Basic Mechanisms of Disease and Potential Therapeutic Strategies

With $245,000 in grants from FRAXA Research Foundation, Dr. Stephen Warren and his lab at Emory University study all aspects of fragile X syndrome, from the mechanisms of repeat expansion to high-throughput drug screens in the Drosophila model of fragile X. Dr. Warren made the original discovery of the fragile X gene, FMR1, in collaboration with the Nelson and Oostra labs. Recent projects include establishment of induced pluripotent stem cell lines from fragile X patients, and determination of other forms of mutation in the fragile X gene, other than the most common trinucleotide repeat expansion.

$245,000 Grant
Stephen Warren, PhD
Principal Investigator
Emory University School of Medicine
2000-2009 FRAXA Research Grants
$245,000 over 10 Years

A New Model for Epigenetic Events in Fragile X Syndrome: Induced Pluripotent Stem Cells

by Joshua Suhl and Stephen Warren, 3/31/2010

We were developing induced pluripotent stem (iPS) cells to serve as a stem cell model and screening tool for potential treatments of Fragile X. This new technology “reprograms” adult cells (for example, skin cells) to stem cells, which are similar to human embryonic stem cells (hESCs), can be cultured indefinitely, and provide a virtually limitless source of fragile X cells for research purposes. hESCs that contain the CGG repeat expansion have been shown to express FMR1. At some point after stem cell differentiation into other cell types, the gene is silenced. iPS cells will provide a pre-differentiation cellular model, permitting research into the timing and mechanism of the FMR1 silencing event.

Additionally, our team recently identified several novel mutations in the FMR1 gene from a group of developmentally delayed males that may affect the function of FMRP. Several of these variants were analyzed to determine if any can be identified as a new cause of Fragile X, highlighting the possibility of alternative etiologies other than the typical repeat expansion and opening new avenues of fragile X research.

Characterizing Phosphorylation as a Regulator of FMRP Translational Suppression in Response to Group 1 mGluR Activity

by Usha Narayan and Stephen Warren, 8/1/2006

Previous work has shown that FMRP is an RNA binding protein that specifically recognizes target mRNA transcripts and influences their translation status. Although FMRP is known to be synthesized/ transported in response to group 1 mGluR activity, little was known about its role in translation in a similar context.

Post-translational modifications (such as phosphorylation) are good candidates for the regulation of activity-dependent protein synthesis. We had recently identified protein phosphatase 2A (PP2A) as a major FMRP phosphatase in both primary neurons and non-neuronal cells. Alterations in PP2A activity were found to mediate rapid and dynamic changes in FMRP phosphorylation upon group 1 mGluR stimulation in primary hippocampal neurons. This mGluR activity-dependent FMRP phosphorylation also correlates with the translational changes of dendrite-localized target transcripts.

Together, these data link group 1 mGluR activation, PP2A enzymatic activity and FMRP phosphorylation with downstream translation. Efforts were directed at investigating the therapeutic potential of this novel signaling cascade.

Identification of Novel Compounds for the Treatment of Fragile X Syndrome

by Shuang Chang and Stephen T. Warren, 3/1/2006

We discovered that Drosophila embryos deficient in dfmr1, the fly model of fragile X syndrome, die when placed on a commercial food source but survive on a standard laboratory food preparation. The commercial food source was found to contain excess glutamate when compared to the lab food and adding glutamate to the lab food resulted in lethality. The mGluR antagonist MPEP rescued the lethality on commercial food, consistent with the mGluR theory of fragile X syndrome.

Using this lethal phenotype on commercial food as a drug screen, we evaluated a panel of 2,000 drugs and natural products, identifying compounds that could rescue the phenotype. This screen identified 20 drugs that could rescue the phenotype and further replication showed four lead drugs to be superior to MPEP in reversing the lethality. Those flies that survived with drug treatment also showed significantly correction the morphological brain abnormality (mushroom body lobe defects) seen in dfmr1-deficient flies growing on lab food. Three of these four drugs appear to modulate the GABAergic inhibitory pathway. Since this inhibitory pathway could temper the excess signaling from the mGluR excitatory pathway in fragile X, we have identified the GABAergic pathway as a druggable target for fragile X syndrome. We were planning expanding the drug-screening panel and testing these four lead drugs in mice deficient in Fmr1.

Dissection of the Fragile X Protein Binding Domains

Report 7/1/2004

This group used novel strategies to examine the binding activity of FMR1 protein, to see which target mRNAs it associates with and presumably regulates. They also investigated the RNA targets of two similar proteins, FXR1 and FXR2, which are thought to work with FMRP in most, if not all, of its functions. They planned to look at the different binding patterns of different isoforms of FMRP; one important fact which is seldom discussed is that FMRP can exist in cells in at least 12 distinct forms, depending on how it is spliced by various cells. It is entirely possible that each of these forms has somewhat different characteristics, which need to be better understood. Furthermore, the investigators hoped that by comparing FMRP from different species, such as chicken and frog, they could learn more about which parts of the Fragile X protein perform specific functions (such as binding RNA, engaging transport mechanisms, etc.)

This is an important area of research because greater understanding of FMRP’s targets will enable us to identify other genes and other proteins which may be causing the pathology in Fragile X. These may, in turn, be potential targets for drug development.

Joshua Suhl, PhD
FRAXA Postdoctoral Fellow (2009)

Shuang Chang, PhD
FRAXA Postdoctoral Fellow (2006)

Usha Narayan, PhD
FRAXA Postdoctoral Fellow (2005)

Reid Alisch, PhD
FRAXA Postdoctoral Fellow (2004)