Noise-induced hearing loss alters potassium-chloride cotransporter KCC2 and GABA inhibition in the auditory centers.

Homeostatic plasticity, the ability of neurons to maintain their averaged activity constant around a set point value, is thought to account for the central hyperactivity after hearing loss. Here, we investigated the putative role of GABAergic neurotransmission in this mechanism after a noise-induced hearing loss larger than 50 dB in high frequencies in guinea pigs. The effect of GABAergic inhibition is linked to the normal functioning of K + -Cl- co-transporter isoform 2 (KCC2) which maintains a low intracellular concentration of chloride. The expression of membrane KCC2 were investigated before and after noise trauma in the ventral and dorsal cochlear nucleus (VCN and DCN, respectively) and in the inferior colliculus (IC). Moreover, the effect of gabazine (GBZ), a GABA antagonist, was also studied on the neural activity in IC. We show that KCC2 is downregulated in VCN, DCN and IC 3 days after noise trauma, and in DCN and IC 30 days after the trauma. As expected, GBZ application in the IC of control animals resulted in an increase of spontaneous and stimulus-evoked activity. In the noise exposed animals, on the other hand, GBZ application decreased the stimulus-evoked activity in IC neurons. The functional implications of these central changes are discussed.

LPS-induced monocarboxylate transporter-1 inhibition facilitates lactate accumulation triggering epithelial-mesenchymal transformation and pulmonary fibrosis.

The epithelial-mesenchymal transformation (EMT) process of alveolar epithelial cells is recognized as involved in the development of pulmonary fibrosis. Recent evidence has shown that lipopolysaccharide (LPS)-induced aerobic glycolysis of lung tissue and elevated lactate concentration are associated with the pathogenesis of sepsis-associated pulmonary fibrosis. However, it is uncertain whether LPS promotes the development of sepsis-associated pulmonary fibrosis by promoting lactate accumulation in lung tissue, thereby initiating EMT process. We hypothesized that monocarboxylate transporter-1 (MCT1), as the main protein for lactate transport, may be crucial in the pathogenic process of sepsis-associated pulmonary fibrosis. We found that high concentrations of lactate induced EMT while moderate concentrations did not. Besides, we demonstrated that MCT1 inhibition enhanced EMT process in MLE-12 cells, while MCT1 upregulation could reverse lactate-induced EMT. LPS could promote EMT in MLE-12 cells through MCT1 inhibition and lactate accumulation, while this could be alleviated by upregulating the expression of MCT1. In addition, the overexpression of MCT1 prevented LPS-induced EMT and pulmonary fibrosis in vivo. Altogether, this study revealed that LPS could inhibit the expression of MCT1 in mouse alveolar epithelial cells and cause lactate transport disorder, which leads to lactate accumulation, and ultimately promotes the process of EMT and lung fibrosis.

Enhancing the anticancer effect of androgen deprivation therapy by monocarboxylate transporter 1 inhibitor in prostate cancer cells.

BACKGROUND: Tumor initiation and progression necessitate a metabolic shift in cancer cells. Consequently, the progression of prostate cancer (PCa), a leading cause of cancer-related deaths in males globally, involves a shift from lipogenic to glycolytic metabolism. Androgen deprivation therapy (ADT) serves as the standard treatment for advanced-stage PCa. However, despite initial patient responses, castrate resistance emerges ultimately, necessitating novel therapeutic approaches. Therefore, in this study, we aimed to investigate the role of monocarboxylate transporters (MCTs) in PCa post-ADT and evaluate their potential as therapeutic targets. METHODS: PCa cells (LNCaP and C4-2 cell line), which has high prostate-specific membrane antigen (PSMA) and androgen receptor (AR) expression among PCa cell lines, was used in this study. We assessed the expression of MCT1 in PCa cells subjected to ADT using charcoal-stripped bovine serum (CSS)-containing medium or enzalutamide (ENZ). Furthermore, we evaluated the synergistic anticancer effects of combined treatment with ENZ and SR13800, an MCT1 inhibitor. RESULTS: Short-term ADT led to a significant upregulation in folate hydrolase 1 (FOLH1) and solute carrier family 16 member 1 (SLC16A1) gene levels, with elevated PSMA and MCT1 protein levels. Long-term ADT induced notable changes in cell morphology with further upregulation of FOLH1/PSMA and SLC16A1/MCT1 levels. Treatment with ENZ, a nonsteroidal anti-androgen, also increased PSMA and MCT1 expression. However, combined therapy with ENZ and SR13800 led to reduced PSMA level, decreased cell viability, and suppressed expression of cancer stem cell markers and migration indicators. Additionally, analysis of human PCa tissues revealed a positive correlation between PSMA and MCT1 expression in tumor regions. CONCLUSIONS: Our results demonstrate that ADT led to a significant upregulation in MCT1 levels. However, the combination of ENZ and SR13800 demonstrated a promising synergistic anticancer effect, highlighting a potential therapeutic significance for patients with PCa undergoing ADT.

Discovery of Highly Potent Solute Carrier 13 Member 5 (SLC13A5) Inhibitors for the Treatment of Hyperlipidemia.

In the face of escalating metabolic disease prevalence, largely driven by modern lifestyle factors, this study addresses the critical need for novel therapeutic approaches. We have identified the sodium-coupled citrate transporter (NaCT or SLC13A5) as a target for intervention. Utilizing rational drug design, we developed a new class of SLC13A5 inhibitors, anchored by the hydroxysuccinic acid scaffold, refining the structure of PF-06649298. Among these, LBA-3 emerged as a standout compound, exhibiting remarkable potency with an IC50 value of 67 nM, significantly improving upon PF-06649298. In vitro assays demonstrated LBA-3's efficacy in reducing triglyceride levels in OPA-induced HepG2 cells. Moreover, LBA-3 displayed superior pharmacokinetic properties and effectively lowered triglyceride and total cholesterol levels in diverse mouse models (PCN-stimulated and starvation-induced), without detectable toxicity. These findings not only spotlight LBA-3 as a promising candidate for hyperlipidemia treatment but also exemplify the potential of targeted molecular design in advancing metabolic disorder therapeutics.

Monocarboxylate Transporters 1 and 2 Are Responsible for L-Lactate Uptake in Differentiated Human Neuroblastoma SH-SY5Y Cells.

L-Lactate transport via monocarboxylate transporters (MCTs) in the central nervous system, represented by the astrocyte-neuron lactate shuttle (ANLS), is crucial for the maintenance of brain functions, including memory formation. Previously, we have reported that MCT1 contributes to L-lactate transport in normal human astrocytes. Therefore, in this study, we aimed to identify transporters that contribute to L-lactate transport in human neurons. SH-SY5Y cells, which are used as a model for human neurons, were differentiated using all-trans-retinoic acid. L-Lactate uptake was measured using radiolabeled L-lactate, and the expression of MCT proteins was confirmed Western blotting. L-Lactate transport was pH-dependent and saturated at high concentrations. Kinetic analysis suggested that L-lactate uptake was biphasic. Furthermore, MCT1, 2 selective inhibitors inhibited L-lactate transport. In addition, the expression of MCT1 and 2 proteins, but not MCT4, was confirmed. In this study, we demonstrated that MCT1 and 2 are major contributors to L-lactate transport in differentiated human neuroblastoma SH-SY5Y cells from the viewpoint of kinetic analysis. These results lead to a better understanding of ANLS in humans, and further exploration of the factors that can promote MCT1 and 2 functions is required.

Oncolytic Adenovirus for the Targeting of Paclitaxel-Resistant Breast Cancer Stem Cells.

Adjuvant systemic therapies effectively reduce the risk of breast cancer recurrence and metastasis, but therapy resistance can develop in some patients due to breast cancer stem cells (BCSCs). Oncolytic adenovirus (OAd) represents a promising therapeutic approach as it can specifically target cancer cells. However, its potential to target BCSCs remains unclear. Here, we evaluated a Cox-2 promoter-controlled, Ad5/3 fiber-modified OAd designed to encode the human sodium iodide symporter (hNIS) in breast cancer models. To confirm the potential of OAds to target BCSCs, we employed BCSC-enriched estrogen receptor-positive (ER+) paclitaxel-resistant (TaxR) cells and tumorsphere assays. OAd-hNIS demonstrated significantly enhanced binding and superior oncolysis in breast cancer cells, including ER+ cells, while exhibiting no activity in normal mammary epithelial cells. We observed improved NIS expression as the result of adenovirus death protein deletion. OAd-hNIS demonstrated efficacy in targeting TaxR BCSCs, exhibiting superior killing and hNIS expression compared to the parental cells. Our vector was capable of inhibiting tumorsphere formation upon early infection and reversing paclitaxel resistance in TaxR cells. Importantly, OAd-hNIS also destroyed already formed tumorspheres seven days after their initiation. Overall, our findings highlight the promise of OAd-hNIS as a potential tool for studying and targeting ER+ breast cancer recurrence and metastasis.

Spatiotemporal expression of thyroid hormone transporter MCT8 and THRA mRNA in human cerebral organoids recapitulating first trimester cortex development.

Thyroid hormones (TH) play critical roles during nervous system development and patients carrying coding variants of MCT8 (monocarboxylate transporter 8) or THRA (thyroid hormone receptor alpha) present a spectrum of neurological phenotypes resulting from perturbed local TH action during early brain development. Recently, human cerebral organoids (hCOs) emerged as powerful in vitro tools for disease modelling recapitulating key aspects of early human cortex development. To begin exploring prospects of this model for thyroid research, we performed a detailed characterization of the spatiotemporal expression of MCT8 and THRA in developing hCOs. Immunostaining showed MCT8 membrane expression in neuronal progenitor cell types including early neuroepithelial cells, radial glia cells (RGCs), intermediate progenitors and outer RGCs. In addition, we detected robust MCT8 protein expression in deep layer and upper layer neurons. Spatiotemporal SLC16A2 mRNA expression, detected by fluorescent in situ hybridization (FISH), was highly concordant with MCT8 protein expression across cortical cell layers. FISH detected THRA mRNA expression already in neuroepithelium before the onset of neurogenesis. THRA mRNA expression remained low in the ventricular zone, increased in the subventricular zone whereas strong THRA expression was observed in excitatory neurons. In combination with a robust up-regulation of known T3 response genes following T3 treatment, these observations show that hCOs provide a promising and experimentally tractable model to probe local TH action during human cortical neurogenesis and eventually to model the consequences of impaired TH function for early cortex development.

Preconditioning-Induced Facilitation of Lactate Release from Astrocytes Is Essential for Brain Ischemic Tolerance.

A sublethal ischemic episode [termed preconditioning (PC)] protects neurons in the brain against a subsequent severe ischemic injury. This phenomenon is known as brain ischemic tolerance and has received much attention from researchers because of its robust neuroprotective effects. We have previously reported that PC activates astrocytes and subsequently upregulates P2X7 receptors, thereby leading to ischemic tolerance. However, the downstream signals of P2X7 receptors that are responsible for PC-induced ischemic tolerance remain unknown. Here, we show that PC-induced P2X7 receptor-mediated lactate release from astrocytes has an indispensable role in this event. Using a transient focal cerebral ischemia model caused by middle cerebral artery occlusion, extracellular lactate levels during severe ischemia were significantly increased in mice who experienced PC; this increase was dependent on P2X7 receptors. In addition, the intracerebroventricular injection of lactate protected against cerebral ischemic injury. In in vitro experiments, although stimulation of astrocytes with the P2X7 receptor agonist BzATP had no effect on the protein levels of monocarboxylate transporter (MCT) 1 and MCT4 (which are responsible for lactate release from astrocytes), BzATP induced the plasma membrane translocation of these MCTs via their chaperone CD147. Importantly, CD147 was increased in activated astrocytes after PC, and CD147-blocking antibody abolished the PC-induced facilitation of astrocytic lactate release and ischemic tolerance. Taken together, our findings suggest that astrocytes induce ischemic tolerance via P2X7 receptor-mediated lactate release.

A potassium-chloride co-transporter with altered genome architecture functions as a suppressor in glioma.

Gliomas, the most lethal tumours in brain, have a poor prognosis despite accepting standard treatment. Limited benefits from current therapies can be attributed to genetic, epigenetic and microenvironmental cues that affect cell programming and drive tumour heterogeneity. Through the analysis of Hi-C data, we identified a potassium-chloride co-transporter SLC12A5 associated with disrupted topologically associating domain which was downregulated in tumour tissues. Multiple independent glioma cohorts were included to analyse the characterization of SLC12A5 and found it was significantly associated with pathological features, prognostic value, genomic alterations, transcriptional landscape and drug response. We constructed two SLC12A5 overexpression cell lines to verify the function of SLC12A5 that suppressed tumour cell proliferation and migration in vitro. In addition, SLC12A5 was also positively associated with GABAA receptor activity and negatively associated with pro-tumour immune signatures and immunotherapy response. Collectively, our study provides a comprehensive characterization of SLC12A5 in glioma and supports SLC12A5 as a potential suppressor of disease progression.

Placental MFSD2A expression in fetal growth restriction and maternal and fetal DHA status.

INTRODUCTION: Fetal growth restriction (FGR) may affect placental transfer of key nutrients to the fetus, such as the fatty acid docosahexaenoic acid (DHA). Major facilitator superfamily domain containing 2A (MFSD2A) has been described as a specific DHA carrier in placenta, but its expression has not been studied in FGR. The aim of this study was to evaluate for the first time the placental MFSD2A levels in late-FGR pregnancies and the maternal and cord plasma DHA. METHODS: 87 pregnant women from a tertial reference center were classified into late-FGR (N = 18) or control (N = 69). Fatty acid profile was determined in maternal and cord venous plasma, as well as placental levels of MFSD2A and of insulin mediators like phospho-protein kinase B (phospho-AKT) and phospho-extracellular regulated kinase (phospho-ERK). RESULTS: Maternal fatty acid profile did not differ between groups. Nevertheless, late-FGR cord vein presented higher content of saturated fatty acids than control, producing a concomitant decrease in the percentage of some unsaturated fatty acids. In the late-FGR group, a lower DHA fetal/maternal ratio was observed when using percentages, but not with concentrations. No alterations were found in the expression of MFSD2A in late-FGR placentas, nor in phospho-AKT or phospho-ERK. DISCUSSION: MFSD2A protein expression was not altered in late-FGR placentas, in line with no differences in cord DHA concentration between groups. The increase in the saturated fatty acid content of late-FGR cord might be a compensatory mechanism to ensure fetal energy supply, decreasing other fatty acids percentage. Future studies are warranted to elucidate if altered saturated fatty acid profile in late-FGR fetuses might predispose them to postnatal catch-up and to long-term health consequences.

Sodium iodide symporter immunolabelling as a predictor of clinical iodide uptake in canine thyroid carcinoma: A preliminary study.

Thyroid follicular tumours may take up iodide via the sodium-iodide symporter. Knowledge of iodide uptake could then allow treatment with I-131 in dogs with high-risk tumours. The objective of this study was to determine the relationship between clinically detectable iodide uptake (as determined by scintigraphy and/or thyroxine concentrations) and sodium iodide symporter immunohistochemical labelling on histologically fixed thyroid tumours. Nineteen dogs were identified who were diagnosed with thyroid carcinoma and underwent surgery from November 2017 to July 2021. All had recorded thyroid hormone concentrations and were hyperthyroid and/or underwent preoperative nuclear imaging using planar scintigraphy (technetium-99m or I-123), or I-124 PET-CT. All dogs subsequently underwent surgery to remove the thyroid mass. Twenty-two tumours were submitted for histopathologic analysis immediately following surgery, which confirmed a diagnosis of thyroid carcinoma for each tumour. Images and/or thyroid hormone concentrations were reviewed for the included cases, and tumours were sorted into an avid/functional group (group 1) and a non-avid/functional group (group 2). The tumour tissues were re-examined histologically using sodium iodide symporter (NIS) immunohistochemistry (IHC). Group 1 contained 15 avid/functional tumours. Twelve of these tumours had membranous NIS IHC labelling. Group 2 contained 7 non-avid tumours. One of these tumours had membranous NIS IHC labelling. This resulted in an overall sensitivity and specificity for identification of avid/functional tumours with membranous NIS of 80.0% and 85.7%, respectively. NIS IHC may predict ion trapping in canine follicular thyroid tumours. Further studies using iodide-based imaging are warranted to better determine the clinical utility of this diagnostic modality.

Structure of antiviral drug bulevirtide bound to hepatitis B and D virus receptor protein NTCP.

Cellular entry of the hepatitis B and D viruses (HBV/HDV) requires binding of the viral surface polypeptide preS1 to the hepatobiliary transporter Na+-taurocholate co-transporting polypeptide (NTCP). This interaction can be blocked by bulevirtide (BLV, formerly Myrcludex B), a preS1 derivative and approved drug for treating HDV infection. Here, to elucidate the basis of this inhibitory function, we determined a cryo-EM structure of BLV-bound human NTCP. BLV forms two domains, a plug lodged in the bile salt transport tunnel of NTCP and a string that covers the receptor's extracellular surface. The N-terminally attached myristoyl group of BLV interacts with the lipid-exposed surface of NTCP. Our structure reveals how BLV inhibits bile salt transport, rationalizes NTCP mutations that decrease the risk of HBV/HDV infection, and provides a basis for understanding the host specificity of HBV/HDV. Our results provide opportunities for structure-guided development of inhibitors that target HBV/HDV docking to NTCP.

Characterisation of anhydro-sialic acid transporters from mucosa-associated bacteria.

Sialic acid (Sia) transporters are critical to the capacity of host-associated bacteria to utilise Sia for growth and/or cell surface modification. While N-acetyl-neuraminic acid (Neu5Ac)-specific transporters have been studied extensively, little is known on transporters dedicated to anhydro-Sia forms such as 2,7-anhydro-Neu5Ac (2,7-AN) or 2,3-dehydro-2-deoxy-Neu5Ac (Neu5Ac2en). Here, we used a Sia-transport-null strain of Escherichia coli to investigate the function of members of anhydro-Sia transporter families previously identified by computational studies. First, we showed that the transporter NanG, from the Glycoside-Pentoside-Hexuronide:cation symporter family, is a specific 2,7-AN transporter, and identified by mutagenesis a crucial functional residue within the putative substrate-binding site. We then demonstrated that NanX transporters, of the Major Facilitator Superfamily, also only transport 2,7-AN and not Neu5Ac2en nor Neu5Ac. Finally, we provided evidence that SiaX transporters, of the Sodium-Solute Symporter superfamily, are promiscuous Neu5Ac/Neu5Ac2en transporters able to acquire either substrate equally well. The characterisation of anhydro-Sia transporters expands our current understanding of prokaryotic Sia metabolism within host-associated microbial communities.

Expression of the monocarboxylate transporter MCT1 is required for virus-specific mouse CD8+ T cell memory development.

Lactate-proton symporter monocarboxylate transporter 1 (MCT1) facilitates lactic acid export from T cells. Here, we report that MCT1 is mandatory for the development of virus-specific CD8+ T cell memory. MCT1-deficient T cells were exposed to acute pneumovirus (pneumonia virus of mice, PVM) or persistent γ-herpesvirus (Murid herpesvirus 4, MuHV-4) infection. MCT1 was required for the expansion of virus-specific CD8+ T cells and the control of virus replication in the acute phase of infection. This situation prevented the subsequent development of virus-specific T cell memory, a necessary step in containing virus reactivation during γ-herpesvirus latency. Instead, persistent active infection drove virus-specific CD8+ T cells toward functional exhaustion, a phenotype typically seen in chronic viral infections. Mechanistically, MCT1 deficiency sequentially impaired lactic acid efflux from activated CD8+ T cells, caused an intracellular acidification inhibiting glycolysis, disrupted nucleotide synthesis in the upstream pentose phosphate pathway, and halted cell proliferation which, ultimately, promoted functional CD8+ T cell exhaustion instead of memory development. Taken together, our data demonstrate that MCT1 expression is mandatory for inducing T cell memory and controlling viral infection by CD8+ T cells.

Cryo-EM structures of the human NaS1 and NaDC1 transporters revealed the elevator transport and allosteric regulation mechanism.

The solute carrier 13 (SLC13) family comprises electrogenic sodium ion-coupled anion cotransporters, segregating into sodium ion-sulfate cotransporters (NaSs) and sodium ion-di- and-tricarboxylate cotransporters (NaDCs). NaS1 and NaDC1 regulate sulfate homeostasis and oxidative metabolism, respectively. NaS1 deficiency affects murine growth and fertility, while NaDC1 affects urinary citrate and calcium nephrolithiasis. Despite their importance, the mechanisms of substrate recognition and transport remain insufficiently characterized. In this study, we determined the cryo-electron microscopy structures of human NaS1, capturing inward-facing and combined inward-facing/outward-facing conformations within a dimer both in apo and sulfate-bound states. In addition, we elucidated NaDC1's outward-facing conformation, encompassing apo, citrate-bound, and N-(p-amylcinnamoyl) anthranilic acid (ACA) inhibitor-bound states. Structural scrutiny illuminates a detailed elevator mechanism driving conformational changes. Notably, the ACA inhibitor unexpectedly binds primarily anchored by transmembrane 2 (TM2), Loop 10, TM11, and TM6a proximate to the cytosolic membrane. Our findings provide crucial insights into SLC13 transport mechanisms, paving the way for future drug design.

Design and development of novel self-assembled catechol-modified bile acid conjugates as pH-responsive apical sodium-dependent bile acid transporter targeting nanoparticles.

Catechol-based biomaterials demonstrate biocompatibility, making them suitable for a wide range of therapeutic applications when integrated into various molecular frameworks. However, the development of orally available catechol-based biomaterials has been hindered by significant pH variations and complex interactions in the gastrointestinal (GI) tract. In this study, we introduce a novel catechol-modified bile acid (CMBA), which is synthesized by anchoring the FDA-approved drug, ursodeoxycholic acid to the neurotransmitter dopamine. This modification could form a new apical sodium-dependent bile acid transporter (ASBT) inhibitor (ASBTi) due to the bile acid moiety. The computational analysis using the TRAnsient Pockets in Proteins (TRAPP) module, coupled with MD simulations, revealed that CMBA exhibits a strong binding affinity at residues 51-55 of ASBT with a low inhibitory constant (Ki) value. Notably, in slightly alkaline biological conditions, CMBA molecules self-assemble into carrier-free nanoparticles with an average size of 240.2 ± 44.2 nm, while maintaining their ability to bind with ASBT. When administered orally, CMBA accumulates in the ileum and liver over 24 h, exhibiting significant therapeutic effects on bile acid (BA) metabolism in a high-fat diet (HFD)-fed mouse model. This study underscores the therapeutic potential of the newly developed catechol-based, pH-responsive ASBT-inhibiting nanoparticles presenting a promising avenue for advancing therapy.

Elevation of hypothalamic ketone bodies induces a decrease in energy expenditures and an increase risk of metabolic disorder.

OBJECTIVE: Ketone bodies (such as ß-hydroxybutyrate or BHB) have been recently proposed as signals involved in brain regulation of energy homeostasis and obesity development. However, the precise role of ketone bodies sensing by the brain, and its impact on metabolic disorder development remains unclear. Nevertheless, partial deletion of the ubiquitous ketone bodies transporter MCT1 in mice (HE mice) results in diet-induced obesity resistance, while there is no alteration under normal chow diet. These results suggest that ketone bodies produced during the high fat diet would be important signals involved in obesity onset. METHODS: In the present study we used a specific BHB infusion of the hypothalamus and analyzed the energy homeostasis of WT or HE mice fed a normal chow diet. RESULTS: Our results indicate that high BHB levels sensed by the hypothalamus disrupt the brain regulation of energy homeostasis. This brain control dysregulation leads to peripheral alterations of energy expenditure mechanisms. CONCLUSIONS: Altogether, the changes induced by high ketone bodies levels sensed by the brain increase the risk of obesity onset in mice.

Sodium-iodide symporter and its related solute carriers in thyroid cancer.

The solute carrier (SLC) family is a large group of membrane transport proteins. Their dysfunction plays an important role in the pathogenesis of thyroid cancer. The most well-known SLC is the sodium-iodide symporter (NIS), also known as sodium/iodide co-transporter or solute carrier family 5 member 5 (SLC5A5) in thyroid cancer. The dysregulation of NIS in thyroid cancer is well documented. The role of NIS in the uptake of iodide is critical in the treatment of thyroid cancer, radioactive iodide (RAI) therapy in particular. In addition to NIS, other SLC members may affect the autophagy, proliferation, and apoptosis of thyroid cancer cells, indicating that an alteration in SLC members may affect different cellular events in the evolution of thyroid cancer. The expression of the SLC members may impact the uptake of chemicals by the thyroid, suggesting that targeting SLC members may be a promising therapeutic strategy in thyroid cancer.

The expression system influences stability, maturation efficiency, and oligomeric properties of the potassium-chloride co-transporter KCC2.

The neuron-specific K+/Cl- co-transporter 2, KCC2, which is critical for brain development, regulates γ-aminobutyric acid-dependent inhibitory neurotransmission. Consistent with its function, mutations in KCC2 are linked to neurodevelopmental disorders, including epilepsy, schizophrenia, and autism. KCC2 possesses 12 transmembrane spans and forms an intertwined dimer. Based on its complex architecture and function, reduced cell surface expression and/or activity have been reported when select disease-associated mutations are present in the gene encoding the protein, SLC12A5. These data suggest that KCC2 might be inherently unstable, as seen for other complex polytopic ion channels, thus making it susceptible to cellular quality control pathways that degrade misfolded proteins. To test these hypotheses, we examined KCC2 stability and/or maturation in five model systems: yeast, HEK293 cells, primary rat neurons, and rat and human brain synaptosomes. Although studies in yeast revealed that KCC2 is selected for endoplasmic reticulum-associated degradation (ERAD), experiments in HEK293 cells supported a more subtle role for ERAD in maintaining steady-state levels of KCC2. Nevertheless, this system allowed for an analysis of KCC2 glycosylation in the ER and Golgi, which serves as a read-out for transport through the secretory pathway. In turn, KCC2 was remarkably stable in primary rat neurons, suggesting that KCC2 folds efficiently in more native systems. Consistent with these data, the mature glycosylated form of KCC2 was abundant in primary rat neurons as well as in rat and human brain. Together, this work details the first insights into the influence that the cellular and membrane environments have on several fundamental KCC2 properties, acknowledges the advantages and disadvantages of each system, and helps set the stage for future experiments to assess KCC2 in a normal or disease setting.

Cryptochrome PtCPF1 regulates high temperature acclimation of marine diatoms through coordination of iron and phosphorus uptake.

Increasing ocean temperatures threaten the productivity and species composition of marine diatoms. High temperature response and regulation are important for the acclimation of marine diatoms to such environments. However, the molecular mechanisms behind their acclimation to high temperature are still largely unknown. In this study, the abundance of PtCPF1 homologs (a member of the cryptochrome-photolyase family in the model diatom Phaeodactylum tricornutum) transcripts in marine phytoplankton is shown to increase with rising temperature based on Tara Oceans datasets. Moreover, the expression of PtCPF1 in P. tricornutum at high temperature (26 °C) was much higher than that at optimum temperature (20 °C). Deletion of PtCPF1 in P. tricornutum disrupted the expression of genes encoding two phytotransferrins (ISIP2A and ISIP1) and two Na+/P co-transporters (PHATRDRAFT_47667 and PHATRDRAFT_40433) at 26 °C. This further impacted the uptake of Fe and P, and eventually caused the arrest of cell division. Gene expression, Fe and P uptake, and cell division were restored by rescue with the native PtCPF1 gene. Furthermore, PtCPF1 interacts with two putative transcription factors (BolA and TF IIA) that potentially regulate the expression of genes encoding phytotransferrins and Na+/P co-transporters. To the best of our knowledge, this is the first study to reveal PtCPF1 as an essential regulator in the acclimation of marine diatoms to high temperature through the coordination of Fe and P uptake. Therefore, these findings help elucidate how marine diatoms acclimate to high temperature.