The aim of our study was to detect differentially regulated proteins and specific signaling pathways in Mongolian gerbil brains during chronic Toxoplasma gondii (T.gondii) PRU strain infection. We use a iTRAQ-based strategy to detecte 4935 proteins, out of which 110 proteins were differentially expressed (>/=2.0-fold, p value <0.05) when the brain of gerbils infected with T.gondii was compared to control brain tissues. We confirmed the authenticity and the accuracy of iTRAQ results through quantitative real-time PCR and western blot (WB), which was consistent with mass spectrometry analysis. Pathway analysis and GO (Gene Ontology) annotations indicated the deregulation of several pathways related to immune response, metabolism and neurological processes, like neuronal growth and neurotransmitter transport. Through the iTRAQ-based strategy, we obtained a comparative proteome profile of brain tissues from Mongolian gerbils with chronic infection of T.gondii. Several differentially expressed proteins involved in neurological pathways, like Parvalbumin, Drebrin or Synaptotagmin, can be further investigated to enhance our understanding of central nervous system (CNS) injury caused by T.gondii.

mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here, we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70K to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes, which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.

Circular RNAs are highly stable single-stranded circular RNAs and enriched in the brain. Previous studies showed that circRNAs, as part of competing endogenous RNAs (ceRNAs) network, play an important role in neurodegenerative and psychiatric diseases. However, the mechanism of circRNA-related ceRNA networks in postoperative cognitive dysfunction (POCD) has not been elucidated yet. POCD usually occurs in elderly patients and is characterized by hippocampal dysfunction. Here, aged C57BL/6 mice were subjected to exploratory laparotomy under sevoflurane anesthesia, and this POCD model was verified by Morris water maze test. Whole-transcriptome sequencing was performed on the hippocampus of control group (Con) and surgery group. One hundred and seventy-seven DEcircRNAs, 221 DEmiRNAs and 2,052 DEmRNAs were identified between two groups. A ceRNA network was established with 92 DEcircRNAs having binding sites with 76 DEmiRNAs and 549 target DEmRNAs. In functional enrichment analysis, a pathological pattern of POCD was highlighted in the ceRNA network: Abnormal metabolic process in neural cells, including oxygen metabolism, could promote apoptosis and then affect the synaptic function, which may undermine the neural plasticity and eventually lead to changes in cognitive function and other behavioral patterns. In conclusion, this specific ceRNA network of circRNAs-miRNAs-mRNAs has provided novel insights into the regulatory mechanisms of POCD and revealed potential therapeutic gene targets.

Subretinal fibrosis (SF) is an important cause of submacular neovascularization that leads to permanent vision loss, but has no effective clinical treatment. The present study examined the influence of metformin on SF, and investigated whether the mechanism involves the microRNA (miR)-140-3p/LIN28B/JNK/STAT3-mediated regulation of oxidative stress, angiogenesis and fibrosis-associated indicators. A mouse model of laser-induced SF was established. In addition, an ARPE-19 fibrotic cell model was established using TGF-β1. A Cell Counting Kit-8 assay was used to examine cell viability. Flow cytometry was used to measure reactive oxygen species levels, and western blotting was used to detect the levels of proteins associated with epithelial-mesenchymal transition (EMT), signaling and fibrosis. The levels of superoxide dismutase, malondialdehyde, glutathione-peroxidase and catalase were measured using kits. Scratch assays and Transwell assays were used to assess cell migration and invasion, respectively, and reverse transcription-quantitative PCR was used to determine the levels of miR-140-3p and LIN28B. Dual-luciferase assays were used to verify the targeting relationship between miR-140-3p and LIN28B, and coimmunoprecipitation was used to confirm the interaction between LIN28B and JNK. Masson staining and hematoxylin and eosin staining were used to examine collagenous fibers and the histopathology of eye tissue. In ARPE-19 cells induced by TGF-β1, metformin promoted miR-140-3p expression and inhibited LIN28B expression and JNK/STAT3 pathway activation, thereby inhibiting oxidative stress, EMT and fibrosis in ARPE-19 cells. The overexpression of LIN28B or treatment with the JNK/STAT3 agonist anisomycin partially reversed the inhibitory effect of metformin on oxidative stress and fibrosis in ARPE-19 cells. The dual-luciferase reporter assay and coimmunoprecipitation assay showed that miR-140-3p targeted the 3' untranslated region of LIN28B mRNA and inhibited LIN28B expression. LIN28B targeted and bound to JNK and regulated the JNK/STAT3 pathway. Therefore, it may be concluded that metformin can promote miR-140-3p expression, inhibit LIN28B and then inhibit the JNK/STAT3 pathway to alleviate SF.

PARK2 is a gene implicated in disease states with opposing responses in cell fate determination, yet its contribution in pro-survival signaling is largely unknown. Here we show that PARK2 is altered in over a third of all human cancers, and its depletion results in enhanced phosphatidylinositol 3-kinase/Akt (PI3K/Akt) activation and increased vulnerability to PI3K/Akt/mTOR inhibitors. PARK2 depletion contributes to AMPK-mediated activation of endothelial nitric oxide synthase (eNOS), enhanced levels of reactive oxygen species, and a concomitant increase in oxidized nitric oxide levels, thereby promoting the inhibition of PTEN by S-nitrosylation and ubiquitination. Notably, AMPK activation alone is sufficient to induce PTEN S-nitrosylation in the absence of PARK2 depletion. Park2 loss and Pten loss also display striking cooperativity to promote tumorigenesis in vivo. Together, our findings reveal an important missing mechanism that might account for PTEN suppression in PARK2-deficient tumors, and they highlight the importance of PTEN S-nitrosylation in supporting cell survival and proliferation under conditions of energy deprivation.

We used ultraperformance liquid chromatography coupled with quadrupole/time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS/MS) to analyze the metabolic profile of reflex tears obtained from patients with dry eye disorders.

Serine/one-carbon metabolism provides critical resources for nucleotide biosynthesis and epigenetic maintenance and is thus necessary in cancer cell growth, although the detailed regulatory mechanisms remain unclear. We uncover a critical role of glycogen synthase kinase 3 (GSK3) in regulating the expression of serine/one-carbon metabolic enzymes. Nuclear enrichment of GSK3 significantly suppresses genes that mediate de novo serine synthesis, including PHGDH, PSAT1, PSPH, and one-carbon metabolism, including SHMT2 and MTHFD2. FRAT1 promotes nuclear exclusion of GSK3, enhances serine/one-carbon metabolism, and, as a result, confers cell vulnerability to inhibitors that target this metabolic process such as SHIN1, a specific SHMT1/2 inhibitor. Furthermore, pharmacological or genetic suppression of GSK3 promotes serine/one-carbon metabolism and exhibits a significant synergistic effect in combination with SHIN1 in suppressing cancer cell proliferation in cultured cells and in vivo. Our observations indicate that inhibition of nuclear GSK3 signaling creates a vulnerability, which results in enhanced efficacy of serine/one-carbon metabolism inhibitors for the treatment of cancer.

Mammalian folate metabolism is comprised of cytosolic and mitochondrial pathways with nearly identical core reactions, yet the functional advantages of such an organization are not well understood. Using genome-editing and biochemical approaches, we find that ablating folate metabolism in the mitochondria of mammalian cell lines results in folate degradation in the cytosol. Mechanistically, we show that QDPR, an enzyme in tetrahydrobiopterin metabolism, moonlights to repair oxidative damage to tetrahydrofolate (THF). This repair capacity is overwhelmed when cytosolic THF hyperaccumulates in the absence of mitochondrially produced formate, leading to THF degradation. Unexpectedly, we also find that the classic antifolate methotrexate, by inhibiting its well-known target DHFR, causes even more extensive folate degradation in nearly all tested cancer cell lines. These findings shed light on design features of folate metabolism, provide a biochemical basis for clinically observed folate deficiency in QDPR-deficient patients, and reveal a hitherto unknown and unexplored cellular effect of methotrexate.

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the irreversible conversion of 5,10-methylene-tetrahydrofolate (THF) to 5-methyl-THF, thereby committing one-carbon units to the methionine cycle. While MTHFR has long been known to be allosterically inhibited by S-adenosylmethionine (SAM), only relatively recently has N-terminal multisite phosphorylation been shown to provide an additional layer of regulation. In vitro, the multiply phosphorylated form of MTHFR is more sensitive to allosteric inhibition by SAM. Here we sought to investigate the kinases responsible for MTHFR multisite phosphorylation and the physiological function of MTHFR phosphorylation in cells. We identified DYRK1A/2 and GSK3A/B among the kinases that phosphorylate MTHFR. In addition, we found that MTHFR phosphorylation is maintained by adequate cellular SAM levels, which are sensed through the C-terminal SAM binding domain of MTHFR. To understand the function of MTHFR phosphorylation in cells, we generated MTHFR CRISPR knockin mutant lines that effectively abolished MTHFR phosphorylation and compared them with the parental cell lines. Whereas the parental cell lines showed increased 5-methyl-THF production in response to homocysteine treatment, the knockin cell lines had high basal levels of 5-methyl-THF and did not respond to homocysteine treatment. Overall, our results suggest that MTHFR multisite phosphorylation coordinates with SAM binding to inhibit MTHFR activity in cells.