Tet2 acts in the lateral habenula to regulate social preference in mice.

The lateral habenula (LHb) has been considered a moderator of social behaviors. However, it remains unknown how LHb regulates social interaction. Here, we show that the hydroxymethylase Tet2 is highly expressed in the LHb. Tet2 conditional knockout (cKO) mice exhibit impaired social preference; however, replenishing Tet2 in the LHb rescues social preference impairment in Tet2 cKO mice. Tet2 cKO alters DNA hydroxymethylation (5hmC) modifications in genes that are related to neuronal functions, as is confirmed by miniature two-photon microscopy data. Further, Tet2 knockdown in the glutamatergic neurons of LHb causes impaired social behaviors, but the inhibition of glutamatergic excitability restores social preference. Mechanistically, we identify that Tet2 deficiency reduces 5hmC modifications on the Sh3rf2 promoter and Sh3rf2 mRNA expression. Interestingly, Sh3rf2 overexpression in the LHb rescues social preference in Tet2 cKO mice. Therefore, Tet2 in the LHb may be a potential therapeutic target for social behavior deficit-related disorders such as autism.

5hmC modification regulates R-loop accumulation in response to stress.

R-loop, an RNA-DNA hybrid structure, arises as a transcriptional by-product and has been implicated in DNA damage and genomic instability when excessive R-loop is accumulated. Although previous study demonstrated that R-loop is associated with ten-eleven translocation (Tet) proteins, which oxidize 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), the sixth base of DNA. However, the relationship between R-loop and DNA 5hmC modification remains unclear. In this study, we found that chronic restraint stress increased R-loop accumulation and decreased 5hmC modification in the prefrontal cortex (PFC) of the stressed mice. The increase of DNA 5hmC modification by vitamin C was accompanied with the decrease of R-loop levels; on the contrary, the decrease of DNA 5hmC modification by a small compound SC-1 increased the R-loop levels, indicating that 5hmC modification inversely regulates R-loop accumulation. Further, we showed that Tet deficiency-induced reduction of DNA 5hmC promoted R-loop accumulation. In addition, Tet proteins immunoprecipitated with Non-POU domain-containing octamer-binding (NONO) proteins. The deficiency of Tet proteins or NONO increased R-loop levels, but silencing Tet proteins and NONO did not further increase the increase accumulation, suggesting that NONO and Tet proteins formed a complex to inhibit R-loop formation. It was worth noting that NONO protein levels decreased in the PFC of stressed mice with R-loop accumulation. The administration of antidepressant fluoxetine to stressed mice increased NONO protein levels, and effectively decreased R-loop accumulation and DNA damage. In conclusion, we showed that DNA 5hmC modification negatively regulates R-loop accumulation by the NONO-Tet complex under stress. Our findings provide potential therapeutic targets for depression.

KRAS gene status in gastric signet-ring cell carcinoma patients and acts as biomarker of MEK inhibitor.

BACKGROUND: Signet-ring cell carcinoma (SRCC) is a specific subtype of stomach cancer with unique epidemiology. Here, we sought to explore the role of KRAS in SRCC. METHODS: KRAS status was studied both in The Cancer Genome Atlas (TCGA) and internal cohorts. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were performed in formalin-fixed and paraffin-embedded (FFPE) samples. We explored patients' survival and clinicopathological characteristics in terms of KRAS mutation and expression. We also explored KRAS status and drug response curve of MEK/mTOR inhibitors in SRCC cell lines. RESULTS: Patients with KRAS mutations and copy number variation (CNV) showed higher mRNA level compared to non-mutant cases (P=0.003 and P<0.001). In internal cohort, 15 samples harbored KRAS mutations. Survival analysis showed that these patients had significantly lower overall survival (OS) (P=0.048). We further analyzed 75 patients with sufficient FFPE samples. Eight patients showed KRAS mutations and one patient showed KRAS amplification. The median OS was 12.5 months for patients with KRAS mutation, and 19.5 months for patients without KRAS mutation (P=0.005). Positive expression of KRAS as shown by IHC was detected in majority of SRCC samples, which was higher than our intestinal cohort (28% vs. 12.6%, P=0.033). We further explored the correlation between KRAS status and drug sensitivity in 4 SRCC cell lines. SNU601 and SNU668, which harbored KRAS mutation, were hypersensitive to MEK and mTOR inhibitors than KRAS wide type cell lines KATO-III and NUGC-4. CONCLUSIONS: Our findings demonstrate that KRAS gene plays an important role in SRCC and reveals therapeutic potential of targeting tumors by inhibiting MEK and mTOR pathways.

Epigenetic Mechanisms of Paternal Stress in Offspring Development and Diseases.

The major biological function of the sperm cell is to transmit the paternal genetic and epigenetic information to the embryo as well as the following offspring. Sperm has a unique epigenome. An increasing body of epidemiological study supports that paternal stress induced by environmental exposures and lifestyle can modulate the sperm epigenome (including histone modification, DNA methylation, and noncoding RNA expression), sperm-egg fusion, embryo development, and offspring health. Based on the existing literature, we have summarized the paternal exposure on sperm epigenome along with the representative phenotypes of offspring and the possible mechanism involved.

DJ-1 regulates tyrosine hydroxylase expression through CaMKKß/CaMKIV/CREB1 pathway in vitro and in vivo.

Lack of dopamine production and neurodegeneration of dopaminergic neurons in the substantia nigra are considered as the major characteristics of Parkinson's disease, a prevalent movement disorder worldwide. DJ-1 mutation leading to loss of its protein functions is a genetic factor of PD. In this study, our results illustrated that DJ-1 can directly interact with Ca2+ /calmodulin-dependent protein kinase kinase ß (CaMKKß) and modifies the cAMP-responsive element binding protein 1 (CREB1) activity, thus regulates tyrosine hydroxylase (TH) expression. In Dj-1 knockout mouse substantia nigra, the levels of TH and the phosphorylation of CREB1 Ser133 are significantly decreased. Moreover, Dj-1 deficiency suppresses the phosphorylation of CaMKIV (Thr196/200) and CREB1 (Ser133), subsequently inhibits TH expression in vitro. Furthermore, Knockdown of Creb1 abolishes the effects of DJ-1 on TH regulation. Our data reveal a novel pathway in which DJ-1 regulates CaMKKß/CaMKIV/CREB1 activities to facilitate TH expression.

Poly-PR in C9ORF72-Related Amyotrophic Lateral Sclerosis/Frontotemporal Dementia Causes Neurotoxicity by Clathrin-Dependent Endocytosis.

GGGGCC repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). It has been reported that hexanucleotide repeat expansions in C9ORF72 produce five dipeptide repeat (DPR) proteins by an unconventional repeat-associated non-ATG (RAN) translation. Within the five DPR proteins, poly-PR and poly-GR that contain arginine are more toxic than the other DPRs (poly-GA, poly-GP, and poly-PA). Here, we demonstrated that poly-PR peptides transferred into cells by endocytosis in a clathrin-dependent manner, leading to endoplasmic reticulum stress and cell death. In SH-SY5Y cells and primary cortical neurons, poly-PR activated JUN amino-terminal kinase (JNK) and increased the levels of p53 and Bax. The uptake of poly-PR peptides by cells was significantly inhibited by knockdown of clathrin or by chlorpromazine, an inhibitor that blocks clathrin-mediated endocytosis. Inhibition of clathrin-dependent endocytosis by chlorpromazine significantly blocked the transfer of poly-PR peptides into cells, and attenuated poly-PR-induced JNK activation and cell death. Our data revealed that the uptake of poly-PR undergoes clathrin-dependent endocytosis and blockade of this process prevents the toxic effects of synthetic poly-PR peptides.

Pharmacological activation of REV-ERBα represses LPS-induced microglial activation through the NF-κB pathway.

REV-ERBα, the NR1D1 (nuclear receptor subfamily 1, group D, member 1) gene product, is a dominant transcriptional silencer that represses the expression of genes involved in numerous physiological functions, including circadian rhythm, inflammation, and metabolism, and plays a crucial role in maintaining immune functions. Microglia-mediated neuroinflammation is tightly associated with various neurodegenerative diseases and psychiatric disorders. However, the role of REV-ERBα in neuroinflammation is largely unclear. In this study, we investigated whether and how pharmacological activation of REV-ERBα affected lipopolysaccharide (LPS)-induced neuroinflammation in mouse microglia in vitro and in vivo. In BV2 cells or primary mouse cultured microglia, application of REV-ERBα agonist GSK4112 or SR9011 dose-dependently suppressed LPS-induced microglial activation through the nuclear factor kappa B (NF-κB) pathway. In BV2 cells, pretreatment with GSK4112 inhibited LPS-induced phosphorylation of the inhibitor of NF-κB alpha (IκBα) kinase (IκK), thus restraining the phosphorylation and degradation of IκBα, and blocked the nuclear translocation of p65, a NF-κB subunit, thereby suppressing the expression and secretion of the proinflammatory cytokines, such as interleukin 6 (IL-6) and tumor necrosis factor α (TNFα). Moreover, REV-ERBα agonist-induced inhibition on neuroinflammation protected neurons from microglial activation-induced damage, which were also demonstrated in mice with their ventral midbrain microinjected with GSK4112, and then stimulated with LPS. Our results reveal that enhanced REV-ERBα activity suppresses microglial activation through the NF-κB pathway in the central nervous system.

Tyrosine hydroxylase down-regulation after loss of Abelson helper integration site 1 (AHI1) promotes depression via the circadian clock pathway in mice.

Abelson helper integration site 1 (AHI1) is associated with several neuropsychiatric and brain developmental disorders, such as schizophrenia, depression, autism, and Joubert syndrome. Ahi1 deficiency in mice leads to behaviors typical of depression. However, the mechanisms by which AHI1 regulates behavior remain to be elucidated. Here, we found that down-regulation of expression of the rate-limiting enzyme in dopamine biosynthesis, tyrosine hydroxylase (TH), in the midbrains of Ahi1-knockout (KO) mice is responsible for Ahi1-deficiency-mediated depressive symptoms. We also found that Rev-Erbα, a TH transcriptional repressor and circadian regulator, is up-regulated in the Ahi1-KO mouse midbrains and Ahi1-knockdown Neuro-2a cells. Moreover, brain and muscle Arnt-like protein 1 (BMAL1), the Rev-Erbα transcriptional regulator, is also increased in the Ahi1-KO mouse midbrains and Ahi1-knockdown cells. Our results further revealed that AHI1 decreases BMAL1/Rev-Erbα expression by interacting with and repressing retinoic acid receptor-related orphan receptor α, a nuclear receptor and transcriptional regulator of circadian genes. Of note, Bmal1 deficiency reversed the reduction in TH expression induced by Ahi1 deficiency. Moreover, microinfusion of the Rev-Erbα inhibitor SR8278 into the ventral midbrain of Ahi1-KO mice significantly increased TH expression in the ventral tegmental area and improved their depressive symptoms. These findings provide a mechanistic explanation for a link between AHI1-related behaviors and the circadian clock pathway, indicating an involvement of circadian regulatory proteins in AHI1-regulated mood and behavior.

[Effects of levonorgestrel intrauterine system on the expressions of estrogen receptor, progesterone receptor and insulin-like growth factor-1].

OBJECTIVE: To explore the effects of levonorgestrel intrauterine system on estrogen receptor (ER) and progesterone receptor (PR), insulin-like growth factor-1 (IGF-1) of endometrial polyps and surrounding endometrial tissue. METHODS: A total of 250 cases of hysteroscopic resection for endometrial polyps were divided into study group (n = 125) with placement of levonorgestrel intrauterine system after hysteroscopic electroresection and control group (n = 125) without placement. The intraoperative and postoperative expressions of ER, PR and IGF-1 in endometrial biopsy specimens were measured by immunohistochemistry. They were followed up for 2 years to analyze the recurrence of endometrial polyp. RESULTS: All patients had successful surgery. When intraoperative and postoperative specimens were compared with regards to the expressions of ER, PR and IGF-1, significant differences existed between two groups. During a 2-year follow-up, the recurrence of endometrial polyp was 10.89% in control group versus 0 in study group. CONCLUSION: Levonorgestrel intrauterine system may inhibit the recurrence and formation of endometrial polyp through lowering the expressions of ER, PR and IGF-1.

[Clinical significance of combined detection of thinprep cytology test and high risk human papillomavirus hybrid capture 2 assay in the screening and recurrence predication of cervical intraepithelial neoplasia].

OBJECTIVE: To evaluate the clinical significance of combined detection of TCT and HR-HPV-HC2 assay in the screening and recurrence prediction of CIN. METHODS: A total of 725 patients with suspected cervical abnormalities received TCT plus HR-HPV-HC2 detection and histopathological examinations.203 CINII-III patients received TCT and HC2 detection and histopathological examinations after treated with loop electrosurgical excision procedure or cold knife conization operation. The results of pathological examinations were gold standard. The sensitivity and specificity of TCT, HR-HPV-HC2 and TCT plus HR-HPV-HC2 were analyzed. RESULTS: The missed diagnosis and misdiagnosis rate of TCT and HC2 alone were 19.23%, 12.50% and 48.15%, 74.07% and sensitivity and specificity 90.02%, 97.01% and 51.85%, 17.76% respectively. The sensitivity, missed diagnosis rate and negative predictive value were 98.25%, 1.75% and 89.70% when TCT or HR-HPV-HC2 met the CIN diagnostic criteria. The specificity and positive predictive value were 58.95% and 72.80% when TCT and HR-HPV-HC2 met the CIN diagnostic criteria. The specificity of TCT or HR-HPV-HC2 alone was 80% for the prediction of recurrence at Month 6 postoperation. The specificity of TCT plus HR-HPV-HC2 was 98.27% for the prediction of recurrence at Month 3 postoperation and the negative predictive value was 98.75% at Month 6 postoperation. The sensitivity of TCT or HR-HPV-HC2 was 97.22% for the prediction of recurrence at Month 3/6. And the negative predictive value was 99.17% at Month 6 postoperation. CONCLUSION: The combined detection of TCT and HR-HPV-HC2 may improve the sensitivity and specificity of CIN and the predication of its postoperative recurrence.

Breast cancer resistance protein expression and 5-fluorouracil resistance.

OBJECTIVE: To filtrate breast cancer resistance protein (BCRP)-mediated resistant agents and to investigate clinical relationship between BCRP expression and drug resistance. METHODS: MTT assay was performed to filtrate BCRP-mediated resistant agents with BCRP expression cell model and to detect chemosensitivity of breast cancer tissue specimens to these agents. A high performance liquid chromatography (HPLC) assay was established, and was used to measure the relative dose of intracellular retention resistant agents. RT-PCR and immunohistochemistry (IHC) were employed to investigate the BCRP expression in breast cancer tissue specimens. RESULTS: MTT assay showed that the expression of BCRP increased with the increasing resistance of 5-fluorouracil (5-Fu) (P<0.05, n=3) in the cell model, while HPLC assay indicated that the intracellular retention dose of 5-Fu was significantly correlated with the expression of BCRP (r=-0.897, P<0.05, n=3). A total of 140 breast cancer tissue specimens were collected. BCRP-positive expression was detected in forty-seven specimens by both RT-PCR and IHC. As shown by MTT assay subsequently, the resistance index (RI) of 47 BCRP-positive breast cancer tissue specimens to 5-Fu was 7-12 times as high as that of adjacent normal tissue samples. BCRP expression was related to 5-Fu resistance (R2=0.8124, P<0.01). CONCLUSION: Resistance to 5-Fu can be mediated by BCRP. Clinical chemotherapy for breast cancer patients can be optimized based on BCRP-positive expression.