Lung-Sen Kao

In vitro differentiation of size-sieved stem cells into electrically active neural cells.

Size‐sieved stem (SS) cells isolated from human bone marrow and propagated in vitro are a population of cells with consistent marker typing, and can form bone, fat, and cartilage. In this experiment, we demonstrated that SS cells could be induced to differentiate into neural cells under experimental cell culture conditions. Five hours after exposure to antioxidant agents (β‐mercaptoethanol ± retinoic acid) in serum‐free conditions, SS cells expressed the protein for nestin, neuron‐specific enolase (NSE), neuron‐specific nuclear protein (NeuN), and neuron‐specific tubulin‐1 (TuJ‐1), and the mRNA for NSE and Tau. Immunofluorescence showed that almost all the cells (>98%) expressed NeuN and TuJ‐1. After 5 days of β‐mercaptoethanol treatment, the SS cells expressed neurofilament high protein but not mitogen‐activated protein‐2, glial filament acidic protein, and galactocerebroside. For such long‐term‐treated cells, voltage‐sensitive ionic current could be detected by electrophysiological recording, and the intracellular calcium ion, Ca2+, concentration can be elevated by high potassium (K+) buffer and glutamate. These findings suggest that SS cells may be an alternative source of undifferentiated cells for cell therapy and gene therapy in neural dysfunction.

Involvement of nitric oxide in dopaminergic transmission in rat striatum: an in vivo electrochemical study.

Abstract: In vivo electrochemical detection with a Nafion‐coated carbon fiber working electrode, which provides information on the spatial and temporal dynamics of dopamine overflow, was used to investigate the involvement of nitric oxide (NO) in the dopaminergic transmission in the striatum of urethane‐anesthetized Sprague‐Dawley rats. A mixture of N‐methyl‐d‐aspartate (NMDA) and nomifensine, a dopamine uptake blocker, was locally pressure‐ejected to elicit a transient dopamine overflow from the dopamine‐containing nerve terminals in the striatum. Local application of Nω‐nitro‐l‐arginine methyl ester (l‐NAME), which blocks endogenous NO formation, increased the magnitude of dopamine release evoked by a subsequent NMDA and nomifensine application but resulted in no significant alteration in the time course. Furthermore, microejection of l‐arginine, an NO precursor, or sodium nitroprusside (SNP), an NO generator, did not cause detectable changes in dopamine level in the striatal extracellular space. However, NMDA‐induced dopamine release was profoundly inhibited with l‐arginine or SNP pretreatment. In addition, NO affects dopamine uptake in rat striatum. Exogenous dopamine applied through a micropipette, reversibly and reproducibly, elicited an electrochemical signal. The time course of these signals was significantly prolonged by l‐NAME treatment. These data suggest that NO is diversely involved in regulating dopaminergic transmission in rat striatum.

Catecholamine secretion from bovine adrenal chromaffin cells: the role of the Na+/Ca2+ exchanger and the intracellular Ca2+ pool.

Abstract: The role of the Na+/Ca2+ exchanger and intracellular nonmitochondrial Ca2+ pool in the regulation of cytosolic free calcium concentration ([Ca2+]i) during catecholamine secretion was investigated. Catecholamine secretion and [Ca2+]i were simultaneously monitored in a single chromaffin cell. After high‐K+ stimulation, control cells and cells in which the Na+/Ca2+ exchange activity was inhibited showed similar rates of [Ca2+]i elevation. However, the recovery of [Ca2+]i to resting levels was slower in the inhibited cells. Inhibition of the exchanger increased the total catecholamine secretion by prolonging the secretion. Inhibition of the Ca2+ pump of the intracellular Ca2+ pool with thapsigargin caused a significant delay in the recovery of [Ca2+]i and greatly enhanced the secretory events. These data suggest that both the Na+/Ca2+ exchanger and the thapsigargin‐sensitive Ca2+ pool are important in the regulation of [Ca2+]i and, by modulating the time course of secretion, are important in determining the extent of secretion.

Reverse mode Na+/Ca2+ exchangers trigger the release of Ca2+ from intracellular Ca2+ stores in cultured rat embryonic cortical neurons.

The importance of Na+/Ca2+ exchangers in the regulation of the physiological and pathological functions of the nervous system has been widely recognized. In this study, we used primary cultured E14.5 cortical neurons as a model system to study the possible roles of the reverse mode Na+/Ca2+ exchange activity in neurotransmission. Using RT-PCR, several exchanger isoforms, ncx1, ncx3 and nckx2-4 were found to be expressed in freshly isolated and cultured cortical neurons. Expression of ncx2 was undetectable in freshly isolated neurons but increased with time in culture. Neurons were treated with ouabain to increase the intracellular Na+ concentration and the extracellular Na+ was replaced by N-methyl-D-glucamine to activate reverse mode Na+/Ca2+ exchange. During the maturation of the neurons, the exchange activity shifted from mostly K+-dependent exchange to both K+-dependent and K+-independent exchange. The [Ca2+]i rises were mostly suppressed by ryanodine and thapsigargin treatments, indicating contributions from the intracellular Ca2+ stores. This [Ca2+]i elevation could propagate to the axon terminal and resulted in elevated [Ca2+]i at the postsynaptic neurons based on the fact that the elevation in the postsynaptic neuron was inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione and tetanus toxin. When neurons were stimulated by AMPA to increase the intracellular Na+ concentration, the [Ca2+]i elevations were significantly inhibited by thapsigargin pretreatment and by KB-R7943. These results demonstrate that, in cultured cortical neurons, the influx of Na+ through the ionotropic glutamate receptor activates reverse Na+/Ca2+ exchange, which then triggers the release of Ca2+ from intracellular Ca2+ stores to enhance Ca2+ signaling and neurotransmitter release.

Exome Sequencing Identifies GNB4 Mutations as a Cause of Dominant Intermediate Charcot-Marie-Tooth Disease

Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of inherited neuropathies. Mutations in approximately 45 genes have been identified as being associated with CMT. Nevertheless, the genetic etiologies of at least 30% of CMTs have yet to be elucidated. Using a genome-wide linkage study, we previously mapped a dominant intermediate CMT to chromosomal region 3q28-q29. Subsequent exome sequencing of two affected first cousins revealed heterozygous mutation c.158G>A (p.Gly53Asp) in GNB4, encoding guanine-nucleotide-binding protein subunit beta-4 (Gβ4), to cosegregate with the CMT phenotype in the family. Further analysis of GNB4 in an additional 88 unrelated CMT individuals uncovered another de novo mutation, c.265A>G (p.Lys89Glu), in this gene in one individual. Immunohistochemistry studies revealed that Gβ4 was abundant in the axons and Schwann cells of peripheral nerves and that expression of Gβ4 was significantly reduced in the sural nerve of the two individuals carrying the c.158G>A (p.Gly53Asp) mutation. Inxa0vitro studies demonstrated that both the p.Gly53Asp and p.Lys89Glu altered proteins impaired bradykinin-induced G-protein-coupled-receptor (GPCR) signaling, which was facilitated by the wild-type Gβ4. This study identifies GNB4 mutations as a cause of CMT and highlights the importance of Gβ4-related GPCR signaling in peripheral-nerve function in humans.

Tracking of secretory vesicles of PC12 cells by total internal reflection fluorescence microscopy.

Total internal reflection fluorescence microscopy is used to detect cellular events near the plasma membrane. Behaviours of secretory vesicles near the cell surface of living PC12 cells, a neuroendocrine cell line, are studied. The secretory vesicles are labelled by over‐expression of enhanced green fluorescent protein‐tagged Rab3A, one of the small G proteins involved in the fusion of secretory vesicles to plasma membrane in PC12 cells. Images acquired by a fast cooled charge‐coupled device camera using conventional fluorescence microscopy and total internal reflection fluorescence microscopy are compared and analysed. Within the small evanescent range (< 200 nm), the movements of the secretory vesicles of PC12 cells before and after stimulation by high K+ are examined. The movements of one vesicle relative to another already docked on the membrane are detected. Total internal reflection fluorescence microscopy provides a novel optical method to trace and analyse the exocytotic events and vesicle specifically near a cell membrane without interference of signals from other parts of the cell.

A novel TFG mutation causes Charcot-Marie-Tooth disease type 2 and impairs TFG function

Objective: To describe a novel mutation in TRK-fused gene (TFG) as a new cause of dominant axonal Charcot-Marie-Tooth disease (CMT) identified by exome sequencing and further characterized by in vitro functional studies. Methods: Exome sequencing and linkage analysis were utilized to investigate a large Taiwanese family with a dominantly inherited adult-onset motor and sensory axonal neuropathy in which mutations in common CMT2-implicated genes had been previously excluded. Functional effects of the mutant gene products were investigated in vitro. Results: Exome sequencing of 2 affected individuals in this family revealed a novel heterozygous mutation, c.806G>T (p.Gly269Val), in TFG that perfectly cosegregates with the CMT2 phenotype in all 27 family members. This mutation occurs at an evolutionarily conserved residue and is absent in the 1,140 ethnically matched control chromosomes. Genome-wide linkage study also supported its disease-causative role. Cell transfection studies showed that the TFG p.Gly269Val mutation increased the propensity of TFG proteins to form aggregates, resulting in sequestration of both mutant and wild-type TFG proteins and might thus deplete functional TFG molecules. The secreted Gaussia luciferase reporter assay demonstrated that inhibition of endogenous TFG compromised the protein secretion pathways, which could only be rescued by expressing wild-type TFG but not the p.Gly269Val altered proteins. TFG mutation was not found in 55 additional unrelated patients with CMT2, suggesting its rarity. Conclusion: This study identifies a new cause of dominant CMT2 and highlights the importance of TFG in the protein secretory pathways that are essential for proper functioning of the human peripheral nervous system.

Regulation of sodium-calcium exchanger activity by creatine kinase under energy-compromised conditions.

Na+/Ca2+ exchanger (NCX) is one of the major mechanisms for removing Ca2+ from the cytosol especially in cardiac myocytes and neurons, where their physiological activities are triggered by an influx of Ca2+. NCX contains a large intracellular loop (NCXIL) that is responsible for regulating NCX activity. Recent evidence has shown that proteins, including kinases and phosphatases, associate with NCX1IL to form a NCX1 macromolecular complex. To search for the molecules that interact with NCX1IL and regulate NCX1 activity, we used the yeast two-hybrid method to screen a human heart cDNA library and found that the C-terminal region of sarcomeric mitochondrial creatine kinase (sMiCK) interacted with NCX1IL. Moreover, both sMiCK and the muscle-type creatine kinase (CKM) coimmunoprecipitated with NCX1 using lysates of cardiacmyocytes and HEK293T cells that transiently expressed NCX1 and various creatine kinases. Both sMiCK and CKM were able to produce a recovery in the decreased NCX1 activity that was lost under energy-compromised conditions. This regulation is mediated through a putative PKC phosphorylation site of sMiCK and CKM. The autophosphorylation and the catalytic activity of sMiCK and CKM are not required for their regulation of NCX1 activity. Our results suggest a novel mechanism for the regulation of NCX1 activity.Na(+)/Ca(2+) exchanger (NCX) is one of the major mechanisms for removing Ca(2+) from the cytosol especially in cardiac myocytes and neurons, where their physiological activities are triggered by an influx of Ca(2+). NCX contains a large intracellular loop (NCXIL) that is responsible for regulating NCX activity. Recent evidence has shown that proteins, including kinases and phosphatases, associate with NCX1IL to form a NCX1 macromolecular complex. To search for the molecules that interact with NCX1IL and regulate NCX1 activity, we used the yeast two-hybrid method to screen a human heart cDNA library and found that the C-terminal region of sarcomeric mitochondrial creatine kinase (sMiCK) interacted with NCX1IL. Moreover, both sMiCK and the muscle-type creatine kinase (CKM) coimmunoprecipitated with NCX1 using lysates of cardiacmyocytes and HEK293T cells that transiently expressed NCX1 and various creatine kinases. Both sMiCK and CKM were able to produce a recovery in the decreased NCX1 activity that was lost under energy-compromised conditions. This regulation is mediated through a putative PKC phosphorylation site of sMiCK and CKM. The autophosphorylation and the catalytic activity of sMiCK and CKM are not required for their regulation of NCX1 activity. Our results suggest a novel mechanism for the regulation of NCX1 activity.

Characterization of Ca2+ signaling pathways in mouse adrenal medullary chromaffin cells.

J. Neurochem. (2010) 112, 1210–1222.

Butyl Benzyl Phthalate Blocks Ca2+ Signaling and Catecholamine Secretion Coupled with Nicotinic Acetylcholine Receptors in Bovine Adrenal Chromaffin Cells

Butyl benzyl phthalate (BBP), a plasticizer and an environmental pollutant, exerts genomic estrogenic-like effects via estrogen receptors. In addition to exerting genomic effects via intracellular steroid receptors, estrogen exerts non-genomic effects through interactions with membrane ion channels to lead the rapid alteration of neuronal excitability. Estradiol is known as to have modulating role on nicotinic acetylcholine receptors (nAChR). We investigated the possibility of BBP exerting non-genomic estrogenic-like effects on nAChR in bovine adrenal chromaffin cells. Our results show that BBP inhibited Ca2+ signaling induced by the nicotinic ligands carbachol, 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) and epibatidine (IC50 levels of 4.3, 4.1, 5.4 microM, respectively) as well as high K+ solution (IC50 50.9 microM). Additionally, in the electrophysiological observations, BBP blocked the inward current coupled with nAChR under the stimulation of carbachol. We, therefore, suggest that nAChR and voltage-gated Ca2+ channels are major and minor sites, respectively, of BBP action on the plasma membrane. The inhibitory effect of BBP on nAChR was found to be both noncompetitive and reversible, remaining unchanged as nAChR ligand concentration increased and decreased after washing. BBP was 10 times more potent than estradiol in inhibiting nAChR-coupled Ca2+ signals. We conclude that BBP exerts a novel rapidly inhibitory effect on nAChR.