A Double-Blind, Randomized, Placebo-Controlled Trial of Bumetanide in Parkinson's Disease.

BACKGROUND: Acting on the main target of dopaminergic cells, the striatal γ-aminobutyric acid (GABA)-ergic cells, might be a new way to treat persons with Parkinson's disease (PD). OBJECTIVE: The objective of this study was to assess the efficacy of bumetanide, an Na-K-Cl cotransporter (NKCC1) inhibitor, to improve motor symptoms in PD. METHODS: This was a 4-month double-blind, randomized, parallel-group, placebo-controlled trial of 1.75 to 3 mg/day bumetanide as an adjunct to levodopa in 44 participants with PD and motor fluctuations. RESULTS: Compared to the baseline, the mean change in OFF Movement Disorder Society Unified Parkinson's Disease Rating Scale Part III score after 4 months of treatment (primary endpoint) did not improve significantly compared with placebo. No changes between participants treated with bumetanide and those treated with placebo were observed for most other outcome measures. Despite no relevant safety signals, bumetanide was poorly tolerated. CONCLUSIONS: There was no evidence in this study that bumetanide has efficacy in improving motor symptoms of PD. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Bumetanide oral solution for the treatment of children and adolescents with autism spectrum disorder: Results from two randomized phase III studies.

The efficacy and safety of bumetanide oral solution for the treatment of autism spectrum disorder (ASD) in children and adolescents was evaluated in two international, multi-center, randomized, double-blind, placebo-controlled phase III trials; one enrolled patients aged 7-17 years (SIGN 1 trial) and the other enrolled younger patients aged 2-6 years (SIGN 2). In both studies, patients were randomized to receive bumetanide oral solution twice daily (BID) or placebo BID during a 6-month double-blind treatment period. The primary endpoint was change in Childhood Autism Rating Scale 2 (CARS2) total raw score from baseline to Week 26. Key secondary endpoints included changes in Social Responsiveness Scale-2, Clinical Global Impression Scale, and Vineland Adaptive Behavior Scale. Each study enrolled 211 patients (bumetanide, n = 107; placebo, n = 104). Both studies were terminated early due to absence of any significant difference between bumetanide and placebo in the overall studied populations. In both studies, CARS2 total raw score decreased from baseline to Week 26 in the bumetanide and placebo groups, with no statistically significant difference between groups. No differences were observed between treatment groups for any of the secondary efficacy endpoints in either study. In both studies, treatment-emergent adverse events that occurred more frequently with bumetanide than placebo included thirst, polyuria, hypokalemia, and dry mouth. These large phase III trials failed to demonstrate a benefit of bumetanide for the treatment of pediatric ASD compared with placebo. Consequently, the sponsor has discontinued the development of bumetanide for the treatment of this condition. Trial registration: https://clinicaltrials.gov: SIGN 1: NCT03715166; SIGN 2: NCT03715153.

GABA Signaling: Therapeutic Targets for Neurodegenerative and Neurodevelopmental Disorders.

This Special Issue, "GABA Signaling: Therapeutic Targets for Neurodegenerative and Neurodevelopmental Disorders", focuses on a fundamental property of the neurotransmitter γ-aminobutyric acid (GABA), namely its capacity to shift, in particular conditions, from the hyperpolarizing to the depolarizing direction [...].

A Wholistic View of How Bumetanide Attenuates Autism Spectrum Disorders.

The specific NKCC1 cotransporter antagonist, bumetanide, attenuates the severity of Autism Spectrum Disorders (ASD), and many neurodevelopmental or neurodegenerative disorders in animal models and clinical trials. However, the pervasive expression of NKCC1 in many cell types throughout the body is thought to challenge the therapeutic efficacy of bumetanide. However, many peripheral functions, including intestinal, metabolic, or vascular, etc., are perturbed in brain disorders contributing to the neurological sequels. Alterations of these functions also increase the incidence of the disorder suggesting complex bidirectional links with the clinical manifestations. We suggest that a more holistic view of ASD and other disorders is warranted to account for the multiple sites impacted by the original intra-uterine insult. From this perspective, large-spectrum active repositioned drugs that act centrally and peripherally might constitute a useful approach to treating these disorders.

A quantitative cholinergic and catecholaminergic 3D Atlas of the developing mouse brain.

The complex organization of brain regions during development requires a three-dimensional approach to facilitate the visualization and quantification of dynamic changes taking place throughout this important period. Using the tissue clearing method combined with immunohistochemistry, three-dimensional (3D) lightsheet microscopy and a multiresolution registration technique, we provide the first 3D atlases of the main cholinergic (CH) and catecholaminergic (CA) systems in the mouse brain from embryonic day 12 (E12) to post-natal day 8 (P8). We report that in several brain structures, there is a logarithmic scale increase of choline acetyltransferase and tyrosine hydroxylase positive neurons from E18 to P8. In addition, a detailed voxel-wise analysis revealed abrupt modifications in the developmental trajectory of many brain structures during the transition from E18 to P0. Our atlases will not only facilitate developmental studies aimed at quantitatively determining the fate of CH or CA neurons in utero but also be used as an anatomical reference to quantify other neuronal populations present in the annotated regions. In the future, these maps will be a reliable tool to study developmental malformations associated with neurological and psychiatric disorders.

Prenatal reduction of E14.5 embryonically fate-mapped pyramidal neurons in a mouse model of autism.

Although several observations suggest that the constitutive biological, genetic or physiological changes leading to autism spectrum disorders (ASD) start in utero, their early impact on the number and density of neurons in the brain remains unknown. Using genetic fate mapping associated with the immunollabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO) clearing method we identified and counted a selective population of neocortical and hippocampal pyramidal neurons in the in utero valproate (VPA) mouse model of autism. We report that 1 day before birth, the number of pyramidal neurons born at E14.5 in the neocortex and hippocampus of VPA mice is smaller than in age-matched controls. VPA also induced a reduction of the neocortical-but not hippocampal-volume 1 day before birth. Interestingly, VPA mice present an increase in both neocortical and hippocampal volumes 2 days after birth compared with controls. These results suggest that the VPA-exposed hippocampus and neocortex differ substantially from controls during the highly complex perinatal period, and specially 1 day before birth, reflecting the early pathogenesis of ASD.

[Early prognostic of ASD: A challenge]. / Pronostiquer tôt les troubles du spectre autistique : Un défi ?

Autism Spectrum Disorders (ASD) are born in the womb generated by intrauterine genetic or environmental insult. ASD diagnostic is made at the age of 3-5 years in Europe and in the US. Relying on this, we have tested the hypothesis of identifying already at birth babies who might be diagnosed later with ASD, thereby facilitating an early use of psychoeducative techniques to attenuate the severity of the symptoms. Here, we discuss the various approaches that have been used to enable an early diagnosis. We have ourselves used an approach based on a "without a priori" machine learning analysis of all maternity biological and ultrasound data available in French maternities (around 116) in utero and after birth. This program made it possible to identify at birth almost all (96%) of babies who will be later neurotypical and around half of those who will be diagnosed with ASD. Some of the parameters allowing this identification were largely unexpected with no known links with ASD. This approach will enable an early identification of babies at risk, but also might be used to diagnose ASD later on, and perhaps could help to get a better understanding of the heterogeneity of ASD.

Title: Pronostiquer tôt les troubles du spectre autistique : Un défi ? Abstract: Les troubles du spectre de l'autisme (TSA) « naissent ¼ in utero à la suite d'évènements pathologiques génétiques ou environnementaux. Le diagnostic des TSA n'est cependant effectué que vers l'âge de 3-5 ans en Europe et aux États-Unis. Un pronostic précoce permettrait pourtant d'atténuer la sévérité des atteintes cognitives, grâce à des approches psycho-éducatives. Une large panoplie d'approches a été suggérée pour établir un pronostic précoce des TSA, se fondant sur l'imagerie cérébrale, sur des enregistrements EEG, sur des biomarqueurs sanguins ou sur l'analyse des contacts visuels. Nous avons développé une approche fondée sur l'analyse par machine learning des données biologiques et échographiques recueillies en routine, du début de la grossesse au lendemain de la naissance, dans les maternités françaises. Ce programme qui permet d'identifier la presque totalité des bébés neurotypiques et la moitié des bébés qui auront un diagnostic de TSA quelques années plus tard, permet aussi d'identifier les paramètres ayant un impact sur le pronostic. Si quelques-uns d'entre eux étaient attendus, d'autres n'ont aucun lien avec les TSA. L'étude sans a priori des données de maternité devrait ainsi permettre un pronostic des TSA dès la naissance, ainsi que de mieux comprendre la pathogenèse de ces syndromes et de les traiter plus tôt.

The GABA Polarity Shift and Bumetanide Treatment: Making Sense Requires Unbiased and Undogmatic Analysis.

GABA depolarizes and often excites immature neurons in all animal species and brain structures investigated due to a developmentally regulated reduction in intracellular chloride concentration ([Cl-]i) levels. The control of [Cl-]i levels is mediated by the chloride cotransporters NKCC1 and KCC2, the former usually importing chloride and the latter exporting it. The GABA polarity shift has been extensively validated in several experimental conditions using often the NKCC1 chloride importer antagonist bumetanide. In spite of an intrinsic heterogeneity, this shift is abolished in many experimental conditions associated with developmental disorders including autism, Rett syndrome, fragile X syndrome, or maternal immune activation. Using bumetanide, an EMA- and FDA-approved agent, many clinical trials have shown promising results with the expected side effects. Kaila et al. have repeatedly challenged these experimental and clinical observations. Here, we reply to the recent reviews by Kaila et al. stressing that the GABA polarity shift is solidly accepted by the scientific community as a major discovery to understand brain development and that bumetanide has shown promising effects in clinical trials.

Kresimir Krnjevic (1927-2021) and GABAergic inhibition: a lifetime dedication.

After over seven decades of neuroscience research, it is now well established that γ-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain. In this paper dedicated to Kresimir Krnjevic (1927-2021), a pioneer and leader in neuroscience, we briefly highlight the fundamental contributions he made in identifying GABA as an inhibitory neurotransmitter in the brain and our personal interactions with him. Of note, between 1972 and 1978 Dr. Krnjevic was a highly reputed Chief Editor of the Canadian Journal of Physiology and Pharmacology.

GABA and glutamate in the preterm neonatal brain: In-vivo measurement by magnetic resonance spectroscopy.

Cognitive and behavioral disabilities in preterm infants, even without obvious brain injury on conventional neuroimaging, underscores a critical need to identify the subtle underlying microstructural and biochemical derangements. The gamma-aminobutyric acid (GABA) and glutamatergic neurotransmitter systems undergo rapid maturation during the crucial late gestation and early postnatal life, and are at-risk of disruption after preterm birth. Animal and human autopsy studies provide the bulk of current understanding since non-invasive specialized proton magnetic resonance spectroscopy (1H-MRS) to measure GABA and glutamate are not routinely available for this vulnerable population due to logistical and technical challenges. We review the specialized 1H-MRS techniques including MEscher-GArwood Point Resolved Spectroscopy (MEGA-PRESS), special challenges and considerations needed for interpretation of acquired data from the developing brain of preterm infants. We summarize the limited in-vivo preterm data, highlight the gaps in knowledge, and discuss future directions for optimal integration of available in-vivo approaches to understand the influence of GABA and glutamate on neurodevelopmental outcomes after preterm birth.

Machine learning analysis of pregnancy data enables early identification of a subpopulation of newborns with ASD.

To identify newborns at risk of developing ASD and to detect ASD biomarkers early after birth, we compared retrospectively ultrasound and biological measurements of babies diagnosed later with ASD or neurotypical (NT) that are collected routinely during pregnancy and birth. We used a supervised machine learning algorithm with a cross-validation technique to classify NT and ASD babies and performed various statistical tests. With a minimization of the false positive rate, 96% of NT and 41% of ASD babies were identified with a positive predictive value of 77%. We identified the following biomarkers related to ASD: sex, maternal familial history of auto-immune diseases, maternal immunization to CMV, IgG CMV level, timing of fetal rotation on head, femur length in the 3rd trimester, white blood cell count in the 3rd trimester, fetal heart rate during labor, newborn feeding and temperature difference between birth and one day after. Furthermore, statistical models revealed that a subpopulation of 38% of babies at risk of ASD had significantly larger fetal head circumference than age-matched NT ones, suggesting an in utero origin of the reported bigger brains of toddlers with ASD. Our results suggest that pregnancy follow-up measurements might provide an early prognosis of ASD enabling pre-symptomatic behavioral interventions to attenuate efficiently ASD developmental sequels.

Brain Volumes in Mice are Smaller at Birth After Term or Preterm Cesarean Section Delivery.

The rate of cesarean section (CS) delivery has steadily increased over the past decades despite epidemiological studies reporting higher risks of neonatal morbidity and neurodevelopmental disorders. Yet, little is known about the immediate impact of CS birth on the brain, hence the need of experimental studies to evaluate brain parameters following this mode of delivery. Using the solvent clearing method iDISCO and 3D imaging technique, we report that on the day of birth, whole-brain, hippocampus, and striatum volumes are reduced in CS-delivered as compared to vaginally-born mice, with a stronger effect observed in preterm CS pups. These results stress the impact of CS delivery, at term or preterm, during parturition and at birth. In contrast, cellular activity and apoptosis are reduced in mice born by CS preterm but not term, suggesting that these early-life processes are only impacted by the combination of preterm birth and CS delivery.

Phenobarbital, midazolam, bumetanide, and neonatal seizures: The devil is in the details.

Kaila, Löscher, and colleagues report that phenobarbital (PHB) and midazolam (MDZ) attenuate neonatal seizures following birth asphyxia, but the former only when applied before asphyxia and the latter before or after the triggering insult. In contrast, the NKCC1 chloride importer antagonist bumetanide (BUM) had no effect whether applied alone or with PHB. The observations are compelling and in accord with earlier studies. However, there are several general issues that deserve discussion. What is the clinical relevance of these data and the validity of animal models of encephalopathic seizures? Why is it that although they act on similar targets, these agents have different efficacy? Are both PHB and MDZ actions restricted to γ-aminobutyric acidergic (GABAergic) mechanisms? Why is BUM inefficient in attenuating seizures but capable of reducing the severity of other brain disorders? We suggest that the relative failure of antiepileptic drugs (AEDs) to treat this severe life-threatening condition is in part explicable by the recurrent seizures that shift the polarity of GABA, thereby counteracting their effects on their target. AEDs might be efficient after a few seizures but not recurrent ones. In addition, PHB and MDZ actions are not limited to GABA signals. BUM efficiently attenuates autism symptomatology notably in patients with tuberous sclerosis but does not reduce the recurrent seizures, illustrating the uniqueness of epilepsies. Therefore, the efficacy of AEDs to treat babies with encephalopathic seizures will depend on the history and severity of the seizures prior to their administration, challenging a universal common underlying mechanism.

Treating Autism With Bumetanide: Are Large Multicentric and Monocentric Trials on Selected Populations Complementary?

In their article in the Journal, Sprengers et al.1 conclude that bumetanide does not attenuate autism spectrum disorder (ASD) despite a nominally significant treatment effect in repetitive behaviors, which is a core symptom of ASD but was defined as a secondary measure in this trial. Four earlier studies performed by 3 independent institutes, including 2 studies2,3 not mentioned by Sprengers et al., testing a total of 169 children (versus 122 placebo) showed a significant amelioration of ASD symptoms. Bumetanide also significantly attenuated behavioral features of patients with tuberous sclerosis according to another study by Sprengers' same group.4.

Alteration in the time and/or mode of delivery differentially modulates early development in mice.

Delivery is a complex biological process involving hormonal and mechanical stimuli that together condition the survival and development of the fetus out of the womb. Accordingly, changes in the time or way of being born are associated with an alteration of fundamental biological functions and hypothesized to promote the emergence of neurodevelopmental disorders. Hence, the steadily rise in preterm birth and cesarean section (CS) delivery rates over the past years has become a worldwide health concern. In our previous work, we reported that even though no long-term autistic-like deficits were observed, mice born preterm by CS presented early transient neuronal and communicative defects. However, understanding if these alterations were due to an early birth combined with CS delivery, or if prematurity solely could lead to a similar outcome remained to be evaluated. Using mice born either at term or preterm by vaginal or CS delivery, we assessed early life ultrasonic vocalizations and the onset of eye opening. We report that alterations in communicative behaviors are finely attuned and specifically affected either by preterm birth or by the association between CS delivery and preterm birth in mice, while delayed onset of eye opening is due to prematurity. Moreover, our work further underlies a gender-dependent vulnerability to changes in the time and/or way of being born with distinct outcomes observed in males and females. Thus, our results shed light on the intricacy of birth alterations and might further explain the disparities reported in epidemiological studies.

Enhanced Glutamatergic Currents at Birth in Shank3 KO Mice.

Autism spectrum disorders (ASD) are neurodevelopmental disorders induced by genetic and environmental factors. In our recent studies, we showed that the GABA developmental shifts during delivery and the second postnatal week are abolished in two rodent models of ASD. Maternal treatment around birth with bumetanide restored the GABA developmental sequence and attenuated the autism pathogenesis in offspring. Clinical trials conducted in parallel confirmed the usefulness of bumetanide treatment to attenuate the symptoms in children with ASD. Collectively, these observations suggest that an alteration of the GABA developmental sequence is a hallmark of ASD. Here, we investigated whether similar alterations occur in the Shank3 mouse model of ASD. We report that in CA3 pyramidal neurons, the driving force and inhibitory action of GABA are not different in naïve and Shank3-mutant age-matched animals at birth and during the second postnatal week. In contrast, the frequency of spontaneous excitatory postsynaptic currents is already enhanced at birth and persists through postnatal day 15. Therefore, in CA3 pyramidal neurons of Shank3-mutant mice, glutamatergic but not GABAergic activity is affected at early developmental stages, hence reflecting the heterogeneity of mechanisms underlying the pathogenesis of ASD.