VCP regulates early tau seed amplification via specific cofactors.

Background Neurodegenerative tauopathies may progress based on seeding by pathological tau assemblies, whereby an aggregate is released from one cell, gains entry to an adjacent or connected cell, and serves as a specific template for its own replication in the cytoplasm. In vitro seeding reactions typically take days, yet seeding into the complex cytoplasmic milieu happens within hours, implicating a machinery with unknown players that controls this process in the acute phase. Methods We used proximity labeling to identify factors that control seed amplification within 5h of seed exposure. We fused split-APEX2 to the C-terminus of tau repeat domain (RD) to reconstitute peroxidase activity 5h after seeded intracellular tau aggregation. Valosin containing protein (VCP/p97) was the top hit. VCP harbors dominant mutations that underlie two neurodegenerative diseases, multisystem proteinopathy and vacuolar tauopathy, but its mechanistic role is unclear. We used immortalized cells and human neurons to study the effects of VCP on tau seeding. We exposed cells to fibrils or brain homogenates in cell culture media and measured effects on uptake and induction of intracellular tau aggregation following various genetic and chemical manipulations of VCP. Results VCP knockdown reduced tau seeding. Chemical inhibitors had opposing effects on aggregation in HEK293T tau biosensor cells and human neurons alike: ML-240 increased seeding efficiency, whereas NMS-873 decreased it. The inhibitors were effective only when administered within 8h of seed exposure, indicating a role for VCP early in seed processing. We screened 30 VCP co-factors in HEK293T biosensor cells by genetic knockout or knockdown. Reduction of ATXN3, NSFL1C, UBE4B, NGLY1, and OTUB1 decreased tau seeding, as did NPLOC4, which also uniquely increased soluble tau levels. By contrast, reduction of FAF2 increased tau seeding. Conclusions Divergent effects on tau seeding of chemical inhibitors and cofactor reduction indicate that VCP regulates this process. This is consistent with a dedicated cytoplasmic processing complex based on VCP that directs seeds acutely towards degradation vs. amplification.

Cryo-EM structure of Alzheimer's disease tau filaments with PET ligand MK-6240.

Positron Emission Tomography (PET) ligands have advanced Alzheimer's disease (AD) diagnosis and treatment. Using autoradiography and cryo-EM, we identified AD brain tissue with elevated tau burden, purified filaments, and determined the structure of second-generation high avidity PET ligand MK-6240 at 2.31 Å resolution, which bound at a 1:1 ratio within the cleft of tau paired-helical filament (PHF), engaging with glutamine 351, lysine K353, and isoleucine 360. This information elucidates the basis of MK-6240 PET in quantifying PHF deposits in AD and may facilitate the structure-based design of superior ligands against tau amyloids.

VCP increases or decreases tau seeding using specific cofactors.

Background: Neurodegenerative tauopathies may progress based on seeding by pathological tau assemblies, whereby an aggregate is released from one cell, gains entry to an adjacent or connected cell, and serves as a specific template for its own replication in the cytoplasm. In vitro seeding reactions typically take days, yet seeding into the complex cytoplasmic milieu can happen within hours. A cellular machinery might regulate this process, but potential players are unknown. Methods: We used proximity labeling to identify factors that control seed amplification. We fused split-APEX2 to the C-terminus of tau repeat domain (RD) to reconstitute peroxidase activity upon seeded intracellular tau aggregation. We identified valosin containing protein (VCP/p97) 5h after seeding. Mutations in VCP underlie two neurodegenerative diseases, multisystem proteinopathy and vacuolar tauopathy, but its mechanistic role is unclear. We utilized tau biosensors, a cellular model for tau aggregation, to study the effects of VCP on tau seeding. Results: VCP knockdown reduced tau seeding. However, distinct chemical inhibitors of VCP and the proteasome had opposing effects on aggregation, but only when given <8h of seed exposure. ML-240 increased seeding efficiency ~40x, whereas NMS-873 decreased seeding efficiency by 50%, and MG132 increased seeding ~10x. We screened VCP co-factors in HEK293 biosensor cells by genetic knockout or knockdown. Reduction of ATXN3, NSFL1C, UBE4B, NGLY1, and OTUB1 decreased tau seeding, as did NPLOC4, which also uniquely increased soluble tau levels. Reduction of FAF2 and UBXN6 increased tau seeding. Conclusions: VCP uses distinct cofactors to determine seed replication efficiency, consistent with a dedicated cytoplasmic processing complex that directs seeds towards dissolution vs. amplification.

FTD-tau S320F mutation stabilizes local structure and allosterically promotes amyloid motif-dependent aggregation.

Amyloid deposition of the microtubule-associated protein tau is associated with neurodegenerative diseases. In frontotemporal dementia with abnormal tau (FTD-tau), missense mutations in tau enhance its aggregation propensity. Here we describe the structural mechanism for how an FTD-tau S320F mutation drives spontaneous aggregation, integrating data from in vitro, in silico and cellular experiments. We find that S320F stabilizes a local hydrophobic cluster which allosterically exposes the 306VQIVYK311 amyloid motif; identify a suppressor mutation that destabilizes S320F-based hydrophobic clustering reversing the phenotype in vitro and in cells; and computationally engineer spontaneously aggregating tau sequences through optimizing nonpolar clusters surrounding the S320 position. We uncover a mechanism for regulating tau aggregation which balances local nonpolar contacts with long-range interactions that sequester amyloid motifs. Understanding this process may permit control of tau aggregation into structural polymorphs to aid the design of reagents targeting disease-specific tau conformations.

Network of hotspot interactions cluster tau amyloid folds.

Cryogenic electron microscopy has revealed unprecedented molecular insight into the conformations of ß-sheet-rich protein amyloids linked to neurodegenerative diseases. It remains unknown how a protein can adopt a diversity of folds and form multiple distinct fibrillar structures. Here we develop an in silico alanine scan method to estimate the relative energetic contribution of each amino acid in an amyloid assembly. We apply our method to twenty-seven ex vivo and in vitro fibril structural polymorphs of the microtubule-associated protein tau. We uncover networks of energetically important interactions involving amyloid-forming motifs that stabilize the different fibril folds. We evaluate our predictions in cellular and in vitro aggregation assays. Using a machine learning approach, we classify the structures based on residue energetics to identify distinguishing and unifying features. Our energetic profiling suggests that minimal sequence elements control the stability of tau fibrils, allowing future design of protein sequences that fold into unique structures.

VCP suppresses proteopathic seeding in neurons.

BACKGROUND: Neuronal uptake and subsequent spread of proteopathic seeds, such as αS (alpha-synuclein), Tau, and TDP-43, contribute to neurodegeneration. The cellular machinery participating in this process is poorly understood. One proteinopathy called multisystem proteinopathy (MSP) is associated with dominant mutations in Valosin Containing Protein (VCP). MSP patients have muscle and neuronal degeneration characterized by aggregate pathology that can include αS, Tau and TDP-43. METHODS: We performed a fluorescent cell sorting based genome-wide CRISPR-Cas9 screen in αS biosensors. αS and TDP-43 seeding activity under varied conditions was assessed using FRET/Flow biosensor cells or immunofluorescence for phosphorylated αS or TDP-43 in primary cultured neurons. We analyzed in vivo seeding activity by immunostaining for phosphorylated αS following intrastriatal injection of αS seeds in control or VCP disease mutation carrying mice. RESULTS: One hundred fifty-four genes were identified as suppressors of αS seeding. One suppressor, VCP when chemically or genetically inhibited increased αS seeding in cells and neurons. This was not due to an increase in αS uptake or αS protein levels. MSP-VCP mutation expression increased αS seeding in cells and neurons. Intrastriatal injection of αS preformed fibrils (PFF) into VCP-MSP mutation carrying mice increased phospho αS expression as compared to control mice. Cells stably expressing fluorescently tagged TDP-43 C-terminal fragment FRET pairs (TDP-43 biosensors) generate FRET when seeded with TDP-43 PFF but not monomeric TDP-43. VCP inhibition or MSP-VCP mutant expression increases TDP-43 seeding in TDP-43 biosensors. Similarly, treatment of neurons with TDP-43 PFFs generates high molecular weight insoluble phosphorylated TDP-43 after 5 days. This TDP-43 seed dependent increase in phosphorlyated TDP-43 is further augmented in MSP-VCP mutant expressing neurons. CONCLUSION: Using an unbiased screen, we identified the multifunctional AAA ATPase VCP as a suppressor of αS and TDP-43 aggregate seeding in cells and neurons. VCP facilitates the clearance of damaged lysosomes via lysophagy. We propose that VCP's surveillance of permeabilized endosomes may protect against the proteopathic spread of pathogenic protein aggregates. The spread of distinct aggregate species may dictate the pleiotropic phenotypes and pathologies in VCP associated MSP.

Ultrasensitive tau biosensor cells detect no seeding in Alzheimer's disease CSF.

Tau protein forms self-replicating assemblies (seeds) that may underlie progression of pathology in Alzheimer's disease (AD) and related tauopathies. Seeding in recombinant protein preparations and brain homogenates has been quantified with "biosensor" cell lines that express tau with a disease-associated mutation (P301S) fused to complementary fluorescent proteins. Quantification of induced aggregation in cells that score positive by fluorescence resonance energy transfer (FRET) is accomplished by cell imaging or flow cytometry. Several groups have reported seeding activity in antemortem cerebrospinal fluid (CSF) using various methods, but these findings are not yet widely replicated. To address this question, we created two improved FRET-based biosensor cell lines based on tau expression, termed version 2 low (v2L) and version 2 high (v2H). We determined that v2H cells are ~ 100-fold more sensitive to AD-derived tau seeds than our original lines, and coupled with immunoprecipitation reliably detect seeding from samples containing as little as 100 attomoles of recombinant tau fibrils or ~ 32 pg of total protein from AD brain homogenate. We tested antemortem CSF from 11 subjects with a clinical diagnosis of AD, 9 confirmed by validated CSF biomarkers. We used immunoprecipitation coupled with seed detection in v2H cells and detected no tau seeding in any sample. Thus we cannot confirm prior reports of tau seeding activity in the CSF of AD patients. This next generation of ultra-sensitive tau biosensors may nonetheless be useful to the research community to quantify tau pathology as sensitively and specifically as possible.

Tau strains shape disease.

Tauopathies consist of over 25 different neurodegenerative diseases that include argyrophilic grain disease (AGD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Pick's disease (PiD). Tauopathies are defined by brain accumulation of microtubule-associated protein tau in fibrillar aggregates, whose prevalence strongly correlates with dementia. Dominant mutations in tau cause neurodegenerative diseases, and most increase its aggregation propensity. Pathogenesis of tauopathies may involve pathological tau conformers that serve as templates to recruit native protein into growing assemblies and also move between brain cells to cause disease progression, similar to prions. Prions adopt pathological conformations, termed "strains," that stably propagate in living systems, and create unique patterns of neuropathology. Data from multiple laboratories now suggest that tau acts as a prion. It propagates unique strains indefinitely in cultured cells, and when these are inoculated into mouse models, they create defined neuropathological patterns, which establish a direct link between conformation and disease. In humans, distinct fibril structures are associated with different diseases, but causality has not been established as in mice. Cryo-EM structures of tau fibrils isolated from tauopathy brains reveal distinct fibril cores across disease. Interestingly, the conformation of the tau monomer unit within different fibril subtypes from the same patient appears relatively preserved. This is consistent with data that the tau monomer samples an ensemble of conformations that act as distinct pathologic templates in the formation of restricted numbers of strains. The propensity of a tau monomer to adopt distinct conformations appears to be linked to defined local motifs that expose different patterns of amyloidogenic amino acid sequences. The prion hypothesis, which predicts that protein structure dictates resultant disease, has proved particularly useful to understand the diversity of human tauopathies. The challenge now is to develop methods to rapidly classify patients according to the structure of the underlying pathological protein assemblies to achieve more accurate diagnosis and effective therapy.

Regulatory inter-domain interactions influence Hsp70 recruitment to the DnaJB8 chaperone.

The Hsp40/Hsp70 chaperone families combine versatile folding capacity with high substrate specificity, which is mainly facilitated by Hsp40s. The structure and function of many Hsp40s remain poorly understood, particularly oligomeric Hsp40s that suppress protein aggregation. Here, we used a combination of biochemical and structural approaches to shed light on the domain interactions of the Hsp40 DnaJB8, and how they may influence recruitment of partner Hsp70s. We identify an interaction between the J-Domain (JD) and C-terminal domain (CTD) of DnaJB8 that sequesters the JD surface, preventing Hsp70 interaction. We propose a model for DnaJB8-Hsp70 recruitment, whereby the JD-CTD interaction of DnaJB8 acts as a reversible switch that can control the binding of Hsp70. These findings suggest that the evolutionarily conserved CTD of DnaJB8 is a regulatory element of chaperone activity in the proteostasis network.

Propagation of Protein Aggregation in Neurodegenerative Diseases.

Most common neurodegenerative diseases feature deposition of protein amyloids and degeneration of brain networks. Amyloids are ordered protein assemblies that can act as templates for their own replication through monomer addition. Evidence suggests that this characteristic may underlie the progression of pathology in neurodegenerative diseases. Many different amyloid proteins, including Aß, tau, and α-synuclein, exhibit properties similar to those of infectious prion protein in experimental systems: discrete and self-replicating amyloid structures, transcellular propagation of aggregation, and transmissible neuropathology. This review discusses the contribution of prion phenomena and transcellular propagation to the progression of pathology in common neurodegenerative diseases such as Alzheimer's and Parkinson's. It reviews fundamental events such as cell entry, amplification, and transcellular movement. It also discusses amyloid strains, which produce distinct patterns of neuropathology and spread through the nervous system. These concepts may impact the development of new diagnostic and therapeutic strategies.

Specific glycosaminoglycan chain length and sulfation patterns are required for cell uptake of tau versus α-synuclein and ß-amyloid aggregates.

Transcellular propagation of protein aggregate "seeds" has been proposed to mediate the progression of neurodegenerative diseases in tauopathies and α-synucleinopathies. We previously reported that tau and α-synuclein aggregates bind heparan sulfate proteoglycans (HSPGs) on the cell surface, promoting cellular uptake and intracellular seeding. However, the specificity and binding mode of these protein aggregates to HSPGs remain unknown. Here, we measured direct interaction with modified heparins to determine the size and sulfation requirements for tau, α-synuclein, and ß-amyloid (Aß) aggregate binding to glycosaminoglycans (GAGs). Varying the GAG length and sulfation patterns, we next conducted competition studies with heparin derivatives in cell-based assays. Tau aggregates required a precise GAG architecture with defined sulfate moieties in the N- and 6-O-positions, whereas the binding of α-synuclein and Aß aggregates was less stringent. To determine the genes required for aggregate uptake, we used CRISPR/Cas9 to individually knock out the major genes of the HSPG synthesis pathway in HEK293T cells. Knockouts of the extension enzymes exostosin 1 (EXT1), exostosin 2 (EXT2), and exostosin-like 3 (EXTL3), as well as N-sulfotransferase (NDST1) or 6-O-sulfotransferase (HS6ST2) significantly reduced tau uptake, consistent with our biochemical findings, and knockouts of EXT1, EXT2, EXTL3, or NDST1, but not HS6ST2 reduced α-synuclein uptake. In summary, tau aggregates display specific interactions with HSPGs that depend on GAG length and sulfate moiety position, whereas α-synuclein and Aß aggregates exhibit more flexible interactions with HSPGs. These principles may inform the development of mechanism-based therapies to block transcellular propagation of amyloid protein-based pathologies.

Widespread tau seeding activity at early Braak stages.

Transcellular propagation of tau aggregates may underlie the progression of pathology in Alzheimer's disease (AD) and other tauopathies. Braak staging (B1, B2, B3) is based on phospho-tau accumulation within connected brain regions: entorhinal cortex (B1); hippocampus/limbic system (B2); and frontal and parietal lobes (B3). We previously developed a specific and sensitive assay that uses flow cytometry to quantify tissue seeding activity based on fluorescence resonance energy transfer (FRET) in cells that stably express tau reporter proteins. In a tauopathy mouse model, we have detected seeding activity far in advance of histopathological changes. It remains unknown whether individuals with AD also develop seeding activity prior to accumulation of phospho-tau. We measured tau seeding activity across four brain regions (hippocampus, frontal lobe, parietal lobe, and cerebellum) in 104 fresh-frozen human AD brain samples from all Braak stages. We observed widespread seeding activity, notably in regions predicted to be free of phospho-tau deposition, and in detergent-insoluble fractions that lacked tau detectable by ELISA. Seeding activity correlated positively with Braak stage and negatively with MMSE. Our results are consistent with early transcellular propagation of tau seeds that triggers subsequent development of neuropathology. The FRET-based seeding assay may also complement standard neuropathological classification of tauopathies.

Tau Prion Strains Dictate Patterns of Cell Pathology, Progression Rate, and Regional Vulnerability In Vivo.

Tauopathies are neurodegenerative disorders that affect distinct brain regions, progress at different rates, and exhibit specific patterns of tau accumulation. The source of this diversity is unknown. We previously characterized two tau strains that stably maintain unique conformations in vitro and in vivo, but did not determine the relationship of each strain to parameters that discriminate between tauopathies such as regional vulnerability or rate of spread. We have now isolated and characterized 18 tau strains in cells based on detailed biochemical and biological criteria. Inoculation of PS19 transgenic tau (P301S) mice with these strains causes strain-specific intracellular pathology in distinct cell types and brain regions, and induces different rates of network propagation. In this system, strains alone are sufficient to account for diverse neuropathological presentations, similar to those that define human tauopathies. Further study of these strains can thus establish a structural logic that governs these biological effects.

Novel autoimmune response in a tauopathy mouse model.

Molecular diagnostic tools with non-invasive properties that allow detection of pathological events in Alzheimer's disease (AD) and other neurodegenerative tauopathies are essential for the development of therapeutics. Several diagnostic strategies based on the identification of biomarkers have been proposed. However, its specificity among neurodegenerative disorders is disputable as the association with pathological events remains elusive. Recently, we showed that Amphiphysin-1 (AMPH1) protein's abundance is reduced in the central nervous system (CNS) of the tauopathy mouse model JNPL3 and AD brains. AMPH1 is a synaptic protein that plays an important role in clathrin-mediated endocytosis and associates with BIN1, one of the most important risk loci for AD. Also, it has been associated with a rare neurological disease known as Stiff-Person Syndrome (SPS). Auto-antibodies against AMPH1 are used as diagnostic biomarkers for a paraneoplastic variant of SPS. Therefore, we set up to evaluate the presence and abundance of auto-AMPH1 antibodies in tau-mediated neurodegeneration. Immunoblots and enzyme-linked immunosorbent assays (ELISA) were conducted to detect the presence of auto-AMPH1 antibodies in sera from euthanized mice that developed neurodegeneration (JNPL3) and healthy control mice (NTg). Results showed increased levels of auto-AMPH1 antibodies in JNPL3 sera compared to NTg controls. The abundance of auto-AMPH1 antibodies correlated with motor impairment and AMPH1 protein level decrease in the CNS. The results suggest that auto-AMPH1 antibodies could serve as a biomarker for the progression of tau-mediated neurodegeneration in JNPL3 mice.

EFhd2 is a novel amyloid protein associated with pathological tau in Alzheimer's disease.

EFhd2 is a conserved calcium-binding protein, abundant within the central nervous system. Previous studies identified EFhd2 associated with pathological forms of tau proteins in the tauopathy mouse model JNPL3, which expresses the human tau(P301L) mutant. This association was validated in human tauopathies, such as Alzheimer's disease (AD). However, the role that EFhd2 may play in tauopathies is still unknown. Here, we show that EFhd2 formed amyloid structures in vitro, a capability that is reduced by calcium ions. Electron microscopy (EM) analyses demonstrated that recombinant EFhd2 formed filamentous structures. EM analyses of sarkosyl-insoluble fractions derived from human AD brains also indicated that EFhd2 co-localizes with aggregated tau proteins and formed granular structures. Immunohistological analyses of brain slices demonstrated that EFhd2 co-localizes with pathological tau proteins in AD brains, confirming the co-aggregation of EFhd2 and pathological tau. Furthermore, EFhd2's coiled-coil domain mediated its self-oligomerization in vitro and its association with tau proteins in JNPL3 mouse brain extracts. The results demonstrate that EFhd2 is a novel amyloid protein associated with pathological tau proteins in AD brain and that calcium binding may regulate the formation of EFhd2's amyloid structures. Hence, EFhd2 may play an important role in the pathobiology of tau-mediated neurodegeneration.