RESUMO
Current therapies for Alzheimer's disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936-a potential candidate for the treatment of memory loss in Alzheimer's disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Desenho de Fármacos , Receptor Muscarínico M1/agonistas , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/patologia , Doença de Alzheimer/complicações , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Animais , Pressão Sanguínea/efeitos dos fármacos , Células CHO , Inibidores da Colinesterase/farmacologia , Cricetulus , Cristalização , Modelos Animais de Doenças , Cães , Donepezila/farmacologia , Eletroencefalografia , Feminino , Células HEK293 , Frequência Cardíaca/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Modelos Moleculares , Simulação de Dinâmica Molecular , Degeneração Neural/complicações , Degeneração Neural/patologia , Primatas , Ratos , Receptor Muscarínico M1/química , Transdução de Sinais , Homologia Estrutural de ProteínaRESUMO
Many organisms evolved strategies to survive desiccation. Plant seeds protect dehydrated embryos from various stressors and can lay dormant for millennia. Hydration is the key trigger to initiate germination, but the mechanism by which seeds sense water remains unresolved. We identified an uncharacterized Arabidopsis thaliana prion-like protein we named FLOE1, which phase separates upon hydration and allows the embryo to sense water stress. We demonstrate that biophysical states of FLOE1 condensates modulate its biological function in vivo in suppressing seed germination under unfavorable environments. We find intragenic, intraspecific, and interspecific natural variation in FLOE1 expression and phase separation and show that intragenic variation is associated with adaptive germination strategies in natural populations. This combination of molecular, organismal, and ecological studies uncovers FLOE1 as a tunable environmental sensor with direct implications for the design of drought-resistant crops, in the face of climate change.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Germinação , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Príons/metabolismo , Sementes/crescimento & desenvolvimento , Água/metabolismo , Arabidopsis/genética , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/ultraestrutura , Desidratação , Imageamento Tridimensional , Peptídeos e Proteínas de Sinalização Intercelular/química , Mutação/genética , Dormência de Plantas , Plantas Geneticamente Modificadas , Domínios Proteicos , Isoformas de Proteínas/metabolismo , Sementes/ultraestruturaRESUMO
Covalent modifications to histones are essential for development, establishing distinct and functional chromatin domains from a common genetic sequence. Whereas repressed chromatin is robustly inherited, no mechanism that facilitates inheritance of an activated domain has been described. Here, we report that the Set3C histone deacetylase scaffold Snt1 can act as a prion that drives the emergence and transgenerational inheritance of an activated chromatin state. This prion, which we term [ESI+] for expressed sub-telomeric information, is triggered by transient Snt1 phosphorylation upon cell cycle arrest. Once engaged, the prion reshapes the activity of Snt1 and the Set3C complex, recruiting RNA pol II and interfering with Rap1 binding to activate genes in otherwise repressed sub-telomeric domains. This transcriptional state confers broad resistance to environmental stress, including antifungal drugs. Altogether, our results establish a robust means by which a prion can facilitate inheritance of an activated chromatin state to provide adaptive benefit.
Assuntos
Cromatina/genética , Histona Desacetilases/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Ligação a Telômeros/genética , Fatores de Transcrição/genética , Pontos de Checagem do Ciclo Celular/genética , Código das Histonas/genética , Histonas/genética , Fosforilação/genética , Príons/genética , RNA Polimerase II/genética , Saccharomyces cerevisiae , Complexo Shelterina , Telômero/genética , Transcrição GênicaRESUMO
To elucidate the role of Tau isoforms and post-translational modification (PTM) stoichiometry in Alzheimer's disease (AD), we generated a high-resolution quantitative proteomics map of 95 PTMs on multiple isoforms of Tau isolated from postmortem human tissue from 49 AD and 42 control subjects. Although Tau PTM maps reveal heterogeneity across subjects, a subset of PTMs display high occupancy and frequency for AD, suggesting importance in disease. Unsupervised analyses indicate that PTMs occur in an ordered manner, leading to Tau aggregation. The processive addition and minimal set of PTMs associated with seeding activity was further defined by analysis of size-fractionated Tau. To summarize, features in the Tau protein critical for disease intervention at different stages of disease are identified, including enrichment of 0N and 4R isoforms, underrepresentation of the C terminus, an increase in negative charge in the proline-rich region (PRR), and a decrease in positive charge in the microtubule binding domain (MBD).
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Processamento de Proteína Pós-Traducional , Proteínas tau/metabolismo , Estudos de Casos e Controles , Estudos de Coortes , Progressão da Doença , Humanos , Análise de Componente Principal , Isoformas de Proteínas/metabolismoRESUMO
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.
Assuntos
Doenças Neurodegenerativas/metabolismo , Agregação Patológica de Proteínas , Amiloide , Animais , Humanos , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/patologia , Proteínas tauRESUMO
Prion-like proteins can assume distinct conformational and physical states in the same cell. Sequence analysis suggests that prion-like proteins are prevalent in various species; however, it remains unclear what functional space they occupy in multicellular organisms. Here, we report the identification of a prion-like protein, Herzog (CG5830), through a multimodal screen in Drosophila melanogaster. Herzog functions as a membrane-associated phosphatase and controls embryonic patterning, likely being involved in TGF-ß/BMP and FGF/EGF signaling pathways. Remarkably, monomeric Herzog is enzymatically inactive and becomes active upon amyloid-like assembly. The prion-like domain of Herzog is necessary for both its assembly and membrane targeting. Removal of the prion-like domain impairs activity, while restoring assembly on the membrane using a heterologous prion-like domain and membrane-targeting motif can restore phosphatase activity. This study provides an example of a prion-like domain that allows an enzyme to gain essential functionality via amyloid-like assembly to control animal development.
Assuntos
Proteínas Amiloidogênicas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Desenvolvimento Embrionário , Fosfoproteínas Fosfatases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/genética , Animais , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Príons/química , Domínios ProteicosRESUMO
Proteins such as FUS phase separate to form liquid-like condensates that can harden into less dynamic structures. However, how these properties emerge from the collective interactions of many amino acids remains largely unknown. Here, we use extensive mutagenesis to identify a sequence-encoded molecular grammar underlying the driving forces of phase separation of proteins in the FUS family and test aspects of this grammar in cells. Phase separation is primarily governed by multivalent interactions among tyrosine residues from prion-like domains and arginine residues from RNA-binding domains, which are modulated by negatively charged residues. Glycine residues enhance the fluidity, whereas glutamine and serine residues promote hardening. We develop a model to show that the measured saturation concentrations of phase separation are inversely proportional to the product of the numbers of arginine and tyrosine residues. These results suggest it is possible to predict phase-separation properties based on amino acid sequences.
Assuntos
Proteína FUS de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Sequência de Aminoácidos , Aminoácidos/química , Animais , Arginina/química , Simulação por Computador , Células HeLa , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/fisiologia , Transição de Fase , Proteínas Priônicas/química , Proteínas Priônicas/genética , Príons/genética , Príons/fisiologia , Domínios Proteicos , Proteína FUS de Ligação a RNA/fisiologia , Proteínas de Ligação a RNA/isolamento & purificação , Células Sf9 , Tirosina/químicaRESUMO
Myriad experiences produce transient memory, yet, contingent on the internal state of the organism and the saliency of the experience, only some memories persist over time. How experience and internal state influence the duration of memory at the molecular level remains unknown. A self-assembled aggregated state of Drosophila Orb2A protein is required specifically for long-lasting memory. We report that in the adult fly brain the mRNA encoding Orb2A protein exists in an unspliced non-protein-coding form. The convergence of experience and internal drive transiently increases the spliced protein-coding Orb2A mRNA. A screen identified pasilla, the fly ortholog of mammalian Nova-1/2, as a mediator of Orb2A mRNA processing. A single-nucleotide substitution in the intronic region that reduces Pasilla binding and intron removal selectively impairs long-term memory. We posit that pasilla-mediated processing of unspliced Orb2A mRNA integrates experience and internal state to control Orb2A protein abundance and long-term memory formation.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Íntrons , Memória de Longo Prazo , Ribonucleoproteínas/metabolismo , Fatores de Transcrição/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Animais , Sequência de Bases , Comportamento Animal , Encéfalo/metabolismo , Condicionamento Psicológico , Proteínas de Drosophila/química , Drosophila melanogaster/genética , Aprendizagem , Modelos Animais , Motivação , Mutação , Isoformas de Proteínas/metabolismo , Splicing de RNA , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/química , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Alterations in transcriptional regulators can orchestrate oncogenic gene expression programs in cancer. Here, we show that the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, which is mutated in over 20% of human tumors, interacts with EWSR1, a member of a family of proteins with prion-like domains (PrLD) that are frequent partners in oncogenic fusions with transcription factors. In Ewing sarcoma, we find that the BAF complex is recruited by the EWS-FLI1 fusion protein to tumor-specific enhancers and contributes to target gene activation. This process is a neomorphic property of EWS-FLI1 compared to wild-type FLI1 and depends on tyrosine residues that are necessary for phase transitions of the EWSR1 prion-like domain. Furthermore, fusion of short fragments of EWSR1 to FLI1 is sufficient to recapitulate BAF complex retargeting and EWS-FLI1 activities. Our studies thus demonstrate that the physical properties of prion-like domains can retarget critical chromatin regulatory complexes to establish and maintain oncogenic gene expression programs.
Assuntos
Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Sarcoma de Ewing/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Repetições de Microssatélites , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Priônicas/metabolismo , Domínios Proteicos , Sarcoma de Ewing/patologiaRESUMO
Manifestation of aggregate pathology in Huntington's disease is thought to be facilitated by a preferential vulnerability of affected brain cells to age-dependent proteostatic decline. To understand how specific cellular backgrounds may facilitate pathologic aggregation, we utilized the yeast model in which polyQ-expanded Huntingtin forms aggregates only when the endogenous prion-forming protein Rnq1 is in its amyloid-like prion [PIN+] conformation. We employed optogenetic clustering of polyQ protein as an orthogonal method to induce polyQ aggregation in prion-free [pin-] cells. Optogenetic aggregation circumvented the prion requirement for the formation of detergent-resistant polyQ inclusions but bypassed the formation of toxic polyQ oligomers, which accumulated specifically in [PIN+] cells. Reconstitution of aggregation in vitro suggested that these polyQ oligomers formed through direct templating on Rnq1 prions. These findings shed light on the mechanism of prion-mediated formation of oligomers, which may play a role in triggering polyQ pathology in the patient brain.
Assuntos
Príons , Proteínas de Saccharomyces cerevisiae , Humanos , Príons/genética , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismoRESUMO
Within the extensive range of self-propagating pathologic protein aggregates of mammals, prions are the most clearly infectious (e.g., â¼109 lethal doses per milligram). The structures of such lethal assemblies of PrP molecules have been poorly understood. Here we report a near-atomic core structure of a brain-derived, fully infectious prion (263K strain). Cryo-electron microscopy showed amyloid fibrils assembled with parallel in-register intermolecular ß sheets. Each monomer provides one rung of the ordered fibril core, with N-linked glycans and glycolipid anchors projecting outward. Thus, single monomers form the templating surface for incoming monomers at fibril ends, where prion growth occurs. Comparison to another prion strain (aRML) revealed major differences in fibril morphology but, like 263K, an asymmetric fibril cross-section without paired protofilaments. These findings provide structural insights into prion propagation, strains, species barriers, and membrane pathogenesis. This structure also helps frame considerations of factors influencing the relative transmissibility of other pathologic amyloids.
Assuntos
Encéfalo/metabolismo , Microscopia Crioeletrônica/métodos , Polissacarídeos/química , Príons/química , Príons/ultraestrutura , Amiloide/química , Animais , Glicolipídeos/química , Humanos , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Camundongos , Fenótipo , Proteínas Priônicas/química , Ligação Proteica , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
Populations of the Eastern Highlands of Papua New Guinea (EHPNG, area 11,157 km2) lived in relative isolation from the rest of the world until the mid-20th century, and the region contains a wealth of linguistic and cultural diversity. Notably, several populations of EHPNG were devastated by an epidemic prion disease, kuru, which at its peak in the mid-twentieth century led to some villages being almost depleted of adult women. Until now, population genetic analyses to learn about genetic diversity, migration, admixture, and the impact of the kuru epidemic have been restricted to a small number of variants or samples. Here, we present a population genetic analysis of the region based on genome-wide genotype data of 943 individuals from 21 linguistic groups and 68 villages in EHPNG, including 34 villages in the South Fore linguistic group, the group most affected by kuru. We find a striking degree of genetic population structure in the relatively small region (average FST between linguistic groups 0.024). The genetic population structure correlates well with linguistic grouping, with some noticeable exceptions that reflect the clan system of community organization that has historically existed in EHPNG. We also detect the presence of migrant individuals within the EHPNG region and observe a significant excess of females among migrants compared to among non-migrants in areas of high kuru exposure (p = 0.0145, chi-squared test). This likely reflects the continued practice of patrilocality despite documented fears and strains placed on communities as a result of kuru and its associated skew in female incidence.
Assuntos
Kuru , Príons , Adulto , Feminino , Humanos , Kuru/epidemiologia , Kuru/genética , Kuru/história , Papua Nova Guiné/epidemiologia , Príons/genética , Genótipo , AprendizagemRESUMO
Mammalian prion diseases are a group of neurodegenerative conditions caused by infection of the central nervous system with proteinaceous agents called prions, including sporadic, variant, and iatrogenic Creutzfeldt-Jakob disease; kuru; inherited prion disease; sheep scrapie; bovine spongiform encephalopathy; and chronic wasting disease. Prions are composed of misfolded and multimeric forms of the normal cellular prion protein (PrP). Prion diseases require host expression of the prion protein gene (PRNP) and a range of other cellular functions to support their propagation and toxicity. Inherited forms of prion disease are caused by mutation of PRNP, whereas acquired and sporadically occurring mammalian prion diseases are controlled by powerful genetic risk and modifying factors. Whereas some PrP amino acid variants cause the disease, others confer protection, dramatically altered incubation times, or changes in the clinical phenotype. Multiple mechanisms, including interference with homotypic protein interactions and the selection of the permissible prion strains in a host, play a role. Several non-PRNP factors have now been uncovered that provide insights into pathways of disease susceptibility or neurotoxicity.
Assuntos
Mamíferos/genética , Doenças Priônicas/genética , Proteínas Priônicas/genética , Animais , Bovinos , Modelos Animais de Doenças , Estudos de Associação Genética , Predisposição Genética para Doença , Testes Genéticos , Cabras/genética , Humanos , Camundongos , Polimorfismo Genético , Doenças Priônicas/etiologia , Proteínas Priônicas/metabolismo , Seleção Genética , Ovinos/genéticaRESUMO
Prion-like spread of disease-specific tau conformers is a hallmark of all tauopathies. A 19-residue probe peptide containing a P301L mutation and spanning the R2/R3 splice junction of tau folds and stacks into seeding-competent fibrils and induces aggregation of 4R, but not 3R tau. These tau peptide fibrils propagate aggregated intracellular tau over multiple generations, have a high ß-sheet content, a colocalized lipid signal, and adopt a well-defined U-shaped fold found in 4R tauopathy brain-derived fibrils. Fully atomistic replica exchange molecular dynamics (MD) simulations were used to compute the free energy landscapes of the conformational ensemble of the peptide monomers. These identified an aggregation-prohibiting ß-hairpin structure and an aggregation-competent U-fold unique to 4R tauopathy fibrils. Guided by MD simulations, we identified that the N-terminal-flanking residues to PHF6, which slightly vary between 4R and 3R isoforms, modulate seeding. Strikingly, when a single amino acid switch at position 305 replaced the serine of 4R tau with a lysine from the corresponding position in the first repeat of 3R tau, the seeding induced by the 19-residue peptide was markedly reduced. Conversely, a 4R tau mimic with three repeats, prepared by replacing those amino acids in the first repeat with those amino acids uniquely present in the second repeat, recovered aggregation when exposed to the 19-residue peptide. These peptide fibrils function as partial prions to recruit naive 4R tau-ten times the length of the peptide-and serve as a critical template for 4R tauopathy propagation. These results hint at opportunities for tau isoform-specific therapeutic interventions.
Assuntos
Príons , Tauopatias , Humanos , Proteínas tau/metabolismo , Tauopatias/metabolismo , Isoformas de Proteínas/metabolismo , Príons/metabolismo , Peptídeos , AminoácidosRESUMO
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 cellular host-factors that can modify 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 majority of the mouse protein-coding transcriptome. We identified 1,191 modulators of prion propagation. While 1,151 modified the expression of both the pathological prion protein, PrPSc , and its cellular counterpart, PrPC , 40 genes selectively affected PrPSc . Of the latter 40 genes, 20 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 PrPC . Hence, genome-wide QUIPPER-based perturbations can discover actionable cellular pathways involved in prion propagation. Further, the unexpected identification of a prion-controlling ribonucleoprotein suggests a role for RNA in the generation of infectious prions.
Assuntos
Doenças Priônicas , Príons , Camundongos , Animais , Ovinos/genética , Príons/genética , Príons/metabolismo , Drosophila melanogaster/genética , Ribonucleoproteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Doenças Priônicas/genética , Doenças Priônicas/patologia , Mamíferos/genéticaRESUMO
A class of long noncoding RNAs (lncRNAs) has architectural functions in nuclear body construction; however, specific RNA domains dictating their architectural functions remain uninvestigated. Here, we identified the domains of the architectural NEAT1 lncRNA that construct paraspeckles. Systematic deletion of NEAT1 portions using CRISPR/Cas9 in haploid cells revealed modular domains of NEAT1 important for RNA stability, isoform switching, and paraspeckle assembly. The middle domain, containing functionally redundant subdomains, was responsible for paraspeckle assembly. Artificial tethering of the NONO protein to a NEAT1_2 mutant lacking the functional subdomains rescued paraspeckle assembly, and this required the NOPS dimerization domain of NONO. Paraspeckles exhibit phase-separated properties including susceptibility to 1,6-hexanediol treatment. RNA fragments of the NEAT1_2 subdomains preferentially bound NONO/SFPQ, leading to phase-separated aggregates in vitro. Thus, we demonstrate that the enrichment of NONO dimers on the redundant NEAT1_2 subdomains initiates construction of phase-separated paraspeckles, providing mechanistic insights into lncRNA-based nuclear body formation.
Assuntos
RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Sequência de Bases , Sistemas CRISPR-Cas , Núcleo Celular/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Domínios Proteicos , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Estabilidade de RNA , Fatores de Transcrição/metabolismoRESUMO
Microglia are resident macrophages of the brain parenchyma and play an essential role in various aspects of brain development, plasticity, and homeostasis. With recent advances in single-cell RNA-sequencing, heterogeneous microglia transcriptional states have been identified in both animal models of neurodegenerative disorders and patients. However, the functional roles of these microglia states remain unclear; specifically, the question of whether individual states or combinations of states are protective or detrimental (or both) in the context of disease progression. To attempt to answer this, the field has largely relied on studies employing mouse models, human in vitro and chimeric models, and human post-mortem tissue, all of which have their caveats, but used in combination can enable new biological insight and validation of candidate disease pathways and mechanisms. In this review, we summarize our current understanding of disease-associated microglia states and phenotypes in neurodegenerative disorders, discuss important considerations when comparing mouse and human microglia states and functions, and identify areas of microglia biology where species differences might limit our understanding of microglia state.
Assuntos
Doenças Neurodegenerativas , Humanos , Animais , Camundongos , Doenças Neurodegenerativas/metabolismo , Microglia , Macrófagos/metabolismo , Modelos Animais de Doenças , EncéfaloRESUMO
Prions are proteinaceous infectious particles that replicate by structural conversion of the host-encoded cellular prion protein (PrPC), causing fatal neurodegenerative diseases in mammals. Species-specific amino acid substitutions (AAS) arising from single nucleotide polymorphisms within the prion protein gene (Prnp) modulate prion disease pathogenesis, and, in several instances, reduce susceptibility of homo- or heterozygous AAS carriers to prion infection. However, a mechanistic understanding of their protective effects against clinical disease is missing. We generated gene-targeted mouse infection models of chronic wasting disease (CWD), a highly contagious prion disease of cervids. These mice express wild-type deer or PrPC harboring the S138N substitution homo- or heterozygously, a polymorphism found exclusively in reindeer (Rangifer tarandus spp.) and fallow deer (Dama dama). The wild-type deer PrP-expressing model recapitulated CWD pathogenesis including fecal shedding. Encoding at least one 138N allele prevented clinical CWD, accumulation of protease-resistant PrP (PrPres) and abnormal PrP deposits in the brain tissue. However, prion seeding activity was detected in spleens, brains, and feces of these mice, suggesting subclinical infection accompanied by prion shedding. 138N-PrPC was less efficiently converted to PrPres in vitro than wild-type deer (138SS) PrPC. Heterozygous coexpression of wild-type deer and 138N-PrPC resulted in dominant-negative inhibition and progressively diminished prion conversion over serial rounds of protein misfolding cyclic amplification. Our study indicates that heterozygosity at a polymorphic Prnp codon can confer the highest protection against clinical CWD and highlights the potential role of subclinical carriers in CWD transmission.
Assuntos
Cervos , Doenças Priônicas , Príons , Rena , Doença de Emaciação Crônica , Camundongos , Animais , Príons/metabolismo , Proteínas Priônicas/genética , Cervos/genética , Doença de Emaciação Crônica/genética , Camundongos Transgênicos , Doenças Priônicas/genéticaRESUMO
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.
Assuntos
Doença de Alzheimer , Anticorpos de Domínio Único , Paralisia Supranuclear Progressiva , Humanos , Animais , Camundongos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Proteínas tau/metabolismo , Anticorpos de Domínio Único/farmacologia , Anticorpos de Domínio Único/metabolismo , Emaranhados Neurofibrilares/metabolismo , Paralisia Supranuclear Progressiva/metabolismo , Anticorpos/metabolismo , Encéfalo/metabolismoRESUMO
Retrotransposons and retroviruses shape genome evolution and can negatively impact genome function. Saccharomyces cerevisiae and its close relatives harbor several families of LTR-retrotransposons, the most abundant being Ty1 in several laboratory strains. The cytosolic foci that nucleate Ty1 virus-like particle (VLP) assembly are not well understood. These foci, termed retrosomes or T-bodies, contain Ty1 Gag and likely Gag-Pol and the Ty1 mRNA destined for reverse transcription. Here, we report an intrinsically disordered N-terminal prion-like domain (PrLD) within Gag that is required for transposition. This domain contains amino acid composition similar to known yeast prions and is sufficient to nucleate prionogenesis in an established cell-based prion reporter system. Deleting the Ty1 PrLD results in dramatic VLP assembly and retrotransposition defects but does not affect Gag protein level. Ty1 Gag chimeras in which the PrLD is replaced with other sequences, including yeast and mammalian prionogenic domains, display a range of retrotransposition phenotypes from wild type to null. We examine these chimeras throughout the Ty1 replication cycle and find that some support retrosome formation, VLP assembly, and retrotransposition, including the yeast Sup35 prion and the mouse PrP prion. Our interchangeable Ty1 system provides a useful, genetically tractable in vivo platform for studying PrLDs, complete with a suite of robust and sensitive assays. Our work also invites study into the prevalence of PrLDs in additional mobile elements.