Probes for INS

One negative emotional state from morphine protracted abstinence is anxiety which can drive craving and relapse risk in opioid addicts. Although the orexinergic system has been reported to be important in mediating emotion processing and addiction, the role of orexinergic system in anxiety from drug protracted abstinence remains elusive. In this study, by using behavioral test, western blot, electrophysiology and virus-mediated regulation of orexin receptor 1 (OX1R), we found that: (1) Intraperitoneal and intra-VTA administration of a selective OX1R antagonist SB334867 alleviated anxiety-like behaviors in open field test (OFT) but not in elevated plus maze test (EPM) in morphine protracted abstinent male mice. (2) OX1R expression in the VTA was upregulated by morphine withdrawal. (3) Virus-mediated knockdown of OX1R in the VTA prevented morphine abstinence-induced anxiety-like behaviors and virus-mediated overexpression of OX1R in the VTA was sufficient to produce anxiety-like behaviors in male mice. (4) The VTA neuronal activity was increased significantly induced by morphine protracted abstinence, which was mediated by OX1R. (5) OX1R was widely distributed in the neuronal soma and processes of dopaminergic and non-dopaminergic neurons in the VTA. The findings revealed that the OX1R mediates morphine abstinence-induced anxiety-like behaviors and the VTA plays a critical role in this effect.

PRATTA:_Incyte Corporation:_ Current Employment, Current equity holder in private company, Current holder of _stock options_ in a privately-held company. BURKE:_Incyte Corporation:_ Current Employment, Current equity holder in private company, Current holder of _stock options_ in a privately-held company. DIPERSIO:_BioLineRx, Ltd.:_ Research Funding; _Macrogenics:_ Research Funding; _NeoImmune Tech:_ Research Funding; _Amphivena Therapeutics:_ Research Funding; _hC Bioscience, Inc.:_ Membership on an entity's Board of Directors or advisory committees; _RiverVest Venture Partners:_ Consultancy, Membership on an entity's Board of Directors or advisory committees; _Incyte:_ Consultancy, Research Funding; _WUGEN:_ Current equity holder in private company, Research Funding; _CAR-T cell Product with Washington University and WUGEN:_ Patents & Royalties; _VLA-4 Inhibitor with Washington University and Magenta Therapeutics:_ Patents & Royalties; _Magenta Therapeutics:_ Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. MAZIARZ:_ASTCT:_ Membership on an entity's Board of Directors or advisory committees; _CRISPR Therapeutics:_ Consultancy, Honoraria; _Novartis:_ Other: Support for research on CART; _Allovir:_ Other: Support for research on Allo HCT costs of care of infectious related complications; _Orca Bio:_ Other: Support for research analysis and for medical writing. FELDMAN:_Incyte Corporation:_ Current Employment, Current equity holder in private company, Current holder of _stock options_ in a privately-held company. BRODEUR:_Incyte Corporation:_ Current Employment, Current equity holder in private company, Current holder of _stock options_ in a privately-held company. TIMMERS:_Incyte Corporation:_ Current Employment, Current equity holder in private company, Current holder of _stock options_ in a privately-held company. IVANOVA:_Incyte Corporation:_ Current Employment, Current equity holder in private company, Current holder of _stock options_ in a privately-held company. BARBOUR:_Incyte Corporation:_ Ended employment in the past 24 months; _Karyopharm:_ Current Employment, Current equity holder in publicly-traded company. MORARIU-ZAMFIR:_Incyte Corporation:_ Current Employment, Current equity holder in private company, Current holder of _stock options_ in a privately-held company. FRIGAULT:_Cytoagents:_ Consultancy; _Iovance:_ Consultancy; _Novartis:_ Consultancy, Research Funding; _Kite/Gilead:_ Consultancy, Research Funding; _Arcellx:_ Research Funding; _JnJ/Legend:_ Consultancy; _BMS:_ Consultancy.

RESULTS Microscopic analysis revealed that the full-length _HTT_ mRNA (_FL-HTT_) was retained in RNA nuclear clusters together with the incompletely spliced _HTT1a_ transcript. These clusters were not observed in zQ175 HD mouse model where, instead, _FL-Htt_ and _Htt1a_ mRNAs were detected as mostly cytoplasmic molecules. Immunohistochemistry showed a progressive appearance of aggregated HTT in nuclei in the cortex, striatum, hippocampus and cerebellum. HTRF indicated that the level of exon 1 HTT was highest in the cerebellum. Soluble mutant exon 1 HTT decreased with age, with concomitant increase in aggregated HTT. In YAC128 MEFs, _HTT1a_ was detected and ASOs targeting _HTT_ were efficient in lowering _HTT_ levels in this model system.

Endocrine disrupting chemicals (EDCs) are raising concerns about adverse effects on fertility in women as they have been shown to disrupt steroidogenesis and ovarian function in animal studies, and they associate to reduced fertility in human cohort studies. However, there is a lack of information regarding mechanisms of action and effects in humans. Our study aims to identify molecular mechanisms of endocrine disruption using two well-known human EDCs, diethylstilbestrol (DES) and ketoconazole (KTZ), via controlled exposure studies in ovarian cell lines and human ovarian tissue culture in vitro. Ovarian cortical tissue slices obtained from tissue collected from Caesarean section (c-section) patients at Karolinska University Hospital was exposed to 10-9 M to 10-5 M KTZ and 10-10 M to 10-6 M DES in vitro for 6 days. Follicle survival and growth were studied using histology, steroid production by liquid-chromatography-mass spectrometry (LC-MS/MS), and tissue viability by cytotoxicity and fibrosis assays. RNA sequencing was performed on primary ovarian cells and ovarian granulosa cell cancer cell lines COV434 and KGN that were exposed for 24 hours to the same concentrations of DES and KTZ as the tissue culture. Selected potential biomarkers were validated using real-time quantitative polymerase chain reaction (RT-qPCR) in the cells, and by in situ RNA hybridization in exposed tissue. Significantly lower non-growing follicle densities (i.e. primordial, intermediary, and primary follicles) were observed in DES 10-10 M group compared to vehicle control. A decrease trend was also observed in DES high dose group and low level KTZ exposed group. On the other hand, slightly higher growing follicle density was shown in high level KTZ exposed group. Levels of pregnenolone and progesterone were significantly reduced in KTZ 10-5 M exposed group. RNA sequencing showed that 445 and 233 differential expressed genes (DEGs) (FDR< 0.1) were affected in DES and KTZ exposed group, respectively, in the cell culture. Gene set variation analysis (GSVA) showed that both DES and KTZ modulated MTORC1 signaling, which was critical for primordial follicle activation and steroidogenesis. We selected stear-oyl-CoA desaturase (SCD), a gene that was shown to involved in cholesterol homeostasis, oocyte maturation and steroidogenesis, for validation as a potential biomarker. Up-regulation of was confirmed in response to KTZ by PCR and RNAscope. In conclusion, DES and KTZ affected folliculogenesis and steroidogenesis in human adult ovarian cortex and SCD may serve as a potential biomarker in response to exposure. Further validation of this potential biomarker may help improve the existing testing guideline and subsequently, contributing to better regulation of chemical in our global market.

Significance Molecules regulated by neuronal activity are necessary for circuits to adapt to changing inputs. Specific classical major histocompatibility class I (MHCI) molecules play roles in circuit and synaptic plasticity, but the function of most members of this family remains unexplored in brain. Here, we show that a nonclassical MHCI molecule, Qa-1 (H2-T23), is expressed in a subset of excitatory neurons and regulated by visually driven activity in the cerebral cortex. Moreover, CD94/NKG2 heterodimers, cognate receptors for Qa-1, are expressed in microglia. A functional interaction between Qa-1 and CD94/NKG2 is necessary for regulating the magnitude of ocular dominance plasticity during the critical period in the visual cortex, implying an interaction in which activity-dependent changes in neurons may be monitored by microglia.

PURPOSE : ADOA is the most common inherited optic neuropathy, starting in the first decade of life and resulting in severe and progressive visual decline due to loss of RGCs. Most patients harbor loss-of-function mutations in the _OPA1 _gene that lead to haploinsufficiency. Reduced OPA1 protein levels result in impaired mitochondrial function in RGCs leading to cell death. Currently, there is no treatment for patients with ADOA. Targeted Augmentation of Nuclear Gene Output (TANGO) ASOs, such as STK-002, reduce the inclusion of a non-productive, alternatively spliced exon in _OPA1, _and leverage the wild-type allele to increase productive _OPA1_ mRNA and protein. We previously demonstrated that TANGO ASOs can increase OPA1 protein levels in human cell lines, rabbit retina, and ADOA patient fibroblasts. In this study, we evaluated ASO localization and OPA1 protein levels in the retina following intravitreal administration of STK-002 to NHPs.

Oncostatin M (OSM) is one of the least studied cytokines in the interleukin-6 family especially considering that its expression correlates with hallmarks of chronic itch, rheumatoid arthritis, irritable bowel syndrome, and more recently neuropathic pain. This gap in knowledge is attributed to numerous species differences in the protein structure of OSM, and its receptor usage both of which affect physiological function. Here we uncover some of these discrepancies across mouse, rat, and human models, further underpinning the importance of studying OSM in human context. We characterized the receptors expression profile of OSMR in human dorsal root ganglia (hDRG) from healthy organ donors and confirmed its presence in small-diameter neurons and surrounding glial-like cells via RNAScope in situ hybridization. To investigate OSM-mediated signaling in hDRG, we treated acutely sliced explants with 10ng/ml OSM for 30min and immunoassayed with markers of translation regulation via the Mitogen activated protein kiNase interacting Kinase (MNK) pathway and its downstream target eukaryotic translation Initiation Factor 4E (eIF4E). We noted significant increases in the p-eIF4E intensity signal in small-diameter neurons and glial-like cells suggesting that OSM activates MNK-eIF4E signaling in these cell types. Our findings cumulatively suggest that blocking OSM signaling in hDRG may attenuate nociceptive hyperexcitability and presents a viable therapeutic target for the treatment of pain. NIH NS065926, NIH NS111929.

Background: Most new HIV infections result from sexual interactions with infected but untreated individuals. Semen is the main vector for viral transmission globally, however, little is known regarding the anatomic origin and form of virus in semen. Methods: In this study, we were able to combine numerous new technologies to characterize the virus present in the semen during SIV infection. Six rhesus macaques (RM) were challenged intravenously with barcoded virus SIVmac239M. Semen and blood samples were collected longitudinally for 17 days post-infection with all male genital tract (MGT) and multiple lymphoid tissues collected at necropsy and subjected to quantitative PCR, next generation sequencing of the viral barcode, and tissue analysis (RNAscope, DNAscope and immunophenotyping). Semen was also collected from 6 animals chronically infected with SIVmac251 and in five CD4 depleted animals in acute phase and 2 weeks post ART initiation. Results: Extremely high levels of viral RNA (vRNA) were detected in seminal plasma (up to 10^9cp/ml) as well as comparable levels of cell associated vRNA and vDNA in seminal cells with detection starting as early as 4 days post-infection. RNAscope and immunophenotyping of seminal cells and MGT tissues revealed myeloid cells as the main source of virus (Fig. 1), while CD4+T cells were harboring vRNA in lymphoid tissues. Sequences show evidence of an early compartment between seminal and blood plasma and no difference in the env gene of virus present in semen/MGT and in Lymph Nodes. Finally, multinuclear giant cells harboring vRNA were the only source of virus in semen in chronically infected and in CD4 depleted RM. Moreover, vRNA + myeloid cells were highly present in semen after 2 weeks on ART.

Controlling primary afferent nociceptive input is a known route to analgesia. This path is exemplified by agonists of the mu opioid receptors that are expressed in nociceptive DRG neurons and can be activated by intrathecal or systemic opioid agonists. For other peripheral analgesic targets, the hypothesis is that similar neuronal expression would validate their development as analgesic agents. In this study, two potential candidates, the kappa opioid and adenosine A3 receptors, are evaluated in human DRG for transcript levels, across species expression, and cell type localization using RNA Scope multiplex fluorescence microscopy. The Kappa receptor shows strong species variation in expression. No expression is detectable in dog DRG, low expression in mouse and rat, and relatively high expression in human DRG. This prompted an anatomical localization study to determine which cell types in the human DRG express the Kappa receptor. In situ hybridization disclosed Kappa receptor expression in satellite glial cells (SGCs) and most neurons were surrounded by fluorescent signal. This result was verified using a second independent probe to a distinct region of the OPRK1 transcript. Essentially no signal was seen in DRG neurons. The adenosine A3 receptor (ADORA3) is another GPCR suggested as a peripheral analgesic drug target. ADORA3 shows strong species expression variation, being absent in mouse, and expressed at low levels in rat and dog DRG. In contrast, ADORA3 was abundant in human DRG, but restricted to SGCs. These data suggest peripherally targeted agonists for either receptor may not be effective analgesic agents. Intramural Research Program, Clinical Center, NIH.

Hypothalamic-pituitary-adrenal (HPA) axis dynamics are disrupted by opioids and may be involved in substance abuse; this persists during withdrawal and abstinence and is associated with co-morbid sleep disruption leading to vulnerability to relapse. We hypothesized that chronic sleep restriction (SR) alters the HPA axis diurnal rhythm and the sexually dimorphic response to acute stressor during opioid abstinence. We developed a rat model to evaluate the effect of persistent sleep loss during opioid abstinence on HPA axis dynamics in male and female rats. Plasma ACTH and corticosterone were measured diurnally and in response to acute restraint stress in rats Before (control) compared to During subsequent opioid abstinence without or with SR. Abstinence, regardless of sleep state, led to an increase in plasma ACTH and corticosterone in the morning in males. There was a tendency for higher PM plasma ACTH during abstinence in SR males (p = 0.076). ACTH and corticosterone responses to restraint were reduced in male SR rats whereas there was a failure to achieve the post-restraint nadir in female SR rats. There was no effect of the treatments or interventions on adrenal weight normalized to body weight. SR resulted in a dramatic increase in hypothalamic PVN AVP mRNA and plasma copeptin in male but not female rats. This corresponded to the attenuation of the HPA axis stress response in SR males during opioid abstinence. We have identified a potentially unique, sexually dimorphic role for magnocellular vasopressin in the control of the HPA axis during opioid abstinence and sleep restriction.

Menopause is associated with the loss of LH ovulatory surges and enhanced visceral adiposity. Visceral fat depots increase from 5-8% at premenopause to 15-20% of total body fat at postmenopause. Here, we report that high-dose LH, hCG, or small molecule LH/CGR agonist ORG43553 injected twice-a-week into 14-weeks-old C57BL/6 male mice protects them from diet-induced obesity. Testosterone levels were elevated in mice treated with LH or hCG, but not with ORG43553. Notably, the anti-obesity action of LH/hCG is independent of testosterone, as blocking the androgen receptor using flutamide yielded similar results. Importantly, male Lhcgr knockout mice on a high-fat diet treated with LH failed to display a reduction in adiposity, confirming the in vivo specificity of action. Furthermore, our data phenocopied Lhcgr haploinsufficiency in mice. We confirmed the presence of Lhcgr in mouse genital and inguinal fat pads, adipose-derived stromal vascular cells, as well as in differentiated and undifferentiated 3T3-L1 murine adipocytes by qPCR, RNAscope in situ hybridization, and immunohistochemistry. Sanger sequencing showed that the extracellular domain of Lhcgr in genital fat depot was identical to the ovarian receptor. Similarly, we identified LHCGR in human subcutaneous and visceral fat depots. Binding of intraperitoneally injected AlexaFluor-488-labeled hCG was found not only in mouse ovary, but also in genital and subcutaneous fat pad, further confirming the presence of LHCGR in adipose tissue. This binding could be competitively displaced in 3T3-L1 cells using unlabeled hCG. LH, hCG and ORG43553 activated ERK1/2 in a dose-dependent manner in undifferentiated and differentiated 3T3-L1 cells, suggesting that the adipose LHCGR is fully functional. LH, hCG, and ORG43553 reduced adipogenic differentiation in 3T3-L1 cells, which is further confirmed by RNA sequencing. Moreover, we observed, that LH and hCG also alters several aspects of immune response in adipose tissue, including inflammatory response and adaptive immunity. In conclusion, we demonstrated that LH/CG receptors are present and fully functional in adipose tissue, and that high-dose intermittent activation of LHCGR in mouse fat depots protects mice from diet-induced obesity and modifies adipose tissue immune response. Presentation: Saturday, June 11, 2022 1:42 p.m. - 1:47 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Located in the frontline against the largest population of microbiota, the intestinal mucosa of mammals has evolved to become an effective immune system. γδ T cells, a unique T cell subpopulation, are rare in circulation blood and lymphoid tissues, but rich in the intestinal mucosa, particularly in the epithelium. Via rapid production of cytokines and growth factors, intestinal γδ T cells are key contributors to epithelial homeostasis and immune surveillance of infection. Intriguingly, recent studies have revealed that the intestinal γδ T cells may play novel exciting functions ranging from epithelial plasticity and remodeling in response to carbohydrate diets to the recovery of ischemic stroke. In this review article, we update regulatory molecules newly defined in lymphopoiesis of the intestinal γδ T cells and their novel functions locally in the intestinal mucosa, such as epithelial remodeling, and distantly in pathological setting, e.g., ischemic brain injury repair, psychosocial stress responses, and fracture repair. The challenges and potential revenues in intestinal γδ T cell studies are discussed.