The circadian transcription factor neuronal PAS domain 2 (NPAS2) is linked to psychiatric disorders associated with altered reward sensitivity. The expression of Npas2 is preferentially enriched in the mammalian forebrain, including the nucleus accumbens (NAc), a major neural substrate of motivated and reward behavior. Previously, we demonstrated that down-regulation of NPAS2 in the NAc reduces the conditioned behavioral response to cocaine in mice. We also showed that Npas2 is preferentially enriched in dopamine receptor 1 containing medium spiny neurons (D1R-MSNs) of the striatum. To extend these studies, we investigated the impact of NPAS2 disruption on accumbal excitatory synaptic transmission and strength, along with the behavioral sensitivity to cocaine reward in a cell-type specific manner. Viral-mediated knockdown of Npas2 in the NAc of male and female C57BL/6J mice increased the excitatory drive onto MSNs. Using Drd1a-tdTomato mice in combination with viral knockdown, we determined these synaptic adaptations were specific to D1R-MSNs relative to non-D1R-MSNs. Interestingly, NAc-specific knockdown of Npas2 blocked cocaine-induced enhancement of synaptic strength and glutamatergic transmission specifically onto D1R-MSNs. Lastly, we designed, validated, and employed a novel Cre-inducible short-hairpin RNA virus for MSN-subtype specific knockdown of Npas2 Cell-type specific Npas2 knockdown in D1R-MSNs, but not D2R-MSNs, in the NAc reduced cocaine conditioned place preference. Together, our results demonstrate that NPAS2 regulates excitatory synapses of D1R-MSNs in the NAc and cocaine reward-related behavior.SIGNIFICANCE STATEMENTDrug addiction is a widespread public health concern often comorbid with other psychiatric disorders. Disruptions of the circadian clock can predispose or exacerbate substance abuse in vulnerable individuals. We demonstrate a role for the core circadian protein, NPAS2, in mediating glutamatergic neurotransmission at medium spiny neurons (MSNs) in the nucleus accumbens (NAc), a region critical for reward processing. We find that NPAS2 negatively regulates functional excitatory synaptic plasticity in the NAc and is necessary for cocaine-induced plastic changes in MSNs expressing the dopamine 1 receptor (D1R). We further demonstrate disruption of NPAS2 in D1R-MSNs produces augmented cocaine preference. These findings highlight the significance of cell-type specificity in mechanisms underlying reward regulation by NPAS2 and extend our knowledge of its function.

An intact renin-angiotensin-system involving ANGII-AT1 receptors is crucial for normal kidney development. It is still unclear in which cell types AT1 receptor signaling is required for normal kidney development, maturation and function. Since all kidney cells deriving from stroma progenitor cells express AT1 receptors and since stromal cells fundamentally influence nephrogenesis and tubular maturation, we investigated the relevance of AT1 receptors in stromal progenitors and their descendants for renal development and function. For this aim, we have generated and analyzed mice with a conditional deletion of AT1a receptor in the FoxD1 cell lineage in combination with a global disruption of the AT1b gene. Those FoxD1-AT1ko mice developed normally. Their kidneys showed neither structural nor functional abnormalities in comparison with wildtype mice, whilst in isolated perfused FoxD1-AT1ko kidneys the vasoconstrictor and renin inhibitory effects of ANGII were absent. In vivo, however, plasma renin concentration and renal renin expression were normal in FoxD1-AT1ko mice, as were blood pressure and glomerular filtration rate. These findings suggest that a strong reduction of AT1 receptors in renal stromal progenitors and their descendants does not disturb normal kidney development.

Abstract

RATIONALE:

Given the paucity of effective treatments for Idiopathic Pulmonary Fibrosis (IPF), new insights into the deleterious mechanisms controlling lung fibroblast activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies. Transforming growth factor β (TGF-β) is the main pro-fibrotic factor, but its inhibition is associated with severe side effects due to its pleiotropic role.

OBJECTIVES:

We hypothesized that downstream non-coding effectors of TGF-β in fibroblasts may represent new effective therapeutic targets whose modulation may be well-tolerated.

METHODS:

We investigated the whole non-coding fraction of TGF-β-stimulated lung fibroblast transcriptome to identify new genomic determinants of lung fibroblast differentiation into myofibroblast. Differential expression of the long non-coding RNA DNM3OS and its associated miRNAs was validated in a murine model of pulmonary fibrosis and in IPF tissue samples. Distinct and complementary antisense oligonucleotide-based strategies aiming at interfering with DNM3OS were used to elucidate the role of DNM3OS and its associated miRNAs in IPF pathogenesis.

MEASUREMENTS AND MAIN RESULTS:

We identified DNM3OS as a fibroblast-specific critical downstream effector of TGF-β-induced lung myofibroblast activation. Mechanistically, DNM3OS regulates this process in trans by giving rise to three distinct profibrotic mature miRNAs (i.e. miR-199a-5p/3p and miR-214-3p), which influence both SMAD and non-SMAD components of TGF-β signaling in a multifaceted way. In vivo, we showed that interfering with DNM3OS function not only prevents lung fibrosis but also improves established pulmonary fibrosis.

CONCLUSION:

Pharmacological approaches aiming at interfering with DNM3OS may represent new effective therapeutic strategies in IPF.

It is well established that DNA-damaging chemotherapies can cause infertility and ovarian endocrine failure by depleting the ovarian reserve of primordial follicles. Currently, no effective pharmacological therapies exist for the preservation of long-term fertility and ovarian function in female cancer patients, due to a limited understanding of the mechanisms of chemotherapy-induced follicle depletion. This study investigated the cellular targets, molecular mechanisms, and temporal course of ovarian reserve depletion following treatment with commonly used chemotherapeutic drugs. Adult female C57BL/6 mice were injected i.p. with saline, cisplatin (5mg/kg), or cyclophosphamide (300mg/kg); ovaries were harvested after 8 or 24 hours. Follicle counts showed depletion of all follicular stages 24 hours after administration of cisplatin or cyclophosphamide. Eight hours post-treatment, H2A histone family member X (γH2AX) immunofluorescence showed DNA double-stranded breaks at all follicular stages, including within primordial follicle oocytes. This staining was resolving by 24 hours, indicating that primordial follicle oocytes begin to undergo either apoptosis or repair in this timeframe. γH2AX-positive follicles were further examined to identify the specific cell types damaged. In primordial, transitional, and primary follicles, only oocytes sustained DNA damage, whereas in secondary and antral follicles, only somatic cells were affected. TUNEL staining confirmed that apoptosis occurs in these targeted cell types. Whilst multi-drug and multi-dose regimens were not examined, this study conclusively shows that cyclophosphamide and cisplatin cause direct damage to primordial follicle oocytes, which then undergo apoptosis. Therefore, future pharmacological strategies to prevent chemotherapy-induced infertility in females must specifically prevent primordial follicle oocyte death.

Actinic keratosis (AK) is a precancerous skin lesion that is common in HIV-positive patients. Without effective treatment, AKs can progress to squamous cell carcinoma. Ingenol mebutate, a PKC agonist, is a US Food and Drug Administration-approved (FDA-approved) topical treatment for AKs. It can induce reactivation of latent HIV transcription in CD4+ T cells both in vitro and ex vivo. Although PKC agonists are known to be potent inducers of HIV expression from latency, their effects in vivo are not known because of the concerns of toxicity. Therefore, we sought to determine the effects of topical ingenol mebutate gel on the HIV transcription profile in HIV-infected individuals with AKs, specifically in the setting of suppressive antiretroviral therapy (ART). We found that AKs cleared following topical application of ingenol mebutate and detected marginal changes in immune activation in the peripheral blood and in skin biopsies. An overall increase in the level of HIV transcription initiation, elongation, and complete transcription was detected only in skin biopsies after the treatment. Our data demonstrate that application of ingenol mebutate to AKs in ART-suppressed HIV-positive patients can effectively cure AKs as well as disrupt HIV latency in the skin tissue microenvironment in vivo without causing massive immune activation.

Sleep is essential to emotional health. Sleep disturbance, particularly REM sleep disturbance, profoundly impacts emotion regulation, but the underlying neural mechanisms remain elusive. Here we show that chronic REM sleep disturbance, achieved in mice by chronic sleep fragmentation (SF), enhanced neural activity in the medial habenula (mHb), a brain region increasingly implicated in negative affect. Specifically, after a 5-day SF procedure that selectively fragmented REM sleep, cholinergic output neurons (ChNs) in the mHb exhibited increased spontaneous firing rate and enhanced firing regularity in brain slices. The SF-induced firing changes remained intact upon inhibition of glutamate, GABA, acetylcholine, and histamine receptors, suggesting cell-autonomous mechanisms independent of synaptic transmissions. Moreover, the SF-induced hyperactivity was not because of enhanced intrinsic membrane excitability, but was accompanied by depolarized resting membrane potential in mHb ChNs. Furthermore, inhibition of TASK-3 (KCNK9) channels, a subtype of two-pore domain K+ channels, mimicked the SF effects by increasing the firing rate and regularity, as well as depolarizing the resting membrane potential in mHb ChNs in control-sleep mice. These effects of TASK-3 inhibition were absent in SF mice, suggesting reduced TASK-3 activity following SF. By contrast, inhibition of small-conductance Ca2+-activated K+ (SK) channels did not produce similar effects. Thus, SF compromised TASK-3 function in mHb ChNs, which likely led to depolarized resting membrane potential and increased spontaneous firing. These results not only demonstrate that selective REM sleep disturbance leads to hyperactivity of mHb ChNs, but also identify a key molecular substrate through which REM sleep disturbance may alter affect regulation.

Abstract

BACKGROUND:

Neuropathic pain is caused by sensory nerve injury, but effective treatments are currently lacking. Microglia are activated in the spinal dorsal horn after sensory nerve injury and contribute to neuropathic pain. Accordingly, molecules expressed by these cells are considered potential targets for therapeutic strategies. Our previous gene screening study using a mouse model of motor nerve injury showed that the G-protein-coupled receptor 34 gene (GPR34) is induced by nerve injury. Because GPR34 is now considered a microglia-enriched gene, we explored the possibility that it might be involved in microglial activation in the dorsal horn in a mouse model of neuropathic pain.

METHODS:

mRNA expression of GPR34 and pro-inflammatory molecules was determined by quantitative real-time PCR in wild-type and GPR34-deficient mice with L4 spinal nerve injury. In situ hybridization was used to identify GPR34 expression in microglia, and immunohistochemistry with the microglial marker Iba1 was performed to examine microglial numbers and morphology. Mechanical sensitivity was evaluated by the von Frey hair test. Liquid chromatography-tandem mass spectrometry quantified expression of the ligand for GPR34, lysophosphatidylserine (LysoPS), in the dorsal horn, and a GPR34 antagonist was intrathecally administrated to examine the effect of inhibiting LysoPS-GPR34 signaling on mechanical sensitivity.

RESULTS:

GPR34 was predominantly expressed by microglia in the dorsal horn after L4 nerve injury. There were no histological differences in microglial numbers or morphology between WT and GPR34-deficient mice. However, nerve injury-induced pro-inflammatory cytokine expression levels in microglia and pain behaviors were significantly attenuated in GPR34-deficient mice. Furthermore, the intrathecal administration of the GPR34 antagonist reduced neuropathic pain.

CONCLUSIONS:

Inhibition of GPR34-mediated signal by GPR34 gene deletion reduced nerve injury-induced neuropathic pain by suppressing pro-inflammatory responses of microglia without affecting their morphology. Therefore, the suppression of GPR34 activity may have therapeutic potential for alleviating neuropathic pain.

Osteoblast-lineage cells in bone human were recently shown to colonize eroded bone surfaces and to closely interact with osteoclasts. They proved identical with reversal cells and are believed to differentiate into bone forming osteoblasts thereby coupling resorption and formation. However, they also exert catabolic activity that contributes to osteoclastic bone resorption, but this has not received much attention. Herein, we used co-cultures of primary human osteoblast-lineage cells and human osteoclasts derived from peripheral blood monocytes to investigate whether a catabolic activity of osteoblast-lineage cells may impact on osteoclastic bone resorption. Through a combination of immunofluorescence, in-situ hybridization, and time-lapse we show that MMP-13 expressing osteoblast-lineage cells are attracted to and closely interact with bone resorbing osteoclasts. This close interaction results in a strong and significant increase in the bone resorptive activity of osteoclasts - especially those making trenches. Importantly, we show that osteoclastic bone resorption becomes sensitive to inhibition of matrix metalloproteinases in the presence, but not in the absence, of osteoblast-lineage cells. We propose that this may be due to the direct action of osteoblast-lineage-derived MMP-13 on bone resorption.

Vascular dementia (VaD) is cognitive decline linked to reduced cerebral blood perfusion, yet there are few therapeutic options to protect cognitive function following cerebrovascular accidents. The purpose of this study was to profile gene expression changes unique to VaD to identify and characterize disease relevant changes that could offer clues for future therapeutic direction. Microarray-based profiling and validation studies of postmortem frontal cortex samples from VaD, Alzheimer disease, and age-matched control subjects revealed that the oxytocin receptor (OXTR) was strongly and differentially upregulated in VaD. Further characterization in fixed tissue from the same cases showed that OXTR upregulation occurs de novo around and within microinfarcts in peri-infarct reactive astrocytes as well as within vascular profiles, likely on microvascular endothelial cells. These results indicate that increased OXTR expression in peri-infarct regions may be a specific response to microvascular insults. Given the established OXTR signaling cascades that elicit antioxidant, anti-inflammatory, and pro-angiogenic responses, the present findings suggest that de novo OXTR expression in the peri-infarct space is a tissue-protective response by astroglial and vascular cells in the wake of ischemic damage that could be exploited as a therapeutic option for the preservation of cognition following cerebrovascular insults.

Glioblastomas are highly aggressive brain tumors with a particularly poor prognosis. Glucose transporter-1 (GLUT1/SLC2A1), a uniporter that is expressed by various carcinomas and may be involved in malignant neoplasm glycometabolism, may also be related to prognosis in glioblastomas. GLUT1 is essential to central nervous system glycometabolism. To clarify the exact role of GLUT1 in glioblastoma, we assessed the expression and localization of GLUT1 in patient samples by immunohistochemistry and in situ RNA hybridization. This revealed that GLUT1 was mainly expressed on perivascular and pseudopalisaded tumor cell membranes. All samples expressed GLUT1 to some degree, with 30.8% showing stronger staining. On the basis of these data, samples were divided into high and low expression groups, although SLC2A1 mRNA expression was also higher in the high GLUT1 expression group. Kaplan-Meier survival curves revealed that high GLUT1 expression associated with lower overall survival (log-rank test, p = 0.001) and worse patient prognoses (p = 0.001). Finally, MIB-1 staining was stronger in high GLUT1 expression samples (p = 0.0004), suggesting a link with proliferation. We therefore hypothesize that GLUT1 expression in glioblastomas may enhance glycolysis, affecting patient prognosis. Examination of GLUT1 in patients with glioblastomas may provide a new prognostic tool to improve outcome.

Abstract

RATIONALE:

The goal was to connect elements of IPF pathogenesis, including: chronic endoplasmic reticulum stress in respiratory epithelia associated with injury/inflammation and remodeling; distal airway mucus obstruction and honeycomb cyst formation with accumulation of MUC5B; and associations between IPF risk and polymorphisms in the MUC5B promoter.

OBJECTIVES:

Test whether the ER stress sensor protein ER-to-nucleus signaling 2 (ERN2) and its downstream effector, the spliced form of x-box binding protein 1 (XBP1S), regulate MUC5B expression and differentially activate the MUC5B promoter variant in respiratory epithelia.

METHODS AND MEASUREMENTS:

Primary human airway epithelia (HAE), transgenic mouse models, human IPF lung tissues, and cell lines expressing XBP1S and MUC5B promoters were used to explore relationships between the ERN2/XBP1S pathway and MUC5B. An inhibitor of the pathway, KIRA6, and XBP1 CRISPR-Cas9 were used in HAE to explore therapeutic potential.

RESULTS:

ERN2 regulated both MUC5B and MUC5AC mRNAs. Downstream XBP1S selectively promoted MUC5B expression in vitro and in distal murine airway epithelia in vivo. XBP1S bound to the proximal region of the MUC5B promoter and differentially up-regulated MUC5B expression in the context of the MUC5B promoter rs35705950 variant. High levels of ERN2 and XBP1S were associated with excessive MUC5B mRNAs in distal airways of human IPF lungs. Cytokine-induced MUC5B expression in HAE was inhibited by KIRA6 and XBP1 CRISPR-Cas9.

CONCLUSION:

A positive feedback bistable ERN2-XBP1S pathway regulates MUC5B-dominated mucus obstruction in IPF, providing a UPR-dependent mechanism linking the MUC5B promoter rs35705950 polymorphism with IPF pathogenesis. Inhibiting ERN2-dependent pathways/elements may provide a therapeutic option for IPF.

Abstract

Purpose/Aim: Many genes have been associated with primary open-angle glaucoma (POAG). Knowing exactly where they are expressed in the eye helps to unravel POAG pathology and to select optimal targets for intervention. We investigated whether RNA in-situ hybridization (RNA-ISH) is a convenient technique to obtain detailed pan-ocular expression data of these genes. We tested this for four diverse candidate POAG genes, selected because of unclear ocular distribution (F5 and Dusp1) and relevance for potential new therapies (Tnf, Tgfβr3). Optn, a POAG gene with well-known ocular expression pattern served as control.

METHODS:

We made a list of candidate glaucoma genes reported in genetic studies. A table of their ocular expression at the tissue level was compiled using publicly available microarray data (the ocular tissue database). To add cellular detail we performed RNA-ISH for Optn, Tnf, Tgfβr3, F5, and Dusp1 on eyes of healthy, 2-month-old, pigmented and albino mice.

RESULTS:

Expression of the Optn control matched with published immunohistochemistry data. Ocular expression of Tnf was generally low, with patches of higher Tnf expression, superficially in the corneal epithelium. F5 had a restricted expression pattern with high expression in the non-pigmented ciliary body epithelium and moderate expression in the peripapillary region. Tgfβr3 and Dusp1 showed ubiquitous expression.

CONCLUSIONS:

RNA-ISH is a suitable technique to determine the ocular expression pattern of POAG genes, adding meaningful cellular detail to existing microarray expression data. For instance, the high expression of F5 in the non-pigmented ciliary body epithelium suggests a role of this gene in aqueous humor dynamics and intraocular pressure. In addition, the ubiquitous expression of Tgfβr3 has implications for designing TGF-β related glaucoma therapies, with respect to side effects. Creating pan-ocular expression maps of POAG genes with RNA-ISH will help to identify POAG pathways in specific cell types and to select targets for drug development.