Globular domain of the prion protein needs to be unlocked by domain swapping to support prion protein conversion

Prion diseases are fatal transmissible neurodegenerative diseases affecting many mammalian species. The normal prion protein (PrP) converts into a pathological aggregated form, PrPSc, which is enriched in the β-sheet structure. While the high resolution structure of the normal PrP was determined, the structure of the converted form of PrP remains inaccessible to high resolution techniques. In order to map the PrP conversion process we introduced disulfide bridges into different positions within the globular domain of PrP, tethering selected secondary structure elements. The majority of tethered PrP mutants exhibited increased thermodynamic stability, nevertheless they converted efficiently. Only the disulfides which tether subdomain B1-H1-B2 to subdomain H2-H3 prevented PrP conversion in vitro and in prion infected cell cultures. Reduction of disulfides recovered the ability of these mutants to convert, demonstrating that the separation of subdomains is an essential step in conversion. Formation of disulfide-linked proteinase K-resistant dimers in fibrils composed of a pair of single cysteine mutants supports the model based on domain-swapped dimers as the building blocks of prion fibrils. In contrast to previously proposed structural models of PrPSc suggesting conversion of large secondary structure segments, we provide evidence for the conservation of secondary structure elements of the globular domain upon PrP conversion. Previous studies already showed that dimerization is the rate-limiting step in PrP conversion. We show that separation and swapping of subdomains of the globular domain is necessary for conversion. Therefore, we propose that domain-swapped dimer of PrP precedes amyloid formation and represents a potential target for therapeutic intervention

The POM Monoclonals: A Comprehensive Set of Antibodies to Non-Overlapping Prion Protein Epitopes

PrPSc, a misfolded and aggregated form of the cellular prion protein PrPC, is the only defined constituent of the transmissible agent causing prion diseases. Expression of PrPC in the host organism is necessary for prion replication and for prion neurotoxicity. Understanding prion diseases necessitates detailed structural insights into PrPC and PrPSc. Towards this goal, we have developed a comprehensive collection of monoclonal antibodies denoted POM1 to POM19 and directed against many different epitopes of mouse PrPC. Three epitopes are located within the N-terminal octarepeat region, one is situated within the central unstructured region, and four epitopes are discontinuous within the globular C-proximal domain of PrPC. Some of these antibodies recognize epitopes that are resilient to protease digestion in PrPSc. Other antibodies immunoprecipitate PrPC, but not PrPSc. A third group was found to immunoprecipitate both PrP isoforms. Some of the latter antibodies could be blocked with epitope-mimicking peptides, and incubation with an excess of these peptides allowed for immunochromatography of PrPC and PrPSc. Amino-proximal antibodies were found to react with repetitive PrPC epitopes, thereby vastly increasing their avidity. We have also created functional single-chain miniantibodies from selected POMs, which retained the binding characteristics despite their low molecular mass. The POM collection, thus, represents a unique set of reagents allowing for studies with a variety of techniques, including western blotting, ELISA, immunoprecipitation, conformation-dependent immunoassays, and plasmon surface plasmon resonance-based assays

FTLD-TDP assemblies seed neoaggregates with subtype-specific features via a prion-like cascade

Morphologically distinct TDP-43 aggregates occur in clinically different FTLD-TDP subtypes, yet the mechanism of their emergence and contribution to clinical heterogeneity are poorly understood. Several lines of evidence suggest that pathological TDP-43 follows a prion-like cascade, but the molecular determinants of this process remain unknown. We use advanced microscopy techniques to compare the seeding properties of pathological FTLD-TDP-A and FTLD-TDP-C aggregates. Upon inoculation of patient-derived aggregates in cells, FTLD-TDP-A seeds amplify in a template-dependent fashion, triggering neoaggregation more efficiently than those extracted from FTLD-TDP-C patients, correlating with the respective disease progression rates. Neoaggregates are sequentially phosphorylated with N-to-C directionality and with subtype-specific timelines. The resulting FTLD-TDP-A neoaggregates are large and contain densely packed fibrils, reminiscent of the pure compacted fibrils present within cytoplasmic inclusions in postmortem brains. In contrast, FTLD-TDP-C dystrophic neurites show less dense fibrils mixed with cellular components, and their respective neoaggregates are small, amorphous protein accumulations. These cellular seeding models replicate aspects of the patient pathological diversity and will be a useful tool in the quest for subtype-specific therapeutics

An Arrayed Genome-Wide Perturbation Screen Identifies the Ribonucleoprotein hnRNP K As Rate-Limiting for Prion Propagation

A defining characteristic of mammalian prions is their capacity for self-sustained propagation. Theoretical considerations and experimental evidence suggest that prion propagation is modulated by cell-autonomous and non-autonomous modifiers. Using a novel quantitative phospholipase protection assay (QUIPPER) for high-throughput prion measurements, we performed an arrayed genome-wide RNA interference (RNAi) screen aimed at detecting modifiers of prion propagation. We exposed prion-infected cells in high-density microplates to 35’364 ternary pools of 52’746 siRNAs targeting 17’582 genes representing the mouse protein-coding transcriptome. We identified 1191 modulators of prion propagation. While 1151 of these modified the expression of both the pathological prion protein, PrP$^{Sc}$, and its cellular counterpart PrP$^{C}$, 40 genes affected selectively PrP$^{Sc}$. Of the latter, 20 genes augmented prion production when suppressed. A prominent limiter of prion propagation was the heterogeneous nuclear ribonucleoprotein Hnrnpk. Psammaplysene A (PSA), which binds Hnrnpk, reduced prion levels in cultured cells and protected them from cytotoxicity. PSA also reduced prion levels in infected cerebellar organotypic slices and alleviated locomotor deficits in prion-infected Drosophila melanogaster expressing ovine PrP$^{C}$. Hence, genome-wide QUIPPER-based perturbations can discover actionable cellular pathways involved in prion propagation. Finally, the unexpected identification of a prioncontrolling ribonucleoprotein suggests a role for RNA in the generation of infectious prions

Comprehensive evaluation of human-derived anti-poly-GA antibodies in cellular and animal models of C9orf72 disease

Hexanucleotide G4C2 repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Dipeptide repeat proteins (DPRs) generated by translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intra-cellular poly-GA and reduced aggregate formation in a poly-GA overexpressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing G4C2 repeats by systemic antibody delivery for up to 16 mo was well-tolerated and led to measurable brain penetration of antibodies. Long-term treatment with anti-GA antibodies produced improvement in an open-field movement test in aged C9orf72450 mice. However, chronic administration of anti-GA antibodies in AAV-(G4C2)149 mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model. Keywords: C9orf72; amyotrophic lateral sclerosis; dipeptide repeat proteins; frontotemporal dementia; immunotherap

Repetitive Immunization Enhances the Susceptibility of Mice to Peripherally Administered Prions

The susceptibility of humans and animals to prion infections is determined by the virulence of the infectious agent, by genetic modifiers, and by hitherto unknown host and environmental risk factors. While little is known about the latter two, the activation state of the immune system was surmised to influence prion susceptibility. Here we administered prions to mice that were repeatedly immunized by two initial injections of CpG oligodeoxynucleotides followed by repeated injections of bovine serum albumin/alum. Immunization greatly reduced the required dosage of peripherally administered prion inoculum necessary to induce scrapie in 50% of mice. No difference in susceptibility was observed following intracerebral prion challenge. Due to its profound impact onto scrapie susceptibility, the host immune status may determine disease penetrance after low-dose prion exposure, including those that may give rise to iatrogenic and variant Creutzfeldt-Jakob disease

TDP-43 oligomerization and RNA binding are codependent but their loss elicits distinct pathologies

Aggregation of the RNA-binding protein TDP-43 is the main common neuropathological feature of TDP-43 proteinopathies. In physiological conditions, TDP-43 is predominantly nuclear and contained in biomolecular condensates formed via liquid-liquid phase separation (LLPS). However, in disease, TDP-43 is depleted from these compartments and forms cytoplasmic or, sometimes, intranuclear inclusions. How TDP-43 transitions from physiological to pathological states remains poorly understood. Here, we show that self-oligomerization and RNA binding cooperatively govern TDP-43 stability, functionality, LLPS and cellular localization. Importantly, our data reveal that TDP-43 oligomerization is connected to, and conformationally modulated by, RNA binding. Mimicking the impaired proteasomal activity observed in patients, we found that TDP-43 forms nuclear aggregates via LLPS and cytoplasmic aggregates via aggresome formation. The favored aggregation pathway depended on the TDP-43 state –monomeric/oligomeric, RNA-bound/-unbound– and the subcellular environment –nucleus/cytoplasm. Our work unravels the origins of heterogeneous pathological species occurring in TDP-43 proteinopathies

Loss of TDP-43 oligomerization or RNA binding elicits distinct aggregation patterns

Aggregation of the RNA-binding protein TAR DNA-binding protein 43 (TDP-43) is the key neuropathological feature of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). In physiological conditions, TDP-43 is predominantly nuclear, forms oligomers, and is contained in biomolecular condensates assembled by liquid-liquid phase separation (LLPS). In disease, TDP-43 forms cytoplasmic or intranuclear inclusions. How TDP-43 transitions from physiological to pathological states remains poorly understood. Using a variety of cellular systems to express structure-based TDP-43 variants, including human neurons and cell lines with near-physiological expression levels, we show that oligomerization and RNA binding govern TDP-43 stability, splicing functionality, LLPS, and subcellular localization. Importantly, our data reveal that TDP-43 oligomerization is modulated by RNA binding. By mimicking the impaired proteasomal activity observed in ALS/FTLD patients, we found that monomeric TDP-43 forms inclusions in the cytoplasm, whereas its RNA binding-deficient counterpart aggregated in the nucleus. These differentially localized aggregates emerged via distinct pathways: LLPS-driven aggregation in the nucleus and aggresome-dependent inclusion formation in the cytoplasm. Therefore, our work unravels the origins of heterogeneous pathological species reminiscent of those occurring in TDP-43 proteinopathy patients

A short purification process for quantitative isolation of PrP(Sc) from naturally occurring and experimental transmissible spongiform encephalopathies

BACKGROUND: Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases affecting both humans and animals. They are associated with post-translational conversion of the normal cellular prion protein (PrP(C)) into a heat- and protease-resistant abnormal isoform (PrP(Sc)). Detection of PrP(Sc) in individuals is widely utilized for the diagnosis of prion diseases. METHODS: TSE brain tissue samples have been processed in order to quantitatively isolate PrP(Sc). The protocol includes an initial homogenization, digestion with proteinase K and salt precipitation. RESULTS: Here we show that over 97 percent of the PrP(Sc) present can be precipitated from infected brain material using this simple salting-out procedure for proteins. No chemically harsh conditions are used during the process in order to conserve the native quality of the isolated protein. CONCLUSION: The resulting PrP(Sc)-enriched preparation should provide a suitable substrate for analyzing the structure of the prion agent and for scavenging for other molecules with which it may associate. In comparison with most methods that exist today, the one described in this study is rapid, cost-effective and does not demand expensive laboratory equipment

Functionally Relevant Domains of the Prion Protein Identified In Vivo

The prion consists essentially of PrPSc, a misfolded and aggregated conformer of the cellular protein PrPC. Whereas PrPC deficient mice are clinically healthy, expression of PrPC variants lacking its central domain (PrPΔCD), or of the PrP-related protein Dpl, induces lethal neurodegenerative syndromes which are repressed by full-length PrP. Here we tested the structural basis of these syndromes by grafting the amino terminus of PrPC (residues 1–134), or its central domain (residues 90–134), onto Dpl. Further, we constructed a soluble variant of the neurotoxic PrPΔCD mutant that lacks its glycosyl phosphatidyl inositol (GPI) membrane anchor. Each of these modifications abrogated the pathogenicity of Dpl and PrPΔCD in transgenic mice. The PrP-Dpl chimeric molecules, but not anchorless PrPΔCD, ameliorated the disease of mice expressing truncated PrP variants. We conclude that the amino proximal domain of PrP exerts a neurotrophic effect even when grafted onto a distantly related protein, and that GPI-linked membrane anchoring is necessary for both beneficial and deleterious effects of PrP and its variants