{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T12:16:07Z","timestamp":1792671367883,"version":"4.5.0"},"reference-count":29,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2019,5,28]],"date-time":"2019-05-28T00:00:00Z","timestamp":1559001600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2019,5,28]],"date-time":"2019-05-28T00:00:00Z","timestamp":1559001600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Methyl-CpG-binding protein 2 (MeCP2) is a multifunctional chromosomal protein that plays a key role in the central nervous system. Its levels need to be tightly regulated, as both deficiency and excess of the protein can lead to severe neuronal dysfunction. Loss-of-function mutations affecting MeCP2 are the primary cause of Rett syndrome (RTT), a severe neurological disorder that is thought to result from absence of functional protein in the brain. Several therapeutic strategies for the treatment of RTT are currently being developed. One of them is the use of stable and native TAT-MeCP2 fusion proteins to replenish its levels in neurons after permeation across the blood-brain barrier (BBB). Here we describe the expression and purification of various transactivator of transcription (TAT)-MeCP2 variants and the development of an electrochemiluminescence based assay (ECLIA) that is able to measure endogenous MeCP2 and recombinant TAT-MeCP2 fusion protein levels in a 96-well plate format. The MeCP2 ECLIA produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error throughout a wide working range. To underline its broad applicability, this assay was used to analyze brain tissue and study the transport of TAT-MeCP2 variants across an <jats:italic>in vitro<\/jats:italic> model of the blood-brain barrier.<\/jats:p>","DOI":"10.1038\/s41598-019-44372-3","type":"journal-article","created":{"date-parts":[[2019,5,28]],"date-time":"2019-05-28T10:04:40Z","timestamp":1559037880000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["An electrochemiluminescence based assay for quantitative detection of endogenous and exogenously applied MeCP2 protein variants"],"prefix":"10.1038","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4438-6043","authenticated-orcid":false,"given":"Hannes","family":"Steinkellner","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna","family":"Sch\u00f6negger","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julia","family":"Etzler","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Prakasha","family":"Kempaiah","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8490-9630","authenticated-orcid":false,"given":"Anna","family":"Huber","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kathrin","family":"Hahn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katrin","family":"Rose","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mark","family":"Duerr","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John","family":"Christodoulou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander V.","family":"Beribisky","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Winfried","family":"Neuhaus","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Franco","family":"Laccone","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2019,5,28]]},"reference":[{"key":"44372_CR1","doi-asserted-by":"publisher","DOI":"10.1186\/s13073-017-0411-7","volume":"9","author":"RR Shah","year":"2017","unstructured":"Shah, R. R. & Bird, A. P. MeCP2 mutations: progress towards understanding and treating Rett syndrome. Genome medicine 9, 17, https:\/\/doi.org\/10.1186\/s13073-017-0411-7 (2017).","journal-title":"Genome medicine"},{"key":"44372_CR2","doi-asserted-by":"publisher","first-page":"1224","DOI":"10.1126\/science.1153252","volume":"320","author":"M Chahrour","year":"2008","unstructured":"Chahrour, M. et al. MeCP2, a key contributor to neurological disease, activates and represses transcription. Science 320, 1224\u20131229, https:\/\/doi.org\/10.1126\/science.1153252 (2008).","journal-title":"Science"},{"key":"44372_CR3","doi-asserted-by":"publisher","first-page":"1818","DOI":"10.1093\/nar\/gkh349","volume":"32","author":"S Kriaucionis","year":"2004","unstructured":"Kriaucionis, S. & Bird, A. The major form of MeCP2 has a novel N-terminus generated by alternative splicing. Nucleic acids research 32, 1818\u20131823, https:\/\/doi.org\/10.1093\/nar\/gkh349 (2004).","journal-title":"Nucleic acids research"},{"key":"44372_CR4","doi-asserted-by":"publisher","first-page":"261","DOI":"10.1038\/nrg3897","volume":"16","author":"MJ Lyst","year":"2015","unstructured":"Lyst, M. J. & Bird, A. Rett syndrome: a complex disorder with simple roots. Nature reviews. Genetics 16, 261\u2013275, https:\/\/doi.org\/10.1038\/nrg3897 (2015).","journal-title":"Nature reviews. Genetics"},{"key":"44372_CR5","doi-asserted-by":"publisher","first-page":"944","DOI":"10.1002\/ana.22124","volume":"68","author":"JL Neul","year":"2010","unstructured":"Neul, J. L. et al. Rett syndrome: revised diagnostic criteria and nomenclature. Annals of neurology 68, 944\u2013950, https:\/\/doi.org\/10.1002\/ana.22124 (2010).","journal-title":"Annals of neurology"},{"key":"44372_CR6","doi-asserted-by":"publisher","first-page":"1093","DOI":"10.1086\/320109","volume":"68","author":"R Trappe","year":"2001","unstructured":"Trappe, R. et al. MECP2 mutations in sporadic cases of Rett syndrome are almost exclusively of paternal origin. American journal of human genetics 68, 1093\u20131101, https:\/\/doi.org\/10.1086\/320109 (2001).","journal-title":"American journal of human genetics"},{"key":"44372_CR7","doi-asserted-by":"publisher","first-page":"417","DOI":"10.1136\/jmg.2007.049452","volume":"44","author":"L Villard","year":"2007","unstructured":"Villard, L. MECP2 mutations in males. Journal of medical genetics 44, 417\u2013423, https:\/\/doi.org\/10.1136\/jmg.2007.049452 (2007).","journal-title":"Journal of medical genetics"},{"key":"44372_CR8","doi-asserted-by":"publisher","first-page":"4389","DOI":"10.1093\/hmg\/ddw269","volume":"25","author":"PD Ross","year":"2016","unstructured":"Ross, P. D. et al. Exclusive expression of MeCP2 in the nervous system distinguishes between brain and peripheral Rett syndrome-like phenotypes. Human molecular genetics 25, 4389\u20134404, https:\/\/doi.org\/10.1093\/hmg\/ddw269 (2016).","journal-title":"Human molecular genetics"},{"key":"44372_CR9","doi-asserted-by":"publisher","first-page":"100","DOI":"10.1016\/j.tins.2015.12.008","volume":"39","author":"DM Katz","year":"2016","unstructured":"Katz, D. M. et al. Rett Syndrome: Crossing the Threshold to Clinical Translation. Trends in neurosciences 39, 100\u2013113, https:\/\/doi.org\/10.1016\/j.tins.2015.12.008 (2016).","journal-title":"Trends in neurosciences"},{"key":"44372_CR10","doi-asserted-by":"publisher","first-page":"1143","DOI":"10.1126\/science.1138389","volume":"315","author":"J Guy","year":"2007","unstructured":"Guy, J., Gan, J., Selfridge, J., Cobb, S. & Bird, A. Reversal of neurological defects in a mouse model of Rett syndrome. Science 315, 1143\u20131147, https:\/\/doi.org\/10.1126\/science.1138389 (2007).","journal-title":"Science"},{"key":"44372_CR11","unstructured":"Laccone, F. A. Synthetic MeCP2 sequence for protein substitution therapy (2007)."},{"key":"44372_CR12","doi-asserted-by":"publisher","first-page":"46490","DOI":"10.1074\/jbc.M408284200","volume":"279","author":"RJ Klose","year":"2004","unstructured":"Klose, R. J. & Bird, A. P. MeCP2 behaves as an elongated monomer that does not stably associate with the Sin3a chromatin remodeling complex. The Journal of biological chemistry 279, 46490\u201346496, https:\/\/doi.org\/10.1074\/jbc.M408284200 (2004).","journal-title":"The Journal of biological chemistry"},{"key":"44372_CR13","doi-asserted-by":"publisher","first-page":"1051","DOI":"10.2217\/epi.15.39","volume":"7","author":"Q Du","year":"2015","unstructured":"Du, Q., Luu, P. L., Stirzaker, C. & Clark, S. J. Methyl-CpG-binding domain proteins: readers of the epigenome. Epigenomics 7, 1051\u20131073, https:\/\/doi.org\/10.2217\/epi.15.39 (2015).","journal-title":"Epigenomics"},{"key":"44372_CR14","doi-asserted-by":"publisher","first-page":"316","DOI":"10.3389\/fnmol.2017.00316","volume":"10","author":"XR Jin","year":"2017","unstructured":"Jin, X. R., Chen, X. S. & Xiao, L. MeCP2 Deficiency in Neuroglia: New Progress in the Pathogenesis of Rett Syndrome. Frontiers in molecular neuroscience 10, 316, https:\/\/doi.org\/10.3389\/fnmol.2017.00316 (2017).","journal-title":"Frontiers in molecular neuroscience"},{"key":"44372_CR15","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1042\/BJ20110648","volume":"439","author":"KK Gadalla","year":"2011","unstructured":"Gadalla, K. K., Bailey, M. E. & Cobb, S. R. MeCP2 and Rett syndrome: reversibility and potential avenues for therapy. The Biochemical journal 439, 1\u201314, https:\/\/doi.org\/10.1042\/BJ20110648 (2011).","journal-title":"The Biochemical journal"},{"key":"44372_CR16","doi-asserted-by":"publisher","first-page":"1043","DOI":"10.1080\/21678707.2016.1229181","volume":"4","author":"WE Kaufmann","year":"2016","unstructured":"Kaufmann, W. E., Stallworth, J. L., Everman, D. B. & Skinner, S. A. Neurobiologically-based treatments in Rett syndrome: opportunities and challenges. Expert opinion on orphan drugs 4, 1043\u20131055, https:\/\/doi.org\/10.1080\/21678707.2016.1229181 (2016).","journal-title":"Expert opinion on orphan drugs"},{"key":"44372_CR17","doi-asserted-by":"publisher","first-page":"640","DOI":"10.1038\/90242","volume":"19","author":"H Xia","year":"2001","unstructured":"Xia, H., Mao, Q. & Davidson, B. L. The HIV Tat protein transduction domain improves the biodistribution of beta-glucuronidase expressed from recombinant viral vectors. Nature biotechnology 19, 640\u2013644, https:\/\/doi.org\/10.1038\/90242 (2001).","journal-title":"Nature biotechnology"},{"key":"44372_CR18","doi-asserted-by":"publisher","first-page":"1569","DOI":"10.1126\/science.285.5433.1569","volume":"285","author":"SR Schwarze","year":"1999","unstructured":"Schwarze, S. R., Ho, A., Vocero-Akbani, A. & Dowdy, S. F. In vivo protein transduction: delivery of a biologically active protein into the mouse. 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Journal of neuroimmunology 179, 37\u201345, https:\/\/doi.org\/10.1016\/j.jneuroim.2006.06.019 (2006).","journal-title":"Journal of neuroimmunology"},{"key":"44372_CR29","doi-asserted-by":"publisher","first-page":"352","DOI":"10.3389\/fncel.2014.00352","volume":"8","author":"W Neuhaus","year":"2014","unstructured":"Neuhaus, W. et al. The pivotal role of astrocytes in an in vitro stroke model of the blood-brain barrier. Frontiers in cellular neuroscience 8, 352, https:\/\/doi.org\/10.3389\/fncel.2014.00352 (2014).","journal-title":"Frontiers in cellular neuroscience"}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-019-44372-3.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-019-44372-3","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-019-44372-3.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,17]],"date-time":"2022-12-17T09:58:11Z","timestamp":1671271091000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-019-44372-3"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,5,28]]},"references-count":29,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2019,12]]}},"alternative-id":["44372"],"URL":"https:\/\/doi.org\/10.1038\/s41598-019-44372-3","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,5,28]]},"assertion":[{"value":"9 May 2018","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"15 May 2019","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"28 May 2019","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare no competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing Interests"}}],"article-number":"7929"}}