Changes In Receptor Expression Research Articles

Relationship between ENaC Regulators and SARS-CoV-2 Virus Receptor (ACE2) Expression in Cultured Adult Human Fungiform (HBO) Taste Cells.

In addition to the α, β, and γ subunits of ENaC, human salt-sensing taste receptor cells (TRCs) also express the δ-subunit. At present, it is not clear if the expression and function of the ENaC δ-subunit in human salt-sensing TRCs is also modulated by the ENaC regulatory hormones and intracellular signaling effectors known to modulate salt responses in rodent TRCs. Here, we used molecular techniques to demonstrate that the G-protein-coupled estrogen receptor (GPER1), the transient receptor potential cation channel subfamily V member 1 (TRPV1), and components of the renin-angiotensin-aldosterone system (RAAS) are expressed in δ-ENaC-positive cultured adult human fungiform (HBO) taste cells. Our results suggest that RAAS components function in a complex with ENaC and TRPV1 to modulate salt sensing and thus salt intake in humans. Early, but often prolonged, symptoms of COVID-19 infection are the loss of taste, smell, and chemesthesis. The SARS-CoV-2 spike protein contains two subunits, S1 and S2. S1 contains a receptor-binding domain, which is responsible for recognizing and binding to the ACE2 receptor, a component of RAAS. Our results show that the binding of a mutated S1 protein to ACE2 decreases ACE2 expression in HBO cells. We hypothesize that changes in ACE2 receptor expression can alter the balance between the two major RAAS pathways, ACE1/Ang II/AT1R and ACE2/Ang-(1–7)/MASR1, leading to changes in ENaC expression and responses to NaCl in salt-sensing human fungiform taste cells.

Racing and Pacing in the Reward System: A Multi-Clock Circadian Control Over Dopaminergic Signalling.

Level of motivation, responsiveness to rewards and punishment, invigoration of exploratory behaviours, and motor performance are subject to daily fluctuations that emerge from circadian rhythms in neuronal activity of the midbrain’s dopaminergic system. While endogenous circadian rhythms are weak in the ventral tegmental area and substantia nigra pars compacta, daily changes in expression of core clock genes, ion channels, neurotransmitter receptors, dopamine-synthesising enzymes, and dopamine transporters, accompanied by changes in electrical activity, are readily observed in these nuclei. These processes cause dopamine levels released in structures innervated by midbrain dopaminergic neurons (e.g., the striatum) to oscillate in a circadian fashion. Additionally, growing evidence show that the master circadian clock located in the suprachiasmatic nucleus of the hypothalamus (SCN) rhythmically influences the activity of the dopaminergic system through various intermediate targets. Thus, circadian changes in the activity of the dopaminergic system and concomitant dopamine release observed on a daily scale are likely to be generated both intrinsically and entrained by the master clock. Previous studies have shown that the information about the value and salience of stimuli perceived by the animal is encoded in the neuronal activity of brain structures innervating midbrain dopaminergic centres. Some of these structures themselves are relatively autonomous oscillators, while others exhibit a weak endogenous circadian rhythm synchronised by the SCN. Here, we place the dopaminergic system as a hub in the extensive network of extra-SCN circadian oscillators and discuss the possible consequences of its daily entrainment for animal physiology and behaviour.

Effect of chronic stress on the relative level of dopamine receptor gene expression

Background. The regulation of the central dopaminergic system under the influence of chronic stress is disturbed, however, the dynamics of changes in the dopamine receptors expression in the periphery remains poorly understood.
 Aim. Evaluation of the different models of chronic stress influence on changes in the relative level of dopamine receptor gene expression in peripheral blood cells of rats during immobilization and intense physical activity.
 Material and methods. For 270 days on 88 Wistar rats, the study on the effect of different models of chronic stress on the change in the relative level of Drd15 genes expression was performed in four groups: the first control group; the second group was subjected to intensive physical activity in the Forced swimming with a load test (7-minute swimming with a load of 8% of body weight 2 times a week); the third group experienced daily 90-minute immobilization for 14 days; the fourth group had combined exposure of physical activity and immobilization. The relative level of dopamine receptor gene expression was determined by real-time polymerase chain reaction after 90, 180, and 270 days of the experiment in peripheral blood cells of the tail vein. The calculation of the relative level of gene expression was carried out based on the Livak method (2Ct); the assessment of the difference significance using a two-sample t-test for independent samples.
 Results. The analysis of the relative level of genes encoding D1-type dopamine receptors expression showed that a decrease in the Drd1 gene expression level after 90 days of the experiment was detected only in male rats from immobilization stress and control groups [RQ 0.35 (p=0.003) and 0.21 (p=0.002), respectively], while in males from other groups and females, the activity of this gene did not change significantly throughout the course of the experiment. The relative expression level of Drd5 gene changed only in female rats. In females subjected to intense physical activity, the level of this gene expression increased almost 4 times (RQ 3.82, p=0.005) 90 days after the start of the experiment, and in females of the control group, the transcriptional activity of the gene decreased 4 times after 180 days of the experiment (RQ 0.25, p=0.015). When assessing changes in the activity of genes encoding D2-type receptors for the Drd3 and Drd4 genes, a significant increase in the relative expression level was revealed in all experimental groups, both in males and females, on the 180th day of exposure to stress factors. At the same time, activation of both genes was occurred after 90 days in the control group only in females and persisted up to another 90 days, after which it returned to the initial level. Expression of the Drd2 gene wasn't detected in rat blood cells.
 Conclusion. The relative level of expression of D1- and D2-like receptor genes in rat peripheral blood cells depends on the type of chronic stress and has pronounced sexual dimorphism.

Downregulation of surface AMPA receptor expression in the striatum following prolonged social isolation, a role of mGlu5 receptors.

Major depressive disorder is a common and serious mood illness. The molecular mechanisms underlying the pathogenesis and symptomatology of depression are poorly understood at present. Multiple neurotransmitter systems are believed to be implicated in depression. Increasing evidence supports glutamatergic transmission as a critical element in depression and antidepressant activity. In this study, we investigated adaptive changes in expression of AMPA receptors in a key limbic reward structure, the striatum, in response to an anhedonic model of depression. Prolonged social isolation in adult rats caused anhedonic/depression- and anxiety-like behavior. In these depressed rats, surface levels of AMPA receptors, mainly GluA1 and GluA3 subunits, were reduced in the nucleus accumbens (NAc). Surface GluA1/A3 expression was also reduced in the caudate putamen (CPu) following chronic social isolation. No change was observed in expression of presynaptic synaptophysin, postsynaptic density-95, and dendritic microtubule-associated protein 2 in the striatum. Noticeably, chronic treatment with the metabotropic glutamate (mGlu) receptor 5 antagonist MTEP reversed the reduction of AMPA receptors in the NAc and CPu. MTEP also prevented depression- and anxiety-like behavior induced by social isolation. These data indicate that adulthood prolonged social isolation induces the adaptive downregulation of GluA1/A3-containing AMPA receptor expression in the limbic striatum. mGlu5 receptor activity is linked to this downregulation, and antagonism of mGlu5 receptors produces an antidepressant effect in this anhedonic model of depression.

Mesenchymal stem cells and their conditioned medium as potential therapeutic strategies in managing comorbid anxiety in rat sepsis induced by cecal ligation and puncture.

Objective(s):Sepsis-associated encephalopathy (SAE) is a common brain dysfunction following sepsis. Due to the beneficial effects of mesenchymal stem cells (MSCs) therapy on anxiety, an extreme and early manifestation of SAE, we hypothesized that MSCs-derived conditioned medium (CM) may be able to attenuate anxiety in cecal ligation and puncture (CLP)-induced sepsis.Materials and Methods:Rats were assigned into 4 groups: sham, CLP, MSC, and CM. All animals, except in the sham group, underwent the CLP procedure to induce sepsis. Two hours after sepsis induction, the rats in MSC and CM groups, received 1×106 MSCs and CM derived from the same number of cells, respectively. 48 hr after the treatments, anxiety-related behaviors were assessed, and brain and right hippocampal tissues were collected.Results:MSCs and CM enhanced the percentages of open arm entries and time spent in the open arms of the elevated plus-maze and the time spent in the light side of the light-dark box. MSCs and CM decreased the Evans blue content and decreased the IL-6 and TNF-α levels in the brain tissue samples. Reductions in the expression of 5-HT2A receptors and phosphorylation of ERK1/2 and an increase in the expression of 5-HT1A receptors in the hippocampal tissue samples were observed in the MSC and CM groups.Conclusion:MSCs and MSCs-derived CM attenuated anxiety-related behaviors to an equal extent by reducing inflammation, modifying 5-HT receptor expression changes, and inhibiting the ERK pathway. Therefore, MSCs-derived CM may be considered a promising therapy for comorbid anxiety in septic patients.

Lifelong changes of neurotransmitter receptor expression and debilitation of hippocampal synaptic plasticity following early postnatal blindness

In the weeks immediately after onset of sensory loss, extensive reorganization of both the cortex and hippocampus occurs. Two fundamental characteristics comprise widespread changes in the relative expression of GABA and glutamate receptors and debilitation of hippocampal synaptic plasticity. Here, we explored whether recovery from adaptive changes in the expression of plasticity-related neurotransmitter receptors and hippocampal synaptic plasticity occurs in the time-period of up to 12 months after onset of sensory loss. We compared receptor expression in CBA/J mice that develop hereditary blindness, with CBA/CaOlaHsd mice that have intact vision and no deficits in other sensory modalities throughout adulthood. GluN1-subunit expression was reduced and the GluN2A:GluN2B ratio was persistently altered in cortex and hippocampus. GABA-receptor expression was decreased and metabotropic glutamate receptor expression was altered. Hippocampal synaptic plasticity was persistently compromised in vivo. But although LTP in blind mice was chronically impaired throughout adulthood, a recovery of the early phase of LTP became apparent when the animals reached 12 months of age. These data show that cortical and hippocampal adaptation to early postnatal blindness progresses into advanced adulthood and is a process that compromises hippocampal function. A partial recovery of hippocampal synaptic plasticity emerges in advanced adulthood, however.

Maternal opioids decrease mu‐opioid receptor expression in the neonatal preBötzinger Complex

An understudied population in the opioid crisis are infants exposed to in utero opioids experiencing severe respiratory deficits. Our recent work demonstrated maternal opioids destabilized neonatal breathing, increased apneas, and blunted opioid‐induced respiratory frequency depression; however, the mechanism(s) underlying these neonatal breathing deficits remains unclear. We hypothesized deficits in neonatal breathing after maternal opioids are due to downregulation of mu‐opioid receptors on key cell types (e.g. neurons and astrocytes), essential for respiratory rhythm generation and modulation in the preBötzinger Complex. While opioid receptor expression is downregulated in the whole brain after maternal opioids, it remains unclear if opioid receptor expression changes in the preBötzinger Complex after in utero opioids to induce neonatal breathing deficits. To assess the typical developmental changes in opioid receptors in the preBötzinger Complex, mu‐opioid receptor expression was first assessed using immunohistochemistry and confocal microscopy on neonatal neurons and astrocytes in the preBötzinger Complex at three ages: the day of birth (postnatal day 0, P0) when neonates must begin robust rhythmic breathing, an early neonatal age (postnatal day 4, P4), and during a critical developmental window (postnatal day 11, P11). At all ages, mu‐opioid receptors were expressed on both neurons and astrocytes in the preBötzinger Complex; however, mu‐opioid receptor expression decreased from P0 (n=6) to P4 (n=6) and was lowest at P11 (n=6). Preliminary data after maternal opioids suggest mu‐opioid receptor expression was lower in preBötzinger Complexes at P0 (n=2) and P4 (n=2), but returned to control levels by P11 (n=2). These results are further supported by in vitro brainstem‐spinal cord recordings of the cervical rootlets (C4) from neonates after maternal opioids, demonstrating decreased opioid sensitivity of isolated respiratory control circuitry after maternal opioids (n=2). Results from these studies will contribute to our understanding of the mechanisms contributing to impaired neonatal breathing after maternal opioids and may lead to better treatments for infants suffering breathing deficits after maternal opioids.

DHT Induces Arterial Stiffening in Female Wild Type Mice

Testosterone is the predominant sex hormone in men and is increased in women with polycystic ovarian syndrome (PCOS). PCOS patients also experience an increased risk for cardiovascular diseases including hypertension and arterial stiffness. Our previous work showed significant downregulation of the G protein‐coupled estrogen receptor (GPER) as well as the nuclear estrogen receptor ERα in cultured vascular smooth muscle cells in response to the testosterone metabolite dihydrotestosterone (DHT), which cannot be aromatized to estrogen. Therefore, we hypothesized that DHT may induce arterial stiffness in female mice via downregulation of vascular estrogen receptors. Two groups of female wildtype mice were treated with DHT via silastic capsule for 4 weeks; one group received a dose designed to mimic DHT levels in men while the other group received twice this dose. While post‐treatment systolic blood pressure was not different between the control, single dose, and double dose groups (111 ± 10, 106 ± 3, 110 ± 7, n=8 per group; P=0.22), intracarotid vascular stiffness measured via pulse wave velocity showed a more than two‐fold increase in single dose (4.3 ± 0.8 m/s) and double dose (4.8 ± 1.0 m/s) DHT treated mice when compared with non‐treated controls (1.9 ± 0.3 m/s; P<0.0001). These findings suggest that high testosterone levels in females may promote arterial stiffening and cardiovascular damage independent of changes in blood pressure. Future studies will include histological analysis of aortic sections using Masson’s trichrome to assess changes in the extracellular matrix as well as droplet digital polymerase chain reaction to assess changes in estrogen receptor expression. In conclusion, these findings of arterial stiffening in response to increased DHT levels in females are important for not only PCOS patients but also women using testosterone for fitness, gender transitioning, or reduced libido.

The Role of Meningeal Immune Cells in the Efficacy of CGRP-based Migraine Therapies

Migraine is a painful, chronic neurological disorder that represents the second most disabling illness worldwide. There are two prominent mechanisms thought to contribute to migraine pain: immune cell activation and calcitonin gene-related peptide (CGRP) signaling. Cortical spreading depression (CSD), thought to underlie the aura that precedes migraine attack in a subpopulation of migraineurs, is associated with increased meningeal macrophage activation. Macrophages can activate meningeal primary afferent neurons and contribute to migraine pain through release of pro-inflammatory cytokines. CSD also increases CGRP synthesis and release, and single-cell RNA sequencing data support the expression of CGRP receptor subunit mRNA in mouse meningeal immune cells. The efficacy of CGRP receptor antagonists as migraine therapies support the critical role of CGRP in the development of migraine pain. However, the mechanism of action of these drugs is incompletely understood. Given the effect of CSD on immune cell activation and CGRP release, the presence of CGRP receptor on immune cells, and the use of CGRP receptor antagonists to treat migraine, the following study uses anatomical, functional, and behavioral approaches to assess the role of meningeal immune cells in the efficacy of CGRP-based migraine therapies. This is done in the context of a minimally invasive model of migraine with aura (optogenetic spreading depression, OSD). I plan to characterize OSD-dependent changes in CGRP receptor expression in meningeal immune cells, assess the ability of CGRP receptor antagonists to prevent OSD-induced changes in macrophage activation, and determine the contribution of macrophage CGRP receptors to OSD-induced pain. The outcomes of these experiments may not only reveal a mechanism underlying the therapeutic efficacy of CGRP antagonists but potentially novel targets and mechanisms to improve existing migraine therapies. Grant support from R01 NS122784.

Age‐ and sex‐specific differences in activation of the cardiac adrenergic cascade

Heart failure (HF) is a costly and devastating public health problem, which differentially impacts male and female patients of all ages across the life‐course. The adrenergic system is dysregulated with HF and is the target of front‐line therapeutics. However, the majority of the work aimed at elucidating the adrenergic signaling response has been conducted in adult males. Consequently, mechanisms of adrenergic signaling that differ by sex and age remain unclear. Here, we set out to identify mechanistic differences in the adrenergic pathway in response to isoproterenol (ISO) stimulation in juvenile (4 weeks), adult (4‐6 months) and aged (18 months) C57Bl6 mice of both sexes. ISO was delivered by mini‐osmotic pump (30 mg/kg/day) for one week. We assessed changes in β‐adrenergic receptor (β‐AR) expression, protein kinase activity, and myofilament protein phosphorylation and function. β1‐AR and β2‐AR protein expression were significantly lower in response to ISO in juvenile males with no difference in expression in juvenile females. In contrast, ISO stimulation in aged males significantly upregulated β1‐AR and β2‐AR protein expression without changes in expression in aged females. Analysis of sarcomeric proteins indicated that phosphorylation of troponin varied in an age‐ and sex‐dependent manner. Phosphorylation of the inhibitory subunit of troponin (TnI) at Ser23/24 was upregulated in response to ISO in females of all ages with no differences in males. These changes in phosphorylation decreased myofilament Ca2+ sensitivity in female hearts in response to ISO. Together, this work suggests that male hearts responded robustly to ISO stimulation as evident by activation throughout the adrenergic cascade while female hearts demonstrated activation of downstream mechanisms at the level of the sarcomere. Ongoing experiments will characterize calcium handling and myofilament mechanics to mechanistically link upstream signals to downstream changes in contractility. These data demonstrate that age and sex are significant biological factors which contribute to adrenergic‐mediated changes in cardiac performance. Understanding these differences is critical for successful therapeutic intervention within the adrenergic signaling cascade for male and female patients of all ages.

VGLUT3 Ablation Differentially Modulates Glutamate Receptor Densities in Mouse Brain.

Type 3 vesicular glutamate transporter (VGLUT3) represents a unique modulator of glutamate release from both nonglutamatergic and glutamatergic varicosities within the brain. Despite its limited abundance, VGLUT3 is vital for the regulation of glutamate signaling and, therefore, modulates the activity of various brain microcircuits. However, little is known about how glutamate receptors are regulated by VGLUT3 across different brain regions. Here, we used VGLUT3 constitutive knock-out (VGLUT3–/–) mice and explored how VGLUT3 deletion influences total and cell surface expression of different ionotropic and metabotropic glutamate receptors. VGLUT3 deletion upregulated the overall expression of metabotropic glutamate receptors mGluR5 and mGluR2/3 in the cerebral cortex. In contrast, no change in the total expression of ionotropic NMDAR glutamate receptors were observed in the cerebral cortex of VGLUT3–/– mice. We noted significant reduction in cell surface levels of mGluR5, NMDAR2A, NMDAR2B, as well as reductions in dopaminergic D1 receptors and muscarinic M1 acetylcholine receptors in the hippocampus of VGLUT3–/– mice. Furthermore, mGluR2/3 total expression and mGluR5 cell surface levels were elevated in the striatum of VGLUT3–/– mice. Last, AMPAR subunit GluA1 was significantly upregulated throughout cortical, hippocampal, and striatal brain regions of VGLUT3–/– mice. Together, these findings complement and further support the evidence that VGLUT3 dynamically regulates glutamate receptor densities in several brain regions. These results suggest that VGLUT3 may play an intricate role in shaping glutamatergic signaling and plasticity in several brain areas.

Aging modifies receptor expression but not muscular contractile response to angiotensin II in rat jejunum

The involvement of renin-angiotensin system in the modulation of gut motility and age-related changes in mRNA expression of angiotensin (Ang II) receptors (ATR) are well accepted. We aimed to characterize, in vitro, the contractile responses induced by Ang II, in jejunum from young (3–6 weeks old) and old rats (≥ 1 year old), to evaluate possible functional differences associated to changes in receptor expression. Mechanical responses to Ang II were examined in vitro as changes in isometric tension. ATR expression was assessed by qRT-PCR. Ang II induced a contractile effect, antagonized by losartan, AT1R antagonist, and increased by PD123319, AT2R antagonist, as well by neural blocker ω-conotoxin and by nitric oxide (NO) synthase inhibitor. No difference in the response was observed between young and old groups. AT1 receptor-mediated contractile response was decreased by U-73122, phospholipase C (PLC) inhibitor; or 2-aminoethoxy-diphenylborate (2-APB), inositol triphosphate (IP3) receptor inhibitor; or nifedipine, l-type calcium channel blocker. Age-related changes in the expression of both AT1 receptor subtypes, AT1a and AT1b, and of AT2 receptors were detected. In conclusion, Ang II modulates the spontaneous contractility of rat jejunum via postjunctional AT1 receptors, involving Ca2+ mobilization from intracellular stores, via PLC/IP3 pathway, and Ca2+ influx from extracellular space, via l-type channels. Prejunctional AT2 receptors would counteract AT1 receptor effects, via NO synthesis. The observed age-related differences in the expression of all AT receptor subtypes are not reflected in the muscular contractile response to Ang II.

The Scaffold Protein PICK1 as a Target in Chronic Pain.

Well-tolerated and effective drugs for treating chronic pain conditions are urgently needed. Most chronic pain patients are not effectively relieved from their pain and suffer from debilitating drug side effects. This has not only drastic negative consequences for the patients’ quality of life, but also constitute an enormous burden on society. It is therefore of great interest to explore new potent targets for effective pain treatment with fewer side effects and without addiction liability. A critical component of chronic pain conditions is central sensitization, which involves the reorganization and strengthening of synaptic transmission within nociceptive pathways. Such changes are considered as maladaptive and depend on changes in the surface expression and signaling of AMPA-type glutamate receptors (AMPARs). The PDZ-domain scaffold protein PICK1 binds the AMPARs and has been suggested to play a key role in these maladaptive changes. In the present paper, we review the regulation of AMPARs by PICK1 and its relation to pain pathology. Moreover, we highlight other pain-relevant PICK1 interactions, and we evaluate various compounds that target PICK1 and have been successfully tested in pain models. Finally, we evaluate the potential on-target side effects of interfering with the action of PICK1 action in CNS and beyond. We conclude that PICK1 constitutes a valid drug target for the treatment of inflammatory and neuropathic pain conditions without the side effects and abuse liability associated with current pain medication.

Changes in P2Y6 receptor-mediated vasoreactivity following focal and global ischemia.

Ischemia, both in the form of focal thromboembolic stroke and following subarachnoid hemorrhage (SAH), causes upregulation of vasoconstrictive receptor systems within the cerebral vasculature. Descriptions regarding changes in purinergic signaling following ischemia are lacking, especially when the importance of purinergic signaling in regulating vascular tone is taken into consideration. This prompted us to evaluate changes in P2Y6‐mediated vasomotor reactivity in two different stroke models in rat. We used wire myography to measure changes in cerebral vasoreactivity to the P2Y6 agonist UDP‐β‐S following either experimental SAH or transient middle cerebral artery occlusion. Changes in receptor localization or receptor expression were evaluated using immunohistochemistry and quantitative flow cytometry. Transient middle cerebral artery occlusion caused an increase in Emax when compared to sham (233.6 [206.1–258.5]% vs. 161.1 [147.1–242.6]%, p = 0.0365). No such change was seen following SAH. Both stroke models were associated with increased levels of P2Y6 receptor expression in the vascular smooth muscle cells (90.94 [86.99–99.15]% and 93.79 [89.96–96.39]% vs. 80.31 [70.80–80.86]%, p = 0.021) and p = 0.039 respectively. There was no change in receptor localization in either of the stroke models. Based on these findings, we conclude that focal ischemic stroke increases vascular sensitivity to UDP‐β‐S by upregulating P2Y6 receptors on vascular smooth muscle cells while experimental SAH did not induce changes in vasoreactivity in spite of increased P2Y6 receptor expression.

Robust In Vitro Pharmacology of Tmod, a Synthetic Dual-Signal Integrator for Cancer Cell Therapy.

Progress toward improved solid-tumor treatment has long been hindered by the lack of truly tumor-specific targets. We have developed an approach to T cell therapy based on a dual-receptor system called Tmod™ that addresses this problem. The Tmod system exploits one of the few common genetic differences between tumor and normal cells: loss of heterozygosity (LOH). It utilizes the basic mechanistic logic that evolved in early vertebrates to mediate self vs. non-self discrimination, where an activation stimulus is blocked by self-ligands. Tmod constructs employ a chimeric antigen receptor (CAR) or T cell receptor (TCR) as activator component and a modified LIR-1 inhibitory receptor (blocker) to achieve high selectivity based on expression of the blocker antigen (Ag). Here we explore the in vitro pharmacology of a blocker directed at the HLA-A*02 Ag paired with either a mesothelin CAR or an HLA-A*11-restricted KRAS peptide TCR. While more sensitive to receptor expression changes on effector cells, we show that Tmod response is well-buffered against variations in Ag levels on target cells. In addition, the data reveal at least two distinguishable pharmacologic mechanisms of Tmod blocker function: (1) reducing activator sensitivity and (2) decreasing activation magnitude.

Epidermal growth factor-targeted fluorescence is unaffected by standard neoadjuvant therapies in human sarcomas.

Curative surgery for other many cancers requires that the tumor be removed with a zone of normal tissue surrounding the tumor with 'negative' margins. Sarcomas, cancers of the bones, muscles, and fat, require WLE for cure. Unfortunately, 'positive' margins occur in 20-25% of sarcoma surgeries, associated with cancer recurrence and reduced survival. Our group successfully tested a small-molecule fluorophore (ABY-029) in sarcomas that targets the epidermal growth factor receptor. We sought to evaluate human sarcoma xenografts for epidermal growth factor receptor expression and binding of ABY-029 with and without exposure to standard presurgical chemotherapy and radiation. We inoculated groups of 24 NSG mice with five cell lines (120 mice total). Eight mice from each cell line received: 1) radiation alone; 2) chemotherapy alone; or 3) chemotherapy and radiation. We administered ABY-029 2-4 hours before surgery. Tumor and biopsy portions of background tissues were removed. All tissues were imaged on a LI-COR Odyssey and processed in pathology. There were no significant reductions in epidermal growth factor receptor expression or in ABY-029-mediated fluorescence in tumors exposed to chemotherapy, radiation, or both. fluorescence-guided surgery demonstrates strong promise to improve curative surgical cancer care, particularly for sarcomas where the positive margin rate is substantial. Fluorophore performance must be evaluated under circumstances that duplicate accurately the biological milieu relevant to a particular cancer. This work shows that human sarcoma xenografts subjected to standard therapies do not demonstrate a change in epidermal growth factor receptor expression or in epidermal growth factor receptor-targeted fluorescence, thereby indicating that epidermal growth factor receptor-targeted fluorescence-guided surgery should be feasible under normal therapeutic conditions in the clinic.

Cervical spinal hemisection alters phrenic motor neuron glutamatergic mRNA receptor expression

Upper cervical spinal cord injuries (SCI) disrupt descending inputs to phrenic motor neurons (PhMNs), impairing respiratory function. Unilateral spinal hemisection at C2 (C2SH) results in loss of ipsilateral rhythmic diaphragm muscle (DIAm) EMG activity associated with lower force behaviors accomplished by recruitment of smaller PhMNs in rats. Activity during higher force, non-ventilatory behaviors that recruit larger PhMNs is minimally impaired following C2SH. We previously showed neuroplasticity in glutamatergic receptor expression in PhMN post-C2SH with changes in NMDA receptor expression reflecting functional recovery over time. We hypothesize that C2SH-induced changes in glutamatergic receptor (AMPA and NMDA) mRNA expression in PhMNs vary with motor neuron size, with more pronounced changes in smaller PhMNs. Retrogradely-labelled PhMNs were classified in tertiles according to somal surface area and mRNA expression was measured using single-cell, multiplex fluorescence in situ hybridization. Ipsilateral to C2SH, a pronounced reduction in NMDA mRNA expression in PhMNs was evident at 3 days post-injury with similar impact on PhMNs in the lower size tertile (~68% reduction) and upper tertile (~60%); by 21 days, there was near complete restoration of NMDA receptor mRNA expression across all PhMNs. There were no changes in NMDA mRNA expression contralateral to C2SH. There were no changes in AMPA mRNA expression at PhMNs on either side of the spinal cord or at any time-point post-C2SH. In summary, following C2SH there is ipsilateral reduction in PhMN NMDA mRNA expression at 3 days that is not limited to smaller PhMN recruited in the generation of lower force ventilatory behaviors. The recovery of NMDA mRNA expression by 21 days post-C2SH is consistent with evidence of spontaneous recovery of ipsilateral DIAm activity at this timepoint. These findings suggest a possible role for NMDA receptor mediated glutamatergic signaling in mechanisms supporting postsynaptic neuroplasticity at the PhMN pool and recovery of DIAm activity after cervical SCI.

A20 INTESTINAL SMOOTH MUSCLE AND FIBROSTENOSIS: TARGETING NR4A1 TO MODULATE PROLIFERATIVE SIGNALLING

BackgroundFibrostenotic Crohn’s Disease (CD), presenting with intestinal fibrosis and stricture formation, has a substantial impact on patient quality of life. Given our poor understanding of its etiology, we lack viable preventative and therapeutic methods. While much focus has been on the fibrotic component, recent studies have implicated the role of intestinal smooth muscle cell (SMC) hyperplasia/hypertrophy in stricture formation. These data suggest targeting SMC proliferation may provide benefit for fibrostenotic CD patients. NR4A1 (nuclear receptor subfamily 4 group A member 1) is an orphan nuclear receptor that has shown to regulate inflammation in experimental models of colitis and dampen SMC proliferation and fibrotic signalling in intestinal and non-intestinal systems. Thus, we sought to characterize the role of NR4A1 in regulating proliferative signalling in intestinal SMCs to determine whether it could be a therapeutic target for fibrostenotic CD.AimsTo determine how NR4A1 regulates intestinal SMC proliferative responses to mitogenic signalling.MethodsPrimary intestinal SMCs were isolated from the colonic tissue of Nr4a1+/+ and Nr4a1-/- mice. A commercially available human colonic SMC line was also used. EdU incorporation assays were used to quantify the relative proliferation of Nr4a1+/+ and Nr4a1-/- SMCs in their basal or stimulated state (with platelet-derived growth factor (PDGF)-BB). In addition, NR4A1 was activated using selective agonists, cytosporone-B (Csn-B) and 6-mercaptopurine (6-MP). Differences in PDGF-BB-induced intracellular signalling was determined using western blotting of phosphorylated proteins after stimulation. Quantification of PDGF receptor transcript expression in Nr4a1+/+ and Nr4a1-/- SMCs was done using qPCR. Finally, immunofluorescence was used to determine the localization of NR4A1 when stimulated with Csn-B, 6-MP, and/or PDGF-BB.ResultsThe proliferation assays showed that Nr4a1-/- SMCs exhibit greater proliferation at baseline and when stimulated with PDGF-BB, compared to Nr4a1+/+ SMCs. However, this was not associated with any differences in the intracellular signalling directly downstream of PDGF receptor activation. Specifically, there were no differences in the intensity and temporal characteristics of Akt- and Erk1/2-phosphorylation between Nr4a1+/+ and Nr4a1-/- SMCs. Interestingly, Nr4a1-/- SMCs had less expression of Pdgfrb, the gene encoding PDGF receptor beta, when compared to Nr4a1+/+ SMCs. However, no changes in receptor expression were observed when SMCs were stimulated with Csn-B and 6-MP.ConclusionsWe show that NR4A1 regulates basal and PDGF-BB-induced SMC proliferation, without directly altering the intracellular signalling cascades induced by receptor activation. Our data supports NR4A1 as a target to control the aberrant SMC proliferation that contributes to fibrostenosis.Funding AgenciesCIHR

Quality Control for Single Cell Imaging Analytics Using Endocrine Disruptor-Induced Changes in Estrogen Receptor Expression.

Background:Diverse toxicants and mixtures that affect hormone responsive cells [endocrine disrupting chemicals (EDCs)] are highly pervasive in the environment and are directly linked to human disease. They often target the nuclear receptor family of transcription factors modulating their levels and activity. Many high-throughput assays have been developed to query such toxicants; however, single-cell analysis of EDC effects on endogenous receptors has been missing, in part due to the lack of quality control metrics to reproducibly measure cell-to-cell variability in responses.Objective:We began by developing single-cell imaging and informatic workflows to query whether the single cell distribution of the estrogen (ER), used as a model system, can be used to measure effects of EDCs in a sensitive and reproducible manner.Methods:We used high-throughput microscopy, coupled with image analytics to measure changes in single cell ER nuclear levels on treatment with toxicants, over a large number of biological and technical replicates.Results:We developed a two-tiered quality control pipeline for single cell analysis and tested it against a large set of biological replicates, and toxicants from the EPA and Agency for Toxic Substances and Disease Registry lists. We also identified a subset of potentially novel EDCs that were active only on the endogenous ER level and activity as measured by single molecule RNA fluorescence in situ hybridization (RNA FISH).Discussion:We demonstrated that the distribution of ER levels per cell, and the changes upon chemical challenges were remarkably stable features; and importantly, these features could be used for quality control and identification of endocrine disruptor toxicants with high sensitivity. When coupled with orthogonal assays, ER single cell distribution is a valuable resource for high-throughput screening of environmental toxicants. https://doi.org/10.1289/EHP9297

GABA-B receptors enhance GABA-A receptor currents by modulation of membrane trafficking in dentate gyrus granule cells

Activation of postsynaptic GABA-B receptors enhances tonic inhibition mediated by high-affinity extrasynaptic GABAA receptors in dentate gyrus granule cells (DGGCs), thalamocortical neurons, and cerebellar granule cells. We investigated the mechanism(s) of GABA current modulation by GABAB receptors in DGGCs using a combination of electrophysiological and biochemical approaches. In acute hippocampal brain slices the GABAB receptor agonist baclofen increased GABA-evoked currents in ∼2/3rds of DGGCs, significantly increasing GABAA currents by 41% on average. Nonstationary noise analysis was performed to estimate the effects of baclofen on single channel conductance, mean open time, and channel number; these estimates suggest that GABAB receptor activation increases receptor number but does not modify single channel properties of GABAA receptors. To directly assess baclofen-induced changes in plasma membrane expression of GABAA receptors, biotinylated western blots were performed. Treatment of hippocampal slices with baclofen significantly increased the surface expression of GABAA receptor subunits (both δ and γ2 subunits) and this effect was inhibited by the GABAB receptor antagonist CGP55845. These data indicate that changes in membrane trafficking and increased number of GABAA receptors in plasma membrane contribute to the enhancement of GABA currents produced by GABAB receptor activation in DGGCs.