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Brain : a Journal of Neurology Oct 2023Although the Na-K-Cl cotransporter (NKCC1) inhibitor bumetanide has prominent positive effects on the pathophysiology of many neurological disorders, the mechanism of...
Although the Na-K-Cl cotransporter (NKCC1) inhibitor bumetanide has prominent positive effects on the pathophysiology of many neurological disorders, the mechanism of action is obscure. Attention paid to elucidating the role of Nkcc1 has mainly been focused on neurons, but recent single cell mRNA sequencing analysis has demonstrated that the major cellular populations expressing NKCC1 in the cortex are non-neuronal. We used a combination of conditional transgenic animals, in vivo electrophysiology, two-photon imaging, cognitive behavioural tests and flow cytometry to investigate the role of Nkcc1 inhibition by bumetanide in a mouse model of controlled cortical impact (CCI). Here, we found that bumetanide rescues parvalbumin-positive interneurons by increasing interneuron-microglia contacts shortly after injury. The longitudinal phenotypic changes in microglia were significantly modified by bumetanide, including an increase in the expression of microglial-derived BDNF. These effects were accompanied by the prevention of CCI-induced decrease in hippocampal neurogenesis. Treatment with bumetanide during the first week post-CCI resulted in significant recovery of working and episodic memory as well as changes in theta band oscillations 1 month later. These results disclose a novel mechanism for the neuroprotective action of bumetanide mediated by an acceleration of microglial activation dynamics that leads to an increase in parvalbumin interneuron survival following CCI, possibly resulting from increased microglial BDNF expression and contact with interneurons. Salvage of interneurons may normalize ambient GABA, resulting in the preservation of adult neurogenesis processes as well as contributing to bumetanide-mediated improvement of cognitive performance.
Topics: Mice; Animals; Bumetanide; Sodium Potassium Chloride Symporter Inhibitors; Microglia; Brain-Derived Neurotrophic Factor; Parvalbumins; Solute Carrier Family 12, Member 2; Interneurons; Neurogenesis
PubMed: 37082944
DOI: 10.1093/brain/awad132 -
Frontiers in Pharmacology 2023Therapeutics discovery and development for Alzheimer's disease (AD) has been an area of intense research to alleviate memory loss and the underlying pathogenic... (Review)
Review
Therapeutics discovery and development for Alzheimer's disease (AD) has been an area of intense research to alleviate memory loss and the underlying pathogenic processes. Recent drug discovery approaches have utilized computational strategies for drug candidate selection which has opened the door to repurposing drugs for AD. Computational analysis of gene expression signatures of patients stratified by the APOE4 risk allele of AD led to the discovery of the FDA-approved drug bumetanide as a top candidate agent that reverses APOE4 transcriptomic brain signatures and improves memory deficits in APOE4 animal models of AD. Bumetanide is a loop diuretic which inhibits the kidney Na-K-2Cl cotransporter isoform, NKCC2, for the treatment of hypertension and edema in cardiovascular, liver, and renal disease. Electronic health record data revealed that patients exposed to bumetanide have lower incidences of AD by 35%-70%. In the brain, bumetanide has been proposed to antagonize the NKCC1 isoform which mediates cellular uptake of chloride ions. Blocking neuronal NKCC1 leads to a decrease in intracellular chloride and thus promotes GABAergic receptor mediated hyperpolarization, which may ameliorate disease conditions associated with GABAergic-mediated depolarization. NKCC1 is expressed in neurons and in all brain cells including glia (oligodendrocytes, microglia, and astrocytes) and the vasculature. In consideration of bumetanide as a repurposed drug for AD, this review evaluates its pharmaceutical properties with respect to its estimated brain levels across doses that can improve neurologic disease deficits of animal models to distinguish between NKCC1 and non-NKCC1 mechanisms. The available data indicate that bumetanide efficacy may occur at brain drug levels that are below those required for inhibition of the NKCC1 transporter which implicates non-NKCC1 brain mechansims for improvement of brain dysfunctions and memory deficits. Alternatively, peripheral bumetanide mechanisms may involve cells outside the central nervous system (e.g., in epithelia and the immune system). Clinical bumetanide doses for improved neurological deficits are reviewed. Regardless of mechanism, the efficacy of bumetanide to improve memory deficits in the APOE4 model of AD and its potential to reduce the incidence of AD provide support for clinical investigation of bumetanide as a repurposed AD therapeutic agent.
PubMed: 37601062
DOI: 10.3389/fphar.2023.1190402 -
Journal of Alzheimer's Disease : JAD 2024Alzheimer's disease (AD) is a chronic neurodegenerative disease needing effective therapeutics urgently. Sildenafil, one of the approved phosphodiesterase-5 inhibitors,...
Sildenafil as a Candidate Drug for Alzheimer's Disease: Real-World Patient Data Observation and Mechanistic Observations from Patient-Induced Pluripotent Stem Cell-Derived Neurons.
BACKGROUND
Alzheimer's disease (AD) is a chronic neurodegenerative disease needing effective therapeutics urgently. Sildenafil, one of the approved phosphodiesterase-5 inhibitors, has been implicated as having potential effect in AD.
OBJECTIVE
To investigate the potential therapeutic benefit of sildenafil on AD.
METHODS
We performed real-world patient data analysis using the MarketScan® Medicare Supplemental and the Clinformatics® databases. We conducted propensity score-stratified analyses after adjusting confounding factors (i.e., sex, age, race, and comorbidities). We used both familial and sporadic AD patient induced pluripotent stem cells (iPSC) derived neurons to evaluate the sildenafil's mechanism-of-action.
RESULTS
We showed that sildenafil usage is associated with reduced likelihood of AD across four new drug compactor cohorts, including bumetanide, furosemide, spironolactone, and nifedipine. For instance, sildenafil usage is associated with a 54% reduced incidence of AD in MarketScan® (hazard ratio [HR] = 0.46, 95% CI 0.32- 0.66) and a 30% reduced prevalence of AD in Clinformatics® (HR = 0.70, 95% CI 0.49- 1.00) compared to spironolactone. We found that sildenafil treatment reduced tau hyperphosphorylation (pTau181 and pTau205) in a dose-dependent manner in both familial and sporadic AD patient iPSC-derived neurons. RNA-sequencing data analysis of sildenafil-treated AD patient iPSC-derived neurons reveals that sildenafil specifically target AD related genes and pathobiological pathways, mechanistically supporting the beneficial effect of sildenafil in AD.
CONCLUSIONS
These real-world patient data validation and mechanistic observations from patient iPSC-derived neurons further suggested that sildenafil is a potential repurposable drug for AD. Yet, randomized clinical trials are warranted to validate the causal treatment effects of sildenafil in AD.
Topics: Aged; United States; Humans; Alzheimer Disease; Induced Pluripotent Stem Cells; Sildenafil Citrate; Neurodegenerative Diseases; Spironolactone; tau Proteins; Medicare; Neurons
PubMed: 38427489
DOI: 10.3233/JAD-231391 -
Cells Jul 2023Human cerebral organoids resemble the 3D complexity of the human brain and have the potential to augment current drug development pipelines for neurological disease....
Human cerebral organoids resemble the 3D complexity of the human brain and have the potential to augment current drug development pipelines for neurological disease. Epilepsy is a complex neurological condition characterized by recurrent seizures. A third of people with epilepsy do not respond to currently available pharmaceutical drugs, and there is not one drug that treats all subtypes; thus, better models of epilepsy are needed for drug development. Cerebral organoids may be used to address this unmet need. In the present work, human cerebral organoids are used along with electrophysiological methods to explore oxygen-glucose deprivation as a hyperexcitability agent. This activity is investigated in its response to current antiseizure drugs. Furthermore, the mechanism of action of the drug candidates is probed with qPCR and immunofluorescence. The findings demonstrate OGD-induced hyperexcitable changes in the cerebral organoid tissue, which is treated with cannabidiol and bumetanide. There is evidence for NKCC1 and KCC2 gene expression, as well as other genes and proteins involved in the complex development of GABAergic signaling. This study supports the use of organoids as a platform for modelling cerebral cortical hyperexcitability that could be extended to modelling epilepsy and used for drug discovery.
Topics: Humans; Solute Carrier Family 12, Member 2; Glucose; Epilepsy; Brain; Organoids
PubMed: 37566028
DOI: 10.3390/cells12151949 -
Frontiers in Aging 2023To investigate whether exposure history to two common loop diuretics, bumetanide and furosemide, affects the risk of developing Alzheimer's disease (AD) after...
To investigate whether exposure history to two common loop diuretics, bumetanide and furosemide, affects the risk of developing Alzheimer's disease (AD) after accounting for socioeconomic status and congestive heart failure. Individuals exposed to bumetanide or furosemide were identified in the Stanford University electronic health record using the de-identified Observational Medical Outcomes Partnership platform. We matched the AD case cohort to a control cohort (1:20 case:control) on gender, race, ethnicity, and hypertension, and controlled for variables that could potentially be collinear with bumetanide exposure and/or AD diagnosis. Among individuals older than 65 years, 5,839 AD cases and 116,103 matched controls were included. A total of 1,759 patients (54 cases and 1,705 controls) were exposed to bumetanide. After adjusting for socioeconomic status and other confounders, the exposure of bumetanide and furosemide was significantly associated with reduced AD risk (respectively, bumetanide odds ratio [OR] = 0.23; 95% confidence interval [CI], 0.15-0.36; = 4.0 × 10; furosemide OR = 0.42; 95% CI, 0.38-0.47; < 2.0 × 10). Our study replicates in an independent sample that a history of bumetanide exposure is associated with reduced AD risk while also highlighting an association of the most common loop diuretic (furosemide) with reduced AD risk. These associations need to be additionally replicated, and the mechanism of action remains to be investigated.
PubMed: 37822457
DOI: 10.3389/fragi.2023.1211571 -
CNS Drugs Sep 2023The inhibitory neurotransmitter γ-aminobutyric acid (GABA) plays an important role in the modulation of neuronal excitability, and a disruption of GABAergic... (Review)
Review
The inhibitory neurotransmitter γ-aminobutyric acid (GABA) plays an important role in the modulation of neuronal excitability, and a disruption of GABAergic transmission contributes to the pathogenesis of some seizure disorders. Although many currently available antiseizure medications do act at least in part by potentiating GABAergic transmission, there is an opportunity for further research aimed at developing more innovative GABA-targeting therapies. The present article summarises available evidence on a number of such treatments in clinical development. These can be broadly divided into three groups. The first group consists of positive allosteric modulators of GABA receptors and includes Staccato alprazolam (an already marketed benzodiazepine being repurposed in epilepsy as a potential rescue inhalation treatment for prolonged and repetitive seizures), the α2/3/5 subtype-selective agents darigabat and ENX-101, and the orally active neurosteroids ETX155 and LPCN 2101. A second group comprises two drugs already marketed for non-neurological indications, which could be repurposed as treatments for seizure disorders. These include bumetanide, a diuretic agent that has undergone clinical trials in phenobarbital-resistant neonatal seizures and for which the rationale for further development in this indication is under debate, and ivermectin, an antiparasitic drug currently investigated in a randomised double-blind trial in focal epilepsy. The last group comprises a series of highly innovative therapies, namely GABAergic interneurons (NRTX-001) delivered via stereotactic cerebral implantation as a treatment for mesial temporal lobe epilepsy, an antisense oligonucleotide (STK-001) aimed at upregulating NaV1.1 currents and restoring the function of GABAergic interneurons, currently tested in a trial in patients with Dravet syndrome, and an adenoviral vector-based gene therapy (ETX-101) scheduled for investigation in Dravet syndrome. Another agent, a subcutaneously administered neuroactive peptide (NRP2945) that reportedly upregulates the expression of GABA receptor α and β subunits is being investigated, with Lennox-Gastaut syndrome and other epilepsies as proposed indications. The diversity of the current pipeline underscores a strong interest in the GABA system as a target for new treatment development in epilepsy. To date, limited clinical data are available for these investigational treatments and further studies are required to assess their potential value in addressing unmet needs in epilepsy management.
Topics: Infant, Newborn; Humans; Epilepsy; gamma-Aminobutyric Acid; Epilepsies, Myoclonic; Epilepsies, Partial; Lennox Gastaut Syndrome; Randomized Controlled Trials as Topic
PubMed: 37603261
DOI: 10.1007/s40263-023-01025-4 -
Stem Cell Reports Jul 2023Quiescence is a hallmark of adult neural stem cells (NSCs) in the mammalian brain, and establishment and maintenance of quiescence is essential for life-long continuous...
Quiescence is a hallmark of adult neural stem cells (NSCs) in the mammalian brain, and establishment and maintenance of quiescence is essential for life-long continuous neurogenesis. How NSCs in the dentate gyrus (DG) of the hippocampus acquire their quiescence during early postnatal stages and continuously maintain quiescence in adulthood is poorly understood. Here, we show that Hopx-CreER-mediated conditional deletion of Nkcc1, which encodes a chloride importer, in mouse DG NSCs impairs both their quiescence acquisition at early postnatal stages and quiescence maintenance in adulthood. Furthermore, PV-CreER-mediated deletion of Nkcc1 in PV interneurons in the adult mouse brain leads to activation of quiescent DG NSCs, resulting in an expanded NSC pool. Consistently, pharmacological inhibition of NKCC1 promotes NSC proliferation in both early postnatal and adult mouse DG. Together, our study reveals both cell-autonomous and non-cell-autonomous roles of NKCC1 in regulating the acquisition and maintenance of NSC quiescence in the mammalian hippocampus.
Topics: Animals; Mice; Hippocampus; Neural Stem Cells; Neurogenesis; Cell Division; Dentate Gyrus; Mammals
PubMed: 37390823
DOI: 10.1016/j.stemcr.2023.05.021 -
Neuropharmacology Aug 2023Early life stress (ELS) alters the excitation-inhibition-balance (EI-balance) in various rodent brain areas and may be responsible for behavioral impairment later in...
Early life stress (ELS) alters the excitation-inhibition-balance (EI-balance) in various rodent brain areas and may be responsible for behavioral impairment later in life. The EI-balance is (amongst others) influenced by the switch of GABAergic transmission from excitatory to inhibitory, the so-called "GABA-switch". Here, we investigated how ELS affects the GABA-switch in mouse infralimbic Prefrontal Cortex layer 2/3 neurons, using the limited-nesting-and-bedding model. In ELS mice, the GABA-switch occurred already between postnatal day (P) 6 and P9, as opposed to P15-P21 in controls. This was associated with increased expression of the inward chloride transporter NKCC1, compared to the outward chloride transporter KCC2, both of which are important for the intracellular chloride concentration and, hence, the GABA reversal potential (Erev). Chloride transporters are not only important for regulating chloride concentration postsynaptically, but also presynaptically. Depending on the Erev of GABA, presynaptic GABA receptor stimulation causes a depolarization or hyperpolarization, and thereby enhanced or reduced fusion of glutamate vesicles respectively, in turn changing the frequency of miniature postsynaptic currents (mEPSCs). In accordance, bumetanide, a blocker of NKCC1, shifted the Erev GABA towards more hyperpolarized levels in P9 control mice and reduced the mEPSC frequency. Other modulators of chloride transporters, e.g. VU0463271 (a KCC2 antagonist) and aldosterone -which increases NKCC1 expression-did not affect postsynaptic Erev in ELS P9 mice, but did increase the mEPSC frequency. We conclude that the mouse GABA-switch is accelerated after ELS, affecting both the pre- and postsynaptic chloride homeostasis, the former altering glutamatergic transmission. This may considerably affect brain development.
Topics: Animals; Mice; Acceleration; Chlorides; gamma-Aminobutyric Acid; Membrane Transport Proteins; Receptors, GABA-A; Symporters; Stress, Physiological
PubMed: 37061088
DOI: 10.1016/j.neuropharm.2023.109543