Role of mutant SOD1 disulfide oxidation and aggregation in the pathogenesis of familial ALS

Transgenic mice that model familial (f)ALS, caused by mutations in superoxide dismutase (SOD)1, develop paralysis with pathology that includes the accumulation of aggregated forms of the mutant protein. Using a highly sensitive detergent extraction assay, we traced the appearance and abundance of detergent-insoluble and disulfide cross-linked aggregates of SOD1 throughout the disease course of SOD1-fALS mice (G93A, G37R, and H46R/H48Q). We demonstrate that the accumulation of disulfide cross-linked, detergent-insoluble, aggregates of mutant SOD1 occurs primarily in the later stages of the disease, concurrent with the appearance of rapidly progressing symptoms. We find no evidence for a model in which aberrant intermolecular disulfide bonding has an important role in promoting the aggregation of mutant SOD1, instead, such cross-linking appears to be a secondary event. Also, using both cell culture and mouse models, we find that mutant protein lacking the normal intramolecular disulfide bond is a major component of the insoluble SOD1 aggregates. Overall, our findings suggest a model in which soluble forms of mutant SOD1 initiate disease with larger aggregates implicated only in rapidly progressing events in the final stages of disease. Within the final stages of disease, abnormalities in the oxidation of a normal intramolecular disulfide bond in mutant SOD1 facilitate the aggregation of mutant protein.     

Mechanism of misfolding of the human prion protein revealed by a pathological mutation

The misfolding and aggregation of the human prion protein (PrP) is associated with transmissible spongiform encephalopathies (TSEs). Intermediate conformations forming during the conversion of the cellular form of PrP into its pathological scrapie conformation are key drivers of the misfolding process. Here, we analyzed the properties of the C-terminal domain of the human PrP (huPrP) and its T183A variant, which is associated with familial forms of TSEs. We show that the mutation significantly enhances the aggregation propensity of huPrP, such as to uniquely induce amyloid formation under physiological conditions by the sole C-terminal domain of the protein. Using NMR spectroscopy, biophysics, and metadynamics simulations, we identified the structural characteristics of the misfolded intermediate promoting the aggregation of T183A huPrP and the nature of the interactions that prevent this species to be populated in the wild-type protein. In support of these conclusions, POM antibodies targeting the regions that promote PrP misfolding were shown to potently suppress the aggregation of this amyloidogenic mutant.

The misfolding and aggregation of the human prion protein (PrP) is associated with a number of fatal neurodegenerative disorders designated as transmissible spongiform encephalopathies (TSEs), including Kuru, Creutzfeldt–Jakob disease, fatal familial insomnia, and mad cow disease (1). In its physiological form, the cellular PrP (PrP^C) is a 23-kDa monomeric glycosylated protein that is linked to the outer surface of the neuronal plasma membrane via a glycophosphatidylinositol (GPI) anchor (2). Under conditions associated with TSEs, PrP^C misfolds into a nonnative conformation (scrapie PrP^Sc) that is prone to aggregation into insoluble amyloid fibrils (3). The conversion into PrP^Sc has been proposed to be able to propagate from one cell to another (4). Genetic traits also exist linking point mutations of PrP and familial forms of TSEs. These pathological mutations are mostly located in the C-terminal globular domain of the PrP^C and have been shown to generally enhance the aggregation propensity of PrP (5, 6).While the pathological relevance of PrP is now established, its function remains highly debated. PrP^C has been shown to be involved in the maintenance of myelin on peripheral nerves (7), and evidence exists for a number of other putative physiological roles, including calcium modulation, copper sensing, long-term potentiation, and long-term memory (8). A well-characterized property of the physiological PrP^C is its native structure, which is composed of a disordered N-terminal flexible tail (residues 23–124) and a structured C-terminal region (125–230) composed of three α-helices and a short antiparallel β-sheet (9–11). Recently, structures of amyloid states of PrP generated in vitro have been resolved, providing insights about the possible in vivo form of PrP^Sc (12, 13). Despite the progress made in the structural characterization of PrP^Sc, there are major gaps in our understanding of the mechanisms that trigger the misfolding and aggregation of PrP^C under conditions associated with the insurgence and development of TSEs. A major challenge in this context is the study of metastable misfolding intermediates, which are elusive to conventional experimental techniques for structure determination because of their transient and heterogeneous nature (14). It is now generally acknowledged that the initial step for the misfolding of the C-terminal domain of PrP^C is the disruption of the native packing of the region formed by the two β-strands S1 and S2 and the α-helix H1, against the region composed of the helices H2–H3 (15). The detachment of these two subdomains, denoted as S1–H1–S2 and H2–H3, respectively, is prevented under native conditions by four main intramolecular “gatekeeper” interactions (D178–R164, T183–Y162, H187–R156, D202–R156). Each of these interactions is altered by specific pathological mutations associated with inherited forms of TSEs (D178N, T183A, H187R, D202N), suggesting a destabilization of the native interface between the two subdomains (15). Among these PrP variants, a mutation associated with very early-onset dementia and spongiform encephalopathy in patients, namely T183A (16), abolishes a crucial hydrogen bond between Y162 from the β-strand S2 and T183 from the α-helix H3. Under physiological conditions, this H-bond stabilizes the packing at the interface between the subdomains S1–H1–S2 and H2–H3 (SI Appendix, Fig. S1) (17), and its depletion by T183A induces the strongest destabilization of the PrP^C structure among the TSE-associated PrP variants (18).We addressed in the present study the fundamental early molecular mechanism of human PrP^C (huPrP^C) misfolding induced by T183A using NMR experiments in combination with biophysical investigations and enhanced molecular metadynamics simulations. The study compared the properties of wild-type (WT) and T183A huPrP^C and identified a misfolded intermediate species that acts as a precursor to the formation of amyloid aggregates by the C-terminal domain of the mutant (residues 125–230) despite the fact that this construct lacks the amyloidogenic region 106–126 (19). We provide conclusive evidence of this aggregation mechanism using POM antibodies (Abs) targeting the specific epitope that was here found to initiate the misfolding of T183A huPrP^C. Taken together, these results generate a new detailed understanding of the structural transitions in huPrP^C that trigger its amyloid formation and provide proof of principle for a structure-based identification of targeted molecular strategies to prevent huPrP^C misfolding and aggregation.     

Non-glycosylphosphatidylinositol (GPI)-anchored recombinant prion protein with dominant-negative mutation inhibits PrPSc replication in vitro

Dominant-negative mouse prion protein (PrP) with a lysine mutation at codon 218 (Q218K) is known to inhibit prion replication. In order to gain further mechanistic insight into such dominant negative inhibition, non-glycosylphosphatidylinositol (GPI)-anchored recombinant PrP with Q218K (rPrP-Q218K) was investigated. When applied into scrapie-infected mouse neuroblastoma (ScN2a) cells, rPrP-Q218K but not wild-type rPrP (rPrP-WT) exclusively inhibited abnormal protease-resistant pathogenic isoform (PrP^Sc) replication without reducing the viability of the cells. It was even more efficient than quinacrine, which has already been prescribed for sporadic Creutzfeldt-Jakob disease (CJD) patients; 50% effective concentration (EC₅₀)?=?0.20?μM, 99% effective concentration (EC₉₉)?=?0.86?μM vs. EC₅₀?=?0.45?μM, EC₉₉?=?1.5?μM. Besides, no apparent cell damage was observed at the concentration of up to 4.3?μM (100?μg/ml). In combination treatment with 0.43?μM (10?μg/ml) of rPrP-Q218K, EC₉₉ of quinacrine was decreased from 1.5?μM to 0.5?μM, and the cell viability was recovered from 50% to over 90% as inversely proportional to the concentration of quinacrine. Such combination could alleviate the side effects of quinacrine by reducing its effective concentration without changing or even acceleration the inhibition efficacy. Since homogeneous, high-quality rPrPs could be easily prepared from Escherichia coli in large quantities, rPrP-Q218K is a good candidate for a prion replication antagonist.