A new animal model of fragile X syndrome suggests that a short time window very early in life is critical for effectively treating the condition.
The study, “A DNAzyme based knockdown model for Fragile-X syndrome in zebrafish reveals a critical window for therapeutic intervention,” was published in the Journal of Pharmacological and Toxicological Methods.
Fragile X is the most frequent single genetic cause of autism. It is caused by mutations in the FMR1 gene, which provides instructions for making an RNA binding protein called fragile X mental retardation protein (FMRP).
FMRP can be found in many tissues, including the brain, testes, and ovaries. In the brain, it controls the production of several proteins at the synapse — the site where nerve cells communicate. The protein is thought to act primarily via a subtype of receptors of the neurotransmitter glutamate (mGluR5). This receptor is found at higher levels in fragile X, leading to changes at the synapse and characteristic fragile X manifestations.
For this reason, many studies have focused on mGluR5 blockers as a potential treatment for fragile X. However, none of these compounds led to significant improvements in the clinical setting.
“This puts forward the need to look for novel targets, alternate treatments and treatment windows for improved clinical efficacy,” researchers at Dr. Reddy’s Institute of Life Sciences, in India, said.
The team set out to create a new animal model of fragile X that would enable the study of early events triggered by the loss of FMRP — and also investigate early critical windows for treatment.
The zebrafish, a small species of freshwater fish, was selected as the animal model. A major advantage of using zebrafish, the scientists said, is the access to embryos to explore early abnormalities and screen for new therapies.
“With this goal, we have used a novel, high-throughput, inexpensive approach to create a transient knockdown [temporary inactivation] of FMRP,” the researchers said.
The team inserted a DNA-based enzyme, or DNAzyme, that specifically recognized and destroyed the messenger RNA (mRNA) of fmr1, which is the zebrafish equivalent of the human FMR1 gene. RNA is generated from DNA and then used to produce a protein. Inserting the DNAzyme prevented the fmr1 from being translated into a functional FMRP protein in zebrafish embryos.
Then, during the embryos’ first seven days of life, the scientists performed behavioral tests to measure anxiety, cognitive impairments, and irritability. Levels of FMRP and other biomarkers of disease also were measured.
At different time points, the investigators also treated embryos with mavoglurant (AFQ056), a mGluR5 blocker currently being developed by Novartis. The goal was to identify an optimal time window for therapeutic intervention.
The results revealed that, within the first 24 hours after inserting the DNAzyme in embryos, fmr1 mRNA levels dropped by 60-80%. Meanwhile, FMRP protein levels dropped to 20%. Both mRNA and protein levels tended to normalize after two days.
Following FMRP knockdown, the zebrafish larvae showed signs of anxiety, irritability, and cognitive impairment during their first seven days of life, which validated the approach as a new animal model of fragile X.
Treatment with mavoglurant restored normal behavior and was more effective when given during the first three days of life than within days 5 to 7.
Likewise, the anxyolitic KU046, with known efficacy in mice with fragile X, led to a significant reduction in anxiety and irritability when administered during the same period.
“Our results in this study appear to indicate that the early window is critical for treatment, and missing this window may have irreversible consequences on the FMRP/mGluR5 axis … precluding a favourable response to treatment at a later stage,” the investigators said.
If the findings are validated in other models of fragile X, “drug discovery may need to be redirected towards developing safe and appropriate therapies which can be administered during pregnancy … as opposed to later in the life of the fetus/child,” they added.
“This may cause a paradigm shift in the way therapeutic strategies are planned for [fragile X], and possibly other neuro-developmental disorders,” the researchers said.
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