Abstract
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.
Highlights
Human methyl-CpG-binding protein 2 (MeCP2) is a chromosomal protein that acts as a transcriptional repressor and global modulator of gene-expression programs in a DNA-methylation-dependent manner[1]
Given that the disease-causing gene is subject to X chromosome inactivation (XCI), girls affected with Rett syndrome (RTT) syndrome are somatic mosaics for cells expressing wild-type and mutant Methyl-CpG-binding protein 2 (MeCP2)
The TAT-MeCP2 and TAT-MeCP2-eGFP constructs were designed to encode for the TAT-fusion protein of interest along with sequences encoding for the Strep-tag for Strep-Tactin affinity chromatography (Fig. 1A,B)
Summary
Human methyl-CpG-binding protein 2 (MeCP2) is a chromosomal protein that acts as a transcriptional repressor and global modulator of gene-expression programs in a DNA-methylation-dependent manner[1]. Deficiency and an excess of the protein causes neuronal dysfunction: while mice overexpressing MeCP2 display seizures and hypoactivity, boys affected with MECP2 duplication syndrome show severe neurological symptoms that can overlap with those of RTT9, highlighting the need for tight regulation of MeCP2 levels in the central nervous system. Given the tight regulation of MECP2 expression, all of the latter approaches face the challenge of normalizing MeCP2 protein levels within the central nervous system without resulting in a detrimental overdose To address this need for highly sensitive and accurate quantification of MeCP2 protein levels, we set out to develop an electrochemiluminescence-based immunoassay (ECLIA) that allows for precise measurement of endogenous as well as exogenous MeCP2 levels in nuclear extracts from different cell lines and mouse tissue samples in a high-throughput format. We used the ECLIA assay described here to test our hypothesis that a recombinant fusion protein consisting of the human MeCP2 isoform B protein and a minimal N-terminal HIV-TAT transduction domain (TAT-MeCP2)[11] has the potential to cross the blood-brain barrier and to raise the nuclear level of MeCP2 protein in neuronal cells, contributing to the further development of a potential protein replacement therapy for RTT
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