Impaired wakefulness and rapid eye movement sleep in dopamine-deficient mice

Despite the established roles of the dopaminergic system in promoting arousal, the effects of loss of dopamine on the patterns of sleep and wakefulness remain elusive. Here, we examined the sleep architecture of dopamine-deficient (DD) mice, which were previously developed by global knockout of tyrosine hydroxylase and its specific rescue in noradrenergic and adrenergic neurons. We found that DD mice have reduced time spent in wakefulness. Unexpectedly, DD mice also exhibited a marked reduction in the time spent in rapid eye movement (REM) sleep. The electroencephalogram power spectrum of all vigilance states in DD mice were also affected. These results support the current understanding of the critical roles of the dopaminergic system in maintaining wakefulness and also implicate its previously unknown effects on REM sleep.

Increase in excitability of hippocampal neurons during novelty-induced hyperlocomotion in dopamine-deficient mice

Dopamine is involved in many important brain functions, including voluntary motor movement. Dysfunction of the dopaminergic system can induce motor impairments, including Parkinson’s disease. We previously found that dopamine-deficient (DD) mice became hyperactive in a novel environment 72 h after the last injection of L-3,4-dihydroxyphenylalanine (L-DOPA) when dopamine was almost completely depleted. In the present study, we investigated neuronal activity in hippocampal subregions during hyperactivity by measuring Fos expression levels using immunohistochemistry. Dopamine-deficient mice were maintained on daily intraperitoneal injections of 50 mg/kg L-DOPA. Seventy-two hours after the last L-DOPA injection, DD mice were exposed to a novel environment for 1, 2, or 4 h, and then brains were collected. In wildtype mice, the number of Fos-immunopositive neurons significantly increased in the hippocampal CA1 region after 1 h of exposure to the novel environment and then decreased. In DD mice, the number of Fos-immunopositive neurons gradually increased and then significantly increased after 4 h of exposure to the novel environment. The number of Fos-immunopositive neurons also significantly increased in the CA3 region and dentate gyrus in DD mice after 4 h of exposure to the novel environment. These results indicate that the delayed and prolonged excitation of hippocampal neurons in the CA1, CA3, and dentate gyrus that is caused by dopamine depletion might be involved in hyperactivity in DD mice.

Risk of Developing Hepatocellular Carcinoma following Depressive Disorder Based on the Expression Level of Oatp2a1 and Oatp2b1

Background. Accumulating evidence from prospective epidemiological studies has showed that depression disorder (DD) is a risk factor for cancer. The aim of this study is to explore the association of DD and the overall occurrence risk of hepatocellular carcinoma (HCC) and the mechanism. Methods. In this study, 60 mice were randomly divided into four groups: Control group, DD group, HCC group, HCC-DD group. Mice received a chronic dose of reserpine to establish depression model, followed by Diethylnitrosamine and Carbon tetrachloride administration to establish HCC models. Behavioral depression was assessed by sucrose preference test (SPT) and the expression of Serotonin 1A (5-HT1A) receptor in the hippocampal. The expression of Oatp2a1 and Oatp2b1 in the digestive system tissues was detected by PCR and western blotting. Results. Reserpine-administrated mice had a reducing sucrose preference at Day 14 compared with blank mice (P<0.05). The expression of 5-HT1A receptor in the hippocampal was decreased in DD mice compared with blank mice. The survival analysis indicated that the HCC mice with DD have poorer survival rate compared with the HCC mice. Compared with HCC mice, the expression of Oatp2a1 and Oatp2b1 was lower in liver and stomach tissue and higher in hepatic carcinoma and colon tissue of HCC-DD mice (P<0.05), and the expression of Oatp2a1 was higher in the spleen tissue of HCC-DD mice while the expression of Oatp2b1 was lower (P<0.05). However, no difference was found in the expression of Oatp2a1 and Oatp2b1 in the small intestine tissue between HCC group and HCC-DD group. Conclusions. DD was the adverse factors for the overall occurrence risk of HCC. Mechanistically, be the downregulation of Oatp2a1 and Oatp2b1 in liver tissue induced by DD might be involved.

Abstract 414: Ath28 Congenic Mice Confirm Atherosclerosis Modifying Gene on the Distal End of Chromosome 2

Objective: Strain intercrosses between apoE-deficient mice on the AKR (athero-resistant) and DBA/2 (athero-sensitive) strains identified the Ath28 quantitative trait locus (QTL) on the distal end of chromosome 2 (chr 2). We bred congenic mice on the DBA/2 background containing AKR alleles on chr 2 from 172.5 to 180 Mb in order to confirm the presence of atherosclerosis modifying genes in this region. Methods and Results: We used marker assisted backcrossing to generate DBA/2.AKR (chr 2) apoE-deficient congenic mice. High density genotyping using the MegaMuga array revealed that the AKR strain donated DNA on chr 2 from 171.5 Mb to the end of the chromosome, with the most distal marker tested at 180 Mb, while DBA/2 markers were found elsewhere in the genome. Mice heterozygote for the congenic interval were intercrossed to generate progeny homozygous for the AKR allele (AA) or the DBA/2 allele (DD). Female and male mice were fed a chow diet and sacrificed at 16 weeks of age. In the females, there was no effect on body weight; the total cholesterol levels trended 13% lower for the DD vs. AA mice (p=0.07); and, the HDL-C levels were 55% higher for the DD vs. AA mice (p=0.05). Despite having lower total cholesterol levels, the DD mice had 68% larger aortic root lesion areas (p=0.05, N=10 and 14 for AA and DD mice, respectively). In the males, the body weight was 12% higher in the DD vs. AA mice (p<0.01); the total cholesterol levels were similar in the DD and AA mice; and, the HDL-C levels were 73% higher for the DD vs. AA mice (p<0.05). The male DD mice had 27% larger aortic root lesion areas (NS), but we are adding more mice to this study to determine if this is significant (N=6 and 11 for AA and DD mice, respectively). Conclusions: Ath28 congenic mice confirm the presence of an atherosclerosis modifying gene on the distal end of chr 2 in chow-fed females. This interval in chr 2 contains over 100 genes. Our prior identification of missense variants between these strains, as well as transcriptomic and eQTL analyses have identified some candidate genes in this interval including Cstf1, Ctcfl, Zbp1, Ankrd60, Gnas, Cdh4, Ctsz, and Rae1 . Further functional genomic studies and creating mice with smaller congenic intervals will be needed in order to narrow the list of candidate genes for in vivo testing.

Abstract 284: Ovariectomy Exacerbates Mitochondrial Dysfunction, Oxidative Stress and Fibrosis to Cause Diastolic Dysfunction

Heart failure with preserved ejection fraction, characterized by diastolic dysfunction (DD) is most common in post-menopausal women, suggesting a link with estrogen (E2). However, the underlying mechanisms are not well understood. In this study we tested how ovariectomy (OVX)-induced E2-deficiency exacerbates DD. Female C57BL6/J mice (12 weeks old) underwent a sham or OVX surgery, and were administered either normal drinking water or L-N G -Nitroarginine methyl ester (L-NAME, 0.3 mg/ml in 1% NaCl solution) in the drinking water and Angiotensin II (AngII, 1.2 mg/kg/day) via subcutaneous osmotic pumps for 5 weeks to induce DD. Subsets of the OVX-DD mice were treated with E2, E2-receptor (ER) α agonist PPT, ERβ agonist DPN or G-protein ER-1 agonist G1. Echocardiography revealed reduced left ventricular (LV) internal diameter, LV volume, cardiac output but increased LV posterior wall thickness in OVX-DD mice compared with sham-DD mice. However, ejection fractions and fractional shortening were normal in all groups. Doppler studies showed E/A ratios dramatically suppressed and E/e’ ratios increased in mice treated with L-NAME+AngII, with larger changes in OVX mice. Extensive fibrosis was seen in OVX-DD hearts than sham-DD hearts. ADP-supported cardiac mitochondrial function with pyruvate-malate was moderately reduced in OVX-DD mice along with greater production of reactive oxygen species. Ionoptix studies revealed delayed relaxation but maintained contraction in cardiomyocytes isolated from OVX-DD mice, similar to effects of low-dose oligomycin in control mice. These data suggest that E2 deficiency is associated with impaired mitochondrial function to cause small decreases in ATP formation, which impair diastolic relaxation but not contraction. Treatment with E2, PPT, DPN and G1 reduced E/e’ ratios in OVX-DD mice, with the greatest effects of G1. Treatment of mice with L-NAME in this model excludes the previously described action of G1 through nitric oxide. In conclusion, OVX exacerbates mitochondrial dysfunction, oxidative stress and fibrosis in non-ischemic HF, leading to exacerbated DD. These may be some of the mechanisms by which E2 protects the myocardium in females. Activation of GPER1 offers a novel therapeutic target for DD after menopause.

Transcription Factor Bach1 and Bach2 Control Common Myeloid Progenitor Cell Differentiation Under Infectious Stimuli

Background: Erythrocyte and granulocyte/macrophage develop from common myeloid progenitor (CMP) (Akashi et al., 2000). Differentiation of hematopoietic progenitor cells is precisely controlled by multiple transcription factors, among which GATA1, C/EBPα, C/EBPβ and Spi-C play pivotal roles in erythrocyte and granulocyte/macrophage differentiation (Mancini et al., 2012; Pongubala et al., 2008; Hirai et al., 2006; Haldar et al., 2014). However, the mechanism by which the differentiation of CMP controlled under infectious condition has been unclear. Bach1 and Bach2 belong to the basic region-leucine zipper family and recognize Maf-recognition elements (Oyake et al., 1996). They promote B cell development by repressing the myeloid genes such as Cebpb and Spic in common lymphoid progenitor cells (Itoh-Nakadai et al., 2014). In addition, Bach1 regulates several target genes related to iron/heme homeostasis such as globin genes and hemeoxygenase-1, and Bach2 may similarly regulate these genes (Igarashi, 2014). Therefore, it is expected that both Bach1 and Bach2 play redundant roles in erythropoiesis. To figure out their roles in erythroid and myeloid cell differentiation, we performed hematological and transcriptomics analyses using Bach1-/- Bach2-/- (double-deficient; DD) mice. Methods: The generation of DD mice on the C57BL/6J background and Bach2 reporter mice with red fluorescent protein coding cDNA inserted in the Bach2 locus were described previously (Itoh-Nakadai et al., 2014). Mice between 8-12 weeks old were analyzed in the present study. Bone marrow (BM) cells were stained with specific combinations of antibodies to identify erythroid/myeloid progenitor and mature cells (Sheila et al., 2008; Cornelis et al., 2007; Socolovsky et al., 2001). Flow cytometry analysis and cell sorting were performed by using FACSAriaⅡ(BD) and FlowJo software (TreeStar). For infectious simulation of CMP, sorted CMPs were incubated with 1μg/ml LPS (Sigma) for 48h and RNA was purified with RNeasy micro kit (Qiagen). Quantitative PCR by using SuperscriptⅢ reverse transcriptase (Invitrogen) and Light Cycler system (Roche) was performed according to manufacturer's instructions. Microarray analysis by using Sure-Print G3 mouse GE microarray slide (Agilent) was performed as previously described (Itoh-Nakadai et al., 2014) and the results were analyzed by using GeneSpring software (Agilent). We used Gene Set Enrichment Analysis (GSEA) to interpret gene expression data (Subramanian et al., 2005; Mootha et al., 2003). LPS stimulation (50 μg/body) of mice was performed as previously described (Ryan et al., 2008). Data were analyzed by the two-sided Student's t-test and p - values of <0.05 were considered statistically significant. Results: DD mice show mild normocytic anemia compered to wild-type (WT), Bach1-/-, and Bach2-/- mice (hemoglobin; 14.4±0.2, 14.0±0.3, 13.5±0.3 and 11.9±0.7 g/dl, for WT, Bach1-/-, Bach2-/- and DD, respectively, p<0.05 for comparison between DD and other genotypes, n=7). Immature and mature erythroblast populations were significantly decreased in BM of DD (immature; 25.8±1.78, 15.6±1.4, mature; 27.6±3.3, 17.4±2.3×106/body for WT and DD, respectively, p<0.05, n=6). Megakaryocyte-erythroid progenitor (MEP)/granulocyte-monocyte progenitor (GMP) ratio was significantly decreased in BM of DD (MEP/GMP: 0.13±0.01, 0.07±0.01 for WT and DD, respectively, p<0.05, n=5). Bach2 expression was detected in CMP, MEP and even GMP by using Bach2-RFP mice. LPS stimulation of WT CMP significantly decreased mRNA levels of Bach1, Bach2 and Gata1. On the other hand, Cebpb and Spic mRNA levels were significantly increased. LPS stimulation of WT mice induced significant increase of granulocyte and decrease of erythrocyte and B lymphocyte in BM, which was consistent with previous reports. It was also shown that LPS stimulation significantly decreased MEP/ GMP ratio. According to the clustering analysis of the microarray data of CMP sorted from WT and DD mice, they showed clearly different expression profiles. GSEA showed that CMP of DD skewed to myeloid cell lineage and lost the erythroid gene expression compared to WT. Conclusions: Bach1 and Bach2 control the differentiation of CMP to erythroid cell or myeloid cell by repressing myeloid genes such as Cebpb and Spic. Infectious stimuli may promote myeloid cell differentiation by reducing the expression of Bach1 and Bach2 in CMP. Disclosures Fujiwara: Chugai Pharmaceutical CO., LTD: Research Funding. Harigae:Chugai Pharmaceutical CO., LTD: Research Funding.

Liver nuclear hormone receptor PPAR, LXR and RXR expression and blood lipid and glucose levels in susceptible and resistent to hepatocarcinogenesis strain of mice

Earlier it was shown that male mice of the DD/He strain were highly susceptible to ortho-aminoasotoluene (OAT) induced hepatocarcinogenesis, and resistant to spontaneous liver tumor development as compared to the СС57BR/Mv strain. In the present work we have made a comparative investigation of peroxisome proliferator-activated receptor (PPAR), liver X-receptor (LXR) and retinoic X-receptor (RXR) mRNA levels in liver as well as concentrations of corticosterone, glucose, lipids and insulin in blood of male DD/He and СС57BR/Mv mice. Using the multiplex RT-PCR method it was found that PPAR-α, PPAR-γ, RXR-α and RXR-β mRNA content was essentially decreased in the liver of DD mice as compared to mice of the СС57BR strain. No significant interstrain differences of LXR-α and LXR-β mRNA content were found. In DD micetere was more then the 3-fold decrease of blood content of corticosterone, which is involved in PPAR and RXR regulation. DD mice demonstrated a significant decrease in blood serum glucose and insulin concentrations as well as higher reactivity to insulin as compared with СС57BR mice. Elevated blood total cholesterol and cholesterol HDL level were found in DD mice whereas triglyceride content was basically the same in both mouse strains. It is known that glucocorticoids, PPAR and RXR play crucial role in transcription regulation of inflammation response. Therefore our data allow to suggest that decreased corticosterone level in blood, PPAR and RXR mRNA content in liver of the DD strain may lead to induction of inflammation by OAT exposure, resulting in a high incidence of tumorigenesis in this strain.

H-2D Ligand Expression by Ly49A+ Natural Killer (NK) Cells Precludes Ligand Uptake from Environmental Cells

To study the adaptation of natural killer (NK) cells to their major histocompatibility complex (MHC) class I environment we have established a novel mouse model with mosaic expression of H-2Dd using a Cre/loxP system. In these mice, we noticed that NK cells expressing the inhibitory receptor for Dd, Ly49A, were specifically underrepresented among cells with low Dd levels. That was due to the acquisition of Dd molecules by the Ly49A+ NK cells that have lost their Dd transgene. The uptake of H-2D molecules via the Ly49A receptor was restricted to strong ligands of Ly49A. Surprisingly, when Ly49A+ NK cells were Dd+, uptake of the alternative ligand Dk was not detectable. Similarly, one anti-Ly49A mAb (A1) bound inefficiently when Ly49A was expressed on Dd+ NK cells. Concomitantly, functional assays demonstrated a reduced capacity of Ly49A to inhibit H-2bDd as compared with H-2b NK cells, rendering Ly49A+ NK cells in Dd+ mice particularly reactive. Minor reductions of Dd levels and/or increases of activating ligands on environmental cells may thus suffice to abrogate Ly49A-mediated NK cell inhibition. The mechanistic explanation for all these phenomena is likely the partial masking of Ly49A by Dd on the same cell via a lateral binding site in the H-2Dd molecule.