Identification of a region from the human cholinergic gene locus that targets expression of the vesicular acetylcholine transporter to a subset of neurons in the medial habenular nucleus in transgenic mice

We use a transgenic mouse model system to elucidate the regulatory regions within the human cholinergic gene locus responsible for vesicular acetylcholine transporter gene expression in vivo. In this report we characterized two transgenes for their ability to confer cholinergic-specific expression of the encoded vesicular acetylcholine transporter. An 11.2 kb transgene (named hV11.2) that spanned from about 5 kb upstream of the start of vesicular acetylcholine transporter translation down to the first choline acetyltransferase coding exon gave expression in the somatomotor neurons and a subpopulation of cholinergic neurons in the medial habenular nucleus. The second transgene (named hV6.7), a 5-prime truncated version of hV11.2 that was devoid of 4.5 kb of gene-regulatory sequences completely lacked vesicular acetylcholine transporter expression in vivo. Our data indicate that vesicular acetylcholine transporter expression in somatomotor neurons and in the medial habenular nucleus is uniquely specified within the cholinergic gene locus, and separable from cholinergic expression elsewhere. The identification of these two subdivisions of the cholinergic nervous system suggests that other cholinergic neurons in the CNS and PNS are similarly regulated by additional discrete domains within the cholinergic gene locus.     

Unusual Regulation of Splicing of the Cholinergic Locus in Caenorhabditis elegans

The essential neurotransmitter acetylcholine functions throughout the animal kingdom. In Caenorhabditis elegans, the acetylcholine biosynthetic enzyme [choline acetyltransferase (ChAT)] and vesicular transporter [vesicular acetylcholine transporter (VAChT)] are encoded by the cha-1 and unc-17 genes, respectively. These two genes compose a single complex locus in which the unc-17 gene is nested within the first intron of cha-1, and the two gene products arise from a common pre-messenger RNA (pre-mRNA) by alternative splicing. This genomic organization, known as the cholinergic gene locus (CGL), is conserved throughout the animal kingdom, suggesting that the structure is important for the regulation and function of these genes. However, very little is known about CGL regulation in any species. We now report the identification of an unusual type of splicing regulation in the CGL of C. elegans, mediated by two pairs of complementary sequence elements within the locus. We show that both pairs of elements are required for efficient splicing to the distal acceptor, and we also demonstrate that proper distal splicing depends more on sequence complementarity within each pair of elements than on the sequences themselves. We propose that these sequence elements are able to form stem-loop structures in the pre-mRNA; such structures would favor specific splicing alternatives and thus regulate CGL splicing. We have identified complementary elements at comparable locations in the genomes of representative species of other animal phyla; we suggest that this unusual regulatory mechanism may be a general feature of CGLs.     

Acetylcholine release and the cholinergic genomic locus

Choline acetyltransferase and vesicular acetylcholine-transporter genes are adjacent and coregulated. They define a cholinergic locus that can be turned on under the control of several factors, including the neurotrophins and the cytokines. Hirschprung's disease, or congenital megacolon, is characterized by agenesis of intramural cholinergic ganglia in the colorectal region. It results from mutations of the RET (GDNF-activated) and the endothelin-receptor genes, causing a disregulation in the cholinergic locus. Using cultured cells, it was shown that the cholinergic locus and the proteins involved in acetylcholine (ACh) release can be expressed separately ACh release could be demonstrated by means of biochemical and electrophysiological assays even in noncholinergic cells following preloading with the transmitter. Some noncholinergic or even nonneuronal cell types were found to be capable of releasing ACh quanta. In contrast, other cells were incompetent for ACh release. Among them, neuroblastoma N18TG-2 cells were rendered release-competent by transfection with the mediatophore gene. Mediatophore is an ACh-translocating protein that has been purified from plasma membranes ofTorpedo nerve terminal; it confers a specificity for ACh to the release process. The mediatophores are activated by Ca²⁺; but with a slower time course, they can be desensitized by Ca²⁺. A strictly regulated calcium microdomain controls the synchronized release of ACh quanta at the active zone. In addition to ACh and ATP, synaptic vesicles have an ATP-dependent Ca²⁺ uptake system; they transiently accumulate Ca²⁺ after a brief period of stimulation. Those vesicles that are docked close to Ca²⁺ channels are therefore in the best position to control the profile and dynamics of the Ca²⁺ microdomains. Thus, vesicles and their whole set of associated proteins (SNAREs and others) are essential for the regulation of the release mechanism in which the mediatophore seems to play a key role.     

Activation of GSK‐3 disrupts cholinergic homoeostasis in nucleus basalis of Meynert and frontal cortex of rats

The cholinergic impairment is an early marker in Alzheimer's disease (AD), while the mechanisms are not fully understood. We investigated here the effects of glycogen synthase kinse‐3 (GSK‐3) activation on the cholinergic homoeostasis in nucleus basalis of Meynert (NBM) and frontal cortex, the cholinergic enriched regions. We activated GSK‐3 by lateral ventricular infusion of wortmannin (WT) and GF‐109203X (GFX), the inhibitors of phosphoinositol‐3 kinase (PI3‐K) and protein kinase C (PKC), respectively, and significantly decreased the acetylcholine (ACh) level via inhibiting choline acetyl transferase (ChAT) rather than regulating acetylcholinesterase (AChE). Neuronal axonal transport was disrupted and ChAT accumulation occurred in NBM and frontal cortex accompanied with hyperphosphorylation of tau and neurofilaments. Moreover, ChAT expression decreased in NBM attributing to cleavage of nuclear factor‐κB/p100 into p52 for translocation into nucleus to lower ChAT mRNA level. The cholinergic dysfunction could be mimicked by overexpression of GSK‐3 and rescued by simultaneous administration of LiCl or SB216763, inhibitors of GSK‐3. Our data reveal the molecular mechanism that may underlie the cholinergic impairments in AD patients.     

Localization of cholinergic innervation and neurturin receptors in adult mouse heart and expression of the neurturin gene

Neurturin (NRTN) is a neurotrophic factor required during development for normal cholinergic innervation of the heart, but whether NRTN continues to function in the adult heart is unknown. We have therefore evaluated NRTN expression in adult mouse heart and the association of NRTN receptors with intracardiac cholinergic neurons and nerve fibers. Mapping the regional distribution and density of cholinergic nerves in mouse heart was an integral part of this goal. Analysis of RNA from adult C57BL/6 mouse hearts demonstrated NRTN expression in atrial and ventricular tissue. Virtually all neurons in the cardiac parasympathetic ganglia exhibited the cholinergic phenotype, and over 90% of these cells contained both components of the NRTN receptor, Ret tyrosine kinase and GDNF family receptor α2 (GFRα2). Cholinergic nerve fibers, identified by labeling for the high affinity choline transporter, were abundant in the sinus and atrioventricular nodes, ventricular conducting system, interatrial septum, and much of the right atrium, but less abundant in the left atrium. The right ventricular myocardium contained a low density of cholinergic nerves, which were sparse in other regions of the working ventricular myocardium. Some cholinergic nerves were also associated with coronary vessels. GFRα2 was present in most cholinergic nerve fibers and in Schwann cells and their processes throughout the heart. Some cholinergic nerve fibers, such as those in the sinus node, also exhibited Ret immunoreactivity. These findings provide the first detailed mapping of cholinergic nerves in mouse heart and suggest that the neurotrophic influence of NRTN on cardiac cholinergic innervation continues in mature animals.This study was supported by the National Heart, Lung, and Blood Institute (grant HL-54633).     

Neurotrophins differentially enhance acetylcholine release, acetylcholine content and choline acetyltransferase activity in basal forebrain neurons

Several lines of evidence indicate that nerve growth factor is important for the development and maintenance of the basal forebrain cholinergic phenotype. In the present study, using rat primary embryonic basal forebrain cultures, we demonstrate the differential regulation of functional cholinergic markers by nerve growth factor treatment (24–96 h). Following a 96‐h treatment, nerve growth factor (1–100 ng/mL) increased choline acetyltransferase activity (168–339% of control), acetylcholine content (141–185%), as well as constitutive (148–283%) and K⁺‐stimulated (162–399%) acetylcholine release, but increased release was not accompanied by increased high‐affinity choline uptake. Enhancement of ACh release was attenuated by vesamicol (1 µm ), suggesting a vesicular source, and was abolished under choline‐free conditions, emphasizing the importance of extracellular choline as the primary source for acetylcholine synthesized for release. A greater proportion of acetylcholine released from nerve growth factor‐treated cultures than from nerve growth factor‐naïve cultures was blocked by voltage‐gated Ca²⁺ channel antagonists, suggesting that nerve growth factor modified this parameter of neurotransmitter release. Cotreatment of NGF (20 ng/mL) with K252a (200 nm ) abolished increases in ChAT activity and prevented enhancement of K⁺‐stimulated ACh release beyond the level associated with K252a, suggesting the involvement of TrkA receptor signaling. Also, neurotrophin‐3, neurotrophin‐4 and brain‐derived neurotrophic factor (all at 5–200 ng/mL) increased acetylcholine release, although they were not as potent as nerve growth factor and higher concentrations were required. High brain‐derived neurotrophic factor concentrations (100 and 200 ng/mL) did, however, increase release to a level similar to nerve growth factor. In summary, long‐term exposure (days) of basal forebrain cholinergic neurons to nerve growth factor, and in a less‐potent fashion the other neurotrophins, enhanced the release of acetylcholine, which was dependent upon a vesicular pool and the availability of extracellular choline.     

Inflammatory and lipid regulation by cholinergic activity in epicardial stromal cells from patients who underwent open‐heart surgery

The modulation of acetylcholine (ACh) release by botulinum toxin injection into epicardial fat diminishes atrial fibrillation (AF) recurrence. These results suggest an interaction between autonomic imbalance and epicardial fat as risk factors of AF. Our aim was to study the inflammatory, lipidic and fibroblastic profile of epicardial stroma from patients who underwent open‐heart surgery, their regulation by cholinergic activity and its association with AF. We performed in vitro and ex vivo assays from paired subcutaneous and epicardial stromal cells or explants from 33 patients. Acute ACh effects in inflammation and lipid‐related genes were analysed by qPCR, in intracellular calcium mobilization were performed by Fluo‐4 AM staining and in neutrophil migration by trans‐well assays. Chronic ACh effects on lipid accumulation were visualized by AdipoRed. Plasma protein regulation by parasympathetic denervation was studied in vagotomized rats. Our results showed a higher pro‐inflammatory profile in epicardial regarding subcutaneous stromal cells. Acute ACh treatment up‐regulated monocyte chemoattractant protein 1 levels. Chronic ACh treatment improved lipid accumulation within epicardial stromal cells (60.50% [22.82‐85.13] vs 13.85% [6.17‐23.16], P < .001). Additionally, patients with AF had higher levels of fatty acid‐binding protein 4 (1.54 ± 0.01 vs 1.47 ± 0.01, P = .005). Its plasma levels were pronouncedly declined in vagotomized rats (2.02 ± 0.21 ng/mL vs 0.65 ± 0.23 ng/mL, P < .001). Our findings support the characterization of acute or chronic cholinergic activity on epicardial stroma and its association with AF.     

Kinesin‐2 differentially regulates the anterograde axonal transports of acetylcholinesterase and choline acetyltransferase in Drosophila

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) are involved in acetylcholine synthesis and degradation at pre‐ and postsynaptic compartments, respectively. Here we show that their anterograde transport in Drosophila larval ganglion is microtubule‐dependent and occurs in two different time profiles. AChE transport is constitutive while that of ChAT occurs in a brief pulse during third instar larva stage. Mutations in the kinesin‐2 motor subunit Klp64D and separate siRNA‐mediated knock‐outs of all the three kinesin‐2 subunits disrupt the ChAT and AChE transports, and these antigens accumulate in discrete nonoverlapping punctae in neuronal cell bodies and axons. Quantification analysis further showed that mutations in Klp64D could independently affect the anterograde transport of AChE even before that of ChAT. Finally, ChAT and AChE were coimmunoprecipitated with the kinesin‐2 subunits but not with each other. Altogether, these suggest that kinesin‐2 independently transports AChE and ChAT within the same axon. It also implies that cargo availability could regulate the rate and frequency of transports by kinesin motors. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

In vitro and in vivo efficacy of anti‐chikungunya virus monoclonal antibodies produced in wild‐type and glycoengineered Nicotiana benthamiana plants

Chikungunya virus (CHIKV) is a mosquito‐transmitted alphavirus, and its infection can cause long‐term debilitating arthritis in humans. Currently, there are no licensed vaccines or therapeutics for human use to combat CHIKV infections. In this study, we explored the feasibility of using an anti‐CHIKV monoclonal antibody (mAb) produced in wild‐type (WT) and glycoengineered (?XFT) Nicotiana benthamiana plants in treating CHIKV infection in a mouse model. CHIKV mAb was efficiently expressed and assembled in plant leaves and enriched to homogeneity by a simple purification scheme. While mAb produced in ?XFT carried a single N‐glycan species at the Fc domain, namely GnGn structures, WT produced mAb exhibited a mixture of N‐glycans including the typical plant GnGnXF₃ glycans, accompanied by incompletely processed and oligomannosidic structures. Both WT and ?XFT plant‐produced mAbs demonstrated potent in vitro neutralization activity against CHIKV. Notably, both mAb glycoforms showed in vivo efficacy in a mouse model, with a slight increased efficacy by the ?XFT‐produced mAbs. This is the first report of the efficacy of plant‐produced mAbs against CHIKV, which demonstrates the ability of using plants as an effective platform for production of functionally active CHIKV mAbs and implies optimization of in vivo activity by controlling Fc glycosylation.     

Comparison of patient‐derived high and low phosphatidylserine‐exposing colorectal carcinoma cells in their interaction with anti‐cancer peptides

Current cancer treatment is frequently compromised by severe adverse effects on healthy cells and tissues as well as by the increasing burden of (multi‐)drug resistances. Some representatives of small, amphipathic peptides known as host defense peptides possess the potential to overcome these limitations and to evolve as future anti‐cancer therapeutics. Peptide NK‐2, derived from porcine NK‐lysin, was originally discovered due to its broad‐spectrum antimicrobial activities. Today, also potent anti‐cancer activity is proven and accompanied by low toxicity towards normal human cells. The molecular basis underlying this target selectivity remains rather elusive. Nevertheless, it is presumptive that preferential peptide interactions with surface factors non‐abundant on healthy human cells play a key role. Here, we investigated the cytotoxicity of peptide NK‐2 and structurally improved anti‐cancer variants thereof against two patient‐derived colorectal cancer cell lines, exposing high and low levels of phosphatidylserine on their cell surfaces, respectively. Concluding from a range of in vitro tests involving cellular as well as lipid vesicle‐based methods, it is proposed that the magnitude of the accessible membrane surface charge is not a primarily decisive factor for selective peptide interactions. Instead, it is suggested that the level of membrane surface‐exposed phosphatidylserine is of crucial importance for the activity of peptide NK‐2 and enhanced variants thereof in terms of their cancer cell selectivity, the overall efficacy, as well as the underlying mode of action and kinetics. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

3,4‐dimethoxystilbene,a resveratrol derivative with anti‐angiogenic effect,induces both macroautophagy and apoptosis in endothelial cells

Angiogenesis plays an important role in many pathological processes. Identification of novel anti‐angiogenic agents will provide new insights into the mechanisms for angiogenesis as well as potential lead compounds for developing new drugs. In the present study, a series of resveratrol methylated derivatives have been synthesized and screened. We found trans‐3,4‐dimethoxystilbene (3,4‐DMS) with the fullest potential to develop as an anti‐angiogenic agent. In vitro and in vivo analyses suggested that 3,4‐DMS could effectively inhibit endothelial cell proliferation, migration, tube formation, and endogenous neovascularization. Our results showed that 3,4‐DMS exerted its anti‐angiogenic effect likely through induction of endothelial cell apoptosis via a pathway involving p53, Bax, cytochrome c, and caspase proteases. Moreover, 3,4‐DMS also induced macroautophagy in endothelial cells through activation of AMPK and the downstream inhibition of mTOR signaling pathway. Further studies indicated that intracellular calcium ([Ca²⁺]_i) might bridge the 3,4‐DMS‐induced apoptosis and macroautophagy through modulating reactive oxygen species (ROS) levels in endothelial cells. Combination of 3,4‐DMS with inhibitor of autophagy, such as 3‐methyladenine (3‐MA) and autophagy‐related gene (ATG) 5 small interfering RNA (siRNA), potentiated the pro‐apoptotic and anti‐angiogenic effects of 3,4‐DMS. Our study provides a novel angiogenic inhibitor and a useful tool in exploring the molecular mechanisms for the crosstalk between apoptosis and macroautophagy in endothelial cells. 3,4‐DMS could be served as a potential lead compound for developing a class of new drugs targeting angiogenesis‐related diseases. J. Cell. Biochem. 114: 697–707, 2013. © 2012 Wiley Periodicals, Inc.     

Differentiated NSC-34 motoneuron-like cells as experimental model for cholinergic neurodegeneration

Alpha-motoneurons appear to be exceedingly affected in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Morphological and physiological degeneration of this neuronal phenotype is typically characterized by a marked decrease of neuronal markers and by alterations of cholinergic metabolism such as reduced choline acetyltransferase (ChAT) expression. The motoneuron-like cell line NSC-34 is a hybrid cell line produced by fusion of neuroblastoma with mouse motoneuron-enriched primary spinal cord cells. In order to further establish this cell line as a valid model system to investigate cholinergic neurodegeneration, NSC-34 cells were differentiated by serum deprivation and additional treatment with all-trans retinoic acid (atRA). Cell maturation was characterized by neurite outgrowth and increased expression of neuronal and cholinergic markers, including MAP2, GAP-43 and ChAT. Subsequently, we used differentiated NSC-34 cells to study early degenerative responses following exposure to various neurotoxins (H₂O₂, TNF-α, and glutamate). Susceptibility to toxin-induced cell death was determined by means of morphological changes, expression of neuronal marker proteins, and the ratio of pro-(Bax) to anti-(Bcl-2) apoptotic proteins. NSC-34 cells respond to low doses of neurotoxins with increased cell death of remaining undifferentiated cells with no obvious adverse effects on differentiated cells. Thus, the different vulnerability of differentiated and undifferentiated NSC-34 cells to neurotoxins is a key characteristic of NSC-34 cells and has to be considered in neurotoxic studies. Nonetheless, application of atRA induced differentiation of NSC-34 cells and provides a suitable model to investigate molecular events linked to neurodegeneration of differentiated neurons.     

Production and characterization of active recombinant interleukin‐12/eGFP fusion protein in stably‐transfected DF1 chicken cells

The adjuvant activity of chicken interleukin‐12 (chIL‐12) protein has been described as similar to that of mammalian IL‐12. Recombinant chIL‐12 can be produced using several methods, but chIL‐12 production in eukaryotic cells is lower than that in prokaryotic cells. Stimulating compounds, such as dimethyl sulfoxide (DMSO), can be added to animal cell cultures to overcome this drawback. In this study, we constructed a cell line, DF1/chIL‐12 which stably expressed a fusion protein, chIL‐12 and enhanced green fluorescent protein (eGFP) connected by a (G₄S)₃ linker sequence. Fusion protein production was increased when cells were cultured in the presence of DMSO. When 1 × 10⁶ DF1/chIL‐12 cells were inoculated in a T‐175 flask containing 30 mL of media, incubated for 15 h, and further cultivated in the presence of 4% DMSO for 48 h, the production of total fusion protein was mostly enhanced compared with the production of total fusion protein by using cell lysates induced with DMSO at other concentrations. The concentrations of the unpurified and purified total fusion proteins in cell lysates were 2,781 ± 2.72 ng mL^?1 and 2,207 ± 3.28 ng mL^?1, respectively. The recovery rate was 79%. The fusion protein stimulated chicken splenocytes to produce IFN‐γ, which was measured using an enzyme‐linked immunosorbent assay, in the culture supernatant, indicating that treating DF1/chIL‐12 cells with DMSO or producing chIL‐12 in a fusion protein form does not have adverse effects on the bioactivity of chIL‐12. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:641–649, 2015