D-peptide-magnetic nanoparticles fragment tau fibrils and rescue behavioral deficits in a mouse model of Alzheimer's disease.

Amyloid fibrils of tau are increasingly accepted as a cause of neuronal death and brain atrophy in Alzheimer's disease (AD). Diminishing tau aggregation is a promising strategy in the search for efficacious AD therapeutics. Previously, our laboratory designed a six-residue, nonnatural amino acid inhibitor D-TLKIVW peptide (6-DP), which can prevent tau aggregation in vitro. However, it cannot block cell-to-cell transmission of tau aggregation. Here, we find D-TLKIVWC (7-DP), a d-cysteine extension of 6-DP, not only prevents tau aggregation but also fragments tau fibrils extracted from AD brains to neutralize their seeding ability and protect neuronal cells from tau-induced toxicity. To facilitate the transport of 7-DP across the blood-brain barrier, we conjugated it to magnetic nanoparticles (MNPs). The MNPs-DP complex retains the inhibition and fragmentation properties of 7-DP alone. Ten weeks of MNPs-DP treatment appear to reverse neurological deficits in the PS19 mouse model of AD. This work offers a direction for development of therapies to target tau fibrils.

Cryo-EM structures of the D290V mutant of the hnRNPA2 low-complexity domain suggests how D290V affects phase separation and aggregation.

Heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2) is a human ribonucleoprotein that transports RNA to designated locations for translation via its ability to phase separate. Its mutated form, D290V, is implicated in multisystem proteinopathy known to afflict two families, mainly with myopathy and Paget's disease of bone. Here, we investigate this mutant form of hnRNPA2 by determining cryo-EM structures of the recombinant D290V low complexity domain. We find that the mutant form of hnRNPA2 differs from the WT fibrils in four ways. In contrast to the WT fibrils, the PY-nuclear localization signals in the fibril cores of all three mutant polymorphs are less accessible to chaperones. Also, the mutant fibrils are more stable than WT fibrils as judged by phase separation, thermal stability, and energetic calculations. Similar to other pathogenic amyloids, the mutant fibrils are polymorphic. Thus, these structures offer evidence to explain how a D-to-V missense mutation diverts the assembly of reversible, functional amyloid-like fibrils into the assembly of pathogenic amyloid, and may shed light on analogous conversions occurring in other ribonucleoproteins that lead to neurological diseases such as amyotrophic lateral sclerosis and frontotemporal dementia.

Fibril structures of TFG protein mutants validate the identification of TFG as a disease-related amyloid protein by the IMPAcT method.

We previously presented a bioinformatic method for identifying diseases that arise from a mutation in a protein's low-complexity domain that drives the protein into pathogenic amyloid fibrils. One protein so identified was the tropomyosin-receptor kinase-fused gene protein (TRK-fused gene protein or TFG). Mutations in TFG are associated with degenerative neurological conditions. Here, we present experimental evidence that confirms our prediction that these conditions are amyloid-related. We find that the low-complexity domain of TFG containing the disease-related mutations G269V or P285L forms amyloid fibrils, and we determine their structures using cryo-electron microscopy (cryo-EM). These structures are unmistakably amyloid in nature and confirm the propensity of the mutant TFG low-complexity domain to form amyloid fibrils. Also, despite resulting from a pathogenic mutation, the fibril structures bear some similarities to other amyloid structures that are thought to be nonpathogenic and even functional, but there are other factors that support these structures' relevance to disease, including an increased propensity to form amyloid compared with the wild-type sequence, structure-stabilizing influence from the mutant residues themselves, and double-protofilament amyloid cores. Our findings elucidate two potentially disease-relevant structures of a previously unknown amyloid and also show how the structural features of pathogenic amyloid fibrils may not conform to the features commonly associated with pathogenicity.

Structure-based design of nanobodies that inhibit seeding of Alzheimer's patient-extracted tau fibrils.

Despite much effort, antibody therapies for Alzheimer's disease (AD) have shown limited efficacy. Challenges to the rational design of effective antibodies include the difficulty of achieving specific affinity to critical targets, poor expression, and antibody aggregation caused by buried charges and unstructured loops. To overcome these challenges, we grafted previously determined sequences of fibril-capping amyloid inhibitors onto a camel heavy chain antibody scaffold. These sequences were designed to cap fibrils of tau, known to form the neurofibrillary tangles of AD, thereby preventing fibril elongation. The nanobodies grafted with capping inhibitors blocked tau aggregation in biosensor cells seeded with postmortem brain extracts from AD and progressive supranuclear palsy (PSP) patients. The tau capping nanobody inhibitors also blocked seeding by recombinant tau oligomers. Another challenge to the design of effective antibodies is their poor blood-brain barrier (BBB) penetration. In this study, we also designed a bispecific nanobody composed of a nanobody that targets a receptor on the BBB and a tau capping nanobody inhibitor, conjoined by a flexible linker. We provide evidence that the bispecific nanobody improved BBB penetration over the tau capping inhibitor alone after intravenous administration in mice. Our results suggest that the design of synthetic antibodies that target sequences that drive protein aggregation may be a promising approach to inhibit the prion-like seeding of tau and other proteins involved in AD and related proteinopathies.

CRISPR screens in iPSC-derived neurons reveal principles of tau proteostasis.

A hallmark of age-associated neurodegenerative diseases is the aggregation of proteins. Aggregation of the protein tau defines tauopathies, which include Alzheimer's disease and frontotemporal dementia. Specific neuronal subtypes are selectively vulnerable to the accumulation of tau aggregates, and subsequent dysfunction and death. The mechanisms underlying cell type-selective vulnerability are unknown. To systematically uncover the cellular factors controlling the accumulation of tau aggregates in human neurons, we conducted a genome-wide CRISPRi-based modifier screen in iPSC-derived neurons. The screen uncovered expected pathways, including autophagy, but also unexpected pathways including UFMylation and GPI anchor synthesis, that control tau oligomer levels. We identify the E3 ubiquitin ligase CUL5 as a tau interactor and potent modifier of tau levels. In addition, disruption of mitochondrial function increases tau oligomer levels and promotes proteasomal misprocessing of tau. These results reveal new principles of tau proteostasis in human neurons and pinpoint potential therapeutic targets for tauopathies.

Low complexity domains of the nucleocapsid protein of SARS-CoV-2 form amyloid fibrils.

The self-assembly of the Nucleocapsid protein (NCAP) of SARS-CoV-2 is crucial for its function. Computational analysis of the amino acid sequence of NCAP reveals low-complexity domains (LCDs) akin to LCDs in other proteins known to self-assemble as phase separation droplets and amyloid fibrils. Previous reports have described NCAP's propensity to phase-separate. Here we show that the central LCD of NCAP is capable of both, phase separation and amyloid formation. Within this central LCD we identified three adhesive segments and determined the atomic structure of the fibrils formed by each. Those structures guided the design of G12, a peptide that interferes with the self-assembly of NCAP and demonstrates antiviral activity in SARS-CoV-2 infected cells. Our work, therefore, demonstrates the amyloid form of the central LCD of NCAP and suggests that amyloidogenic segments of NCAP could be targeted for drug development.

Small molecules disaggregate alpha-synuclein and prevent seeding from patient brain-derived fibrils.

The amyloid aggregation of alpha-synuclein within the brain is associated with the pathogenesis of Parkinson's disease (PD) and other related synucleinopathies, including multiple system atrophy (MSA). Alpha-synuclein aggregates are a major therapeutic target for treatment of these diseases. We identify two small molecules capable of disassembling preformed alpha-synuclein fibrils. The compounds, termed CNS-11 and CNS-11g, disaggregate recombinant alpha-synuclein fibrils in vitro, prevent the intracellular seeded aggregation of alpha-synuclein fibrils, and mitigate alpha-synuclein fibril cytotoxicity in neuronal cells. Furthermore, we demonstrate that both compounds disassemble fibrils extracted from MSA patient brains and prevent their intracellular seeding. They also reduce in vivo alpha-synuclein aggregates in C. elegans. Both compounds also penetrate brain tissue in mice. A molecular dynamics-based computational model suggests the compounds may exert their disaggregating effects on the N terminus of the fibril core. These compounds appear to be promising therapeutic leads for targeting alpha-synuclein for the treatment of synucleinopathies.

Structure-based discovery of small molecules that disaggregate Alzheimer's disease tissue derived tau fibrils in vitro.

Alzheimer's disease (AD) is the consequence of neuronal death and brain atrophy associated with the aggregation of protein tau into fibrils. Thus disaggregation of tau fibrils could be a therapeutic approach to AD. The small molecule EGCG, abundant in green tea, has long been known to disaggregate tau and other amyloid fibrils, but EGCG has poor drug-like properties, failing to fully penetrate the brain. Here we have cryogenically trapped an intermediate of brain-extracted tau fibrils on the kinetic pathway to EGCG-induced disaggregation and have determined its cryoEM structure. The structure reveals that EGCG molecules stack in polar clefts between the paired helical protofilaments that pathologically define AD. Treating the EGCG binding position as a pharmacophore, we computationally screened thousands of drug-like compounds for compatibility for the pharmacophore, discovering several that experimentally disaggregate brain-derived tau fibrils in vitro. This work suggests the potential of structure-based, small-molecule drug discovery for amyloid diseases.

De novo designed protein inhibitors of amyloid aggregation and seeding.

Neurodegenerative diseases are characterized by the pathologic accumulation of aggregated proteins. Known as amyloid, these fibrillar aggregates include proteins such as tau and amyloid-ß (Aß) in Alzheimer's disease (AD) and alpha-synuclein (αSyn) in Parkinson's disease (PD). The development and spread of amyloid fibrils within the brain correlates with disease onset and progression, and inhibiting amyloid formation is a possible route toward therapeutic development. Recent advances have enabled the determination of amyloid fibril structures to atomic-level resolution, improving the possibility of structure-based inhibitor design. In this work, we use these amyloid structures to design inhibitors that bind to the ends of fibrils, "capping" them so as to prevent further growth. Using de novo protein design, we develop a library of miniprotein inhibitors of 35 to 48 residues that target the amyloid structures of tau, Aß, and αSyn. Biophysical characterization of top in silico designed inhibitors shows they form stable folds, have no sequence similarity to naturally occurring proteins, and specifically prevent the aggregation of their targeted amyloid-prone proteins in vitro. The inhibitors also prevent the seeded aggregation and toxicity of fibrils in cells. In vivo evaluation reveals their ability to reduce aggregation and rescue motor deficits in Caenorhabditis elegans models of PD and AD.

Cryo-EM structure of RNA-induced tau fibrils reveals a small C-terminal core that may nucleate fibril formation.

In neurodegenerative diseases including Alzheimer's and amyotrophic lateral sclerosis, proteins that bind RNA are found in aggregated forms in autopsied brains. Evidence suggests that RNA aids nucleation of these pathological aggregates; however, the mechanism has not been investigated at the level of atomic structure. Here, we present the 3.4-Å resolution structure of fibrils of full-length recombinant tau protein in the presence of RNA, determined by electron cryomicroscopy (cryo-EM). The structure reveals the familiar in-register cross-ß amyloid scaffold but with a small fibril core spanning residues Glu391 to Ala426, a region disordered in the fuzzy coat in all previously studied tau polymorphs. RNA is bound on the fibril surface to the positively charged residues Arg406 and His407 and runs parallel to the fibril axis. The fibrils dissolve when RNase is added, showing that RNA is necessary for fibril integrity. While this structure cannot exist simultaneously with the tau fibril structures extracted from patients' brains, it could conceivably account for the nucleating effects of RNA cofactors followed by remodeling as fibrils mature.

Bioinformatic identification of previously unrecognized amyloidogenic proteins.

Low-complexity domains (LCDs) of proteins have been shown to self-associate, and pathogenic mutations within these domains often drive the proteins into amyloid aggregation associated with disease. These domains may be especially susceptible to amyloidogenic mutations because they are commonly intrinsically disordered and function in self-association. The question therefore arises whether a search for pathogenic mutations in LCDs of the human proteome can lead to identification of other proteins associated with amyloid disease. Here, we take a computational approach to identify documented pathogenic mutations within LCDs that may favor amyloid formation. Using this approach, we identify numerous known amyloidogenic mutations, including several such mutations within proteins previously unidentified as amyloidogenic. Among the latter group, we focus on two mutations within the TRK-fused gene protein (TFG), known to play roles in protein secretion and innate immunity, which are associated with two different peripheral neuropathies. We show that both mutations increase the propensity of TFG to form amyloid fibrils. We therefore conclude that TFG is a novel amyloid protein and propose that the diseases associated with its mutant forms may be amyloidoses.

Amyloid fibrils in FTLD-TDP are composed of TMEM106B and not TDP-43.

Frontotemporal lobar degeneration (FTLD) is the third most common neurodegenerative condition after Alzheimer's and Parkinson's diseases1. FTLD typically presents in 45 to 64 year olds with behavioural changes or progressive decline of language skills2. The subtype FTLD-TDP is characterized by certain clinical symptoms and pathological neuronal inclusions with TAR DNA-binding protein (TDP-43) immunoreactivity3. Here we extracted amyloid fibrils from brains of four patients representing four of the five FTLD-TDP subclasses, and determined their structures by cryo-electron microscopy. Unexpectedly, all amyloid fibrils examined were composed of a 135-residue carboxy-terminal fragment of transmembrane protein 106B (TMEM106B), a lysosomal membrane protein previously implicated as a genetic risk factor for FTLD-TDP4. In addition to TMEM106B fibrils, we detected abundant non-fibrillar aggregated TDP-43 by immunogold labelling. Our observations confirm that FTLD-TDP is associated with amyloid fibrils, and that the fibrils are formed by TMEM106B rather than TDP-43.

Cryo-EM structures of hIAPP fibrils seeded by patient-extracted fibrils reveal new polymorphs and conserved fibril cores.

Amyloidosis of human islet amyloid polypeptide (hIAPP) is a pathological hallmark of type II diabetes (T2D), an epidemic afflicting nearly 10% of the world's population. To visualize disease-relevant hIAPP fibrils, we extracted amyloid fibrils from islet cells of a T2D donor and amplified their quantity by seeding synthetic hIAPP. Cryo-EM studies revealed four fibril polymorphic atomic structures. Their resemblance to four unseeded hIAPP fibrils varies from nearly identical (TW3) to non-existent (TW2). The diverse repertoire of hIAPP polymorphs appears to arise from three distinct protofilament cores entwined in different combinations. The structural distinctiveness of TW1, TW2 and TW4 suggests they may be faithful replications of the pathogenic seeds. If so, the structures determined here provide the most direct view yet of hIAPP amyloid fibrils formed during T2D.

Inhibition of amyloid formation of the Nucleoprotein of SARS-CoV-2.

The SARS-CoV-2 Nucleoprotein (NCAP) functions in RNA packaging during viral replication and assembly. Computational analysis of its amino acid sequence reveals a central low-complexity domain (LCD) having sequence features akin to LCDs in other proteins known to function in liquid-liquid phase separation. Here we show that in the presence of viral RNA, NCAP, and also its LCD segment alone, form amyloid-like fibrils when undergoing liquid-liquid phase separation. Within the LCD we identified three 6-residue segments that drive amyloid fibril formation. We determined atomic structures for fibrils formed by each of the three identified segments. These structures informed our design of peptide inhibitors of NCAP fibril formation and liquid-liquid phase separation, suggesting a therapeutic route for Covid-19. ONE SENTENCE SUMMARY: Atomic structures of amyloid-driving peptide segments from SARS-CoV-2 Nucleoprotein inform the development of Covid-19 therapeutics.

Crystal structure of a conformational antibody that binds tau oligomers and inhibits pathological seeding by extracts from donors with Alzheimer's disease.

Soluble oligomers of aggregated tau accompany the accumulation of insoluble amyloid fibrils, a histological hallmark of Alzheimer disease (AD) and two dozen related neurodegenerative diseases. Both oligomers and fibrils seed the spread of Tau pathology, and by virtue of their low molecular weight and relative solubility, oligomers may be particularly pernicious seeds. Here, we report the formation of in vitro tau oligomers formed by an ionic liquid (IL15). Using IL15-induced recombinant tau oligomers and a dot blot assay, we discovered a mAb (M204) that binds oligomeric tau, but not tau monomers or fibrils. M204 and an engineered single-chain variable fragment (scFv) inhibited seeding by IL15-induced tau oligomers and pathological extracts from donors with AD and chronic traumatic encephalopathy. This finding suggests that M204-scFv targets pathological structures that are formed by tau in neurodegenerative diseases. We found that M204-scFv itself partitions into oligomeric forms that inhibit seeding differently, and crystal structures of the M204-scFv monomer, dimer, and trimer revealed conformational differences that explain differences among these forms in binding and inhibition. The efficiency of M204-scFv antibodies to inhibit the seeding by brain tissue extracts from different donors with tauopathies varied among individuals, indicating the possible existence of distinct amyloid polymorphs. We propose that by binding to oligomers, which are hypothesized to be the earliest seeding-competent species, M204-scFv may have potential as an early-stage diagnostic for AD and tauopathies, and also could guide the development of promising therapeutic antibodies.

Methyl arachidonyl fluorophosphonate inhibits Mycobacterium tuberculosis thioesterase TesA and globally affects vancomycin susceptibility.

Phthiocerol dimycocerosates and phenolic glycolipids (PGL) are considered as major virulence elements of Mycobacterium tuberculosis, in particular because of their involvement in cell wall impermeability and drug resistance. The biosynthesis of these waxy lipids involves multiple enzymes, including thioesterase A (TesA). We observed that purified recombinant M. tuberculosis TesA is able to dimerize in the presence of palmitoyl-CoA and our 3D structure model of TesA with this acyl-CoA suggests hydrophobic interaction requirement for dimerization. Furthermore, we identified that methyl arachidonyl fluorophosphonate, which inhibits TesA by covalently modifying the catalytic serine, also displays a synergistic antimicrobial activity with vancomycin further warranting the development of TesA inhibitors as valuable antituberculous drug candidates.

Structural evidence for the critical role of the prion protein hydrophobic region in forming an infectious prion.

Prion or PrPSc is the proteinaceous infectious agent causing prion diseases in various mammalian species. Despite decades of research, the structural basis for PrPSc formation and prion infectivity remains elusive. To understand the role of the hydrophobic region in forming infectious prion at the molecular level, we report X-ray crystal structures of mouse (Mo) prion protein (PrP) (residues 89-230) in complex with a nanobody (Nb484). Using the recombinant prion propagation system, we show that the binding of Nb484 to the hydrophobic region of MoPrP efficiently inhibits the propagation of proteinase K resistant PrPSc and prion infectivity. In addition, when added to cultured mouse brain slices in high concentrations, Nb484 exhibits no neurotoxicity, which is drastically different from other neurotoxic anti-PrP antibodies, suggesting that the Nb484 can be a potential therapeutic agent against prion disease. In summary, our data provides the first structure-function evidence supporting a crucial role of the hydrophobic region of PrP in forming an infectious prion.

Correction to: In Vitro Seeding Activity of Glycoform-Deficient Prions from Variably Protease-Sensitive Prionopathy and Familial CJD Associated with PrPV180I Mutation.

The original version of this article unfortunately contained a mistake. The email address Dr. Wen-Quan Zou, one of the corresponding authors should be written as "wxz6@case.edu" instead of "wxz@case.edu".

In Vitro Seeding Activity of Glycoform-Deficient Prions from Variably Protease-Sensitive Prionopathy and Familial CJD Associated with PrPV180I Mutation.

Both sporadic variably protease-sensitive prionopathy (VPSPr) and familial Creutzfeldt-Jakob disease linked to the prion protein (PrP) V180I mutation (fCJDV180I) have been found to share a unique pathological prion protein (PrPSc) that lacks the protease-resistant PrPSc glycosylated at residue 181 because two of four PrP glycoforms are apparently not converted into the PrPSc from their cellular PrP (PrPC). To investigate the seeding activity of these unique PrPSc molecules, we conducted in vitro prion conversion experiments using serial protein misfolding cyclic amplification (sPMCA) and real-time quaking-induced conversion (RT-QuIC) assays with different PrPC substrates. We observed that the seeding of PrPSc from VPSPr or fCJDV180I in the sPMCA reaction containing normal human or humanized transgenic (Tg) mouse brain homogenates generated PrPSc molecules that unexpectedly exhibited a dominant diglycosylated PrP isoform along with PrP monoglycosylated at residue 181. The efficiency of PrPSc amplification was significantly higher in non-CJDMM than in non-CJDVV human brain homogenate, whereas it was higher in normal TgVV than in TgMM mouse brain homogenate. PrPC from the mixture of normal TgMM and Tg mouse brain expressing PrPV180I mutation (Tg180) but not TgV180I alone was converted into PrPSc by seeding with the VPSPr or fCJDV180I. The RT-QuIC seeding activity of PrPSc from VPSPr and fCJDV180I was significantly lower than that of sCJD. Our results suggest that the formation of glycoform-selective prions may be associated with an unidentified factor in the affected brain and the glycoform-deficiency of PrPSc does not affect the glycoforms of in vitro newly amplified PrPSc.

Prion infectivity is encoded exclusively within the structure of proteinase K-resistant fragments of synthetically generated recombinant PrPSc.

Transmissible spongiform encephalopathies, also known as prion diseases, are a group of fatal neurodegenerative disorders affecting both humans and animals. The central pathogenic event in prion disease is the misfolding of normal prion protein (PrPC) into the pathogenic conformer, PrPSc, which self-replicates by converting PrPC to more of itself. The biochemical hallmark of PrPSc is its C-terminal resistance to proteinase K (PK) digestion, which has been historically used to define PrPSc and is still the most widely used characteristic for prion detection. We used PK-resistance as a biochemical measure for the generation of recombinant prion from bacterially expressed recombinant PrP. However, the existence of both PK- resistant and -sensitive PrPSc forms in animal and human prion disease led to the question of whether the in vitro-generated recombinant prion infectivity is due to the PK-resistant or -sensitive recombinant PrP forms. In this study, we compared undigested and PK-digested recombinant prions for their infectivity using both the classical rodent bioassay and the cell-based prion infectivity assay. Similar levels of infectivity were detected in PK-digested and -undigested samples by both assays. A time course study of recombinant prion propagation showed that the increased capability to seed the conversion of endogenous PrP in cultured cells coincided with an increase of the PK-resistant form of recombinant PrP. Moreover, prion infectivity diminished when recombinant prion was subjected to an extremely harsh PK digestion. These results demonstrated that the infectivity of recombinant prion is encoded within the structure of the PK-resistant PrP fragments. This characteristic of recombinant prion, that a simple PK digestion is able to eliminate all PK-sensitive (non-infectious) PrP species, makes possible a more homogenous material that will be ideal for dissecting the molecular basis of prion infectivity.