Involvement of the miRNA Pathway in Fragile X Syndrome

With $304,000 in funding from FRAXA Research Foundation, Dr. Oostra and colleagues at Erasmus University studied miRNA and fragile X. miRNAs are RNAs that can repress the translation of target mRNAs – therefore they can play a role in protein synthesis within the neuron. Preliminary results showed large differences in miRNA expression in the fragile X mouse brain compared to the wild type.

$304,000 Grant
Ben Oostra, PhD
Principal Investigator
Femke de Vrij, PhD
Co-Principal Investigator
Erasmus University
FRAXA Research Grant
$120,000 (2008-2009)
$119,000 (2004-2005)
$35,000 (2000)
$30,000 (1996)

Final Report: Understanding the Mechanism of mGluR5 Directed Therapy and the Involvement of the miRNA Pathway in Fragile X Syndrome

by Femke deVrij, 11/15/2010

We have established a rescue of PPI (prepulse inhibition) deficits and abnormal spine morphology in Fmr1 KO mice with mGluR5 antagonists fenobam and AFQ056 in collaboration with Novartis. Our results have been published Levenga et al 2010.

To study the spine morphology in adult mice, we set up a Dioloistic labeling protocol to stain neurons in slices with use of a Genegun. By shooting DiI coated bullets into hippocampal brain slices of perfused animals, we are now able to quantify neurons in their original surroundings (ex vivo) in a region-specific manner. Interestingly, a fragile X related spine phenotype in the hippocampus seemed restricted to the CA1 area, while no differences compared with wildtype neurons were found in the CA3 hippocampal area.

In the miRNA project, much time and effort was spent on the validation of miRNA data from previous experiments. Theoretically, overexpression of miRNAs that are downregulated in Fmr1 KO neurons, might have a rescue effect on protrusion morphology. Quantification of protrusion morphology after overexpression of these miRNAs is still ongoing.

Understanding the Mechanism of mGluR5 Directed Therapy and the Involvement of the miRNA Pathway in Fragile X Syndrome

by Femke deVrij , 5/1/2008

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.

Role of the Cerebellum in the Dysfunction of Fragile X

Bas Koekkoek, MD, PhD Postdoctoral Fellow


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.

A Novel Therapeutic Approach: FMRP Conjugated with the PTD Segment of the HIV-1 TAT Protein

by Ben Oostra, January 2000

It is generally agreed that mental impairment in fragile X patients is caused by lack of the fragile X protein (FMRP) in the neurons of the central nervous system. Our project is aimed at restoring FMRP to neurons, by using an new tool, the HIV-1 TAT protein. Our work will have three phases:

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.

Update… October 2004:
It has been reported that a protein transduction domain (TAT) is able to deliver macromolecules into cells and even into the brain when fused to the protein in question. Upon production of a TAT-FMRP fusion protein in a baculovirus-expression system, we used immunohistochemistry to verify TAT-mediated uptake of FMRP in fibroblasts. However, uptake efficiency and velocity was lower than expected. Neuronal uptake was highly inefficient and the fusion protein demonstrated toxicity.
Reis SA, Willemsen R, van Unen L, Hoogeveen AT, Oostra BA. Prospects of TAT-mediated protein therapy for fragile X syndrome. J Mol Histol. 2004 May;35(4):389-95.

Mouse Models for Fragile X Syndrome

Report 4/1/1996

The primary objective for this grant is to understand the function of the FMR1 gene in the brain and the deleterious effect on brain functioning (mental retardation) if the FMR1 gene is not working properly. The studies on the transgenic mouse which is a good model to study fragile X mental retardation, might also contribute on our understanding of behavior, learning and memory.