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Human iPSC neurons display activity-dependent neurotransmitter secretion: aberrant catecholamine levels in schizophrenia neurons.

ABSTRACT: This study investigated human-induced pluripotent stem cell (hiPSC) -derived neurons for their ability to secrete neurotransmitters in an activity-dependent manner, the fundamental property required for chemical neurotransmission. Cultured hiPSC neurons showed KCl stimulation of activity-dependent secretion of catecholamines--dopamine (DA), norepinephrine (NE), and epinephrine (Epi)--and the peptide neurotransmitters dynorphin and enkephlain. hiPSC neurons express the biosynthetic enzymes for catecholamines and neuropeptides. Because altered neurotransmission contributes to schizophrenia (SZ), we compared SZ to control cultures of hiPSC neurons and found that SZ cases showed elevated levels of secreted DA, NE, and Epi. Consistent with increased catecholamines, the SZ neuronal cultures showed a higher percentage of tyrosine hydroxylase (TH)-positive neurons, the first enzymatic step for catecholamine biosynthesis. These findings show that hiPSC neurons possess the fundamental property of activity-dependent neurotransmitter secretion and can be advantageously utilized to examine regulation of neurotransmitter release related to brain disorders.

SUBMITTER: Hook V

PROVIDER: S-EPMC4223699 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Human iPSC neurons display activity-dependent neurotransmitter secretion: aberrant catecholamine levels in schizophrenia neurons.

Hook Vivian V Brennand Kristen J KJ Kim Yongsung Y Toneff Thomas T Funkelstein Lydiane L Lee Kelly C KC Ziegler Michael M Gage Fred H FH

Stem cell reports 20140911 4

This study investigated human-induced pluripotent stem cell (hiPSC) -derived neurons for their ability to secrete neurotransmitters in an activity-dependent manner, the fundamental property required for chemical neurotransmission. Cultured hiPSC neurons showed KCl stimulation of activity-dependent secretion of catecholamines--dopamine (DA), norepinephrine (NE), and epinephrine (Epi)--and the peptide neurotransmitters dynorphin and enkephlain. hiPSC neurons express the biosynthetic enzymes for ca ...[more]

PMID: 25358781

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Project description:Reelin (Reln) is a glycoprotein that in postnatal and adult mammalian brain is believed to be secreted from telencephalic GABAergic interneurons and cerebellar glutamatergic granule neurons into the extracellular matrix. To address the question of whether Reln neurosecretion occurs via a regulated or a constitutive process, we exposed postnatal rat cerebellar granule neurons (CGNs) maintained in culture for 7-9 days to: (i) 100 microM N-methyl-D-aspartate (NMDA) in a Mg(+2)-free medium to stimulate NMDA-selective glutamate receptors and Ca(2+)-dependent neurotransmitter release, (ii) 50 mM KCl to depolarize the cells and elicit Ca(2+)-dependent exocytosis, (iii) 10-100 microM nicotine to activate excocytosis by nicotinic receptors present in these cells, (iv) 10 microM 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxo-benzo[f]quinoxaline-7-sulfonamide in combination with 10 microM dizocilpine to block alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- and NMDA-preferring glutamate receptors activated by endogenously released glutamate, or (v) EGTA (5 mM) to virtually eliminate extracellular Ca(2+) and block Ca(2+)-dependent exocytosis. Although, CGNs express and secrete Reln (measured by quantitative immunoblotting), none of the above-mentioned conditions that control regulated exocytosis alters the stores or the rate of Reln release. In contrast, application of either: (i) a Reln antisense oligonucleotide (5'-GCAATGTGCAGGGAAATG-3') (10 microM) that reduces Reln biosynthesis or (ii) brefeldin A (5 x 10(-5) M), an inhibitor of the traffic of proteins between the endoplasmic reticulum and the Golgi network, sharply curtail the rate of Reln secretion. Because, in subcellular fractionation studies, we have shown that Reln is not contained in synaptic vesicles, these data suggest that Reln secretion from CGNs does not require Ca(2+)-dependent exocytosis, but probably is related to a Reln pool stored in Golgi secretory vesicles mediating a constitutive secretory pathway.

Project description:BackgroundAcetylcholine (ACh) and norepinephrine (NE) are representative neurotransmitters of parasympathetic and sympathetic nerves, respectively, that antagonize each other to coregulate internal body functions. This also includes the control of different kinds of hormone secretion from pancreatic islets. However, the molecular mechanisms have not been fully elucidated, and whether innervation in islets is abnormal in diabetes mellitus also remains unclear.Methods and resultsImmunofluorescence colocalization and islet perfusion were performed and the results demonstrated that ACh/NE and their receptors were highly expressed in islet and rapidly regulated different hormones secretion. Phosphorylation is considered an important posttranslational modification in islet innervation and it was identified by quantitative proteomic and phosphoproteomic analyses in this study. The phosphorylated islet proteins were found involved in many biological and pathological processes, such as synaptic signalling transduction, calcium channel opening and insulin signalling pathway. Then, the kinases were predicted by motif analysis and further screened and verified by kinase-specific siRNAs in different islet cell lines (αTC1-6, Min6 and TGP52). After functional verification, Ksr2 and Pkacb were considered the key kinases of ACh and NE in insulin secretion, and Cadps, Mlxipl and Pdcd4 were the substrates of these kinases measured by immunofluorescence co-staining. Then, the decreased expression of receptors, kinases and substrates of ACh and NE were found in diabetic mice and the aberrant rhythm in insulin secretion could be improved by combined interventions on key receptors (M3 (pilocarpine) or α2a (guanfacine)) and kinases (Ksr2 or Pkacb).ConclusionsAbnormal innervation was closely associated with the degree of islet dysfunction in diabetic mice and the aberrant rhythm in insulin secretion could be ameliorated significantly after intervention with key receptors and kinases in the early stage of diabetes mellitus, which may provide a promising therapeutic strategy for diabetes mellitus in the future.

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Human iPSC neurons display activity-dependent neurotransmitter secretion: aberrant catecholamine levels in schizophrenia neurons.

Publications

Human iPSC neurons display activity-dependent neurotransmitter secretion: aberrant catecholamine levels in schizophrenia neurons.

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