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...

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. Expression of G127X and G85R in mouse cell lines did not induce misfolding of murine wtSOD1, and a species restriction element for human wtSOD1 conversion was mapped to a region of sequence divergence in loop II and β-strand 3 of the SOD1 β-barrel (residues 24-36), then further refined surprisingly to a single tryptophan residue at codon 32 (W32) in human SOD1. Time course experiments enabled by W32 restriction revealed that G127X and misfolded wtSOD1 can induce misfolding of cell-endogenous wtSOD1. Finally, aggregated recombinant G127X is capable of inducing misfolding and protease sensitivity of recombinant human wtSOD1 in a cell-free system containing reducing and chelating agents; cell-free wtSOD1 conversion was also restricted by W32. These observations demonstrate that misfolded SOD1 can induce misfolding of natively structured wtSOD1 in a physiological intracellular milieu, consistent with a direct protein-protein interaction.neurodegeneration | protein misfolding | prion | template-directed misfolding | seeded polymerization

Necdin and Magel2 are related proteins inactivated in Prader-Willi syndrome (PWS), a sporadic chromosomal deletion disorder. We demonstrate that necdin and Magel2 bind to and prevent proteasomal degradation of Fez1, a fasciculation and elongation protein implicated in axonal outgrowth and kinesin-mediated transport, and also bind to the Bardet-Biedl syndrome (BBS) protein BBS4 in co-transfected cells. The interactions among necdin, Magel2, Fez1 and BBS4 occur at or near centrosomes. Centrosomal or pericentriolar dysfunction has previously been implicated in BBS and may also be important in the features of PWS that overlap with BBS, such as learning disabilities, hypogonadism and obesity. Morphological abnormalities in axonal outgrowth and fasciculation manifest in several regions of the nervous system in necdin null mouse embryos, including axons of sympathetic, retinal ganglion cell, serotonergic and catecholaminergic neurons. These data demonstrate that necdin mediates intracellular processes essential for neurite outgrowth and that loss of necdin impinges on axonal outgrowth. We further suggest that loss of necdin contributes to the neurological phenotype of PWS, and raise the possibility that co-deletion of necdin and the related protein Magel2 may explain the lack of single gene mutations in PWS.