J Mol Neurosci DOI 10.1007/s12031-013-0176-4

Cholinesterases as Biomarkers for Parasympathetic Dysfunction and Inflammation-Related Disease Shani Shenhar-Tsarfaty & Shlomo Berliner & Natan M. Bornstein & Hermona Soreq

Received: 15 October 2013 / Accepted: 5 November 2013 # Springer Science+Business Media New York 2013

Abstract Accumulating evidence suggests parasympathetic dysfunction and elevated inflammation as underlying processes in multiple peripheral and neurological diseases. Acetylcholine, the main parasympathetic neurotransmitter and inflammation regulator, is hydrolyzed by the two closely homologous enzymes, acetylcholinesterase and butyrylcholinesterase (AChE and BChE, respectively), which are also expressed in the serum. Here, we consider the potential value of both enzymes as possible biomarkers in diseases associated with parasympathetic malfunctioning. We cover the modulations of cholinesterase activities in inflammation-related events as well as by cholinesterase-targeted microRNAs. We further discuss epigenetic control over cholinesterase gene expression and the impact of single-nucleotide polymorphisms on the corresponding physiological and pathological processes. In particular, we focus on measurements of circulation cholinesterases as a readily quantifiable readout for changes in the sympathetic/parasympathetic balance and the implications of changes in this readout in health and disease. Taken together, this cumulative know-how calls for expanding S. Shenhar-Tsarfaty : H. Soreq (*) The Edmond and Lily Safra Center for Brain Science and Department of Biological Chemistry, The Life Sciences Institute, The Hebrew University of Jerusalem, Jerusalem 91904, Israel e-mail: [email protected] S. Shenhar-Tsarfaty Internal Medicine “E” and Neurology Departments, Tel Aviv Medical Center, affiliated to the Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel S. Berliner Internal Medicine “E” Department, Tel Aviv Medical Center, affiliated to the Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel N. M. Bornstein Neurology Department, Tel Aviv Medical Center, affiliated to the Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

the use of cholinesterase activity measurements for both basic research and as a clinical assessment tool. Keywords Cholinesterases . Acetylcholinesterase . Butyrylcholinesterase . Biomarkers . Diseases . Inflammation

Introduction Acetylcholine (ACh), the first neurotransmitter discovered, was originally described as “vagus stuff” by Otto Loewi due to its ability to mimic the electrical stimulation of the vagus nerve (Loewi 1921). Today, ACh is known as the principal neurotransmitter in brain cholinergic neurons and postganglionic parasympathetic and cholinergic sympathetic nerves, in the periphery, and of both sympathetic and parasympathetic preganglionic fibers (Schwartz 2000). In addition, various peripheral cells such as pancreatic alpha cells (RodriguezDiaz et al. 2011), endothelial cells (Wessler et al. 1998), and placenta cells were found to express non-neuronal ACh (Bhuiyan et al. 2006). Cholinergic molecules have also been detected in the circulation, in thrombocytes (Lev-Lehman et al. 1997), and in lymphocytes (Kawashima and Fujii 2003). Correspondingly, cholinergic signaling is notably involved in central cognitive processes (Conner et al. 2003), in controlling peripheral homeostasis through activation of the parasympathetic system, and in blockade of inflammatory responses (Tracey 2010). The traditional view of ACh acting solely as neurotransmitter has to be revised based on the findings demonstrating the non-neuronal cholinergic system. Acetylcholinesterase (AChE) has been identified in human blood mononuclear leukocytes, human leukemic T cell lines, and rat lymphocytes. Stimulation of T lymphocytes with phytohemagglutinin activates the lymphoid cholinergic system, as evidenced by increased synthesis and release of ACh, increased AChE activity, and increased expression of mRNA encoding choline acetyltransferase and ACh receptors (Kawashima and Fujii

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2000). The widespread synthesis of ACh beyond the nervous system has changed the paradigm of ACh acting merely as a neurotransmitter (Tracey 2010; Wessler and Kirkpatrick 2008). In neuronal elements, in peripheral cells and organs, and in body fluids, the action of ACh is terminated by its degradation into choline and acetate by two closely homologous enzymes, AChE and butyrylcholinesterase (BChE) (Soreq and Seidman 2001). AChE is the major cholinesterase in the brain and performs ACh hydrolysis considerably faster than BChE (Loewenstein-Lichtenstein et al. 1995). To enable continuous surveillance over the ACh hydrolysis process, AChE gene expression is continuously subjected to several regulation levels. Thus, the AChE promoter includes many motifs for transcription factors (Meshorer et al. 2004; Meshorer et al. 2006) and is subject to epigenetic regulation (Sailaja et al. 2012) at the posttranscriptional level; alternative splicing allows the production of three C-terminally distinct AChE variants, the “synaptic” (S) (or “tailed,” AChE-T; Massoulie et al. 2008), “erythrocytic” (E), and “readthrough” (R) AChE isoforms. Yet more recently, the resultant AChE mRNA transcript was found to be suppressed by microRNA (miRNA)132 (Shaked et al. 2009) and predictably by other miRNAs with both neuronal and immune functions (Soreq and Wolf 2011; Hanin and Soreq 2011). Balanced cholinergic signaling depends on the concerted expression of multiple receptors, enzymes, and transporters, and imbalanced response can lead to disease (Ofek and Soreq 2013). For example, cholinergic involvement in the pathogenesis of myasthenia gravis, Sjögren’s syndrome, asthma, and inflammatory bowel disease (IBD) is clearly apparent and possible treatment modalities are aimed to restore cholinergic balance (Ofek and Soreq 2013). Consequently, over the last century, much effort has been devoted to develop reliable methods for manipulating cholinergic signaling and measuring its effect and, yet more specifically, to develop biomarkers for distinguishing between health and the various diseases where cholinergic signaling is impaired.

Cholinergic Status as Biomarker for Disease Diagnosis and Risk Stratification Biomarkers are defined as “measurable characteristics that reflect physiological, pharmacological, or disease processes” according to the European Medicines Agency (Biomarkers Definitions Working Group 2001). Biomarkers are basically biological substances that can be used to indicate the presence or onset of a certain disorder. In line with this, biomarkers are very important indicators of normal and abnormal biological processes and could serve not only as indicators but also as significant players in the pathological pathway. Therefore, a good biomarker should be precise and reliable, distinguishable

between normal and disease tissues, and differentiable between different diseases (Rachakonda et al. 2004). Biochemistry-based approaches for biomarker investigations can be employed in different aspects of medicine, such as elucidation of pathways affected in disease, identification of individuals who are at a high risk of developing disease for prognosis and prediction of response, identification of individuals who are most likely to react to specific therapeutic interventions, and prediction of which patients will develop specific side effects (Guest et al. 2013). Tremendous efforts have been made in recent years to identify the pathological, biochemical, and genetic biomarkers of diverse diseases so that the diagnosis could be established at earlier stages. All of this equates to improvement in patient care by using biomarkers in so-called personalized medicine approaches (Honda et al. 2013). In practice, a good biomarker is defined as a characteristic that is objectively measured and evaluated as an indicator of normal processes or pharmacologic responses to therapeutic intervention (Frank and Hargreaves 2003). Sensitivity, specificity, ease of use, standardization, and proper clinical evaluations are the most important factors that ultimately define the diagnostic utility of a biomarker. The progress in the translational application of a biomarker from basic research to clinical setting is long, challenging, and requires multiple validations using different biological methods and separate clinical cohorts. In our current review, we focus on the most recent findings of cholinergic biomarkers and their biomedical implications as possible prognostic, diagnostic, or therapeutic efficacy biomarkers. Imbalanced sympathetic/parasympathetic activity has been associated with poor cardiovascular outcome (Cole et al. 1999), as well as all-cause mortality (Adabag et al. 2008a), calling for identifying measurable biomarkers of parasympathetic activity for predicting future risks. Indirect measures of cardiac parasympathetic dysfunction such as elevated resting heart rate, delayed heart rate recovery following exercise, and attenuated heart rate increase during exercise have all been shown to be independent predictors for adverse cardiovascular outcome (Cole et al. 1999; Jouven et al. 2005; Leeper et al. 2007). Abnormalities in these parameters (Lahiri et al. 2008) have been shown in diverse study populations to be associated with sudden cardiac death (Jouven et al. 2005; Adabag et al. 2008b), as well as all-cause mortality (Cole et al. 1999; Leeper et al. 2007; Arena et al. 2006; Savonen et al. 2008), but clinically validated biomarkers to assess diseasespecific impairments in the parasympathetic system are not yet available. Of note, the parasympathetic neurotransmitter ACh is extremely labile and difficult to use for clinical measurements (Soreq and Seidman 2001), which is why the use of its hydrolyzing enzymes as an indirect measurement for parasympathetic dysfunction can be beneficial in many disease settings.

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Two Cholinesterases and Multiple miRNAs may Contribute to the Cholinergic Status and Parasympathetic Dysfunction ACh is hydrolyzed in the serum by two homologous enzymes with unique features: AChE and BChE. BChE is the major ACh-hydrolyzing enzyme in the circulation (LoewensteinLichtenstein et al. 1995). Correspondingly, most previous measurements of ACh-hydrolyzing capacity in the serum used butyrylthiocholine (BTCh), a butyrylcholine (BCh) analog as a substrate. A recent study demonstrated a strong inverse relation between serum BTCh-hydrolyzing activity and long-term mortality in a cohort of stable coronary artery disease (CAD) patients (Goliasch et al. 2012a). However, BCh is not physiologically available in the body and is only hydrolyzed by BChE but not AChE. Moreover, AChE is 20-fold faster than BChE in hydrolyzing ACh, and studies demonstrate a causal link between inflammatory pathways and cholinergic signaling (Shaked et al. 2009; Metz and Tracey 2005). Accumulating findings suggest another important feature of the cholinergic pathway that supports its capacity to restore homoeostasis, namely, the cholinergic anti-inflammatory pathway, which inhibits cytokine synthesis and release (Borovikova et al. 2000; Ofek et al. 2007). This predicts yet more direct associations between cholinesterase activities and inflammation both in the brain and in peripheral lymphocytes, where ACh shows anti-inflammatory properties that inhibit innate immune responses. This mechanism depends on the α7 nicotinic ACh receptor (α7nAChR), which inhibits NF-κB nuclear translocation and suppresses cytokine release by monocytes and macrophages as well. Hence, parasympathetic vagus activation initiates as an anti-inflammatory reflex-like process (Tracey 2010). Activation of this “cholinergic reflex” has been shown to alleviate inflammatory disease, including endotoxemia and septic peritonitis (Tracey 2010). A recently emerging family of posttranscriptional controllers of most genes, including cholinesterases, are miRNAs. Those are small RNA molecules each of which may target many mRNA transcripts by interacting with short “seed” sequences, leading to suppressed protein levels (Bartel 2009). Many mRNAs can be silenced by multiple miRNAs, and miRNAs often target more than one mRNA, thereby potentially controlling or participating in an entire battery of biological functions (Bartel 2009). As many as 244 miRNAs were identified as potentially targeting the 3′-untranslated regions of different cholinesterase transcripts: 116 for BChE, 47 for the “synaptic” AChE-S splice variant, and 81 for the normally rare “readthrough” splice variant AChE-R (Hanin and Soreq 2011). BChE and AChE show very little overlapping miRNAs, suggesting that they are subject to distinct modes of miRNA regulation. To evaluate the importance of the cholinesterase-targeted miRNAs, additional experimentally

validated target transcripts that were identified for the cholinesterase-targeted miRNAs need to be analyzed. Based on these data, we predict that miRNAs targeting cholinesterases can attenuate inflammation and verified it for miR-132 (Shaked et al. 2009).

Relevance of Cholinesterase Activity to Diseases Serum cholinesterase activities were found to differ from control values in a number of disease phenotypes. Figure 1 summarizes the disease-associated differences in cholinesterase activities and Table 1 details the corresponding studies and prospects for treatment, as is briefly detailed in the succeeding subsections. Neurodegenerative Diseases Alzheimer’s disease (AD) is the most common cause of cognitive deterioration in elderly people. Early work established the “cholinergic hypothesis of AD” in which the pathogenesis of AD cognitive symptoms is linked to a deficiency in the brain’s ACh. Neocortical deficits in AChsynthesizing choline acetyltransferase as well as reduced choline uptake and ACh release were also observed in AD patients, confirming a substantial presynaptic cholinergic deficit. Furthermore, cholinergic system abnormalities were correlated with senile plaques and cognitive test scores (Perry et al. 1978), as well as with RNA metabolism impairment in sporadic AD patients (Berson et al. 2012). These data together Parasympathetic / sympathetic ACh

Stroke

AChE

BChE

AD PD Anxiety MI IBD metabolic syndrome Diabetes

AChE

BChE

Fig. 1 Disease-associated difference in cholinesterase activities. Shown is the sympathetic/parasympathetic balance, where ACh reflects parasympathetic activities and cholinesterases, which hydrolyze ACh, reflect sympathetic activity. In each of the listed diseases, the arrows represent the direction of change in AChE or BChE enzymatic activity. Diseases marked with circles reflect biomarker utility to distinguish between disease states to control

J Mol Neurosci Table 1 Cholinergic-associated central and peripheral diseases and proposed therapies Disease

Risk for

Biomarker AChE

BChE

Stroke

Survival

Decrease

Increase

Increase

AD

Cognitive decline Cognitive decline

Increase Increase (brain)

na

Increase Decrease

Mutation

Inflammation

BChE-K variant

CRP, interleukin-6, interlukin-1, fibrinogen, ESR

Relevant miRs Reference

miR-132 Parkinson Myocardial infarction IBD Anxiety MetS

Diabetes

Mortality

State Trait Diabetes, cardiovascular disease Cardiovascular disease

Ben Assayag et al. (2010); Shenhar-Tsarfaty et al. (2010) Ben Assayag et al. (2010) Podoly et al. (2009); Shaked et al. (2009) Goliasch et al. (2012a)

Decrease Increase Decrease na

Decrease

CRP, TNF-α

na Increase

BChE-K variant

na

Decrease

BChE-K variant

miR-132

Maharshak et al. (2013) Sklan et al. (2004) Podoly et al. (2009); Shenhar-Tsarfaty et al. (2011b) Shenhar-Tsarfaty et al. (2011b)

ESR (eruthrocyte sedimentation rate) NA (not applicable)

established the basis for AChE inhibitor (AChEIs) therapeutics as the first licensed medications and the most prevalent ones to date for the symptomatic treatment of AD patients. Yet, cerebrospinal fluid (CSF) BChE activity does not change after short-term or long-term treatment with ChE inhibitors (donepezil, galantamine, and rivastigmine) in AD patients, while a positive correlation was found between plasma concentration and AChE activity. So far, AChE and BChE activities in the CSF were not correlated with clinical outcome in any group considered (Parnetti and Chiasserini 2011). The analysis of postmortem brain tissue is necessary to verify AD by immunohistochemical analysis of plaques (Aβ) and tangles (tau). Postmortem analysis or, alternatively, brain biopsies might also allow screening for general pathological changes in the AD brain, but are not useful for routine biomarker analysis. CSF is a very useful fluid for AD diagnosis because it reflects metabolic processes in the brain owing to direct contact between the brain and CSF. Its diagnostic use is however limited because of invasive collection by lumbar puncture. Currently, serum measurements are the gold standard in clinics because they are minimally invasive, as compared with CSF. For AD biomarker discovery, the use of plasma is still limited because changes are very small and heterogeneous and not all necessarily related to AD. A major advantage of blood samples is that patients can be followed up. The disadvantage is the more complex and time-consuming processing of blood cells. The use of body fluids requires very sensitive methods to detect low-level proteins and the correlation to AD pathologies is unclear (Humpel 2011).

Increasing evidence links aberrant expression of miRNAs to neurodegenerative disorders including AD (Lau and de Strooper 2010). However, the small number of patients analyzed represents a major issue when identifying which miRNAs are deregulated during disease. Confounding factors, in particular the technologies used for miRNA profiling which provide deviating results (Pritchard et al. 2012), have to be considered as well. But clearly miRNAs, such as miR-132-3p (Lau et al. 2013), deserve further functional exploration to deepen our understanding of molecular mechanisms driving not only late onset AD but also other neurodegenerative disorders. Parkinson’s disease is classically characterized as a motor neurodegenerative disorder. The motor symptoms in Parkinson’s are secondary to an altered dopamine–ACh balance due to reduced striatal dopaminergic tone and subsequent cholinergic overactivity (Calabresi et al. 2006). In the past, anticholinergic drugs were given to improve motor aspects of the disease. Moreover, serum AChE activity is reduced in Israeli Parkinson’s disease patients, as compared with controls, but the AChE homologous enzyme, BChE, was not (Benmoyal-Segal et al. 2005). Cerebrovascular Diseases Stroke continues to present a significant public health challenge, being not only the second leading cause of death but also a leading cause of adult disability (Roger et al. 2012). Despite extended care in dedicated stroke units, bacterial

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pneumonia remains the main medical complication after stroke, accounting for almost 20 % of in-hospital deaths and poor outcomes at discharge (Koennecke et al. 2011). There is growing evidence that acute injury to the central nervous system (CNS), including stroke, directly impairs the antibacterial host defense (Meisel and Meisel 2011). Focal cerebral ischemia induced by occlusion of the middle cerebral artery in mice, a model of human stroke, is associated with the development of spontaneous bacterial infections within 24 h after the onset of stroke, leading to high mortality (Prass et al. 2003). Infections are preceded by rapid suppression of peripheral cellular immune responses, which is mainly characterized by lymphocyte apoptosis and altered lymphocytic cytokine production; this appears to be triggered by a stroke-induced overactivation of the sympathetic nervous system (Prass et al. 2003). The vagus nerve stimulates celiac ganglion adrenergic neurons that innervate the spleen, leading to the release of ACh and the activation of nAChR on splenic macrophages. This blocks the production of the proinflammatory cytokine tumor necrosis factor-α (TNF-α). Recently, Rosas-Ballina et al. (2011) identified a subpopulation of CD4+ T cells that secrete ACh, express β-adrenergic receptors, and are located adjacent to adrenergic nerve endings in the spleen. Transplanting these T cells into mutant mice devoid of T cells exposed to endotoxemia-inducing insults rescued the attenuation of TNF-α by vagus nerve stimulation (Rosas-Ballina et al. 2011). Furthermore, reducing expression by small interfering RNA of the ACh biosynthesis regulator ChAT in these T cells before transplantation blocked the rescue of TNF-α attenuation after vagus nerve stimulation. Thus, ACh secretion by these T cells is required in this inflammatory reflex (Trakhtenberg and Goldberg 2011). Given that ACh is rapidly and efficiently hydrolyzed by the hydrolytic enzymes AChE and BChE, measuring these enzymes’ activity provides an effective biomarker of the immunosuppressive power of the autonomous nervous system. In our own study, we demonstrated that, in patients after acute ischemic stroke, declined serum AChE activity predicts the neurological outcome, survival, and inflammatory reactions (Ben Assayag et al. 2010). Moreover, in an experimental model of stroke, occlusion of the middle cerebral artery in mice deficient in invariant natural killer T cells (achieved by ablating CD1d) lead to increased bacterial burden in the lungs, greater pulmonary inflammatory damage, and decreased survival as compared with wild-type mice, while stroke severity was similar in both strains. Prophylactic antibiotic treatment completely prevented stroke-associated death in the Cd1d −/− mice, suggesting that their lack of invariant natural killer T cells rendered them more susceptible to death from infections after stroke (Wong et al. 2011). Additionally, Sykora et al. (2011) reported decreases in the autonomous system’s measure of baroreflex sensitivity (BRS, another marker for cholinergic imbalance) as an independent predictor for poststroke infections, and both

BRS and serum AChE activity correlated with multiple inflammatory biomarkers (Shenhar-Tsarfaty et al. 2011a). Together, these different yet interrelated approaches to estimate the cholinergic suppression of inflammation demonstrate its value for assessing the consequent risk of infection (Table 1). Taken together, both brain mapping (Alkalay et al. 2013) and clinical diagnostics (Ben Assayag et al. 2010) should be monitored in comparison to cholinesterase measurements in body fluids. Anxiety Anxiety is one of the most prevalent of all psychiatric disorders in the general population. Anxiety feelings can become a pathologic disorder when it is excessive and uncontrollable, requires no specific external stimulus, and manifests with a wide range of physical and affective symptoms as well as changes in behavior and cognition. Anxiety provokes cholinergic hyperarousal (e.g., sweating, intestinal or gastric constrictions, etc.) (Mayer et al. 2001; Pohjavaara et al. 2003). Polymorphisms in the corresponding AChE and BChE genes could, hence, affect both the environmental and the experience-related elements of anxiety. In mice, acute stress causes modulation of the genetic regulation of ACh availability in the CNS (Kaufer et al. 1998). This gene–environment interaction involves overproduction of the readthrough monomeric AChE (AChE-R) splice variant. Such overproduction acts in the short term to reduce excess ACh after stress. At a longer term, this overproduction is associated with glucocorticoid-regulated neuronal hyperarousal and extreme sensitivity to anti-AChEs (Meshorer et al. 2002). Transgenic overexpression of AChE-R in mice intensifies conflict behavior (Birikh et al. 2003), a phenomenon associated with anxiety. An inverse correlation was found between serum AChE, but not BChE, activity with trait anxiety, but not state anxiety (Sklan et al. 2004). More recently, we showed, in a mild model of predator scent-induced anxiety, long-lasting hippocampal elevation of miR-132 accompanied by and associated with reduced AChE activity. This attributes the stressinducible cognitive impairments to cholinergic-mediated induction of miR-132 and consequently suppressed AChE, opening venues for intercepting these miR-132-mediated damages (Shaltiel et al. 2013). Coronary Artery Disease Serum BChE has been implicated in the development of CAD (Alcantara et al. 2002), and Calderon-Margalit et al. (2006) demonstrated that individuals in the lowest quintile of BChE activity had significantly higher rates of all-cause and cardiovascular mortality. More recently, Goliasch et al. (2012b) demonstrated a strong association between decreased serum cholinesterase and long-term adverse outcome in patients with

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known CAD, which was stronger in stable CAD patients than in those with acute coronary syndrome. Further work will be required to quantify AChE and offer mechanistic explanation to this variable. Inflammatory Bowel Disease IBD, ulcerative colitis (UC), and Crohn’s disease are all characterized by chronic inflammation that primarily involves the gut and are believed to result from a dysregulated immune response towards enteric microbial antigens in a genetically predisposed host. Multileveled evidence suggests a role for the anti-inflammatory reflex in the pathogenesis of IBD. Electrical stimulation of the vagus nerve attenuated dextran sodium sulfate (DSS)-induced colitis in rats (Meregnani et al. 2011). Similarly, in vagotomized mice, DSS-induced colitis was more severe compared to control mice (Ghia et al. 2006) and sympathectomy improved experimental colitis (McCafferty et al. 1997). Administration of a cholinergic agonist (anabaseine) to mice resulted in attenuation of trinitrobenzenesulfonic acid-induced colitis and decreased colonic TNF-α production. Opposite effects were demonstrated when the mice were exposed to a nicotinic receptor antagonist (chlorisondamine diiodide) (Bai et al. 2007). Although the anti-inflammatory reflex has not been studied in human IBD, enteric nervous system abnormalities in IBD patients may support the potential role of this reflex in IBD pathogenesis. Structurally, changes in ganglia size and number as well as axonal necrosis in autonomic nerves in the gut have been observed. Up to 35 % of patients with UC suffer from impaired parasympathetic function, resulting in sympathetic dominance (Ghia et al. 2006; Ganguli et al. 2007). We found the cholinergic status, as well as AChE activity, to be reduced in IBD patients suffering from moderate–severe disease as compared to healthy controls or IBD patients presenting low disease severity. Additionally, we found inversely directed correlations between C-reactive protein (CRP) measures and cholinergic enzyme activities in IBD patients and controls as well as higher miR-132 levels in inflamed compared to apparently quiescent intestinal biopsies from IBD patients (Maharshak et al. 2013). Metabolic Syndrome and Diabetes Mellitus The metabolic syndrome (MetS), characterized by abdominal obesity, hypertriglyceridemia, low HDL cholesterol, hypertension, and elevated fasting glucose levels (Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults 2001), is a major health risk in developed countries, with a prevalence of 25 % among adults in the USA (Ford et al. 2002). MetS is associated with an increased risk for cardiovascular disease in both genders (Najarian et al. 2006). Biochemical and neurophysiological

measurements point to enhanced sympathetic and impaired parasympathetic functioning in MetS patients (Straznicky et al. 2008). More specifically, decreased vagal activity was found in individuals with obesity (Alvarez et al. 2002), dysglycemia (Thalamas et al. 2000), hypertension (Greenwood et al. 1999), and hyperlipidemia (Shishehbor et al. 2004), suggesting perturbation of the cholinergic pathways in MetS. One possible cause of impaired vagal activity is the increase in BChE-mediated hydrolysis of ACh observed in early type 2 diabetes mellitus (T2DM) patients, followed by BChE decreases in established patients (Rao et al. 2007). This temporary BChE elevation would reduce parasympathetic signals, increasing the sympathetic to parasympathetic ratio. We have previously shown that, in MetS subjects, serum BChE activity is elevated compared to T2DM patients and healthy controls. The BChE-K genotype, reflecting a single amino acid substitution (A to G), which changes protein interaction properties (Podoly et al. 2009), showed similar prevalence in T2DM and healthy volunteers, excluding this genotype as a risk factor for T2DM. However, the activity differences remained unexplained (Podoly et al. 2009; Shenhar-Tsarfaty et al. 2011b), perhaps alluding to miRNA involvement. One possible limitation for the use of BChE as a biomarker for MetS is that its origin is mainly synthesis in the liver; therefore, its serum levels may be a prognostic marker or functional indicator of acute or chronic liver disease and malnutrition rather than CNS cholinergic conditions.

Summary Accumulating evidence in basic research and clinically oriented studies suggests that cholinergic parameters became more important and relevant as disease biomarkers than ever. The cholinergic system is subject to aging-related impairments, which will predictably increase with the rapidly extending life expectancy; AChEIs became the leading therapeutics for AD (Giacobini 2003); and ACh was found to regulate inflammation through interaction with α7nAChR on macrophages, inhibition of the nuclear translocation of NF-κB, and interception of the production of proinflammatory mediators. Moreover, the need to evaluate cholinergic parameters is in constant increase given the growing knowledge of the sympathetic/parasympathetic shift and the inflammatory background contribution to a large variety of central and peripheral disorders. However, the establishment of cholinesterase measurements as diagnostic markers should precede their implementation for evaluating therapeutic efficacy. Taken together, these recent developments call for devoting more attention to cholinergic involvement in multiple diseases where it can participate both as a valid biomarker and as a significant player.

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Our current review has been focused on AChE and BChE involvement in multiple illnesses, such as cardiovascular, neurological, and inflammatory diseases. We cover cholinergic modulators, the cholinergic anti-inflammatory reflex, cholinesterase-targeted miRNAs, and single-nucleotide polymorphisms and their relevance for disease processes. In particular, we focus on measurements of circulation cholinesterases (AChE and BChE) as a readily amenable readout for cholinergic events. Altogether, this diverse collection of diseases calls for expanding the use of such measurements wherever possible, both as a pharmacological assessment tool and as a basic research tool. Acknowledgments S.S-T is grateful to the Edmond and Lily Safra Center for Brain Science for post-doctoral fellowship.

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