Central Channelopathies in Obesity.

As obesity has raised heightening awareness, researchers have attempted to identify potential targets that can be treated for therapeutic intervention. Focusing on the central nervous system (CNS), the key organ in maintaining energy balance, a plethora of ion channels that are expressed in the CNS have been inspected and determined through manipulation in different hypothalamic neural subpopulations for their roles in fine-tuning neuronal activity on energy state alterations, possibly acting as metabolic sensors. However, a remaining gap persists between human clinical investigations and mouse studies. Despite having delineated the pathways and mechanisms of how the mouse study-identified ion channels modulate energy homeostasis, only a few targets overlap with the obesity-related risk genes extracted from human genome-wide association studies. Here, we present the most recently discovered CNS-specific metabolism-correlated ion channels using reverse and forward genetics approaches in mice and humans, respectively, in the hope of illuminating the prospects for future therapeutic development.

Genotype-phenotype correlation in Taiwanese children with diazoxide-unresponsive congenital hyperinsulinism.

Objective: Congenital hyperinsulinism (CHI) is a group of clinically and genetically heterogeneous disorders characterized by dysregulated insulin secretion. The aim of the study was to elucidate genetic etiologies of Taiwanese children with the most severe diazoxide-unresponsive CHI and analyze their genotype-phenotype correlations. Methods: We combined Sanger with whole exome sequencing (WES) to analyze CHI-related genes. The allele frequency of the most common variant was estimated by single-nucleotide polymorphism haplotype analysis. The functional effects of the ATP-sensitive potassium (KATP) channel variants were assessed using patch clamp recording and Western blot. Results: Nine of 13 (69%) patients with ten different pathogenic variants (7 in ABCC8, 2 in KCNJ11 and 1 in GCK) were identified by the combined sequencing. The variant ABCC8 p.T1042QfsX75 identified in three probands was located in a specific haplotype. Functional study revealed the human SUR1 (hSUR1)-L366F KATP channels failed to respond to intracellular MgADP and diazoxide while hSUR1-R797Q and hSUR1-R1393C KATP channels were defective in trafficking. One patient had a de novo dominant mutation in the GCK gene (p.I211F), and WES revealed mosaicism of this variant from another patient. Conclusion: Pathogenic variants in KATP channels are the most common underlying cause of diazoxide-unresponsive CHI in the Taiwanese cohort. The p.T1042QfsX75 variant in the ABCC8 gene is highly suggestive of a founder effect. The I211F mutation in the GCK gene and three rare SUR1 variants associated with defective gating (p.L366F) or traffic (p.R797Q and p.R1393C) KATP channels are also associated with the diazoxide-unresponsive phenotype.

Distinct features of the host-parasite interactions between nonadherent and adherent Trichomonas vaginalis isolates.

Cytoadherence of Trichomonas vaginalis to human vaginal epithelial cells (hVECs) was previously shown to involve surface lipoglycans and several reputed adhesins on the parasite. Herein, we report some new observations on the host-parasite interactions of adherent versus nonadherent T. vaginalis isolates to hVECs. The binding of the TH17 adherent isolate to hVECs exhibited an initial discrete phase followed by an aggregation phase inhibited by lactose. T. vaginalis infection immediately induced surface expression of galectin-1 and -3, with extracellular amounts in the spent medium initially decreasing and then increasing thereafter over the next 60 min. Extracellular galectin-1 and -3 were detected on the parasite surface but only the TH17 adherent isolate could uptake galectin-3 via the lysosomes. Only the adherent isolate could morphologically transform from the round-up flagellate with numerous transient protrusions into a flat amoeboid form on contact with the solid surface. Cytochalasin D challenge revealed that actin organization was essential to parasite morphogenesis and cytoadherence. Real-time microscopy showed that parasite exploring and anchoring on hVECs via the axostyle may be required for initial cytoadherence. Together, the parasite cytoskeleton behaviors may collaborate with cell surface adhesion molecules for cytoadherence. The nonadherent isolate migrated faster than the adherent isolate, with motility transiently increasing in the presence of hVECs. Meanwhile, differential histone acetylation was detected between the two isolates. Also, TH17 without Mycoplasma symbiosis suggests that symbiont might not determine TH17 innate cytoadherence. Our findings regarding distinctive host-parasite interactions of the isolates may provide novel insights into T. vaginalis infection.

pH-Dependence of Glucose-Dependent Activity of Beta Cell Networks in Acute Mouse Pancreatic Tissue Slice.

Extracellular pH has the potential to affect various aspects of the pancreatic beta cell function. To explain this effect, a number of mechanisms was proposed involving both extracellular and intracellular targets and pathways. Here, we focus on reassessing the influence of extracellular pH on glucose-dependent beta cell activation and collective activity in physiological conditions. To this end we employed mouse pancreatic tissue slices to perform high-temporally resolved functional imaging of cytosolic Ca2+ oscillations. We investigated the effect of either physiological H+ excess or depletion on the activation properties as well as on the collective activity of beta cell in an islet. Our results indicate that lowered pH invokes activation of a subset of beta cells in substimulatory glucose concentrations, enhances the average activity of beta cells, and alters the beta cell network properties in an islet. The enhanced average activity of beta cells was determined indirectly utilizing cytosolic Ca2+ imaging, while direct measuring of insulin secretion confirmed that this enhanced activity is accompanied by a higher insulin release. Furthermore, reduced functional connectivity and higher functional segregation at lower pH, both signs of a reduced intercellular communication, do not necessary result in an impaired insulin release.

Kv1.1 preserves the neural stem cell pool and facilitates neuron maturation during adult hippocampal neurogenesis.

Adult hippocampal neurogenesis is critical for learning and memory, and aberrant adult neurogenesis has been implicated in cognitive decline associated with aging and neurological diseases [J. T. Gonçalves, S. T. Schafer, F. H. Gage, Cell 167, 897­914 (2016)]. In previous studies, we observed that the delayed-rectifier voltage-gated potassium channel Kv1.1 controls the membrane potential of neural stem and progenitor cells and acts as a brake on neurogenesis during neonatal hippocampal development [S. M. Chou et al., eLife 10, e58779 (2021)]. To assess the role of Kv1.1 in adult hippocampal neurogenesis, we developed an inducible conditional knockout mouse to specifically remove Kv1.1 from adult neural stem cells via tamoxifen administration. We determined that Kv1.1 deletion in adult neural stem cells causes overproliferation and depletion of radial glia-like neural stem cells, prevents proper adult-born granule cell maturation and integration into the dentate gyrus, and moderately impairs hippocampus-dependent contextual fear learning and memory. Taken together, these findings support a critical role for this voltage-gated ion channel in adult neurogenesis.

Allosteric coupling between transmembrane segment 4 and the selectivity filter of TALK1 potassium channels regulates their gating by extracellular pH.

Opening of two-pore domain K+ channels (K2Ps) is regulated by various external cues, such as pH, membrane tension, or temperature, which allosterically modulate the selectivity filter (SF) gate. However, how these cues cause conformational changes in the SF of some K2P channels remains unclear. Herein, we investigate the mechanisms by which extracellular pH affects gating in an alkaline-activated K2P channel, TALK1, using electrophysiology and molecular dynamics (MD) simulations. We show that R233, located at the N-terminal end of transmembrane segment 4, is the primary pHo sensor. This residue distally regulates the orientation of the carbonyl group at the S1 potassium-binding site through an interacting network composed of residues on transmembrane segment 4, the pore helix domain 1, and the SF. Moreover, in the presence of divalent cations, we found the acidic pH-activated R233E mutant recapitulates the network interactions of protonated R233. Intriguingly, our data further suggested stochastic coupling between R233 and the SF gate, which can be described by an allosteric gating model. We propose that this allosteric model could predict the hybrid pH sensitivity in heterodimeric channels with alkaline-activated and acidic-activated K2P subunits.

Carbamazepine promotes surface expression of mutant Kir6.2-A28V ATP-sensitive potassium channels by modulating Golgi retention and autophagy.

Pancreatic ß-cells express ATP-sensitive potassium (KATP) channels, consisting of octamer complexes containing four sulfonylurea receptor 1 (SUR1) and four Kir6.2 subunits. Loss of KATP channel function causes persistent hyperinsulinemic hypoglycemia of infancy (PHHI), a rare but debilitating condition if not treated. We previously showed that the sodium-channel blocker carbamazepine (Carb) corrects KATP channel surface expression defects induced by PHHI-causing mutations in SUR1. In this study, we show that Carb treatment can also ameliorate the trafficking deficits associated with a recently discovered PHHI-causing mutation in Kir6.2 (Kir6.2-A28V). In human embryonic kidney 293 or INS-1 cells expressing this mutant KATP channel (SUR1 and Kir6.2-A28V), biotinylation and immunostaining assays revealed that Carb can increase surface expression of the mutant KATP channels. We further examined the subcellular distributions of mutant KATP channels before and after Carb treatment; without Carb treatment, we found that mutant KATP channels were aberrantly accumulated in the Golgi apparatus. However, after Carb treatment, coimmunoprecipitation of mutant KATP channels and Golgi marker GM130 was diminished, and KATP staining was also reduced in lysosomes. Intriguingly, Carb treatment also simultaneously increased autophagic flux and p62 accumulation, suggesting that autophagy-dependent degradation of the mutant channel was not only stimulated but also interrupted. In summary, our data suggest that surface expression of Kir6.2-A28V KATP channels is rescued by Carb treatment via promotion of mutant KATP channel exit from the Golgi apparatus and reduction of autophagy-mediated protein degradation.

Potential cross-species correlations in social hierarchy and memory between mice and young children.

Social hierarchy is associated with various phenotypes. Although memory is known to be important for hierarchy formation, the difference in memory abilities between dominant and subordinate individuals remains unclear. In this study, we examined memory performance in mice with different social ranks and found better memory abilities in dominant mice, along with greater long-term potentiation and higher memory-related gene expression in the hippocampus. Daily injection of memory-improving drugs could also enhance dominance. To validate this correlation across species, through inventory, behavioral and event-related potential studies, we identified better memory abilities in preschool children with higher social dominance. Better memory potentially helped children process dominance facial cues and learn social strategies to acquire higher positions. Our study shows a remarkable similarity between humans and mice in the association between memory and social hierarchy and provides valuable insight into social interactions in young animals, with potential implications for preschool education.

Fragment-Directed Random Mutagenesis by the Reverse Kunkel Method.

Two fundamentally different approaches are routinely used for protein engineering: user-defined mutagenesis and random mutagenesis, each with its own strengths and weaknesses. Here, we invent a unique mutagenesis protocol, which combines the advantages of user-defined mutagenesis and random mutagenesis. The new method, termed the reverse Kunkel method, allows the user to create random mutations at multiple specified regions in a one-pot reaction. We demonstrated the reverse Kunkel method by mimicking the somatic hypermutation in antibodies that introduces random mutations concentrated in complementarity-determining regions. Coupling with the phage display and yeast display selections, we successfully generated dramatically improved antibodies against a model protein and a neurotransmitter peptide in terms of affinity and immunostaining performance. The reverse Kunkel method is especially suitable for engineering proteins whose activities are determined by multiple variable regions, such as antibodies and adeno-associated virus capsids, or whose functional domains are composed of several discontinuous sequences, such as Cas9 and Cas12a.

Kv1.1 channels regulate early postnatal neurogenesis in mouse hippocampus via the TrkB signaling pathway.

In the postnatal brain, neurogenesis occurs only within a few regions, such as the hippocampal sub-granular zone (SGZ). Postnatal neurogenesis is tightly regulated by factors that balance stem cell renewal with differentiation, and it gives rise to neurons that participate in learning and memory formation. The Kv1.1 channel, a voltage-gated potassium channel, was previously shown to suppress postnatal neurogenesis in the SGZ in a cell-autonomous manner. In this study, we have clarified the physiological and molecular mechanisms underlying Kv1.1-dependent postnatal neurogenesis. First, we discovered that the membrane potential of neural progenitor cells is highly dynamic during development. We further established a multinomial logistic regression model for cell-type classification based on the biophysical characteristics and corresponding cell markers. We found that the loss of Kv1.1 channel activity causes significant depolarization of type 2b neural progenitor cells. This depolarization is associated with increased tropomyosin receptor kinase B (TrkB) signaling and proliferation of neural progenitor cells; suppressing TrkB signaling reduces the extent of postnatal neurogenesis. Thus, our study defines the role of the Kv1.1 potassium channel in regulating the proliferation of postnatal neural progenitor cells in mouse hippocampus.

Augmented Insulin and Leptin Resistance of High Fat Diet-Fed APPswe/PS1dE9 Transgenic Mice Exacerbate Obesity and Glycemic Dysregulation.

Alzheimer's disease (AD), a progressive neurodegenerative disease is highly associated with metabolic syndromes. We previously demonstrated that glycemic dysregulation and obesity are augmented in high fat diet (HFD)-treated APPswe/PS1dE9 (APP/PS1) transgenic mice. In the current study, the underlying mechanism mediating exacerbated metabolic stresses in HFD APP/PS1 transgenic mice was further examined. APP/PS1 mice developed insulin resistance and, consequently, impaired glucose homeostasis after 10 weeks on HFD. [18F]-2-fluoro-2-deoxy-d-glucose ([18F]-FDG) positron emission tomography showed that interscapular brown adipose tissue is vulnerable to HFD and AD-related pathology. Chronic HFD induced hyperphagia, with limited effects on basal metabolic rates in APP/PS1 transgenic mice. Excessive food intake may be caused by impairment of leptin signaling in the hypothalamus because leptin failed to suppress the food intake of HFD APP/PS1 transgenic mice. Leptin-induced pSTAT3 signaling in the arcuate nucleus was attenuated. Dysregulated energy homeostasis including hyperphagia and exacerbated obesity was elicited prior to the presence of the amyloid pathology in the hypothalamus of HFD APP/PS1 transgenic mice; nevertheless, cortical neuroinflammation and the level of serum Aß and IL-6 were significantly elevated. Our study demonstrates the pivotal role of AD-related pathology in augmenting HFD-induced insulin and leptin resistance and impairing hypothalamic regulation of energy homeostasis.

A new familial form of a late-onset, persistent hyperinsulinemic hypoglycemia of infancy caused by a novel mutation in KCNJ11.

The ATP-sensitive potassium channel (KATP) functions as a metabo-electric transducer in regulating insulin secretion from pancreatic ß-cells. The pancreatic KATP channel is composed of a pore-forming inwardly-rectifying potassium channel, Kir6.2, and a regulatory subunit, sulphonylurea receptor 1 (SUR1). Loss-of-function mutations in either subunit often lead to the development of persistent hyperinsulinemic hypoglycemia of infancy (PHHI). PHHI is a rare genetic disease and most patients present with immediate onset within the first few days after birth. In this study, we report an unusual form of PHHI, in which the index patient developed hyperinsulinemic hypoglycemia after 1 year of age. The patient failed to respond to routine medication for PHHI and underwent a complete pancreatectomy. Genotyping of the index patient and his immediate family members showed that the patient and other family members with hypoglycemic episodes carried a heterozygous novel mutation in KCNJ11 (C83T), which encodes Kir6.2 (A28V). Electrophysiological and cell biological experiments revealed that A28V hKir6.2 is a dominant-negative, loss-of-function mutation and that KATP channels carrying this mutation failed to reach the cell surface. De novo protein structure prediction indicated that this A28V mutation reoriented the ER retention motif located at the C-terminal of the hKir6.2, and this result may explain the trafficking defect caused by this point mutation. Our study is the first report of a novel form of late-onset PHHI that is caused by a dominant mutation in KCNJ11 and exhibits a defect in proper surface expression of Kir6.2.

N-linked glycosylation of Kv1.2 voltage-gated potassium channel facilitates cell surface expression and enhances the stability of internalized channels.

KEY POINTS: Kv1.2 and related voltage-gated potassium channels have a highly conserved N-linked glycosylation site in the first extracellular loop, with complex glycosylation in COS-7 cells similar to endogenous Kv1.2 glycosylation in hippocampal neurons. COS-7 cells expressing Kv1.2 show a crucial role of this N-linked glycosylation in the forward trafficking of Kv1.2 to the cell membrane. Although both wild-type and non-glycosylated mutant Kv1.2 channels that have reached the cell membrane are internalized at a comparable rate, mutant channels are degraded at a faster rate. Treatment of wild-type Kv1.2 channels on the cell surface with glycosidase to remove sialic acids also results in the faster degradation of internalized channels. Glycosylation of Kv1.2 is important with respect to facilitating trafficking to the cell membrane and enhancing the stability of channels that have reached the cell membrane. ABSTRACT: Studies in cultured hippocampal neurons and the COS-7 cell line demonstrate important roles for N-linked glycosylation of Kv1.2 channels in forward trafficking and protein degradation. Kv1.2 channels can contain complex N-linked glycans, which facilitate cell surface expression of the channels. Additionally, the protein stability of cell surface-expressed Kv1.2 channels is affected by glycosylation via differences in the degradation of internalized channels. The present study reveals the importance of N-linked complex glycosylation in boosting Kv1.2 channel density. Notably, sialic acids at the terminal sugar branches play an important role in dampening the degradation of Kv1.2 internalized from the cell membrane to promote its stability.

Spindle-F Is the Central Mediator of Ik2 Kinase-Dependent Dendrite Pruning in Drosophila Sensory Neurons.

During development, certain Drosophila sensory neurons undergo dendrite pruning that selectively eliminates their dendrites but leaves the axons intact. How these neurons regulate pruning activity in the dendrites remains unknown. Here, we identify a coiled-coil protein Spindle-F (Spn-F) that is required for dendrite pruning in Drosophila sensory neurons. Spn-F acts downstream of IKK-related kinase Ik2 in the same pathway for dendrite pruning. Spn-F exhibits a punctate pattern in larval neurons, whereas these Spn-F puncta become redistributed in pupal neurons, a step that is essential for dendrite pruning. The redistribution of Spn-F from puncta in pupal neurons requires the phosphorylation of Spn-F by Ik2 kinase to decrease Spn-F self-association, and depends on the function of microtubule motor dynein complex. Spn-F is a key component to link Ik2 kinase to dynein motor complex, and the formation of Ik2/Spn-F/dynein complex is critical for Spn-F redistribution and for dendrite pruning. Our findings reveal a novel regulatory mechanism for dendrite pruning achieved by temporal activation of Ik2 kinase and dynein-mediated redistribution of Ik2/Spn-F complex in neurons.

Rapamycin ameliorates age-dependent obesity associated with increased mTOR signaling in hypothalamic POMC neurons.

VIDEO ABSTRACT: The prevalence of obesity in older people is the leading cause of metabolic syndromes. Central neurons serving as homeostatic sensors for body-weight control include hypothalamic neurons that express pro-opiomelanocortin (POMC) or neuropeptide-Y (NPY) and agouti-related protein (AgRP). Here, we report an age-dependent increase of mammalian target of rapamycin (mTOR) signaling in POMC neurons that elevates the ATP-sensitive potassium (K(ATP)) channel activity cell-autonomously to silence POMC neurons. Systemic or intracerebral administration of the mTOR inhibitor rapamycin causes weight loss in old mice. Intracerebral rapamycin infusion into old mice enhances the excitability and neurite projection of POMC neurons, thereby causing a reduction of food intake and body weight. Conversely, young mice lacking the mTOR-negative regulator TSC1 in POMC neurons, but not those lacking TSC1 in NPY/AgRP neurons, were obese. Our study reveals that an increase in mTOR signaling in hypothalamic POMC neurons contributes to age-dependent obesity.

Kv1.1-dependent control of hippocampal neuron number as revealed by mosaic analysis with double markers.

Megencephaly, or mceph, is a spontaneous frame-shift mutation of the mouse Kv1.1 gene. This mceph mutation results in a truncated Kv1.1 channel α-subunit without the channel pore domain or the voltage sensor. Interestingly, mceph/mceph mouse brains are enlarged and ­ unlike wild-type mouse brains ­ they keep growing throughout adulthood, especially in the hippocampus and ventral cortex. We used mosaic analysis with double markers (MADM) to identify the underlying mechanism. In mceph-MADM6 mice with only a small fraction of neurons homozygous for the mceph mutation, those homozygous mceph/mceph neurons in the hippocampus are more abundant than wild-type neurons produced by sister neural progenitors. In contrast, neither mceph/mceph astrocytes, nor neurons in the adjacent dorsal cortex (including the entorhinal and parietal cortex) exhibited overgrowth in the adult brain. The sizes of mceph/mceph hippocampal neurons were comparable to mceph/+ or wild-type neurons. Our mosaic analysis reveals that loss of Kv1.1 function causes an overproduction of hippocampal neurons, leading to an enlarged brain phenotype.

Rapamycin induces glucose intolerance in mice by reducing islet mass, insulin content, and insulin sensitivity.

Rapamycin, a specific inhibitor for mTOR complex 1, is an FDA-approved immunosuppressant for organ transplant. Recent developments have raised the prospect of using rapamycin to treat cancer or diabetes and to delay aging. It is therefore important to assess how rapamycin treatment affects glucose homeostasis. Here, we show that the same rapamycin treatment reported to extend mouse life span significantly impaired glucose homeostasis of aged mice. Moreover, rapamycin treatment of lean C57B/L6 mice reduced glucose-stimulated insulin secretion in vivo and ex vivo as well as the insulin content and beta cell mass of pancreatic islets. Confounding the diminished capacity for insulin release, rapamycin decreased insulin sensitivity. The multitude of rapamycin effects thus all lead to glucose intolerance. As our findings reveal that chronic rapamycin treatment could be diabetogenic, monitoring glucose homeostasis is crucial when using rapamycin as a therapeutic as well as experimental reagent.

Altered ultrasonic vocalizations in a tuberous sclerosis mouse model of autism.

Tuberous sclerosis (TSC) is an autosomally dominant neurocutaneous disease notable for its high comorbidity with autism in human patients. Studies of murine models of tuberous sclerosis have found defects in cognition and learning, but thus far have not uncovered deficits in social behaviors relevant to autism. To explore social communication and interaction in TSC2 heterozygous mice, we recorded ultrasonic vocalizations (USV) and found that although both wild-type (WT) and heterozygous pups born to WT dams showed similar call rates and patterns, baseline vocalization rates were elevated in pups born to heterozygous dams. Further analysis revealed several robust features of maternal potentiation in all but WT pups born to heterozygous dams. This lack of potentiation is suggestive of defects in mother-pup social interaction during or before the reunion period between WT pups and heterozygous dams. Intriguingly, male pups of both genotypes born to heterozygous dams showed particularly heightened call rates and burst patterns. Because our maternal retrieval experiments revealed that TSC2(+/-) dams exhibited improved defensive reactions against intruders and highly efficient pup retrieval performance, the alterations in their pups' USVs and maternal potentiation do not appear to result from poor maternal care. These findings suggest that a pup's interaction with its mother strongly influences the pup's vocal communication, revealing an intriguing dependence of this social behavior on TSC2 gene dosage of both parties involved. Our study of this murine model thus uncovers social abnormalities that arise from TSC haploinsufficiency and are suggestive of autism.

GC-MS analysis of liposoluble constituents from the stems of Cynomorium songaricum.

In order to evaluate the differences and similarities between the liposoluble constituents in Cynomorium songaricum populations, stem liposoluble constituents in five populations of C. songaricum collected from three different geographic regions and four different hosts were obtained by solvent extraction and analyzed by GC-MS. Cluster analysis of the percentage composition of 80 compounds showed differences in chemical composition which were related to the geographic origin rather than the host. Hexadecanoic acid was the most abundant compound in the essential oils of C. songaricum from hosts Nitraria sibirica and Nitraria tanguticum. Whereas (Z)-9-octadecenoic acid was accumulated in the oils of C. songaricum from Zygophyllum xanthoxylum and Peganum harmala. Four of the five populations had characteristic components, which were specific to each population.

Exocytosis of insulin: in vivo maturation of mouse endocrine pancreas.

The aim of this study was to define when an insulin-positive cell becomes functional in vivo and starts to exocytose insulin in a regulated nutrient-dependent manner. Insulin-positive cells appear in embryonic life (midgestation) and complete their maturation, presumably around birth. In order to work with embryonic and newborn endocrine pancreas, we used organotypic slices. The mouse embryonic pancreas slices show high basal insulin release that is not further elevated by high glucose levels. Despite the presence of functional voltage-activated ion channels, the cells are not electrically active in the presence of secretagogues. At birth, the high basal insulin release drops and, after postnatal day 2, the insulin-positive cells show both adult-like bursting electrical activity and hormone release induced by high glucose levels. These properties allowed us to define them as beta cells. Despite the apparent stability of the transcription factor profile reported in insulin-positive cells during late-embryonic life, functional beta cells appear only 2 days after birth.