Improvement of Cognitive Function in Ovariectomized Rats by Human Neural Stem Cells Overexpressing Choline Acetyltransferase via Secretion of NGF and BDNF

Menopause is associated with memory deficits attributed to reduced serum estrogen levels. We evaluated whether an increase in brain-derived neurotrophic factor (BDNF) and nerve-growth factor (NGF) levels, through transplantation of choline acetyltransferase (ChAT)-overexpressing neural stem cells (F3.ChAT), improved learning and memory in ovariectomized rats. PD13 mouse neuronal primary culture cells were treated with estradiol or co-cultured with F3.ChAT cells; choline transporter1 (CHT1), ChAT, and vesicular acetylcholine transporter (VAChT) expression was evaluated using real-time PCR. The relationship between estrogen receptors (ERs) and neurotrophin family members was analyzed using immunohistochemistry. After the transplantation of F3.ChAT cells into OVx rats, we evaluated the memory, ACh level, and the expression of ER, neurotrophin family proteins, and cholinergic system. Estradiol upregulated CHT1, ChAT, and VAChT expression in ER; they were co-localized with BDNF, NGF, and TrkB. Co-culture with F3.ChAT upregulated CHT1, ChAT, and VAChT by activating the neurotrophin signalling pathway. Transplantation of F3.ChAT cells in OVX animals increased the ACh level in the CSF and improved memory deficit. In addition, it increased the expression of ERs, neurotrophin signaling, and the cholinergic system in the brains of OVX animals. Therefore, the estradiol deficiency induced memory loss by the down-regulation of the neurotrophin family and F3.ChAT could ameliorate the cognitive impairment owing to the loss or reduction of estradiol.     

Blocking Muscarinic Receptor 3 Attenuates Tumor Growth and Decreases Immunosuppressive and Cholinergic Markers in an Orthotopic Mouse Model of Colorectal Cancer

Tumor cells have evolved to express immunosuppressive molecules allowing their evasion from the host’s immune system. These molecules include programmed death ligands 1 and 2 (PD-L1 and PD-L2). Cancer cells can also produce acetylcholine (ACh), which plays a role in tumor development. Moreover, tumor innervation can stimulate vascularization leading to tumor growth and metastasis. The effects of atropine and muscarinic receptor 3 (M3R) blocker, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP), on cancer growth and spread were evaluated in vitro using murine colon cancer cell line, CT-26, and in vivo in an orthotopic mouse model of colorectal cancer. In the in vitro model, atropine and 4-DAMP significantly inhibited CT-26 cell proliferation in a dose dependent manner and induced apoptosis. Atropine attenuated immunosuppressive markers and M3R via inhibition of EGFR/AKT/ERK signaling pathways. However, 4-DAMP showed no effect on the expression of PD-L1, PD-L2, and choline acetyltransferase (ChAT) on CT-26 cells but attenuated M3R by suppressing the phosphorylation of AKT and ERK. Blocking of M3R in vivo decreased tumor growth and expression of immunosuppressive, cholinergic, and angiogenic markers through inhibition of AKT and ERK, leading to an improved immune response against cancer. The expression of immunosuppressive and cholinergic markers may hold potential in determining prognosis and treatment regimens for colorectal cancer patients. This study’s results demonstrate that blocking M3R has pronounced antitumor effects via several mechanisms, including inhibition of immunosuppressive molecules, enhancement of antitumor immune response, and suppression of tumor angiogenesis via suppression of the AKT/ERK signaling pathway. These findings suggest a crosstalk between the cholinergic and immune systems during cancer development. In addition, the cholinergic system influences cancer evasion from the host’s immunity.     

Effect of cholineacetyltransferase activity and choline recycle ratio on diffusion-reaction modeling,bifurcation and chaotic behavior of acetylcholine neurocycle and their relation to Alzheimer's and Parkinson's diseases

This paper is an extension of the previous two papers (Mustafa et al., 2009a, Mustafa et al., 2009b) on the modeling, simulation and analysis of bifurcation, dynamics, and chaotic characteristics of the acetylcholine (ACh) neurocycle. The effects of cholineacetyltransferase (ChAT) activity and choline recycle ratio as bifurcation parameters, on the system performance are studied through modifying the two-compartment model. It is found that as ChAT activity increases, ACh concentration in the model compartments increases continuously. The effect of choline recycle ratio shows that choline uptake represents the key role for providing choline as a substrate for ACh synthesis. It is found that the choline recycle ratio has a great influence on all state variables in both compartments. A detailed bifurcation and sensitive analyses over a wide range of ChAT activity and choline recycle ratio parameters are carried out in order to highlight the characteristics of ACh cholinergic system like non-linear dynamics and different solutions such as multiplicity of: stationery states as well as periodic and chaotic states. The relations between the results and the functions of ACh cholinergic neurons are investigated. A comparison between the results and other experimental and physiological measurements is performed. It is found that there is a good relation between cholinergic diseases such as Alzheimer's and Parkinson's diseases, and level of ACh in brains from one side and current results extracted from the disturbances in ChAT activity and choline recycle from the other side. It is found that choline recycled is the most critical factor in ACh processes in comparison with any of the following parameters: ChAT activity, as well as feed acetate and ACh concentrations.     

Cholinergic Modulation of Neuroinflammation: Focus on α7 Nicotinic Receptor

All nervous system pathologies (e.g., neurodegenerative/demyelinating diseases and brain tumours) develop neuroinflammation, a beneficial process during pathological events, aimed at removing damaged cells, toxic agents, and/or pathogens. Unfortunately, excessive inflammation frequently occurs during nervous system disorders, becoming a detrimental event capable of enhancing neurons and myelinating glial cell impairment, rather than improving their survival and activity. Consequently, targeting the neuroinflammation could be relevant for reducing brain injury and rescuing neuronal and glial cell functions. Several studies have highlighted the role of acetylcholine and its receptors in the regulation of central and peripheral inflammation. In particular, α7 nicotinic receptor has been described as one of the main regulators of the “brain cholinergic anti-inflammatory pathway”. Its expression in astrocytes and microglial cells and the ability to modulate anti-inflammatory cytokines make this receptor a new interesting therapeutic target for neuroinflammation regulation. In this review, we summarize the distribution and physiological functions of the α7 nicotinic receptor in glial cells (astrocytes and microglia) and its role in the modulation of neuroinflammation. Moreover, we explore how its altered expression and function contribute to the development of different neurological pathologies and exacerbate neuroinflammatory processes.     

The Contribution of TSLP Activation to Hyperalgesia in Dorsal Root Ganglia Neurons of a Rat

Peripheral nerve injury involves divergent alterations within dorsal root ganglia (DRG) neurons sensitized by persistent inflammation. Thymic stromal lymphopoietin (TSLP) production is crucial in the development of chronic inflammatory responses. Herein, we investigate the changes of TSLP expression in rats’ DRG neurons between injured and uninjured sides in the same rat. Linalyl acetate (LA) was served as a TSLP inhibitor and given intraperitoneally. Rats were assigned to be group of chronic constriction injury (CCI) of the sciatic nerve and the group of CCI of the sciatic nerve administrated with LA. Over 14 days, the rats were measured for paw withdrawal thresholds. DRGs were collected to assess morphological changes via immunofluorescence study. After receiving CCI, the rats rapidly developed mechanical hyperalgesia. TSLP expression at DRG, on the ipsilateral injured side, was consistent with changes in pain behaviors. TSLP appeared in nerve fibers with both small diameters and large diameters. Additionally, TSLP was expressed mostly in transient receptor potential vanilloid-1 (TRPV1)-positive nociceptive neurons. Administration with LA can attenuate the pain behaviors and expression of TSLP in DRG neurons, and in apoptotic neurons at the injured side, but not in the contra-lateral uninjured side. Overall, these results imply that altered expressions of TSLP in nociceptive DRG neurons contributed to mechanical hyperalgesia in a CCI rat model.     

Exendin-4 Promotes Schwann Cell Survival/Migration and Myelination In Vitro

Besides its insulinotropic actions on pancreatic β cells, neuroprotective activities of glucagon-like peptide-1 (GLP-1) have attracted attention. The efficacy of a GLP-1 receptor (GLP-1R) agonist exendin-4 (Ex-4) for functional repair after sciatic nerve injury and amelioration of diabetic peripheral neuropathy (DPN) has been reported; however, the underlying mechanisms remain unclear. In this study, the bioactivities of Ex-4 on immortalized adult rat Schwann cells IFRS1 and adult rat dorsal root ganglion (DRG) neuron–IFRS1 co-culture system were investigated. Localization of GLP-1R in both DRG neurons and IFRS1 cells were confirmed using knockout-validated monoclonal Mab7F38 antibody. Treatment with 100 nM Ex-4 significantly enhanced survival/proliferation and migration of IFRS1 cells, as well as stimulated the movement of IFRS1 cells toward neurites emerging from DRG neuron cell bodies in the co-culture with the upregulation of myelin protein 22 and myelin protein zero. Because Ex-4 induced phosphorylation of serine/threonine-specific protein kinase AKT in these cells and its effects on DRG neurons and IFRS1 cells were attenuated by phosphatidyl inositol-3′-phosphate-kinase (PI3K) inhibitor LY294002, Ex-4 might act on both cells to activate PI3K/AKT signaling pathway, thereby promoting myelination in the co-culture. These findings imply the potential efficacy of Ex-4 toward DPN and other peripheral nerve lesions.     

Changes in the Neurochemical Coding of the Anterior Pelvic Ganglion Neurons Supplying the Male Pig Urinary Bladder Trigone after One-Sided Axotomy of Their Nerve Fibers

The present study investigated the effect of unilateral axotomy of urinary bladder trigone (UBT)-projecting nerve fibers from the right anterior pelvic ganglion (APG) on changes in the chemical coding of their neuronal bodies. The study was performed using male pigs with immunohistochemistry and quantitative real-time PCR (qPCR). The animals were divided into a control (C), a morphological (MG) or a molecular biology group (MBG). APG neurons supplying UBT were revealed using the retrograde tracing technique with Fast Blue (FB). Unilateral axotomy resulted in an over 50% decrease in the number of FB+ neurons in both APG ganglia. Immunohistochemistry revealed significant changes in the chemical coding of FB+ cells only in the right ganglion: decreased expression of dopamine-B-hydroxylase (DBH)/tyrosine hydroxylase (TH) and up-regulation of the vesicular acetylcholine transporter (VAChT)/choline acetyltransferase (ChAT), galanin (GAL), vasoactive intestinal polypeptide (VIP) and brain nitric oxide synthase (bNOS). The qPCR results partly corresponded with immunofluorescence findings. In the APGs, genes for VAChT and ChAT, TH and DBH, VIP, and NOS were distinctly down-regulated, while the expression of GAL was up-regulated. Such data may be the basis for further studies concerning the plasticity of these ganglia under experimental or pathological conditions.     

Activation of Kinin B1R Upregulates ADAM17 and Results in ACE2 Shedding in Neurons

Angiotensin converting enzyme 2 (ACE2) is a critical component of the compensatory axis of the renin angiotensin system. Alterations in ACE2 gene and protein expression, and activity mediated by A Disintegrin And Metalloprotease 17 (ADAM17), a member of the “A Disintegrin And Metalloprotease” (ADAM) family are implicated in several cardiovascular and neurodegenerative diseases. We previously reported that activation of kinin B1 receptor (B1R) in the brain increases neuroinflammation, oxidative stress and sympathoexcitation, leading to the development of neurogenic hypertension. We also showed evidence for ADAM17-mediated ACE2 shedding in neurons. However, whether kinin B1 receptor (B1R) activation has any role in altering ADAM17 activity and its effect on ACE2 shedding in neurons is not known. In this study, we tested the hypothesis that activation of B1R upregulates ADAM17 and results in ACE2 shedding in neurons. To test this hypothesis, we stimulated wild-type and B1R gene-deleted mouse neonatal primary hypothalamic neuronal cultures with a B1R-specific agonist and measured the activities of ADAM17 and ACE2 in neurons. B1R stimulation significantly increased ADAM17 activity and decreased ACE2 activity in wild-type neurons, while pretreatment with a B1R-specific antagonist, R715, reversed these changes. Stimulation with specific B1R agonist Lys-Des-Arg⁹-Bradykinin (LDABK) did not show any effect on ADAM17 or ACE2 activities in neurons with B1R gene deletion. These data suggest that B1R activation results in ADAM17-mediated ACE2 shedding in primary hypothalamic neurons. In addition, stimulation with high concentration of glutamate significantly increased B1R gene and protein expression, along with increased ADAM17 and decreased ACE2 activities in wild-type neurons. Pretreatment with B1R-specific antagonist R715 reversed these glutamate-induced effects suggesting that indeed B1R is involved in glutamate-mediated upregulation of ADAM17 activity and ACE2 shedding.     

Pretreatment with Zonisamide Mitigates Oxaliplatin-Induced Toxicity in Rat DRG Neurons and DRG Neuron–Schwann Cell Co-Cultures

Oxaliplatin (OHP) is a platinum-based agent that can cause peripheral neuropathy, an adverse effect in which the dorsal root ganglion (DRG) neurons are targeted. Zonisamide has exhibited neuroprotective activities toward adult rat DRG neurons in vitro and therefore, we aimed to assess its potential efficacy against OHP-induced neurotoxicity. Pretreatment with zonisamide (100 μM) alleviated the DRG neuronal death caused by OHP (75 μM) and the protective effects were attenuated by a co-incubation with 25 μM of the mitogen-activated protein kinase (MAPK; MEK/ERK) inhibitor, U0126, or the phosphatidyl inositol-3′-phosphate-kinase (PI3K) inhibitor, LY294002. Pretreatment with zonisamide also suppressed the OHP-induced p38 MAPK phosphorylation in lined DRG neurons, ND7/23, while the OHP-induced DRG neuronal death was alleviated by pretreatment with the p38 MAPK inhibitor, SB239063 (25 μM). Although zonisamide failed to protect the immortalized rat Schwann cells IFRS1 from OHP-induced cell death, it prevented neurite degeneration and demyelination-like changes, as well as the reduction of the serine/threonine-specific protein kinase (AKT) phosphorylation in DRG neuron–IFRS1 co-cultures exposed to OHP. Zonisamide’s neuroprotection against the OHP-induced peripheral sensory neuropathy is possibly mediated by a stimulation of the MEK/ERK and PI3K/AKT signaling pathways and suppression of the p38 MAPK pathway in DRG neurons. Future studies will allow us to solidify zonisamide as a promising remedy against the neurotoxic adverse effects of OHP.     

Kisspeptin-10 Rescues Cholinergic Differentiated SHSY-5Y Cells from α-Synuclein-Induced Toxicity In Vitro

The neuropathological substrate of dementia with Lewy bodies (DLB) is defined by the inextricable cross-seeding accretion of amyloid-β (Aβ) and α-synuclein (α-syn)-laden deposits in cholinergic neurons. The recent revelation that neuropeptide kisspeptin-10 (KP-10) is able to mitigate Aβ toxicity via an extracellular binding mechanism may provide a new horizon for innovative drug design endeavors. Considering the sequence similarities between α-syn’s non-amyloid-β component (NAC) and Aβ’s C-terminus, we hypothesized that KP-10 would enhance cholinergic neuronal resistance against α-syn’s deleterious consequences through preferential binding. Here, human cholinergic SH-SY5Y cells were transiently transformed to upsurge the mRNA expression of α-syn while α-syn-mediated cholinergic toxicity was quantified utilizing a standardized viability-based assay. Remarkably, the E46K mutant α-syn displayed elevated α-syn mRNA levels, which subsequently induced more cellular toxicity compared with the wild-type α-syn in choline acetyltransferase (ChAT)-positive cholinergic neurons. Treatment with a high concentration of KP-10 (10 µM) further decreased cholinergic cell viability, while low concentrations of KP-10 (0.01–1 µM) substantially suppressed wild-type and E46K mutant α-syn-mediated toxicity. Correlating with the in vitro observations are approximations from in silico algorithms, which inferred that KP-10 binds favorably to the C-terminal residues of wild-type and E46K mutant α-syn with CDOCKER energy scores of −118.049 kcal/mol and −114.869 kcal/mol, respectively. Over the course of 50 ns simulation time, explicit-solvent molecular dynamics conjointly revealed that the docked complexes were relatively stable despite small-scale fluctuations upon assembly. Taken together, our findings insinuate that KP-10 may serve as a novel therapeutic scaffold with far-reaching implications for the conceptualization of α-syn-based treatments.     

Nogo-A Induced Polymerization of Microtubule Is Involved in the Inflammatory Heat Hyperalgesia in Rat Dorsal Root Ganglion Neurons

The microtubule, a major constituent of cytoskeletons, was shown to bind and interact with transient receptor potential vanilloid subfamily member 1 (TRPV1), and serves a pivotal role to produce thermal hyperalgesia in inflammatory pain. Nogo-A is a modulator of microtubule assembly and plays a key role in maintaining the function of TRPV1 in inflammatory heat pain. However, whether the microtubule dynamics modulated by Nogo-A in dorsal root ganglion (DRG) neurons participate in the inflammatory pain is not elucidated. Here we reported that the polymerization of microtubules in the DRG neurons, as indicated by the acetylated α-tubulin, tubulin polymerization-promoting protein 3 (TPPP3), and microtubule numbers, was significantly elevated in the complete Freund’s adjuvant (CFA) induced inflammatory pain. Consistent with our previous results, knock-out (KO) of Nogo-A protein significantly attenuated the heat hyperalgesia 72 h after CFA injection and decreased the microtubule polymerization via up-regulation of phosphorylation of collapsin response mediator protein 2 (CRMP2) in DRG. The colocalization of acetylated α-tubulin and TRPV1 in DRG neurons was also reduced dramatically in Nogo-A KO rats under inflammatory pain. Moreover, the down-regulation of TRPV1 in DRG of Nogo-A KO rats after injection of CFA was reversed by intrathecal injection of paclitaxel, a microtubule stabilizer. Furthermore, intrathecal injection of nocodazole (a microtubule disruptor) attenuated significantly the CFA-induced inflammatory heat hyperalgesia and the mechanical pain in a rat model of spared nerve injury (SNI). In these SNI cases, the Nogo-A and acetylated α-tubulin in DRG were also significantly up-regulated. We conclude that the polymerization of microtubules promoted by Nogo-A in DRG contributes to the development of inflammatory heat hyperalgesia mediated by TRPV1.     

Structural Identification of the Pacemaker Cells and Expression of Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channels in the Heart of the Wild Atlantic Cod,Gadus morhua (Linnaeus, 1758)

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are proteins that contain highly conserved functional domains and sequence motifs that are correlated with their unique biophysical activities, to regulate cardiac pacemaker activity and synaptic transmission. These pacemaker proteins have been studied in mammalian species, but little is known now about their heart distribution in lower vertebrates and c-AMP modulation. Here, we characterized the pacemaker system in the heart of the wild Atlantic cod (Gadus morhua), with respect to primary pacemaker molecular markers. Special focus is given to the structural, ultrastructural and molecular characterization of the pacemaker domain, through the expression of HCN channel genes and the immunohistochemistry of HCN isoforms, including the location of intracardiac neurons that are adjacent to the sinoatrial region of the heart. Similarly to zebrafish and mammals, these neurons are immunoreactive to ChAT, VAChT and nNOS. It has been shown that cardiac pacemaking can be modulated by sympathetic and parasympathetic pathways, and the existence of intracardiac neurons projecting back to the central nervous system provide a plausible link between them.     

In Vitro Model to Investigate Communication between Dorsal Root Ganglion and Spinal Cord Glia

Chronic discogenic back pain is associated with increased inflammatory cytokine levels that can influence the proximal peripheral nervous system, namely the dorsal root ganglion (DRG). However, transition to chronic pain is widely thought to involve glial activation in the spinal cord. In this study, an in vitro model was used to evaluate the communication between DRG and spinal cord glia. Primary neonatal rat DRG cells were treated with/without inflammatory cytokines (TNF-α, IL-1β, and IL-6). The conditioned media were collected at two time points (12 and 24 h) and applied to spinal cord mixed glial culture (MGC) for 24 h. Adult bovine DRG and spinal cord cell cultures were also tested, as an alternative large animal model, and results were compared with the neonatal rat findings. Compared with untreated DRG-conditioned medium, the second cytokine-treated DRG-conditioned medium (following medium change, thus containing solely DRG-derived molecules) elevated CD11b expression and calcium signal in neonatal rat microglia and enhanced Iba1 expression in adult bovine microglia. Cytokine treatment induced a DRG-mediated microgliosis. The described in vitro model allows the use of cells from large species and may represent an alternative to animal pain models (3R principles).     

Acrylamide Neurotoxicity as a Possible Factor Responsible for Inflammation in the Cholinergic Nervous System

Acrylamide (ACR) is a chemical compound that exhibits neurotoxic and genotoxic effects. It causes neurological symptoms such as tremors, general weakness, numbness, tingling in the limbs or ataxia. Numerous scientific studies show the effect of ACR on nerve endings and its close connection with the cholinergic system. The cholinergic system is part of the autonomic nervous system that regulates higher cortical functions related to memory, learning, concentration and attention. Within the cholinergic system, there are cholinergic neurons, anatomical cholinergic structures, the neurotransmitter acetylcholine (ACh) and cholinergic receptors. Some scientific reports suggest a negative effect of ACR on the cholinergic system and inflammatory reactions within the body. The aim of the study was to review the current state of knowledge on the influence of acrylamide on the cholinergic system and to evaluate its possible effect on inflammatory processes. The cholinergic anti-inflammatory pathway (CAP) is a neuroimmunomodulatory pathway that is located in the blood and mucous membranes. The role of CAP is to stop the inflammatory response in the appropriate moment. It prevents the synthesis and the release of pro-inflammatory cytokines and ultimately regulates the local and systemic immune response. The cellular molecular mechanism for inhibiting cytokine synthesis is attributed to acetylcholine (ACh), the major vagal neurotransmitter, and the α7 nicotinic receptor (α7nAChR) subunit is a key receptor for the cholinergic anti-inflammatory pathway. The combination of ACh with α7nAChR results in inhibition of the synthesis and release of pro-inflammatory cytokines. The blood AChE is able to terminate the stimulation of the cholinergic anti-inflammatory pathway due to splitting ACh. Accordingly, cytokine production is essential for pathogen protection and tissue repair, but over-release of cytokines can lead to systemic inflammation, organ failure, and death. Inflammatory responses are precisely regulated to effectively protect against harmful stimuli. The central nervous system dynamically interacts with the immune system, modulating inflammation through the humoral and nervous pathways. The stress-induced rise in acetylcholine (ACh) level acts to ease the inflammatory response and restore homeostasis. This signaling process ends when ACh is hydrolyzed by acetylcholinesterase (AChE). There are many scientific reports indicating the harmful effects of ACR on AChE. Most of them indicate that ACR reduces the concentration and activity of AChE. Due to the neurotoxic effect of acrylamide, which is related to the disturbance of the secretion of neurotransmitters, and its influence on the disturbance of acetylcholinesterase activity, it can be concluded that it disturbs the normal inflammatory response.     

Effect of Bladder Injection of OnabotulinumtoxinA on the Central Expression of Genes Associated with the Control of the Lower Urinary Tract: A Study in Normal Rats

To investigate a possible central mechanism of action of Botulinum toxin A (BoNT/A) following injection in the bladder, complementary to the acknowledged peripheral bladder effect, we studied changes in the expression of neuropeptides and receptors involved in lower urinary tract function in the spinal cord (SC) and dorsal root ganglia (DRG) of normal rats following BoNT/A bladder injection. Thirty-six Sprague-Dawley rats, divided into three groups of n = 12, received bladder injections of 2U or 5U OnabotulinumtoxinA (BOTOX^®), or saline. Six animals from each group were sacrificed on days 7 and 14. Expression of Tachykinin 1 (Tac1), capsaicin receptor (TRPV1), neuropeptide Y (NPY), proenkephalin (PENK) and muscarinic receptors M1, M2, M3, was evaluated in the bladder, L6-S1 DRG, and SC segments using real-time PCR and Western blotting. Real-time PCR revealed increased expression of NPY in all tissues except for SC, and increased TRPV1 and PENK expression in DRG and SC, whereas expression of Tac1, M1 and M2 was decreased. Less significant changes were noted in protein levels. These findings suggest that bladder injections of OnabotulinumtoxinA may be followed by changes in the expression of sensory, sympathetic and cholinergic bladder function regulators at the DRG/SC level.     

Excess Heritability Contribution of Alcohol Consumption Variants in the “Missing Heritability” of Type 2 Diabetes Mellitus

We aim to compare the relative heritability contributed by variants of behavior-related environmental phenotypes and elucidate the role of these factors in the conundrum of “missing heritability” of type 2 diabetes. Methods: We used Linkage-Disequilibrium Adjusted Kinships (LDAK) and LDAK-Thin models to calculate the relative heritability of each variant and compare the relative heritability for each phenotype. Biological analysis was carried out for the phenotype whose variants made a significant contribution. Potential hub genes were prioritized based on topological parameters of the protein-protein interaction network. We included 16 behavior-related phenotypes and 2607 valid variants. In the LDAK model, we found the variants of alcohol consumption and caffeine intake were identified as contributing higher relative heritability than that of the random variants. Compared with the relative expected heritability contributed by the variants associated with type 2 diabetes, the relative expected heritability contributed by the variants associated with these two phenotypes was higher. In the LDAK-Thin model, the relative heritability of variants of 11 phenotypes was statistically higher than random variants. Biological function analysis showed the same distributions among type 2 diabetes and alcohol consumption. We eventually screened out 31 hub genes interacting intensively, four of which were validated and showed the upregulated expression pattern in blood samples seen in type 2 diabetes cases. Conclusion: We found that alcohol consumption contributed higher relative heritability. Hub genes may influence the onset of type 2 diabetes by a mediating effect or a pleiotropic effect. Our results provide new insight to reveal the role of behavior-related factors in the conundrum of “missing heritability” of type 2 diabetes.     

Ca2+ Signalling Induced by NGF Identifies a Subset of Capsaicin-Excitable Neurons Displaying Enhanced Chemo-Nociception in Dorsal Root Ganglion Explants from Adult pirt-GCaMP3 Mouse

Nociceptors sense hazards via plasmalemmal cation channels, including transient receptor potential vanilloid 1 (TRPV1). Nerve growth factor (NGF) sensitises TRPV1 to capsaicin (CAPS), modulates nociceptor excitability and induces thermal hyperalgesia, but cellular mechanisms remain unclear. Confocal microscopy was used to image changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) across neuronal populations in dorsal root ganglia (DRG) explants from pirt-GCaMP3 adult mice, which express a fluorescent reporter in their sensory neurons. Raised [Ca²⁺]_i was detected in 84 neurons of three DRG explants exposed to NGF (100 ng/mL) and most (96%) of these were also excited by 1 μM CAPS. NGF elevated [Ca²⁺]_i in about one-third of the neurons stimulated by 1 μM CAPS, whether applied before or after the latter. In neurons excitable by NGF, CAPS-evoked [Ca²⁺]_i signals appeared significantly sooner (e.g., respective lags of 1.0 ± 0.1 and 1.9 ± 0.1 min), were much (>30%) brighter and lasted longer (6.6 ± 0.4 vs. 3.9 ± 0.2 min) relative to those non-responsive to the neurotrophin. CAPS tachyphylaxis lowered signal intensity by ~60% but was largely prevented by NGF. Increasing CAPS from 1 to 10 μM nearly doubled the number of cells activated but only modestly increased the amount co-activated by NGF. In conclusion, a sub-population of the CAPS-sensitive neurons in adult mouse DRG that can be excited by NGF is more sensitive to CAPS, responds with stronger signals and is further sensitised by transient exposure to the neurotrophin.     

TFG-β Nuclear Staining as a Potential Relapse Risk Factor in Early-Stage Non-Small-Cell Lung Cancer

Nowadays, the impact of the tumor-immune microenvironment (TME) in non-small-cell lung cancer (NSCLC) prognosis and treatment response remains unclear. Thus, we evaluated the expression of PD-L1, tumor-infiltrating lymphocytes (TILs), and transforming growth factor beta (TGF-β) in NSCLC to identify differences in TME, detect possible new prognostic factors, and assess their relationship. We retrospectively analyzed 55 samples from patients who underwent NSCLC surgery and had over a 5-year follow-up. PD-L1 expression was determined by immunohistochemistry following standard techniques. The presence of TILs was evaluated at low magnification and classified into two categories, “intense” and “non-intense”. Cytoplasmic TGF-β staining visualization was divided into four categories, and unequivocal nuclear staining in >1% of viable tumor cells was defined as “present” or “absent”. Our aim was to identify differences in disease-free survival (DFS) and overall survival (OS). Tumor stage was the only objective prognostic factor for OS. PD-L1 expression and the presence of TILs had no prognostic impact, neither their combination. There seems to be a lower expression of PD-L1 and a higher expression of TILs in early stages of the disease. Our TGF-β nuclear staining analysis was promising, since it was associated with worse DFS, revealing this protein as a possible prognostic biomarker of recurrence for resectable NSCLC.