DEFINING THE TARGET PATHWAYS FOR A PHARMACOLOGICAL TREATMENT IN RETT SYNDROME

Rett syndrome (RTT) is a postnatal, progressive neurodevelopmental disorder that affects approximately 1 in 10,000 newborn girls worldwide. In most cases, it is caused by loss-of-function mutations in the MECP2 gene on the X chromosome, which encodes the methyl-CpG-binding protein 2 (MeCP2). RTT patients typically develop normally for the first 6–18 months of life, after which they undergo regression in motor, communication, and cognitive skills. Anatomically, RTT neurons exhibit dendritic atrophy and a marked reduction in dendritic spine density, leading to neuronal network dysfunction. The discovery that re-expression of MECP2 in Mecp2-/y mice can largely reverse RTT-like phenotypes, and that neurons lacking MECP2 do not undergo degeneration, supports the notion that RTT is a potentially reversible disorder. However, a definitive cure remains unavailable. Gene therapy, although promising, faces challenges due to MECP2 mosaicism, which can result in inefficiency and potential toxicity from MECP2 overexpression in cells carrying the normal allele. Pharmacological strategies therefore represent an attractive alternative. Currently, Trofinetide (DAYBLUE) is the only FDA-approved drug for RTT treatment. In our laboratory, we developed an in vitro model of primary hippocampal neurons to identify developmental stages affected in RTT. Mecp2-/y neurons showed dendritic atrophy at days-in-vitro (DIV) 6 and 12, identifying these as potential therapeutic windows. The first aim of my PhD project was to identify FDA-approved multifunctional drugs capable of reversing neuronal atrophy in Mecp2-/y neurons. A preliminary screening of 640 FDA-approved drugs at DIV 6 identified 14 compounds that rescued neuronal morphology. During my PhD, I further tested these 14 drugs (10 μM) at DIV 12, when neurons are mature, form synapses, and exhibit spontaneous activity. Morphological analysis included five parameters: total dendritic length (TDL), polarization index (Lm/Lsym), Sholl crossings (TC), distal dendrites (SO1), and maximal dendritic length (MDL). Among the 14 compounds plus mirtazapine (positive control, 1 μM), five emerged as the most promising: D1, D3, and D5 rescued all morphological parameters, similar to mirtazapine, while D2 and D4 recovered three of five. Functional assessment of synaptic activity revealed that D2, D4, and D5 restored calcium transient frequency in Mecp2-/y hippocampal neurons, indicating normalization of network activity. The second part of my PhD focused on elucidating the cellular mechanisms and biochemical networks underlying these drug effects. In collaboration with Prof. Carosati (University of Trieste, Department of Pharmaceutical and Chemical Sciences), we developed a BioGPS ligand–pocket database comprising 25,717 human protein pockets. The five active drugs showed affinity for more binding pockets than inactive controls, suggesting a polypharmacological profile and the ability to modulate multiple pathways. Notably, these drugs shared 12 common protein targets, including soluble epoxide hydrolase (sEH), an enzyme in the arachidonic acid (AA) cascade. In hippocampi of Mecp2-/y mice at postnatal day 35 (early symptomatic stage), both sEH gene expression and protein levels were upregulated, and drug D5 partially inhibited its activity. Given the link between sEH and cholesterol metabolism, I quantified unesterified cholesterol in human RTT fibroblasts (R255X and R168X mutations). Compared with healthy controls, RTT fibroblasts showed excessive free cholesterol accumulation, which was significantly reduced by treatment with the five lead drugs. Overall, this study identified FDA-approved compounds with polypharmacological properties that rescue morphological, functional, and metabolic deficits in RTT cellular models. Their effects on dendritic arborization, calcium signalling, and cholesterol metabolism provide new insights into their mechanisms of action, indicating sEH as a promising therapeutic target.

Rett syndrome (RTT) is a postnatal, progressive neurodevelopmental disorder that affects approximately 1 in 10,000 newborn girls worldwide. In most cases, it is caused by loss-of-function mutations in the MECP2 gene on the X chromosome, which encodes the methyl-CpG-binding protein 2 (MeCP2). RTT patients typically develop normally for the first 6–18 months of life, after which they undergo regression in motor, communication, and cognitive skills. Anatomically, RTT neurons exhibit dendritic atrophy and a marked reduction in dendritic spine density, leading to neuronal network dysfunction. The discovery that re-expression of MECP2 in Mecp2-/y mice can largely reverse RTT-like phenotypes, and that neurons lacking MECP2 do not undergo degeneration, supports the notion that RTT is a potentially reversible disorder. However, a definitive cure remains unavailable. Gene therapy, although promising, faces challenges due to MECP2 mosaicism, which can result in inefficiency and potential toxicity from MECP2 overexpression in cells carrying the normal allele. Pharmacological strategies therefore represent an attractive alternative. Currently, Trofinetide (DAYBLUE) is the only FDA-approved drug for RTT treatment. In our laboratory, we developed an in vitro model of primary hippocampal neurons to identify developmental stages affected in RTT. Mecp2-/y neurons showed dendritic atrophy at days-in-vitro (DIV) 6 and 12, identifying these as potential therapeutic windows. The first aim of my PhD project was to identify FDA-approved multifunctional drugs capable of reversing neuronal atrophy in Mecp2-/y neurons. A preliminary screening of 640 FDA-approved drugs at DIV 6 identified 14 compounds that rescued neuronal morphology. During my PhD, I further tested these 14 drugs (10 μM) at DIV 12, when neurons are mature, form synapses, and exhibit spontaneous activity. Morphological analysis included five parameters: total dendritic length (TDL), polarization index (Lm/Lsym), Sholl crossings (TC), distal dendrites (SO1), and maximal dendritic length (MDL). Among the 14 compounds plus mirtazapine (positive control, 1 μM), five emerged as the most promising: D1, D3, and D5 rescued all morphological parameters, similar to mirtazapine, while D2 and D4 recovered three of five. Functional assessment of synaptic activity revealed that D2, D4, and D5 restored calcium transient frequency in Mecp2-/y hippocampal neurons, indicating normalization of network activity. The second part of my PhD focused on elucidating the cellular mechanisms and biochemical networks underlying these drug effects. In collaboration with Prof. Carosati (University of Trieste, Department of Pharmaceutical and Chemical Sciences), we developed a BioGPS ligand–pocket database comprising 25,717 human protein pockets. The five active drugs showed affinity for more binding pockets than inactive controls, suggesting a polypharmacological profile and the ability to modulate multiple pathways. Notably, these drugs shared 12 common protein targets, including soluble epoxide hydrolase (sEH), an enzyme in the arachidonic acid (AA) cascade. In hippocampi of Mecp2-/y mice at postnatal day 35 (early symptomatic stage), both sEH gene expression and protein levels were upregulated, and drug D5 partially inhibited its activity. Given the link between sEH and cholesterol metabolism, I quantified unesterified cholesterol in human RTT fibroblasts (R255X and R168X mutations). Compared with healthy controls, RTT fibroblasts showed excessive free cholesterol accumulation, which was significantly reduced by treatment with the five lead drugs. Overall, this study identified FDA-approved compounds with polypharmacological properties that rescue morphological, functional, and metabolic deficits in RTT cellular models. Their effects on dendritic arborization, calcium signalling, and cholesterol metabolism provide new insights into their mechanisms of action, indicating sEH as a promising therapeutic target.