Aberrant Splicing of tau Pre-mRNA Caused by Intronic Mutations Associated with the Inherited Dementia Frontotemporal Dementia with Parkinsonism Linked to Chromosome 17

Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 07/2000; 20(11):4036-48. DOI: 10.1128/MCB.20.11.4036-4048.2000
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Frontotemporal dementia accounts for a significant fraction of dementia cases. Frontotemporal dementia with parkinsonism linked to chromosome 17 is associated with either exonic or intronic mutations in the tau gene. This highlights the involvement of aberrant pre-mRNA splicing in the pathogenesis of neurodegenerative disorders. Little is known about the molecular mechanisms of the splicing defects underlying these diseases. To establish a model system for studying the role of pre-mRNA splicing in neurodegenerative diseases, we have constructed a tau minigene that reproduces tau alternative splicing in both cultured cells and in vitro biochemical assays. We demonstrate that mutations in a nonconserved intronic region of the human tau gene lead to increased splicing between exon 10 and exon 11. Systematic biochemical analyses indicate the importance of U1 snRNP and, to a lesser extent, U6 snRNP in differentially recognizing wild-type versus intron mutant tau pre-mRNAs. Gel mobility shift assays with purified U1 snRNP and oligonucleotide-directed RNase H cleavage experiments support the idea that the intronic mutations destabilize a stem-loop structure that sequesters the 5' splice site downstream of exon 10 in tau pre-mRNA, leading to increases in U1 snRNP binding and in splicing between exon 10 and exon 11. Thus, mutations in nonconserved intronic regions that increase rather than decrease alternative splicing can be an important pathogenic mechanism for the development of human diseases.

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Available from: Jocelyn Côté, Oct 05, 2015
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    • "Thermal stability studies of oligonucleotides demonstrated that disease-associated mutations within the putative stem-loop lower the melting temperature of the RNA duplex (i.e., where the double-stranded RNA dissociates with the single-stranded form) [35, 36]. A minigene construct encoding exons 9–11 recapitulates normal tau exon 10 splicing for the wild-type sequence and increased exon 10 inclusion for disease-causing mutations [37]. This minigene has been used in our lab to demonstrate that other mutations specifically designed to enhance stability of the stem-loop (and located distal to the U1 snRNP binding site) reduce exon 10 inclusion to decrease 4R/3R as predicted [36], validating the stem-loop as a bona fide structure involved in the regulation of tau exon 10 splicing and worthy of consideration as a therapeutic target. "
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    ABSTRACT: Brain deposition of the amyloid beta-protein (A β ) and tau are characteristic features in Alzheimer's disease (AD). Mutations in the A β precursor protein (APP) and a protease involved in A β production from APP strongly argue for a pathogenic role of A β in AD, while mutations in tau are associated with related disorders collectively called frontotemporal lobar degeneration (FTLD). Despite intense effort, therapeutic strategies that target A β or tau have not yet yielded medications, suggesting that alternative approaches should be pursued. In recent years, our laboratory has studied the role of mRNA in AD and FTLD, specifically those encoding tau and the A β -producing protease BACE1. As many FTLD-causing tau mutations destabilize a hairpin structure that regulates RNA splicing, we have targeted this structure with small molecules, antisense oligonucleotides, and small molecule-antisense conjugates. We have also discovered that microRNA interaction with the 3'-untranslated region of tau regulates tau expression. Regarding BACE1, we found that alternative splicing leads to inactive splice isoforms and antisense oligonucleotides shift splicing toward these inactive isoforms to decrease A β production. In addition, a G-quadruplex structure in the BACE1 mRNA plays a role in splice regulation. The prospects for targeting tau and BACE1 mRNAs as therapeutic strategies will be discussed.
    04/2014; 2014(5):757549. DOI:10.1155/2014/757549
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    • "We next examined the effect of the K298E tau mutation on exon 10 alternative mRNA splicing in cultured cells. A minigene construct with A892>G mutation was engineered based on the previously described human tau minigene model [17]. Human NSCs were transiently transfected at day 20 of differentiation with mutant K298E or the wild-type tau minigene, as described [1]. "
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    ABSTRACT: Frontotemporal lobar degeneration (FTLD) consists of a group of neurodegenerative diseases characterized by behavioural and executive impairment, language disorders and motor dysfunction. About 20-30 % of cases are inherited in a dominant manner. Mutations in the microtubule-associated protein tau gene (MAPT) cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17T). Here we report a novel MAPT mutation (K298E) in exon 10 in a patient with FTDP-17T. Neuropathological studies of post-mortem brain showed widespread neuronal loss and gliosis and abundant deposition of hyperphosphorylated tau in neurons and glia. Molecular studies demonstrated that the K298E mutation affects both protein function and alternative mRNA splicing. Fibroblasts from a skin biopsy of the proband taken at post-mortem were directly induced into neurons (iNs) and expressed both 3-repeat and 4-repeat tau isoforms. As well as contributing new knowledge on MAPT mutations in FTDP-17T, this is the first example of the successful generation of iNs from skin cells retrieved post-mortem.
    Acta Neuropathologica 11/2013; 127(2). DOI:10.1007/s00401-013-1219-1 · 10.76 Impact Factor
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    • "The potential of lentivirus-delivered TauPTM6 to promote trans-splicing at the level of E10 was assessed in HeLa cells first transduced with LV-TauPTM6 and then transfected with the TauEx9-11WT minigene consisting of MAPT exons 9, 10 and 11 and minimal intronic sequences flanking E10 (16). An 866 bp trans-spliced product containing minigene-derived exon 9 and TauPTM6-derived exons 10–13 and FLAG epitope sequences was detected by RT–PCR using a minigene-specific forward primer and the PTM-specific reverse primer, FLAGR, complementary to the FLAG sequence (Fig. 2, top panel). "
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    ABSTRACT: Abnormal metabolism of the tau protein is central to the pathogenesis of a number of dementias, including Alzheimer's disease. Aberrant alternative splicing of exon 10 in the tau pre-mRNA resulting in an imbalance of tau isoforms is one of the molecular causes of the inherited tauopathy, FTDP-17. We showed previously in heterologous systems that exon 10 inclusion in tau mRNA could be modulated by spliceosome-mediated RNA trans-splicing (SMaRT). Here, we evaluated the potential of trans-splicing RNA reprogramming to correct tau mis-splicing in differentiated neurons in a mouse model of tau mis-splicing, the htau transgenic mouse line, expressing the human MAPT gene in a null mouse Mapt background.Trans-splicing molecules designed to increase exon 10 inclusion were delivered to neurons using lentiviral vectors. We demonstrate reprogramming of tau transcripts at the RNA level after transduction of cultured neurons or after direct delivery and long-term expression of viral vectors into the brain of htau mice in vivo. Tau RNA trans-splicing resulted in an increase in exon 10 inclusion in the mature tau mRNA. Importantly, we also show that the trans-spliced product is translated into a full-length chimeric tau protein. These results validate the potential of SMaRT to correct tau mis-splicing and provide a framework for its therapeutic application to neurodegenerative conditions linked to aberrant RNA processing.
    Human Molecular Genetics 03/2013; 22(13). DOI:10.1093/hmg/ddt108 · 6.39 Impact Factor
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