SOD1 aggregation is specific to ALS and results in large inclusions in cases of SOD1-mutant fALS (73), but smaller inclusions and misfolded forms of SOD1 can be observed in all forms of ALS (74). Disease-associated mutations decrease the solubility of SOD1, resulting in higher supersaturation levels than is the case for the wild-type protein.…”

Prion-like Properties of Tau Protein: The Importance of Extracellular Tau as a Therapeutic Target

“…Although much contention exists with regard to mechanisms of Tau secretion (52,53,(73)(74)(75)(76)(77), a recent study on SOD1 aggregates suggests a dual mode of aggregate release. Under this paradigm, healthy cells release SOD1 into the medium in association with exosomes, whereas dying cells release free aggregates (78). However, there is clearly no consensus about release mechanisms involved in normal versus pathophysiology.…”