Ben Oostra, PhD
Principal Investigator
Erasmus University
Rotterdam, The Netherlands

FRAXA Awards:
  $60,000 in 2009 
  $60,000 in 2008 
  $64,000 in 2005
  $55,000 in 2004
  $35,000 in 2000 

Understanding the mechanism of mGluR5 directed therapy and the involvement of the miRNA pathway in Fragile X syndrome

Femke de Vrij, PhD
Principal Investigator

By Femke deVrij 5/2008

FMRP is thought to be involved in the transport of dendritic mRNAs and the regulation of local mRNA translation at synaptic sites. The presumed loss of translational regulation at synaptic sites might underlie the cognitive impairment in FXS. FXS patients do not show gross brain abnormalities. However, when studied in detail, both FXS patients and Fmr1 KO mice show structural malformations of dendritic protrusions, which partly form the synaptic connections between neurons. These malformations generally correspond to an immature protrusion phenotype.

Our research is focused on therapeutic targets for Fragile X syndrome. mGluR5 based therapeutic approaches have already proven to be successful in several mouse and fruit fly studies. Our own results recently began to unravel the mechanism behind these therapeutic strategies. We have developed both an in vitro model to study the structural rescue of dendritic protrusions in primary hippocampal neurons and an in vivo model to study the rescue of a prepulse inhibition behavioral defect in Fmr1 KO mice. By combining therapy based studies in Fmr1 KO mice using these two models, we want to test several new mGluR5 antagonists and study the underlying molecular mechanism of mGluR5 targeted treatment.

Moreover, we want to pursue preliminary results that implicate the miRNA pathway as mediator of the Fragile X related protrusion morphology phenotype. miRNAs are small single-stranded regulatory RNAs that can repress the translation of target mRNAs. Our preliminary results show large differences in miRNA expression in Fmr1 KO brain compared to wild type mouse brain. We plan to further characterize these miRNAs by studying their function in relation to FXS at a cellular level in primary hippocampal neurons and biochemically in synaptosomal fractions of wild type and Fmr1 KO mouse brain material. We hypothesize that the differentially regulated miRNAs that we identified in Fmr1 KO neurons will play a role in regulating local translation at synaptic sites. Therefore, we will also study the effect of mGluR5 antagonists on the levels of these specific miRNAs. In addition, artificial regulation of miRNAs by up- or downregulating their expression in primary hippocampal neurons will allow us to study the effect of miRNA expression on protrusion morphology in Fmr1 KO neurons.

In the August 4, 2005, issue of Neuron, Ben Oostra and Chris De Zeeuw and colleagues at Erasmus University Rotterdam reported that they have pinpointed a specific cause of defects in motor learning in Fragile X patients. Their work is the first investigation of the role of abnormalities in the brain's cerebellum in Fragile X. Fragile X syndrome occurs when a defect in the Fragile X mental retardation 1 (FMR1) gene renders it unable to produce its normal protein, FMRP. A decade ago, Dr. Oostra and colleagues generated mice which lack FMRP. These so-called "knockout mice" are now used worldwide by the fragile X research community to study symptoms of - and test potential treatments for - the disorder.

This study evaluated the behavioral effects of the Fragile X mutation on motor learning in both the knockout mice and human patients.

They found that mice lacking the FMR1 gene showed deficits in a motor learning task known to be largely controlled by the cerebellum. In this "eyeblink conditioning" task, the mice were taught to associate a tone with a puff of air on their eye, and the blink response was measured as an indication of how well the animals could learn the task. The researchers found that mice completely lacking the FMR1 gene showed deficits in this motor learning task. But most importantly, the researchers also found that mice lacking the FMR1 gene only in specific neurons, called Purkinje cells, in the cerebellum also showed the deficit.

Detailed electrophysiological studies of Purkinje cells in such mutant mice revealed that the cells showed an enhanced weakening of their signaling connections -- called long-term depression (LTD). When the researchers conducted similar eyeblink tests in Fragile X patients, they found the same severe deficits.

Recently, a hypothesis (called the mGluR theory) was proposed by Mark Bear and colleagues which suggests possible treatments for Fragile X. The mGluR theory proposes that lack of FMRP leads to excessive long-term depression and that compounds that dampen mGluR activation may reverse symptoms of Fragile X. To test this theory, proper quantitative tests are needed for measuring cognitive deficits in both the animal model and fragile X patients.

Drs. Oostra and De Zeeuw will now begin testing whether this phenotype and related cognitive and motor learning deficits in the mouse model can be treated with compounds that dampen mGluRs. They aim to identify a robust phenotype in Fragile X patients and develop a quantitative evaluation method that can be used in clinical trials of new treatments for Fragile X syndrome.

This work was in part sponsored by a grant from FRAXA.

1) We will produce large amounts of FMRP in insect cells. We will modify this FMRP so that it contains a short sequence taken from the HIV-1 TAT protein, which has been shown to help facilitate the uptake of proteins in cells.

2) We will test whether this modified protein can be taken up by cells cultured in vitro. These can be cell lines from fragile X patients which are completely devoid of the FMR1 protein. We will monitor the uptake, hoping that the cells will take in large amounts of protein.

3) Finally, these studies will be followed in vivo by injecting the modified protein into FMR1 knockout mice. The uptake of protein will be tested biochemically and by testing the behavior of the mice, to see if their fragile X symptoms are ameliorated by the introduced protein.