RESUMO
Although complex inflammatory-like alterations are observed around the amyloid plaques of Alzheimer's disease (AD), little is known about the molecular changes and cellular interactions that characterize this response. We investigate here, in an AD mouse model, the transcriptional changes occurring in tissue domains in a 100-µm diameter around amyloid plaques using spatial transcriptomics. We demonstrate early alterations in a gene co-expression network enriched for myelin and oligodendrocyte genes (OLIGs), whereas a multicellular gene co-expression network of plaque-induced genes (PIGs) involving the complement system, oxidative stress, lysosomes, and inflammation is prominent in the later phase of the disease. We confirm the majority of the observed alterations at the cellular level using in situ sequencing on mouse and human brain sections. Genome-wide spatial transcriptomics analysis provides an unprecedented approach to untangle the dysregulated cellular network in the vicinity of pathogenic hallmarks of AD and other brain diseases.
Assuntos
Doença de Alzheimer/patologia , Análise de Sequência de DNA/métodos , Transcriptoma , Doença de Alzheimer/genética , Amiloide/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas do Sistema Complemento/genética , Proteínas do Sistema Complemento/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Bainha de Mielina/genética , Bainha de Mielina/metabolismo , Estresse Oxidativo/genéticaRESUMO
Reduction of amyloid beta (Aß) has been shown to be effective in treating Alzheimer's disease (AD), but the underlying assumption that neurons are the main source of pathogenic Aß is untested. Here, we challenge this prevailing belief by demonstrating that oligodendrocytes are an important source of Aß in the human brain and play a key role in promoting abnormal neuronal hyperactivity in an AD knock-in mouse model. We show that selectively suppressing oligodendrocyte Aß production improves AD brain pathology and restores neuronal function in the mouse model in vivo. Our findings suggest that targeting oligodendrocyte Aß production could be a promising therapeutic strategy for treating AD.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Modelos Animais de Doenças , Camundongos Transgênicos , Neurônios , Oligodendroglia , Animais , Feminino , Humanos , Masculino , Camundongos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Técnicas de Introdução de Genes , Neurônios/metabolismo , Oligodendroglia/metabolismoRESUMO
Ten years of remarkable progress in understanding the fundamental biochemistry of Alzheimer's disease have been followed by ten years of remarkable and increasing clinical insight into the natural progression of the disorder. The concept of a long, intermediary, prodromal phase between the first appearance of amyloid plaques and tangles and the manifestation of dementia is now well established. The major challenge for the next decade is to chart the many cellular processes that underlie this phase and link the biochemical alterations to the clinical manifestation of Alzheimer's disease. We discuss here how genetics, new cell culture systems, and improved animal models will fuel this work. We anticipate that the resulting novel insights will provide a basis for further drug development for this terrible disease.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Placa Amiloide/metabolismo , Doença de Alzheimer/fisiopatologia , Doença de Alzheimer/terapia , Animais , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Humanos , Proteínas tau/metabolismoRESUMO
Recently, we reported oligoadenylate synthetase 1 (OAS1) contributed to the risk of Alzheimer's disease, by its enrichment in transcriptional networks expressed by microglia. However, the function of OAS1 within microglia was not known. Using genotyping from 1313 individuals with sporadic Alzheimer's disease and 1234 control individuals, we confirm the OAS1 variant, rs1131454, is associated with increased risk for Alzheimer's disease. The same OAS1 locus has been recently associated with severe coronavirus disease 2019 (COVID-19) outcomes, linking risk for both diseases. The single nucleotide polymorphisms rs1131454(A) and rs4766676(T) are associated with Alzheimer's disease, and rs10735079(A) and rs6489867(T) are associated with severe COVID-19, where the risk alleles are linked with decreased OAS1 expression. Analysing single-cell RNA-sequencing data of myeloid cells from Alzheimer's disease and COVID-19 patients, we identify co-expression networks containing interferon (IFN)-responsive genes, including OAS1, which are significantly upregulated with age and both diseases. In human induced pluripotent stem cell-derived microglia with lowered OAS1 expression, we show exaggerated production of TNF-α with IFN-γ stimulation, indicating OAS1 is required to limit the pro-inflammatory response of myeloid cells. Collectively, our data support a link between genetic risk for Alzheimer's disease and susceptibility to critical illness with COVID-19 centred on OAS1, a finding with potential implications for future treatments of Alzheimer's disease and COVID-19, and development of biomarkers to track disease progression.
Assuntos
2',5'-Oligoadenilato Sintetase/genética , Doença de Alzheimer/genética , COVID-19/genética , Ligação Genética/genética , Predisposição Genética para Doença/genética , Gravidade do Paciente , Adolescente , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/epidemiologia , COVID-19/diagnóstico , COVID-19/epidemiologia , Células Cultivadas , Feminino , Redes Reguladoras de Genes/genética , Predisposição Genética para Doença/epidemiologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Adulto JovemRESUMO
Amyloid ß-protein (Aß) sequence length variants with varying aggregation propensity coexist in vivo, where coaggregation and cross-catalysis phenomena may affect the aggregation process. Until recently, naturally occurring amyloid ß-protein (Aß) variants were believed to begin at or after the canonical ß-secretase cleavage site within the amyloid ß-protein precursor. However, N-terminally extended forms of Aß (NTE-Aß) were recently discovered and may contribute to Alzheimer's disease. Here, we have used thioflavin T fluorescence to study the aggregation kinetics of Aß42 variants with N-terminal extensions of 5-40 residues, and transmission electron microscopy to analyze the end states. We find that all variants form amyloid fibrils of similar morphology as Aß42, but the half-time of aggregation (t1/2) increases exponentially with extension length. Monte Carlo simulations of model peptides suggest that the retardation is due to an underlying general physicochemical effect involving reduced frequency of productive molecular encounters. Indeed, global kinetic analyses reveal that NTE-Aß42s form fibrils via the same mechanism as Aß42, but all microscopic rate constants (primary and secondary nucleation, elongation) are reduced for the N-terminally extended variants. Still, Aß42 and NTE-Aß42 coaggregate to form mixed fibrils and fibrils of either Aß42 or NTE-Aß42 catalyze aggregation of all monomers. NTE-Aß42 monomers display reduced aggregation rate with all kinds of seeds implying that extended termini interfere with the ability of monomers to nucleate or elongate. Cross-seeding or coaggregation may therefore represent an important contribution in the in vivo formation of assemblies believed to be important in disease.
Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Dicroísmo Circular , Cinética , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Método de Monte CarloRESUMO
INTRODUCTION: The cause of sporadic Alzheimer's disease (AD) remains unclear. Given the growing evidence that protein aggregates can spread in a "prion-like" fashion, we reasoned that a small population of brain cells producing such "prion-like" particles due to a postzygotic acquired mutation would be sufficient to trigger the disease. Deep DNA sequencing technology should in principle allow the detection of such mosaics. METHODS: To detect the somatic mutations of genes causing AD present in a small number of cells, we developed a targeted deep sequencing approach to scrutinize the genomic loci of APP, PSEN1, and PSEN2 genes in DNA extracted from the entorhinal cortex, one of the brain regions showing the earliest signs of AD pathology. We also included the analysis of the MAPT gene because mutations may promote tangle formation. We validated candidate mutations with an independent targeted ultradeep amplicon sequencing technique. RESULTS: We demonstrate that our approach can detect single-nucleotide mosaic variants with a 1% allele frequency and copy number mosaic variants present in as few as 10% of cells. We screened 72 AD and 58 control brain samples and identified three mosaic variants with low allelic frequency (â¼1%): two novel MAPT variants in sporadic AD patients and a known PSEN2 variant in a Braak II control subject. Moreover, we detected both novel and known pathogenic nonmosaic heterozygous variants in PSEN1 and PSEN2 in this cohort of sporadic AD patients. DISCUSSION: Our results show that mosaic mutations with low allelic frequencies in AD-relevant genes can be detected in brain-derived DNA, but larger samples need to be investigated before a more definitive conclusion with regard to the pathogenicity of such mosaics can be made.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Córtex Entorrinal/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mosaicismo , Análise de Sequência de DNA/métodos , Idoso , Idoso de 80 Anos ou mais , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Estudos de Coortes , Variações do Número de Cópias de DNA , Frequência do Gene , Humanos , Mutação , Projetos Piloto , Polimorfismo de Nucleotídeo Único , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/genética , Presenilina-2/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Alzheimer's disease (AD) is characterized by synaptic loss, which can result from dysfunctional microglial phagocytosis and complement activation. However, what signals drive aberrant microglia-mediated engulfment of synapses in AD is unclear. Here we report that secreted phosphoprotein 1 (SPP1/osteopontin) is upregulated predominantly by perivascular macrophages and, to a lesser extent, by perivascular fibroblasts. Perivascular SPP1 is required for microglia to engulf synapses and upregulate phagocytic markers including C1qa, Grn and Ctsb in presence of amyloid-ß oligomers. Absence of Spp1 expression in AD mouse models results in prevention of synaptic loss. Furthermore, single-cell RNA sequencing and putative cell-cell interaction analyses reveal that perivascular SPP1 induces microglial phagocytic states in the hippocampus of a mouse model of AD. Altogether, we suggest a functional role for SPP1 in perivascular cells-to-microglia crosstalk, whereby SPP1 modulates microglia-mediated synaptic engulfment in mouse models of AD.
Assuntos
Doença de Alzheimer , Camundongos , Animais , Doença de Alzheimer/metabolismo , Microglia/metabolismo , Osteopontina/metabolismo , Fagócitos/metabolismo , Macrófagos/metabolismo , Fagocitose , Modelos Animais de Doenças , Peptídeos beta-Amiloides/metabolismoRESUMO
Single-nucleus RNA sequencing (snRNA-seq) is used as an alternative to single-cell RNA-seq, as it allows transcriptomic profiling of frozen tissue. However, it is unclear whether snRNA-seq is able to detect cellular state in human tissue. Indeed, snRNA-seq analyses of human brain samples have failed to detect a consistent microglial activation signature in Alzheimer's disease. Our comparison of microglia from single cells and single nuclei of four human subjects reveals that, although most genes show similar relative abundances in cells and nuclei, a small population of genes (â¼1%) is depleted in nuclei compared to whole cells. This population is enriched for genes previously implicated in microglial activation, including APOE, CST3, SPP1, and CD74, comprising 18% of previously identified microglial-disease-associated genes. Given the low sensitivity of snRNA-seq to detect many activation genes, we conclude that snRNA-seq is not suited for detecting cellular activation in microglia in human disease.
Assuntos
Perfilação da Expressão Gênica/métodos , Microglia/fisiologia , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , HumanosRESUMO
Inhibition of gamma-secretase cleavage of the amyloid precursor protein (APP) is a prime target for the development of therapeutics for treating Alzheimer's disease; however, complete inhibition of this activity would also impair the processing of many other proteins, including the APP homologues, amyloid precursor-like protein (APLP) 1 and 2. To prevent unwanted side effects, therapeutically useful gamma-secretase inhibitors should specifically target APP processing while sparing cleavage of other gamma-substrates. Thus, since APLP1 and APLP2 are more similar to APP than any of the other known gamma-secretase substrates and have important physiological roles in their own right, we reasoned that comparison of the effect of gamma-secretase inhibitors on APLP processing should provide a sensitive indicator of the selectivity of putative inhibitors. To address this issue, we have optimized microsome and cell culture assays to monitor the gamma-secretase proteolysis of APP and APLPs. Production of the gamma-secretase-generated intracellular domain (ICD) occurs more rapidly from APLP1 than from either APLP2 or APP, suggesting that APLP1 is a better gamma-substrate and that substrate recognition is not restricted to the highly conserved amino acid sequences surrounding the epsilon-site. As expected, the well-characterized gamma-secretase modulator, fenofibrate, did not inhibit ICD release, whereas a related compound, FT-9, inhibited gamma-secretase both in microsomes and in whole cells. Importantly, FT-9 displayed a preferential effect, inhibiting cleavage of APP much more effectively than cleavage of APLP1. These findings suggest that selective inhibitors can be developed and that screening of compounds against APP and APLPs should assist in this process.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Fenofibrato/análogos & derivados , Inibidores de Proteases/farmacologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Benzodiazepinonas/farmacologia , Biocatálise/efeitos dos fármacos , Células CHO , Cricetinae , Cricetulus , Dipeptídeos/farmacologia , Fenofibrato/farmacologia , Flurbiprofeno/farmacologia , Humanos , Cinética , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Microssomos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato/efeitos dos fármacos , TransfecçãoRESUMO
RNA analysis at the cellular resolution in the human brain is challenging. Here, we describe an optimised approach for detecting single RNA transcripts in a cell-type specific manner in frozen human brain tissue using multiplexed fluorescent RNAscope probes. We developed a new robust analytical approach for RNAscope quantification. Our method shows that low RNA integrity does not significantly affect RNAscope signal, recapitulates bulk RNA analysis and provides spatial context to transcriptomic analysis of human post-mortem brain at single-cell resolution. In summary, our optimised method allows the usage of frozen human samples from brain banks to perform quantitative RNAscope analysis.
Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica , RNA Mensageiro/genética , Análise de Célula Única , Doença de Alzheimer/genética , Congelamento , Células HeLa , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Microglia/metabolismo , RNA Mensageiro/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Regulação para CimaRESUMO
Gene expression profiles of more than 10,000 individual microglial cells isolated from cortex and hippocampus of male and female AppNL-G-F mice over time demonstrate that progressive amyloid-ß accumulation accelerates two main activated microglia states that are also present during normal aging. Activated response microglia (ARMs) are composed of specialized subgroups overexpressing MHC type II and putative tissue repair genes (Dkk2, Gpnmb, and Spp1) and are strongly enriched with Alzheimer's disease (AD) risk genes. Microglia from female mice progress faster in this activation trajectory. Similar activated states are also found in a second AD model and in human brain. Apoe, the major genetic risk factor for AD, regulates the ARMs but not the interferon response microglia (IRMs). Thus, the ARMs response is the converging point for aging, sex, and genetic AD risk factors.
Assuntos
Envelhecimento/patologia , Doença de Alzheimer/patologia , Biomarcadores/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Microglia/patologia , Placa Amiloide/patologia , Envelhecimento/genética , Envelhecimento/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/fisiologia , Animais , Biomarcadores/análise , Encéfalo/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Camundongos Transgênicos , Microglia/metabolismo , Placa Amiloide/genética , Placa Amiloide/metabolismo , Presenilinas/fisiologia , Caracteres SexuaisRESUMO
Although genetics highlights the role of microglia in Alzheimer's disease, one-third of putative Alzheimer's disease risk genes lack adequate mouse orthologs. Here we successfully engraft human microglia derived from embryonic stem cells in the mouse brain. The cells recapitulate transcriptionally human primary microglia ex vivo and show expression of human-specific Alzheimer's disease risk genes. Oligomeric amyloid-ß induces a divergent response in human versus mouse microglia. This model can be used to study the role of microglia in neurological diseases.
Assuntos
Doença de Alzheimer/genética , Células-Tronco Embrionárias/citologia , Microglia/metabolismo , Microglia/transplante , Transcriptoma , Peptídeos beta-Amiloides/farmacologia , Animais , Diferenciação Celular , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Microglia/efeitos dos fármacosRESUMO
Radial glial progenitors play pivotal roles in the development and patterning of the spinal cord, and their fate is controlled by Notch signaling. How Notch is shaped to regulate their crucial transition from expansion toward differentiation remains, however, unknown. miR-132 in the developing zebrafish dampens Notch signaling via a cascade involving the transcriptional corepressor Ctbp2 and the Notch suppressor Sirt1. At early embryonic stages, high Ctbp2 levels sustain Notch signaling and radial glial expansion and concomitantly induce miR-132 expression via a double-negative feedback loop involving Rest inhibition. The changing balance in miR-132 and Ctbp2 interaction gradually drives the switch in Notch output and radial glial progenitor fate as part of the larger developmental program involved in the transition from embryonic to larval spinal cord.
Assuntos
Diferenciação Celular , MicroRNAs/metabolismo , Microglia/metabolismo , Células-Tronco Neurais/metabolismo , Receptores Notch/metabolismo , Proteínas Repressoras/metabolismo , Medula Espinal/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Proteínas do Olho , Retroalimentação Fisiológica , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Microglia/citologia , Células-Tronco Neurais/citologia , Receptores Notch/genética , Proteínas Repressoras/genética , Sirtuína 1/genética , Sirtuína 1/metabolismo , Medula Espinal/citologia , Medula Espinal/embriologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
OBJECTIVE: We evaluated microRNAs (miRNAs) as potential biomarkers for Alzheimer disease (AD) by analyzing the expression level of miRNAs in CSF of patients with AD dementia and nonaffected control subjects. METHODS: Using quantitative PCR, we profiled the expression level of 728 miRNAs in CSF of nonaffected control subjects and patients with clinically ascertained AD dementia, and we further compared the expression level of candidate miRNAs in 37 control subjects and 35 patients with AD dementia. RESULTS: The level of hsa-miR-27a-3p in CSF is reduced in patients with dementia due to AD in 2 different cohorts of subjects (cohort 1: p = 0.008; cohort 2: p = 0.015; 2-tailed unpaired Welch t test). Moreover, low levels of hsa-miR-27a-3p were accompanied by high CSF tau levels and low CSF ß-amyloid levels. CONCLUSIONS: Our pilot study highlights hsa-miR-27a-3p as a candidate biomarker for AD and provides the groundwork for further confirmation studies in larger cohorts and in other hospitals.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Regulação da Expressão Gênica , MicroRNAs/antagonistas & inibidores , MicroRNAs/líquido cefalorraquidiano , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/metabolismo , Biomarcadores/líquido cefalorraquidiano , Estudos de Coortes , Feminino , Humanos , Masculino , MicroRNAs/biossíntese , Pessoa de Meia-Idade , Projetos PilotoRESUMO
Aggregation of the amyloid ß-protein (Aß) is believed to play a central role in initiating the molecular cascade that culminates in Alzheimer-type dementia (AD), a disease which in its early stage is characterized by synaptic loss and impairment of episodic memory. Here we show that intracerebroventricular injection of Aß-containing water-soluble extracts of AD brain inhibits consolidation of the memory of avoidance learning in the rat and that this effect is highly dependent on the interval between learning and administration. When injected at 1 hour post training extracts from 2 different AD brains significantly impaired recall tested at 48 hours. Ultrastructural examination of hippocampi from animals perfused after 48 hours revealed that Aß-mediated impairment of avoidance memory was associated with lower density of synapses and altered synaptic structure in the dentate gyrus and CA1 fields. These behavioral and ultrastructural data suggest that human brain-derived Aß impairs formation of long-term memory by compromising the structural plasticity essential for consolidation and that Aß targets processes initiated very early in the consolidation pathway.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Aprendizagem da Esquiva/efeitos dos fármacos , Encéfalo/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Masculino , Memória Episódica , Ratos , Ratos WistarRESUMO
The amyloid precursor family of proteins are of considerable interest, both because of their role in Alzheimer's disease pathogenesis and because of their normal physiological functions. In mammals, the amyloid precursor protein (APP) has two homologs, amyloid precursor-like protein (APLP) 1 and APLP2. All three proteins undergo ectodomain shedding and regulated intramembrane proteolysis, and important functions have been attributed to the full-length proteins, shed ectodomains, C-terminal fragments and intracellular domains (ICDs). One of the proteases that is known to cleave APP and that is essential for generation of the amyloid beta-protein is the beta-site APP-cleaving enzyme 1 (BACE1). Here, we investigated the effects of genetic manipulation of BACE1 on the processing of the APP family of proteins. BACE1 expression regulated the levels and species of full-length APLP1, APP and APLP2, of their shed ectodomains, and of their membrane-bound C-terminal fragments. In particular, APP processing appears to be tightly regulated, with changes in beta-cleaved APPs (APPsbeta) being compensated for by changes in alpha-cleaved APPs (APPsalpha). In contrast, the total levels of soluble cleaved APLP1 and APLP2 species were less tightly regulated, and fluctuated with BACE1 expression. Importantly, the production of ICDs for all three proteins was not decreased by loss of BACE1 activity. These results indicate that BACE1 is involved in regulating ectodomain shedding, maturation and trafficking of the APP family of proteins. Consequently, whereas inhibition of BACE1 is unlikely to adversely affect potential ICD-mediated signaling, it may alter other important facets of amyloid precursor-like protein/APP biology.