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The nuclear RNA-binding protein TDP43 is integrally involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Previous studies uncovered N-terminal TDP43 isoforms that are predominantly cytosolic in localization, highly prone to aggregation, and enriched in susceptible spinal motor neurons. In healthy cells, however, these shortened (s)TDP43 isoforms are difficult to detect in comparison to full-length (fl)TDP43, raising questions regarding their origin and selective regulation. Here, we show that sTDP43 is created as a byproduct of TDP43 autoregulation and cleared by nonsense mediated RNA decay (NMD). The sTDP43-encoding transcripts that escape NMD can lead to toxicity but are rapidly degraded post-translationally. Circumventing these regulatory mechanisms by overexpressing sTDP43 results in neurodegeneration in vitro and in vivo via N-terminal oligomerization and impairment of flTDP43 splicing activity, in addition to RNA binding-dependent gain-of-function toxicity. Collectively, these studies highlight endogenous mechanisms that tightly regulate sTDP43 expression and provide insight into the consequences of aberrant sTDP43 accumulation in disease.
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Copy number variation (CNV) may lead to pathological traits, and Charcot-Marie-Tooth disease type 1A (CMT1A), the commonest inherited peripheral neuropathy, is due to a genomic duplication encompassing the dosage-sensitive PMP22 gene. MicroRNAs act as repressors on post-transcriptional regulation of gene expression and in rodent models of CMT1A, overexpression of one such microRNA (miR-29a) has been shown to reduce the PMP22 transcript and protein level. Here we present genomic and functional evidence, for the first time in a human CNV-associated phenotype, of the 3' untranslated region (3'-UTR)-mediated role of microRNA repression on gene expression. The proband of the family presented with an early-onset, severe sensorimotor demyelinating neuropathy and harboured a novel de novo deletion in the PMP22 3'-UTR. The deletion is predicted to include the miR-29a seed binding site and transcript analysis of dermal myelinated nerve fibres using a novel platform, revealed a marked increase in PMP22 transcript levels. Functional evidence from Schwann cell lines harbouring the wild-type and mutant 3'-UTR showed significantly increased reporter assay activity in the latter, which was not ameliorated by overexpression of a miR-29a mimic. This shows the importance of miR-29a in regulating PMP22 expression and opens an avenue for therapeutic drug development.
Assuntos
Doença de Charcot-Marie-Tooth , MicroRNAs , Humanos , Doença de Charcot-Marie-Tooth/patologia , MicroRNAs/genética , Variações do Número de Cópias de DNA , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Expressão GênicaRESUMO
Cells elaborate transcriptional programs in response to external signals. In the peripheral nerves, Schwann cells (SC) sort axons of given caliber and start the process of wrapping their membrane around them. We identify Actin-like protein 6a (ACTL6a), part of SWI/SNF chromatin remodeling complex, as critical for the integration of axonal caliber recognition with the transcriptional program of myelination. Nuclear levels of ACTL6A in SC are increased by contact with large caliber axons or nanofibers, and result in the eviction of repressive histone marks to facilitate myelination. Without Actl6a the SC are unable to coordinate caliber recognition and myelin production. Peripheral nerves in knockout mice display defective radial sorting, hypo-myelination of large caliber axons, and redundant myelin around small caliber axons, resulting in a clinical motor phenotype. Overall, this suggests that ACTL6A is a key component of the machinery integrating external signals for proper myelination of the peripheral nerve.
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Charcot-Marie-Tooth 1A (CMT1A) is the most common form of hereditary peripheral neuropathies, characterized by genetic duplication of the critical myelin gene Peripheral Myelin Protein 22 (PMP22). PMP22 overexpression results in abnormal Schwann cell differentiation, leading to axonal loss and muscle wasting. Since regulation of PMP22 expression is a major target of therapeutic discovery for CMT1A, we sought to establish unbiased approaches that allow the identification of therapeutic agents for this disease. Using genome editing, we generated a coincidence reporter assay that accurately monitors Pmp22 transcript levels in the S16 rat Schwann cell line, while reducing reporter-based false positives. A quantitative high-throughput screen (qHTS) of 42â¯577 compounds using this assay revealed diverse novel chemical classes that reduce endogenous Pmp22 transcript levels. Moreover, some of these classes show pharmacological specificity in reducing Pmp22 over another major myelin-associated gene, Mpz (Myelin protein zero). Finally, to investigate whether compound-mediated reduction of Pmp22 transcripts translates to reduced PMP22 protein levels, we edited the S16 genome to generate a reporter assay that expresses a PMP22-HiBiT fusion protein using CRISPR/Cas9. Overall, we present a screening platform that combines genome edited cell lines encoding reporters that monitor transcriptional and post-translational regulation of PMP22 with titration-based screening (e.g., qHTS), which could be efficiently incorporated into drug discovery campaigns for CMT1A.
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The induction of nerve injury response genes in Schwann cells depends on both transcriptional and epigenomic reprogramming. The nerve injury response program is regulated by the repressive histone mark H3K27 trimethylation (H3K27me3), deposited by Polycomb repressive complex 2 (PRC2). Loss of PRC2 function leads to early and augmented induction of the injury response gene network in peripheral nerves, suggesting H3K27 demethylases are required for derepression of Polycomb-regulated nerve injury genes. To determine the function of H3K27 demethylases in nerve injury, we generated Schwann cell-specific knockouts of H3K27 demethylase Kdm6b and double knockouts of Kdm6b/Kdm6a (encoding JMJD3 and UTX). We found that H3K27 demethylases are largely dispensable for Schwann cell development and myelination. In testing the function of H3K27 demethylases after injury, we found early induction of some nerve injury genes was diminished compared with control, but most injury genes were largely unaffected at 1 and 7 days post injury. Although it was proposed that H3K27 demethylases are required to activate expression of the cyclin-dependent kinase inhibitor Cdkn2a in response to injury, Schwann cell-specific deletion of H3K27 demethylases affected neither the expression of this gene nor Schwann cell proliferation after nerve injury. To further characterize the regulation of nerve injury response genes, we found that injury genes are associated with repressive histone H2AK119 ubiquitination catalyzed by PRC1, which declines after injury. Overall, our results indicate H3K27 demethylation is not required for induction of injury response genes and that other mechanisms likely are involved in activating Polycomb-repressed injury genes in peripheral nerve.
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Inibidor p16 de Quinase Dependente de Ciclina/genética , Histona Desmetilases/genética , Histona Desmetilases com o Domínio Jumonji/genética , Traumatismos dos Nervos Periféricos/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Código das Histonas/genética , Histonas/genética , Humanos , Metilação , Camundongos , Traumatismos dos Nervos Periféricos/patologia , Complexo Repressor Polycomb 2/genética , Células de Schwann/metabolismo , Células de Schwann/patologia , Ubiquitinação/genéticaRESUMO
Anterior cruciate ligament (ACL) rupture is an important condition of the human knee. Second ruptures are common and societal costs are substantial. Canine cranial cruciate ligament (CCL) rupture closely models the human disease. CCL rupture is common in the Labrador Retriever (5.79% prevalence), ~100-fold more prevalent than in humans. Labrador Retriever CCL rupture is a polygenic complex disease, based on genome-wide association study (GWAS) of single nucleotide polymorphism (SNP) markers. Dissection of genetic variation in complex traits can be enhanced by studying structural variation, including copy number variants (CNVs). Dogs are an ideal model for CNV research because of reduced genetic variability within breeds and extensive phenotypic diversity across breeds. We studied the genetic etiology of CCL rupture by association analysis of CNV regions (CNVRs) using 110 case and 164 control Labrador Retrievers. CNVs were called from SNPs using three different programs (PennCNV, CNVPartition, and QuantiSNP). After quality control, CNV calls were combined to create CNVRs using ParseCNV and an association analysis was performed. We found no strong effect CNVRs but found 46 small effect (max(T) permutation P<0.05) CCL rupture associated CNVRs in 22 autosomes; 25 were deletions and 21 were duplications. Of the 46 CCL rupture associated CNVRs, we identified 39 unique regions. Thirty four were identified by a single calling algorithm, 3 were identified by two calling algorithms, and 2 were identified by all three algorithms. For 42 of the associated CNVRs, frequency in the population was <10% while 4 occurred at a frequency in the population ranging from 10-25%. Average CNVR length was 198,872bp and CNVRs covered 0.11 to 0.15% of the genome. All CNVRs were associated with case status. CNVRs did not overlap previous canine CCL rupture risk loci identified by GWAS. Associated CNVRs contained 152 annotated genes; 12 CNVRs did not have genes mapped to CanFam3.1. Using pathway analysis, a cluster of 19 homeobox domain transcript regulator genes was associated with CCL rupture (P = 6.6E-13). This gene cluster influences cranial-caudal body pattern formation during embryonic limb development. Clustered genes were found in 3 CNVRs on chromosome 14 (HoxA), 28 (NKX6-2), and 36 (HoxD). When analysis was limited to deletion CNVRs, the association was strengthened (P = 8.7E-16). This study suggests a component of the polygenic risk of CCL rupture in Labrador Retrievers is associated with small effect CNVs and may include aspects of stifle morphology regulated by homeobox domain transcript regulator genes.
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Lesões do Ligamento Cruzado Anterior/genética , Ligamento Cruzado Anterior , Variações do Número de Cópias de DNA , Polimorfismo de Nucleotídeo Único , Animais , Cães , Estudo de Associação Genômica AmplaRESUMO
Copy number variation of the peripheral nerve myelin gene Peripheral Myelin Protein 22 (PMP22) causes multiple forms of inherited peripheral neuropathy. The duplication of a 1.4 Mb segment surrounding this gene in chromosome 17p12 (c17p12) causes the most common form of Charcot-Marie-Tooth disease type 1A, whereas the reciprocal deletion of this gene causes a separate neuropathy termed hereditary neuropathy with liability to pressure palsies (HNPP). PMP22 is robustly induced in Schwann cells in early postnatal development, and several transcription factors and their cognate regulatory elements have been implicated in coordinating the gene's proper expression. We previously found that a distal super-enhancer domain was important for Pmp22 expression in vitro, with particular impact on a Schwann cell-specific alternative promoter. Here, we investigate the consequences of deleting this super-enhancer in vivo. We find that loss of the super-enhancer in mice reduces Pmp22 expression throughout development and into adulthood, with greater impact on the Schwann cell-specific promoter. Additionally, these mice display tomacula formed by excessive myelin folding, a pathological hallmark of HNPP, as have been previously observed in heterozygous Pmp22 mice as well as sural biopsies from patients with HNPP. Our findings demonstrate a mechanism by which smaller copy number variations, not including the Pmp22 gene, are sufficient to reduce gene expression and phenocopy a peripheral neuropathy caused by the HNPP-associated deletion encompassing PMP22.
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Artrogripose/genética , Doença de Charcot-Marie-Tooth/genética , Elementos Facilitadores Genéticos/genética , Neuropatia Hereditária Motora e Sensorial/genética , Proteínas da Mielina/genética , Adulto , Animais , Artrogripose/metabolismo , Artrogripose/patologia , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Variações do Número de Cópias de DNA/genética , Neuropatia Hereditária Motora e Sensorial/metabolismo , Neuropatia Hereditária Motora e Sensorial/patologia , Heterozigoto , Humanos , Camundongos , Bainha de Mielina/genética , Bainha de Mielina/metabolismo , Nervos Periféricos/metabolismo , Nervos Periféricos/patologia , Fenótipo , Células de Schwann/metabolismo , Células de Schwann/patologiaRESUMO
OBJECTIVE: Development of biomarkers for Charcot-Marie-Tooth (CMT) disease is critical for implementing effective clinical trials. The most common form of CMT, type 1A, is caused by a genomic duplication surrounding the PMP22 gene. A recent report (Neurology 2018;90:e518-3524) showed elevation of neurofilament light (NfL) in plasma of CMT1A disease patients, which correlated with disease severity. However, no plasma/serum biomarker has been identified that is specific to Schwann cells, the most directly affected cells in CMT1A. METHODS: We used the Olink immuno PCR platform to profile CMT1A patient (n = 47, 2 cohorts) and normal control plasma (n = 41, two cohorts) on five different Olink panels to screen 398 unique proteins. RESULTS: The TMPRSS5 protein (Transmembrane protease serine 5) was elevated 2.07-fold (P = <0.0001) in two independent cohorts of CMT1A samples relative to controls. TMPRSS5 is most highly expressed in Schwann cells of peripheral nerve. Consistent with early myelination deficits in CMT1A, TMPRSS5 was not significantly correlated with disease score (CMTES-R, CMTNS-R), nerve conduction velocities (Ulnar CMAP, Ulnar MNCV), or with age. TMPRSS5 was not significantly elevated in smaller sample sets from patients with CMT2A, CMT2E, CMT1B, or CMT1X. The Olink immuno PCR assays confirmed elevated levels of NfL (average 1.58-fold, P < 0.0001), which correlated with CMT1A patient disease score. INTERPRETATION: These data identify the first Schwann cell-specific protein that is elevated in plasma of CMT1A patients, and may provide a disease marker and a potentially treatment-responsive biomarker with good disease specificity for clinical trials.
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Doença de Charcot-Marie-Tooth/sangue , Doença de Charcot-Marie-Tooth/diagnóstico , Proteínas de Membrana/sangue , Proteínas Mitocondriais/sangue , Células de Schwann , Serina Endopeptidases/sangue , Adulto , Animais , Biomarcadores/sangue , Células Cultivadas , Doença de Charcot-Marie-Tooth/fisiopatologia , Estudos de Coortes , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Condução Nervosa/fisiologia , Reação em Cadeia da Polimerase , RatosRESUMO
BACKGROUND: Charcot-Marie-Tooth disease type 1A (CMT1A) is caused by a uniform 1.5-Mb duplication on chromosome 17p, which includes the PMP22 gene. Patients often present the classic neuropathy phenotype, but also with high clinical variability. OBJECTIVE: We aimed to identify genetic variants that are potentially associated with specific clinical outcomes in CMT1A. METHODS: We genotyped over 600,000 genomic markers using DNA samples from 971 CMT1A patients and performed a case-only genome-wide association study (GWAS) to identify potential genetic association in a subset of 644 individuals of European ancestry. A total of 14 clinical outcomes were analyzed in this study. RESULTS: The analyses yielded suggestive association signals in four clinical outcomes: difficulty with eating utensils (lead SNP rs4713376, chr6â:â30773314, Pâ=â9.91×10-7, odds ratioâ=â3.288), hearing loss (lead SNP rs7720606, chr5â:â126551732, Pâ=â2.08×10-7, odds ratioâ=â3.439), decreased ability to feel (lead SNP rs17629990, chr4â:â171224046, Pâ=â1.63×10-7, odds ratioâ=â0.336), and CMT neuropathy score (lead SNP rs12137595, chr1â:â4094068, Pâ=â1.14×10-7, betaâ=â3.014). CONCLUSIONS: While the results require validation in future genetic and functional studies, the detected association signals may point to novel genetic modifiers in CMT1A.
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Doença de Charcot-Marie-Tooth/genética , Genes Modificadores/genética , Estudo de Associação Genômica Ampla , Genótipo , HumanosRESUMO
OBJECTIVE: Charcot-Marie-Tooth (CMT) disease is most commonly caused by duplication of a chromosomal segment surrounding Peripheral Myelin Protein 22, or PMP22 gene, which is classified as CMT1A. Several candidate therapies reduce Pmp22 mRNA levels in CMT1A rodent models, but development of biomarkers for clinical trials in CMT1A is a challenge given its slow progression and difficulty in obtaining nerve samples. Quantitative PCR measurements of PMP22 mRNA in dermal nerves were performed using skin biopsies in human clinical trials for CMT1A, but this approach did not show increased PMP22 mRNA in CMT1A patients compared to controls. One complicating factor is the variable amounts of Schwann cells (SCs) in skin. The objective of the study was to develop a novel method for precise evaluation of PMP22 levels in skin biopsies that can discriminate CMT1A patients from controls. METHODS: We have developed methods to normalize PMP22 transcript levels to SC-specific genes that are not altered by CMT1A status. Several CMT1A-associated genes were assembled into a custom Nanostring panel to enable precise transcript measurements that can be normalized to variable SC content. RESULTS: The digital expression data from Nanostring analysis showed reproducible elevation of PMP22 levels in CMT1A versus control skin biopsies, particularly after normalization to SC-specific genes. INTERPRETATION: This platform should be useful in clinical trials for CMT1A as a biomarker of target engagement that can be used to optimize dosing, and the same normalization framework is applicable to other types of CMT. ANN NEUROL 2019;85:887-898.
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Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Proteínas da Mielina/genética , Células de Schwann/patologia , Pele/patologia , Adolescente , Adulto , Idoso , Animais , Biomarcadores/metabolismo , Biópsia , Doença de Charcot-Marie-Tooth/metabolismo , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas da Mielina/biossíntese , Células de Schwann/metabolismo , Pele/metabolismo , Adulto JovemRESUMO
OBJECTIVE: Genetic modifiers in rare disease have long been suspected to contribute to the considerable variance in disease expression, including Charcot-Marie-Tooth disease type 1A (CMT1A). To address this question, the Inherited Neuropathy Consortium collected a large standardized sample of such rare CMT1A patients over a period of 8 years. CMT1A is caused in most patients by a uniformly sized 1.5 Mb duplication event involving the gene PMP22. METHODS: We genotyped DNA samples from 971 CMT1A patients on Illumina BeadChips. Genome-wide analysis was performed in a subset of 330 of these patients, who expressed the extremes of a hallmark symptom: mild and severe foot dorsiflexion strength impairment. SIPA1L2 (signal-induced proliferation-associated 1 like 2), the top identified candidate modifier gene, was expressed in the peripheral nerve, and our functional studies identified and confirmed interacting proteins using coimmunoprecipitation analysis, mass spectrometry, and immunocytochemistry. Chromatin immunoprecipitation and in vitro siRNA experiments were used to analyze gene regulation. RESULTS: We identified significant association of 4 single nucleotide polymorphisms (rs10910527, rs7536385, rs4649265, rs1547740) in SIPA1L2 with foot dorsiflexion strength (p < 1 × 10-7 ). Coimmunoprecipitation and mass spectroscopy studies identified ß-actin and MYH9 as SIPA1L2 binding partners. Furthermore, we show that SIPA1L2 is part of a myelination-associated coexpressed network regulated by the master transcription factor SOX10. Importantly, in vitro knockdown of SIPA1L2 in Schwannoma cells led to a significant reduction of PMP22 expression, hinting at a potential strategy for drug development. INTERPRETATION: SIPA1L2 is a potential genetic modifier of CMT1A phenotypic expressions and offers a new pathway to therapeutic interventions. ANN NEUROL 2019;85:316-330.
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Doença de Charcot-Marie-Tooth/genética , Pé/fisiopatologia , Proteínas Ativadoras de GTPase/genética , Genes Modificadores/genética , Debilidade Muscular/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Doença de Charcot-Marie-Tooth/fisiopatologia , Criança , Pré-Escolar , Feminino , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Debilidade Muscular/fisiopatologia , Proteínas da Mielina/genética , Neurilemoma/genética , Fenótipo , Polimorfismo de Nucleotídeo Único , Ratos , Índice de Gravidade de Doença , Adulto JovemRESUMO
The transition of differentiated Schwann cells to support of nerve repair after injury is accompanied by remodeling of the Schwann cell epigenome. The EED-containing polycomb repressive complex 2 (PRC2) catalyzes histone H3K27 methylation and represses key nerve repair genes such as Shh, Gdnf, and Bdnf, and their activation is accompanied by loss of H3K27 methylation. Analysis of nerve injury in mice with a Schwann cell-specific loss of EED showed the reversal of polycomb repression is required and a rate limiting step in the increased transcription of Neuregulin 1 (type I), which is required for efficient remyelination. However, mouse nerves with EED-deficient Schwann cells display slow axonal regeneration with significantly low expression of axon guidance genes, including Sema4f and Cntf. Finally, EED loss causes impaired Schwann cell proliferation after injury with significant induction of the Cdkn2a cell cycle inhibitor gene. Interestingly, PRC2 subunits and CDKN2A are commonly co-mutated in the transition from benign neurofibromas to malignant peripheral nerve sheath tumors (MPNST's). RNA-seq analysis of EED-deficient mice identified PRC2-regulated molecular pathways that may contribute to the transition to malignancy in neurofibromatosis.
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Proliferação de Células/fisiologia , Regulação da Expressão Gênica/genética , Regeneração Nervosa/efeitos dos fármacos , Complexo Repressor Polycomb 2/metabolismo , Células de Schwann/fisiologia , Neuropatia Ciática/fisiopatologia , Animais , Proliferação de Células/efeitos dos fármacos , Imunoprecipitação da Cromatina , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Regeneração Nervosa/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurregulinas/metabolismo , Proteína Oncogênica v-akt/metabolismo , Complexo Repressor Polycomb 2/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Células de Schwann/efeitos dos fármacos , Células de Schwann/ultraestrutura , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Peripheral nerve myelination is adversely affected in the most common form of the hereditary peripheral neuropathy called Charcot-Marie-Tooth Disease. This form, classified as CMT1A, is caused by a 1.4 Mb duplication on chromosome 17, which includes the abundantly expressed Schwann cell myelin gene, Peripheral Myelin Protein 22 (PMP22). This is one of the most common copy number variants causing neurological disease. Overexpression of Pmp22 in rodent models recapitulates several aspects of neuropathy, and reduction of Pmp22 in such models results in amelioration of the neuropathy phenotype. Recently we identified a potential super-enhancer approximately 90-130 kb upstream of the Pmp22 transcription start sites. This super-enhancer encompasses a cluster of individual enhancers that have the acetylated histone H3K27 active enhancer mark, and coincides with smaller duplications identified in patients with milder CMT1A-like symptoms, where the PMP22 coding region itself was not part of the duplication. In this study, we have utilized genome editing to create a deletion of this super-enhancer to determine its role in Pmp22 regulation. Our data show a significant decrease in Pmp22 transcript expression using allele-specific internal controls. Moreover, the P2 promoter of the Pmp22 gene, which is used in other cell types, is affected, but we find that the Schwann cell-specific P1 promoter is disproportionately more sensitive to loss of the super-enhancer. These data show for the first time the requirement of these upstream enhancers for full Pmp22 expression.
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Doença de Charcot-Marie-Tooth/genética , Variações do Número de Cópias de DNA/genética , Elementos Facilitadores Genéticos/genética , Proteínas da Mielina/genética , Animais , Linhagem Celular , Doença de Charcot-Marie-Tooth/patologia , Duplicação Cromossômica/genética , Cromossomos Humanos Par 17/genética , Edição de Genes , Regulação da Expressão Gênica/genética , Humanos , Regiões Promotoras Genéticas , Ratos , Células de Schwann/patologiaRESUMO
Measurement of gene expression for high-throughput screening is an increasingly used technique that has been developed for not only gene dosage disorders resulting from disease-associated copy number variations, but also for induction/repression of genes modulating the severity of a disease phenotype. Traditional methods have employed transient or stable transfection of reporter constructs in which a single reporter is driven by selected regulatory elements from the candidate gene. However, individual regulatory elements are inherently unable to capture the integrated regulation of multiple enhancers at the endogenous locus, and random reporter insertion can result in neighborhood effects that impact the physiological responsiveness of the reporter. Therefore, we outline a general method of employing genome editing to insert reporters into the 3' UTR of a candidate gene, which has been used successfully in our studies of the Pmp22 gene associated with Charcot-Marie-Tooth disease. The method employs genome editing to insert two nonhomologous reporters that maximize the efficiency of identification of biologically active molecules through concordant responses in small molecule screening. We include a number of aspects of the design and construction of these reporter assays that will be applicable to creation of similar assays in a variety of cell types.
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Doença de Charcot-Marie-Tooth/genética , Edição de Genes/métodos , Genes Reporter/genética , Ensaios de Triagem em Larga Escala/métodos , Proteínas da Mielina/genética , Bioensaio/instrumentação , Bioensaio/métodos , Sistemas CRISPR-Cas/genética , Linhagem Celular , Edição de Genes/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Luciferases de Vaga-Lume/química , Luciferases de Vaga-Lume/genética , Medições Luminescentes/instrumentação , Medições Luminescentes/métodos , Transfecção/instrumentação , Transfecção/métodosRESUMO
OBJECTIVE: Our goal was to define the genetic cause of the profound hypomyelination in the taiep rat model and determine its relevance to human white matter disease. METHODS: Based on previous localization of the taiep mutation to rat chromosome 9, we tested whether the mutation resided within the Tubb4a (ß-tubulin 4A) gene, because mutations in the TUBB4A gene have been described in patients with central nervous system hypomyelination. To determine whether accumulation of microtubules led to progressive demyelination, we analyzed the spinal cord and optic nerves of 2-year-old rats by light and electron microscopy. Cerebral white matter from a patient with TUBB4A Asn414Lys mutation and magnetic resonance imaging evidence of severe hypomyelination were studied similarly. RESULTS: As the taiep rat ages, there is progressive loss of myelin in the brain and dorsal column of the spinal cord associated with increased oligodendrocyte numbers with accumulation of microtubules. This accumulation involved the entire cell body and distal processes of oligodendrocytes, but there was no accumulation of microtubules in axons. A single point mutation in Tubb4a (p.Ala302Thr) was found in homozygous taiep samples. A similar hypomyelination associated with increased oligodendrocyte numbers and arrays of microtubules in oligodendrocytes was demonstrated in the human patient sample. INTERPRETATION: The taiep rat is the first animal model of TUBB4 mutations in humans and a novel system in which to test the mechanism of microtubule accumulation. The finding of microtubule accumulation in a patient with a TUBB4A mutation and leukodystrophy confirms the usefulness of taiep as a model of the human disease. Ann Neurol 2017;81:690-702.
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Doenças Desmielinizantes , Modelos Animais de Doenças , Microtúbulos/metabolismo , Nervo Óptico/diagnóstico por imagem , Medula Espinal/diagnóstico por imagem , Tubulina (Proteína)/genética , Substância Branca/diagnóstico por imagem , Animais , Pré-Escolar , Doenças Desmielinizantes/diagnóstico por imagem , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/fisiopatologia , Humanos , Imageamento por Ressonância Magnética , Microscopia Eletrônica , RatosRESUMO
Schwann cells are myelinating glia in the peripheral nervous system that form the myelin sheath. A major cause of peripheral neuropathy is a copy number variant involving the Peripheral Myelin Protein 22 (PMP22) gene, which is located within a 1.4-Mb duplication on chromosome 17 associated with the most common form of Charcot-Marie-Tooth Disease (CMT1A). Rodent models of CMT1A have been used to show that reducing Pmp22 overexpression mitigates several aspects of a CMT1A-related phenotype. Mechanistic studies of Pmp22 regulation identified enhancers regulated by the Sox10 (SRY sex determining region Y-box 10) and Egr2/Krox20 (Early growth response protein 2) transcription factors in myelinated nerves. However, relatively little is known regarding how other transcription factors induce Pmp22 expression during Schwann cell development and myelination. Here, we examined Pmp22 enhancers as a function of cell type-specificity, nerve injury and development. While Pmp22 enhancers marked by active histone modifications were lost or remodeled after injury, we found that these enhancers were permissive in early development prior to Pmp22 upregulation. Pmp22 enhancers contain binding motifs for TEA domain (Tead) transcription factors of the Hippo signaling pathway. We discovered that Tead1 and co-activators Yap and Taz are required for Pmp22 expression, as well as for the expression of Egr2 Tead1 directly binds Pmp22 and Egr2 enhancers early in development and Tead1 binding is induced during myelination, correlating with Pmp22 expression. The data identify Tead1 as a novel regulator of Pmp22 expression during development in concert with Sox10 and Egr2.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Proteínas de Ligação a DNA/genética , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteínas da Mielina/genética , Doenças do Sistema Nervoso Periférico/genética , Fatores de Transcrição SOXE/genética , Fatores de Transcrição/genética , Animais , Doença de Charcot-Marie-Tooth/patologia , Variações do Número de Cópias de DNA/genética , Proteínas de Ligação a DNA/biossíntese , Modelos Animais de Doenças , Proteína 2 de Resposta de Crescimento Precoce/biossíntese , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Bainha de Mielina/genética , Bainha de Mielina/patologia , Neurogênese/genética , Doenças do Sistema Nervoso Periférico/patologia , Fenótipo , Células de Schwann/metabolismo , Células de Schwann/patologia , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/biossínteseRESUMO
BACKGROUND: Although pre-transplant immunization is routinely recommended, this recommendation is based on little data. The primary objective of this study was to compare antibody responses in lung transplant patients who received influenza vaccine before the transplant, within the first six months of transplant, between 13 and 60 months post-transplant, and 110 months or beyond transplant. METHODS: This prospective cohort study included 357 total immunization events performed over five yr to measure H1N1, H3N2, and B antibody responses to the influenza vaccine in pre- and post-lung transplant patients. Geometric mean titers, seroprotection (antibody titer at least 1:40), seroconversion (fourfold increase between pre and post), and mean fold increases were compared. RESULTS: The geometric mean titer distributions were different for H3N2 and B (ANOVA; p = 0.002 for both). Pre-transplant antibody concentrations were higher compared to the 13- to 60-month group for H3N2 (corrected p = 0.002) and the healthy group for B (corrected p = 0.001). The ≥110-month group had higher seroconversion rates compared to the 13- to 60-month group for H3N2 and B viruses. CONCLUSION: Lung pre-transplant patients and the long-term survivors have higher responses to the influenza vaccine than early post-transplant and the transplant control groups.
Assuntos
Anticorpos Antivirais/sangue , Formação de Anticorpos/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/prevenção & controle , Pneumopatias/imunologia , Transplante de Pulmão , Anticorpos Antivirais/imunologia , Estudos de Casos e Controles , Feminino , Seguimentos , Sobrevivência de Enxerto , Humanos , Vacinas contra Influenza/administração & dosagem , Influenza Humana/imunologia , Influenza Humana/virologia , Pneumopatias/cirurgia , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Fatores de Risco , VacinaçãoRESUMO
BACKGROUND: Lung transplant recipients are among those with the highest risk of influenza infection and complications each year. A few studies show adequate responses after influenza immunization; no studies examined the season-long protection. METHODS: Influenza antibody concentrations were measured using hemagglutination inhibition assays before immunization, 2 to 4 weeks after immunization, and 6 months after immunization in 25 healthy controls and 54 lung transplant patients. Two definitions of seroprotection (40 hemagglutination units (HAU) and 160 HAU which confers about 95% protection) were used. RESULTS: Influenza vaccine responses were high in both groups postimmunization (100% at 40 HAU and 60% healthy and 61% lung transplant at 160 HAU; P = 1.0; chi-square). At 6 months after immunization, seroprotection rates at 40 HAU (95% healthy and 97% lung transplant; P = 1.0) and at 160 HAU (24% healthy and 36% lung transplant; P = 0.40) were observed. CONCLUSION: Seroprotection rates do not differ between healthy and transplant groups over 6 months when 40 HAU or 160 HAU is used. However, the seroprotection rates are disappointingly low when 160 HAU (the antibody concentration associated with 95% protection from infection) is used. Annual influenza vaccine should continue to be a high priority for lung transplant patients.
Assuntos
Anticorpos Antivirais/sangue , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Transplante de Pulmão , Adulto , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Testes de Inibição da Hemaglutinação , Humanos , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/administração & dosagem , Influenza Humana/sangue , Influenza Humana/imunologia , Influenza Humana/virologia , Transplante de Pulmão/efeitos adversos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estações do Ano , Fatores de Tempo , Resultado do Tratamento , VacinaçãoRESUMO
Copy number variation resulting in excess PMP22 protein causes the peripheral neuropathy Charcot-Marie-Tooth disease, type 1A. To broadly interrogate chemically sensitive transcriptional pathways controlling PMP22 protein levels, we used the targeting precision of TALEN-mediated genome editing to embed reporters within the genetic locus harboring the Peripheral Myelin Protein 22 (Pmp22) gene. Using a Schwann cell line with constitutively high endogenous levels of Pmp22, we obtained allelic insertion of secreted bioluminescent reporters with sufficient signal to enable a 1536-well assay. Our findings from the quantitative high-throughput screening (qHTS) of several thousand drugs and clinically investigated compounds using this assay design both overlapped and expanded results from a previous assay using a randomly inserted reporter gene controlled by a single regulatory element of the Pmp22 gene. A key difference was the identification of a kinase-controlled inhibitory pathway of Pmp22 transcription revealed by the activity of the Protein kinase C (PKC)-modulator bryostatin.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , HumanosRESUMO
BACKGROUND: The timing of influenza vaccination and susceptibility to re-circulating virus in the population is influenced by the persistence of seroprotection. Immunosuppressed transplant patients are known to have lower antibody response rates than healthy individuals, but acceptable antibody concentrations are achieved. The duration of this seroprotection beyond a single season has not been evaluated in either healthy or immunosuppressed populations. METHODS: Influenza antibody concentrations against viruses no longer included in the vaccine were measured in serum by hemagglutination inhibition assay annually following vaccination of 73 lung transplant participants and 27 healthy controls. Seroprotection was defined as a titer of ≥ 1:40 and was compared between groups over the measured term using Fisher's exact tests. RESULTS: Seroprotection rates for influenza A and B strains at one year following immunization were 100% for lung transplant and healthy controls. Rates at two years for the influenza A strains were 65-74% for lung transplant vs. 77-100% in healthy controls. Rates for influenza B strains two years following immunization were 27-50% for lung transplant vs. 16-38% in healthy controls. (Fisher's exact test; not significant for between group comparisons; p < 0.05 for between season comparisons) CONCLUSIONS: Vaccine-induced antibody persistence appears to be influenced more by the vaccine virus strain than the immune status of the vaccinated individuals. Seroprotection rates are high 12 mo following influenza vaccination but wane over the second year, particularly for influenza B viruses. Annual influenza immunization is indicated, even for healthy individuals and even when the vaccine viruses do not change.