Young-Jin Kang

Potential Hypoglycemic Effect of an Ethanol Extract of Gynostemma pentaphyllum in C57BL/KsJ-db/db Mice

This study was conducted to evaluate the antihyperglycemic effect of an extract of Gynostemma pentaphyllum Makino, containing standardized concentrations of gypenosides, in C57BL/KSJ-db/db mice. For 5 weeks, animals were provided a standard AIN-76 diet (normal control) with rosiglitazone (0.005%, wt/wt) or two different doses of G. pentaphyllum ethanol extract (GPE) of the plant leaves (0.0025% and 0.01%, wt/wt). After the experimental period, the blood glucose levels of the high-dose GPE- and rosiglitazone-supplemented groups were significantly lower than that of the control group. The plasma insulin concentrations of the GPE-supplemented mice were significantly elevated compared to the control group. The GPE and rosiglitazone treatments profoundly affected the intraperitoneal insulin tolerance test compared to the control group, but not the intraperitoneal glucose tolerance test. In the evaluation of effects on hepatic glucose metabolism, the ratios of glucokinase/glucose-6-phosphatase activities in the high-dose GPE- and rosiglitazone-supplemented groups were prominently higher than that of the control group. The histology of the pancreatic islets revealed that the insulin-positive beta-cell numbers were higher in the high-dose GPE- and rosiglitazone-supplemented groups than in the control group. These results suggest that the supplementation of high-dose GPE (0.01%) in the diet lowers the blood glucose level by altering the hepatic glucose metabolic enzyme activities.

Sparc Protein Is Required for Normal Growth of Zebrafish Otoliths

Otoliths and the homologous otoconia in the inner ear are essential for balance. Their morphogenesis is less understood than that of other biominerals, such as bone, and only a small number of their constituent proteins have been characterized. As a novel approach to identify unknown otolith proteins, we employed shotgun proteomics to analyze crude extracts from trout and catfish otoliths. We found three proteins that had not been associated previously with otolith or otoconia formation: ‘Secreted acidic cysteine rich glycoprotein’ (Sparc), an important bone protein that binds collagen and Ca2+; precerebellin-like protein, which contains a C1q domain and may associate with the collagenous otolin-1 during its assembly into a framework; and neuroserpin, a serine protease inhibitor that may regulate local protease activity during framework assembly. We then used the zebrafish to investigate whether Sparc plays a role in otolith morphogenesis. Immunodetection demonstrated that Sparc is a true constituent of otoliths. Knockdown of Sparc expression in morphant zebrafish resulted in four principal types of defective otoliths: smaller, extra and ectopic, missing and fused, or completely absent. Smaller size was the predominant phenotype and independent of the severity of otic-vesicle defects. These results suggested that Sparc is directly required for normal otolith growth.

Spatially Heterogeneous Choroid Plexus Transcriptomes Encode Positional Identity and Contribute to Regional CSF Production

A sheet of choroid plexus epithelial cells extends into each cerebral ventricle and secretes signaling factors into the CSF. To evaluate whether differences in the CSF proteome across ventricles arise, in part, from regional differences in choroid plexus gene expression, we defined the transcriptome of lateral ventricle (telencephalic) versus fourth ventricle (hindbrain) choroid plexus. We find that positional identities of mouse, macaque, and human choroid plexi derive from gene expression domains that parallel their axial tissues of origin. We then show that molecular heterogeneity between telencephalic and hindbrain choroid plexi contributes to region-specific, age-dependent protein secretion in vitro. Transcriptome analysis of FACS-purified choroid plexus epithelial cells also predicts their cell-type-specific secretome. Spatial domains with distinct protein expression profiles were observed within each choroid plexus. We propose that regional differences between choroid plexi contribute to dynamic signaling gradients across the mammalian cerebroventricular system.

Fusion of Regionally Specified hPSC-Derived Organoids Models Human Brain Development and Interneuron Migration

Organoid techniques provide unique platforms to model brain development and neurological disorders. Whereas several methods for recapitulating corticogenesis have been described, a system modeling human medial ganglionic eminence (MGE) development, a critical ventral brain domain producing cortical interneurons and related lineages, has been lacking until recently. Here, we describe the generation of MGE and cortex-specific organoids from human pluripotent stem cells that recapitulate the development of MGE and cortex domains, respectively. Population and single-cell RNA sequencing (RNA-seq) profiling combined with bulk assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) analyses revealed transcriptional and chromatin accessibility dynamics and lineage relationships during MGE and cortical organoid development. Furthermore, MGE and cortical organoids generated physiologically functional neurons and neuronal networks. Finally, fusing region-specific organoids followed by live imaging enabled analysis of human interneuron migration and integration. Together, our study provides a platform for generating domain-specific brain organoids and modeling human interneuron migration and offers deeper insight into molecular dynamics during human brain development.

BMP4 Sufficiency to Induce Choroid Plexus Epithelial Fate from Embryonic Stem Cell-Derived Neuroepithelial Progenitors

Choroid plexus epithelial cells (CPECs) have essential developmental and homeostatic roles related to the CSF and blood–CSF barrier they produce. Accordingly, CPEC dysfunction has been implicated in many neurological disorders, such as Alzheimers disease, and transplant studies have provided proof-of-concept for CPEC-based therapies. However, such therapies have been hindered by the inability to expand or generate CPECs in culture. During development, CPECs differentiate from preneurogenic neuroepithelial cells and require bone morphogenetic protein (BMP) signaling, but whether BMPs suffice for CPEC induction is unknown. Here we provide evidence for BMP4 sufficiency to induce CPEC fate from neural progenitors derived from mouse embryonic stem cells (ESCs). CPEC specification by BMP4 was restricted to an early time period after neural induction in culture, with peak CPEC competency correlating to neuroepithelial cells rather than radial glia. In addition to molecular, cellular, and ultrastructural criteria, derived CPECs (dCPECs) had functions that were indistinguishable from primary CPECs, including self-assembly into secretory vesicles and integration into endogenous choroid plexus epithelium following intraventricular injection. We then used BMP4 to generate dCPECs from human ESC-derived neuroepithelial cells. These findings demonstrate BMP4 sufficiency to instruct CPEC fate, expand the repertoire of stem cell-derived neural derivatives in culture, and herald dCPEC-based therapeutic applications aimed at the unique interface between blood, CSF, and brain governed by CPECs.

Neurophysiology of space travel: energetic solar particles cause cell type-specific plasticity of neurotransmission

In the not too distant future, humankind will embark on one of its greatest adventures, the travel to distant planets. However, deep space travel is associated with an inevitable exposure to radiation fields. Space-relevant doses of protons elicit persistent disruptions in cognition and neuronal structure. However, whether space-relevant irradiation alters neurotransmission is unknown. Within the hippocampus, a brain region crucial for cognition, perisomatic inhibitory control of pyramidal cells (PCs) is supplied by two distinct cell types, the cannabinoid type 1 receptor (CB1)-expressing basket cells (CB1BCs) and parvalbumin (PV)-expressing interneurons (PVINs). Mice subjected to low-dose proton irradiation were analyzed using electrophysiological, biochemical and imaging techniques months after exposure. In irradiated mice, GABA release from CB1BCs onto PCs was dramatically increased. This effect was abolished by CB1 blockade, indicating that irradiation decreased CB1-dependent tonic inhibition of GABA release. These alterations in GABA release were accompanied by decreased levels of the major CB1 ligand 2-arachidonoylglycerol. In contrast, GABA release from PVINs was unchanged, and the excitatory connectivity from PCs to the interneurons also underwent cell type-specific alterations. These results demonstrate that energetic charged particles at space-relevant low doses elicit surprisingly selective long-term plasticity of synaptic microcircuits in the hippocampus. The magnitude and persistent nature of these alterations in synaptic function are consistent with the observed perturbations in cognitive performance after irradiation, while the high specificity of these changes indicates that it may be possible to develop targeted therapeutic interventions to decrease the risk of adverse events during interplanetary travel.

Cell Type-specific Intrinsic Perithreshold Oscillations in Hippocampal GABAergic Interneurons

The hippocampus plays a critical role in learning, memory, and spatial processing through coordinated network activity including theta and gamma oscillations. Recent evidence suggests that hippocampal subregions (e.g., CA1) can generate these oscillations at the network level, at least in part, through GABAergic interneurons. However, it is unclear whether specific GABAergic interneurons generate intrinsic theta and/or gamma oscillations at the single-cell level. Since major types of CA1 interneurons (i.e., parvalbumin-positive basket cells (PVBCs), cannabinoid type 1 receptor-positive basket cells (CB1BCs), Schaffer collateral-associated cells (SCAs), neurogliaform cells and ivy cells) are thought to play key roles in network theta and gamma oscillations in the hippocampus, we tested the hypothesis that these cells generate intrinsic perithreshold oscillations at the single-cell level. We performed whole-cell patch-clamp recordings from GABAergic interneurons in the CA1 region of the mouse hippocampus in the presence of synaptic blockers to identify intrinsic perithreshold membrane potential oscillations. The majority of PVBCs (83%), but not the other interneuron subtypes, produced intrinsic perithreshold gamma oscillations if the membrane potential remained above -45 mV. In contrast, CB1BCs, SCAs, neurogliaform cells, ivy cells, and the remaining PVBCs (17%) produced intrinsic theta, but not gamma, oscillations. These oscillations were prevented by blockers of persistent sodium current. These data demonstrate that the major types of hippocampal interneurons produce distinct frequency bands of intrinsic perithreshold membrane oscillations.

Group I metabotropic glutamate receptors generate two types of intrinsic membrane oscillations in hippocampal oriens/alveus interneurons

ABSTRACT GABAergic interneurons in the hippocampus are critically involved in almost all hippocampal circuit functions including coordinated network activity. Somatostatin‐expressing oriens‐lacunosum moleculare (O‐LM) interneurons are a major subtype of dendritically projecting interneurons in hippocampal subregions (e.g., CA1), and express group I metabotropic glutamate receptors (mGluRs), specifically mGluR1 and mGluR5. Group I mGluRs are thought to regulate hippocampal circuit functions partially through GABAergic interneurons. Previous studies suggest that a group I/II mGluR agonist produces slow supra‐threshold membrane oscillations (<0.1Hz), which are associated with high‐frequency action potential (AP) discharges in O‐LM interneurons. However, the properties and underlying mechanisms of these slow oscillations remain largely unknown. We performed whole‐cell patch‐clamp recordings from mouse interneurons in the stratum oriens/alveus (O/A interneurons) including CA1 O‐LM interneurons. Our study revealed that the selective mGluR1/5 agonist (S)‐3,5‐dihydroxyphenylglycine (DHPG) induced slow membrane oscillations (<0.1Hz), which were associated with gamma frequency APs followed by AP‐free perithreshold gamma oscillations. The selective mGluR1 antagonist (S)‐(+)‐&agr;‐Amino‐4‐carboxy‐2‐methylbenzeneacetic acid (LY367385) reduced the slow oscillations, and the selective mGluR5 antagonist 2‐methyl‐6‐(phenylethynyl)pyridine hydrochloride (MPEP) partially blocked them. Blockade of nonselective cation‐conducting transient receptor potential channels, L‐type Ca2+ channels, or ryanodine receptors all abolished the slow oscillations, suggesting the involvement of multiple mechanisms. Our findings suggest that group I mGluR activation in O/A interneurons may play an important role in coordinated network activity, and O/A interneuron vulnerability to excitotoxicity, in disease states like seizures, is at least in part due to an excessive rise in intracellular Ca2+. HIGHLIGHTSGroup I mGluR activation in O/A interneurons produced slow membrane oscillations.Slow oscillations were associated with gamma oscillations.Slow oscillations were mediated by both mGluR1 and mGluR5.TRP channels and L‐type Ca2+ channels were involved in the slow oscillations.Ryanodine receptors were also involved in the slow oscillations.

Accurate, strong, and stable reporting of choroid plexus epithelial cells in transgenic mice using a human transthyretin BAC

BackgroundChoroid plexus epithelial cells express high levels of transthyretin, produce cerebrospinal fluid and many of its proteins, and make up the blood-cerebrospinal fluid barrier. Choroid plexus epithelial cells are vital to brain health and may be involved in neurological diseases. Transgenic mice containing fluorescent and luminescent reporters of these cells would facilitate their study in health and disease, but prior transgenic reporters lost expression over the early postnatal period.MethodsHuman bacterial artificial chromosomes in which the transthyretin coding sequence was replaced with DNA for tdTomato or luciferase 2 were used in pronuclear injections to produce transgenic mice. These mice were characterized by visualizing red fluorescence, immunostaining, real-time reverse transcription polymerase chain reaction, and luciferase enzyme assay.ResultsReporters were faithfully expressed in cells that express transthyretin constitutively, including choroid plexus epithelial cells, retinal pigment epithelium, pancreatic islets, and liver. Expression of tdTomato in choroid plexus began at the appropriate embryonic age, being detectable by E11.5. Relative levels of tdTomato transcript in the liver and choroid plexus paralleled relative levels of transcripts for transthyretin. Expression remained robust over the first postnatal year, although choroid plexus transcripts of tdTomato declined slightly with age whereas transthyretin remained constant. TdTomato expression patterns were consistent across three founder lines, displayed no sex differences, and were stable across several generations. Two of the tdTomato lines were bred to homozygosity, and homozygous mice are healthy and fertile. The usefulness of tdTomato reporters in visualizing and analyzing live Transwell cultures was demonstrated. Luciferase activity was very high in homogenates of choroid plexus and continued to be expressed through adulthood. Luciferase also was detectable in eye and pancreas.ConclusionsTransgenic mice bearing fluorescent and luminescent reporters of transthyretin should prove useful for tracking transplanted choroid plexus epithelial cells, for purifying the cells, and for reporting their derivation from stem cells. They also should prove useful for studying transthyretin synthesis by other cell types, as transthyretin has been implicated in many functions and conditions, including clearance of β-amyloid peptides associated with Alzheimer’s disease, heat shock in neurons, processing of neuropeptides, nerve regeneration, astrocyte metabolism, and transthyretin amyloidosis.