University of Wisconsin–Madison

Qiang Chang, PhD

Qiang Chang, PhD Slide of the Week

Title: CREB signaling is involved in Rett syndrome pathogenesis

Legend: (A) Representative Western blot images of the levels of CREB and pCREB in H9 and MECP2T158M/T158M neurons. (B) Quantification of the levels of CREB and pCREB in H9 and MECP2T158M/T158 neurons. n=4 independent differentiation experiments (*p<0.05). (C) Representative images of Synapsin-CREB-Synapsin-GFP (green) labeled MECP2T158M/T158M neurons counterstained with DAPI (blue) and immunostained with antibodies specific against CREB (top) or pCREB (bottom), Scale bar = 20 µm. (D) Representative images of neuronal morphology in Synapsin-GFP (left) and Synapsin-CREB-Synapsin-GFP (right) expressing MECP2T158M/T158M neurons. Scale bar = 20 µm. (E) Sholl analysis of dendritic complexity in Synapsin-GFP or Synapsin-CREB-Synapsin-GFP expressing MECP2T158M/T158M neurons. (F) Quantification of total neurite length in Synapsin-GFP or Synapsin-CREB-Synapsin-GFP expressing MECP2T158M/T158M neurons. Data from 3 independent differentiation experiments were included in analyses in (E-F) ****p < 0.0001, ***p < 0.001, **p < 0.01.

Citation: Bu, Q., Wang, A., Hamzah, H., Waldman, A., Jiang, K., Dong, Q., Li, R., Kim, J., Turner, D. and Chang, Q., 2017. CREB signaling is involved in Rett syndrome pathogenesis. Journal of Neuroscience, 37(13), pp.3671-3685.

Abstract: Rett syndrome (RTT) is a debilitating neurodevelopmental disorder caused by mutations in the MECP2 gene.  To facilitate the study of cellular mechanisms in human cells, we established several human stem cell lines: human embryonic stem cell (hESC) line carrying the common T158M mutation (MECP2T158M/T158M), hESC line expressing no MECP2 (MECP2-KO), congenic pair of wild type and mutant RTT patient-specific induced pluripotent stem cell (iPSC) line carrying the V247fs mutation (V247fs-WT and V247fs-MT), and iPSC line in which the V247fs mutation was corrected by CRISPR/Cas9-based genome editing (V247fs-MT-correction). Detailed analyses of forebrain neurons differentiated from these human stem cell lines revealed genotype-dependent quantitative phenotypes in neurite growth, dendritic complexity, and mitochondrial function.  At the molecular level, we found a significant reduction in the level of CREB and phosphorylated CREB in forebrain neurons differentiated from MECP2T158M/T158M, MECP2-KO, and V247fs-MT stem cell lines.  Importantly, overexpression of CREB or pharmacological activation of CREB signaling in those forebrain neurons rescued the phenotypes in neurite growth, dendritic complexity, and mitochondrial function. Finally, pharmacological activation of CREB in the female Mecp2 heterozygous mice rescued several behavioral defects. Together, our study establishes a robust in vitro platform for consistent quantitative evaluation of genotype-dependent RTT phenotypes, reveals a previously unappreciated role of CREB signaling in RTT pathogenesis, and identifies a potential therapeutic target for RTT.

About the investigator: Chang’s long-term goal is to understand the molecular mechanism underlying DNA methylation-dependent epigenetic regulation of brain functions. His current focus is on the central role of MeCP2 (methyl-CpG binding protein 2), a molecular linker between DNA methylation and chromatin remodeling and transcriptional control, in the development and function of the nervous system. The functional significance of such a molecular linker is highlighted by the fact that mutations in the MECP2 gene cause Rett syndrome (RTT), a debilitating neurodevelopmental disorder that shares many features with autism. His studies include basic research aimed at understanding the function of MeCP2 and translational research aimed at understanding disease pathology and developing effective treatment. These two types of research are tightly interwoven—the need to solve a practical problem in translational research will always influence the direction of basic research; and fundamental mechanisms revealed by basic research will ultimately guide the effort in treating/curing the disease.

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