Acetylation discriminates disease-specific tau deposition.

Pathogenic aggregation of the protein tau is a hallmark of Alzheimer's disease and several other tauopathies. Tauopathies are characterized by the deposition of specific tau isoforms as disease-related tau filament structures. The molecular processes that determine isoform-specific deposition of tau are however enigmatic. Here we show that acetylation of tau discriminates its isoform-specific aggregation. We reveal that acetylation strongly attenuates aggregation of four-repeat tau protein, but promotes amyloid formation of three-repeat tau. We further identify acetylation of lysine 298 as a hot spot for isoform-specific tau aggregation. Solid-state NMR spectroscopy demonstrates that amyloid fibrils formed by unmodified and acetylated three-repeat tau differ in structure indicating that site-specific acetylation modulates tau structure. The results implicate acetylation as a critical regulator that guides the selective aggregation of three-repeat tau and the development of tau isoform-specific neurodegenerative diseases.

Reactivated endogenous retroviruses promote protein aggregate spreading.

Prion-like spreading of protein misfolding is a characteristic of neurodegenerative diseases, but the exact mechanisms of intercellular protein aggregate dissemination remain unresolved. Evidence accumulates that endogenous retroviruses, remnants of viral germline infections that are normally epigenetically silenced, become upregulated in neurodegenerative diseases such as amyotrophic lateral sclerosis and tauopathies. Here we uncover that activation of endogenous retroviruses affects prion-like spreading of proteopathic seeds. We show that upregulation of endogenous retroviruses drastically increases the dissemination of protein aggregates between cells in culture, a process that can be inhibited by targeting the viral envelope protein or viral protein processing. Human endogenous retrovirus envelopes of four different clades also elevate intercellular spreading of proteopathic seeds, including pathological Tau. Our data support a role of endogenous retroviruses in protein misfolding diseases and suggest that antiviral drugs could represent promising candidates for inhibiting protein aggregate spreading.

Viruses in neurodegenerative diseases: More than just suspects in crimes.

Neurodegenerative diseases (NDs) such as Alzheimer's and Parkinson's disease are fatal neurological diseases that can be of idiopathic, genetic, or even infectious origin, as in the case of transmissible spongiform encephalopathies. The etiological factors that lead to neurodegeneration remain unknown but likely involve a combination of aging, genetic risk factors, and environmental stressors. Accumulating evidence hints at an association of viruses with neurodegenerative disorders and suggests that virus-induced neuroinflammation and perturbation of neuronal protein quality control can be involved in the early steps of disease development. In this review, we focus on emerging evidence for a correlation between NDs and viral infection and discuss how viral manipulations of cellular processes can affect the formation and dissemination of disease-associated protein aggregates.

Möglicher Einfluss von Viren auf die Ausbreitung von Proteinaggregaten.

Neurodegenerative diseases are associated with misfolding of proteins into highly-ordered amyloid fibrils. These protein aggregates can be transmitted to other cells in which they induce aggregation of proteins of the same kind. Mechanisms of intercellular transfer include direct cell contact or transfer of aggregates within extracellular vesicles. Recent research suggests that viral proteins can increase the intercellular spreading of protein aggregation by promoting the required membrane interactions.

Propagation and Dissemination Strategies of Transmissible Spongiform Encephalopathy Agents in Mammalian Cells.

Transmissible spongiform encephalopathies or prion disorders are fatal infectious diseases that cause characteristic spongiform degeneration in the central nervous system. The causative agent, the so-called prion, is an unconventional infectious agent that propagates by converting the host-encoded cellular prion protein PrP into ordered protein aggregates with infectious properties. Prions are devoid of coding nucleic acid and thus rely on the host cell machinery for propagation. While it is now established that, in addition to PrP, other cellular factors or processes determine the susceptibility of cell lines to prion infection, exact factors and cellular processes remain broadly obscure. Still, cellular models have uncovered important aspects of prion propagation and revealed intercellular dissemination strategies shared with other intracellular pathogens. Here, we summarize what we learned about the processes of prion invasion, intracellular replication and subsequent dissemination from ex vivo cell models.

Highly efficient intercellular spreading of protein misfolding mediated by viral ligand-receptor interactions.

Protein aggregates associated with neurodegenerative diseases have the ability to transmit to unaffected cells, thereby templating their own aberrant conformation onto soluble homotypic proteins. Proteopathic seeds can be released into the extracellular space, secreted in association with extracellular vesicles (EV) or exchanged by direct cell-to-cell contact. The extent to which each of these pathways contribute to the prion-like spreading of protein misfolding is unclear. Exchange of cellular cargo by both direct cell contact or via EV depends on receptor-ligand interactions. We hypothesized that enabling these interactions through viral ligands enhances intercellular proteopathic seed transmission. Using different cellular models propagating prions or pathogenic Tau aggregates, we demonstrate that vesicular stomatitis virus glycoprotein and SARS-CoV-2 spike S increase aggregate induction by cell contact or ligand-decorated EV. Thus, receptor-ligand interactions are important determinants of intercellular aggregate dissemination. Our data raise the possibility that viral infections contribute to proteopathic seed spreading by facilitating intercellular cargo transfer.

Extracellular Vesicle Separation Techniques Impact Results from Human Blood Samples: Considerations for Diagnostic Applications.

Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable source of biomarkers. However, for EVs to be used as biomarkers in clinical practice, simple, comparable, and reproducible analytical methods must be applied. Although progress is being made in EV separation methods for human biofluids, the implementation of EV assays for clinical diagnosis and common guidelines are still lacking. We conducted a comprehensive analysis of established EV separation techniques from human serum and plasma, including ultracentrifugation and size exclusion chromatography (SEC), followed by concentration using (a) ultracentrifugation, (b) ultrafiltration, or (c) precipitation, and immunoaffinity isolation. We analyzed the size, number, protein, and miRNA content of the obtained EVs and assessed the functional delivery of EV cargo. Our results demonstrate that all methods led to an adequate yield of small EVs. While no significant difference in miRNA content was observed for the different separation methods, ultracentrifugation was best for subsequent flow cytometry analysis. Immunoaffinity isolation is not suitable for subsequent protein analyses. SEC + ultracentrifugation showed the best functional delivery of EV cargo. In summary, combining SEC with ultracentrifugation gives the highest yield of pure and functional EVs and allows reliable analysis of both protein and miRNA contents. We propose this combination as the preferred EV isolation method for biomarker studies from human serum or plasma.

Co-factor-free aggregation of tau into seeding-competent RNA-sequestering amyloid fibrils.

Pathological aggregation of the protein tau into insoluble aggregates is a hallmark of neurodegenerative diseases. The emergence of disease-specific tau aggregate structures termed tau strains, however, remains elusive. Here we show that full-length tau protein can be aggregated in the absence of co-factors into seeding-competent amyloid fibrils that sequester RNA. Using a combination of solid-state NMR spectroscopy and biochemical experiments we demonstrate that the co-factor-free amyloid fibrils of tau have a rigid core that is similar in size and location to the rigid core of tau fibrils purified from the brain of patients with corticobasal degeneration. In addition, we demonstrate that the N-terminal 30 residues of tau are immobilized during fibril formation, in agreement with the presence of an N-terminal epitope that is specifically detected by antibodies in pathological tau. Experiments in vitro and in biosensor cells further established that co-factor-free tau fibrils efficiently seed tau aggregation, while binding studies with different RNAs show that the co-factor-free tau fibrils strongly sequester RNA. Taken together the study provides a critical advance to reveal the molecular factors that guide aggregation towards disease-specific tau strains.

Human ORC/MCM density is low in active genes and correlates with replication time but does not delimit initiation zones.

Eukaryotic DNA replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with gene expression, replication timing, and fork directionality profiles obtained by RNA-seq, Repli-seq, and OK-seq. Both, the origin recognition complex (ORC) and the minichromosome maintenance complex (MCM) are significantly and homogeneously depleted from transcribed genes, enriched at gene promoters, and more abundant in early- than in late-replicating domains. Surprisingly, after controlling these variables, no difference in ORC/MCM density is detected between initiation zones, termination zones, unidirectionally replicating regions, and randomly replicating regions. Therefore, ORC/MCM density correlates with replication timing but does not solely regulate the probability of replication initiation. Interestingly, H4K20me3, a histone modification proposed to facilitate late origin licensing, was enriched in late-replicating initiation zones and gene deserts of stochastic replication fork direction. We discuss potential mechanisms specifying when and where replication initiates in human cells.

Reverse engineering synthetic antiviral amyloids.

Human amyloids have been shown to interact with viruses and interfere with viral replication. Based on this observation, we employed a synthetic biology approach in which we engineered virus-specific amyloids against influenza A and Zika proteins. Each amyloid shares a homologous aggregation-prone fragment with a specific viral target protein. For influenza we demonstrate that a designer amyloid against PB2 accumulates in influenza A-infected tissue in vivo. Moreover, this amyloid acts specifically against influenza A and its common PB2 polymorphisms, but not influenza B, which lacks the homologous fragment. Our model amyloid demonstrates that the sequence specificity of amyloid interactions has the capacity to tune amyloid-virus interactions while allowing for the flexibility to maintain activity on evolutionary diverging variants.

Intercellular Transmission of a Synthetic Bacterial Cytotoxic Prion-Like Protein in Mammalian Cells.

RepA is a bacterial protein that builds intracellular amyloid oligomers acting as inhibitory complexes of plasmid DNA replication. When carrying a mutation enhancing its amyloidogenesis (A31V), the N-terminal domain (WH1) generates cytosolic amyloid particles that are inheritable within a bacterial lineage. Such amyloids trigger in bacteria a lethal cascade reminiscent of mitochondrial impairment in human cells affected by neurodegeneration. To fulfill all the criteria to qualify as a prion-like protein, horizontal (intercellular) transmissibility remains to be demonstrated for RepA-WH1. Since this is experimentally intractable in bacteria, here we transiently expressed in a murine neuroblastoma cell line the soluble, barely cytotoxic RepA-WH1 wild type [RepA-WH1(WT)] and assayed its response to exposure to in vitro-assembled RepA-WH1(A31V) amyloid fibers. In parallel, murine cells releasing RepA-WH1(A31V) aggregates were cocultured with human neuroblastoma cells expressing RepA-WH1(WT). Both the assembled fibers and donor-derived RepA-WH1(A31V) aggregates induced, in the cytosol of recipient cells, the formation of cytotoxic amyloid particles. Mass spectrometry analyses of the proteomes of both types of injured cells pointed to alterations in mitochondria, protein quality triage, signaling, and intracellular traffic. Thus, a synthetic prion-like protein can be propagated to, and become cytotoxic to, cells of organisms placed at such distant branches of the tree of life as bacteria and mammalia, suggesting that mechanisms of protein aggregate spreading and toxicity follow default pathways.IMPORTANCE Proteotoxic amyloid seeds can be transmitted between mammalian cells, arguing that the intercellular exchange of prion-like protein aggregates can be a common phenomenon. RepA-WH1 is derived from a bacterial intracellular functional amyloid protein, engineered to become cytotoxic in Escherichia coli Here, we have studied if such bacterial aggregates can also be transmitted to, and become cytotoxic to, mammalian cells. We demonstrate that RepA-WH1 is capable of entering naive cells, thereby inducing the cytotoxic aggregation of a soluble RepA-WH1 variant expressed in the cytosol, following the same trend that had been described in bacteria. These findings highlight the universality of one of the central principles underlying prion biology: No matter the biological origin of a given prion-like protein, it can be transmitted to a phylogenetically unrelated recipient cell, provided that the latter expresses a soluble protein onto which the incoming protein can readily template its amyloid conformation.

Entropic Bristles Tune the Seeding Efficiency of Prion-Nucleating Fragments.

Prions of lower eukaryotes are self-templating protein aggregates with cores formed by parallel in-register beta strands. Short aggregation-prone glutamine (Q)- and asparagine (N)-rich regions embedded in longer disordered domains have been proposed to act as nucleation sites that initiate refolding of soluble prion proteins into highly ordered fibrils, termed amyloid. We demonstrate that a short Q/N-rich peptide corresponding to a proposed nucleation site in the prototype Saccharomyces cerevisiae prion protein Sup35 is sufficient to induce infectious cytosolic prions in mouse neuroblastoma cells ectopically expressing the soluble Sup35 NM prion domain. Embedding this nucleating core in a non-native N-rich sequence that does not form amyloid but acts as an entropic bristle quadruples seeding efficiency. Our data suggest that large disordered sequences flanking an aggregation core in prion proteins act as not only solubilizers of the monomeric protein but also breakers of the formed amyloid fibrils, enhancing infectivity of the prion seeds.

Fibril-induced glutamine-/asparagine-rich prions recruit stress granule proteins in mammalian cells.

Prions of lower eukaryotes are self-templating protein aggregates that replicate by converting homotypic proteins into stable, tightly packed beta-sheet-rich protein assemblies. Propagation is mediated by prion domains, low-complexity regions enriched in polar and devoid of charged amino acid residues. In mammals, compositionally similar domains modulate the assembly of dynamic stress granules (SGs) that associate via multivalent weak interactions. Dysregulation of SGs composed of proteins with prion-like domains has been proposed to underlie the formation of pathological inclusions in several neurodegenerative diseases. The events that drive prion-like domains into transient or solid assemblies are not well understood. We studied the interactors of the prototype prion domain NM of Saccharomyces cerevisiae Sup35 in its soluble or fibril-induced prion conformation in the mammalian cytosol. We show that the interactomes of soluble and prionized NM overlap with that of SGs. Prion induction by exogenous seeds does not cause SG assembly, demonstrating that colocalization of aberrant protein inclusions with SG components does not necessarily reveal SGs as initial sites of protein misfolding.

All the Same? The Secret Life of Prion Strains within Their Target Cells.

Prions are infectious ß-sheet-rich protein aggregates composed of misfolded prion protein (PrPSc) that do not possess coding nucleic acid. Prions replicate by recruiting and converting normal cellular PrPC into infectious isoforms. In the same host species, prion strains target distinct brain regions and cause different disease phenotypes. Prion strains are associated with biophysically distinct PrPSc conformers, suggesting that strain properties are enciphered within alternative PrPSc quaternary structures. So far it is unknown how prion strains target specific cells and initiate productive infections. Deeper mechanistic insight into the prion life cycle came from cell lines permissive to a range of different prion strains. Still, it is unknown why certain cell lines are refractory to infection by one strain but permissive to another. While pharmacologic and genetic manipulations revealed subcellular compartments involved in prion replication, little is known about strain-specific requirements for endocytic trafficking pathways. This review summarizes our knowledge on how prions replicate within their target cells and on strain-specific differences in prion cell biology.

Experimental models to study prion disease pathogenesis and identify potential therapeutic compounds.

Prion diseases are devastating neurodegenerative disorders for which no drugs are available. The successful development of therapeutics depends on drug screening platforms and preclinical models that recapitulate key molecular and pathological features of the disease. Innovative experimental tools have been developed over the last few years that might facilitate drug discovery, including cell-free prion replication assays and prion-infected flies. However, there is still room for improvement. Animal models of genetic prion disease are few, and only partially recapitulate the complexity of the human disorder. Moreover, we still lack a human cell culture model suitable for high-content anti-prion drug screening. This review provides an overview of the models currently used in prion research, and discusses their promise and limitations for drug discovery.

Deregulated Splicing Is a Major Mechanism of RNA-Induced Toxicity in Huntington's Disease.

Huntington's disease (HD) is caused by an expanded CAG repeat in the huntingtin (HTT) gene, translating into an elongated polyglutamine stretch. In addition to the neurotoxic mutant HTT protein, the mutant CAG repeat RNA can exert toxic functions by trapping RNA-binding proteins. While few examples of proteins that aberrantly bind to mutant HTT RNA and execute abnormal function in conjunction with the CAG repeat RNA have been described, an unbiased approach to identify the interactome of mutant HTT RNA is missing. Here, we describe the analysis of proteins that preferentially bind mutant HTT RNA using a mass spectrometry approach. We show that (I) the majority of proteins captured by mutant HTT RNA belong to the spliceosome pathway, (II) expression of mutant CAG repeat RNA induces mis-splicing in a HD cell model, (III) overexpression of one of the splice factors trapped by mutant HTT ameliorates the HD phenotype in a fly model and (VI) deregulated splicing occurs in human HD brain. Our data suggest that deregulated splicing is a prominent mechanism of RNA-induced toxicity in HD.

Prion Replication in the Mammalian Cytosol: Functional Regions within a Prion Domain Driving Induction, Propagation, and Inheritance.

Prions of lower eukaryotes are transmissible protein particles that propagate by converting homotypic soluble proteins into growing protein assemblies. Prion activity is conferred by so-called prion domains, regions of low complexity that are often enriched in glutamines and asparagines (Q/N). The compositional similarity of fungal prion domains with intrinsically disordered domains found in many mammalian proteins raises the question of whether similar sequence elements can drive prion-like phenomena in mammals. Here, we define sequence features of the prototype Saccharomyces cerevisiae Sup35 prion domain that govern prion activities in mammalian cells by testing the ability of deletion mutants to assemble into self-perpetuating particles. Interestingly, the amino-terminal Q/N-rich tract crucially important for prion induction in yeast was dispensable for the prion life cycle in mammalian cells. Spontaneous and template-assisted prion induction, growth, and maintenance were preferentially driven by the carboxy-terminal region of the prion domain that contains a putative soft amyloid stretch recently proposed to act as a nucleation site for prion assembly. Our data demonstrate that preferred prion nucleation domains can differ between lower and higher eukaryotes, resulting in the formation of prions with strikingly different amyloid cores.

Inhibition of the MID1 protein complex: a novel approach targeting APP protein synthesis.

Alzheimer's disease (AD) is characterized by two neuropathological hallmarks: senile plaques, which are composed of amyloid-ß (Aß) peptides, and neurofibrillary tangles, which are composed of hyperphosphorylated tau protein. Aß peptides are derived from sequential proteolytic cleavage of the amyloid precursor protein (APP). In this study, we identified a so far unknown mode of regulation of APP protein synthesis involving the MID1 protein complex: MID1 binds to and regulates the translation of APP mRNA. The underlying mode of action of MID1 involves the mTOR pathway. Thus, inhibition of the MID1 complex reduces the APP protein level in cultures of primary neurons. Based on this, we used one compound that we discovered previously to interfere with the MID1 complex, metformin, for in vivo experiments. Indeed, long-term treatment with metformin decreased APP protein expression levels and consequently Aß in an AD mouse model. Importantly, we have initiated the metformin treatment late in life, at a time-point where mice were in an already progressed state of the disease, and could observe an improved behavioral phenotype. These findings together with our previous observation, showing that inhibition of the MID1 complex by metformin also decreases tau phosphorylation, make the MID1 complex a particularly interesting drug target for treating AD.