|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
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
Bas Koekkoek, MD, PhD
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.
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
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.
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.