FRAXA - Fragile X Research Foundation

Role of the Cerebellum in the Dysfunction of Fragile X

Ben Oostra, PhD, Principal Investigator
Bas Koekkoek, MD, PhD, Postdoctoral Fellow
Erasmus University, Rotterdam, The Netherlands

FRAXA Awards:
$64,000 in 2005
$55,000 in 2004

Update 8/9/2005
Understanding Fragile X with the blink of an eye

While researchers have long known that a single gene defect causes Fragile X syndrome, they are still tracing how that defect creates the complex mix of mental impairment, autistic behaviors, attention deficits, and other problems in the disorder. Fragile X is particularly important because it is the most common single-gene cause of mental retardation and the most common known cause of autism.

In the August 4, 2005, issue of Neuron, Ben Oostra and Chris De Zeeuw and colleagues at Erasmus University Rotterdam report 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.

Their current study evaluates 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 novel hypothesis (called the mGluR theory) has been 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, the Fragile X Research Foundation.

by Michael Tranfaglia, MD, FRAXA Medical Director, 7/2004

The mGluR Theory of Huber, Bear, and Warren predicts that excessive function of signaling pathways associated with mGluRs causes most of the symptoms of Fragile X. It appears that one of the mGluRs -- mGluR5 -- is responsible for most of the problem in the brain overall. However, mGluR5 is not present at all in the cerebellum, a part of the brain associated with coordination of movement and sensory integration, and the area which is known to express very large quantities of FMRP in the normal brain. Clearly, the cerebellum is important in the pathogenesis of Fragile X (and autism, too!), but here it is another mGluR receptor -- mGluR1 -- which regulates the activity of these pathways. Dr. Oostra’s lab has demonstrated altered synaptic plasticity in the cerebellum of FMR1 knockout mice and correlated this with changes in the shape of dendritic spines in the neurons of the cerebellum. This change is also correlated with changes in a specific behavior in mice: eye-blink response. Furthermore, eye-blink response can be tested in humans, and Dr. Oostra’s group will attempt this test with Fragile X patients. They will also attempt to treat these abnormalities in mice with mGluR1 and mGluR5 antagonists. Additionally, they will study another critical brain region, the amygdala, which mediates the startle response and is probably very important for causing many of the psychiatric symptoms seen in Fragile X.

A novel therapeutic approach: FMRP conjugated with the PTD segment of the HIV-1 TAT protein

funded for $35,000 in January 2000

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

Recently 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.
Reference:
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.