Heterogeneous subpopulations of GABAAR-responding neurons coexist across neuronal network scales and developmental stages in health and disease.

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in adults. Depolarizing GABA responses have been well characterized at neuronal-population average level during typical neurodevelopment and partially in brain disorders. However, no investigation has specifically assessed whether a mosaicism of cells with either depolarizing or hyperpolarizing/inhibitory GABAergic responses exists in animals in health/disease at diverse developmental stages, including adulthood. Here, we showed that such mosaicism is present in wild-type (WT) and down syndrome (DS) neuronal networks, as assessed at increasing scales of complexity (cultures, brain slices, behaving mice). Nevertheless, WT mice presented a much lower percentage of cells with depolarizing GABA than DS mice. Restoring the mosaicism of hyperpolarizing and depolarizing GABA-responding neurons to WT levels rescued anxiety behavior in DS mice. Moreover, we found heterogeneous GABAergic responses in developed control and trisomic human induced-pluripotent-stem-cells-derived neurons. Thus, a heterogeneous subpopulation of GABA-responding cells exists in physiological/pathological conditions in mouse and human neurons, possibly contributing to disease-associated behaviors.

Early IGF-1 receptor inhibition in mice mimics preterm human brain disorders and reveals a therapeutic target.

Besides recent advances in neonatal care, preterm newborns still develop sex-biased behavioral alterations. Preterms fail to receive placental insulin-like growth factor-1 (IGF-1), a major fetal growth hormone in utero, and low IGF-1 serum levels correlate with preterm poor neurodevelopmental outcomes. Here, we mimicked IGF-1 deficiency of preterm newborns in mice by perinatal administration of an IGF-1 receptor antagonist. This resulted in sex-biased brain microstructural, functional, and behavioral alterations, resembling those of ex-preterm children, which we characterized performing parallel mouse/human behavioral tests. Pharmacological enhancement of GABAergic tonic inhibition by the U.S. Food and Drug Administration-approved drug ganaxolone rescued functional/behavioral alterations in mice. Establishing an unprecedented mouse model of prematurity, our work dissects the mechanisms at the core of abnormal behaviors and identifies a readily translatable therapeutic strategy for preterm brain disorders.

Cation Chloride Cotransporter NKCC1 Operates through a Rocking-Bundle Mechanism.

The sodium, potassium, and chloride cotransporter 1 (NKCC1) plays a key role in tightly regulating ion shuttling across cell membranes. Lately, its aberrant expression and function have been linked to numerous neurological disorders and cancers, making it a novel and highly promising pharmacological target for therapeutic interventions. A better understanding of how NKCC1 dynamically operates would therefore have broad implications for ongoing efforts toward its exploitation as a therapeutic target through its modulation. Based on recent structural data on NKCC1, we reveal conformational motions that are key to its function. Using extensive deep-learning-guided atomistic simulations of NKCC1 models embedded into the membrane, we captured complex dynamical transitions between alternate open conformations of the inner and outer vestibules of the cotransporter and demonstrated that NKCC1 has water-permeable states. We found that these previously undefined conformational transitions occur via a rocking-bundle mechanism characterized by the cooperative angular motion of transmembrane helices (TM) 4 and 9, with the contribution of the extracellular tip of TM 10. We found these motions to be critical in modulating ion transportation and in regulating NKCC1's water transporting capabilities. Specifically, we identified interhelical dynamical contacts between TM 10 and TM 6, which we functionally validated through mutagenesis experiments of 4 new targeted NKCC1 mutants. We conclude showing that those 4 residues are highly conserved in most Na+-dependent cation chloride cotransporters (CCCs), which highlights their critical mechanistic implications, opening the way to new strategies for NKCC1's function modulation and thus to potential drug action on selected CCCs.

Preclinical Development of the Na-K-2Cl Co-transporter-1 (NKCC1) Inhibitor ARN23746 for the Treatment of Neurodevelopmental Disorders.

Alterations in the expression of the Cl- importer Na-K-2Cl co-transporter-1 (NKCC1) and the exporter K-Cl co-transporter 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.

A rat model of a focal mosaic expression of PCDH19 replicates human brain developmental abnormalities and behaviours.

Protocadherin 19 gene-related epilepsy or protocadherin 19 clustering epilepsy is an infantile-onset epilepsy syndrome characterized by psychiatric (including autism-related), sensory, and cognitive impairment of varying degrees. Protocadherin 19 clustering epilepsy is caused by X-linked protocadherin 19 protein loss of function. Due to random X-chromosome inactivation, protocadherin 19 clustering epilepsy-affected females present a mosaic population of healthy and protocadherin 19-mutant cells. Unfortunately, to date, no current mouse model can fully recapitulate both the brain histological and behavioural deficits present in people with protocadherin 19 clustering epilepsy. Thus, the search for a proper understanding of the disease and possible future treatment is hampered. By inducing a focal mosaicism of protocadherin 19 expression using in utero electroporation in rats, we found here that protocadherin 19 signalling in specific brain areas is implicated in neuronal migration, heat-induced epileptic seizures, core/comorbid behaviours related to autism and cognitive function.

Structural basis for inhibition of the Cation-chloride cotransporter NKCC1 by the diuretic drug bumetanide.

Cation-chloride cotransporters (CCCs) NKCC1 and NKCC2 catalyze electroneutral symport of 1 Na+, 1 K+, and 2 Cl- across cell membranes. NKCC1 mediates trans-epithelial Cl- secretion and regulates excitability of some neurons and NKCC2 is critical to renal salt reabsorption. Both transporters are inhibited by the so-called loop diuretics including bumetanide, and these drugs are a mainstay for treating edema and hypertension. Here, our single-particle electron cryo-microscopy structures supported by functional studies reveal an outward-facing conformation of NKCC1, showing bumetanide wedged into a pocket in the extracellular ion translocation pathway. Based on these and the previously published inward-facing structures, we define the translocation pathway and the conformational changes necessary for ion translocation. We also identify an NKCC1 dimer with separated transmembrane domains and extensive transmembrane and C-terminal domain interactions. We further define an N-terminal phosphoregulatory domain that interacts with the C-terminal domain, suggesting a mechanism whereby (de)phosphorylation regulates NKCC1 by tuning the strength of this domain association.

Cation-coupled chloride cotransporters: chemical insights and disease implications.

Cation-coupled chloride cotransporters (CCCs) modulate the transport of sodium and/or potassium cations coupled with chloride anions across the cell membrane. CCCs thus help regulate intracellular ionic concentration and consequent cell volume homeostasis. This has been largely exploited in the past to develop diuretic drugs that act on CCCs expressed in the kidney. However, a growing wealth of evidence has demonstrated that CCCs are also critically involved in a great variety of other pathologies, motivating most recent drug discovery programs targeting CCCs. Here, we examine the structure-function relationship of CCCs. By linking recent high-resolution cryogenic electron microscopy (cryo-EM) data with older biochemical/functional studies on CCCs, we discuss the mechanistic insights and opportunities to design selective CCC modulators to treat diverse pathologies.

Pharmacological tools to target NKCC1 in brain disorders.

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.

Design, Synthesis, In Vitro and In Vivo Characterization of Selective NKCC1 Inhibitors for the Treatment of Core Symptoms in Down Syndrome.

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.

Restoring neuronal chloride homeostasis with anti-NKCC1 gene therapy rescues cognitive deficits in a mouse model of Down syndrome.

A common feature of diverse brain disorders is the alteration of GABA-mediated inhibition because of aberrant, intracellular chloride homeostasis induced by changes in the expression and/or function of chloride transporters. Notably, pharmacological inhibition of the chloride importer NKCC1 is able to rescue brain-related core deficits in animal models of these pathologies and in some human clinical studies. Here, we show that reducing NKCC1 expression by RNA interference in the Ts65Dn mouse model of Down syndrome (DS) restores intracellular chloride concentration, efficacy of gamma-aminobutyric acid (GABA)-mediated inhibition, and neuronal network dynamics in vitro and ex vivo. Importantly, adeno-associated virus (AAV)-mediated, neuron-specific NKCC1 knockdown in vivo rescues cognitive deficits in diverse behavioral tasks in Ts65Dn animals. Our results highlight a mechanistic link between NKCC1 expression and behavioral abnormalities in DS mice and establish a molecular target for new therapeutic approaches, including gene therapy, to treat brain disorders characterized by neuronal chloride imbalance.

Modelling genetic mosaicism of neurodevelopmental disorders in vivo by a Cre-amplifying fluorescent reporter.

Genetic mosaicism, a condition in which an organ includes cells with different genotypes, is frequently present in monogenic diseases of the central nervous system caused by the random inactivation of the X-chromosome, in the case of X-linked pathologies, or by somatic mutations affecting a subset of neurons. The comprehension of the mechanisms of these diseases and of the cell-autonomous effects of specific mutations requires the generation of sparse mosaic models, in which the genotype of each neuron is univocally identified by the expression of a fluorescent protein in vivo. Here, we show a dual-color reporter system that, when expressed in a floxed mouse line for a target gene, leads to the creation of mosaics with tunable degree. We demonstrate the generation of a knockout mosaic of the autism/epilepsy related gene PTEN in which the genotype of each neuron is reliably identified, and the neuronal phenotype is accurately characterized by two-photon microscopy.

Rescuing Over-activated Microglia Restores Cognitive Performance in Juvenile Animals of the Dp(16) Mouse Model of Down Syndrome.

Microglia are brain-resident immune cells and regulate mechanisms essential for cognitive functions. Down syndrome (DS), the most frequent cause of genetic intellectual disability, is caused by a supernumerary chromosome 21, containing also genes related to the immune system. In the hippocampus of the Dp(16) mouse model of DS and DS individuals, we found activated microglia, as assessed by their morphology; activation markers; and, for DS mice, electrophysiological profile. Accordingly, we found increased pro-inflammatory cytokine levels and altered interferon signaling in Dp(16) hippocampi. DS mice also showed decreased spine density and activity of hippocampal neurons and hippocampus-dependent cognitive behavioral deficits. Depletion of defective microglia or treatment with a commonly used anti-inflammatory drug rescued the neuronal spine and activity impairments and cognitive deficits in juvenile Dp(16) mice. Our results suggest an involvement of microglia in Dp(16)-mouse cognitive deficits and identify a new potential therapeutic approach for cognitive disabilities in DS individuals.

Discovery of a Small Molecule Drug Candidate for Selective NKCC1 Inhibition in Brain Disorders.

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.

A Localized Scaffold for cGMP Increase Is Required for Apical Dendrite Development.

Studies in cultured neurons have established that axon specification instructs neuronal polarization and is necessary for dendrite development. However, dendrite formation in vivo occurs when axon formation is prevented. The mechanisms promoting dendrite development remain elusive. We find that apical dendrite development is directed by a localized cyclic guanosine monophosphate (cGMP)-synthesizing complex. We show that the scaffolding protein Scribble associates with cGMP-synthesizing enzymes soluble-guanylate-cyclase (sGC) and neuronal nitric oxide synthase (nNOS). The Scribble scaffold is preferentially localized to and mediates cGMP increase in dendrites. These events are regulated by kinesin KifC2. Knockdown of Scribble, sGC-ß1, or KifC2 or disrupting their associations prevents cGMP increase in dendrites and causes severe defects in apical dendrite development. Local cGMP elevation or sGC expression rescues the effects of Scribble knockdown on dendrite development, indicating that Scribble is an upstream regulator of cGMP. During neuronal polarization, dendrite development is directed by the Scribble scaffold that might link extracellular cues to localized cGMP increase.

Gene Editing Preserves Visual Functions in a Mouse Model of Retinal Degeneration.

Inherited retinal dystrophies (IRDs) are a large and heterogeneous group of degenerative diseases caused by mutations in various genes. Given the favorable anatomical and immunological characteristics of the eye, gene therapy holds great potential for their treatment. Our goal is to validate the preservation of visual functions by viral-free homology directed repair (HDR) in an autosomal recessive loss of function mutation. We used a tailored gene editing system based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to prevent retinal photoreceptor death in the retinal degeneration 10 (Rd10) mouse model of retinitis pigmentosa. We tested the gene editing tool in vitro and then used in vivo subretinal electroporation to deliver it to one of the retinas of mouse pups at different stages of photoreceptor differentiation. Three months after gene editing, the treated eye exhibited a higher visual acuity compared to the untreated eye. Moreover, we observed preservation of light-evoked responses both in explanted retinas and in the visual cortex of treated animals. Our study validates a CRISPR/Cas9-based therapy as a valuable new approach for the treatment of retinitis pigmentosa caused by autosomal recessive loss-of-function point mutations.

Synaptogenesis Stimulates a Proteasome-Mediated Ribosome Reduction in Axons.

Ribosomes and a subset of cellular mRNAs are trafficked into axons of developing neurons. The axonal localization of translational machinery allows new proteins to be rapidly and locally synthesized during axonal growth and pathfinding. However, in mature neurons, axonal ribosomes are significantly reduced or even absent. The mechanism that elicits this removal is currently unknown. Here, we demonstrate that synapse formation is the trigger for ribosome reduction in mature axons. In vivo analysis shows that axonal ribosome levels decrease in rat brain at a developmental stage coincident with synapse formation. Next, we observe in vitro that different synaptogenic inducers trigger an overall decrease of ribosomal proteins and rRNA in the axons of spinal motor neurons. We further observe that this process is dependent on the ubiquitin-proteasome system but not on autophagy. Together, these data identify synaptogenesis as the long missing biological trigger that leads to ribosome disappearance during axonal maturation.

Oxytocin Signaling in the Central Amygdala Modulates Emotion Discrimination in Mice.

Recognition of other's emotions influences the way social animals interact and adapt to the environment. The neuropeptide oxytocin (OXT) has been implicated in different aspects of emotion processing. However, the role of endogenous OXT brain pathways in the social response to different emotional states in conspecifics remains elusive. Here, using a combination of anatomical, genetic, and chemogenetic approaches, we investigated the contribution of endogenous OXT signaling in the ability of mice to discriminate unfamiliar conspecifics based on their emotional states. We found that OXTergic projections from the paraventricular nucleus of the hypothalamus (PVN) to the central amygdala (CeA) are crucial for the discrimination of both positively and negatively valenced emotional states. In contrast, blocking PVN OXT release into the nucleus accumbens, prefrontal cortex, and hippocampal CA2 did not alter this emotion discrimination. Furthermore, silencing each of these PVN OXT pathways did not influence basic social interaction. These findings were further supported by the demonstration that virally mediated enhancement of OXT signaling within the CeA was sufficient to rescue emotion discrimination deficits in a genetic mouse model of cognitive liability. Our results indicate that CeA OXT signaling plays a key role in emotion discrimination both in physiological and pathological conditions.

The development of synaptic transmission is time-locked to early social behaviors in rats.

The development of functional synapses is a sequential process preserved across many brain areas. Here, we show that glutamatergic postsynaptic currents anticipated GABAergic currents in Layer II/III of the rat neocortex, in contrast to the pattern described for other brain areas. The frequencies of both glutamatergic and GABAergic currents increased abruptly at the beginning of the second postnatal week, supported by a serotonin upsurge. Integrative behaviors arose on postnatal day (P)9, while most motor and sensory behaviors, which are fundamental for pup survival, were already in place at approximately P7. A reduction in serotonin reuptake accelerated the development of functional synapses and integrative huddling behavior, while sparing motor and sensory function development. A decrease in synaptic transmission in Layer II/III induced by a chemogenetic approach only inhibited huddling. Thus, precise developmental sequences mediate early, socially directed behaviors for which neurotransmission and its modulation in supragranular cortical layers play key roles.

KCC2 Regulates Dendritic Spine Formation in a Brain-Region Specific and BDNF Dependent Manner.

KCC2 is the major chloride extruder in neurons. The spatiotemporal regulation of KCC2 expression orchestrates the developmental shift towards inhibitory GABAergic drive and the formation of glutamatergic synapses. Whether KCC2's role in synapse formation is similar in different brain regions is unknown. First, we found that KCC2 subcellular localization, but not overall KCC2 expression levels, differed between cortex and hippocampus during the first postnatal week. We performed site-specific in utero electroporation of KCC2 cDNA to target either hippocampal CA1 or somatosensory cortical pyramidal neurons. We found that a premature expression of KCC2 significantly decreased spine density in CA1 neurons, while it had the opposite effect in cortical neurons. These effects were cell autonomous, because single-cell biolistic overexpression of KCC2 in hippocampal and cortical organotypic cultures also induced a reduction and an increase of dendritic spine density, respectively. In addition, we found that the effects of its premature expression on spine density were dependent on BDNF levels. Finally, we showed that the effects of KCC2 on dendritic spine were dependent on its chloride transporter function in the hippocampus, contrary to what was observed in cortex. Altogether, these results demonstrate that KCC2 regulation of dendritic spine development, and its underlying mechanisms, are brain-region specific.