Pharmacological Rescue of Dendritic Pathology in the Ts65Dn Mouse Model of Down Syndrome

Down syndrome (DS) is a genetic pathology characterized by brain hypotrophy and severe cognitive disability. Although defective neurogenesis is an important determinant of cognitive impairment, a severe dendritic pathology appears to be an equally important factor. It is well established that serotonin plays a pivotal role both on neurogenesis and dendritic maturation. Since the serotonergic system is profoundly altered in the DS brain, we wondered whether defects in the hippocampal development can be rescued by treatment with fluoxetine, a selective serotonin reuptake inhibitor and a widely used antidepressant drug. A previous study of our group showed that fluoxetine fully restores neurogenesis in the Ts65Dn mouse model of DS and that this effect is accompanied by a recovery of memory functions. The goal of the current study was to establish whether fluoxetine also restores dendritic development and maturation. In mice aged 45 days, treated with fluoxetine in the postnatal period P3-P15, we examined the dendritic arbor of newborn and mature granule cells of the dentate gyrus (DG). The granule cells of trisomic mice had a severely hypotrophic dendritic arbor, fewer spines and a reduced innervation than euploid mice. Treatment with fluoxetine fully restored all these defects. Moreover the impairment of excitatory and inhibitory inputs to CA3 pyramidal neurons was fully normalized in treated trisomic mice, indicating that fluoxetine can rescue functional connectivity between the DG and CA3. The widespread beneficial effects of fluoxetine on the hippocampal formation suggest that early treatment with fluoxetine can be a suitable therapy, possibly usable in humans, to restore the physiology of the hippocampal networks and, hence, memory functions. These findings may open the way for future clinical trials in children and adolescents with DS.

Role of Shiga toxins in the pathogenesis of hemolytic uremic syndrome

During the pathogenesis of hemolytic uremic syndrome (HUS), a severe sequela of Shiga toxin (Stx)-producing Escherichia coli (STEC) gastrointestinal infections, before the toxin acts on the target endothelial cells of the kidney and brain, several Stx forms are transported in the bloodstream: free Stx; Stx bound to circulating cells through Gb3Cer and TLR4 receptors; and Stx associated to blood cell-derived microvesicles. The latter form is mainly responsible for the development of life-threatening HUS in 15% of STEC-infected patients. Stx consist of five B subunits non-covalently bound to a single A subunit (uncleaved Stx) which can be cleaved in two fragments (A1 and A2) held by a disulfide bond (cleaved Stx). After reduction, the enzymatically active A1 fragment responsible for toxicity is released. Cleaved and uncleaved Stx are biologically active but functionally different, thus their presence in patients’ blood could affect the onset of HUS. Currently, there are no effective therapies for the treatment of STEC-infected patients and the gold standard strategies available for the diagnosis are very expensive and time-consuming. In this thesis, by exploiting the resolving power of SERS technology (Amplified Raman Spectroscopy on Surfaces), a plasmonic biosensor was developed as effective diagnostic tool for early detection of Stx in patients’ sera. An acellular protein synthesis system for detecting cleaved Stx2a in human serum based on its greater translation inhibition after treatment with reducing agents was developed and used to identify cleaved Stx in STEC-infected patients’ sera. Pathogenic microvesicles from Stx2a-challenged blood from healthy donors were isolated and characterized. The antibiotic NAB815, acting as inhibitor of toxin binding to TLR4 expressed by circulating cells, was found to be effective in impairing the formation of blood cell-derived microvesicles containing Stx2a, also having a protective effect in cellular models. This approach could be proposed as an innovative treatment for HUS prevention.

Synthesis and characterization of novel selective NKCC1 inhibitors for the treatment of down syndrome and brain disorders characterized by depolarizing GABAergic transmission.

Proper GABAergic transmission through Cl-permeable GABAA receptors is fundamental for physiological brain development and function. Indeed, defective GABAergic signaling – due to a high NKCC1/KCC2 expression ratio – has been implicated in several neurodevelopmental disorders (e.g., Down syndrome, DS, Autism spectrum disorders, ASD). Interestingly, NKCC1 inhibition by the FDA-approved diuretic drug bumetanide reverts cognitive deficits in the TS65Dn mouse models of DS and core symptoms in other models of brain disorders. However, the required chronic treatment with bumetanide is burdened by its diuretic side effects caused by the antagonization of the kidney Cl importer NKCC2. This may lead to hypokalemia, while jeopardizing drug compliance. Crucially, these issues would be solved by selective NKCC1 inhibitors, thus devoid of the diuretic effect of bumetanide. To this aim, starting from bumetanide’s structure, we applied a ligand-based computational approach to design new molecular entities that we tested in vitro for their capacity to selectively block NKCC1. Extensive synthetic efforts and structure-activity relationships analyses allowed us to improve in vitro potency and overall drug-like properties of the initially identified chemical hits. As a result, we identified a new highly potent NKCC1 inhibitor (ARN23746) that displayed excellent solubility, metabolic stability, and no significant effect on NKCC2 in vitro. Moreover, this novel and selective NKCC1 inhibitor was able to rescue cognitive deficits in DS mice and social/repetitive behaviors in ASD mice, with no diuretic effect and no overt toxicity upon chronic treatment in adult animals. Thus, ARN23746 a selective NKCC1 inhibitor devoid of the diuretic effect – represents a suitable and solid therapeutic strategy for the treatment of Down syndrome and all the brain neurological disorders characterized by depolarizing GABAergic transmission.