Cholinergic leukocytes in sepsis and at the neuroimmune junction in the spleen.

The spleen is a key participant in the pathophysiology of sepsis and inflammatory disease. Many splenocytes exhibit a cholinergic phenotype, but our knowledge regarding their cholinergic biology and how they are affected by sepsis is incomplete. We evaluated effects of acute sepsis on the spleen using the cecal ligation and puncture (CLP) model in C57BL/6 and ChATBAC-eGFP mice. Quantification of cholinergic gene expression showed that choline acetyltransferase and vesicular acetylcholine transporter (VAChT) are present and that VAChT is upregulated in sepsis, suggesting increased capacity for release of acetylcholine (ACh). High affinity choline transporter is not expressed but organic acid transporters are, providing additional mechanisms for release. Flow cytometry studies identified subpopulations of cholinergic T and B cells as well as monocytes/macrophages. Neither abundance nor GFP intensity of cholinergic T cells changed in sepsis, suggesting that ACh synthetic capacity was not altered. Spleens have low acetylcholinesterase activity, and the enzyme is localized primarily in red pulp, characteristics expected to favor cholinergic signaling. For cellular studies, ACh was quantified by mass spectroscopy using d4-ACh internal standard. Isolated splenocytes from male mice contain more ACh than females, suggesting the potential for gender-dependent differences in cholinergic immune function. Isolated splenocytes exhibit basal ACh release, which can be increased by isoproterenol (4 and 24 h) or by T cell activation with antibodies to CD3 and CD28 (24 h). Collectively, these data support the concept that sepsis enhances cholinergic function in the spleen and that release of ACh can be triggered by stimuli via different mechanisms.

Variable expression of GFP in different populations of peripheral cholinergic neurons of ChATBAC-eGFP transgenic mice.

Immunohistochemistry is used widely to identify cholinergic neurons, but this approach has some limitations. To address these problems, investigators developed transgenic mice that express enhanced green fluorescent protein (GFP) directed by the promoter for choline acetyltransferase (ChAT), the acetylcholine synthetic enzyme. Although, it was reported that these mice express GFP in all cholinergic neurons and non-neuronal cholinergic cells, we could not detect GFP in cardiac cholinergic nerves in preliminary experiments. Our goals for this study were to confirm our initial observation and perform a qualitative screen of other representative autonomic structures for the presences of GFP in cholinergic innervation of effector tissues. We evaluated GFP fluorescence of intact, unfixed tissues and the cellular localization of GFP and vesicular acetylcholine transporter (VAChT), a specific cholinergic marker, in tissue sections and intestinal whole mounts. Our experiments identified two major tissues where cholinergic neurons and/or nerve fibers lacked GFP: 1) most cholinergic neurons of the intrinsic cardiac ganglia and all cholinergic nerve fibers in the heart and 2) most cholinergic nerve fibers innervating airway smooth muscle. Most cholinergic neurons in airway ganglia stained for GFP. Cholinergic systems in the bladder and intestines were fully delineated by GFP staining. GFP labeling of input to ganglia with long preganglionic projections (vagal) was sparse or weak, while that to ganglia with short preganglionic projections (spinal) was strong. Total absence of GFP might be due to splicing out of the GFP gene. Lack of GFP in nerve projections from GFP-positive cell bodies might reflect a transport deficiency.

Deletion of neurturin impairs development of cholinergic nerves and heart rate control in postnatal mouse hearts.

The neurotrophic factor neurturin is required for normal cholinergic innervation of adult mouse heart and bradycardic responses to vagal stimulation. Our goals were to determine effects of neurturin deletion on development of cardiac chronotropic and dromotropic functions, vagal baroreflex response, and cholinergic nerve density in nodal regions of postnatal mice. Experiments were performed on postnatal C57BL/6 wild-type (WT) and neurturin knockout (KO) mice. Serial electrocardiograms were recorded noninvasively from conscious pups using an ECGenie apparatus. Mice were treated with atenolol to evaluate and block sympathetic effects on heart rate (HR) and phenylephrine (PE) to stimulate the baroreflex. Immunohistochemistry was used to label cholinergic nerves in paraffin sections. WT and KO mice showed similar age-dependent increases in HR and decreases in PR interval between postnatal days (P) 2.5 and 21. Treatment with atenolol reduced HR significantly in WT and KO pups at P7.5. PE caused a reflex bradycardia that was significantly smaller in KO pups. Cholinergic nerve density was significantly less in nodal regions of P7.5 KO mice. We conclude that cholinergic nerves have minimal influence on developmental changes in HR and PR, QRS, and QTc intervals in mouse pups. However, cholinergic nerves mediate reflex bradycardia by 1 week postnatally. Deletion of neurturin impairs cholinergic innervation of the heart and the vagal efferent component of the baroreflex early during postnatal development.

Effect of obesity and exercise on the expression of the novel myokines, Myonectin and Fibronectin type III domain containing 5.

Metabolic dysfunction in skeletal muscle is a major contributor to the development of type 2 diabetes. Endurance exercise training has long been established as an effective means to directly restore skeletal muscle glucose and lipid uptake and metabolism. However, in addition to the direct effects of skeletal muscle on glucose and lipids, there is renewed interest in the ability of skeletal muscle to coordinate metabolic activity of other tissues, such as adipose tissue and liver. The purpose of this study was to examine the effects of endurance exercise on the expression level of two novel muscle-derived secreted factors, or myokines, Myonectin and Fibronectin type III domain containing 5 (FNDC5), the precursor for Irisin. Methods. We performed immunoblot analysis and quantitative real-time PCR analysis of Myonectin and FNDC5 in the diaphragm muscles of obese Zucker rat (OZR) and lean Zucker rat (LZR) with 9 weeks of aerobic training on a motorized treadmill. Results. We show that myonectin gene expression is increased in the OZR model of obesity and decreases with exercise in both lean and obese Zucker rats. Conversely, myonectin protein concentration was elevated with exercise. Similarly, FNDC5 mRNA levels are significantly higher in the OZR, however exercise training had no effect on the expression level of FNDC5 in either the LZR or OZR. We did not observe any difference in muscle protein content of Irisin with obesity or exercise. Conclusion. Our data shows that exercise training does not increase either FNDC5 or myonectin gene expression, indicating that increased transcriptional regulation of these myokines is not induced by exercise. However, our data also indicates a yet to be explored disconnect between myonectin gene expression and protein content. Further, this report highlights the importance of verifying reference genes when completing gene expression analysis. We found that many commonly used reference genes varied significantly by obesity and/or exercise and would have skewed the results of this study if used to normalize gene expression data. The unstable reference genes include: beta-Actin, beta-2-microglobulin, Non-POU domain containing, octamer-binding, Peptidylprolyl isomerase H, 18S ribosomal RNA, TATA box binding protein and Transferrin receptor.

Effect of neurturin deficiency on cholinergic and catecholaminergic innervation of the murine eye.

Neurturin (NRTN) is a neurotrophic factor required for the development of many parasympathetic neurons and normal cholinergic innervation of the heart, lacrimal glands and numerous other tissues. Previous studies with transgenic mouse models showed that NRTN is also essential for normal development and function of the retina (J. Neurosci. 28:4123-4135, 2008). NRTN knockout (KO) mice exhibit a marked thinning of the outer plexiform layer (OPL) of the retina, with reduced abundance of horizontal cell dendrites and axons, and aberrant projections of horizontal cells and bipolar cells into the outer nuclear layer. The effects of NRTN deletion on specific neurotransmitter systems in the retina and on cholinergic innervation of the iris are unknown. To begin addressing this deficiency, we used immunohistochemical methods to study cholinergic and noradrenergic innervation of the iris and the presence and localization of cholinergic and dopaminergic neurons and nerve fibers in eyes from adult male wild-type (WT) and NRTN KO mice (age 4-6 months). Mice were euthanized, and eyes were removed and fixed in cold neutral buffered formalin or 4% paraformaldehyde. Formalin-fixed eyes were embedded in paraffin, and 5 µm cross-sections were collected. Representative sections were stained with hematoxylin and eosin or processed for fluorescence immunohistochemistry after treatment for antigen retrieval. Whole mount preparations were dissected from paraformaldehyde fixed eyes and used for immunohistochemistry. Cholinergic and catecholaminergic nerve fibers were labeled with primary antibodies to the vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH), respectively. Cholinergic and dopaminergic cell bodies were labeled with antibodies to choline acetyltransferase (ChAT) and TH, respectively. Cholinergic innervation of the mouse iris was restricted to the sphincter region, and noradrenergic fibers occurred throughout the iris and in the ciliary processes. This pattern was unaffected by deletion of NRTN. Furthermore, functional experiments demonstrated that cholinergic regulation of the pupil diameter was retained in NRTN KO mice. Hematoxylin and eosin stains of the retina confirmed a marked thinning of the OPL in KO mice. VAChT and ChAT staining of the retina revealed two bands of cholinergic processes in the inner plexiform layer, and these were unaffected by NRTN deletion. Likewise, NRTN deletion did not affect the abundance of ChAT-positive ganglion and amacrine cells. In marked contrast, staining for TH showed an increased abundance of dopaminergic processes in the OPL of retina from KO mice. Staining of retinal whole mounts for TH showed no difference in the abundance of dopaminergic amacrine cells between WT and KO mice. These findings demonstrate that the neurotrophic factor NRTN is not required for the development or maintenance of cholinergic innervation of the iris, cholinergic control of pupil diameter, or for development of cholinergic and dopaminergic amacrine cells of the retina. However, NRTN deficiency causes a marked reduction in the size of the OPL and aberrant growth of dopaminergic processes into this region.

Evaluation of a technique to identify acetylcholinesterase C-terminal peptides in human serum samples.

A novel theory for neurodegeneration is that non-cholinergic functions of acetylcholinesterase (AChE) are responsible for the progressive death of global neurons. The C-terminal region of AChE has been shown to be responsible for non-cholinergic actions of AChE by binding to an allosteric site on the alpha 7-nicotinic acetylcholine receptor, thereby causing calcium influx; the resultant signal has trophic effects in immature neurons, but toxic effects in mature neurons. Although there is strong in vitro and in vivo evidence for the involvement of this C-terminal region of AChE in neurodegeneration, a cleaved C-terminal peptide has not yet been identified in human brains. This preliminary study aimed to identify the cleaved AChE C-terminal peptide in serum from human Alzheimer's disease patients using immunoaffinity purification. A number of antibodies were tested for sensitivity and specificity towards peptide sequences from the C-terminus. Although the antibodies were able to identify peptide in vitro, peptide was not detected using immunoaffinity purification of human serum, possibly due to insufficient detection limits of the antibody. Therefore more sensitive techniques are required to identify cleaved AChE C-terminal peptides in human samples. None the less, C-terminal AChE peptide might act as a signalling molecule in an as yet unexplored system.

Upregulation of alpha7 Nicotinic Receptors by Acetylcholinesterase C-Terminal Peptides.

BACKGROUND: The alpha-7 nicotinic acetylcholine receptor (alpha7-nAChR) is well known as a potent calcium ionophore that, in the brain, has been implicated in excitotoxicity and hence in the underlying mechanisms of neurodegenerative disorders such as Alzheimer's disease. Previous research implied that the activity of this receptor may be modified by exposure to a peptide fragment derived from the C-terminal region of the enzyme acetylcholinesterase. This investigation was undertaken to determine if the functional changes observed could be attributed to peptide binding interaction with the alpha7-nAChR, or peptide modulation of receptor expression. METHODOLOGY/PRINCIPAL FINDINGS: This study provides evidence that two peptides derived from the C-terminus of acetylcholinesterase, not only selectively displace specific bungarotoxin binding at the alpha7-nAChR, but also alter receptor binding properties for its familiar ligands, including the alternative endogenous agonist choline. Of more long-term significance, these peptides also induce upregulation of alpha7-nAChR mRNA and protein expression, as well as enhancing receptor trafficking to the plasma membrane. CONCLUSIONS/SIGNIFICANCE: The results reported here demonstrate a hitherto unknown relationship between the alpha7-nAChR and the non-enzymatic functions of acetylcholinesterase, mediated independently by its C-terminal domain. Such an interaction may prove valuable as a pharmacological tool, prompting new approaches for understanding, and combating, the process of neurodegeneration.

Non-hydrolytic functions of acetylcholinesterase. The significance of C-terminal peptides.

This review explores the possibility that acetylcholinesterase may play a pivotal, non-hydrolytic role in neurodegeneration. More specifically, C-terminal sequences of acetylcholinesterase may act as signalling molecules in key brain regions characteristically vulnerable to Alzheimer's, Parkinson's and motor neuron disease.

Multiple cascade effects of oxidative stress on astroglia.

Many neurodegenerative diseases share common underlying features, most prominent of which are dysregulation of calcium homeostasis and reactive astrogliosis, ultimately triggered by oxidative stress. Interestingly, an additional feature of the early response to stress conditions is the upregulation and release of acetylcholinesterase (AChE). This study therefore investigates the link between oxidative stress, calcium influx, gene expression, protein synthesis, and AChE release. We report that, in astroglia and in an immortalized cell line, GH4-halpha7, acute oxidative stress causes influx of extracellular calcium through L-type voltage-gated calcium channels (L-VGCC), followed by increased release of AChE into the extracellular medium. Moreover, rapid and sustained changes in mRNA expression of AChE, L-VGCC, and melastatin-like transient receptor potential 2 (TRPM2) accompany profound suppression of global protein synthesis. Application of L-VGCC blockers selectively reduces stress-induced calcium influx and AChE release, mitigates changes in gene expression, and facilitates recovery from protein synthesis suppression. Although glia exhibit greater sensitivity in their responses, the results are comparable in astroglia and GH4-halpha7 cells, and suggest a generalized and integrated cellular response to stress conditions that characterizes changes observed in neurodegeneration.