An electrochemiluminescence based assay for quantitative detection of endogenous and exogenously applied MeCP2 protein variants

• Published in: Scientific reports Vol. 9; no. 1; p. 7929
• Main Authors: Steinkellner, Hannes, Schönegger, Anna, Etzler, Julia, Kempaiah, Prakasha, Huber, Anna, Hahn, Kathrin, Rose, Katrin, Duerr, Mark, Christodoulou, John, Beribisky, Alexander V., Neuhaus, Winfried, Laccone, Franco
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 28-05-2019; Nature Publishing Group
• Subjects: 13; 631/1647/2163; 631/45/612/1229; 82; 82/80; 82/83; Animals; Blood-brain barrier; Blood-Brain Barrier - metabolism; Brain Chemistry; Cells, Cultured; Central nervous system; Electrochemical Techniques - methods; Female; Fibroblasts - chemistry; Fibroblasts - metabolism; Fusion protein; HEK293 Cells; Humanities and Social Sciences; Humans; Luminescent Measurements - methods; Male; MeCP2 protein; Methyl-CpG binding protein; Methyl-CpG-Binding Protein 2 - administration & dosage; Methyl-CpG-Binding Protein 2 - analysis; Methyl-CpG-Binding Protein 2 - pharmacokinetics; Mice; Mice, Inbred C57BL; multidisciplinary; Proteins; Recombinant Fusion Proteins - administration & dosage; Recombinant Fusion Proteins - analysis; Recombinant Fusion Proteins - pharmacokinetics; Rett syndrome; Science; Science (multidisciplinary); Transcription
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-44372-3

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
in vitro
model of the blood-brain barrier.