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.
Pyroglutamate amyloid-? peptides (pGlu-A?) are particularly pernicious forms of amyloid-? peptides (A?) present in Alzheimer's disease (AD) brains. pGlu-A? peptides are N-terminally truncated forms of full-length A? peptides (flA?(1-40/42)) in which the N-terminal glutamate is cyclized to pyroglutamate to generate pGlu-A?(3-40/42). ?-secretase cleavage of amyloid-? precursor protein (A?PP) produces flA?(1-40/42), but it is not yet known whether the ?-secretase BACE1 or the alternative ?-secretase cathepsin B (CatB) participate in the production of pGlu-A?. Therefore, this study examined the effects of gene knockout of these proteases on brain pGlu-A? levels in transgenic A?PPLon mice, which express A?PP isoform 695 and have the wild-type (wt) ?-secretase activity found in most AD patients. Knockout or overexpression of the CatB gene reduced or increased, respectively, pGlu-A?(3-40/42), flA?(1-40/42), and pGlu-A? plaque load, but knockout of the BACE1 gene had no effect on those parameters in the transgenic mice. Treatment of A?PPLon mice with E64d, a cysteine protease inhibitor of CatB, also reduced brain pGlu-A?(3-42), flA?(1-40/42), and pGlu-A? plaque load. Treatment of neuronal-like chromaffin cells with CA074Me, an inhibitor of CatB, resulted in reduced levels of pGlu-A?(3-40) released from the activity-dependent, regulated secretory pathway. Moreover, CatB knockout and E64d treatment has been previously shown to improve memory deficits in the A?PPLon mice. These data illustrate the role of CatB in producing pGlu-A? and flA? that participate as key factors in the development of AD. The advantages of CatB inhibitors, especially E64d and its derivatives, as alternatives to BACE1 inhibitors in treating AD patients are discussed.
N-truncated pyroglutamate (pGlu)-amyloid-? [A?(3-40/42)] peptides are key components that promote A? peptide accumulation, leading to neurodegeneration and memory loss in Alzheimer's disease. Because A? deposition in the brain occurs in an activity-dependent manner, it is important to define the subcellular organelle for pGlu-A?(3-40/42) production by glutaminyl cyclase (QC) and their colocalization with full-length A?(1-40/42) peptides for activity-dependent, regulated secretion. Therefore, the objective of this study was to investigate the hypothesis that pGlu-A? and QC are colocalized with A? in dense-core secretory vesicles (DCSV) for activity-dependent secretion with neurotransmitters.
Cholesteryl ester transfer protein (CETP) plays an important role in reverse cholesterol transport, with decreased CETP activity increasing HDL levels. Formation of an alternative splice form lacking exon 9 (?9-CETP) has been associated with two single nucleotide polymorphisms (SNPs) in high linkage disequilibrium with each other, namely rs9930761 T>C located in intron 8 in a putative splicing branch site and rs5883 C>T in a possible exonic splicing enhancer (ESE) site in exon 9. To assess the relative effect of rs9930761 and rs5883 on splicing, mini-gene constructs spanning CETP exons 8 to 10, carrying all four possible allele combinations, were transfected into HEK293 and HepG2 cells. The minor T allele of rs5883 enhanced splicing significantly in both cell lines whereas the minor C allele of rs9930761 did not. In combination, the two alleles did not yield greater splicing than the rs5883 T allele alone in HepG2 cells. These results indicate that the genetic effect on CETP splicing is largely attributable to rs5883. We also confirm that ?9-CETP protein is expressed in the liver but fails to circulate in the blood.
Beta-amyloid (A?) peptides are secreted from neurons, resulting in extracellular accumulation of A? and neurodegeneration of Alzheimers disease. Because neuronal secretion is fundamental for the release of neurotransmitters, this study assessed the hypothesis that A? undergoes co-release with neurotransmitters. Model neuronal-like chromaffin cells were investigated, and results illustrate regulated, co-secretion of A?(1-40) and A?(1-42) with peptide neurotransmitters (galanin, enkephalin, and NPY) and catecholamine neurotransmitters (dopamine, norepinephrine, and epinephrine). Regulated secretion from chromaffin cells was stimulated by KCl depolarization and nicotine. Forskolin, stimulating cAMP, also induced co-secretion of A? peptides with peptide and catecholamine neurotransmitters. These data suggested the co-localization of A? with neurotransmitters in dense core secretory vesicles (DCSV) that store and secrete such chemical messengers. Indeed, A? was demonstrated to be present in DCSV with neuropeptide and catecholamine transmitters. Furthermore, the DCSV organelle contains APP and its processing proteases, ?- and ?-secretases, that are necessary for production of A?. Thus, A? can be generated in neurotransmitter-containing DCSV. Human IMR32 neuroblastoma cells also displayed regulated secretion of A?(1-40) and A?(1-42) with the galanin neurotransmitter. These findings illustrate that A? peptides are present in neurotransmitter-containing DCSV, and undergo co-secretion with neuropeptide and catecholamine neurotransmitters that regulate brain functions.
The Purkinje cell degeneration (pcd) mouse is a recessive model of neurodegeneration, involving cerebellum and retina. Purkinje cell death in pcd is dramatic, as >99% of Purkinje neurons are lost in 3 weeks. Loss of function of Nna1 causes pcd, and Nna1 is a highly conserved zinc carboxypeptidase. To determine the basis of pcd, we implemented a two-pronged approach, combining characterization of loss-of-function phenotypes of the Drosophila Nna1 ortholog (NnaD) with proteomics analysis of pcd mice. Reduced NnaD function yielded larval lethality, with survivors displaying phenotypes that mirror disease in pcd. Quantitative proteomics revealed expression alterations for glycolytic and oxidative phosphorylation enzymes. Nna proteins localize to mitochondria, loss of NnaD/Nna1 produces mitochondrial abnormalities, and pcd mice display altered proteolytic processing of Nna1 interacting proteins. Our studies indicate that Nna1 loss of function results in altered bioenergetics and mitochondrial dysfunction.
Dynorphin opioid neuropeptides mediate neurotransmission for analgesia and behavioral functions. Dynorphin A, dynorphin B, and alpha-neoendorphin are generated from prodynorphin by proteolytic processing. This study demonstrates the significant role of the cysteine protease cathepsin L for producing dynorphins. Cathepsin L knockout mouse brains showed extensive decreases in dynorphin A, dynorphin B, and alpha-neoendorphin that were reduced by 75%, 83%, and 90%, respectively, compared to controls. Moreover, cathepsin L in brain cortical neurons was colocalized with dynorphins in secretory vesicles, the primary site of neuropeptide production. Cellular coexpression of cathepsin L with prodynorphin in PC12 cells resulted in increased production of dynorphins A and B. Comparative studies of PC1/3 and PC2 convertases showed that PC1/3 knockout mouse brains had a modest decrease in dynorphin A, and PC2 knockout mice showed a minor decrease in alpha-neoendorphin. Overall, these results demonstrate a prominent role for cathepsin L, jointly with PC1/3 and PC2, for production of dynorphins in brain.
The endopin serpin protease inhibitors have been identified by molecular studies as components of secretory vesicles that produce neuropeptides. Endopin 1 inhibits trypsin-like serine proteases, and endopin 2 inhibits cathepsin L that produces neuropeptides in secretory vesicles. To assess the secretory vesicle and neuroendocrine tissue distribution of these endopins, the goal of this study was to define specific antisera for each endopin isoform and to examine their localization with neuropeptides and in neuroendocrine tissues.
Cholecystokinin (CCK) is a peptide neurotransmitter whose production requires proteolytic processing of the proCCK precursor to generate active CCK8 neuropeptide in brain. This study demonstrates the significant role of the cysteine protease cathepsin L for CCK8 production. In cathepsin L knockout (KO) mice, CCK8 levels were substantially reduced in brain cortex by an average of 75%. To evaluate the role of cathepsin L in producing CCK in the regulated secretory pathway of neuroendocrine cells, pituitary AtT-20 cells that stably produce CCK were treated with the specific cathepsin L inhibitor, CLIK-148. CLIK-148 inhibitor treatment resulted in decreased amounts of CCK secreted from the regulated secretory pathway of AtT-20 cells. CLIK-148 also reduced cellular levels of CCK9 (Arg-CCK8), consistent with CCK9 as an intermediate product of cathepsin L, shown by the decreased ratio of CCK9/CCK8. The decreased CCK9/CCK8 ratio also suggests a shift in the production to CCK8 over CCK9 during inhibition of cathepsin L. During reduction of the PC1/3 processing enzyme by siRNA, the ratio of CCK9/CCK8 was increased, suggesting a shift to the cathepsin L pathway for the production of CCK9. The changes in ratios of CCK9 compared to CCK8 are consistent with dual roles of the cathepsin L protease pathway that includes aminopeptidase B to remove NH2-terminal Arg or Lys, and the PC1/3 protease pathway. These results suggest that cathepsin L functions as a major protease responsible for CCK8 production in mouse brain cortex, and participates with PC1/3 for CCK8 production in pituitary cells.
The production of the peptide hormones ACTH, alpha-MSH, and beta-endorphin requires proteolytic processing of POMC which is hypothesized to utilize dual cysteine- and subtilisin-like protease pathways, consisting of the secretory vesicle cathepsin L pathway and the well-known subtilisin-like prohormone convertase (PC) pathway. To gain knowledge of these protease components in human pituitary where POMC-derived peptide hormones are produced, this study investigated the presence of these protease pathway components in human pituitary. With respect to the cathepsin L pathway, human pituitary contained cathepsin L of 27-29 kDa and aminopeptidase B of approximately 64 kDa, similar to those in secretory vesicles of related neuroendocrine tissues. The serpin inhibitor endopin 2, a selective inhibitor of cathepsin L, was also present. With respect to the PC pathway, human pituitary expresses PC1/3 and PC2 of approximately 60-65 kDa, which represent active PC1/3 and PC2; peptide hormone production then utilizes carboxypeptidase E (CPE) which is present as a protein of approximately 55 kDa. Analyses of POMC products in human pituitary showed that they resemble those in mouse pituitary which utilizes cathepsin L and PC2 for POMC processing. These findings suggest that human pituitary may utilize the cathepsin L and prohormone convertase pathways for producing POMC-derived peptide hormones.
Peptide neurotransmitters function as key intercellular signaling molecules in the nervous system. These peptides are generated in secretory vesicles from proneuropeptides by proteolytic processing at dibasic residues, followed by removal of N- and/or C-terminal basic residues to form active peptides. Enkephalin biosynthesis from proenkephalin utilizes the cysteine protease cathepsin L and the subtilisin-like prohormone convertase 2 (PC2). Cathepsin L generates peptide intermediates with N-terminal basic residue extensions, which must be removed by an aminopeptidase. In this study, we identified cathepsin H as an aminopeptidase in secretory vesicles that produces (Met)enkephalin (ME) by sequential removal of basic residues from KR-ME and KK-ME, supported by in vivo knockout of the cathepsin H gene. Localization of cathepsin H in secretory vesicles was demonstrated by immunoelectron microscopy and immunofluorescence deconvolution microscopy. Purified human cathepsin H sequentially removes N-terminal basic residues to generate ME, with peptide products characterized by nano-LC-MS/MS tandem mass spectrometry. Cathepsin H shows highest activities for cleaving N-terminal basic residues (Arg and Lys) among amino acid fluorogenic substrates. Notably, knockout of the cathepsin H gene results in reduction of ME in mouse brain. Cathepsin H deficient mice also show a substantial decrease in galanin peptide neurotransmitter levels in brain. These results illustrate a role for cathepsin H as an aminopeptidase for enkephalin and galanin peptide neurotransmitter production.
Proteases are required for processing precursors into active neuropeptides that function as neurotransmitters for cell-cell communication. This study demonstrates the novel function of human cathepsin V protease for producing the neuropeptides enkephalin and neuropeptide Y (NPY). Cathepsin V is a human-specific cysteine protease gene. Findings here show that expression of cathepsin V in neuroendocrine PC12 cells and human neuronal SK-N-MC cells results in production of (Met)enkephalin from proenkephalin. Gene silencing of cathepsin V by siRNA in human SK-N-MC cells results in reduction of (Met)enkephalin by more than 80%, illustrating the prominent role of cathepsin V for neuropeptide production. In vitro processing of proenkephalin by cathepsin V occurs at dibasic residue sites to generate enkephalin-containing peptides and an ?24-kDa intermediate present in human brain. Cathepsin V is present in human brain cortex and hippocampus where enkephalin and NPY are produced and is present in purified human neuropeptide secretory vesicles. Colocalization of cathepsin V with enkephalin and NPY in secretory vesicles of human neuroblastoma cells was illustrated by confocal microscopy. Furthermore, expression of cathepsin V with proNPY results in NPY production. These findings indicate the unique function of human cathepsin V for producing enkephalin and NPY neuropeptides required for neurotransmission in health and neurological diseases.
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