RESUMO
In neurodegenerative diseases, proteins fold into amyloid structures with distinct conformations (strains) that are characteristic of different diseases. However, there is a need to rapidly identify amyloid conformations in situ. Here, we use machine learning on the full information available in fluorescent excitation/emission spectra of amyloid-binding dyes to identify six distinct different conformational strains in vitro, as well as amyloid-ß (Aß) deposits in different transgenic mouse models. Our EMBER (excitation multiplexed bright emission recording) imaging method rapidly identifies conformational differences in Aß and tau deposits from Down syndrome, sporadic and familial Alzheimer's disease human brain slices. EMBER has in situ identified distinct conformational strains of tau inclusions in astrocytes, oligodendrocytes, and neurons from Pick's disease. In future studies, EMBER should enable high-throughput measurements of the fidelity of strain transmission in cellular and animal neurodegenerative diseases models, time course of amyloid strain propagation, and identification of pathogenic versus benign strains.
Assuntos
Doença de Alzheimer , Doença de Pick , Camundongos , Animais , Humanos , Microscopia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Doença de Pick/metabolismo , Amiloide/metabolismo , Encéfalo/metabolismo , Camundongos Transgênicos , Proteínas tau/metabolismo , Placa Amiloide/metabolismoRESUMO
RATIONALE: Cardiac pacemaker cells (PCs) in the sinoatrial node (SAN) have a distinct gene expression program that allows them to fire automatically and initiate the heartbeat. Although critical SAN transcription factors, including Isl1 (Islet-1), Tbx3 (T-box transcription factor 3), and Shox2 (short-stature homeobox protein 2), have been identified, the cis-regulatory architecture that governs PC-specific gene expression is not understood, and discrete enhancers required for gene regulation in the SAN have not been identified. OBJECTIVE: To define the epigenetic profile of PCs using comparative ATAC-seq (assay for transposase-accessible chromatin with sequencing) and to identify novel enhancers involved in SAN gene regulation, development, and function. METHODS AND RESULTS: We used ATAC-seq on sorted neonatal mouse SAN to compare regions of accessible chromatin in PCs and right atrial cardiomyocytes. PC-enriched assay for transposase-accessible chromatin peaks, representing candidate SAN regulatory elements, were located near established SAN genes and were enriched for distinct sets of TF (transcription factor) binding sites. Among several novel SAN enhancers that were experimentally validated using transgenic mice, we identified a 2.9-kb regulatory element at the Isl1 locus that was active specifically in the cardiac inflow at embryonic day 8.5 and throughout later SAN development and maturation. Deletion of this enhancer from the genome of mice resulted in SAN hypoplasia and sinus arrhythmias. The mouse SAN enhancer also directed reporter activity to the inflow tract in developing zebrafish hearts, demonstrating deep conservation of its upstream regulatory network. Finally, single nucleotide polymorphisms in the human genome that occur near the region syntenic to the mouse enhancer exhibit significant associations with resting heart rate in human populations. CONCLUSIONS: (1) PCs have distinct regions of accessible chromatin that correlate with their gene expression profile and contain novel SAN enhancers, (2) cis-regulation of Isl1 specifically in the SAN depends upon a conserved SAN enhancer that regulates PC development and SAN function, and (3) a corresponding human ISL1 enhancer may regulate human SAN function.
Assuntos
Arritmia Sinusal/metabolismo , Relógios Biológicos , Sequenciamento de Cromatina por Imunoprecipitação , Elementos Facilitadores Genéticos , Frequência Cardíaca , Proteínas com Homeodomínio LIM/metabolismo , Nó Sinoatrial/metabolismo , Fatores de Transcrição/metabolismo , Potenciais de Ação , Animais , Arritmia Sinusal/genética , Arritmia Sinusal/fisiopatologia , Epigênese Genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Humanos , Proteínas com Homeodomínio LIM/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Polimorfismo de Nucleotídeo Único , Nó Sinoatrial/fisiopatologia , Fatores de Tempo , Fatores de Transcrição/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Down syndrome (DS) is a common genetic condition caused by trisomy of chromosome 21. Among the complex clinical features including musculoskeletal, neurological and cardiovascular disabilities, individuals with DS have an increased risk of developing progressive dementia and early onset Alzheimer's Disease (AD). This is attributed to the increased gene dosage of amyloid-ß (Aß) precursor protein gene, the formation of self-propagating Aß and tau prion conformers, and the deposition of neurotoxic Aß plaques and tau neurofibrillary tangles. Tau amyloid fibrils have previously been established to adopt many distinct conformations across different neurodegenerative conditions. Here we report the characterization of brain samples from four DS cases spanning 36 to 63 years of age by spectral confocal imaging with conformation-specific dyes and cryo-electron microscopy (cryo-EM) to determine structures of isolated tau fibrils. High-resolution structures reveal paired helical filament (PHF) and straight filament (SF) conformations of tau that are identical to those determined from AD. The PHFs and SFs are made of two C-shaped protofilaments with a cross-ß/ß-helix motif. Similar to filaments from AD cases, most filaments from the DS cases adopted the PHF form, while a minority (~20%) formed SFs. Samples from the youngest individual with no documented dementia had sparse tau deposits. To isolate tau for cryo-EM from this challenging sample we used a novel affinity-grid method involving a graphene-oxide surface derivatized with anti-tau antibodies. This improved isolation and revealed primarily tau PHFs and a minor population of chronic traumatic encephalopathy type II-like filaments were present in this youngest case. These findings expand the similarities between AD and DS to the molecular level, providing insight into their related pathologies and the potential for targeting common tau filament folds by small-molecule therapeutics and diagnostics.
RESUMO
Down syndrome (DS) is a common genetic condition caused by trisomy of chromosome 21. Among their complex clinical features, including musculoskeletal, neurological, and cardiovascular disabilities, individuals with DS have an increased risk of developing progressive dementia and early-onset Alzheimer's disease (AD). This dementia is attributed to the increased gene dosage of the amyloid-ß (Aß) precursor protein gene, the formation of self-propagating Aß and tau prion conformers, and the deposition of neurotoxic Aß plaques and tau neurofibrillary tangles. Tau amyloid fibrils have previously been established to adopt many distinct conformations across different neurodegenerative conditions. Here, we report the characterization of brain samples from four DS cases spanning 36-63 years of age by spectral confocal imaging with conformation-specific dyes and cryo-electron microscopy (cryo-EM) to determine structures of isolated tau fibrils. High-resolution structures revealed paired helical filament (PHF) and straight filament (SF) conformations of tau that were identical to those determined from AD cases. The PHFs and SFs are made of two C-shaped protofilaments, each containing a cross-ß/ß-helix motif. Similar to filaments from AD cases, most filaments from the DS cases adopted the PHF form, while a minority (approximately 20%) formed SFs. Samples from the youngest individual with no documented dementia had sparse tau deposits. To isolate tau for cryo-EM from this challenging sample we used a novel affinity-grid method involving a graphene oxide surface derivatized with anti-tau antibodies. This method improved isolation and revealed that primarily tau PHFs and a minor population of chronic traumatic encephalopathy type II-like filaments were present in this youngest case. These findings expand the similarities between AD and DS to the molecular level, providing insight into their related pathologies and the potential for targeting common tau filament folds by small-molecule therapeutics and diagnostics.
Assuntos
Doença de Alzheimer , Microscopia Crioeletrônica , Síndrome de Down , Proteínas tau , Humanos , Síndrome de Down/patologia , Síndrome de Down/metabolismo , Proteínas tau/metabolismo , Proteínas tau/ultraestrutura , Microscopia Crioeletrônica/métodos , Pessoa de Meia-Idade , Doença de Alzheimer/patologia , Doença de Alzheimer/metabolismo , Feminino , Adulto , Masculino , Emaranhados Neurofibrilares/patologia , Emaranhados Neurofibrilares/metabolismo , Encéfalo/patologia , Encéfalo/metabolismo , Encéfalo/ultraestruturaRESUMO
In neurodegenerative diseases proteins fold into amyloid structures with distinct conformations (strains) that are characteristic of different diseases. However, there is a need to rapidly identify amyloid conformations in situ . Here we use machine learning on the full information available in fluorescent excitation/emission spectra of amyloid binding dyes to identify six distinct different conformational strains in vitro , as well as Aß deposits in different transgenic mouse models. Our EMBER (excitation multiplexed bright emission recording) imaging method rapidly identifies conformational differences in Aß and tau deposits from Down syndrome, sporadic and familial Alzheimer's disease human brain slices. EMBER has in situ identified distinct conformational strains of tau inclusions in astrocytes, oligodendrocytes, and neurons from Pick's disease. In future studies, EMBER should enable high-throughput measurements of the fidelity of strain transmission in cellular and animal neurodegenerative diseases models, time course of amyloid strain propagation, and identification of pathogenic versus benign strains. Significance: In neurodegenerative diseases proteins fold into amyloid structures with distinct conformations (strains) that are characteristic of different diseases. There is a need to rapidly identify these amyloid conformations in situ . Here we use machine learning on the full information available in fluorescent excitation/emission spectra of amyloid binding dyes to identify six distinct different conformational strains in vitro , as well as Aß deposits in different transgenic mouse models. Our imaging method rapidly identifies conformational differences in Aß and tau deposits from Down syndrome, sporadic and familial Alzheimer's disease human brain slices. We also identified distinct conformational strains of tau inclusions in astrocytes, oligodendrocytes, and neurons from Pick's disease. These findings will facilitate the identification of pathogenic protein aggregates to guide research and treatment of protein misfolding diseases.
RESUMO
Ultraconserved enhancer sequences show perfect conservation between human and rodent genomes, suggesting that their functions are highly sensitive to mutation. However, current models of enhancer function do not sufficiently explain this extreme evolutionary constraint. We subjected 23 ultraconserved enhancers to different levels of mutagenesis, collectively introducing 1,547 mutations, and examined their activities in transgenic mouse reporter assays. Overall, we find that the regulatory properties of ultraconserved enhancers are robust to mutation. Upon mutagenesis, nearly all (19/23, 83%) still functioned as enhancers at one developmental stage, as did most of those tested again later in development (5/9, 56%). Replacement of endogenous enhancers with mutated alleles in mice corroborated results of transgenic assays, including the functional resilience of ultraconserved enhancers to mutation. Our findings show that the currently known activities of ultraconserved enhancers do not necessarily require the perfect conservation observed in evolution and suggest that additional regulatory or other functions contribute to their sequence constraint.