Screening for drugs potentially interfering with MCT8-mediated T3 transport in vitro identifies dexamethasone and some commonly used drugs as inhibitors of MCT8 activity.

BACKGROUND: Monocarboxylate transporter 8 (MCT8) is the first thyroid hormone transporter that has been linked to a human disease. Besides genetic alterations other factors might impair MCT8 activity. AIM: This study aimed at investigating whether some common drugs having a structural similarity with TH and/or whose treatment is associated with thyroid function test abnormalities, or which behave as antagonists of TH action can inhibit MCT8-mediated T3 transport. METHODS: [125I]T3 uptake and efflux were measured in COS-7 cells transiently transfected with hMCT8 before and after exposure to increasing concentrations of hydrocortisone, dexamethasone, prednisone, prednisolone, amiodarone, desethylamiodarone, dronedarone, buspirone, carbamazepine, valproic acid, and L-carnitine. The mode of inhibition was also determined. RESULTS: Dexamethasone significantly inhibited T3 uptake at 10 µM; hydrocortisone reduced T3 uptake only at high concentrations, i.e. at 500 and 1000 µM; prednisone and prednisolone were devoid of inhibitory potential. Amiodarone caused a reduction of T3 uptake by MCT8 only at the highest concentrations used (44% at 50 µM and 68% at 100 µM), and this effect was weaker than that produced by desethylamiodarone and dronedarone; buspirone resulted a potent inhibitor, reducing T3 uptake at 0.1-10 µM. L-Carnitine inhibited T3 uptake only at 500 mM and 1 M. Kinetic experiments revealed a noncompetitive mode of inhibition for all compounds. All drugs inhibiting T3 uptake did not affect T3 release. CONCLUSION: This study shows a novel effect of some common drugs, which is inhibition of T3 transport mediated by MCT8. Specifically, dexamethasone, buspirone, desethylamiodarone, and dronedarone behave as potent inhibitors of MCT8.

An Intramolecular Ionic Interaction Linking Defective Sodium/Iodide Symporter Transport to the Plasma Membrane and Dyshormonogenic Congenital Hypothyroidism.

Background: The sodium/iodide symporter (NIS) mediates active iodide accumulation in the thyroid follicular cell. Autosomal recessive iodide transport defect (ITD)-causing loss-of-function NIS variants lead to dyshormonogenic congenital hypothyroidism due to deficient iodide accumulation for thyroid hormonogenesis. Here, we aimed to identify, and if so to functionally characterize, novel ITD-causing NIS pathogenic variants in a patient diagnosed with severe dyshormonogenic congenital hypothyroidism due to a defect in iodide accumulation in the thyroid follicular cell, as suggested by nondetectable radioiodide accumulation in a normally located thyroid gland, as well as in salivary glands. Methods: The proposita NIS-coding SLC5A5 gene was sequenced using Sanger sequencing. In silico analysis and functional in vitro characterization of the novel NIS variants were performed. Results: Sanger sequencing revealed novel compound heterozygous SLC5A5 gene variants (c.970-3C>A and c.1106A>T, p.D369V). In silico analysis suggested that c.970-3C>A disrupts the canonical splice acceptor site located in intron 7. Splicing minigene reporter assay revealed that c.970-3C>A causes exon 8 skipping during NIS pre-mRNA splicing leading to the NIS pathogenic variant p.Y324Hfs*148. Moreover, in silico analysis indicated p.D369V as pathogenic. Functional in vitro studies demonstrated that p.D369V NIS does not mediate iodide accumulation, as p.D369V causes NIS to be retained in the endoplasmic reticulum. Mechanistically, we propose an intramolecular ionic interaction involving the ß carboxyl group of D369 and the guanidinium group of R130, located in transmembrane segment 4. Of note, an Asp residue at position 369-which is highly conserved in SLC5A family members-is required for functional NIS expression at the plasma membrane. Conclusions: We uncovered a critical intramolecular interaction between R130 and D369 required for NIS maturation and plasma membrane expression. Moreover, we identified the first intronic variant causing aberrant NIS pre-mRNA splicing, thus expanding the mutational landscape in the SLC5A5 gene leading to dyshormonogenic congenital hypothyroidism.

Decreased spasticity of Baishaoluoshi Decoction through the BDNF/TrKB-KCC2 pathway on poststroke spasticity rats.

OBJECTIVE: K+-Cl- cotransporter-2 (KCC2), which primarily extrudes chloride in mature neurons, triggers hemiplegia limb spasticity after ischemic stroke by affecting neuronal excitability. Our previous study revealed that the Chinese herb Baishaoluoshi Decoction decreases hemiplegia limb spasticity in poststroke spasticity (PSS) patients. This study aimed at elucidating on the effects of Baishaoluoshi Decoction on the BDNF/TrKB-KCC2 pathway in PSS rat models. METHODS: Middle cerebral artery occlusion (MCAO) was adopted for the establishment of PSS rat models. Muscle tension was evaluated by Modified Ashworth Scale. Nissl staining and transmission electron microscopy were used to measure the protective effects of Baishaoluoshi Decoction on ischemic injury-induced neuronal damage due to MCAO. Expression levels of BDNF, TrKB, and KCC2 in brain tissues around the infarct and brainstem were detected by immunohistochemical staining. RESULTS: It was found that Baishaoluoshi Decoction suppressed hemiplegia limb spasticity and alleviated the damage in neurons and synapses in PSS rat models. Importantly, the expression of BDNF, TrKB, and KCC2 in brain tissues around the infarct and brainstem were significantly upregulated after treatment with low-dose and high-dose Baishaoluoshi Decoction. CONCLUSION: Suppression of spasticity by Baishaoluoshi Decoction in PSS rat models may be correlated with upregulated BDNF/TrKB-KCC2 pathway, which may be a complementary therapeutic strategy for PSS.

Preliminary study of analgesic effect of bumetanide on neuropathic pain in patients with spinal cord injury.

BACKGROUND/OBJECTIVE: The current study evaluated the analgesic effects of bumetanide as an adjunctive treatment in managing neuropathic pain following spinal cord injury. The peripheral expression level of Na-K-Cl-cotransporter-1 (NKCC1) and K-Cl-cotransporter-2 (KCC2) genes in polymorphonuclear lymphocytes (PMLs) assessed as a possible biomarker indicating central underlying mechanisms. METHODS: This open-label, single-arm, pilot trial of bumetanide (2 mg/day) is an add-on treatment conducted in 14 SCI patients for 19 weeks. The whole duration consisted of three phases: pre-treatment (1 month), titration (3 weeks), and active treatment (4 months). Ultimately, nine patients completed the study. The primary outcome variables were the endpoint pain score measured by the numeric rating scale (NRS), and the short-form McGill Pain Questionnaire. Secondary endpoints included the Short-Form Health Survey that measures the quality of life. Blood samples were collected and used for determining the expression of NKCC1 and KCC2 genes in transcription and translation levels. RESULTS: Bumetanide treatment significantly reduced average pain intensity according to the NRS and the short form of the McGill Pain Questionnaire scores. The baseline expression of KCC2 protein was low between groups and increased significantly following treatment (P < 0.05). Through the current study, pain improvement accompanied by the more significant mean change from the baseline for the overall quality of life. CONCLUSION: These data might be a piece of preliminary evidence for the analgesic effect of bumetanide on neuropathic pain and could support the potential role of the upregulation of KCC2 protein and involvement of GABAergic disinhibition in producing neuropathic pain.

Interference on Iodine Uptake and Human Thyroid Function by Perchlorate-Contaminated Water and Food.

BACKGROUND: Perchlorate-induced natrium-iodide symporter (NIS) interference is a well-recognized thyroid disrupting mechanism. It is unclear, however, whether a chronic low-dose exposure to perchlorate delivered by food and drinks may cause thyroid dysfunction in the long term. Thus, the aim of this review was to overview and summarize literature results in order to clarify this issue. METHODS: Authors searched PubMed/MEDLINE, Scopus, Web of Science, institutional websites and Google until April 2020 for relevant information about the fundamental mechanism of the thyroid NIS interference induced by orally consumed perchlorate compounds and its clinical consequences. RESULTS: Food and drinking water should be considered relevant sources of perchlorate. Despite some controversies, cross-sectional studies demonstrated that perchlorate exposure affects thyroid hormone synthesis in infants, adolescents and adults, particularly in the case of underlying thyroid diseases and iodine insufficiency. An exaggerated exposure to perchlorate during pregnancy leads to a worse neurocognitive and behavioral development outcome in infants, regardless of maternal thyroid hormone levels. DISCUSSION AND CONCLUSION: The effects of a chronic low-dose perchlorate exposure on thyroid homeostasis remain still unclear, leading to concerns especially for highly sensitive patients. Specific studies are needed to clarify this issue, aiming to better define strategies of detection and prevention.

The Protective Effect of Cynara Cardunculus Extract in Diet-Induced NAFLD: Involvement of OCTN1 and OCTN2 Transporter Subfamily.

Hyperlipidemia and insulin-resistance are often associated with Non-Alcoholic Fatty Liver Disease (NAFLD) thereby representing a true issue worldwide due to increased risk of developing cardiovascular and systemic disorders. Although clear evidence suggests that circulating fatty acids contribute to pathophysiological mechanisms underlying NAFLD and hyperlipidemia, further studies are required to better identify potential beneficial approaches for counteracting such a disease. Recently, several artichoke extracts have been used for both reducing hyperlipidemia, insulin-resistance and NAFLD, though the mechanism is unclear. Here we used a wild type of Cynara Cardunculus extract (CyC), rich in sesquiterpens and antioxidant active ingredients, in rats fed a High Fat Diet (HFD) compared to a Normal Fat Diet (NFD). In particular, in rats fed HFD for four consecutive weeks, we found a significant increase of serum cholesterol, triglyceride and serum glucose. This effect was accompanied by increased body weight and by histopathological features of liver steatosis. The alterations of metabolic parameters found in HFDs were antagonised dose-dependently by daily oral supplementation of rats with CyC 10 and 20 mg/kg over four weeks, an effect associated to significant improvement of liver steatosis. The effect of CyC (20 mg/kg) was also associated to enhanced expression of both OCTN1 and OCTN2 carnitine-linked transporters. Thus, present data suggest a contribution of carnitine system in the protective effect of CyC in diet-induced hyperlipidemia, insulin-resistance and NAFLD.

Effect of transforming growth factor-ß1 on functional expression of monocarboxylate transporter 1 in alveolar epithelial A549 cells.

Epithelial-mesenchymal transition (EMT) contributes to the development of severe lung diseases, such as pulmonary fibrosis. Recently, it has been reported that EMT involves complex metabolic reprogramming triggered by several factors including transforming growth factor (TGF-ß1) and that monocarboxylate transporter (MCT1) plays an essential role in these metabolic changes. The aim of the present study was to clarify the functional expression of MCT1 during TGF-ß1-induced EMT in alveolar epithelial A549 cells. The transport function of MCT1 in A549 cells was examined using [3H]γ-hydroxybutyrate (GHB) and [3H] lactic acid (LA) as substrates and α-cyano-4-hydroxycinnamate (CHC), lactic acid, phloretin, and AR-C155858 (AR) as inhibitors of MCT1. EMT was induced by treating the cells with TGF-ß1. mRNA and protein expression levels were analyzed using real-time PCR and Western blotting, respectively. Time-, temperature-, and pH-dependent GHB and LA uptake were observed in A549 cells. CHC, lactic acid, phloretin, and AR significantly inhibited the uptake of GHB in a concentration-dependent manner, suggesting that MCT1 is primarily responsible for transport of monocarboxylates such as GHB and LA in A549 cells. TGF-ß1 treatment significantly enhanced GHB and LA uptake as well as the mRNA and protein expression levels of MCT1 in A549 cells. These changes were neutralized by co-treatment with SB431542, an inhibitor for the TGF-ß1 signaling pathway. CHC and AR had no effect on TGF-ß1-induced EMT-related gene expression changes. Here, we have clearly characterized functional expression of MCT1 in A549 cells and have shown that MCT1 may be upregulated via the TGF-ß1 signaling pathway.

Tamoxifen-induced, intestinal-specific deletion of Slc5a6 in adult mice leads to spontaneous inflammation: involvement of NF-κB, NLRP3, and gut microbiota.

The sodium-dependent multivitamin transporter (SMVT; SLC5A6) is involved in intestinal absorption of vitamin B7 (biotin). We have previously shown that mice with an embryonic intestinal-specific SMVT knockout (KO) develop biotin deficiency and severe spontaneous intestinal inflammation in addition to growth retardation, developmental delays, and death within the first 6-7 wk of life. The profound morbidity and mortality associated with the SMVT-KO has limited our ability to further characterize the intestinal inflammation and other sequelae of this deletion in adult mice with a mature gut microbiota. To overcome this limitation, we generated an intestine-specific, tamoxifen-inducible, conditional SMVT-KO (SMVT-icKO). Our results showed that adult SMVT-icKO mice have reduced body weight, biotin deficiency, shorter colonic length, and bloody diarrhea compared with age- and sex-matched control littermates. All SMVT-icKO mice also developed spontaneous intestinal inflammation associated with induction of calprotectin (S100a8/S100a9), proinflammatory cytokines (IL-1ß, TNF-α, IFN-γ, and IL-6), and an increase in intestinal permeability. Additionally, the intestines of SMVT-icKO showed activation of the NF-κB pathway and the nucleotide-binding domain and leucine-rich repeat pyrin 3 domain (NLRP3) inflammasome. Notably, administration of broad-spectrum antibiotics reduced lethality and led to normalization of intestinal inflammation, proinflammatory cytokines, altered mucosal integrity, and reduced expression of the NLRP3 inflammasome. Overall, these findings support our conclusion that the biotin transport pathway plays an important role in the maintenance of intestinal homeostasis, and that NF-κB and the NLRP3 inflammasome, as well as gut microbiota, drive the development of intestinal inflammation when SMVT is absent.NEW & NOTEWORTHY This study demonstrates that deletion of the intestinal biotin uptake system in adult mice leads to the development of spontaneous gut inflammation and that luminal microbiota plays a role in its development.

Traumatic Brain Injury Temporal Proteome Guides KCC2-Targeted Therapy.

Advancing therapeutics for traumatic brain injury (TBI) remains a challenge, necessitating testable targets with interventions appropriately timed to intercede on evolving secondary insults. Neuroproteomics provides a global molecular approach to deduce the complex post-translational processes that underlie secondary events after TBI. Yet method advancement has outpaced approaches to interrogate neuroproteomic complexity, in particular when addressing the well-recognized temporal evolution of TBI pathobiology. Presented is a detailed account of the temporal neuroproteomic response to mild-moderate rat controlled cortical impact within perilesioned somatosensory neocortex across the first two weeks after injury. Further, this investigation assessed use of artificial neural network and functional enrichment analyses to discretize the temporal response across some 2047 significantly impacted proteins. Results were efficiently narrowed onto ion transporters with phenotypic relevance to abnormal GABAergic transmission and a delayed decline amenable to intervention under managed care conditions. The prototypical target potassium/chloride co-transporter 2 (KCC2 or SLC12A5) was investigated further with the KCC2-selective modulator CLP290. Guided by post-translational processing revealed one-day after insult to precede KCC2 protein loss a day after, CLP290 was highly effective at restoring up to 70% of lost KCC2 localization, which was significantly correlated with recovery of sham-level function in assessed somatosensory behavioral tasks. The timing of administration was important, with no significant improvement observed if given earlier, one-hour after insult, or later when KCC2 protein decline begins. Results portend importance for a detailed post-translational characterization when devising TBI treatments, and support the therapeutic promise of KCC2-targeted CLP290 intervention for positive functional recovery after brain injury.

Pharmaco-resistant Neonatal Seizures: Critical Mechanistic Insights from a Chemoconvulsant Model.

Neonatal seizures are harmful to the developing brain and are associated with mortality and long-term neurological comorbidities. Hypoxic-ischemic encephalopathy (HIE) seizures represent a significant proportion of such seizures. Phenobarbital (PB) remains the first line anti-seizure drug (ASD) treatment but fails ~50% of the time. Translational models of neonatal seizures are crucial to investigating mechanisms underlying PB-resistance. A model of PB-resistant ischemic seizures in postnatal day 7 (P7) CD-1 mice reported K-Cl cotransporter 2 (KCC2) degradation that has been shown to be due to activation of the TrkB pathway. We investigated PB-efficacy in a pentylenetetrazole (PTZ) model of neonatal seizures in the same strain and age using identical treatment protocols to gain insights into mechanisms underlying PB-resistance. A single dose of PTZ (80 mg/kg; IP) consistently induced repetitive seizures that did not progress to status epilepticus (SE). PB (25 mg/kg; IP, single dose) significantly suppressed the PTZ-induced seizures. This was associated with significant KCC2 upregulation and stable Na-K-Cl cotransporter 1 (NKCC1) expression at 24h. The TrkB pathway was not activated. PTZ seizure burdens were significantly higher than those reported for ischemic seizures, indicating seizure severity did not dictate the differences in PB-efficacy. Bumetanide (BTN) (0.1-0.2 mg/kg; IP) did not work as an anti-seizure agent, similar to the ischemic model. When investigating mechanisms underlying the emergence of PB-resistance in translational models, the method by which seizures are induced may dictate mechanisms underlying emergence of PB-resistance.

Effects of combined brief etomidate anesthesia and postnatal stress on amygdala expression of Cl- cotransporters and corticotropin-releasing hormone and alcohol intake in adult rats.

Early life stressors, including general anesthesia, can have adverse effects on adult neural and behavioral outcomes, such as disruptions in inhibitory signaling, stress responsivity and increased risk of psychiatric disorders. Here we used a rat model to determine the effects of combined exposure to etomidate (ET) neonatal anesthesia and maternal separation on adult amygdala expression of genes for corticotropin-releasing hormone (Crh) and the chloride co-transporters Nkcc1 and Kcc2, as well as ethanol intake. Male and female Sprague-Dawley rats were subjected to 2 h of ET anesthesia on postnatal days (P) 4, 5, or 6 followed by maternal separation for 3 h on P10 (ET + SEP). During the P91-P120 period rats had daily 2 h access to three 0.05% saccharin solutions containing 0%, 5%, or 10% ethanol, followed by gene expression analyses. The ET + SEP group had increased Crh mRNA levels and Nkcc1/Kcc2 mRNA ratios in the amygdala, with greater increases in Nkcc1/Kcc2 mRNA ratios in males. A moderate increase in 5% ethanol intake was evident in the ET + SEP males, but not females, after calculation of the ratio of alcohol intake between the last week and first week of exposure. In contrast, control males tended to decrease alcohol consumption during the same period. A brief exposure to ET combined with a subsequent episode of stress early in life induced significant alterations in expression of amygdala Crh, Nkcc1 and Kcc2 with greater changes in the Cl- transporter expression in males. The possibility of increased alcohol intake in the exposed males requires further confirmation using different alcohol intake paradigms.

Phosphoregulation of K+ -Cl- cotransporters during cell swelling: Novel insights.

The K+ -Cl- cotransporters (KCCs) belong to the cation-Cl- cotransporter family and consist of four isoforms and many splice variants. Their main role is to promote electroneutral efflux of K+ and Cl- ions across the surface of many cell types and, thereby, to regulate intracellular ion concentration, cell volume, and epithelial salt movement. These transport systems are induced by an increase in cell volume and are less active at lower intracellular [Cl- ] (Cli ), but the mechanisms at play are still ill-defined. In this work, we have exploited the Xenopus laevis expression system to study the role of lysine-deficient protein kinases (WNKs), protein phosphatases 1 (PP1s), and SPS1-related proline/alanine-rich kinase (SPAK) in KCC4 regulation during cell swelling. We have found that WNK4 and PP1 regulate KCC4 activity as part of a common signaling module, but that they do not exert their effects through SPAK or carrier dephosphorylation. We have also found that the phosphatases at play include PP1α and PP1γ1, but that WNK4 acts directly on the PP1s instead of the opposite. Unexpectedly, however, both cell swelling and a T926A substitution in the C-terminus of full-length KCC4 led to higher levels of heterologous K+ -Cl- cotransport and overall carrier phosphorylation. These results imply that the response to cell swelling must also involve allosteric-sensitive kinase-dependent phosphoacceptor sites in KCC4. They are thus partially inconsistent with previous models of KCC regulation.

Neuroprotection by Propofol Post-Conditioning: Focus on PKMζ/KCC2 Pathway Activity.

Critical and major operations are often accompanied by brain ischemic complications. Previous studies found that propofol post-conditioning provided neuroprotective functions through upregulating the expression of potassium chloride cotransporter 2 (KCC2) in gamma-aminobutyric acid (GABA) interneurons. Membrane expression and phosphorylation represents KCC2 activity, which were modulated by a protein kinase C (PKC)-dependent mechanism. However, the role of propofol in increasing KCC2 phosphorylation and the involvement of protein kinase Mζ (PKMζ), a major subtype of PKC, in the KCC2 pathway remained unclear. In this study, we established middle cerebral artery occlusion model in rats to evaluate the long-term recovery of brain functions using behavioral experiments. KCC2 and PKMζ were assessed via western blot. We used the selective inhibitor, zeta inhibitory peptide (ZIP), to investigate the relationship between KCC2 and PKMζ. Intracellular chloride concentration in the hippocampal CA1 area was measured to determine KCC2 activity. We found that propofol, infused at a speed of 20 mg kg-1 h-1 for 2 h at the onset of reperfusion, improved neurological deficits and cognitive dysfunction following ischemia/reperfusion injury. PKMζ expression was significantly upregulated, which improved KCC2 membrane expression and phosphorylation in the ischemic hippocampal CA1 area, and these effects could last up to 28 days. But ZIP inhibited this process. Ultimately, we showed that propofol increased KCC2 phosphorylation and PKMζ was the upstream of KCC2. Propofol led to long-term recovery of brain functions by upregulating the activity of the PKMζ/KCC2 pathway.

TSH/IGF-1 Receptor Cross-Talk Rapidly Activates Extracellular Signal-Regulated Kinases in Multiple Cell Types.

We previously showed that thyrotropin (TSH)/insulinlike growth factor (IGF)-1 receptor cross-talk appears to be involved in Graves' orbitopathy (GO) pathogenesis and upregulation of thyroid-specific genes in human thyrocytes. In orbital fibroblasts from GO patients, coadministration of TSH and IGF-1 induces synergistic increases in hyaluronan secretion. In human thyrocytes, TSH plus IGF-1 synergistically increased expression of the sodium-iodide symporter that appeared to involve ERK1/2 activation. However, the details of ERK1/2 activation were not known, nor was whether ERK1/2 was involved in this synergism in other cell types. Using primary cultures of GO fibroblasts (GOFs) and human thyrocytes, as well as human embryonic kidney (HEK) 293 cells overexpressing TSH receptors (HEK-TSHRs), we show that simultaneous activation of TSHRs and IGF-1 receptors (IGF-1Rs) causes rapid, synergistic phosphorylation/activation of ERK1 and ERK2 in all three cell types. This effect is partially inhibited by pertussis toxin, an inhibitor of TSHR coupling to Gi/Go proteins. In support of a role for Gi/Go proteins in ERK1/2 phosphorylation, we found that knockdown of Gi(1-3) and Go in HEK-TSHRs inhibited ERK1/2 phosphorylation stimulated by TSH and TSH plus IGF-1. These data demonstrate that the synergistic effects of TSH plus IGF-1 occur early in the TSHR signaling cascade and further support the idea that TSHR/IGF-1R cross-talk is an important mechanism for regulation of human GOFs and thyrocytes.

Prochlorperazine Increases KCC2 Function and Reduces Spasticity after Spinal Cord Injury.

In mature neurons, low intracellular chloride level required for inhibition is maintained by the potassium-chloride cotransporter, KCC2. Impairment of Cl- extrusion after KCC2 dysfunction has been involved in many central nervous system disorders, such as seizures, neuropathic pain, or spasticity, after a spinal cord injury (SCI). This makes KCC2 an appealing drug target for restoring Cl- homeostasis and inhibition in pathological conditions. In the present study, we screen the Prestwick Chemical Library® and identify conventional antipsychotics phenothiazine derivatives as enhancers of KCC2 activity. Among them, prochlorperazine hyperpolarizes the Cl- equilibrium potential in motoneurons of neonatal rats and restores the reciprocal inhibition post-SCI. The compound alleviates spasticity in chronic adult SCI rats with an efficacy equivalent to the antispastic agent, baclofen, and rescues the SCI-induced downregulation of KCC2 in motoneurons below the lesion. These pre-clinical data support prochlorperazine for a new therapeutic indication in the treatment of spasticity post-SCI and neurological disorders involving a KCC2 dysfunction.

K-Cl Cotransporter 2-mediated Cl- Extrusion Determines Developmental Stage-dependent Impact of Propofol Anesthesia on Dendritic Spines.

BACKGROUND: General anesthetics potentiating γ-aminobutyric acid (GABA)-mediated signaling are known to induce a persistent decrement in excitatory synapse number in the cerebral cortex when applied during early postnatal development, while an opposite action is produced at later stages. Here, the authors test the hypothesis that the effect of general anesthetics on synaptogenesis depends upon the efficacy of GABA receptor type A (GABAA)-mediated inhibition controlled by the developmental up-regulation of the potassium-chloride (K-Cl) cotransporter 2 (KCC2). METHODS: In utero electroporation of KCC2 was used to prematurely increase the efficacy of (GABAA)-mediated inhibition in layer 2/3 pyramidal neurons in the immature rat somatosensory cortex. Parallel experiments with expression of the inward-rectifier potassium channel Kir2.1 were done to reduce intrinsic neuronal excitability. The effects of these genetic manipulations (n = 3 to 4 animals per experimental group) were evaluated using iontophoretic injection of Lucifer Yellow (n = 8 to 12 cells per animal). The total number of spines analyzed per group ranged between 907 and 3,371. RESULTS: The authors found a robust effect of the developmental up-regulation of KCC2-mediated Cl transport on the age-dependent action of propofol on dendritic spines. Premature expression of KCC2, unlike expression of a transport-inactive KCC2 variant, prevented a propofol-induced decrease in spine density. In line with a reduction in neuronal excitability, the above result was qualitatively replicated by overexpression of Kir2.1. CONCLUSIONS: The KCC2-dependent developmental increase in the efficacy of GABAA-mediated inhibition is a major determinant of the age-dependent actions of propofol on dendritic spinogenesis.

pKa of Glu325 in LacY.

Lactose permease (LacY), a paradigm for the largest family of membrane transport proteins, catalyzes the coupled translocation of a galactoside and a H+ across the cytoplasmic membrane of Escherichia coli (galactoside/H+ symport). One of the most important aspects of the mechanism is the relationship between protonation and binding of the cargo galactopyranoside. In this regard, it has been shown that protonation is required for binding. Furthermore when galactoside affinity is measured as a function of pH, an apparent pK (pKapp) of ∼10.5 is obtained. Strikingly, when Glu325, a residue long known to be involved in coupling between H+ and sugar translocation, is replaced with a neutral side chain, the pH effect is abolished, and high-affinity binding is observed until LacY is destabilized at alkaline pH. In this paper, infrared spectroscopy is used to identify Glu325 in situ. Moreover, it is demonstrated that this residue exhibits a pKa of 10.5 ± 0.1 that is insensitive to the presence of galactopyranoside. Thus, it is apparent that protonation of Glu325 specifically is required for effective sugar binding to LacY.

Depolarizing γ-aminobutyric acid contributes to glutamatergic network rewiring in epilepsy.

OBJECTIVE: Rewiring of excitatory glutamatergic neuronal circuits is a major abnormality in epilepsy. Besides the rewiring of excitatory circuits, an abnormal depolarizing γ-aminobutyric acidergic (GABAergic) drive has been hypothesized to participate in the epileptogenic processes. However, a remaining clinically relevant question is whether early post-status epilepticus (SE) evoked chloride dysregulation is important for the remodeling of aberrant glutamatergic neuronal circuits. METHODS: Osmotic minipumps were used to infuse intracerebrally a specific inhibitor of depolarizing GABAergic transmission as well as a functionally blocking antibody toward the pan-neurotrophin receptor p75 (p75NTR ). The compounds were infused between 2 and 5 days after pilocarpine-induced SE. Immunohistochemistry for NKCC1, KCC2, and ectopic recurrent mossy fiber (rMF) sprouting as well as telemetric electroencephalographic and electrophysiological recordings were performed at day 5 and 2 months post-SE. RESULTS: Blockade of NKCC1 after SE with the specific inhibitor bumetanide restored NKCC1 and KCC2 expression, normalized chloride homeostasis, and significantly reduced the glutamatergic rMF sprouting within the dentate gyrus. This mechanism partially involves p75NTR signaling, as bumetanide application reduced SE-induced p75NTR expression and functional blockade of p75NTR decreased rMF sprouting. The early transient (3 days) post-SE infusion of bumetanide reduced rMF sprouting and recurrent seizures in the chronic epileptic phase. INTERPRETATION: Our findings show that early post-SE abnormal depolarizing GABA and p75NTR signaling fosters a long-lasting rearrangement of glutamatergic network that contributes to the epileptogenic process. This finding defines promising and novel targets to constrain reactive glutamatergic network rewiring in adult epilepsy. Ann Neurol 2017;81:251-265.

TAZ Induction Directs Differentiation of Thyroid Follicular Cells from Human Embryonic Stem Cells.

OBJECTIVE: The differentiation program for human thyroid follicular cells (TFCs) relies on the interplay between sequence-specific transcription factors and transcriptional co-regulators. Transcriptional co-activator with PDZ-binding motif (TAZ) is a co-activator that regulates several transcription factors, including PAX8 and NKX2-1, which play a central role in thyroid-specific gene transcription. TAZ and PAX8/NKX2-1 are co-expressed in the nuclei of thyroid cells, and TAZ interacts directly with both PAX8 and NKX2-1, leading to their enhanced transcriptional activity on the thyroglobulin (TG) promoter and additional genes. METHODS: The use of a small molecule, ethacridine, recently identified as a TAZ activator, in the differentiation of thyroid cells from human embryonic stem (hES) cells was studied. First, endodermal cells were derived from hES cells using Activin A, followed by induction of differentiation into thyroid cells directed by ethacridine and thyrotropin (TSH). RESULTS: The expression of TAZ was increased in the Activin A-derived endodermal cells by ethacridine in a dose-dependent manner and followed by increases in PAX8 and NKX2-1 when assessed by both quantitative polymerase chain reaction and immunostaining. Following further differentiation with the combination of ethacridine and TSH, the thyroid-specific genes TG, TPO, TSHR, and NIS were all induced in the differentiated hES cells. When these cells were cultured with extracellular matrix-coated dishes, thyroid follicle formation and abundant TG protein expression were observed. Furthermore, such hES cell-derived thyroid follicles showed a marked TSH-induced and dose-dependent increase in radioiodine uptake and protein-bound iodine accumulation. CONCLUSION: These data show that fully functional human thyroid cells can be derived from hES cells using ethacridine, a TAZ activator, which induces thyroid-specific gene expression and promotes thyroid cell differentiation from the hES cells. These studies again demonstrate the importance of transcriptional regulation in thyroid cell development. This approach also yields functional human thyrocytes, without any gene transfection or complex culture conditions, by directly manipulating the transcriptional machinery without interfering with intermediate signaling events.