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...

Cells infected with prions contain both prion protein isoforms cellular prion protein (PrP C ) and scrapie prion protein (PrP Sc ). PrPSc is formed posttranslationally through the pathological refolding of PrP C . In scrapieinfected ScN2a cells, the metabolism of both PrP isoforms involves cholesterol-dependent pathways. We show here that both PrP C and PrP Sc are attached to Triton X-100-insoluble, low-density complexes or "rafts." These complexes are sensitive to saponin and thus probably contain cholesterol. This finding suggests that the transformation PrP C 3 PrP Sc occurs within rafts. It also reveals the existence of rafts in late compartments of the endocytic pathway, where most PrP Sc resides. When Triton X-100 lysates of cells were incubated at 37°C prior to density analysis, PrP C was still found in buoyant complexes, although it now failed to sediment at high speed. This property was shared by another glycophosphatidyl inositol protein, Thy-1, and also by the raft resident GM1. In one ScN2a clone and in the brain of a Syrian hamster with scrapie, Triton X-100 extraction at 37°C permitted resolution of PrP C and PrP Sc into two distinct peaks of different densities. This suggests that there are two populations of PrP-containing rafts and may permit isolation of PrP C -specific rafts from those containing PrP Sc . Our findings reinforce the contention that rafts are involved in various aspects of PrP metabolism and in the "life cycle" of prions.Prions are unique proteinaceous pathogens that cause a series of fatal encephalopathies such as Creutzfeldt-Jakob disease of humans, scrapie of sheep, and bovine spongiform encephalopathy (1). Prions seem to propagate in the host by posttranslationally (2, 3) refolding a normal host protein, the cellular prion protein (PrP C ), 1 to an aberrant conformation (4, 5). The only known component of prions is the misfolded isoform of PrP C , the scrapie prion protein (PrP Sc ) (6, 7). Current evidence argues that direct interaction of PrP Sc with PrP C is a prerequisite for the transformation PrP C ϩ PrP Sc 3 2PrP Sc (8,9). PrP C is a phosphoinositol glycolipid (GPI)-anchored glycoprotein present on the surface of neurons and other cells (10,11). The PrP isoforms appear to be chemically identical (12) but differ in their conformation (4); PrP C contains ϳ40% ␣-helix and is devoid of ␤-sheet, whereas PrP Sc has more than 40% ␤-sheet (4, 13-16). The two PrP isoforms differ considerably in their properties; PrP C is readily soluble in most detergents and is completely degraded by proteases, whereas PrP Sc is insoluble in detergents, possesses a protease-resistant core termed PrP27-30, and polymerizes into amyloidic structures called prion rods (17,18). Since no isoform-specific PrP antibody has yet been developed, the disparate properties of PrP C and PrP Sc serve as the sole ways to differentiate experimentally between these proteins. The subcellular sites where PrP Sc is formed, and the trafficking pathways leading to these sites, remain largely unknown. Scrapie-infecte...

In neurons, posttranslational modification by palmitate regulates the trafficking and function of signaling molecules, neurotransmitter receptors, and associated synaptic scaffolding proteins. However, the enzymatic machinery involved in protein palmitoylation has remained elusive. Here, using biochemical assays, we show that huntingtin (htt) interacting protein, HIP14, is a neuronal palmitoyl transferase (PAT). HIP14 shows remarkable substrate specificity for neuronal proteins, including SNAP-25, PSD-95, GAD65, synaptotagmin I, and htt. Conversely, HIP14 is catalytically invariant toward paralemmin and synaptotagmin VII. Exogenous HIP14 enhances palmitoylation-dependent vesicular trafficking of several acylated proteins in both heterologous cells and neurons. Moreover, interference with endogenous expression of HIP14 reduces clustering of PSD-95 and GAD65 in neurons. These findings define HIP14 as a mammalian palmitoyl transferase involved in the palmitoylation and trafficking of multiple neuronal proteins.