Probes for LONG

Ketamine, an N-methyl-D-aspartate (NMDA)-receptor antagonist, is a recently revitalized treatment for pain and depression, yet its actions at the molecular level remain incompletely defined. In this molecular-pharmacological investigation in the rat, we used short- and longer-term infusions of high dose ketamine to stimulate neuronal transcription processes. We hypothesized that a progressively stronger modulation of neuronal gene networks would occur over time in cortical and limbic pathways. A continuous intravenous administration paradigm for ketamine was developed in rat consisting of short (1 h) and long duration (10 h, and 10 h + 24 h recovery) infusions of anesthetic concentrations to activate or inhibit gene transcription in a pharmacokinetically controlled fashion. Transcription was measured by RNA-Seq in three brain regions: frontal cortex, hippocampus, and amygdala. Cellular level gene localization was performed with multiplex fluorescent in situ hybridization. Induction of a shared transcriptional regulatory network occurred within 1 h in all three brain regions consisting of (a) genes involved in stimulus-transcription factor coupling that are induced during altered synaptic activity (immediate early genes, IEGs, such as c-Fos, 9-12 significant genes per brain region, p < 0.01 per gene) and (b) the Nrf2 oxidative stress-antioxidant response pathway downstream from glutamate signaling (Nuclear Factor Erythroid-Derived 2-Like 2) containing 12-25 increasing genes (p < 0.01) per brain region. By 10 h of infusion, the acute results were further reinforced and consisted of more and stronger gene alterations reflecting a sustained and accentuated ketamine modulation of regional excitation and plasticity. At the cellular level, in situ hybridization localized up-regulation of the plasticity-associated gene Bdnf, and the transcription factors Nr4a1 and Fos, in cortical layers III and V. After 24 h recovery, we observed overshoot of transcriptional processes rather than a smooth return to homeostasis suggesting an oscillation of plasticity occurs during the transition to a new phase of neuronal regulation. These data elucidate critical molecular regulatory actions during and downstream of ketamine administration that may contribute to the unique drug actions of this anesthetic agent. These molecular investigations point to pathways linked to therapeutically useful attributes of ketamine.

Background: Hepatocellular Carcinoma (HCC) is a major form of liver cancer and a leading cause of cancer-related death worldwide. New insights into HCC pathobiology and mechanism of drug actions are urgently needed to improve patient outcomes. HCC undergoes metabolic reprogramming of glucose metabolism from respiration to aerobic glycolysis, a phenomenon known as the 'Warburg Effect' that supports rapid cancer cell growth, survival, and invasion. mTOR is known to promote Warburg Effect, but the underlying mechanism(s) remains poorly defined. The aim of this study is to understand the mechanism(s) and significance of mTOR regulation of aerobic glycolysis in HCC. Methods: We profiled mTORC1-dependent long non-coding RNAs (lncRNAs) by RNA-seq of HCC cells treated with rapamycin. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were used to explore the transcriptional regulation of NEAT1 by mTORC1. [U-13C]-glucose labeling and metabolomic analysis, extracellular acidification Rate (ECAR) by Seahorse XF Analyzer, and glucose uptake assay were used to investigate the role of mTOR-NEAT1-NONO signaling in the regulation of aerobic glycolysis. RNA immunoprecipitation (RIP) and NONO-binding motif scanning were performed to identify the regulatory mechanism of pre-mRNA splicing by mTOR-NEAT1. Myristoylated AKT1 (mAKT1)/NRASV12-driven HCC model developed by hydrodynamic transfection (HDT) was employed to explore the significance of mTOR-NEAT1 signaling in HCC tumorigenesis and mTOR-targeted therapy. Results: mTOR regulates lncRNA transcriptome in HCC and that NEAT1 is a major mTOR transcriptional target. Interestingly, although both NEAT1_1 and NEAT1_2 are down-regulated in HCC, only NEAT1_2 is significantly correlated with poor overall survival of HCC patients. NEAT1_2 is the organizer of nuclear paraspeckles that sequester the RNA-binding proteins NONO and SFPQ. We show that upon oncogenic activation, mTORC1 suppresses NEAT1_2 expression and paraspeckle biogenesis, liberating NONO/SFPQ, which in turn, binds to U5 within the spliceosome, stimulating mRNA splicing and expression of key glycolytic enzymes. This series of actions lead to enhanced glucose transport, aerobic glycolytic flux, lactate production, and HCC growth both in vitro and in vivo. Furthermore, the paraspeckle-mediated mechanism is important for the anticancer action of US FDA-approved drugs rapamycin/temsirolimus. Conclusions: These findings reveal a molecular mechanism by which mTOR promotes the 'Warburg Effect', which is important for the metabolism and development of HCC, and anticancer response of mTOR-targeted therapy.

Background Pain is the most common cause of disability in IBD. What causes inter-individual variability in chronic pain after successful treatment of inflammation remains elusive. We have shown that activation of TRPV1+ colonic nociceptors is essential for the establishment of persistent pain in DSS colitis. Nociceptor development coincides with microbial colonization, while early life dysbiosis can lead to visceral hypersensitivity in adulthood. Whether the microbiota dictates nociceptor development and pain susceptibility remains unknown. Here we test the hypothesis that the microbiota programs nociceptor specification during early development, rendering them more susceptible to sensitization later in life. We have identified the aryl hydrocarbon receptor (AHR) that senses bacterial-derived metabolites as a candidate target that orchestrates transcriptional regulation in nociceptors. Aims We investigated the developmental regulation of nociceptors by the microbiome and how it influences pain sensitivity. We will determine the effects of AHR activation on nociceptor lineage and function as well as the long term impact of AHR signaling on pain sensitivity. Methods We have developed a germ-free (GF) TRPV1-GFP reporter mouse that was used to phenotype and visualise TRPV1+ nociceptors in the absence of a microbiota. We will isolate TRPV1+ neurons by FACS to identify genes that are under the control of the microbiota and to characterise the phosphoproteome of TRPV1+ nociceptors in GF conditions. Finally, we will investigate the role of AHR signaling in nociceptors both acutely and during development. Results We showed a reduction in thermal pain threshold and a reduction in capsaicin test responses in GF mice. The number and size of DRG neurons was unchanged in GF mice. Examination of molecular markers for peptidergic (CGRP) and non-peptidergic (IB4) neurons did not show a difference. Finally, there was no difference in the expression of TRPV1, suggesting post-translational modification of the channel. In cultured DRG neurons, we found a decrease in capsaicin induced action potentials and a decrease in the amplitude of the capsaicin response in GF mice. Using RNAscope, we showed that TRPV1+ neurons express AHR. Conclusions Our results highlight the importance of bacterial composition in regulating the development of nociceptors and pain sensitivity in adulthood. Furthermore, we are the first to demonstrate the expression of AHR in sensory neurons. These findings point to a role of the microbiota in programming nociceptors during development. My work will advance our understanding of the role of commensal bacteria in regulating pain and could lead to recommendations for the treatment of neonates in early life to reduce their risk of developing chronic pain later in life. Funding Agencies CAG, CIHR

Exercise training is well established as the most effective way to enhance muscle performance and muscle building. The composition of skeletal muscle fiber type affects systemic energy expenditures, and perturbations in metabolic homeostasis contribute to the onset of obesity and other metabolic dysfunctions. Long noncoding RNAs (lncRNAs) have been demonstrated to play critical roles in diverse cellular processes and diseases, including human cancers; however, the functional importance of lncRNAs in muscle performance, energy balance, and obesity remains elusive. We previously reported that the lncRNA H19 regulates the poly-ubiquitination and protein stability of dystrophin (DMD) in muscular dystrophy.Here, we identified mouse/human H19-interacting proteins using mouse/human skeletal muscle tissues and liquid chromatography-mass spectrometry (LC-MS). Human induced pluripotent stem-derived skeletal muscle cells (iPSC-SkMC) from a healthy donor and Becker Muscular Dystrophy (BMD) patients were utilized to study DMD post-translational modifications and associated proteins. We identified a gain-of-function (GOF) mutant of H19 and characterized the effects on myoblast differentiation and fusion to myotubes using iPSCs. We then conjugated H19 RNA gain-of-function oligonucleotides (Rgof) with the skeletal muscle enrichment peptide agrin (referred to as AGR-H19-Rgof) and evaluated AGR-H19-Rgof's effects on skeletal muscle performance using wild-type (WT) C57BL/6 J mice and its anti-obesity effects using high-fat diet (HFD)- and leptin deficiency-induced obese mouse models.We demonstrated that both human and mouse H19 associated with DMD and that the H19 GOF exhibited enhanced interaction with DMD compared to WT H19. DMD was found to associate with serine/threonine-protein kinase MRCK alpha (MRCKα) and α-synuclein (SNCA) in iPSC-SkMC derived from BMD patients. Inhibition of MRCKα and SNCA-mediated phosphorylation of DMD antagonized the interaction between H19 and DMD. These signaling events led to improved skeletal muscle cell differentiation and myotube fusion. The administration of AGR-H19-Rgof improved the muscle mass, muscle performance, and base metabolic rate of WT mice. Furthermore, mice treated with AGR-H19-Rgof exhibited resistance to HFD- or leptin deficiency-induced obesity.Our study suggested the functional importance of the H19 GOF mutant in enhancing muscle performance and anti-obesity effects.

Hypophosphatasia (HPP) is caused by loss-of-function mutations in the ALPL gene that encodes tissue-nonspecific alkaline phosphatase (TNAP), whose deficiency results in the accumulation of extracellular inorganic pyrophosphate (PPi ), a potent mineralization inhibitor. Skeletal and dental hypomineralization characterizes HPP, with disease severity varying from life-threatening perinatal or infantile forms to milder forms that manifest in adulthood or only affect the dentition. Enzyme replacement therapy (ERT) using mineral-targeted recombinant TNAP (Strensiq/asfotase alfa) markedly improves the life span, skeletal phenotype, motor function, and quality of life of patients with HPP, though limitations of ERT include frequent injections due to a short elimination half-life of 2.28 days and injection site reactions. We tested the efficacy of a single intramuscular administration of adeno-associated virus 8 (AAV8) encoding TNAP-D10 to increase the life span and improve the skeletal and dentoalveolar phenotypes in TNAP knockout (Alpl-/- ) mice, a murine model for severe infantile HPP. Alpl-/- mice received 3 × 1011 vector genomes/body of AAV8-TNAP-D10 within 5 days postnatal (dpn). AAV8-TNAP-D10 elevated serum ALP activity and suppressed plasma PPi . Treatment extended life span of Alpl-/- mice, and no ectopic calcifications were observed in the kidneys, aorta, coronary arteries, or brain in the 70 dpn observational window. Treated Alpl-/- mice did not show signs of rickets, including bowing of long bones, enlargement of epiphyses, or fractures. Bone microstructure of treated Alpl-/- mice was similar to wild type, with a few persistent small cortical and trabecular defects. Histology showed no measurable osteoid accumulation but reduced bone volume fraction in treated Alpl-/- mice versus controls. Treated Alpl-/- mice featured normal molar and incisor dentoalveolar tissues, with the exceptions of slightly reduced molar enamel and alveolar bone density. Histology showed the presence of cementum and normal periodontal ligament attachment. These results support gene therapy as a promising alternative to ERT for the treatment of HPP.

Introduction: Conventional 2D mono-culture in vitro models using immortalized cell lines are still widely used in experimental nephrology, albeit their limited translatability and predictivity for the in vivo situation. The feasibility of more sophisticated assays is often reduced by complex protocols and long lasting procedures. We aimed to establish and validate an easy-to-use but yet (patho-) physiologically relevant 3D cell culture assay that mimics key aspects of the in vivo situation of renal tubules, including a leak-thight epithelium with a luminal and baso-lateral side, interstitial matrix, a peri-tubular capillary and circulating blood cells inside its lumen. Methods: We utilized the 3-lane OrganoPlate system (Mimetas, Leiden, Netherlands) as a scaffold. After infusing a collagen I matrix in the middle channel (C2), primary human renal progenitor cells are seeded into the upper channel (C1), adhering to the C2-matrix. The plate is put on a perfusion rocker, that facilitates continuous gravity-triggered bidirectional perfusion in all channels. Thereafter the cells form a leaktight tubular structure with a continuous lumen. Next, human endothelial cells are seeded into the bottom channel (C3), which adhered to the opposite site of C2 and formed a vessel-like structure with a continuous lumen, as well. Finally, primary human white blood cells (WBCs) were isolated and seeded into C3 (figure A). Results: The leak-tightness of the 3D-tubule increased significantly over time, as measured by tracing the diffusion of a 150 kDa FITClabeled dextran from C1 to C2 (time-to-leakage day 1: 3.3 2.6 min; day 9: 36.2 10.7 min), indicating the stability of the co-culture system as well as a cellular maturation resulting in significant barrier functionality as seen in vivo (figure B). In accordance with this and other studies, the primary human tubular cells expressed higher levels of functionally relevant proteins in 3D than under 2D, no-flow conditions, as indicated by cell-number normalized mean fluorescence intensity measured by immunofluorescence, e..g ZO-1 (2.1 0.4 vs. 82.2 20.8) and Na-KATPase (2.3 0.3 vs. 52.8 5.4). Additionally, the growth conditions of the OrganoPlate rendered the cells more resilient to stimuli of acute tubular necrosis, e.g. extracellular histones, as compared to standard cell culture, indicated by cell number normalized lactate dehydrogenase release.The primary WBCs seeded inside the endothelial lumen (C3) did not leave the compartment under normal culture conditions, but displayed extravasation and directed migration from C3 through C2 towards C1 when attracted by chemokines released from tubular cells in C1. This effect was inhibitable by pre-emptive treatment of the endothelium with an selective monoclonal anti-P-selectin antibody (percent migrated cells, medium: 0 0, chemokines: 4.59 0.6, chemokines + Pselectin AB: 1.0 0.5, figure C). This serves as a proof of principle, that the system is applicable to study complex cell-cell and cell-substrate interactions, such as chemokine-mediate immune cell homing. Conclusions: The results of this study suggest, that sophisticated 3D co-culture models of a renal tubule including an interstitial compartment, a peri-tubluar capillary and circulating immune cells are feasible and potentially suited for in depth mechanistic studies in vitro.

Purpose: The synovial joint exhibits extraordinary biotribological properties allowing the articular cartilage layers to slide past each other at very low friction even under local pressures of up to 18 MPa (~180 atm). Articular cartilage is exquisitely mechanical sensitive. Compressive mechanical load contributes to articular cartilage homeostasis; however, overuse or destabilizing the joint increases surface shear stress, which promotes cartilage degradation. Our previous Results show that shear stress, induced by joint destabilization, regulates a number of inflammatory genes 6h post surgery, including Mmp3, Il1b, Arg1, Ccl2, and Il6. Immobilizing the joint by prolonged anesthesia or sciatic neurectomy abrogates the regulation of inflammatory genes and prevents development of OA. In this study, we use RNA Scope to identify which cells of the cartilage are activated by surface shear after joint destabilisation, and test whether this is modifiable by injection of a biocompatible phospholipid-based lubricant. Methods: Destabilization of the medial meniscus (DMM) or sham surgery was performed on the right knee of 10-week-old male C57BL/6 mice. 30 ml of lubricant (PMPC: poly(methacryloylphosphsphorylcholine)-functionalized lipid vesicles) or vehicle control (PBS) solution was injected in the joint two days before and at the time of surgery. Cartilage from naïve (no surgery) and DMM-operated knees of four mice per data point was collected by microdissection for bulk mRNA extraction. Expression levels of selected genes including shear-responsive genes Il1b and Mmp3 were tested by RT-PCR using TaqMan Low Density Arrays (TLDA) microfluidic cards. In addition, whole joints were collected and processed following the standard protocol for RNAscope (Advanced Cell Diagnostics). Coronal sections in the middle of the joints were sliced by a cryostat. Consecutive sections were used for Safranin O staining and RNAscope to identify anatomical tissues and detect the expression of genes of interest. Gene expression signals were collated from 11 stacks by confocal microscopy (Zeiss Confocal 880) focusing on the medial tibia cartilage, and were quantified by counting individual mRNA dots in the sham, DMM, vehicle and lubricant groups. Results: We observed the upregulation of injury-responsive genes Il1b, Mmp3, Ccl2, Adamts 4, Nos2, and Timp1 in the articular cartilage of DMM operated joints compared to Naïve (non-operated) animals. The injection of the lubricant in the joint significantly suppressed the expression of shear-responsive genes Il1b and Mmp3 after DMM, but did not influence the increase of other injury-induced inflammatory genes, such as Timp1, Adamts 4, Ccl2, Nos2. For RNAscope, focusing on Mmp3 expression, the number of Mmp3 positive cells increased two-fold in the DMM-vehicle group compared with the sham-vehicle group. Most of Mmp3 signal was expressed in the superficial region of the cartilage. DMM-PMPC groups showed a reduced number of Mmp3 positive cells compared with DMM-vehicle, with levels similar to sham-vehicle and sham-PMPC groups. Conclusions: Our data demonstrate that shear stress-induced inflammatory genes are regulated in the superficial layer of cartilage after joint destabilisation and can be suppressed by joint injection of a biocompatible engineered lubricant. As these lubricants have long retention times in the joint (data not presented), we believe that they may provide a potential novel therapeutic strategy for preventing the development of post-trauma OA. These studies are underway

Purpose: As skeletal maturity is approached, long bone elongation draws to a close and the cartilaginous growth plate (GP) ossifies and fuses as bone bridges form. This is likely a pivotal moment for the appendicular skeleton, but our mechanistic appreciation of how this process is orchestrated is limited. We have been studying how chondrocytes integrate biological cues, such as growth factor signalling, and mechanical forces, and have investigated the mechanosensitivity of epiphyseal fusion and roles for putative mechanotransduction machinery, including the primary cilium, in these contexts. Here we asked whether primary cilia have a mechanotransduction role in the juvenile GP and adolescent epiphyseal fusion. Methods: We used an inducible aggrecan (ACAN) Cre mouse model, enabling temporal deletion of the core ciliary protein IFT88 in cartilage to investigate GP narrowing dynamics and closure from 4 - 10 weeks of age. Both control (Ift88fl/fl) and cKO (Ift88fl/fl;ACANCreERT2) were injected with tamoxifen (I.P.). Cre activity was validated using a ROSA26TdTomato reporter line. Animals were exposed to (i) sciatic and femoral double neurectomy (DN) to off-load the right hind limb (immobilised DN) whilst the left bears full weight (contralateral DN) at 8 weeks of age, or (ii) voluntary wheel exercise between 8 and 10 weeks of age. Joints were scanned by μCT before histomorphometric analyses of tibial GP using Safranin-O/fast green, TUNEL, Collagen type X (ColX) immunohistochemistry, Von Kossa and TRAP. Cryosections of mouse GPs were analysed by confocal microscopy to investigate primary cilia prevalence and RNA scope was used to identify molecular mechanisms in situ. Medians +/- 95% confidence intervals quoted throughout below, Two-way ANOVA statistical comparisons. Results: We have, for the first time, investigated the role of cilia beyond 4 weeks of age. μCT analysis showed GP length in wild-type mice reduces from ∼260 μm to 130 μm between 4 and 10 weeks of age. Deletion of IFT88 in juvenile mice at 4 or 6 weeks of age resulted in longer GPs in cKO mice at every timepoint compared with control mice (Fig. 1A, 1st and 2nd panel and 1B). Thus, two weeks after tamoxifen, cKO GP lengths were not statistically significantly different to controls at time of treatment, indicating inhibition of GP closure. Deleting IFT88 at 8 weeks of age also resulted in longer GPs (p< 0.0001, n=12 controls, n=23 cKO). Interestingly, some cKO mice exhibited extremely elongated GPs at the edges of the tibia, which appeared as large holes by μCT (Fig. 1A), whilst the centre of the GP appeared less affected. Histology confirmed longer GPs were predominantly characterised by increases in hypertrophic chondrocyte populations. The large, often bi-lateral “holes”, observed by μCT were largely filled with disorganised hypertrophic chondrocytes, as indicated by IHC labelling for ColX. Interestingly limb immobilisation, (DN), at 8 weeks of age, rescued the GP phenotype observed in IFT88 cKO mice (Fig. 1A, 2nd and 3rd panels, and 1C), whilst the contralateral, unoperated (increased load-bearing) limb exhibited bi-lateral failure of ossification, similar to that observed in IFT88 cKO mice. Compared with naïve controls, wheel-exercised mice also displayed elongated GP (p< 0.0001, n=12 controls, n=10 wheel exercised) (Fig. 1A, 4th panel, and 1C) at 10 weeks of age. These expanded GP were, again, most pronounced at the edges of the tibia, whilst the centre of the GP appeared less affected and again was largely filled with disorganised, differentiated, ColX positive hypertrophic chondrocytes. In both wheel exercised and IFT88 cKO mice, regions of failed ossification, but not middle regions, were associated with loss of osteoclast activity. Confocal imaging and analysis revealed a statistically significant (p< 0.001) decrease in cilia positive cells in wheel exercised mice (32.9%, n=5) compared with control (40.7%, n=4) and IFT88cKO mice (p< 0.0001, (23.4%, n=4) compared with controls (40.7% n=4) at 10 weeks of age. Ongoing experiments are investigating 3D spatial analysis of fusion mechanisms, and the status of ciliary Hh signalling (Gli1, by RNAscope) within GP from control, DN, exercised, and cKO mice to dissect the apparently negative, regulatory role IFT88 is plays in the mechanical regulation of epiphyseal fusion. Conclusions: We conclude that IFT88 unequivocally plays a role in GP closure, its removal resulting in failed ossification of the GP, without disruption to chondrocytic lineage differentiation. This phenomenon, observed in cKO animals, is mechanosensitive with limb immobilisation rescuing the phenotype, suggesting, paradoxically, that IFT88 is dampening a mechanically-induced signal in the GP. Wheel exercise also resulted in impaired ossification thus these data collectively unveil both the acute response of the adolescent mouse GP to exercise and, through Ift88 deletion (cKO), a novel mechanoregulatory mechanism orchestrated by ciliary IFT. The effects of altered mechanics and mechanotransduction are most pronounced in the hypertrophic zone where cells are apparently trapped short of transdifferentiation. Osteoclast recruitment and/or activity is impaired, and bone formation inhibited. These Results may have implications for our understanding of hypertrophic chondrocyte biology in articular cartilage in OA. Moreover, it has been proposed that changes to mechanical inputs during adolescence and associated cam morphology contribute to hip OA development. In adolescent patient cohorts, high levels of exercise lead to cartilaginous hypertrophy, epiphyseal extension, cam development, and reduced rates of GP closure. Femoral and tibial epiphyseal extension has also been observed in adolescent athletes that sustain repetitive trauma through high intensity exercise. This research is crucial to a holistic understanding of skeletal mechano-biological health, and the effects of exercise, on the maturing appendicular skeleton