Summary Aberrant expression ratio of Cl − transporters, NKCC1 and KCC2, is implicated in several brain conditions. NKCC1 inhibition by the FDA-approved diuretic drug, bumetanide, rescues core symptoms in rodent models and/or clinical trials with patients. However, bumetanide has a strong diuretic effect due to inhibition of the kidney Cl − transporter NKCC2, creating critical drug compliance issues and health concerns. Here, we report the discovery of a new chemical class of selective NKCC1 inhibitors and the lead drug candidate ARN23746. ARN23746 restores the physiological intracellular Cl − in murine Down syndrome neuronal cultures, has excellent solubility and metabolic stability, and displays no issues with off-target activity in vitro . ARN23746 recovers core symptoms in mouse models of Down syndrome and autism, with no diuretic effect, nor overt toxicity upon chronic treatment in adulthood. ARN23746 is ready for advanced preclinical/manufacturing studies toward the first sustainable therapeutics for the neurological conditions characterized by impaired Cl − homeostasis.

Highlights d DS mice display microglia alterations and cognitive impairment d Depletion of microglia rescues cognitive impairment in DS mice d Acetaminophen treatment rescues microglia and cognitive impairments in DS mice d Brain samples of DS people recapitulate microglia alterations

The chloride importer NKCC1 and the chloride exporter KCC2 are key regulators of neuronal chloride concentration. A defective NKCC1/KCC2 expression ratio is associated with several brain disorders. Preclinical/clinical studies have shown that NKCC1 inhibition by the United States FDA-approved diuretic bumetanide is a potential therapeutic strategy in preclinical/clinical studies of multiple neurological conditions. However, bumetanide has poor brain penetration and causes unwanted diuresis by inhibiting NKCC2 in the kidney. To overcome these issues, a growing number of studies have reported more brain-penetrating and/or selective bumetanide prodrugs, analogs, and new molecular entities. Here, we review the evidence for NKCC1 pharmacological inhibition as an effective strategy to manage neurological disorders. We also discuss the advantages and limitations of bumetanide repurposing and the benefits and risks of new NKCC1 inhibitors as therapeutic agents for brain disorders. Neuronal Cl homeostasis: role in brain function and disordersIn neurons, the sodium (Na + )-potassium (K + )-Cltransporter isoform 1 (NKCC1, SLC12A2) and the K + -Cltransporter isoform 2 (KCC2, SLC12A5) [1] are key regulators of intracellular chloride concentration ([Cl -] i ). In the central nervous system (CNS), NKCC1 functions as a Climporter, and is highly expressed in immature neurons during early development [2]. Conversely, KCC2 expression is relatively low in early development (resulting in a high NKCC1/KCC2 ratio), increases during the postnatal period, and is more highly expressed in mature neurons (resulting in a low NKCC1/KCC2 ratio; Box 1). The fine regulation of [Cl -] i by NKCC1 and KCC2 is essential for brain development, including cell proliferation and apoptosis, and neuronal migration and maturation [3]. Moreover, NKCC1 and KCC2 are key for neuronal synaptic plasticity (see Glossary) and for maintaining a proper excitatory/inhibitory balance, which is fundamental for brain functions [3].A defective NKCC1/KCC2 expression ratio is often associated with several neurological and psychiatric disorders [4]. In particular, a large and growing body of literature reporting preclinical and clinical studies has shown that upregulation of NKCC1 and/or downregulation of KCC2 (resulting in an increased NKCC1/KCC2 ratio) underlie neurodevelopmental, insult-induced neurological, and neurodegenerative disorders [4] (Box 1; see Outstanding questions). Restoring neuronal [Cl -] i by inhibiting NKCC1 with bumetanide (an unselective NKCC1 inhibitor) was the first proof of concept for NKCC1 as a valuable target for several neurological conditions in preclinical (animal models) and clinical studies (patients) [5,6] (see Outstanding questions). Bumetanide is an FDAapproved thick ascending loop (TAL) of Henle diuretic, which acts by inhibiting the kidney transporter NKCC2. Due to its potent diuretic effect, bumetanide is currently indicated only to treat edema and swelling caused by congestive heart failure, acute pulmonary congestion, and hepatic an...

Intracellular chloride concentration [Cl – ] i is defective in several neurological disorders. In neurons, [Cl – ] i is mainly regulated by the action of the Na + –K + –Cl – importer NKCC1 and the K + –Cl – exporter KCC2. Recently, we have reported the discovery of ARN23746 as the lead candidate of a novel class of selective inhibitors of NKCC1. Importantly, ARN23746 is able to rescue core symptoms of Down syndrome (DS) and autism in mouse models. Here, we describe the discovery and extensive characterization of this chemical class of selective NKCC1 inhibitors, with focus on ARN23746 and other promising derivatives. In particular, we present compound 40 ( ARN24092 ) as a backup/follow-up lead with in vivo efficacy in a mouse model of DS. These results further strengthen the potential of this new class of compounds for the treatment of core symptoms of brain disorders characterized by the defective NKCC1/KCC2 expression ratio.

Accurate and timely expression of specific genes guarantees the healthy development and function of the brain. Indeed, variations in the correct amount or timing of gene expression lead to improper development and/or pathological conditions. Almost forty years after the first successful gene transfection in in vitro cell cultures, it is currently possible to regulate gene expression in an area-specific manner at any step of central nervous system development and in adulthood in experimental animals in vivo, even overcoming the very poor accessibility of the brain. Here, we will review the diverse approaches for acute gene transfer in vivo, highlighting their advantages and disadvantages with respect to the efficiency and specificity of transfection as well as to brain accessibility. In particular, we will present well-established chemical, physical and virus-based approaches suitable for different animal models, pointing out their current and future possible applications in basic and translational research as well as in gene therapy.

Synaptic transmission is critically dependent on synaptic vesicle (SV) recycling. Although the precise mechanisms of SV retrieval are still debated, it is widely accepted that a fundamental role is played by clathrin-mediated endocytosis, a form of endocytosis that capitalizes on the clathrin/adaptor protein complex 2 (AP2) coat and several accessory factors. Here, we show that the previously uncharacterized protein KIAA1107, predicted by bioinformatics analysis to be involved in the SV cycle, is an AP2-interacting clathrin-endocytosis protein (APache). We found that APache is highly enriched in the CNS and is associated with clathrin-coated vesicles via interaction with AP2. APache-silenced neurons exhibit a severe impairment of maturation at early developmental stages, reduced SV density, enlarged endosome-like structures, and defects in synaptic transmission, consistent with an impaired clathrin/AP2-mediated SV recycling. Our data implicate APache as an actor in the complex regulation of SV trafficking, neuronal development, and synaptic plasticity.

Alterations in the expression of the Cl − importer Na-K-2Cl co-transporter-1 (NKCC1) and the exporter K-Cl cotransporter 2 (KCC2) lead to impaired intracellular chloride concentration in neurons and imbalanced excitation/inhibition in the brain. These alterations have been observed in several neurological disorders (e.g., Down syndrome and autism). Recently, we have reported the discovery of the selective NKCC1 inhibitor "compound ARN23746" for the treatment of Down syndrome and autism in mouse models. Here, we report on an extensive preclinical characterization of ARN23746 toward its development as a clinical candidate. ARN23746 shows an overall excellent metabolism profile and good brain penetration. Moreover, ARN23746 is effective in rescuing cognitive impairment in Down syndrome mice upon per os administration, in line with oral treatment of neurodevelopmental disorders. Notably, ARN23746 does not present signs of toxicity or diuresis even if administered up to 50 times the effective dose. These results further support ARN23746 as a solid candidate for clinical trial-enabling studies.

PCDH19 gene-related epilepsy or PCDH19 clustering epilepsy (PCDH19-CE) is an infantile-onset epilepsy syndrome characterized by psychiatric (including autism-related), sensory and cognitive impairment of varying degrees. PCDH19-CE is caused by X-linked PCDH19 protein loss of function. Due to random X-chromosome inactivation, PCDH19-CE-affected females present a mosaic population of healthy and PCDH19-mutant cells. Unfortunately, to date, no current mouse model can fully recapitulate both the brain histological and behavioral deficits present in people with PCDH19-CE. Thus, the search for a proper understanding of the disease and possible future treatment is hampered. By inducing a focal mosaicism of PCDH19 expression using in utero electroporation in rats, we found here that PCDH19 signaling in specific brain areas is implicated in neuronal migration, heat-induced epileptic seizures, core/comorbid behaviors related to autism and cognitive function.