Proteomic analysis of the Rett syndrome experimental model mecp2^Q63X mutant zebrafish

Highlights

• Effects of mecp2 mutation on whole protein expression in zebrafish

• LC–ESI–MS/MS identified 20 differentially expressed proteins vs. wild-type.

• Identified proteins imply energy depletion, redox imbalance and muscle deficit.

• The majority of protein changes occur already at larval stage.

• Protein expression changes indicate the broad impact of mecp2 mutations.

Abstract

Rett syndrome (RTT) is a severe genetic disorder resulting from mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene. Recently, a zebrafish carrying a mecp2-null mutation has been developed with the resulting phenotypes exhibiting defective sensory and thigmotactic responses, and abnormal motor behavior reminiscent of the human disease. Here, we performed a proteomic analysis to examine protein expression changes in mecp2-null vs. wild-type larvae and adult zebrafish. We found a total of 20 proteins differentially expressed between wild-type and mutant zebrafish, suggesting skeletal and cardiac muscle functional defects, a stunted glycolysis and depleted energy availability. This molecular evidence is directly linked to the mecp2-null zebrafish observed phenotype. In addition, we identified changes in expression of proteins critical for a proper redox balance, suggesting an enhanced oxidative stress, a phenomenon also documented in human patients and RTT murine models. The molecular alterations observed in the mecp2-null zebrafish expand our knowledge on the molecular cascade of events that lead to the RTT phenotype.

Biological significance

We performed a proteomic study of a non-mammalian vertebrate model (zebrafish, Danio rerio) for Rett syndrome (RTT) at larval and adult stages of development. Our results reveal major protein expression changes pointing out to defects in energy metabolism, redox status imbalance, and muscle function, both skeletal and cardiac. Our molecular analysis grants the mecp2-null zebrafish as a valuable RTT model, triggering new research approaches for a better understanding of the RTT pathogenesis and phenotype expression. This non-mammalian vertebrate model of RTT strongly suggests a broad impact of Mecp2 dysfunction.

Introduction

Rett syndrome (RTT, MIM 312750), a progressive neurodevelopmental disorder with a frequency of approximately 1:10,000 live births, is a leading cause of severe intellectual disability in the female [1]. The typical disease shows a period of 6 to 18 months of an apparent normal neurodevelopment followed by regression and progressive loss of acquired cognitive, social and motor skills [2]. De novo mutations, altering the function of the X-linked methyl-CpG binding protein 2 (MECP2) gene, are the main cause of RTT [3]. Although the MECP2 functions are yet to be fully clarified, the encoded protein can act as either a transcriptional repressor or activator by binding to methylated DNA [4], and is able to modulate alternative RNA splicing [5] and miRNA processing [6].

Several experimental mammalian models of RTT recapitulating several features of the disease have been developed [7]. Non-mammalian models of RTT have been recently described, including Drosophila and Danio rerio [8], [9]. In particular, zebrafish has recently gained much attention as a vertebrate model for human neurodevelopmental and neurodegenerative diseases [10], showing a number of unique advantages that include its strong genetics (i.e., amenable to molecular manipulations of its genome) [11], [12], a large repertoire of well-studied behaviors [13], imaging capabilities in larvae and access to early nervous system development [14]. The mecp2-null zebrafish model mirrors the defective motor behavior and sensory response observed in RTT patients [9], [15].

Most studies have focused on defects linked to the development and maintenance of brain networks [16]. However, the fact that MECP2 is broadly expressed, from the early stages of development with expression levels increasing progressively and becoming particularly enriched in neural tissues [17], and the large set of symptoms described in human patients [18], argues for pleiotropic roles of MECP2.

Hence, by using a proteomic approach in whole tissue samples from larvae and adults, we demonstrate that mecp2-null zebrafish exhibits changes in the expression of proteins mainly linked to the balance of the redox status, energy metabolism and muscle function.