Expression of cytokines at baseline correlate/predict in the disc the outcome of surgery after disc degeneration: A 12-month follow-up study.

BACKGROUND: Low back pain (LBP) is a highly prevalent condition that comprise a large portion of outpatient practice, challenging the diagnosis and treatment. However, the diagnostic tools are limited to clinical history, physical examination and imaging. Degenerative disc disease (DDD) is a significant cause of LBP, and emerging literature confirms the elevated levels of biomarkers in the discs. These biomarkers may serve as a tool for diagnosis, but may also be useful in predicting the treatment outcome. Here, we examine the expression of various cytokines on 1-year recovery from patients with LBP. METHODS: Patient-reported outcome (PRO) in terms of pain intensity (VAS), disability (ODI), and quality of life (Eq-5D) is collected from 44 patients at baseline and 12 months after surgery to study the influence of baseline TNF-α, IL-1ß, and IL-6 mRNA expression in both annulus fibrosus (AF) and nucleus pulposus (NP). RESULTS: Between baseline and follow-up, our cohort showed improvement in VAS back pain (p < 0.001), VAS leg pain (p < 0.001), ODI (p = 0.02), and Eq-5D (p = 0.01). Baseline levels of IL-1 ß was positively correlated with VAS back pain scores in AF (p = 0.05) and NP (p = 0.01) at 1-year follow-up. TNF-α expression at baseline was also positively correlated to ODI scores (p = 0.01) at follow-up and inversely correlated to improvements in ODI score between baseline and follow-up, suggesting that high TNF-α expression at baseline is associated with poor outcomes from surgery. CONCLUSION: The results from our study support that TNF-α expression at baseline can serve as a very important predictor of treatment response from lumbar fusion surgery.

Characterizing the binding of TC-5619 and encenicline on the alpha7 nicotinic acetylcholine receptor using PET imaging in the pig.

The alpha7 nicotinic acetylcholine receptor (α7-nAChR) has has long been considered a promising therapeutic target for addressing cognitive impairments associated with a spectrum of neurological and psychiatric disorders, including Alzheimer's disease and schizophrenia. However, despite this potential, clinical trials employing α7-nAChR (partial) agonists such as TC-5619 and encenicline (EVP-6124) have fallen short in demonstrating sufficient efficacy. We here investigate the target engagement of TC-5619 and encenicline in the pig brain by use of the α7-nAChR radioligand 11C-NS14492 to characterize binding both with in vitro autoradiography and in vivo occupancy using positron emission tomography (PET). In vitro autoradiography demonstrates significant concentration-dependent binding of 11C-NS14492, and both TC-5619 and encenicline can block this binding. Of particular significance, our in vivo investigations demonstrate that TC-5619 achieves substantial α7-nAChR occupancy, effectively blocking approximately 40% of α7-nAChR binding, whereas encenicline exhibits more limited α7-nAChR occupancy. This study underscores the importance of preclinical PET imaging and target engagement analysis in informing clinical trial strategies, including dosing decisions.

Binding of the monoacylglycerol lipase (MAGL) radiotracer [3H]T-401 in the rat brain after status epilepticus.

OBJECTIVES: Monoacylglycerol lipase (MAGL) is a cytosolic serine hydrolase considered a potential novel drug target for the treatment of CNS disorders including epilepsy. Here we examined MAGL levels in a rat model of epilepsy. METHODS: Autoradiography has been used to validate the binding properties of the MAGL radiotracer, [3H]T-401, in the rat brain, and to explore spatial and temporal changes in binding levels in a model of temporal lobe epilepsy model using unilateral intra-hippocampal injections of kainic acid (KA) in rats. RESULTS: Specific and saturable binding of [3H]T-401 was detected in both cortical grey and subcortical white matter. Saturation experiments revealed a KD in the range between 15 nM and 17 nM, and full saturation was achieved at concentrations around 30 nM. The binding could be completely blocked with the cold ligand (Ki 44.2 nM) and at higher affinity (Ki 1.27 nM) with another structurally different MAGL inhibitor, ABD 1970. Bilateral reduction in [3H]T-401 binding was observed in the cerebral cortex and the hippocampus few days after status epilepticus that further declined to a level of around 30% compared to the control. No change in binding was observed in either the hypothalamus nor the white matter at any time point. Direct comparison to [3H]UCB-J binding to synaptic vesicle glycoprotein 2 A (SV2A), another protein localized in the pre-synapse, revealed that while binding to MAGL remained low in the chronic phase, SV2A was increased significantly in some cortical areas. SIGNIFICANCE: These data show that MAGL is reduced in the cerebral cortex and hippocampus in a chronic epilepsy model and indicate that MAGL inhibitors may further reduce MAGL activity in the treatment resistant epilepsy patient.

Systemic treatment with a selective TNFR2 agonist alters the central and peripheral immune responses and transiently improves functional outcome after experimental ischemic stroke.

Ischemic stroke often leaves survivors with permanent disabilities and therapies aimed at limiting detrimental inflammation and improving functional outcome are still needed. Tumor necrosis factor (TNF) levels increase rapidly after ischemic stroke, and while signaling through TNF receptor 1 (TNFR1) is primarily detrimental, TNFR2 signaling mainly has protective functions. We therefore investigated how systemic stimulation of TNFR2 with the TNFR2 agonist NewSTAR2 affects ischemic stroke in mice. We found that NewSTAR2 treatment induced changes in peripheral immune cell numbers and transiently affected microglial numbers and neuroinflammation. However, this was not sufficient to improve long-term functional outcome after stroke in mice.

Synaptic vesicle glycoprotein 2A (SV2A) levels in the cerebral cortex in patients with Alzheimer's disease: a radioligand binding study in postmortem brains.

Histological and biochemical analyses in postmortem tissues have demonstrated neurodegenerative changes in the cerebral cortex in patients with Alzheimer's disease (AD), and it has been suggested that this represents a loss of synapses. PET imaging of the (pre)synaptic vesicular glycoprotein 2A (SV2A) has demonstrated a reduction in synapse density in AD in the hippocampus but not consistently in the neocortex. This investigation examines the level of [3H]UCB-J binding in postmortem cortical tissue from patients with AD and matched healthy controls using autoradiography. Among the neocortical areas examined, the binding was significantly lower only in the middle frontal gyrus in AD compared to matched controls. No differences were observed in the parietal, temporal, or occipital cortex. The binding levels in the frontal cortex in the AD cohort displayed large variability among subjects, and this revealed a highly significant negative association with the age of the patient. These results demonstrate low UCB-J binding in the frontal cortex of patients with AD, and this biomarker correlates negatively with age, which may further indicate that SV2A could be an important biomarker in AD patients.

Characterization of the Novel P2X7 Receptor Radioligand [3H]JNJ-64413739 in Human Brain Tissue.

Radioligands targeting microglia cells have been developed to identify and determine neuroinflammation in the living brain. One recently discovered ligand is JNJ-64413739 that binds selectively to the purinergic receptor P2X7R. The expression of P2X7R is increased under inflammation; hence, the ligand is considered useful in the detection of neuroinflammation in the brain. [18F]JNJ-64413739 has been evaluated in healthy subjects with positron emission tomography; however, the in vitro binding properties of the ligand in human brain tissue have not been investigated. Therefore, the purpose of this study was to measure Bmax and Kd of [3H]JNJ-64413739 using autoradiography on human cortical tissue sections resected from a total of 48 patients with treatment-resistant epilepsy. Correlations between the specific binding of [3H]JNJ-64413739 with age, sex, and duration of disease were explored. Finally, to examine the relationship between P2X7R and TSPO availability, specific binding of [3H]JNJ-64413739 and [123I]CLINDE was examined in the same tissue. The binding was measured in both cortical gray and subcortical white matter. Saturation revealed a Kd (5 nM) value similar between gray and white matter but a larger Bmax in the white than in the gray matter. The binding was completely displaced by the cold ligand and structurally different P2X7R ligands. The variability in saturable binding among the samples was found to be 38% in gray and white matter but was not correlated to either age, sex, or the duration of the disease. Interestingly, there was no significant correlation between [3H]JNJ-64413739 and [123I]CLINDE binding. These data demonstrate that [3H]JNJ-64413739 is a suitable radioligand for evaluating the distribution and expression of the P2X7R in the human brain.

Transient receptor potential (TRP) channels mRNA transcripts in the lumbar intervertebral discs: biomarkers for inflammation, pain, disability, and clinical outcome.

Transient receptor potential (TRP) channels are widely expressed cation channels that play an essential role in mediating Ca2+ homeostasis and are considered potential regulators of inflammatory pain. This study investigates the expression of the TRP channel subtypes TRPV1, TRPV4, TRPC6, TRPM2, TRPM8 in lumbar intervertebral disc (IVD) biopsies from patients with chronic low back pain (LBP). We determined the expression of these TRP channel subtypes in the annulus fibrosus (AF) and the nucleus pulposus (NP) from 46 patients with LBP undergoing 1-2 level lumbar fusion surgery for degenerative disc disease. The mRNA transcripts were analyzed using quantitative real-time polymerase chain reaction (RT-qPCR), and the expression levels were compared against visual analog scale (VAS) and oswestry disability index (ODI) scores (0-100) for pain and disability. A significant positive correlation was demonstrated between VAS score and the mRNA expression of TRPV1, TRPC6, TRPM2, TRPM8 in the AF. We also found a significant positive correlation between ODI scores and expression of TRPV1 and TRPM8. Further, there is a significant positive correlation between TNF-α and TRPV1, TRPM2 and TRPM8 expression in the AF, and IL-6 to TRPV1 in the NP. Interestingly, when investigating treatment response via a 12-month postoperative follow-up ODI, we found a significant correlation between only TRPV1 expression at baseline and the follow-up ODI scores, which indicates this marker could predict the effectiveness of surgery. These results strongly suggest an association between pain, inflammatory mediators, and TRP channel expression in lumbar disc biopsies of patients with chronic LBP.

Subacute effects of a single dose of psilocybin on biomarkers of inflammation in healthy humans: An open-label preliminary investigation.

Rationale: Psilocybin is a serotonergic psychedelic that has gained prominent attention recently as a potential therapeutic for neuropsychiatric disorders including Major Depressive Disorder. Pre-clinical and initial studies in humans suggest that serotonin 2A receptor agonists, including serotonergic psychedelics, have anti-inflammatory effects. This may contribute to its therapeutic effects as previous studies indicate a link between neuropsychiatric disorders and inflammatory processes. However, the effect of psilocybin on biomarkers of inflammation has not been evaluated in humans. Objectives: Investigate the effect of a single dose of psilocybin on peripheral biomarkers of inflammation in healthy humans. Methods: Blood samples were collected from 16 healthy participants before and one day after the administration of a single oral dose of psilocybin (mean dose: 0.22 mg/kg) and subsequently analyzed for concentrations of high-sensitivity C-reactive protein (hsCRP), tumor-necrosis-factor (TNF) and soluble urokinase plasminogen activator receptor (suPAR). Change in inflammatory markers was evaluated using a paired t-test where p < 0.05 was considered statistically significant. Results: We did not observe statistically significant changes in any of the above biomarkers of inflammation (all Cohen's d ≤ 0.31; all p ≥ 0.23). Conclusions: Our data do not support that a single dose of psilocybin reduces biomarkers of inflammation in healthy individuals one day after administration. Nevertheless, we suggest that future studies consider additional markers of inflammation, including markers of neuroinflammation, and evaluate potential anti-inflammatory effects of psilocybin therapy in clinical cohorts where more prominent effects may be observable.

The expression of metalloproteinases in the lumbar disc correlates strongly with Pfirrmann MRI grades in lumbar spinal fusion patients.

Introduction: Increased catabolism of the extracellular matrix is observed under degenerative disc disease (DDD). The cleavage of extracellular matrix proteins in the nucleus pulposus (NP) by either matrix metalloproteinases (MMPs) or a disintegrin and metalloproteinases with thrombospondin motifs (ADAMTSs) is believed to be involved in the degeneration, but the mechanisms are not known. Research question: Here, we examine the correlation between expression of several MMPs and ADAMTSs subtypes in lumbar discs from 34 patients with low back pain (LBP) undergoing 1-2 level lumbar fusion surgery (L4/L5 and/or L5/S1) for DDD with or without spondylolisthesis. Materials and Methods: The mRNA levels of MMPs (subtypes 1, 2, 3, 10, and 13) and ADAMTSs (subtypes 1, 4, and 5) were analyzed using quantitative real-time polymerase chain reaction (RT-qPCR) and correlated to the Pfirrmann magnetic resonance imaging classification system (grade I-V) of lumbar DDD. Results: We find a highly significant positive correlation between Pfirrmann grades and the gene expression of MMP1 (r=0.67, p=0.0001), MMP3 (r=0.61, p=0.0002), MMP10 (r=0.6701, p=0.0001), MMP13 (r=0.48, p=0.004), ADAMTS1 (r=0.67, p=0.0001), and ADAMTS5 (r=0.53, p=0.0017). The similar regulation of these transcript suggests their involvement in disc degeneration. Interestingly, a post hoc analysis (uncorrected p-values) also demonstrated a positive correlation between expression of TNF-α, IL-6 and both ADAMTSs/MMPs and the Pfirrmann grades. Discussion and Conclusion: These findings show that disc degradation in DDD is strongly associated with the expression of some metalloproteinases.

Synergistic effect of serotonin 1A and serotonin 1B/D receptor agonists in the treatment of L-DOPA-induced dyskinesia in 6-hydroxydopamine-lesioned rats.

BACKGROUND: The gold standard for symptomatic relief of Parkinson's disease (PD) is L-DOPA. However, long-term treatment often leads to motor complications such as L-DOPA-induced dyskinesia (LID). While amantadine (Gocovri™) is the only approved therapy for dyskinesia in PD patients on the American market, it is associated with neurological side effects and limited efficacy. Thus, there remains a high unmet need for addressing LID in PD patients worldwide. OBJECTIVE: The objective of this study was to evaluate the efficacy, safety and performance compared to approved treatments of the serotonin receptor 1A (5-HT1A) and 5-HT1B/D agonists buspirone and zolmitriptan in the 6-hydroxydopamine unilaterally lesioned rat model for PD. METHODS: The hemiparkinsonian 6-OHDA-lesioned rats underwent chronic treatment with L-DOPA to induce dyskinesia and were subsequently used for efficacy testing of buspirone, zolmitriptan and comparison with amantadine, measured as abnormal involuntary movement (AIM) scores after L-DOPA challenge. Safety testing was performed in model and naïve animals using forelimb adjusting, rotarod and open field tests. RESULTS: 5-HT1A and 5-HT1B/D agonism effectively reduced AIM scores in a synergistic manner. The drug combination of buspirone and zolmitriptan was safe and did not lead to tolerance development following sub-chronic administration. Head-to-head comparison with amantadine showed superior performance of buspirone and zolmitriptan in the model. CONCLUSIONS: The strong anti-dyskinetic effect found with combined 5-HT1A and 5-HT1B/D agonism renders buspirone and zolmitriptan together a meaningful treatment for LID in PD.

Spatio-Temporal Alterations in Synaptic Density During Epileptogenesis in the Rat Brain.

Synaptic vesicle glycoprotein 2A (SV2A) is a transmembrane protein that binds levetiracetam and is involved in neurotransmission via an unknown mechanism. SV2A-immunoreactivity is reduced in animal models of epilepsy, and in postmortem hippocampi from patients with temporal lobe epilepsy. It is not known if other regions outside the hippocampus are affected in epilepsy, and whether SV2A expression is permanently reduced or regulated over time. In this study, we induced a generalized status epilepticus (SE) by systemic administration of lithium-pilocarpine to adult female rats. The brains from all animals experiencing SE were collected at different time points after the treatment. The radiotracer, [11C]-UCB-J, binds to SV2A with high affinity, and has been used for in vivo imaging as an a-proxy marker for synaptic density. Here we determined the level of tritiated UCB-J binding by semiquantitative autoradiography in the cerebral cortex, hippocampus, thalamus, and hypothalamus, and in cortical subregions. A prominent and highly significant reduction in SV2A binding capacity was observed over the first days after SE in the cerebral cortex and the hippocampus, but not in the thalamus and hypothalamus. The magnitude in reduction was larger and occurred earlier in the hippocampus and the piriform cortex, than in other cortical subregions. Interestingly, in all areas examined, the binding capacity returned to control levels 12 weeks after the SE comparable to the chronic epileptic phase. These data indicate that lithium-pilocarpine-induced epileptogenesis involves both loss and gain of synapses in the in a time-dependent manner.

Temporal and spatial changes in synaptic vesicle glycoprotein 2A (SV2A) under kainic acid induced epileptogenesis: An autoradiographic study.

Synaptic Vesicle Glycoprotein 2A (SV2A) has been proposed as a presynaptic marker in several neurological disorders. Not only is SV2A the target for the antiepileptic drug levetiracetam, but also considered a marker of mature pre-synapses. In this study, we aimed to assess the binding of [3H]UCB-J as a selective radioligand for SV2A to visualize and determine changes during different stages of epileptogenesis by in-vitro autoradiography in rat models of temporal lobe epilepsy. Two different kainic acid (KA) injection routes were used to model temporal lobe epilepsy in the rat; a systemic (10 mg/kg KA injected intraperitoneally) and a local model (1.875 mM KA injected intrahippocampally). Brain tissue was sampled at different time points after the initial status epilepticus and semi-quantitative [3H]UCB-J autoradiography was performed to determine temporal and spatial changes under the progression of epileptogenesis. A decrease in [3H]UCB-J binding was observed in many brain areas in the acute phases after both types of kainic acid administration. Peak reductions occurred slightly before in systemic-treated animals (within 3-10 days) than after local-treated animals (within 5-15 days). Interestingly in the systemic model, we observed a full restoration in the binding level 30 days after the treatment in most areas probably reflecting neuronal reorganization. However, after the local injection in the hippocampus, the binding in the hippocampus, and in temporal and piriform cortices did not return to basal levels. The time-course profile displayed lateralization in the local model. These results demonstrate changes in the amount of a presynaptic SV2A binding site after seizures and suggest that SV2A may have importance in eliciting spontaneous seizures and/or be a biomarker for epileptogenesis. The present study shows that SV2A is a biomarker of acute phase epileptogenesis in specific brain regions.

Expression profile of synaptic vesicle glycoprotein 2A, B, and C paralogues in temporal neocortex tissue from patients with temporal lobe epilepsy (TLE).

Synaptic vesicle glycoprotein-2 (SV2) is a family of proteins consisting of SV2A, SV2B, and SV2C. This protein family has attracted attention in recent years after SV2A was shown to be an epileptic drug target and a perhaps a biomarker of synaptic density. So far, the anatomical localization of these proteins in the rodent and human brain have been reported, but co-expression of SV2 genes on a cellular level, their expressions in the human brain, comparison to radioligand binding, any possible regulation in epilepsy are not known. We have here analyzed the expression of SV2 genes in neuronal subtypes in the temporal neocortex in selected specimens by using single nucleus-RNA sequencing, and performed quantitative PCR in populations of temporal lobe epilepsy (TLE) patients and healthy controls. [3H]-UCB-J autoradiography was performed to analyze the correlation between the mRNA transcript and binding capacity to SV2A. Our data showed that the SV2A transcript is expressed in all glutamatergic and GABAergic cortical subtypes, while SV2B expression is restricted to only the glutamatergic neurons and SV2C has very limited expression in a small subgroup of GABAergic interneurons. The level of [3H]-UCB-J binding and the concentration of SV2A mRNA is strongly correlated in each patient, and the expression is lower in the TLE patients. There is no relationship between SV2A expression and age, sex, seizure frequency, duration of epilepsy, or whether patients were recently treated with levetiracetam or not. Collectively, these findings point out a neuronal subtype-specific distribution of the expression of the three SV2 genes, and the lower levels of both radioligand binding and expression further emphasize the significance of these proteins in this disease.

Synaptic Density and Neuronal Metabolic Function Measured by Positron Emission Tomography in the Unilateral 6-OHDA Rat Model of Parkinson's Disease.

Parkinson's disease (PD) is caused by progressive neurodegeneration and characterised by motor dysfunction. Neurodegeneration of dopaminergic neurons also causes aberrations within the cortico-striato-thalamo-cortical (CSTC) circuit, which has been hypothesised to lead to non-motor symptoms such as depression. Individuals with PD have both lower synaptic density and changes in neuronal metabolic function in the basal ganglia, as measured using [11C]UCB-J and [18F]FDG positron emission tomography (PET), respectively. However, the two radioligands have not been directly compared in the same PD subject or in neurodegeneration animal models. Here, we investigate [11C]UCB-J binding and [18F]FDG uptake in the CSTC circuit following a unilateral dopaminergic lesion in rats and compare it to sham lesioned rats. Rats received either a unilateral injection of 6-hydroxydopamine (6-OHDA) or saline in the medial forebrain bundle and rostral substantia nigra (n = 4/group). After 3 weeks, all rats underwent two PET scans using [18F]FDG, followed by [11C]UCB-J on a separate day. [18F]FDG uptake and [11C]UCB-J binding were both lower in the ipsilateral striatal regions compared to the contralateral regions. Using [11C]UCB-J, we could detect an 8.7% decrease in the ipsilateral ventral midbrain, compared to a 2.9% decrease in ventral midbrain using [18F]FDG. Differential changes between hemispheres for [11C]UCB-J and [18F]FDG outcomes were also evident in the CSTC circuit's cortical regions, especially in the orbitofrontal cortex and medial prefrontal cortex where higher synaptic density yet lower neuronal metabolic function was observed, following lesioning. In conclusion, [11C]UCB-J and [18F]FDG PET can detect divergent changes following a dopaminergic lesion in rats, especially in cortical regions that are not directly affected by the neurotoxin. These results suggest that combined [11C]UCB-J and [18F]FDG scans could yield a better picture of the heterogeneous cerebral changes in neurodegenerative disorders.

N1H- and N1-Substituted Phenylguanidines as α7 Nicotinic Acetylcholine (nACh) Receptor Antagonists: Structure-Activity Relationship Studies.

We previously reported that N-(3-chlorophenyl)guanidine (1) represents a novel α7 nicotinic ACh (nACh) receptor antagonist chemotype. In the present study, a small series of compounds was synthesized with the intent to investigate the structure-activity relationship (SAR). Preliminary data suggested that the N-methyl analog of 1, 2, was several times more potent. Therefore, the chloro group at the aryl 3-position of 1 and its N1-methyl counterpart 2 were replaced with a number of substituents considering the electronic, lipophilic, and steric nature of the substituents. The potencies of the compounds to inhibit acetylcholine (ACh)-induced responses were obtained in Xenopus laevis oocytes expressing human α7 nicotinic ACh receptors (nAChRs) using a two-electrode voltage-clamp assay. We found that the nature of the 3-position substituents had relatively little (i.e., <10-fold) effect on potency, and the presence of an N1-isopropyl substituent was tolerated. Here, we report the first SAR investigation of this novel α7 nAChR antagonist chemotype.

In Vitro and In Vivo Characterization of Dibenzothiophene Derivatives [125I]Iodo-ASEM and [18F]ASEM as Radiotracers of Homo- and Heteromeric α7 Nicotinic Acetylcholine Receptors.

The α7 nicotinic acetylcholine receptor (α7 nAChR) is involved in several cognitive and physiologic processes; its expression levels and patterns change in neurologic and psychiatric diseases, such as schizophrenia and Alzheimer's disease, which makes it a relevant drug target. Development of selective radioligands is important for defining binding properties and occupancy of novel molecules targeting the receptor. We tested the in vitro binding properties of [125I]Iodo-ASEM [(3-(1,4-diazabycyclo[3.2.2]nonan-4-yl)-6-(125I-iododibenzo[b,d]thiopentene 5,5-dioxide)] in the mouse, rat and pig brain using autoradiography. The in vivo binding properties of [18F]ASEM were investigated using positron emission tomography (PET) in the pig brain. [125I]Iodo-ASEM showed specific and displaceable high affinity (~1 nM) binding in mouse, rat, and pig brain. Binding pattern overlapped with [125I]α-bungarotoxin, specific binding was absent in α7 nAChR gene-deficient mice and binding was blocked by a range of α7 nAChR orthosteric modulators in an affinity-dependent order in the pig brain. Interestingly, relative to the wild-type, binding in ß2 nAChR gene-deficient mice was lower for [125I]Iodo-ASEM (58% ± 2.7%) than [125I]α-bungarotoxin (23% ± 0.2%), potentially indicating different binding properties to heteromeric α7ß2 nAChR. [18F]ASEM PET in the pig showed high brain uptake and reversible tracer kinetics with a similar spatial distribution as previously reported for α7 nAChR. Blocking with SSR-180,711 resulted in a significant decrease in [18F]ASEM binding. Our findings indicate that [125I]Iodo-ASEM allows sensitive and selective imaging of α7 nAChR in vitro, with better signal-to-noise ratio than previous tracers. Preliminary data of [18F]ASEM in the pig brain demonstrated principal suitable kinetic properties for in vivo quantification of α7 nAChR, comparable to previously published data.

Anti-Inflammatory Effect of Alpha7 Nicotinic Acetylcholine Receptor Modulators on BV2 Cells.

OBJECTIVE: The development of neuroinflammation shares numerous risk factors and involves many complex interactions which contribute to disease pathology. An important cell type in neuroinflammation is the active microglia cell - the resident immune cell of the CNS. There is increasing need to understand how these pathways related to neuroinflammation work and how they can be regulated. Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated receptors and widely distributed in the brain. The α7 nAChR is a penta-homomeric receptor and is one of the nAChRs expressed in microglia. This study was first designed to characterize the effects of lipopolysaccharide (LPS) on BV2 culture cells, a cell line of murine microglia origin, on release of inflammatory markers and to characterize the inhibitory effects of α7 nAChR modulators in these cells. METHODS: First, the BV2 cell cultures were functionally validated by exposing them to LPS for 4-24 h and then examining the release of tumor necrosis factor-alpha (TNF-α) using ELISA and nitric oxide (NO) release using the Griess assay, respectively. Next, α7 nAChR modulators with different pharmacological profiles were applied dose-dependently to study their effects on LPS-induced release of NO and TNF-α. RESULTS: The time-course and dose-response curve revealed that LPS dose-dependently activated (EC50 = 2.5 ng/mL) BV2 cells releasing TNF-α at 4 h, followed by release of NO that occurred first at 8-h time point. The α7 nAChR subunit mRNA was identified in the BV2 cells. The pharmacology studies showed the α7 nAChR selective modulators NS6740 and TQS reduced NO and cytokine release from BV2 cell cultures. CONCLUSION: We here identified the dose- and time-dependent effects of LPS in BV2 cell cultures on several inflammatory readouts and showed that α7 nAChR modulators with little or no ion channel activity inhibited this anti-inflammatory mechanism.

Alpha7 nicotinic acetylcholine receptors and neural network synaptic transmission in human induced pluripotent stem cell-derived neurons.

The α7 nicotinic acetylcholine receptor has been extensively researched as a target for treatment of cognitive impairment in Alzheimer's disease and schizophrenia. Investigation of the α7 receptor is commonly performed in animals but it is critical to increase the biologically relevance of the model systems to fully capture the physiological role of the α7 receptor in humans. For example most humans, in contrast to animals, express the hybrid gene CHRFAM7A, the product of which modulates α7 receptor activity. In the present study, we used human induced pluripotent stem cell (hiPSC) derived neurons to establish a humanized α7 model. We established a cryobank of neural stem cells (NSCs) that could reproducibly be matured into neurons expressing neuronal markers and CHRNA7 and CHRFAM7A. The neurons responded to NMDA, GABA, and acetylcholine and exhibited synchronized spontaneous calcium oscillations. Gene expression studies and application of a range of α7 positive allosteric modulators (PNU-120595, TQS, JNJ-39393406 and AF58801) together with the α7 agonist PNU-282987 during measurement of intracellular calcium levels demonstrated the presence of functional α7 receptors in matured hiPSC-derived neuronal cultures. Pharmacological α7 activation also resulted in intracellular signaling as measured by ERK 1/2 phosphorylation and c-Fos protein expression. Moreover, PNU-120596 increased the frequency of the spontaneous calcium oscillations demonstrating implication of α7 receptors in human synaptic networks activity. Overall, we show that hiPSC derived neurons are an advanced in vitro model for studying human α7 receptor pharmacology and the involvement of this receptor in cellular processes as intracellular signaling and synaptic transmission.

DNA methylation regulates CHRNA7 transcription and can be modulated by valproate.

The CHRNA7 gene encoding the α7 nicotinic acetylcholine receptor (nAChR) has repeatedly been linked with schizophrenia and the P50 sensory gating deficit. The α7 nAChR is considered a promising drug target for treatment of cognitive dysfunction in schizophrenia and improves memory and executive functions in patients and healthy individuals. However, clinical trials with pro-cognitive drugs are challenged by large inter-individual response variations and these have been linked to genotypic variations reducing CHRNA7 expression and α7 nAChR function. Genetic variants as well as environmental conditions may cause epigenetic dysregulation and it has previously been found that DNA methylation of a region surrounding the transcription start site of CHRNA7 is important for tissue specific regulation and gene silencing. In the present study we identify two additional regions involved in epigenetic regulation of the CHRNA7 promoter. In human temporal cortex we find large variations in expression of CHRNA7 and establish evidence for a significant correlation with DNA methylation levels of one region. We then establish evidence that genotypic variations can influence methylation levels of the CHRNA7 promoter. Epigenetic dysregulation can be reversed by pharmacological intervention and in HeLa cells. Valproate, a commonly used mood stabiliser, caused demethylation and increased CHRNA7 expression in HeLA cells. Similar demethylation effect and increased CHRNA7 expression was obtained in SH-SY5Y cells stimulated concomitantly with valproate and nicotine. In summary, both genetic and epigenetic information could be useful to predict treatment outcomes in patients and epigenetic modulation may serve as a mechanism for potentiating the effects of α7 nAChR agonists.

Analysis of gonadotrophin-releasing hormone-1 and kisspeptin neuronal systems in the nonphotoregulated seasonally breeding eastern rock elephant-shrew (Elephantulus myurus).

Of the 18 sub-Saharan elephant-shrew species, only eastern rock elephant-shrews reproduce seasonally throughout their distribution, a process seemingly independent of photoperiod. The present study characterizes gonadal status and location/intensity of gonadotrophin-releasing hormone-1 (GnRH-1) and kisspeptin immunoreactivities in this polyovulating species in the breeding and nonbreeding seasons. GnRH-1-immunoreactive (ir) cell bodies are predominantly in the medial septum, diagonal band, and medial preoptic area; processes are generally sparse except in the external median eminence. Kisspeptin-ir cell bodies are detected only within the arcuate nucleus; the density of processes is generally low, except in the septohypothalamic nucleus, ventromedial bed nucleus of the stria terminalis, arcuate nucleus, and internal and external median eminence. Kisspeptin-ir processes are negligible at locations containing GnRH-1-ir cell bodies. The external median eminence is the only site with conspicuously overlapping distributions of the respective immunoreactivities and, accordingly, a putative site for kisspeptin's regulation of GnRH-1 release in this species. In the nonbreeding season in males, there is an increase in the rostral population of GnRH-1-ir cell bodies and density of GnRH-1-ir processes in the median eminence. In both sexes, the breeding season is associated with increased kisspeptin-ir process density in the rostral periventricular area of the third ventricle and arcuate nucleus; at the latter site, this is positively correlated with gonadal mass. Cross-species comparisons lead us to hypothesize differential mechanisms within these peptidergic systems: that increased GnRH-1 immunoreactivity during the nonbreeding season reflects increased accumulation with reduced release; that increased kisspeptin immunoreactivity during the breeding season reflects increased synthesis with increased release.