With $120,000 in funding from FRAXA Research Foundation over 2008-2009, Dr. Ben Oostra and Dr. Femke deVrij at Erasmus University explored the miRNA pathway in Fragile X syndrome.
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.
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.