Preclinical evaluation of serotonin type 7 (5-HT7) receptor agonists as novel pharmacological tools in Fragile X Syndrome
Michela Spatuzza, PhD
Previous work from this laboratory indicate a potential novel strategy to treat Fragile X: they have recently shown that activating 5-HT7 receptors reversed mGluR-LTD both in normal mice and in Fragile X mice. In the current project, they will see if administration of a selective 5-HT7 receptor agonist to Fragile X mice can reverse abnormal phenotypes, such as excessive mGluR-mediated synaptic plasticity, altered spine morphology and increased audiogenic seizure susceptibility. Positive results would suggest that 5-HT7 receptors are another new treatment target for Fragile X.
Brain neurons communicate by means of synapses. Persistent changes in the efficiency of synaptic transmission, referred to as “synaptic plasticity”, continuously occur in the brain as a result of learning and are considered as the cellular basis for memory. Fragile X patients display alterations in the shape and organization of brain synapses and synapse malfunction was evidenced in animal models of FXS. In Fmr1 KO mice, a model of FXS, synaptic plasticity is pathologically altered in the frontal cortex and hippocampus, the main brain regions responsible for learning and memory. In particular, a form of long-term depression of synaptic efficacy mediated by the neurotransmitter glutamate (mGluR-LTD) is abnormally enhanced due to exaggerated signalling downstream of group I metabotropic glutamate receptors (mGluRs). A lot of experimental data support this view, since many abnormal features in Fmr1 KO mice can be corrected either by pharmacological blockade or by reduced expression of mGlu receptors. The possibility of therapeutic treatment of FXS patients with selective blockers of mGlu receptors is currently being investigated and the first results from clinical trials are encouraging.
We have recently found a new pathway to reduce the signalling through mGlu receptors that ultimately leads to synapse malfunction in the FXS mouse model. We have studied the role of serotonin (5-HT), a neurotransmitter controlling many physiological functions and playing an important modulatory role in learning and memory. Using electrophysiology to record synaptic currents and immunocytochemical and biochemical techniques to measure the amount of glutamate receptors on neuronal membrane surface, we found that serotonin reversed excessive mGluR-LTD in the hippocampus of Fmr1 KO mice and reduced it to a level comparable to wild-type (healthy) mice. In particular, we have identified a receptor named 5-HT7 as responsible for serotonin effect. 5-HT7 receptors were discovered relatively recently; for these receptors, selective blockers are available whereas putative agonists (molecules able to activate the receptor) are still under investigation.
Since synaptic plasticity is altered in the mouse model of Fragile X syndrome and can be rescued by activation of 5-HT7 receptors, we are now facing the following questions: 1) can 5-HT7 receptor activation also correct other excessive consequences of mGluR-activated signalling? 2) Would a systemic treatment of Fmr1 KO mice with a 5-HT7 receptor agonist correct the pathological features of FXS? 3) Which molecules, among new putative 5-HT7 receptor agonists, are able to activate the receptor and are suitable for systemic administration? To address the above issues, we have designed a preclinical study on Fmr1 KO mice to test if administration of a 5-HT7 receptor agonist can reverse FXS abnormal features. We will synthesize and characterize new 5-HT7 receptor agonists to find molecules with improved pharmacokinetic properties (chemical and metabolic stability in vivo and ability to penetrate the brain) and select the most suitable for systemic administration. We will initially test if the selected 5-HT7 agonists are able to reverse the exaggerated mGluR-LTD in the hippocampus of Fmr1 KO mice. Next, we will administer a 5-HT7 agonist to Fmr1 KO mice in vivo and test its effect on altered dendrite morphology and enhanced sensitivity to epilepsy, both typical features of Fmr1 KO mice as well as of FXS patients. Results from these experiments will indicate if selective activation of 5-HT7 receptors can be considered as a novel pharmacological strategy for FXS therapy.
The current project involves three research groups using different and complementary techniques. Prof. Lucia Ciranna (Department of Bio-medical Sciences, Università di Catania, Italy) directs a research group of electrophysiology using patch clamp, a technique that allows the recording of synaptic currents from living brain slices. This group will prepare hippocampal slices from wild-type and Fmr1 KO mice and will test the effects of new 5-HT7 receptor agonists developed by Prof. Leopoldo (see below) on mGluR-LTD, the form of synaptic plasticity that is abnormally enhanced in Fmr1 KO mice. Dr. Maria Vincenza Catania (Institute of Neurological Sciences, CNR, Catania, Italy) leads a research group specialized in the preparation of neuronal cultures, immunocytochemistry using fluorescent antibodies, biochemical techniques, and microscopy. Dr. Catania also collaborates with Dr. Musumeci from IRCSS Oasi Maria Santissima (Troina, EN), a highly specialized centre for research on intellectual disability and for medical care of FXS patients, together with whom she has characterized the epileptic phenotype of FXS mice. The research group of Dr Catania will test the audiogenic seizure susceptibility (AGS) of Fmr1 KO mice following administration of a 5-HT7 receptor agonist and will study dendritic spine morphology in Fmr1 KO mice treated with a 5-HT7 receptor agonist.
Prof. Marcello Leopoldo (Department of Pharmacy, Università di Bari, Italy) directs a group of medicinal chemistry specialized on design and synthesis of 5-HT receptor agonists and antagonists, with particular regard to 5-HT7 receptors. The laboratory of Prof. Leopoldo performs molecular modeling, chemical synthesis, radioligand binding assays and evaluation of structure-activity relationship of new compounds. In the present project, Prof. Marcello Leopoldo will synthesise new selective and high-affinity ligands for 5-HT7 receptors designed in his laboratory. The group will also determine key pharmacokinetic parameters (metabolic stability and brain permeability) of new compounds with promising features. Selected 5-HT7 agonists will be studied in vitro by Prof Ciranna, who will test their effects on mGluR-LTD, and in vivo by Dr Catania, who will test their effects on seizure susceptibility and on brain dendrite morphology in Fmr1 KO mice.
Michela Spatuzza, PhD