Stephen Haggarty, PhD—Harvard/MIT
Small Molecule Modulators of Lithium for treatment of Fragile X Syndrome

Stephen Haggarty, PhD, Principal Investigator
Surya Reis, PhD, FRAXA Postdoctoral Fellow

FRAXA Awards:

$69,500 in 2010
$75,000 in 2009
$75,000 in 2008


Dr. Haggarty has developed a high-throughput drug screen to find compounds that inhibit GSK3, a critical enzyme in fragile X. Looking for compounds that can accomplish this either alone or in combination with lithium, offering the possibility of enhancing the effectiveness of lithium as a treatment. His drug screen uses patient-specific neural progenitor (NP) cells derived from human induced pluripotent stem cells (iPSCs) - which are created from cells in a skin biopsy from people with Fragile X syndrome (FXS) and other autism spectrum disorders.
Publication of Results: Epigenetic characterization of the FMR1 gene and aberrant neurodevelopment in human induced pluripotent stem cell models of fragile x syndrome.

Sheridan SD, Theriault KM, Reis SA, Zhou F, Madison JM, Daheron L, Loring JF, Haggarty SJ., 11/3/2011

PLoS One. 2011;6(10):e26203. Epub 2011 Oct 12.

Abstract

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. In addition to cognitive deficits, FXS patients exhibit hyperactivity, attention deficits, social difficulties, anxiety, and other autistic-like behaviors. FXS is caused by an expanded CGG trinucleotide repeat in the 5' untranslated region of the Fragile X Mental Retardation (FMR1) gene leading to epigenetic silencing and loss of expression of the Fragile X Mental Retardation protein (FMRP). Despite the known relationship between FMR1 CGG repeat expansion and FMR1 silencing, the epigenetic modifications observed at the FMR1 locus, and the consequences of the loss of FMRP on human neurodevelopment and neuronal function remain poorly understood.

To address these limitations, we report on the generation of induced pluripotent stem cell (iPSC) lines from multiple patients with FXS and the characterization of their differentiation into post-mitotic neurons and glia. We show that clones from reprogrammed FXS patient fibroblast lines exhibit variation with respect to the predominant CGG-repeat length in the FMR1 gene. In two cases, iPSC clones contained predominant CGG-repeat lengths shorter than measured in corresponding input population of fibroblasts. In another instance, reprogramming a mosaic patient having both normal and pre-mutation length CGG repeats resulted in genetically matched iPSC clonal lines differing in FMR1 promoter CpG methylation and FMRP expression. Using this panel of patient-specific, FXS iPSC models, we demonstrate aberrant neuronal differentiation from FXS iPSCs that is directly correlated with epigenetic modification of the FMR1 gene and a loss of FMRP expression. Overall, these findings provide evidence for a key role for FMRP early in human neurodevelopment prior to synaptogenesis and have implications for modeling of FXS using iPSC technology. By revealing disease-associated cellular phenotypes in human neurons, these iPSC models will aid in the discovery of novel therapeutics for FXS and other autism-spectrum disorders sharing common pathophysiology.

Full Text of article (free) at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3192166
Characterization of small molecule modulators of lithium using a human neural progenitor cell model of Fragile X syndrome

Figure 1. Overview of generation of neural progenitors (middle; red, nestin; blue, DNA) form human induced pluripotent stem cell clones (left) that can be differentiated (8 weeks) into neuronal cells (right; MAP2, red; SNAP25, green; DNA, blue).

by Stephen J. Haggarty, 4/1/2010

Chemical genomics is a translational research strategy that aims to develop small-molecule therapeutics for human genetic disorders based upon a molecular understanding of disease etiology and pathophysiology. One limitation for the development of new mechanism of action therapeutics has been access to cell models that capture the complexity of human genetic variation associated with disease. However, recent advances in the field of stem cell biology now enable the generation of genetically accurate cellular models of human diseases and the differentiation of these stem cells into defined cell types of the human body, including neurons and glia.

To take advantage of these advances for the study of FXS, we have established, and are actively validating, a process for the generation of self-renewing, patient-specific neural progenitor (NP) cells derived from human induced pluripotent stem cells (iPSCs) created from fibroblasts from patients with Fragile X syndrome (FXS) and other autism spectrum disorders. The ability to derive genetically accurate iPSC-NP cells lines from readily accessible patient cells available through existing cell repositories and collected from fresh patient skin biopsies will facilitate the elucidation of potential inter-patient variability and the influence of factors such as CGG repeat length and other genetic modifiers of FXS.

The overall goal of our project is to develop, characterize, and use a panel of iPSC-NP cells as a platform for chemical genomic characterization of small molecules targeting pathways implicated in FXS pathology. Based upon recent studies from the labs of Jope, Bauchwitz, as well as others, our particular interest is in the role that dysregulation of the kinase glycogen synthase kinase 3 (GSK-3) may play in FXS and other disorders with neuropsychiatric symptoms. On the basis of these findings, we propose to pilot the use of iPSC-NP cells to study FXS by testing the effects of small molecules targeting the GSK-3/b-catenin pathway. We have implemented a miniaturized culture system using iPSC-NP cells and are developing image-based assays measuring neural cell morphology, differentiation, and GSK-3/b-catenin signaling as well as transcriptional reporters to measure GSK-3 signaling. We are now using these assays to look for disease-associated phenotypic differences.

Upon completion of these studies we expect to have identified small molecules that can be tested in other models of FXS for their ability to ameliorate molecular and behavioral symptoms of FXS. These studies may help identify novel targets for the development of therapeutics for the treatment and cure of FXS.