RESUMEN
Approximately half of Alzheimer's disease (AD) brains have concomitant Lewy pathology at autopsy, suggesting that α-synuclein (α-SYN) aggregation is a regulated event in the pathogenesis of AD. Genome-wide association studies revealed that the ε4 allele of the apolipoprotein E (APOE4) gene, the strongest genetic risk factor for AD, is also the most replicated genetic risk factor for Lewy body dementia (LBD), signifying an important role of APOE4 in both amyloid-ß (Aß) and α-SYN pathogenesis. How APOE4 modulates α-SYN aggregation in AD is unclear. In this study, we aimed to determine how α-SYN is associated with AD-related pathology and how APOE4 impacts α-SYN seeding and toxicity. We measured α-SYN levels and their association with other established AD-related markers in brain samples from autopsy-confirmed AD patients (N = 469), where 54% had concomitant LB pathology (AD + LB). We found significant correlations between the levels of α-SYN and those of Aß40, Aß42, tau and APOE, particularly in insoluble fractions of AD + LB. Using a real-time quaking-induced conversion (RT-QuIC) assay, we measured the seeding activity of soluble α-SYN and found that α-SYN seeding was exacerbated by APOE4 in the AD cohort, as well as a small cohort of autopsy-confirmed LBD brains with minimal Alzheimer type pathology. We further fractionated the soluble AD brain lysates by size exclusion chromatography (SEC) ran on fast protein liquid chromatography (FPLC) and identified the α-SYN species (~ 96 kDa) that showed the strongest seeding activity. Finally, using human induced pluripotent stem cell (iPSC)-derived neurons, we showed that amplified α-SYN aggregates from AD + LB brain of patients with APOE4 were highly toxic to neurons, whereas the same amount of α-SYN monomer was not toxic. Our findings suggest that the presence of LB pathology correlates with AD-related pathologies and that APOE4 exacerbates α-SYN seeding activity and neurotoxicity, providing mechanistic insight into how APOE4 affects α-SYN pathogenesis in AD.
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Enfermedad de Alzheimer , Apolipoproteína E4 , Células Madre Pluripotentes Inducidas , Enfermedad por Cuerpos de Lewy , Síndromes de Neurotoxicidad , Enfermedad de Alzheimer/patología , Apolipoproteína E4/genética , Apolipoproteínas E , Estudio de Asociación del Genoma Completo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cuerpos de Lewy/patología , Enfermedad por Cuerpos de Lewy/patología , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismoRESUMEN
Intron retention (IR) has been implicated in the pathogenesis of complex diseases such as cancers; its association with Alzheimer's disease (AD) remains unexplored. We performed genome-wide analysis of IR through integrating genetic, transcriptomic, and proteomic data of AD subjects and mouse models from the Accelerating Medicines Partnership-Alzheimer's Disease project. We identified 4535 and 4086 IR events in 2173 human and 1736 mouse genes, respectively. Quantitation of IR enabled the identification of differentially expressed genes that conventional exon-level approaches did not reveal. There were significant correlations of intron expression within innate immune genes, like HMBOX1, with AD in humans. Peptides with a high probability of translation from intron-retained mRNAs were identified using mass spectrometry. Further, we established AD-specific intron expression Quantitative Trait Loci, and identified splicing-related genes that may regulate IR. Our analysis provides a novel resource for the search for new AD biomarkers and pathological mechanisms.
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Enfermedad de Alzheimer , Autopsia , Encéfalo/patología , Modelos Animales de Enfermedad , Genómica , Intrones/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Proteínas de Homeodominio/genética , Humanos , Ratones , Proteómica , Sitios de Carácter Cuantitativo , TranscriptomaRESUMEN
INTRODUCTION: MAPT H1 haplotype is implicated as a risk factor for neurodegenerative diseases including Alzheimer's disease (AD). METHODS: Using Alzheimer's Disease Genetics Consortium (ADGC) genome-wide association study (GWAS) data (n = 18,841), we conducted a MAPT H1/H2 haplotype-stratified association to discover MAPT haplotype-specific AD risk loci. RESULTS: We identified 11 loci-5 in H2-non-carriers and 6 in H2-carriers-although none of the MAPT haplotype-specific associations achieved genome-wide significance. The most significant H2 non-carrier-specific association was with a NECTIN2 intronic (P = 1.33E-07) variant, and that for H2 carriers was near NKX6-1 (P = 1.99E-06). The GABRG2 locus had the strongest epistasis with MAPT H1/H2 variant rs8070723 (P = 3.91E-06). Eight of the 12 genes at these loci had transcriptome-wide significant differential expression in AD versus control temporal cortex (q < 0.05). Six genes were members of the brain transcriptional co-expression network implicated in "synaptic transmission" (P = 9.85E-59), which is also enriched for neuronal genes (P = 1.0E-164), including MAPT. DISCUSSION: This stratified GWAS identified loci that may confer AD risk in a MAPT haplotype-specific manner. This approach may preferentially enrich for neuronal genes implicated in synaptic transmission.
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Enfermedad de Alzheimer/genética , Predisposición Genética a la Enfermedad , Haplotipos , Polimorfismo de Nucleótido Simple , Proteínas tau/genética , Sitios Genéticos , Estudio de Asociación del Genoma Completo , Genotipo , HumanosRESUMEN
INTRODUCTION: Abnormal gene expression patterns may contribute to the onset and progression of late-onset Alzheimer's disease (LOAD). METHODS: We performed transcriptome-wide meta-analysis (N = 1440) of blood-based microarray gene expression profiles as well as neuroimaging and cerebrospinal fluid (CSF) endophenotype analysis. RESULTS: We identified and replicated five genes (CREB5, CD46, TMBIM6, IRAK3, and RPAIN) as significantly dysregulated in LOAD. The most significantly altered gene, CREB5, was also associated with brain atrophy and increased amyloid beta (Aß) accumulation, especially in the entorhinal cortex region. cis-expression quantitative trait loci mapping analysis of CREB5 detected five significant associations (P < 5 × 10-8 ), where rs56388170 (most significant) was also significantly associated with global cortical Aß deposition measured by [18 F]Florbetapir positron emission tomography and CSF Aß1-42 . DISCUSSION: RNA from peripheral blood indicated a differential gene expression pattern in LOAD. Genes identified have been implicated in biological processes relevant to Alzheimer's disease. CREB, in particular, plays a key role in nervous system development, cell survival, plasticity, and learning and memory.
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Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Proteína de Unión al Elemento de Respuesta al AMP Cíclico/genética , Perfilación de la Expresión Génica , Anciano , Enfermedad de Alzheimer/sangre , Péptidos beta-Amiloides/líquido cefalorraquídeo , Péptidos beta-Amiloides/metabolismo , Compuestos de Anilina , Atrofia/patología , Encéfalo/patología , Corteza Entorrinal/patología , Glicoles de Etileno , Femenino , Técnicas de Genotipaje , Humanos , Masculino , Tomografía de Emisión de PositronesRESUMEN
Misfolding and aggregation of alpha-synuclein (α-synuclein) with concomitant cytotoxicity is a hallmark of Lewy body related disorders such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Although it plays a pivotal role in pathogenesis and disease progression, the function of α-synuclein and the molecular mechanisms underlying α-synuclein-induced neurotoxicity in these diseases are still elusive. Many in vitro and in vivo experimental models mimicking α-synuclein pathology such as oligomerization, toxicity and more recently neuronal propagation have been generated over the years. In particular, cellular models have been crucial for our comprehension of the pathogenic process of the disease and are beneficial for screening of molecules capable of modulating α-synuclein toxicity. Here, we review α-synuclein based cell culture models that reproduce some features of the neuronal populations affected in patients, from basic unicellular organisms to mammalian cell lines and primary neurons, to the cutting edge models of patient-specific cell lines. These reprogrammed cells known as induced pluripotent stem cells (iPSCs) have garnered attention because they closely reproduce the characteristics of neurons found in patients and provide a valuable tool for mechanistic studies. We also discuss how different cell models may constitute powerful tools for high-throughput screening of molecules capable of modulating α-synuclein toxicity and prevention of its propagation. This article is part of the Special Issue "Synuclein".
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Agregación Patológica de Proteínas/metabolismo , alfa-Sinucleína/metabolismo , Técnicas de Cultivo de Célula , Células Cultivadas , Reprogramación Celular , Dopamina/metabolismo , Evaluación Preclínica de Medicamentos , Células HEK293 , Humanos , Técnicas In Vitro , Células Madre Pluripotentes Inducidas/metabolismo , Cuerpos de Lewy/metabolismo , Modelos Neurológicos , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Neuronas/metabolismo , Pliegue de Proteína , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sinucleinopatías/metabolismoRESUMEN
Progressive supranuclear palsy (PSP) is a neurodegenerative parkinsonian disorder characterized by tau pathology in neurons and glial cells. Transcriptional regulation has been implicated as a potential mechanism in conferring disease risk and neuropathology for some PSP genetic risk variants. However, the role of transcriptional changes as potential drivers of distinct cell-specific tau lesions has not been explored. In this study, we integrated brain gene expression measurements, quantitative neuropathology traits and genome-wide genotypes from 268 autopsy-confirmed PSP patients to identify transcriptional associations with unique cell-specific tau pathologies. We provide individual transcript and transcriptional network associations for quantitative oligodendroglial (coiled bodies = CB), neuronal (neurofibrillary tangles = NFT), astrocytic (tufted astrocytes = TA) tau pathology, and tau threads and genomic annotations of these findings. We identified divergent patterns of transcriptional associations for the distinct tau lesions, with the neuronal and astrocytic neuropathologies being the most different. We determined that NFT are positively associated with a brain co-expression network enriched for synaptic and PSP candidate risk genes, whereas TA are positively associated with a microglial gene-enriched immune network. In contrast, TA is negatively associated with synaptic and NFT with immune system transcripts. Our findings have implications for the diverse molecular mechanisms that underlie cell-specific vulnerability and disease risk in PSP.
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Química Encefálica/genética , Expresión Génica/genética , Parálisis Supranuclear Progresiva/genética , Parálisis Supranuclear Progresiva/patología , Tauopatías/genética , Tauopatías/patología , Anciano , Astrocitos/patología , Femenino , Redes Reguladoras de Genes , Estudio de Asociación del Genoma Completo , Humanos , Sistema Inmunológico/patología , Inmunohistoquímica , Masculino , Ovillos Neurofibrilares/genética , Ovillos Neurofibrilares/patología , Neuronas/patología , Proteoma , ARN/biosíntesis , ARN/genética , Sinapsis/patologíaRESUMEN
INTRODUCTION: Comparative transcriptome analyses in Alzheimer's disease (AD) and other neurodegenerative proteinopathies can uncover both shared and distinct disease pathways. METHODS: We analyzed 940 brain transcriptomes including patients with AD, progressive supranuclear palsy (PSP; a primary tauopathy), and control subjects. RESULTS: We identified transcriptional coexpression networks implicated in myelination, which were lower in PSP temporal cortex (TCX) compared with AD. Some of these associations were retained even after adjustments for brain cell population changes. These TCX myelination network structures were preserved in cerebellum but they were not differentially expressed in cerebellum between AD and PSP. Myelination networks were downregulated in both AD and PSP, when compared with control TCX samples. DISCUSSION: Downregulation of myelination networks may underlie both PSP and AD pathophysiology, but may be more pronounced in PSP. These data also highlight conservation of transcriptional networks across brain regions and the influence of cell type changes on these networks.
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Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Parálisis Supranuclear Progresiva/metabolismo , Transcriptoma , Enfermedad de Alzheimer/genética , Estudios de Cohortes , Biología Computacional , Femenino , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Masculino , Vaina de Mielina/metabolismo , Neuronas/metabolismo , Parálisis Supranuclear Progresiva/genéticaRESUMEN
INTRODUCTION: We sought to determine whether a systems biology approach may identify novel late-onset Alzheimer's disease (LOAD) loci. METHODS: We performed gene-wide association analyses and integrated results with human protein-protein interaction data using network analyses. We performed functional validation on novel genes using a transgenic Caenorhabditis elegans Aß proteotoxicity model and evaluated novel genes using brain expression data from people with LOAD and other neurodegenerative conditions. RESULTS: We identified 13 novel candidate LOAD genes outside chromosome 19. Of those, RNA interference knockdowns of the C. elegans orthologs of UBC, NDUFS3, EGR1, and ATP5H were associated with Aß toxicity, and NDUFS3, SLC25A11, ATP5H, and APP were differentially expressed in the temporal cortex. DISCUSSION: Network analyses identified novel LOAD candidate genes. We demonstrated a functional role for four of these in a C. elegans model and found enrichment of differentially expressed genes in the temporal cortex.
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Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Estudio de Asociación del Genoma Completo , Polimorfismo de Nucleótido Simple/genética , Biología de Sistemas , Lóbulo Temporal/metabolismo , Enfermedad de Alzheimer/inducido químicamente , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Animales Modificados Genéticamente , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Caenorhabditis elegans/genética , Modelos Animales de Enfermedad , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Factor de Crecimiento Similar a EGF de Unión a Heparina/genética , Factor de Crecimiento Similar a EGF de Unión a Heparina/metabolismo , Humanos , Masculino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Translocasas Mitocondriales de ADP y ATP/genética , Translocasas Mitocondriales de ADP y ATP/metabolismo , NADH Deshidrogenasa/genética , NADH Deshidrogenasa/metabolismo , Mapas de Interacción de Proteínas , Interferencia de ARN/fisiologíaRESUMEN
INTRODUCTION: We hypothesized that common Alzheimer's disease (AD)-associated variants within the triggering receptor expressed on myeloid (TREM) gene cluster influence disease through gene expression. METHODS: Expression microarrays on temporal cortex and cerebellum from â¼400 neuropathologically diagnosed subjects and two independent RNAseq replication cohorts were used for expression quantitative trait locus analysis. RESULTS: A variant within a DNase hypersensitive site 5' of TREM2, rs9357347-C, associates with reduced AD risk and increased TREML1 and TREM2 levels (uncorrected P = 6.3 × 10-3 and 4.6 × 10-2, respectively). Meta-analysis on expression quantitative trait locus results from three independent data sets (n = 1006) confirmed these associations (uncorrected P = 3.4 × 10-2 and 3.5 × 10-3, Bonferroni-corrected P = 6.7 × 10-2 and 7.1 × 10-3, respectively). DISCUSSION: Our findings point to rs9357347 as a functional regulatory variant that contributes to a protective effect observed at the TREM locus in the International Genomics of Alzheimer's Project genome-wide association study meta-analysis and suggest concomitant increase in TREML1 and TREM2 brain levels as a potential mechanism for protection from AD.
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Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Anciano , Anciano de 80 o más Años , Cerebelo/metabolismo , Femenino , Expresión Génica , Predisposición Genética a la Enfermedad , Variación Genética , Humanos , Desequilibrio de Ligamiento , Masculino , Análisis por Micromatrices , Familia de Multigenes , Sitios de Carácter Cuantitativo , Lóbulo Temporal/metabolismoRESUMEN
To determine the effects of single nucleotide polymorphisms (SNPs) identified in a genome-wide association study of progressive supranuclear palsy (PSP), we tested their association with brain gene expression, CpG methylation and neuropathology. In 175 autopsied PSP subjects, we performed associations between seven PSP risk variants and temporal cortex levels of 20 genes in-cis, within ±100 kb. Methylation measures were collected using reduced representation bisulfite sequencing in 43 PSP brains. To determine whether SNP/expression associations are due to epigenetic modifications, CpG methylation levels of associated genes were tested against relevant variants. Quantitative neuropathology endophenotypes were tested for SNP associations in 422 PSP subjects. Brain levels of LRRC37A4 and ARL17B were associated with rs8070723; MOBP with rs1768208 and both ARL17A and ARL17B with rs242557. Expression associations for LRRC37A4 and MOBP were available in an additional 100 PSP subjects. Meta-analysis revealed highly significant associations for PSP risk alleles of rs8070723 and rs1768208 with higher LRRC37A4 and MOBP brain levels, respectively. Methylation levels of one CpG in the 3' region of ARL17B associated with rs242557 and rs8070723. Additionally, methylation levels of an intronic ARL17A CpG associated with rs242557 and that of an intronic MOBP CpG with rs1768208. MAPT and MOBP region risk alleles also associated with higher levels of neuropathology. Strongest associations were observed for rs242557/coiled bodies and tufted astrocytes; and for rs1768208/coiled bodies and tau threads. These findings suggest that PSP variants at MAPT and MOBP loci may confer PSP risk via influencing gene expression and tau neuropathology. MOBP, LRRC37A4, ARL17A and ARL17B warrant further assessment as candidate PSP risk genes. Our findings have implications for the mechanism of action of variants at some of the top PSP risk loci.
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Alelos , Metilación de ADN , Expresión Génica/fisiología , Estudio de Asociación del Genoma Completo , Parálisis Supranuclear Progresiva/genética , Parálisis Supranuclear Progresiva/metabolismo , Anciano , Anciano de 80 o más Años , Femenino , Expresión Génica/genética , Sitios Genéticos , Humanos , Masculino , Neuropatología/métodos , Polimorfismo de Nucleótido Simple/genética , Riesgo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
BACKGROUND: Radiation therapy is the standard of care for central nervous system tumours. Despite the success of radiation therapy in reducing tumour mass, irradiation (IR)-induced vasculopathies and neuroinflammation contribute to late-delayed complications, neurodegeneration, and premature ageing in long-term cancer survivors. Mesenchymal stromal cells (MSCs) are adult stem cells that facilitate tissue integrity, homeostasis, and repair. Here, we investigated the potential of the iPSC-derived MSC (iMSC) secretome in immunomodulation and vasculature repair in response to radiation injury utilizing human cell lines. METHODS: We generated iPSC-derived iMSC lines and evaluated the potential of their conditioned media (iMSC CM) to treat IR-induced injuries in human monocytes (THP1) and brain vascular endothelial cells (hCMEC/D3). We further assessed factors in the iMSC secretome, their modulation, and the molecular pathways they elicit. RESULTS: Increasing doses of IR disturbed endothelial tube and spheroid formation in hCMEC/D3. When IR-injured hCMEC/D3 (IR ≤ 5 Gy) were treated with iMSC CM, endothelial cell viability, adherence, spheroid compactness, and proangiogenic sprout formation were significantly ameliorated, and IR-induced ROS levels were reduced. iMSC CM augmented tube formation in cocultures of hCMEC/D3 and iMSCs. Consistently, iMSC CM facilitated angiogenesis in a zebrafish model in vivo. Furthermore, iMSC CM suppressed IR-induced NFκB activation, TNF-α release, and ROS production in THP1 cells. Additionally, iMSC CM diminished NF-kB activation in THP1 cells cocultured with irradiated hCMEC/D3, iMSCs, or HMC3 microglial lines. The cytokine array revealed that iMSC CM contains the proangiogenic and immunosuppressive factors MCP1/CCL2, IL6, IL8/CXCL8, ANG (Angiogenin), GROα/CXCL1, and RANTES/CCL5. Common promoter regulatory elements were enriched in TF-binding motifs such as androgen receptor (ANDR) and GATA2. hCMEC/D3 phosphokinome profiling revealed increased expression of pro-survival factors, the PI3K/AKT/mTOR modulator PRAS40 and ß-catenin in response to CM. The transcriptome analysis revealed increased expression of GATA2 in iMSCs and the enrichment of pathways involved in RNA metabolism, translation, mitochondrial respiration, DNA damage repair, and neurodevelopment. CONCLUSIONS: The iMSC secretome is a comodulated composite of proangiogenic and immunosuppressive factors that has the potential to alleviate radiation-induced vascular endothelial cell damage and immune activation.
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Células Endoteliales , Células Madre Pluripotentes Inducidas , Células Madre Mesenquimatosas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Células Endoteliales/metabolismo , Células Endoteliales/efectos de la radiación , Secretoma/metabolismo , Animales , Pez Cebra , Medios de Cultivo Condicionados/farmacología , Neovascularización Fisiológica/efectos de la radiaciónRESUMEN
Background Mesenchymal stromal cells (MSCs) have a dynamic secretome that plays a critical role in tissue repair and regeneration. However, studying the MSC secretome in mixed-culture disease models remains challenging. This study aimed to develop a mutant methionyl-tRNA synthetase-based toolkit (MetRS L274G ) to selectively profile secreted proteins from MSCs in mixed-culture systems and demonstrate its potential for investigating MSC responses to pathological stimulation. Methods We used CRISPR/Cas9 homology-directed repair to stably integrate MetRS L274G into cells, enabling the incorporation of the non-canonical amino acid, azidonorleucine (ANL), and facilitating selective protein isolation using click chemistry. MetRS L274G was integrated into both in H4 cells and induced pluripotent stem cells (iPSCs) for a series of proof-of-concept studies. Following iPSC differentiation into induced-MSCs, we validated their identity and co-cultured MetRS L274G -expressing iMSCs with naïve or lipopolysaccharide- (LPS) treated THP-1 cells. We then profiled the iMSC secretome using antibody arrays. Results Our results showed successful integration of MetRS L274G into targeted cells, allowing specific isolation of proteins from mixed-culture environments. We also demonstrated that the secretome of MetRS L274G -expressing iMSCs can be differentiated from that of THP-1 cells in co-culture, and is altered when co-cultured with LPS-treated THP-1 cells compared to naïve THP-1 cells. Conclusions The MetRS L274G -based toolkit we have generated enables selective profiling of the MSC secretome in mixed-culture disease models. This approach has broad applications for examining not only MSC responses to models of pathological conditions, but any other cell type that can be differentiated from iPSCs. This can potentially reveal novel MSC-mediated repair mechanisms and advancing our understanding of tissue regeneration processes.
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BACKGROUND: Mesenchymal stromal cells (MSCs) have a dynamic secretome that plays a critical role in tissue repair and regeneration. However, studying the MSC secretome in mixed-culture disease models remains challenging. This study aimed to develop a mutant methionyl-tRNA synthetase-based toolkit (MetRSL274G) to selectively profile secreted proteins from MSCs in mixed-culture systems and demonstrate its potential for investigating MSC responses to pathological stimulation. METHODS: We used CRISPR/Cas9 homology-directed repair to stably integrate MetRSL274G into cells, enabling the incorporation of the non-canonical amino acid, azidonorleucine (ANL), and facilitating selective protein isolation using click chemistry. MetRSL274G was integrated into both in H4 cells and induced pluripotent stem cells (iPSCs) for a series of proof-of-concept studies. Following iPSC differentiation into induced-MSCs, we validated their identity and co-cultured MetRSL274G-expressing iMSCs with naïve or lipopolysaccharide (LPS)-treated THP-1 cells. We then profiled the iMSC secretome using antibody arrays. RESULTS: Our results showed successful integration of MetRSL274G into targeted cells, allowing specific isolation of proteins from mixed-culture environments. We also demonstrated that the secretome of MetRSL274G-expressing iMSCs can be differentiated from that of THP-1 cells in co-culture and is altered when co-cultured with LPS-treated THP-1 cells compared to naïve THP-1 cells. CONCLUSIONS: The MetRSL274G-based toolkit we have generated enables selective profiling of the MSC secretome in mixed-culture disease models. This approach has broad applications for examining not only MSC responses to models of pathological conditions, but any other cell type that can be differentiated from iPSCs. This can potentially reveal novel MSC-mediated repair mechanisms and advancing our understanding of tissue regeneration processes.
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Células Madre Mesenquimatosas , Metionina-ARNt Ligasa , Metionina-ARNt Ligasa/genética , Metionina-ARNt Ligasa/metabolismo , Lipopolisacáridos , Secretoma , Células Madre Mesenquimatosas/metabolismo , AminoácidosRESUMEN
Background: Intracytoplasmic inclusions comprised of aggregated alpha-synuclein (αsyn) represent a key histopathological feature of neurological disorders collectively termed "synucleinopathies," which includes Parkinson's disease (PD). Mutations and multiplications in the SNCA gene encoding αsyn cause familial forms of PD and a large body of evidence indicate a correlation between αsyn accumulation and disease. Decreasing αsyn expression is recognized as a valid target for PD therapeutics, with down-regulation of SNCA expression potentially attenuating downstream cascades of pathologic events. Here, we evaluated if Honokiol (HKL), a polyphenolic compound derived from magnolia tree bark with demonstrated neuroprotective properties, can modulate αsyn levels in multiple experimental models. Methods: Human neuroglioma cells stably overexpressing αsyn, mouse primary neurons, and human iPSC-derived neurons were exposed to HKL and αsyn protein and SNCA messenger RNA levels were assessed. The effect of HKL on rotenone-induced overexpression of αsyn levels was further assessed and transcriptional profiling of mouse cortical neurons treated with HKL was performed to identify potential targets of HKL. Results: We demonstrate that HKL can successfully reduce αsyn protein levels and SNCA expression in multiple in vitro models of PD with our data supporting a mechanism whereby HKL acts by post-transcriptional modulation of SNCA rather than modulating αsyn protein degradation. Transcriptional profiling of mouse cortical neurons treated with HKL identifies several differentially expressed genes (DEG) as potential targets to modulate SNCA expression. Conclusion: This study supports a HKL-mediated downregulation of SNCA as a viable strategy to modify disease progression in PD and other synucleinopathies. HKL has potential as a powerful tool for investigating SNCA gene modulation and its downstream effects.
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Traumatic brain injury (TBI) is traditionally characterized by primary and secondary injury phases, both contributing to pathological and morphological changes. The mechanisms of damage and chronic consequences of TBI remain to be fully elucidated, but synaptic homeostasis disturbances and impaired energy metabolism are proposed to be a major contributor. It has been proposed that an increase of extracellular (eATP) adenosine triphosphate (ATP) in the area immediately surrounding impact may play a pivotal role in this sequence of events. After tissue injury, rupture of cell membranes allows release of intracellular ATP into the extracellular space, triggering a cascade of toxic events and inflammation. ATP is a ubiquitous messenger; however, simple and reliable techniques to measure its concentration have proven elusive. Here, we integrate a sensitive bioluminescent eATP sensor known as pmeLUC, with a controlled cortical impact mouse model to monitor eATP changes in a living animal after injury. Using the pmeLUC probe, a rapid increase of eATP is observed proximal to the point of impact within minutes of the injury. This event is significantly attenuated when animals are pretreated with an ATP hydrolyzing agent (apyrase) before surgery, confirming the contribution of eATP. This new eATP reporter could be useful for understanding the role of eATP in the pathogenesis in TBI and may identify a window of opportunity for therapeutic intervention.
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Adenosina Trifosfato/metabolismo , Lesiones Traumáticas del Encéfalo/metabolismo , Espacio Extracelular/metabolismo , Animales , Apirasa , Lesiones Traumáticas del Encéfalo/etiología , Lesiones Traumáticas del Encéfalo/patología , Modelos Animales de Enfermedad , Mediciones Luminiscentes , Ratones , Valor Predictivo de las Pruebas , Factores de TiempoRESUMEN
BACKGROUND: Misfolding and aggregation of the presynaptic protein alpha-synuclein (αsyn) is a hallmark of Parkinson's disease (PD) and related synucleinopathies. Although predominantly localized in the cytosol, a body of evidence has shown that αsyn localizes to mitochondria and contributes to the disruption of key mitochondrial processes. Mitochondrial dysfunction is central to the progression of PD and mutations in mitochondrial-associated proteins are found in familial cases of PD. The sirtuins are highly conserved nicotinamide adenine dinucleotide (NAD+)-dependent enzymes that play a broad role in cellular metabolism and aging. Interestingly, mitochondrial sirtuin 3 (SIRT3) plays a major role in maintaining mitochondrial function and preventing oxidative stress, and is downregulated in aging and age-associated diseases such as neurodegenerative disorders. Herein, we hypothesize that αsyn is associated with decreased SIRT3 levels contributing to impaired mitochondrial dynamics and biogenesis in PD. METHODS: The level of mitochondrial SIRT3 was assessed in cells expressing oligomeric αsyn within the cytosolic and mitochondrial-enriched fractions. Mitochondrial integrity, respiration, and health were examined using several markers of mitochondrial dynamics and stress response and by measuring the rate of oxygen consumption (OCR). Our findings were validated in a rodent model of PD as well as in human post-mortem Lewy body disease (LBD) brain tissue. RESULTS: Here, we demonstrate that αsyn associates with mitochondria and induces a decrease in mitochondrial SIRT3 levels and mitochondrial biogenesis. We show that SIRT3 downregulation is accompanied by decreased phosphorylation of AMPK and cAMP-response element binding protein (CREB), as well as increased phosphorylation of dynamin-related protein 1 (DRP1), indicative of impaired mitochondrial dynamics. OCR was significantly decreased suggesting a mitochondria respiratory deficit. Interestingly treatment with AMPK agonist 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) restores SIRT3 expression, improves mitochondrial function, and decreases αsyn oligomer formation in a SIRT3-dependent manner. CONCLUSIONS: Together, our findings suggest that pharmacologically increasing SIRT3 levels can counteract αsyn-induced mitochondrial dysfunction by reducing αsyn oligomers and normalizing mitochondrial bioenergetics. These data support a protective role for SIRT3 in PD-associated pathways and contribute significant mechanistic insight into the interplay of SIRT3 and αsyn.
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Mitocondrias/metabolismo , Enfermedad de Parkinson/metabolismo , Sirtuina 3/metabolismo , alfa-Sinucleína/metabolismo , Anciano , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Mitocondrias/patología , Enfermedad de Parkinson/patología , Ratas , Ratas Sprague-DawleyRESUMEN
This case report describes an individual with brain calcifications, cognitive decline, motor dysfunction, and hypocalcaemia. Exome sequencing revealed a previously reported variant in the CASR gene and a variant of uncertain significance in PDGFRB. The clinical phenotype is likely explained by the CASR variant, but we discuss how the PDGFRB variant could also participate in the phenotype.
RESUMEN
The main genetic risk factors for progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) are located at chromosome 17q21.31. The identification of risk H1 subhaplotypes suggests that disease-specific variants can be identified by resequencing the 17q21.31 region (1.4 Mb) in carriers of risk H1 subhaplotypes. We hypothesized that PSP/CBD H1 subhaplotype carriers could have undergone a mutational event absent among unaffected carriers leading to the disease risk. We performed this strategy in definite PSP subjects, definite CBD subjects, and healthy controls and tried to replicate the findings in a larger PSP/CBD case-control series. In the resequencing process, 40 candidate variants were identified, but an association between PSP and rs76970862 was replicated only using an unadjusted model. Gene expression association analysis of this variant suggested no potential functional effect. Although our results failed to identify disease-associated variants, it is still possible that the risk of PSP/CBD at chromosome 17 is driven by rare variants, even in PSP/CBD H1 cases or variants located outside the capture regions.
Asunto(s)
Cromosomas Humanos Par 17/genética , Estudios de Asociación Genética , Variación Genética/genética , Tauopatías/genética , Ganglios Basales , Corteza Cerebral , Haplotipos , Heterocigoto , Humanos , Enfermedades Neurodegenerativas/genética , Receptores de Hormona Liberadora de Corticotropina/genética , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Riesgo , Parálisis Supranuclear Progresiva/genética , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
BACKGROUND: Rare coding variants ABI3_rs616338-T and PLCG2_rs72824905-G were identified as risk or protective factors, respectively, for Alzheimer's disease (AD). METHODS: We tested the association of these variants with five neurodegenerative diseases in Caucasian case-control cohorts: 2742 AD, 231 progressive supranuclear palsy (PSP), 838 Parkinson's disease (PD), 306 dementia with Lewy bodies (DLB) and 150 multiple system atrophy (MSA) vs. 3351 controls; and in an African-American AD case-control cohort (181 AD, 331 controls). 1479 AD and 1491 controls were non-overlapping with a prior report. RESULTS: Using Fisher's exact test, there was significant association of both ABI3_rs616338-T (OR = 1.41, p = 0.044) and PLCG2_rs72824905-G (OR = 0.56, p = 0.008) with AD. These OR estimates were maintained in the non-overlapping replication AD-control analysis, albeit at reduced significance (ABI3_rs616338-T OR = 1.44, p = 0.12; PLCG2_rs72824905-G OR = 0.66, p = 0.19). None of the other cohorts showed significant associations that were concordant with those for AD, although the DLB cohort had suggestive findings (Fisher's test: ABI3_rs616338-T OR = 1.79, p = 0.097; PLCG2_rs72824905-G OR = 0.32, p = 0.124). PLCG2_rs72824905-G showed suggestive association with pathologically-confirmed MSA (OR = 2.39, p = 0.050) and PSP (OR = 1.97, p = 0.061), although in the opposite direction of that for AD. We assessed RNA sequencing data from 238 temporal cortex (TCX) and 224 cerebellum (CER) samples from AD, PSP and control patients and identified co-expression networks, enriched in microglial genes and immune response GO terms, and which harbor PLCG2 and/or ABI3. These networks had higher expression in AD, but not in PSP TCX, compared to controls. This expression association did not survive adjustment for brain cell type population changes. CONCLUSIONS: We validated the associations previously reported with ABI3_rs616338-T and PLCG2_rs72824905-G in a Caucasian AD case-control cohort, and observed a similar direction of effect in DLB. Conversely, PLCG2_rs72824905-G showed suggestive associations with PSP and MSA in the opposite direction. We identified microglial gene-enriched co-expression networks with significantly higher levels in AD TCX, but not in PSP, a primary tauopathy. This co-expression network association appears to be driven by microglial cell population changes in a brain region affected by AD pathology. Although these findings require replication in larger cohorts, they suggest distinct effects of the microglial genes, ABI3 and PLCG2 in neurodegenerative diseases that harbor significant vs. low/no amyloid ß pathology.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Mutación Missense , Enfermedades Neurodegenerativas/genética , Fosfolipasa C gamma/genética , Negro o Afroamericano/genética , Anciano , Anciano de 80 o más Años , Encéfalo/metabolismo , Femenino , Humanos , Masculino , Microglía/metabolismo , Factores de Riesgo , Población Blanca/genéticaRESUMEN
There is considerable interest in harnessing innate immunity to treat Alzheimer's disease (AD). Here, we explore whether a decoy receptor strategy using the ectodomain of select TLRs has therapeutic potential in AD. AAV-mediated expression of human TLR5 ectodomain (sTLR5) alone or fused to human IgG4 Fc (sTLR5Fc) results in robust attenuation of amyloid ß (Aß) accumulation in a mouse model of Alzheimer-type Aß pathology. sTLR5Fc binds to oligomeric and fibrillar Aß with high affinity, forms complexes with Aß, and blocks Aß toxicity. Oligomeric and fibrillar Aß modulates flagellin-mediated activation of human TLR5 but does not, by itself, activate TLR5 signaling. Genetic analysis shows that rare protein coding variants in human TLR5 may be associated with a reduced risk of AD. Further, transcriptome analysis shows altered TLR gene expression in human AD. Collectively, our data suggest that TLR5 decoy receptor-based biologics represent a novel and safe Aß-selective class of biotherapy in AD.