RESUMO
X-linked Dystonia-Parkinsonism (XDP) is a Mendelian neurodegenerative disease that is endemic to the Philippines and is associated with a founder haplotype. We integrated multiple genome and transcriptome assembly technologies to narrow the causal mutation to the TAF1 locus, which included a SINE-VNTR-Alu (SVA) retrotransposition into intron 32 of the gene. Transcriptome analyses identified decreased expression of the canonical cTAF1 transcript among XDP probands, and de novo assembly across multiple pluripotent stem-cell-derived neuronal lineages discovered aberrant TAF1 transcription that involved alternative splicing and intron retention (IR) in proximity to the SVA that was anti-correlated with overall TAF1 expression. CRISPR/Cas9 excision of the SVA rescued this XDP-specific transcriptional signature and normalized TAF1 expression in probands. These data suggest an SVA-mediated aberrant transcriptional mechanism associated with XDP and may provide a roadmap for layered technologies and integrated assembly-based analyses for other unsolved Mendelian disorders.
Assuntos
Distúrbios Distônicos/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Genoma Humano , Transcriptoma/genética , Processamento Alternativo/genética , Elementos Alu/genética , Sequência de Bases , Sistemas CRISPR-Cas/genética , Estudos de Coortes , Família , Feminino , Loci Gênicos , Haplótipos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Íntrons/genética , Masculino , Repetições Minissatélites/genética , Modelos Genéticos , Degeneração Neural/genética , Degeneração Neural/patologia , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos Nucleotídeos Curtos e Dispersos , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/genética , Fator de Transcrição TFIID/metabolismoRESUMO
RNA polymerase II (RNAPII) transcription is governed by the pre-initiation complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. After initiation, RNAPII enzymes pause after transcribing less than 100 bases; precisely how RNAPII pausing is enforced and regulated remains unclear. To address specific mechanistic questions, we reconstituted human RNAPII promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pausing, and P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pausing, suggesting RNAPII pausing is an inherent PIC function. In agreement, pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP), and PRO-seq experiments revealed widespread disruption of RNAPII pausing upon acute depletion (t = 60 min) of TFIID subunits in human or Drosophila cells. These results establish a TFIID requirement for RNAPII pausing and suggest pause regulatory factors may function directly or indirectly through TFIID.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/genética , Fator de Transcrição TFIID/metabolismo , Transcrição Gênica , Animais , Drosophila/genética , Proteínas de Drosophila/genética , Células HCT116 , Humanos , Ligação Proteica , RNA Polimerase II/metabolismo , Fator de Transcrição TFIID/genéticaRESUMO
TFIID binds promoter DNA to recruit RNA polymerase II and other basal factors for transcription. Although the TATA-binding protein (TBP) subunit of TFIID is necessary and sufficient for in vitro transcription, the TBP-associated factor (TAF) subunits recognize downstream promoter elements, act as coactivators, and interact with nucleosomes. In yeast nuclear extracts, transcription induces stable TAF binding to downstream promoter DNA, promoting subsequent activator-independent transcription reinitiation. In vivo, promoter responses to TAF mutations correlate with the level of downstream, rather than overall, Taf1 cross-linking. We propose a new model in which TAFs function as reinitiation factors, accounting for the differential responses of promoters to various transcription factor mutations.
Assuntos
Regiões Promotoras Genéticas/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Transcrição Gênica/genética , Acetilação , Histonas/metabolismo , Mutação/genética , Ligação Proteica , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores de Transcrição/metabolismoRESUMO
X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disease caused by a retrotransposon insertion in intron 32 of the TAF1 gene. This insertion causes mis-splicing of intron 32 (TAF1-32i) and reduced TAF1 levels. TAF1-32i transcript is unique to XDP patient cells and can be detected in their extracellular vesicles (EVs). We engrafted patient and control iPSC-derived neural progenitor cells (hNPCs) into the striatum of mice. To track TAF1-32i transcript spread by EVs, we transduced the brain-implanted hNPCs with a lentiviral construct called ENoMi, which consists of a re-engineered tetraspanin scaffold tagged with bioluminescent and fluorescent reporter proteins under an EF-1α promoter. Alongside this improved detection in ENoMi-hNPCs-derived EVs, their surface allows specific immunocapture purification, thereby facilitating TAF1-32i analysis. Using this ENoMi-labeling method, TAF1-32i was demonstrated in EVs released from XDP hNPCs implanted in mouse brains. Post-implantation of ENoMi-XDP hNPCs, TAF1-32i transcript was retrieved in EVs isolated from mouse brain and blood, and levels increased over time in plasma. We compared and combined our EV isolation technique to analyze XDP-derived TAF1-32i with other techniques, including size exclusion chromatography and Exodisc. Overall, our study demonstrates the successful engraftment of XDP patient-derived hNPCs in mice as a tool for monitoring disease markers with EVs.
Assuntos
Vesículas Extracelulares , Doenças Neurodegenerativas , Humanos , Camundongos , Animais , Fator de Transcrição TFIID/genética , Fator de Transcrição TFIID/metabolismo , Biomarcadores , Encéfalo/metabolismo , Vesículas Extracelulares/metabolismoRESUMO
Phenotypic assays detect small-molecule bioactivity at functionally relevant cellular sites, and inherently cover a variety of targets and mechanisms of action. They can uncover new small molecule-target pairs and may give rise to novel biological insights. By means of an osteoblast differentiation assay which employs a Hedgehog (Hh) signaling agonist as stimulus and which monitors an endogenous marker for osteoblasts, we identified a pyrrolo[3,4-g]quinoline (PQ) pseudo-natural product (PNP) class of osteogenesis inhibitors. The most potent PQ, termed Tafbromin, impairs canonical Hh signaling and modulates osteoblast differentiation through binding to the bromodomain 2 of the TATA-box binding protein-associated factor 1 (TAF1). Tafbromin is the most selective TAF1 bromodomain 2 ligand and promises to be an invaluable tool for the study of biological processes mediated by TAF1(2) bromodomains.
Assuntos
Fatores Associados à Proteína de Ligação a TATA , Fator de Transcrição TFIID , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fatores Associados à Proteína de Ligação a TATA/química , Fator de Transcrição TFIID/metabolismo , Fator de Transcrição TFIID/química , Fator de Transcrição TFIID/antagonistas & inibidores , Humanos , Histona Acetiltransferases/metabolismo , Histona Acetiltransferases/antagonistas & inibidores , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/citologia , Diferenciação Celular/efeitos dos fármacos , Quinolinas/química , Quinolinas/farmacologia , Estrutura MolecularRESUMO
High-risk neuroblastoma (HR-NB) is an aggressive childhood cancer that responds poorly to currently available therapies and is associated with only about a 50% 5-year survival rate. MYCN amplification is a critical driver of these aggressive tumors, but so far there have not been any approved treatments to effectively treat HR-NB by targeting MYCN or its downstream effectors. Thus, the identification of novel molecular targets and therapeutic strategies to treat children diagnosed with HR-NB represents an urgent unmet medical need. Here, we conducted a targeted siRNA screening and identified TATA box-binding protein-associated factor RNA polymerase I subunit D, TAF1D, as a critical regulator of the cell cycle and proliferation in HR-NB cells. Analysis of three independent primary NB cohorts determined that high TAF1D expression correlated with MYCN-amplified, high-risk disease and poor clinical outcomes. TAF1D knockdown more robustly inhibited cell proliferation in MYCN-amplified NB cells compared with MYCN-non-amplified NB cells, as well as suppressed colony formation and inhibited tumor growth in a xenograft mouse model of MYCN-amplified NB. RNA-seq analysis revealed that TAF1D knockdown downregulates the expression of genes associated with the G2/M transition, including the master cell-cycle regulator, cell-cycle-dependent kinase 1 (CDK1), resulting in cell-cycle arrest at G2/M. Our findings demonstrate that TAF1D is a key oncogenic regulator of MYCN-amplified HR-NB and suggest that therapeutic targeting of TAF1D may be a viable strategy to treat HR-NB patients by blocking cell-cycle progression and the proliferation of tumor cells.
Assuntos
Neuroblastoma , Humanos , Animais , Camundongos , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/patologia , Proliferação de Células/genética , Divisão Celular , Fase G2 , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão GênicaRESUMO
Triple-negative breast cancer (TNBC) is a heterogeneous breast cancer subtype with poor prognoses and limited therapeutic options. The TATA-box binding protein associated factor 1 (TAF1) is an essential protein involved in the transcriptional regulation of cancer development and progress. However, the therapeutic potential and underlying mechanism of targeting TAF1 in TNBC remain unknown. Here, using chemical probe BAY-299, we identify that TAF1 inhibition leads to the induction of endogenous retrovirus (ERVs) expression and double-stranded RNA (dsRNA) formation, resulting in the activation of interferon responses and cell growth suppression in a subset of TNBC, resembling anti-viral mimicry effect. This correlation between TAF1 and interferon signature was validated in three independent breast cancer patient datasets. Furthermore, we observe heterogeneous responses to TAF1 inhibition across a set of TNBC cell lines. By integrating transcriptome and proteome data, we demonstrate that high levels of proliferating cell nuclear antigen (PCNA) protein serve as a predictive biomarker associated with suppressive tumor immune responses in various cancers, which may limit the efficiency of TAF1 inhibition.
Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Interferons/farmacologia , Transcriptoma , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Chemoresistance remains a major obstacle to the treatment of esophageal cancer (EC). Exosome-mediated transfer of long noncoding RNAs (lncRNAs) has recently been unveiled to correlate with the regulation of drug resistance in EC. This study aimed to investigate the physiological mechanisms by which exosome-encapsulated lncRNA myocardial infarction-associated transcript (MIAT) derived from tumor cells might mediate the paclitaxel (PTX) resistance of EC cells. First, MIAT was experimentally determined to be upregulated in PTX nonresponders and PTX-resistant EC cells. Silencing of MIAT in PTX-resistant EC cells decreased cell viability and enhanced apoptosis, corresponding to a reduced half-maximal inhibitory concentration (IC50 ) value. Next, exosomes were isolated from EC109 and EC109/T cells, and EC109 cells were cocultured with EC109/T-cell-derived exosomes. Accordingly, MIAT was revealed to be transmitted through exosomes from EC109/T cells to EC109 cells. Tumor-derived exosomes carrying MIAT increased the IC50 value of PTX and suppressed apoptosis in EC109 cells to promote PTX resistance. Furthermore, MIAT promoted the enrichment of TATA-box binding protein-associated Factor 1 (TAF1) in the promoter region of sterol regulatory element binding transcription factor 1 (SREBF1), as shown by a chromatin immunoprecipitation assay. This might be the mechanism by which MIAT could promote PTX resistance. Finally, in vivo experiments further confirmed that the knockdown of MIAT attenuated the resistance of EC cells to PTX. Collectively, these results indicate that tumor-derived exosome-loaded MIAT activates the TAF1/SREBF1 axis to induce PTX resistance in EC cells, providing a potential therapeutic target for overcoming PTX resistance in EC.
Assuntos
Neoplasias Esofágicas , Exossomos , MicroRNAs , Infarto do Miocárdio , RNA Longo não Codificante , Humanos , Paclitaxel/farmacologia , Exossomos/metabolismo , Linhagem Celular Tumoral , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , RNA Longo não Codificante/genética , MicroRNAs/genética , Proliferação de Células , Proteína de Ligação a Elemento Regulador de Esterol 1RESUMO
BRD9 and TAF1(2) have been regarded as significant targets of drug design for clinically treating acute myeloid leukemia, malignancies, and inflammatory diseases. In this study, multiple short molecular dynamics simulations combined with the molecular mechanics generalized Born surface area method were employed to investigate the binding selectivity of three ligands, 67B, 67C, and 69G, to BRD9/TAF1(2) with IC50 values of 230/59 nM, 1400/46 nM, and 160/410 nM, respectively. The computed binding free energies from the MM-GBSA method displayed good correlations with that provided by the experimental data. The results indicate that the enthalpic contributions played a critical factor in the selectivity recognition of inhibitors toward BRD9 and TAF1(2), indicating that 67B and 67C could more favorably bind to TAF1(2) than BRD9, while 69G had better selectivity toward BRD9 over TAF1(2). In addition, the residue-based free energy decomposition approach was adopted to calculate the inhibitor-residue interaction spectrum, and the results determined the gatekeeper (Y106 in BRD9 and Y1589 in TAF1(2)) and lipophilic shelf (G43, F44, and F45 in BRD9 and W1526, P1527, and F1528 in TAF1(2)), which could be identified as hotspots for designing efficient selective inhibitors toward BRD9 and TAF1(2). This work is also expected to provide significant theoretical guidance and insightful molecular mechanisms for the rational designs of efficient selective inhibitors targeting BRD9 and TAF1(2).
Assuntos
Desenho de Fármacos , Simulação de Dinâmica Molecular , Entropia , Termodinâmica , Ligação ProteicaRESUMO
Emerging data have highlighted the importance of long noncoding RNAs (lncRNAs) in exerting critical biological functions and roles in different forms of brain cancer, including gliomas. In this study, we sought to investigate the role of lncRNA FOXD2 adjacent opposite strand RNA 1 (FOXD2-AS1) in glioma cells. First, we used sphere formation assay and flow cytometry to select U251 glioma stem cells (GSCs). Then, we quantified the expression of lncRNA FOXD2-AS1, TATA-box binding protein associated factor 1 (TAF-1) and NOTCH1 in glioma tissues and GSCs, as well as the expression of GSC stem markers, OCT4, SOX2, Nanog, Nestin and CD133 in GSCs. Colony formation assay, sphere formation assay, and flow cytometry were used to evaluate GSC stemness. Next, the correlations among lncRNA FOXD2-AS1, TAF-1 and NOTCH1 were investigated. LncRNA FOXD2-AS1, TAF-1 and NOTCH1 were found to be elevated in glioma tissues and GSCs, and silencing lncRNA FOXD2-AS1 inhibited stemness and proliferation, while promoting apoptosis and differentiation of GSCs. LncRNA FOXD2-AS1 overexpression also led to increased NOTCH1 by recruiting TAF-1 to the NOTCH1 promoter region, thereby promoting stemness and proliferation, while impairing cell apoptosis and differentiation. Mechanistically, lncRNA FOXD2-AS1 elevation promoted glioma in vivo by activating the NOTCH signalling pathway via TAF-1 upregulation. Taken together, the key findings of our investigation support the proposition that downregulation of lncRNA FOXD2-AS1 presents a viable and novel molecular candidate for improving glioma treatment.
Assuntos
Glioma , MicroRNAs , RNA Longo não Codificante , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/metabolismo , Humanos , MicroRNAs/genética , Células-Tronco Neoplásicas/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
Despite tremendous advances in the diagnosis and treatment of NSCLC, the morbidity and mortality of NSCLC still rank high worldwide. Epithelial-mesenchymal transition (EMT) is vital to the invasion, metastasis, and recurrence of NSCLC. Unfortunately, the mechanism behind NSCLC cancer cell EMT remains elusive. Therefore, determining the potential key molecules that induce EMT is important. TATA-binding protein-associated factor-1 (TAF1) is an important component of the preinitiation complex (PIC) that is dysregulated in carcinogenesis. However, the role of TAF1 in NSCLC development is unknown. Therefore, we studied the role of TAF1 in the pathogenesis of NSCLC. First, the expression of TAF1 was determined in human NSCLC tissues and cell lines. TAF1-overexpressing and TAF1 knockdown cell lines were established to evaluate the effect of TAF1 on NSCLC cell proliferation, invasion and migration by colony formation and Transwell assays. The target genes of TAF1 were identified by PCR array and verified by luciferase reporter assay. Our data demonstrated that TAF1 is upregulated in NSCLC. Higher TAF1 expression predicted poor outcomes in NSCLC patients. Mechanistically, TAF1 transcriptionally activated TGFß1, thus promoting NSCLC cell EMT and the development of NSCLC. Targeting TAF1/TGFß1 signalling may be potentially helpful as a therapeutic for NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Proteínas Pol1 do Complexo de Iniciação de Transcrição , Humanos , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/patologia , Invasividade Neoplásica/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genéticaRESUMO
BACKGROUND: X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disorder caused by the intronic insertion of a SINE-VNTR-Alu (SVA) retrotransposon carrying an (AGAGGG)n repeat expansion in the TAF1 gene. The molecular mechanisms by which this mutation causes neurodegeneration remain elusive. OBJECTIVES: We investigated whether (AGAGGG)n repeats undergo repeat-associated non-AUG (RAN) translation, a pathogenic mechanism common among repeat expansion diseases. METHODS: XDP-specific RAN translation reporter plasmids were generated, transfected in HEK293 cells, and putative dipeptide repeat proteins (DPRs) were detected by Western blotting. Immunocytochemistry was performed in COS-7 cells to determine the subcellular localization of one DPR. RESULTS: We detected putative DPRs from two reading frames, supporting the translation of poly-(Glu-Gly) and poly-(Arg-Glu) species. XDP RAN translation initiates within the (AGAGGG)n sequence and poly-(Glu-Gly) DPRs formed nuclear inclusions in transfected cells. CONCLUSIONS: In summary, our work provides the first in-vitro proof of principle that the XDP-linked (AGAGGG)n repeat expansions can undergo RAN translation. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Assuntos
Distúrbios Distônicos , Doenças Genéticas Ligadas ao Cromossomo X , Humanos , Células HEK293 , Distúrbios Distônicos/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/genética , Íntrons , Proteína C9orf72/genéticaRESUMO
The TATA-box binding protein associated factor 1 (TAF1) is part of the TFIID complex that plays a key role during the initiation of transcription. Variants of TAF1 are associated with neurodevelopmental disorders. Previously, we found that CRISPR/Cas9 based editing of the TAF1 gene disrupts the morphology of the cerebral cortex and blunts the expression as well as the function of the CaV3.1 (T-type) voltage gated calcium channel. Here, we tested the efficacy of SAK3 (ethyl 8'-methyl-2', 4-dioxo-2-(piperidin-1-yl)-2'H-spiro [cyclopentane-1, 3'-imidazo [1, 2-a] pyridine]-2-ene-3-carboxylate), a T-type calcium channel enhancer, in an animal model of TAF1 intellectual disability (ID) syndrome. At post-natal day 3, rat pups were subjected to intracerebroventricular (ICV) injection of either gRNA-control or gRNA-TAF1 CRISPR/Cas9 viruses. At post-natal day 21, the rat pups were given SAK3 (0.25 mg/kg, p.o.) or vehicle for 14 days (i.e. till post-natal day 35) and then subjected to behavioral, morphological, and molecular studies. Oral administration of SAK3 (0.25 mg/kg, p.o.) significantly rescued locomotion abnormalities associated with TAF1 gene editing. SAK3 treatment prevented the loss of cortical neurons and GFAP-positive astrocytes observed after TAF1 gene editing. In addition, SAK3 protected cells from apoptosis. SAK3 also restored the Brain-derived neurotrophic factor/protein kinase B/Glycogen Synthase Kinase 3 Beta (BDNF/AKT/GSK3ß) signaling axis in TAF1 edited animals. Finally, SAK3 normalized the levels of three GSK3ß substrates - CaV3.1, FOXP2, and CRMP2. We conclude that the T-type calcium channel enhancer SAK3 is beneficial against the deleterious effects of TAF1 gene-editing, in part, by stimulating the BDNF/AKT/GSK3ß signaling pathway.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Modelos Animais de Doenças , Histona Acetiltransferases/deficiência , Imidazóis/administração & dosagem , Deficiência Intelectual/tratamento farmacológico , Deficiência Intelectual/metabolismo , Compostos de Espiro/administração & dosagem , Fatores Associados à Proteína de Ligação a TATA/deficiência , Fator de Transcrição TFIID/deficiência , Animais , Animais Recém-Nascidos , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Histona Acetiltransferases/genética , Injeções Intraventriculares , Deficiência Intelectual/genética , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Gravidez , Ratos , Ratos Sprague-Dawley , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genéticaRESUMO
X-linked Dystonia Parkinsonism (XDP) is a recessive, genetically inherited neurodegenerative disorder endemic to Panay Island in the Philippines. Clinical symptoms include the initial appearance of dystonia, followed by parkinsonian traits after 10-15 years. The basal ganglia, particularly the striatum, is an area of focus in XDP neuropathology research, as the striatum shows marked atrophy that correlates with disease progression. Thus, XDP shares features of Parkinson's disease symptomatology, in addition to the genetic predisposition and presence of striatal atrophy resembling Huntington's disease. However, further research is required to reveal the detailed pathology and indicators of disease in the XDP brain. First, there are limited neuropathological studies that have investigated neuronal changes and neuroinflammation in the XDP brain. However, multiple neuroimaging studies on XDP patients provide clues to other affected brain regions. Furthermore, molecular pathological studies have elucidated that the main genetic cause of XDP is in the TAF-1 gene, but how this mutation relates to XDP neuropathology still remains to be fully investigated. Hence, we aim to provide an extensive overview of the current literature describing neuropathological changes within the XDP brain, and discuss future research avenues, which will provide a better understanding of XDP neuropathogenesis.
Assuntos
Encéfalo/diagnóstico por imagem , Distúrbios Distônicos/diagnóstico por imagem , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico por imagem , Gânglios da Base/diagnóstico por imagem , Gânglios da Base/patologia , Encéfalo/patologia , Imagem de Difusão por Ressonância Magnética , Distúrbios Distônicos/patologia , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Humanos , Imageamento por Ressonância Magnética , Neostriado/diagnóstico por imagem , Neostriado/patologiaRESUMO
BACKGROUND: X-linked dystonia parkinsonism is a generalized, progressive dystonia followed by parkinsonism with onset in adulthood and accompanied by striatal neurodegeneration. Causative mutations are located in a noncoding region of the TATA-box binding protein-associated factor 1 (TAF1) gene and result in aberrant splicing. There are 2 major TAF1 isoforms that may be decreased in symptomatic patients, including the ubiquitously expressed canonical cTAF1 and the neuronal-specific nTAF1. OBJECTIVE: The objective of this study was to determine the behavioral and transcriptomic effects of decreased cTAF1 and/or nTAF1 in vivo. METHODS: We generated adeno-associated viral (AAV) vectors encoding microRNAs targeting Taf1 in a splice-isoform selective manner. We performed intracerebroventricular viral injections in newborn mice and rats and intrastriatal infusions in 3-week-old rats. The effects of Taf1 knockdown were assayed at 4 months of age with evaluation of motor function, histology, and RNA sequencing of the striatum, followed by its validation. RESULTS: We report motor deficits in all cohorts, more pronounced in animals injected at P0, in which we also identified transcriptomic alterations in multiple neuronal pathways, including the cholinergic synapse. In both species, we show a reduced number of striatal cholinergic interneurons and their marker mRNAs after Taf1 knockdown in the newborn. CONCLUSION: This study provides novel information regarding the requirement for TAF1 in the postnatal maintenance of striatal cholinergic neurons, the dysfunction of which is involved in other inherited forms of dystonia. © 2021 International Parkinson and Movement Disorder Society.
Assuntos
Distonia , Distúrbios Distônicos , Histona Acetiltransferases/genética , Transtornos Parkinsonianos , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genética , Adulto , Animais , Colinérgicos , Distúrbios Distônicos/genética , Distúrbios Distônicos/metabolismo , Humanos , Camundongos , Isoformas de Proteínas , RatosRESUMO
BACKGROUND: X-linked dystonia-parkinsonism is a rare neurological disease endemic to the Philippines. Dystonic symptoms appear in males at the mean age of 40 years and progress to parkinsonism with degenerative pathology in the striatum. A retrotransposon inserted in intron 32 of the TAF1 gene leads to alternative splicing in the region and a reduction of the full-length mRNA transcript. OBJECTIVES: The objective of this study was to discover cell-based and biofluid-based biomarkers for X-linked dystonia-parkinsonism. METHODS: RNA from patient-derived neural progenitor cells and their secreted extracellular vesicles were used to screen for dysregulation of TAF1 expression. Droplet-digital polymerase chain reaction was used to quantify the expression of TAF1 mRNA fragments 5' and 3' to the retrotransposon insertion and the disease-specific splice variant TAF1-32i in whole-blood RNA. Plasma levels of neurofilament light chain were measured using single-molecule array. RESULTS: In neural progenitor cells and their extracellular vesicles, we confirmed that the TAF1-3'/5' ratio was lower in patient samples, whereas TAF1-32i expression is higher relative to controls. In whole-blood RNA, both TAF1-3'/5' ratio and TAF1-32i expression can differentiate patient (n = 44) from control samples (n = 18) with high accuracy. Neurofilament light chain plasma levels were significantly elevated in patients (n = 43) compared with both carriers (n = 16) and controls (n = 21), with area under the curve of 0.79. CONCLUSIONS: TAF1 dysregulation in blood serves as a disease-specific biomarker that could be used as a readout for monitoring therapies targeting TAF1 splicing. Neurofilament light chain could be used in monitoring neurodegeneration and disease progression in patients. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Assuntos
Doenças Genéticas Ligadas ao Cromossomo X , Fatores Associados à Proteína de Ligação a TATA , Adulto , Biomarcadores , Distúrbios Distônicos , Doenças Genéticas Ligadas ao Cromossomo X/genética , Histona Acetiltransferases/genética , Humanos , Filamentos Intermediários , Masculino , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genéticaRESUMO
X-Linked Dystonia-Parkinsonism (XDP) is a neurodegenerative disease affecting individuals with ancestry to the island of Panay in the Philippines. In recent years there has been considerable progress at elucidating the genetic basis of XDP and candidate disease mechanisms in patient-derived cellular models, but the neural substrates that give rise to XDP in vivo are still poorly understood. Previous studies of limited XDP postmortem brain samples have reported a selective dropout of medium spiny neurons within the striatum, although neuroimaging of XDP patients has detected additional abnormalities in multiple brain regions beyond the basal ganglia. Given the need to fully define the CNS structures that are affected in this disease, we created a brain bank in Panay to serve as a tissue resource for detailed studies of XDP-related neuropathology. Here we describe this platform, from donor recruitment and consent to tissue collection, processing, and storage, that was assembled within a predominantly rural region of the Philippines with limited access to medical and laboratory facilities. Thirty-six brains from XDP individuals have been collected over an initial 4 years period. Tissue quality was assessed based on histologic staining of cortex, RNA integrity scores, detection of neuronal transcripts in situ by fluorescent hybridization chain reaction, and western blotting of neuronal and glial proteins. The results indicate that this pipeline preserves tissue integrity to an extent compatible with a range of morphologic, molecular, and biochemical analyses. Thus the algorithms that we developed for working in rural communities may serve as a guide for establishing similar brain banks for other rare diseases in indigenous populations.
Assuntos
Distonia , Distúrbios Distônicos , Doenças Neurodegenerativas , Encéfalo/diagnóstico por imagem , Distúrbios Distônicos/genética , Doenças Genéticas Ligadas ao Cromossomo X , HumanosRESUMO
Huntington's disease and X-linked dystonia parkinsonism are two monogenic basal ganglia model diseases. Huntington's disease is caused by a polyglutamine-encoding CAG repeat expansion in the Huntingtin (HTT) gene leading to several toxic interactions of both the expanded CAG-containing mRNA and the polyglutamine-containing protein, while X-linked dystonia parkinsonism is caused by a retrotransposon insertion in the TAF1 gene, which decreases expression of this core scaffold of the basal transcription factor complex TFIID. SRSF6 is an RNA-binding protein of the serine and arginine-rich (SR) protein family that interacts with expanded CAG mRNA and is sequestered into the characteristic polyglutamine-containing inclusion bodies of Huntington's disease brains. Here we report decreased levels of the SRSF6 interactor and regulator SREK1-another SR protein involved in RNA processing-which includes TAF1 as one of its targets. This led us to hypothesize that Huntington's disease and X-linked dystonia parkinsonism pathogeneses converge in TAF1 alteration. We show that diminishing SRSF6 through RNA interference in human neuroblastoma cells leads to a decrease in SREK1 levels, which, in turn, suffices to cause diminished TAF1 levels. We also observed decreased SREK1 and TAF1 levels in striatum of Huntington's disease patients and transgenic model mice. We then generated mice with neuronal transgenic expression of SREK1 (TgSREK1 mice) that, interestingly, showed transcriptomic alterations complementary to those in Huntington's disease mice. Most importantly, by combining Huntington's disease and TgSREK1 mice we verify that SREK1 overexpression corrects TAF1 deficiency and attenuates striatal atrophy and motor phenotype of Huntington's disease mice. Our results therefore demonstrate that altered RNA processing upon SREK1 dysregulation plays a key role in Huntington's disease pathogenesis and pinpoint TAF1 as a likely general determinant of selective vulnerability of the striatum in multiple neurological disorders.
Assuntos
Distúrbios Distônicos/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Histona Acetiltransferases/metabolismo , Doença de Huntington/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/metabolismo , Animais , Distúrbios Distônicos/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Humanos , Doença de Huntington/genética , Camundongos , Camundongos Transgênicos , Fosfoproteínas/genética , Fatores de Processamento de Serina-Arginina/genéticaRESUMO
T-type calcium channels, in the central nervous system, are involved in the pathogenesis of many neurodegenerative diseases, including TAF1 intellectual disability syndrome (TAF1 ID syndrome). Here, we evaluated the efficacy of a novel T-type Ca2+ channel enhancer, SAK3 (ethyl 8'-methyl-2', 4-dioxo-2-(piperidin-1-yl)-2'H-spiro [cyclopentane-1, 3'-imidazo [1, 2-a] pyridine]-2-ene-3-carboxylate) in an animal model of TAF1 ID syndrome. At post-natal day 3, rat pups were subjected to intracerebroventricular (ICV) injection of either gRNA-control or gRNA-TAF1 CRISPR/Cas9 viruses. At post-natal day 21 animals were given SAK3 (0.25 mg/kg, p.o.) or vehicle up to post-natal day 35 (i.e. 14 days). Rats were subjected to behavioral, morphological, electrophysiological, and molecular studies. Oral administration of SAK3 (0.25 mg/kg, p.o.) significantly rescued the behavior abnormalities in beam walking test and open field test caused by TAF1 gene editing. We observed an increase in calbindin-positive Purkinje cells and GFAP-positive astrocytes as well as a decrease in IBA1-positive microglia cells in SAK3-treated animals. In addition, SAK3 protected the Purkinje and granule cells from apoptosis induced by TAF-1 gene editing. SAK3 also restored the excitatory post synaptic current (sEPSCs) in TAF1 edited Purkinje cells. Finally, SAK3 normalized the BDNF/AKT signaling axis in TAF1 edited animals. Altogether, these observations suggest that SAK3 could be a novel therapeutic agent for TAF1 ID syndrome.
Assuntos
Cerebelo/efeitos dos fármacos , Histona Acetiltransferases/genética , Imidazóis/farmacologia , Deficiência Intelectual/fisiopatologia , Neurônios/efeitos dos fármacos , Compostos de Espiro/farmacologia , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genética , Animais , Canais de Cálcio Tipo T/efeitos dos fármacos , Canais de Cálcio Tipo T/metabolismo , Modelos Animais de Doenças , Deficiência Intelectual/genética , Ratos , Ratos Sprague-Dawley , SíndromeRESUMO
BACKGROUND: X-linked dystonia-parkinsonism is a neurodegenerative movement disorder. The underlying molecular basis has still not been completely elucidated, but likely involves dysregulation of TAF1 expression. In X-linked dystonia-parkinsonism, 3 disease-specific single-nucleotide changes (DSCs) introduce (DSC12) or abolish (DSC2 and DSC3) CpG dinucleotides and consequently sites of putative DNA methylation. Because transcriptional regulation tightly correlates with specific epigenetic marks, we investigated the role of DNA methylation in the pathogenesis of X-linked dystonia-parkinsonism. METHODS: DNA methylation at DSC12, DSC3, and DSC2 was quantified by bisulfite pyrosequencing in DNA from peripheral blood leukocytes, fibroblasts, induced pluripotent stem cell-derived cortical neurons and brain tissue from X-linked dystonia-parkinsonism patients and age- and sex-matched healthy Filipino controls in a prospective study. RESULTS: Compared with controls, X-linked dystonia-parkinsonism patients showed striking differences in DNA methylation at the 3 investigated CpG sites. Using methylation-sensitive luciferase reporter gene assays and immunoprecipitation, we demonstrated (1) that lack of DNA methylation because of DSC2 and DSC3 affects gene promoter activity and (2) that methylation at all 3 investigated CpG sites alters DNA-protein interaction. Interestingly, DSC3 decreased promoter activity per se compared with wild type, and promoter activity further decreased when methylation was present. Moreover, we identified specific binding of proteins to the investigated DSCs that are associated with splicing and RNA and DNA binding. CONCLUSIONS: We identified altered DNA methylation in X-linked dystonia-parkinsonism patients as a possible additional mechanism modulating TAF1 expression and putative novel targets for future therapies using DNA methylation-modifying agents. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.