With $184,000 in funding from FRAXA Research Foundation from 1996-2005, Dr. Ben Oostra and his team at Erasmus University have done multiple studies related to Fragile X syndrome. This lab created the first Fragile X mouse models and went on to perform many critical studies in Fragile X mouse models. Results published.
Role of the Cerebellum in the Dysfunction of Fragile X
Bas Koekkoek, MD, PhD Postdoctoral Fellow, 8/9/2005
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 Protein 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
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 if the FMR1 gene is not working properly. The studies on the transgenic mouse which is a good model to study Fragile X , might also contribute on our understanding of behavior, learning and memory.