Retinal and optic nerve degeneration in liver X receptor ß knockout mice.

The retina is an extension of the brain. Like the brain, neurodegeneration of the retina occurs with age and is the cause of several retinal diseases including optic neuritis, macular degeneration, and glaucoma. Liver X receptors (LXRs) are expressed in the brain where they play a key role in maintenance of cerebrospinal fluid and the health of dopaminergic neurons. Herein, we report that LXRs are expressed in the retina and optic nerve and that loss of LXRß, but not LXRα, leads to loss of ganglion cells in the retina. In the retina of LXRß-/- mice, there is an increase in amyloid A4 and deposition of beta-amyloid (Aß) aggregates but no change in the level of apoptosis or autophagy in the ganglion cells and no activation of microglia or astrocytes. However, in the optic nerve there is a loss of aquaporin 4 (AQP4) in astrocytes and an increase in activation of microglia. Since loss of AQP4 and microglial activation in the optic nerve precedes the loss of ganglion cells, and accumulation of Aß in the retina, the cause of the neuronal loss appears to be optic nerve degeneration. In patients with optic neuritis there are frequently AQP4 autoantibodies which block the function of AQP4. LXRß-/- mouse is another model of optic neuritis in which AQP4 antibodies are not detectable, but AQP4 function is lost because of reduction in its expression.

Expression and localization of retinoid receptors in the testis of normal and infertile men.

Retinoic acid (RA), the active metabolite of vitamin A, is one of the most important factors regulating spermatogenesis. RA activates downstream pathways through its receptors (retinoic acid receptor alpha [RARA], retinoic acid receptor beta, and retinoic acid receptor gamma [RARG]) and retinoid X receptors (retinoid X receptor alpha [RXRA], retinoid X receptor beta [RXRB], and retinoid X receptor gamma [RXRG]). These receptors may serve as therapeutic targets for infertile men. However, the localization and expression of retinoid receptors in normal and infertile men were unknown. In this study, we found RARA and RARG were mostly localized in spermatocytes and round spermatids, RXRB was mainly expressed in Sertoli cells, and RXRG was expressed in most cell types in the fertile human testis. The localization of RARA, RARG, RXRB, and RXRG in men with hypospermatogenesis (HYPO) was similar to that of men with normal fertility. In addition, the messenger RNA expression levels of RARA, RARG, RXRA, RXRB, and RXRG were significantly decreased in men with Sertoli cell-only syndrome (SCOS) and maturational arrest (MA), but not in men with HYPO. These results suggest that reduced levels of RARA, RARG, RXRB, RXRA, and RXRG are more closely associated with SCOS and MA spermatogenetic failure. These results could contribute to the development of new molecular indicators of spermatogenic dysfunction and might provide novel therapeutic targets for treating male infertility.

Estrogen receptor ß, a regulator of androgen receptor signaling in the mouse ventral prostate.

As estrogen receptor ß-/- (ERß-/-) mice age, the ventral prostate (VP) develops increased numbers of hyperplastic, fibroplastic lesions and inflammatory cells. To identify genes involved in these changes, we used RNA sequencing and immunohistochemistry to compare gene expression profiles in the VP of young (2-mo-old) and aging (18-mo-old) ERß-/- mice and their WT littermates. We also treated young and old WT mice with an ERß-selective agonist and evaluated protein expression. The most significant findings were that ERß down-regulates androgen receptor (AR) signaling and up-regulates the tumor suppressor phosphatase and tensin homolog (PTEN). ERß agonist increased expression of the AR corepressor dachshund family (DACH1/2), T-cadherin, stromal caveolin-1, and nuclear PTEN and decreased expression of RAR-related orphan receptor c, Bcl2, inducible nitric oxide synthase, and IL-6. In the ERß-/- mouse VP, RNA sequencing revealed that the following genes were up-regulated more than fivefold: Bcl2, clusterin, the cytokines CXCL16 and -17, and a marker of basal/intermediate cells (prostate stem cell antigen) and cytokeratins 4, 5, and 17. The most down-regulated genes were the following: the antioxidant gene glutathione peroxidase 3; protease inhibitors WAP four-disulfide core domain 3 (WFDC3); the tumor-suppressive genes T-cadherin and caveolin-1; the regulator of transforming growth factor ß signaling SMAD7; and the PTEN ubiquitin ligase NEDD4. The role of ERß in opposing AR signaling, proliferation, and inflammation suggests that ERß-selective agonists may be used to prevent progression of prostate cancer, prevent fibrosis and development of benign prostatic hyperplasia, and treat prostatitis.

Ablation of Liver X receptors α and ß leads to spontaneous peripheral squamous cell lung cancer in mice.

The etiology of peripheral squamous cell lung cancer (PSCCa) remains unknown. Here, we show that this condition spontaneously develops in mice in which the genes for two oxysterol receptors, Liver X Receptor (LXR) α (Nr1h3) and ß (Nr1h2), are inactivated. By 1 y of age, most of these mice have to be euthanized because of severe dyspnea. Starting at 3 mo, the lungs of LXRα,ß(Dko) mice, but not of LXRα or LXRß single knockout mice, progressively accumulate foam cells, so that by 1 y, the lungs are covered by a "golden coat." There is infiltration of inflammatory cells and progressive accumulation of lipid in the alveolar wall, type 2 pneumocytes, and macrophages. By 14 mo, there are three histological lesions: one resembling adenomatous hyperplasia, one squamous metaplasia, and one squamous cell carcinoma characterized by expression of transformation-related protein (p63), sex determining region Y-box 2 (Sox2), cytokeratin 14 (CK14), and cytokeratin 13 (CK13) and absence of thyroid transcription factor 1 (TTF1), and prosurfactant protein C (pro-SPC). RNA sequencing analysis at 12 mo confirmed a massive increase in markers of M1 macrophages and lymphocytes. The data suggest a previously unidentified etiology of PSCCa: cholesterol dysregulation and M1 macrophage-predominant lung inflammation combined with damage to, and aberrant repair of, lung tissue, particularly the peripheral parenchyma. The results raise the possibility that components of the LXR signaling may be useful targets in the treatment of PSCCa.

Liver X receptor α induces 17ß-hydroxysteroid dehydrogenase-13 expression through SREBP-1c.

Liver X receptors, including LXRα and LXRß, are known to be master regulators of liver lipid metabolism. Activation of LXRα increases hepatic lipid storage in lipid droplets (LDs). 17ß-Hydroxysteroid dehydrogenase-13 (17ß-HSD13), a recently identified liver-specific LD-associated protein, has been reported to be involved in the development of nonalcoholic fatty liver disease. However, little is known about its transcriptional regulation. In the present study, we aimed at determining whether 17ß-HSD13 gene transcription is controlled by LXRs. We found that treatment with T0901317, a nonspecific LXR agonist, increased both 17ß-HSD13 mRNA and protein levels in cultured hepatocytes. It also significantly upregulated hepatic 17ß-HSD13 expression in wild-type (WT) and LXRß-/- mice but not in LXRα-/- mice. Basal expression of 17ß-HSD13 in the livers of LXRα-/- mice was lower than that in the livers of WT and LXRß-/- mice. Moreover, induction of hepatic 17ß-HSD13 expression by T0901317 was almost completely abolished in SREBP-1c-/- mice. Bioinformatics analysis revealed a consensus sterol regulatory element (SRE)-binding site in the promoter region of the 17ß-HSD13 gene. A 17ß-HSD13 gene promoter-driven luciferase reporter and ChIP assays further confirmed that the 17ß-HSD13 gene was under direct control of SREBP-1c. Collectively, these findings demonstrate that LXRα activation induces 17ß-HSD13 expression in a SREBP-1c-dependent manner. 17ß-HSD13 may be involved in the development of LXRα-mediated fatty liver.

Targeting estrogen receptor ß in microglia and T cells to treat experimental autoimmune encephalomyelitis.

A therapeutic goal in the treatment of certain CNS diseases, including multiple sclerosis, amyotrophic lateral sclerosis, and Parkinson disease, is to down-regulate inflammatory pathways. Inflammatory molecules produced by microglia are responsible for removal of damaged neurons, but can cause collateral damage to normal neurons located close to defective neurons. Although estrogen can inactivate microglia and inhibit the recruitment of T cells and macrophages into the CNS, there is controversy regarding which of the two estrogen receptors (ERs), ERα or ERß, mediates the beneficial effects in microglia. In this study, we found that ERß, but not ERα, is expressed in microglia. Using the experimental autoimmune encephalomyelitis (EAE) model in SJL/J mice, we evaluated the benefit of an ERß agonist as a modulator of neuroinflammation. Treatment of EAE mice with LY3201, a selective ERß agonist provided by Eli Lilly, resulted in marked reduction of activated microglia in the spinal cord. LY3201 down-regulated the nuclear transcription factor NF-κB, as well as the NF-κB-induced gene inducible nitric oxide synthase in microglia and CD3(+) T cells. In addition, LY3201 inhibited T-cell reactivity through regulation of indoleamine-2,3-dioxygenase. In the EAE model, treatment with LY3201 decreased mortality in the first 2 wk after disease onset, and also reduced the severity of symptoms in mice surviving for 4 wk. Our data show that ERß-selective agonists, by modulating the immune system in both microglia and T cells, offer promise as a useful class of drugs for treating degenerative diseases of the CNS.

MicroRNA expression profile of mature dendritic cell in chronic rhinosinusitis.

OBJECTIVE: Chronic rhinosinusitis (CRS), which includes CRS without nasal polyposis (CRSsNP) and with nasal polyposis (CRSwNP), shows imbalance of helper T cells (Th) and regulatory T cells (Treg). The balance of Th and Treg cells is orchestrated by dendritic cells (DCs). Recent studies show functions of DCs can be regulated by microRNAs (miRNAs or miRs). This study is aimed to investigate miRNAs expression profiles of peripheral blood DCs in CRS. METHODS: Peripheral blood samples of 30 patients with CRS and 7 patients with nasal septum deviation alone were collected. CD14(+) monocytes were isolated from these samples and differentiated into dendritic cells (DCs). Small RNAs were extracted from mature DCs and reversely transcribed into cDNA by Mir-XTM miRNA First-Strand synthesis method. MiRNA microarrays were used for miRNA expression analysis. Microarray results were validated by real-time PCR performed on five top list target genes. RESULTS: MiRNA microarrays showed that DCs from different types of patients have different sets of differential expressed miRNAs when comparing with Controls; they also share 31 commonly changed miRNAs among all three groups of CRS patients. Of these 31 miRNAs, 5 miRNAs were up-regulated and 25 miRNAs were down-regulated in all three types of CRS, while MiR-1290 was down-regulated in CRSsNP but up-regulated in both atopic CRSwNP and non-atopic CRSwNP. CONCLUSIONS: By comparing miRNA gene expression patterns in 3 types of CRS patients, we have been able to identify candidate miRNAs that might mediate the core pathogenesis of CRS through regulating dendritic cells. These miRNAs could serve as potential therapeutic targets for CRS.

Liver X receptor ß protects dopaminergic neurons in a mouse model of Parkinson disease.

Parkinson disease (PD) is a progressive neurodegenerative disease whose progression may be slowed, but at present there is no pharmacological intervention that would stop or reverse the disease. Liver X receptor ß (LXRß) is a member of the nuclear receptor super gene family expressed in the central nervous system, where it is important for cortical layering during development and survival of dopaminergic neurons throughout life. In the present study we have used the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD to investigate the possible use of LXRß as a target for prevention or treatment of PD. The dopaminergic neurons of the substantia nigra of LXRß(-/-) mice were much more severely affected by MPTP than were those of their WT littermates. In addition, the number of activated microglia and GFAP-positive astrocytes was higher in the substantia nigra of LXRß(-/-) mice than in WT littermates. Administration of the LXR agonist GW3965 to MPTP-treated WT mice protected against loss of dopaminergic neurons and of dopaminergic fibers projecting to the striatum, and resulted in fewer activated microglia and astroglia. Surprisingly, LXRß was not expressed in the neurons of the substantia nigra but in the microglia and astroglia. We conclude that LXR agonists may have beneficial effects in treatment of PD by modulating the cytotoxic functions of microglia.

Anxiety in liver X receptor ß knockout female mice with loss of glutamic acid decarboxylase in ventromedial prefrontal cortex.

Anxiety disorders are the most prevalent mental disorders in adolescents in the United States. Female adolescents are more likely than males to be affected with anxiety disorders, but less likely to have behavioral and substance abuse disorders. The prefrontal cortex (PFC), amygdala, and dorsal raphe are known to be involved in anxiety disorders. Inhibitory input from the PFC to the amygdala controls fear and anxiety typically originating in the amygdala, and disruption of the inhibitory input from the PFC leads to anxiety, fear, and personality changes. Recent studies have implicated liver X receptor ß (LXRß) in key neurodevelopmental processes and neurodegenerative diseases. In the present study, we used elevated plus-maze, startle and prepulse inhibition, open field, and novel object recognition tests to evaluate behavior in female LXRß KO (LXRß(-/-)) mice. We found that the female LXRß(-/-) mice were anxious with impaired behavioral responses but normal locomotion and memory. Immunohistochemistry analysis revealed decreased expression of the enzyme responsible for GABA synthesis, glutamic acid decarboxylase (65+67), in the ventromedial PFC. Expression of tryptophan hydroxylase 2 in the dorsal raphe was normal. We conclude that the anxiogenic phenotype in female LXRß(-/-) mice is caused by reduced GABAergic input from the ventromedial PFC to the amygdala.

Reduction of dendritic spines and elevation of GABAergic signaling in the brains of mice treated with an estrogen receptor ß ligand.

An estrogen receptor (ER) ß ligand (LY3201) with a preference for ERß over ERα was administered in s.c. pellets releasing 0.04 mg/d. The brains of these mice were examined 3 d after treatment had begun. Although estradiol-17ß is known to increase spine density and glutaminergic signaling, as measured by Golgi staining, a clear reduction in spines was evident on the dendritic branches in LY3201-treated mice but no morphological alteration and no difference in the number of dendritic spines on dendritic stems were observed. In the LY3201-treatment group, there was higher expression of glutamic acid decarboxylase (GAD) in layer V of cortex and in the CA1 of hippocampus, more GAD(+) terminals surrounding the pyramidal neurons and less glutamate receptor (NMDAR) on the neurons in layer V. There were no alterations in expression of Iba1 or in Olig2 or CNPase. However, GFAP(+) astrocytes were increased in the LY3201-treatment group. There were also more projections characteristic of activated astrocytes and increased expression of glutamine synthetase (GS). No expression of ERß was detectable in the nuclei of astrocytes. Clearly, LY3201 caused a shift in the balance between excitatory and inhibitory neurotransmission in favor of inhibition. This shift was due in part to increased synthesis of GABA and increased removal of glutamate from the synaptic cleft by astrocytes. The data reveal that treatment with a selective ERß agonist results in changes opposite to those reported in estradiol-17ß-treated mice and suggests that ERα and ERß play opposing roles in the brain.

[Determination of short- and medium-chain chlorinated paraffins in different components of human blood using gas chromatography-electron capture negative ion-low resolution mass spectrometry].

Short- and medium-chain chlorinated paraffins (SCCPs and MCCPs) have attracted significant attention because of their persistence, biotoxicity, bioaccumulation, and long-range migration. Given their worldwide detection in a variety of environmental matrices, concerns related to the high exposure risks of SCCPs and MCCPs to humans have grown. Thus, knowledge of the contamination patterns of SCCPs and MCCPs and their distribution characteristics in the vivo exposure of humans is of great importance. However, little information is available on the contamination of SCCPs and MCCPs in human blood/plasma/serum, mainly because of the difficulty of sample preparation and quantitative analysis. In this study, a new blood sample pretreatment method based on Percoll discontinuous density gradient centrifugation was developed to separate plasma, red blood cells, white blood cells, and platelets from human whole blood. A series of Percoll sodium chloride buffer solutions with mass concentrations of 1.095, 1.077, and 1.060 g/mL were placed in a centrifuge tube from top to bottom to establish discontinuous density gradients. The dosage for each density gradient was 1.5 mL. Human whole blood samples mixed with 0.85% sodium chloride aqueous solution were then added to the top layer of the Percoll sodium chloride solution. After centrifugation, the whole blood was separated into four components. The plasma was located at the top layer of the centrifuge tube, whereas the platelets, white blood cells, and red blood cells were retained at the junction of the various Percoll sodium chloride solutions. The sampling volume of human whole blood and incubation time were optimized, and results indicated that an excessively long incubation time could lead to hemolysis, resulting in a decrease in the recoveries of SCCPs and MCCPs. Therefore, a sampling volume of 1.5 mL and incubation time of 10 min at 4 ℃ were adopted. The cells of the blood components were further broken and extracted by ultrasonic pretreatment, followed by multilayer silica gel column chromatography for lipid removal. The use of 80 mL of n-hexane-dichloromethane (1∶1, v/v) and 50 mL of dichloromethane as the elution solvents (collected together) for the gel column separated the SCCPs and MCCPs from the lipid molecules in the blood samples. Gas chromatography-electron capture negative ion-low resolution mass spectrometry (GC-ECNI-LRMS) was used to determine the SCCPs and MCCPs. Quantification using the corrected total response factor with degrees of chlorination was achieved with linear corrections (R2=0.912 and 0.929 for the SCCPs and MCCPs, respectively). The method detection limits (MDLs) for the SCCPs and MCCPs were 1.57 and 8.29 ng/g wet weight (ww, n=7), respectively. The extraction internal standard recoveries were 67.0%-126.6% for the SCCPs and 69.5%-120.5% for the MCCPs. The developed method was applied to determine SCCPs and MCCPs in actual human whole blood samples. The contents of SCCPs and MCCPs were 10.81-65.23 and 31.82-105.65 ng/g (ww), respectively. Red blood cells exhibited the highest contents of CPs, followed by plasma, white blood cells, and platelets. The proportions of SCCPs and MCCPs in red blood cells and plasma were 70% and 66%, respectively. In all four components, the MCCP contents were higher than the SCCP contents, and the ratios of MCCPs to SCCPs ranged from 1.04 to 3.78. Similar congener patterns of SCCPs and MCCPs were found in the four components of human whole blood. C10-CPs and C14-CPs were predominantly observed in the SCCPs and MCCPs, respectively. In summary, a simple and efficient method was proposed to determine low concentrations of SCCPs and MCCPs in human blood with high sensitivity and selectivity. This method can meet requirements for the quantitative analysis of SCCPs and MCCPs in human blood components, thereby providing technical support for human health risk assessment.

Ablation of Liver X receptor ß in mice leads to overactive macrophages and death of spiral ganglion neurons.

Age-related hearing loss is the most common type of hearing impairment, and is typically characterized by the loss of spiral ganglion neurons (SGNs). The two Liver X receptors (LXRs) are oxysterol-activated nuclear receptors which in adults, regulate genes involved in cholesterol homeostasis and modulation of macrophage activity. LXRß plays a key role in maintenance of health of dopaminergic neurons in the substantia nigra, large motor neurons in the spinal cord, and retinal ganglion cells in adult mice. We now report that LXRß is expressed in the SGNs of the cochlea and that loss of LXRß leads to age-related cochlea degeneration. We found that in the cochlea of LXRß-/- mice, there is loss of SGNs, activation of macrophages, demyelination in the spiral ganglion, decrease in glutamine synthetase (GS) expression and increase in glutamate accumulation in the cochlea. Part of the cause of damage to the SGNs might be glutamate toxicity which is known to be very toxic to these cells. Our study provides a so far unreported role of LXRß in maintenance of SGNs whose loss is a very common cause of hearing impairment.

Neuroglobin Attenuates Beta Amyloid-Induced Apoptosis Through Inhibiting Caspases Activity by Activating PI3K/Akt Signaling Pathway.

Excessive accumulation and deposition of amyloid-beta (Aß) has been considered as a pivotal event in the pathogenesis of Alzheimer's disease (AD). Neuronal apoptosis is one of the characteristics of AD, which is a possible mechanism underlying Aß-induced neuronal neurotoxicity. Neuroglobin (Ngb) is a newly discovered vertebrate heme protein that exhibits neuroprotective functions against cell death associated with hypoxic and amyloid insult. However, until now, the exact mechanism of neuroglobin's protective action has not been determined. To investigate the potential neuroprotective roles and mechanisms of Ngb, transgenic AD mice (APPswe/PSEN1dE9) and SH-SY5Y cells transfected with pAPPswe were enrolled into the study. In vivo, overexpression of Ngb via intracerebroventricular injection with pNgb attenuated memory, cognitive impairment, and plaque generations. In pAPPswe transfected SH-SY5Y cells, Ngb not only decreased the generation of Aß42, but also attenuated mitochondrial dysfunction and apoptosis through suppressing the activation of caspase-3, caspase-9 by Akt activating phosphorylation, which were restrained by phosphatidylinositol 3-kinase inhibitor (LY294002). Our data indicate the anti-apoptotic property of Ngb may play a neuroprotective role against AD.

An ERß agonist induces browning of subcutaneous abdominal fat pad in obese female mice.

Estrogen, via estrogen receptor alpha (ERα), exerts several beneficial effects on metabolism and energy homeostasis by controlling size, enzymatic activity and hormonal content of adipose tissue. The actions of estrogen on sympathetic ganglia, which are key players in the browning process, are less well known. In the present study we show that ERß influences browning of subcutaneous adipose tissue (SAT) via its actions both on sympathetic ganglia and on the SAT itself. A 3-day-treatment with a selective ERß agonist, LY3201, induced browning of SAT in 1-year-old obese WT and ERα-/- female mice. Browning was associated with increased expression of ERß in the nuclei of neurons in the sympathetic ganglia, increase in tyrosine hydroxylase in both nerve terminals in the SAT and sympathetic ganglia neurons and an increase of ß3-adrenoceptor in the SAT. LY3201 had no effect on browning in young female or male mice. In the case of young females browning was already maximal while in males there was very little expression of ERß in the SAT and very little expression of the ß3-adrenoceptor. The increase in both sympathetic tone and responsiveness of adipocytes to catecholamines reveals a novel role for ERß in controlling browning of adipose tissue.

The Value of Proton Magnetic Resonance Spectroscopy in High-Intensity Focused Ultrasound Treatment of Experimental Liver Cancer.

High-intensity focused ultrasound (HIFU) is a rapidly developing, non-invasive technique for local treatment of solid tumors that produce coagulative tumor necrosis. This study is aimed to investigate the feasibility of proton magnetic resonance spectroscopy (MRS) on early assessing treatment of HIFU ablation in rabbit with VX2 liver tumor. HIFU ablation was performed on normal liver and VX2 tumor in rabbit, and MRS was performed on normal liver and VX2 tumor before and 2 days after 100% HIFU ablation or 80% ablation in tumor volume. Choline (Cho) and choline/lipid (Cho/Lip) ratios between complete and partial HIFU ablation of tumor were compared. Tissues were harvested and sequentially sliced to confirm the necrosis. In normal liver, the Cho value liver was not obviously changed after HIFU (P > .05), but the Cho/Lip ratio was decreased (P < .05). Cho in liver VX2 tumor was much higher than that in normal liver (P < .001). Cho and Cho/Lip ratio were significantly decreased in tumor after complete HIFU ablation and partial HIFU ablation, and the Cho value in complete HIFU tumor ablation did not show any difference from that in normal liver after HIFU (P > .05); however, the Cho value in partial ablation was still higher than that in normal liver before or in tumor after complete HIFU treatment due to the residual part of tumors, and Cho/Lip ratio is lower than that in complete HIFU treatment (P < .001). The changes in MRS parameters were consistent with histopathologic changes of the tumor tissues after treatment. MRS could differentiate the complete tumor necrosis from residual tumor tissue, when combined with magnetic resonance imaging. We conclude that MRS may be applied as an important, non-invasive biomarker for monitoring the thoroughness of HIFU ablation.