Cholinesterase-Targeting microRNAs Identified in silico Affect Specific Biological Processes

MicroRNAs (miRs) have emerged as important gene silencers affecting many target mRNAs. Here, we report the identification of 244 miRs that target the 3′-untranslated regions of different cholinesterase transcripts: 116 for butyrylcholinesterase (BChE), 47 for the synaptic acetylcholinesterase (AChE-S) splice variant, and 81 for the normally rare splice variant AChE-R. Of these, 11 and 6 miRs target both AChE-S and AChE-R, and AChE-R and BChE transcripts, respectively. BChE and AChE-S showed no overlapping miRs, attesting to their distinct modes of miR regulation. Generally, miRs can suppress a number of targets; thereby controlling an entire battery of functions. To evaluate the importance of the cholinesterase-targeted miRs in other specific biological processes we searched for their other experimentally validated target transcripts and analyzed the gene ontology enriched biological processes these transcripts are involved in. Interestingly, a number of the resulting categories are also related to cholinesterases. They include, for BChE, response to glucocorticoid stimulus, and for AChE, response to wounding and two child terms of neuron development: regulation of axonogenesis and regulation of dendrite morphogenesis. Importantly, all of the AChE-targeting miRs found to be related to these selected processes were directed against the normally rare AChE-R splice variant, with three of them, including the neurogenesis regulator miR-132, also directed against AChE-S. Our findings point at the AChE-R splice variant as particularly susceptible to miR regulation, highlight those biological functions of cholinesterases that are likely to be subject to miR post-transcriptional control, demonstrate the selectivity of miRs in regulating specific biological processes, and open new venues for targeted interference with these specific processes

Stressing hematopoiesis and immunity: an acetylcholinesterase window into nervous and immune system interactions

Hematopoietic stem cells (HSCs) differentiate and generate all blood cell lineages while maintaining self-renewal ability throughout life. Systemic responses to stressful insults, either psychological or physical exert both stimulating and down-regulating effects on these dynamic members of the immune system. Stress-facilitated division and re-oriented differentiation of progenitor cells modifies hematopoietic cell type composition, while enhancing cytokine production and promoting inflammation. Inversely, stress-induced increases in the neurotransmitter acetylcholine (ACh) act to mitigate inflammatory response and regain homeostasis. This signaling process is terminated when ACh is hydrolyzed by acetylcholinesterase (AChE). Alternative splicing, which is stress-modified, changes the composition of AChE variants, modifying their terminal sequences, susceptibility for microRNA suppression, and sub-cellular localizations. Intriguingly, the effects of stress and AChE variants on hematopoietic development and inflammation in health and disease are both subject to small molecule as well as oligonucleotide-mediated manipulations in vitro and in vivo. The therapeutic agents can thus be targeted to the enzyme protein, its encoding mRNA transcripts, or the regulator microRNA-132, opening new venues for therapeutic interference with multiple nervous and immune system diseases

MicroRNA regulators of cholinergic signaling link neuromuscular, cardiac and metabolic systems

 The neurotransmitter acetylcholine (ACh) notably regulates many brain and bodily functions, including metabolic as well as heart and muscle activities. Concordantly, acquired changes in ACh signaling lead to significant and widespread systemic effects. Those can be observed both when ACh signaling fails, as in the motor neuron disease amyotrophic lateral sclerosis (ALS) and when it over-reacts, as in the hyper-cholinergic excitation following organophosphate poisoning. To explore the corresponding molecular mechanisms, we focused on regulation of ACh signaling by microRNAs (miRs).  Current research implicates miRs as post-transcriptional modulators of gene expression, playing pivotal, rapid and interactive roles across various systems. To interrogate the systemic role of ACh-regulating miRs (CholinomiRs), we sought evidence for CholinomiRs with dual or triple roles in neuromuscular junctions (NMJ), heart development and functioning, and/or metabolic systems. Here, we report key links between CholinomiRs with known cardiac and metabolic roles, including the nicotinic acetylcholine receptor-targeted miR-1 and the acetylcholinesterase-targeted miR-132, and NMJ-related metabolic regulating miRs such as the histone deacetylase 4-targeted miR-206. Taken together, this information indicates a bridging role for CholinomiRs that may be relevant both for NMJ degeneration and the metabolic changes observed in ALS patients, and for the cardiac irregularities and NMJ degeneration reported following organophosphate poisoning. Uncovering the potentially causal involvement of CholinomiRs in balancing neuromuscular, cardiac and metabolic functions might improve our understanding of the inter-tissue communication and the processes of reaching homeostatic states which are essential for balancing between seemingly separate body systems, allowing a more encompassing look on disorders involving impaired cholinergic signaling.</p

Longitudinal basal forebrain degeneration interacts with TREM2/C3 biomarkers of inflammation in presymptomatic Alzheimer’s disease

Copyright © 2020 the authors Cholinergic inputs originating from the peripheral nervous system regulate the inflammatory immune responses of macrophages during clearance of blood-based pathogens. Because microglia are involved in clearing amyloid and tau pathology from the central nervous system, we hypothesized that cholinergic input originating from the basal forebrain might similarly regulate inflammatory immune responses to these pathologies in the aging brain. To explore this hypothesis, we leveraged the Alzheimer’s Disease Neuroimaging Initiative dataset. Cognitively normal older male and female human adults were differentiated according to the relative concentration of phosphorylated tau and amyloid in their cerebrospinal fluid, yielding neurotypical and preclinical, cognitively healthy, subgroups. We then tracked these two groups longitudinally with structural MRI and biomarkers of inflammation, including soluble sTREM2 levels in the CSF and complement C3 expression in the blood transcriptome. Longitudinal loss of basal forebrain volume was larger in the preclinical compared with the neurotypical subgroup. Across preclinical adults, loss of basal forebrain volume was associated with greater longitudinal accumulation of sTREM2 and higher peripheral blood C3 expression. None of these relationships were attributable to degeneration in the whole-brain gray matter volume. Preclinical APOE e4 carriers exhibited the largest loss of basal forebrain volume and highest C3 expression. Consistent with the known anti-inflammatory influence of the peripheral cholinergic pathways on macrophages, our findings indicate that a loss of central cholinergic input originating from the basal forebrain might remove a key check on microglial inflammation induced by amyloid and tau accumulation

Translational control of recombinant human acetylcholinesterase accumulation in plants

<p>Abstract</p> <p>Background</p> <p>Codon usage differences are known to regulate the levels of gene expression in a species-specific manner, with the primary factors often cited to be mRNA processing and accumulation. We have challenged this conclusion by expressing the human acetylcholinesterase coding sequence in transgenic plants in its native GC-rich sequence and compared to a matched sequence with (dicotyledonous) plant-optimized codon usage and a lower GC content.</p> <p>Results</p> <p>We demonstrate a 5 to 10 fold increase in accumulation levels of the "synaptic" splice variant of human acetylcholinesterase in <it>Nicotiana benthamiana </it>plants expressing the optimized gene as compared to the native human sequence. Both transient expression assays and stable transformants demonstrated conspicuously increased accumulation levels. Importantly, we find that the increase is not a result of increased levels of acetylcholinesterase mRNA, but rather its facilitated translation, possibly due to the reduced energy required to unfold the sequence-optimized mRNA.</p> <p>Conclusion</p> <p>Our findings demonstrate that codon usage differences may regulate gene expression at different levels and anticipate translational control of acetylcholinesterase gene expression in its native mammalian host as well.</p

Activation of the Alternative NFκB Pathway Improves Disease Symptoms in a Model of Sjogren's Syndrome

The purpose of our study was to understand if Toll-like receptor 9 (TLR9) activation could contribute to the control of inflammation in Sjogren's syndrome. To this end, we manipulated TLR9 signaling in non-obese diabetic (NOD) and TLR9−/− mice using agonistic CpG oligonucleotide aptamers, TLR9 inhibitors, and the in-house oligonucleotide BL-7040. We then measured salivation, inflammatory response markers, and expression of proteins downstream to NF-κB activation pathways. Finally, we labeled proteins of interest in salivary gland biopsies from Sjogren's syndrome patients, compared to Sicca syndrome controls. We show that in NOD mice BL-7040 activates TLR9 to induce an alternative NF-κB activation mode resulting in increased salivation, elevated anti-inflammatory response in salivary glands, and reduced peripheral AChE activity. These effects were more prominent and also suppressible by TLR9 inhibitors in NOD mice, but TLR9−/− mice were resistant to the salivation-promoting effects of CpG oligonucleotides and BL-7040. Last, salivary glands from Sjogren's disease patients showed increased inflammatory and decreased anti-inflammatory biomarkers, in addition to decreased levels of alternative NF-κB pathway proteins. In summary, we have demonstrated that activation of TLR9 by BL-7040 leads to non-canonical activation of NF-κB, promoting salivary functioning and down-regulating inflammation. We propose that BL-7040 could be beneficial in treating Sjogren's syndrome and may be applicable to additional autoimmune syndromes

Changes in readthrough acetylcholinesterase expression modulate amyloid-beta pathology

Alzheimer's disease has long been known to involve cholinergic deficits, but the linkage between cholinergic gene expression and the Alzheimer's disease amyloid pathology has remained incompletely understood. One known link involves synaptic acetylcholinesterase (AChE-S), shown to accelerate amyloid fibrils formation. Here, we report that the ‘Readthrough' AChE-R splice variant, which differs from AChE-S in its 26 C-terminal residues, inversely exerts neuroprotective effects from amyloid β (Aβ) induced toxicity. In vitro, highly purified AChE-R dose-dependently suppressed the formation of insoluble Aβ oligomers and fibrils and abolished Aβ toxicity to cultured cells, competing with the prevalent AChE-S protein which facilitates these processes. In vivo, double transgenic APPsw/AChE-R mice showed lower plaque burden, fewer reactive astrocytes and less dendritic damage than single APPsw mice, inverse to reported acceleration of these features in double APPsw/AChE-S mice. In hippocampi from Alzheimer's disease patients (n = 10), dentate gyrus neurons showed significantly elevated AChE-R mRNA and reduced AChE-S mRNA. However, immunoblot analyses revealed drastic reductions in the levels of intact AChE-R protein, suggesting that its selective loss in the Alzheimer's disease brain exacerbates the Aβ-induced damages and revealing a previously unforeseen linkage between cholinergic and amyloidogenic event

Forebrain Deletion of the Vesicular Acetylcholine Transporter Results in Deficits in Executive Function, Metabolic, and RNA Splicing Abnormalities in the Prefrontal Cortex

One of the key brain regions in cognitive processing and executive function is the prefrontal cortex (PFC), which receives cholinergic input from basal forebrain cholinergic neurons. We evaluated the contribution of synaptically released acetylcholine (ACh) to executive function by genetically targeting the vesicular acetylcholine transporter (VAChT) in the mouse forebrain. Executive function was assessed using a pairwise visual discrimination paradigm and the 5-choice serial reaction time task (5-CSRT). In the pairwise test, VAChT-deficient mice were able to learn, but were impaired in reversal learning, suggesting that these mice present cognitive inflexibility. Interestingly, VAChT-targeted mice took longer to reach criteria in the 5-CSRT. Although their performance was indistinguishable from that of control mice during low attentional demand, increased attentional demand revealed striking deficits in VAChT-deleted mice. Galantamine, a cholinesterase inhibitor used in Alzheimer\u27s disease, significantly improved the performance of control mice, but not of VAChT-deficient mice on the 5-CSRT. In vivo magnetic resonance spectroscopy showed altered levels of two neurochemical markers of neuronal function, taurine and lactate, suggesting altered PFC metabolism in VAChT-deficient mice. The PFC of these mice displayed a drastic reduction in the splicing factor heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1), whose cholinergic-mediated reduction was previously demonstrated in Alzheimer\u27s disease. Consequently, several key hnRNPA2/B1 target transcripts involved in neuronal function present changes in alternative splicing in VAChT-deficient mice, including pyruvate kinase M, a key enzyme involved in lactate metabolism. We propose that VAChT-targeted mice can be used to model and to dissect the neurochemical basis of executive abnormalities

Gene expression differences in peripheral blood of Parkinson's disease patients with distinct progression profiles

The prognosis of neurodegenerative disorders is clinically challenging due to the inexistence of established biomarkers for predicting disease progression. Here, we performed an exploratory cross-sectional, case-control study aimed at determining whether gene expression differences in peripheral blood may be used as a signature of Parkinson's disease (PD) progression, thereby shedding light into potential molecular mechanisms underlying disease development. We compared transcriptional profiles in the blood from 34 PD patients who developed postural instability within ten years with those of 33 patients who did not develop postural instability within this time frame. Our study identified >200 differentially expressed genes between the two groups. The expression of several of the genes identified was previously found deregulated in animal models of PD and in PD patients. Relevant genes were selected for validation by real-time PCR in a subset of patients. The genes validated were linked to nucleic acid metabolism, mitochondria, immune response and intracellular-transport. Interestingly, we also found deregulation of these genes in a dopaminergic cell model of PD, a simple paradigm that can now be used to further dissect the role of these molecular players on dopaminergic cell loss. Altogether, our study provides preliminary evidence that expression changes in specific groups of genes and pathways, detected in peripheral blood samples, may be correlated with differential PD progression. Our exploratory study suggests that peripheral gene expression profiling may prove valuable for assisting in prediction of PD prognosis, and identifies novel culprits possibly involved in dopaminergic cell death. Given the exploratory nature of our study, further investigations using independent, well-characterized cohorts will be essential in order to validate our candidates as predictors of PD prognosis and to definitively confirm the value of gene expression analysis in aiding patient stratification and therapeutic intervention

Whole blood microRNA expression associated with stroke: Results from the Framingham Heart Study

Emerging evidence suggests microRNAs (miRNAs) may play an important role in explaining variation in stroke risk and recovery in humans, yet there are still few longitudinal studies examining the association between whole blood miRNAs and stroke. Accounting for multiple testing and adjusting for potentially confounding technical and clinical variables, here we show that whole blood miR-574-3p expression was significantly lower in participants with chronic stroke compared to non-cases. To explore the functional relevance of our findings, we analyzed miRNA-mRNA whole blood co-expression, pathway enrichment, and brain tissue gene expression. Results suggest miR-574-3p is involved in neurometabolic and chronic neuronal injury response pathways, including brain gene expression of DBNDD2 and ELOVL1. These results suggest miR-574-3p plays a role in regulating chronic brain and systemic cellular response to stroke and thus may implicate miR-574-3p as a partial mediator of long-term stroke outcomes