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PDL1 is a protein that induces immunosuppression by binding to PD1 expressed on immune cells. In line with historical studies, we found that membrane-bound PD1 expression was largely restricted to immune cells; PD1 was not detectable at either the mRNA or protein level in peripheral neurons using single neuron qPCR, immunolabeling and flow cytometry. However, we observed widespread expression of PDL1 in both sensory and sympathetic neurons that could have important implications for patients receiving immunotherapies targeting this pathway that include unexpected autonomic and sensory related effects. While signaling pathways downstream of PD1 are well established, little to no information is available regarding the intracellular signaling downstream of membrane-bound PDL1 (also known as reverse signaling). Here, we administered soluble PD1 to engage neuronally expressed PDL1 and found that PD1 significantly reduced nocifensive behaviors evoked by algogenic capsaicin. We used calcium imaging to examine the underlying neural mechanism of this reduction and found that exogenous PD1 diminished TRPV1-dependent calcium transients in dissociated sensory neurons. Furthermore, we observed a reduction in membrane expression of TRPV1 following administration of PD1. Exogenous PD1 had no effect on pain-related behaviors in sensory neuron specific PDL1 knockout mice. These data indicate that neuronal PDL1 activation is sufficient to modulate sensitivity to noxious stimuli and as such, may be an important homeostatic mechanism for regulating acute nociception.

Digestive functions of the colon depend on sensory-motor reflexes in the enteric nervous system (ENS), initiated by intrinsic primary afferent neurons (IPANs). IPAN terminals project to the mucosal layer of the colon, allowing communication with epithelial cells comprising the colon lining. The chemical nature and functional significance of this epithelial-neural communication in regard to secretion and colon motility are of high interest. Colon epithelial cells can produce and release neuroactive substances such as ATP and 5-hydroxytryptamine (5-HT), which can activate receptors on adjacent nerve fibers, including IPAN subtypes. In this study, we examined if stimulation of epithelial cells alone is sufficient to activate neural circuits that control colon motility. Optogenetics and calcium imaging were used in ex vivo preparations of the mouse colon to selectively stimulate the colon epithelium, measure changes in motility, and record activity of neurons within the myenteric plexus. Light-mediated activation of epithelial cells lining the distal, but not proximal, colon caused local contractions and increased the rate of colonic migrating motor complexes. Epithelial-evoked local contractions in the distal colon were reduced by both ATP and 5-HT receptor antagonists. Our findings indicate that colon epithelial cells likely use purinergic and serotonergic signaling to initiate activity in myenteric neurons, produce local contractions, and facilitate large-scale coordination of ENS activity responsible for whole colon motility patterns. NEW & NOTEWORTHY Using an all-optical approach to measure real-time cell-to-cell communication responsible for colon functions, we show that selective optogenetic stimulation of distal colon epithelium produced activity in myenteric neurons, as measured with red genetically encoded calcium indicators. The epithelial-induced neural response led to local contractions, mediated by both purinergic and serotonergic signaling, and facilitated colonic motor complexes that propagate from proximal to distal colon.

Visceral pain is a prevalent symptom of inflammatory bowel disease (IBD) that can be difficult to treat. Pain and hypersensitivity are mediated by extrinsic primary afferent neurons (ExPANs) that innervate the colon. Recent studies indicate that the colon epithelium contributes to initiating ExPAN firing and nociceptive responses. Based on these findings we hypothesized that the epithelium contributes to inflammation-induced hypersensitivity. A key prediction of this hypothesis is that inhibition of the epithelium would attenuate nociceptive signaling and inflammatory hypersensitivity. To test this hypothesis, the inhibitory yellow light activated protein archaerhodopsin was targeted to the intestinal epithelium (villin-Arch) or the ExPANs (TRPV1-Arch) that innervate the colon. Visceral sensitivity was assessed by measuring the visceromotor response (VMR) to colorectal distension (CRD), with and without yellow light illumination of the colon lumen. Inhibition of the colon epithelium in healthy villin-Arch mice significantly diminished the CRD-induced VMR. Direct inhibition of ExPANs during CRD using TRPV1-Arch mice showed that ExPAN and epithelial inhibition were similarly effective in reducing the VMR to CRD. We then investigated the effect of epithelial and ExPAN inhibition in the dextran sulfate sodium (DSS) model of inflammatory bowel disease (IBD). Inhibition of the colon epithelium significantly decreased DSS-induced hypersensitivity and was comparable to inhibition of ExPANS. Together these results reveal the potential of targeting the colon epithelium for treatment of pain.

BACKGROUND & AIMS: The colon is innervated by intrinsic and extrinsic neurons that coordinate functions necessary for digestive health. Sympathetic input suppresses colon motility by acting on intrinsic myenteric neurons, but the extent of sympathetic-induced changes on large-scale network activity in myenteric circuits has not been determined. Compounding the complexity of sympathetic function, there is evidence that sympathetic transmitters can regulate activity in non-neuronal cells (such as enteric glia and innate immune cells). METHODS: We performed anatomical tracing, immunohistochemistry, optogenetic (GCaMP calcium imaging, channelrhodopsin), and colon motility studies in mice and single cell RNA sequencing in human colon to investigate how sympathetic postganglionic neurons modulate colon function. RESULTS: Individual neurons in each sympathetic prevertebral ganglion innervated the proximal or distal colon, with processes closely opposed to multiple cell types. Calcium imaging in semi-intact mouse colon preparations revealed changes in spontaneous and evoked neural activity, as well as activation of non-neuronal cells, induced by sympathetic nerve stimulation. The overall pattern of response to sympathetic stimulation was unique to the proximal or distal colon. Region-specific changes in cellular activity correlated with motility patterns produced by electrical and optogenetic stimulation of sympathetic pathways. Pharmacology experiments (mouse) and RNA sequencing (human) indicated that appropriate receptors were expressed on different cell types to account for the responses to sympathetic stimulation. Regional differences in expression of alpha-1 adrenoceptors in human colon emphasize the translational relevance of our mouse findings. CONCLUSIONS: Sympathetic neurons differentially regulate activity of neurons and non-neuronal cells in proximal and distal colon to promote distinct changes in motility patterns, likely reflecting the distinct roles played by these two regions.

Peptidylarginine deiminase (PAD) enzymes convert histone tail arginine residues to citrulline resulting in chromatin decondensation. Our previous work found that PAD isoforms are expressed in female reproductive tissues in an estrous cycle-dependent fashion, but their role in the anterior pituitary gland is unknown. Thus, we investigated PAD expression and function in gonadotrope cells. The gonadotrope-derived LβT2 cell line strongly expresses PAD2 at the protein level compared with other PAD isoforms. Consistent with this, PAD2 protein expression is highest during the estrous phase of the estrous cycle and colocalizes with the LH β-subunit in the mouse pituitary. Using the GnRH agonist buserelin (GnRHa), studies in LβT2 and mouse primary gonadotrope cells revealed that 30 minutes of stimulation caused distinct puncta of PAD2 to localize in the nucleus. Once in the nucleus, GnRHa stimulated PAD2 citrullinates histone H3 tail arginine residues at sites 2, 8, and 17 within 30 minutes; however, this effect and PAD2 nuclear localization was blunted by incubation of the cells with the pan-PAD inhibitor, biphenyl-benzimidazole-Cl-amidine. Given that PAD2 citrullinates histones in gonadotropes, we next analyzed the functional consequence of PAD2 inhibition on gene expression. Our results show that GnRHa stimulates an increase in LHβ and FSHβ mRNA and that this response is significantly reduced in the presence of the PAD inhibitor biphenyl-benzimidazole-Cl-amidine. Overall, our data suggest that GnRHa stimulates PAD2-catalyzed histone citrullination in gonadotropes to epigenetically regulate gonadotropin gene expression.

Gonadotrope cells of the anterior pituitary are characterized by their ability to mount a cyclical pattern of gonadotropin secretion to regulate gonadal function and fertility. Recent in vitro and in vivo evidence suggests that gonadotropes exhibit dramatic remodeling of the actin cytoskeleton following gonadotropin-releasing hormone (GnRH) exposure. GnRH engagement of actin is critical for gonadotrope function on multiple levels. First, GnRH-induced cell movements lead to spatial repositioning of the in vivo gonadotrope network toward vascular endothelium, presumably to access the bloodstream for effective hormone release. Interestingly, these plasticity changes can be modified depending on the physiological status of the organism. Additionally, GnRH-induced actin assembly appears to be fundamental to gonadotrope signaling at the level of extracellular signal-regulated kinase (ERK) activation, which is a well-known regulator of luteinizing hormone (LH) β-subunit synthesis. Last, GnRH-induced cell membrane projections are capable of concentrating LHβ-containing vesicles and disruption of the actin cytoskeleton reduces LH secretion. Taken together, gonadotrope network positioning and LH synthesis and secretion are linked to GnRH engagement of the actin cytoskeleton. In this review, we will cover the dynamics and organization of the in vivo gonadotrope cell network and the mechanisms of GnRH-induced actin-remodeling events important in ERK activation and subsequently hormone secretion.

Abdominal pain and bowel dysmotility are hallmark symptoms of functional bowel disorders, yet it is unclear how pain and dysmotility are correlated mechanistically. Extrinsic colon afferents (EPANs) provide sensory input to the CNS, and visceral pain is thought to be due to hypersensitivity of these afferents. The enteric nervous system (ENS) acts autonomously to control motility reflexes, but extrinsic nerve pathways coordinate activity between distant regions of the GI tract, and importantly, allow the central nervous system (CNS) to regulate GI functioning. To test whether EPANs can directly influence ENS activity, we used an ex vivo colon-pelvic nerve-L6 DRG preparation, in which the roots were cut from the spinal cord, that allowed selective activation of EPANs via dorsal root stimulation and simultaneous calcium imaging (GCaMP6s) of myenteric neurons in the colon. Electrical stimulation of the dorsal root activated the majority of L6 DRG neurons, but produced no GCaMP responses in myenteric neurons of the colon in any of the visual fields tested (n=8). By contrast, ventral root stimulation led to GCaMP responses in ∼20% of myenteric neurons, followed by smooth muscle contraction, as indicated by movement of the imaging field. Interestingly, when the spinal cord was left intact, dorsal root stimulation produced responses in 12% of myenteric neurons (n=5), suggesting the presence of a sensory-parasympathetic spinal reflex. To elucidate this reflex in vivo, we used optogenetic stimulation to produce visceromotor responses and then processed spinal cord tissue for the immediate early gene, c-fos. There were significantly greater numbers of parasympathetic preganglionic neurons (PPNs) with c-fos staining in lumbosacral spinal segments from TRPV1-ChR2 mice (n=3) compared to controls (n=3), and TRPV1 central terminals were observed in close apposition to PPN. Overall, our data suggests that colon afferents influence ENS activity and colon motility by engaging parasympathetic pathways through a spinal reflex.

The mammalian target of rapamycin (mTOR) assembles into two different multi-protein complexes, mTORC1 and mTORC2. The mTORC2 complex is distinct due to the unique expression of the specific core regulatory protein Rictor (rapamycin-insensitive companion of mTOR). mTORC2 has been implicated in regulating actin cytoskeletal reorganization but its role in gonadotrope function is unknown. Using the gonadotrope-derived LβT2 cell line, we find that the GnRH agonist buserelin (GnRHa) phosphorylates both mTOR and Rictor. Interestingly, inhibition of mTORC2 blunts GnRHa-induced cyto-architectural rearrangements. Coincident with blunting of actin reorganization, inhibition of mTORC2 also attenuates GnRHa-mediated activation of both protein kinase C (PKC) and extracellular signal regulated kinase (ERK). Collectively, our data suggests that GnRHa-mediated mTORC2 activation is important in facilitating actin reorganization events critical for initiating PKC activity and subsequent ERK phosphorylation in the gonadotrope-derived LβT2 cell line.

Gonadotrophin-releasing hormone (GnRH) agonists are used to treat gonadal steroid-dependent disorders in humans and to contracept animals. These agonists are considered to work by desensitising gonadotrophs to GnRH, thereby suppressing follicle-stimulating hormone (FSH) and luteinising hormone (LH) secretion. It is not known whether changes occur in the cellular composition of the pituitary gland after chronic GnRH agonist exposure. Adult male Sprague-Dawley rats were treated with a sham, deslorelin, or deslorelin plus testosterone implant for 41.0 ± 0.6 days. In a second experiment, rats were castrated and treated with deslorelin and/or testosterone. Pituitary sections were labelled immunocytochemically for FSHβ and LHβ, or gonadotrophin α subunit (αGSU). Deslorelin suppressed testis weight by two-thirds and reduced plasma FSH and LH in intact rats. Deslorelin decreased the percentage of gonadotrophs, although the effect was specific to the FSHβ-immunoreactive (-ir) cells. Testosterone did not reverse the deslorelin-induced reduction in the overall gonadotroph population. However, in the presence of testosterone, the proportion of gonadotrophs that was FSHβ-ir increased in the remaining gonadotrophs. There was no effect of treatment on the total LHβ-ir cell population, although the loss of FSHβ in bi-hormonal cells increased the proportion of mono-hormonal LHβ-ir gonadotrophs. The castration-induced plasma LH and FSH increases were suppressed by deslorelin, testosterone or both. Castration increased both LH-ir and FSH-ir without increasing the overall gonadotroph population, thus increasing the proportion of bi-hormonal cells. Deslorelin suppressed these increases. Testosterone increased FSH-ir in deslorelin-treated castrate rats. Deslorelin did not affect αGSU immunoreactivity, suggesting that the gonadotroph population per se is not eliminated by deslorelin, although the ability of gonadotrophs to synthesise FSHβ is compromised. We hypothesise that the FSH dominant suppression may be central to the long-term contraceptive efficacy of deslorelin in the male.

We have shown that GnRH-mediated engagement of the cytoskeleton induces cell movement and is necessary for ERK activation. It also has previously been established that a dominant negative form of the mechano-GTPase dynamin (K44A) attenuates GnRH activation of ERK. At present, it is not clear at what level these cellular events might be linked. To explore this, we used live cell imaging in the gonadotrope-derived αT3–1 cell line to determine that dynamin-green fluorescent protein accumulated in GnRH-induced lamellipodia and plasma membrane protrusions. Coincident with translocation of dynamin-green fluorescent protein to the plasma membrane, we demonstrated that dynamin colocalizes with the actin cytoskeleton and the actin binding protein, cortactin at the leading edge of the plasma membrane. We next wanted to assess the physiological significance of these findings by inhibiting dynamin GTPase activity using dynasore. We find that dynasore suppresses activation of ERK, but not c-Jun N-terminal kinase, after exposure to GnRH agonist. Furthermore, exposure of αT3–1 cells to dynasore inhibited GnRH-induced cyto-architectural rearrangements. Recently it has been discovered that GnRH induced Ca2+ influx via the L-type Ca2+ channels requires an intact cytoskeleton to mediate ERK phosphorylation. Interestingly, not only does dynasore attenuate GnRH-mediated actin reorganization, it also suppresses Ca2+ influx through L-type Ca2+ channels visualized in living cells using total internal reflection fluorescence microscopy. Collectively, our data suggest that GnRH-induced membrane remodeling events are mediated in part by the association of dynamin and cortactin engaging the actin cytoskeleton, which then regulates Ca2+ influx via L-type channels to facilitate ERK phosphorylation.

Gonadotropin-releasing hormone (GnRH) agonists are routinely used to suppress the reproductive axis of many mammals, especially in zoos. Current treatments are reversible. There is a need to develop nonreversible agents, and this study investigates the effects of high-dose and long-duration exposure to the GnRH agonist, deslorelin, in the rat model. Studies indicate that the follicle-stimulating hormone (FSH) gonadotropin is predominantly affected, and following high-dose exposure to deslorelin for a long duration, the ability of gonadotropes to synthesize FSH may be compromised, perhaps permanently. Understanding the mechanisms by which such persistent suppression of FSH occurs may facilitate the development of novel next-generation contraceptives. It is hypothesized that direct testicular effects of GnRH agonists may play a critical role in the efficacy of GnRH agonists in male contraception.