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Epithelial–mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells plays a key role in proliferative retinal diseases such as age-related macular degeneration by contributing to subretinal fibrosis. To investigate the potential role of retinoic acid receptor-α (RAR-α) signaling in this process, we have now examined the effects of the RAR-α agonist Am580 on EMT induced by transforming growth factor-β2 (TGF-β2) in primary mouse RPE cells cultured in a three-dimensional type I collagen gel as well as on subretinal fibrosis in a mouse model. We found that Am580 inhibited TGF-β2-induced collagen gel contraction mediated by RPE cells. It also attenuated the TGF-β2-induced expression of the mesenchymal markers α-smooth muscle actin, fibronectin, and collagen type I; production of pro-matrix metalloproteinase 2 and interleukin-6; expression of the focal adhesion protein paxillin; and phosphorylation of SMAD2 in the cultured RPE cells. Finally, immunofluorescence analysis showed that Am580 suppressed both the TGF-β2-induced translocation of myocardin-related transcription factor-A (MRTF-A) from the cytoplasm to the nucleus of cultured RPE cells as well as subretinal fibrosis triggered by laser-induced photocoagulation in a mouse model. Our observations thus suggest that RAR-α signaling inhibits EMT in RPE cells and might attenuate the development of fibrosis associated with proliferative retinal diseases.

BACKGROUND/AIM We have previously reported that treatment of pancreatic cancer cells with active hexose-correlated compound (AHCC), an extract of a basidiomycete mushroom, decreases the levels of tumor-associated proteins including heat-shock protein 27 (HSP27), heat shock factor 1 (HSF1) and sex-determining region Y-box 2 (SOX2). The transmembrane glycoprotein, CUB domain-containing protein 1 (CDCP1) has been reported to be up-regulated in various cancers, and be associated with invasion and metastasis. The aim of this study was to examine the effect of AHCC on the expression of CDCP1 in KLM1-R cells. MATERIALS AND METHODS Gemcitabine-resistant pancreatic cancer cells (KLM1-R) were treated with AHCC (10 mg/ml) for 48 h. Western blot analysis of cell extracts with anti-CDCP1 or anti-actin antibodies was performed to assess the expression of CDCP1. RESULTS Expression of CDCP1 was reduced by AHCC treatment of KLM1-R cells, whereas expression of actin was not affected. The ratio of intensities of CDCP1/actin in AHCC-treated KLM1-R cells was significantly suppressed (p

Understanding how energy metabolism and related proteins influence neural progenitor cells in adult tissues is critical for developing new strategies in clinical tissue regeneration therapy. We have recently reported that a subtoxic concentration of glutamate-induced neural progenitor cells in the mature ex vivo rat retina. We herein explore changes in the metabolic pathways during the process. We firstly observed an increase in lactate and lactate dehydrogenase concentration in the glutamate-treated retina. We then investigated the levels of glycolytic enzymes and confirmed significant upregulation of pyruvate kinase M type (PKM), especially PKM2, enolase, phosphoglycerate mutase 1 (PGAM1), and inosine-5'-monophosphate dehydrogenase (IMPDH1) in the glutamate-treated retina compared to the untreated retina. An analysis of the subcellular localization of PKM2 revealed nuclear translocation in the treated retina, which has been reported to regulate cell cycle proliferation and glycolytic enzymes. Our findings indicate that the mature rat retina undergoes an increase in aerobic glycolysis. PKM2, both in the cytoplasm and in the nucleus, may thus play an important role during neural progenitor cell induction, as it does in other proliferating cells.

Purpose Epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells is related to the pathogenesis of subretinal fibrosis such as that associated with macular degeneration. The role of myocardin-related transcription factor A (MRTF-A) in EMT of RPE cells and subretinal fibrosis was investigated. Methods The migratory activity of human RPE-1 cells in culture was evaluated using a scratch assay. The subcellular distribution of MRTF-A in RPE-1 cells, as well as the extent of subretinal fibrosis in a mouse model, were determined by immunofluorescence analysis. Expression of α-smooth muscle actin (α-SMA), collagen type I (COL1), connective tissue growth factor (CTGF), and paxillin was examined by immunoblot analysis or reverse transcription and quantitative polymerase chain reaction analysis, whereas that of pro-matrix metalloproteinase-2 (MMP-2) was assessed by gelatin zymography. Results The MRTF-A signaling inhibitor CCG-1423 suppressed RPE-1 cell migration in a concentration-dependent manner. Transforming growth factor-beta (TGF-β2) induced MRTF-A translocation from the cytoplasm to the nucleus of RPE-1 cells, and this effect was attenuated by CCG-1423. TGF-β2 up-regulated the abundance of α-SMA, paxillin, and pro-MMP-2 proteins as well as the amounts of α-SMA, COL1, and CTGF mRNAs in a manner sensitive to inhibition by CCG-1423. Finally, intravitreal injection of CCG-1423 markedly attenuated the development of subretinal fibrosis induced by photocoagulation in vivo. Conclusions Our results implicate MRTF-A in EMT of RPE cells and in the development of subretinal fibrosis in vivo, suggesting that MRTF-A is a potential therapeutic target for retinal diseases characterized by subretinal fibrosis.

Necrosis is a form of cell death that results in rupture of the plasma membrane and the release of cellular contents, and it can give rise to sterile inflammation in the retina and other tissues. The secretion of vascular endothelial growth factor (VEGF) by retinal pigment epithelial (RPE) cells contributes to retinal homeostasis as well as to pathological angiogenesis. We have now examined the effect of a necrotic cell lysate prepared from human RPE cells (NLR) on the release of VEGF by healthy RPE cells. We found that NLR markedly increased the release of VEGF from RPE cells and that this effect was attenuated by nintedanib, a multiple receptor tyrosine kinase inhibitor, whereas it was unaffected by inhibitors of NF-κB signaling or of caspase-1. NLR also induced the phosphorylation of extracellular signal–regulated kinase (Erk) and signal transducer and activator of transcription 3 (Stat3) in a manner sensitive to inhibition by nintedanib, although inhibitors of Erk and Stat3 signaling pathways did not affect NLR-induced VEGF secretion. In addition, nintedanib attenuated the development of choroidal neovascularization in mice. Our results have thus shown that a necrotic lysate of RPE cells induced VEGF secretion from healthy RPE cells and that this effect was mediated by receptor tyrosine kinase signaling. They therefore suggest that VEGF secretion by healthy RPE cells is a potential therapeutic target for retinal diseases associated with sterile inflammation and pathological angiogenesis.

Microtubule-associated protein 1A/1B-light chain 3 (LC3)-II is essential for autophagosome formation and is widely used to monitor autophagic activity. We show that CGK733 induces LC3 II and LC3-puncta accumulation, which are not involved in the activation of autophagy. The treatment of CGK733 did not alter the autophagic flux and was unrelated to p62 degradation. Treatment with CGK733 activated the AMP-activated protein kinase (AMPK) and protein kinase RNA-like endoplasmic reticulum kinase/CCAAT-enhancer-binding protein homologous protein (PERK/CHOP) pathways and elevated the expression of p21Waf1/Cip1. Inhibition of both AMPK and PERK/CHOP pathways by siRNA or chemical inhibitor could block CGK733-induced p21Waf1/Cip1 expression as well as caspase-3 cleavage. Knockdown of LC3 B (but not LC3 A) abolished CGK733-triggered LC3 II accumulation and consequently diminished AMPK and PERK/CHOP activity as well as p21Waf1/Cip1 expression. Our results demonstrate that CGK733-triggered LC3 II formation is an initial event upstream of the AMPK and PERK/CHOP pathways, both of which control p21Waf1/Cip1 expression.

Many fusion genes, which are the result of chromosomal translocation and work as an oncogene, have been recently identified, but their mode of actions is still unclear. Here, we performed a yeast mutant screening for oncogenes of Ewing's sarcoma to easily identify essential regions responsible for fusion protein functions using a yeast genetic system. Three kinds of oncogenes including EWS/FLI1, EWS/ERG, and EWS/E1AF exhibited growth inhibition in yeast. In this screening, we identified 13 single amino acid substitution mutants which could suppress growth inhibition by oncogenes. All of the point mutation positions of the EWS/ETS family proteins were located within the ETS domain, which is responsible for the interaction with a specific DNA motif. Eight-mutated residues within the ETS domain matched to 13 completely conserved amino acid residues in the human ETS domains. Moreover, mutants also showed reduced transcriptional activities on the DKK2 promoter, which is upregulated by the EWS/ETS family, compared to that of the wild type. These results suggest that the ETS domain in the EWS/ETS family proteins may be a primary target for growth inhibition of Ewing's sarcoma and that this yeast screening system can be applied for the functional screening of the oncogenes.

Background/Aim: From the standpoint of cancer therapy, it is valuable to enhance the anticancer effects of chemotherapy. Our previous reports revealed that upregulation of heat-shock protein 27 (HSP27) has been linked to gemcitabine resistance of pancreatic cancer cells. Enzyme-treated asparagus extract (ETAS) is an extract that is produced from asparagus. The purpose of this study was to investigate the effect of ETAS on the expression of HSP27 and other HSPs in the gemcitabine-resistant pancreatic cancer cell line KLM1-R. Materials and Methods: KLM1-R cells were treated with ETAS, and expression levels of HSPs, including HSP27, were investigated by western blotting. Results: ETAS down-regulated HSP27 and pHSP27 (serine 78) in KLM1-R cells, but, HSP70 and GRP78 levels were not altered. Conclusion: This study suggests the potential therapeutic benefit of ETAS in enhancing anticancer effects by its combination with gemcitabine for patients with pancreatic cancer., peer-reviewed

Aim As severe alcohol intoxication impairs memory function, a high concentration of ethanol (60 mmol L−1) acutely inhibits long-term potentiation (LTP), a cellular model of learning and memory, in rat hippocampal slices. Neurosteroids are involved in this LTP inhibition. We recently reported that the inhibitory effects of 60 mmol L−1 ethanol are blocked by 4-methylpyrazole (4MP), an inhibitor of alcohol dehydrogenase, suggesting that acetaldehyde locally generated within the hippocampus participates in LTP inhibition. We investigated whether acetaldehyde generated by ethanol metabolism contributes to neurosteroidogenesis and LTP inhibition. Results Like 60 mmol L−1 ethanol, we found that exogenous acetaldehyde enhanced neurosteroid immunostaining in CA1 pyramidal neurons, and that augmented neurosteroid immunostaining by high ethanol alone was blocked by 4MP, but not by inhibitors of other ethanol metabolism pathways. The inhibitory effects of 60 mmol L−1 ethanol on LTP were mimicked by a lower concentration of ethanol (20 mmol L−1) plus acetaldehyde (60 μmol L−1), although neither agent alone was effective at these concentrations, suggesting that 60 mmol L−1 ethanol inhibits LTP through multiple actions, one of which involves acetaldehyde and the other of which requires just 20 mmol L−1 ethanol. The effects of ethanol and acetaldehyde on neurosteroid staining and LTP were overcome by inhibition of neurosteroid synthesis and by blockade of N-methyl-D-aspartate receptors (NMDAR). Conclusion These observations show that acetaldehyde generated by local ethanol metabolism within the hippocampus serves as a signal for neurosteroid synthesis in pyramidal neurons, and participates in the synaptic dysfunction associated with severe alcohol intoxication.

Consistent with the ability of severe alcohol intoxication to impair memory, high concentrations of ethanol (60 mM) acutely inhibit long-term potentiation (LTP) in the CA1 region of rat hippocampal slices. To account for this, we hypothesized that local metabolism to acetaldehyde may contribute to the effects of high ethanol on synaptic function. However, sodium azide, a catalase inhibitor, and allyl sulfide, an inhibitor of cytochrome P450 2E1 (CYP2E1), failed to overcome LTP inhibition by 60 mM ethanol. In contrast, LTP was successfully induced in the presence of ethanol plus 4-methylpyrazole (4MP), an inhibitor of alcohol dehydrogenase, suggesting that local metabolism via alcohol dehydrogenase contributes to synaptic effects. Furthermore, exogenously administered acetaldehyde overcame the effects of 4MP on LTP inhibition mediated by ethanol. These observations indicate that acetaldehyde generated by local metabolism within the hippocampus participates in the synaptic dysfunction associated with severe alcohol intoxication.

LY294002 and wortmannin are chemical compounds that act as potent inhibitors of phosphoinositide 3-kinases (PI3Ks). Both of them are generally used to inhibit cell proliferation as cancer treatment by inhibiting the PI3K/protein kinase B (AKT) signaling pathway. In this study, LY294002 (but not wortmannin) showed an abnormal ability to enhance AKT phosphorylation (at Ser472) specifically in gemcitabine (GEM)-resistant pancreatic cancer (PC) cell lines PK59 and KLM1-R. LY294002 was shown to activate AKT and accumulate phospho-AKT at the intracellular membrane in PK59, which was abolished by treatment with AKTi-1/2 or wortmannin. Inhibiting AKT phosphorylation by treatment with AKTi-1/2 or wortmannin further enhanced LY294002-induced cell death in PK59 and KLM1-R cells. In addition, treatment with wortmannin alone failed to inhibit cell proliferation in both PK59 and KLM1-R cells. Thus, our results reveal that LY294002 displays the opposite effect on PI3K-dependent AKT phosphorylation, which maintains cell survival from the cytotoxicity introduced by LY294002 itself in GEM-resistant pancreatic cancer cells. We suggest that targeting the PI3K/AKT signaling pathway with inhibitors may be counterproductive for patients with PC who have acquired GEM-resistance.

Children with neurofibromatosis type 1 (NF1) are prone to learning and behavioral abnormalities, including problems with spatial learning and attention. The molecular etiology for these deficits is unclear, as previous studies have implicated defective dopamine, cyclic adenosine monophosphate (cAMP), and Ras homeostasis. Using behavioral, electrophysiological, and primary culture, we now demonstrate that reduced dopamine signaling is responsible for cAMP-dependent defects in neuron function and learning. Collectively, these results establish defective dopaminergic function as a contributing factor underlying impaired spatial learning and memory in children and adults with NF1, and support the use of treatments that restore normal dopamine homeostasis for select individuals.

Oseltamivir (Tamiflu) is now being stockpiled by several governments as a first line treatment for an anticipated outbreak of avian influenza caused by H5N1. However, abnormal behaviors and death associated with the use of Tamiflu have developed into a major issue in Japan where Tamiflu is often prescribed for seasonal influenza. Thus, it is critical to determine neuropsychiatric effects of oseltamivir and to establish methods for safe administration. Using juvenile rats and rat hippocampal slices, we investigated whether oseltamivir has adverse effects on the central nervous system. Systemic injection of oseltamivir (50mg/kg i.p.) produced no change in behavior within 2h. However, prior injection of oseltamivir significantly altered the duration of loss of lightning reflex following ethanol injection (3.3g/kg, i.p.). Ethanol injection in the presence of oseltamivir also resulted in enhanced hypothermia. In the CA1 region of hippocampal slices, oseltamivir (100 microM) induced paired-pulse facilitation in population spikes without changes in excitatory postsynaptic potentials. Similarly, 3 microM oseltamivir carboxylate, the active metabolite of oseltamivir, facilitated neuronal firing, though the facilitation did not involve GABAergic disinhibition. Moreover, oseltamivir carboxylate produced further facilitation following administration of 60mM ethanol. These findings indicate that oseltamivir has effects on the central nervous system, especially when combined with other agents.

Glutamate has been shown to induce neural progenitor cells in the adult vertebrate retina. However, protein dynamics during progenitor cell induction by glutamate are not fully understood. To identify specific proteins involved in the process, we employed two-dimensional electrophoresis-based proteomics on glutamate untreated and treated retinal ex vivo sections. Rat retinal tissues were incubated with 1 mM glutamate for 1 h, followed by incubation in glutamate-free media for a total of 24 h. Consistent with prior reports, it was found that mitotic cells appeared in the outer nuclear layer without any histological damage. Immunohistological evaluations and immunoblotting confirmed the emergence of neuronal progenitor cells in the mature retina treated with glutamate. Proteomic analysis revealed the up-regulation of dihydropyrimidinase-related protein 3 (DRP-3), DRP-2 and stress-induced-phosphoprotein 1 (STIP1) during neural progenitor cell induction by glutamate. Moreover, mRNA expression of DRP-3, especially, its long isoform, robustly increased in the treated retina compared to that in the untreated retina. These results may indicate that glutamate induces neural progenitor cells in the mature rat retina by up-regulating the proteins which mediate cell mitosis and neurite growth.

// Yufeng Wang 1 , Yasuhiro Kuramitsu 1 , Byron Baron 1 , Takao Kitagawa 1 , Junko Akada 1 , Kazuhiro Tokuda 1 , Dan Cui 2 , Kazuyuki Nakamura 1, 3 1 Department of Biochemistry and Functional Proteomics, Yamguchi University Graduate School of Medicine, Ube, Japan 2 Department of Pathology, Yamguchi University Graduate School of Medicine, Ube, Japan 3 Centre of Clinical Laboratories in Tokuyama Medical Association Hospital, Shunan, Japan Correspondence to: Yasuhiro Kuramitsu, e-mail: climates@yamaguchi-u.ac.jp Keywords: CGK733, calcium sequestration, PERK/CHOP, ER stress, non-apoptotic death Received: April 02, 2015 Accepted: June 29, 2015 Published: July 10, 2015 ABSTRACT Calcium ions (Ca 2+ ) are indispensable for the physiology of organisms and the molecular regulation of cells. We observed that CGK733, a synthetic chemical substance, induced non-apoptotic cell death and stimulated reversible calcium sequestration by vesicles in pancreatic cancer cells. The endoplasmic reticulum (ER) stress eukaryotic translation initiation factor 2-alpha kinase 3/C/EBP homologous protein (PERK/CHOP) signaling pathway was shown to be activated by treatment with CGK733. Ionomycin, an ER stress drug and calcium ionophore, can activate PERK/CHOP signaling and accelerate CGK733-induced calcium sequestration. Knockdown of CHOP diminished CGK733-induced vesicular calcium sequestration, but had no effects on the cell death. Proteomic analysis demonstrated that the ER-located calcium-binding proteins, calumenin and protein S100-A11, were altered in CGK733-treated cells compared to non-treated controls. Our study reveals that CGK733-induced intracellular calcium sequestration is correlated with the PERK/CHOP signaling pathway and may also be involved in the dysregulations of calcium-binding proteins.

Purpose: We investigated whether artificial aqueous humours are adequate incubation media compared with artificial cerebrospinal fluid (aCSF), and evaluated the retinal toxicity of two vital stains − trypan blue (TB) and indocyanine green (ICG) − and triamcinolone acetonide (TA) using isolated rat retinas incubated in artificial aqueous humours. Methods: In experiment 1, retinal segments were isolated and incubated in aCSF, BSS plus®, Opeguard® Neo Kit, or phosphate-buffered saline (PBS). In experiment 2, retinal tissues were exposed to one of the agents and incubated in BSS plus®. Retinal damage was assessed by morphological examination and biochemical assay, which measured lactate dehydrogenase (LDH) in the medium once every hour. Results: In experiment 1, BSS plus® was confirmed as a suitable incubation medium. In experiment 2, there were no significant changes in the retinas exposed to TB or TA. Tissues exposed to ICG showed damage in every retinal layer and significantly higher release of LDH. Conclusion: Exposure to ICG caused retinal damage in isolated rat retina tissue in our experimental model (in vitro).

Subretinal fibrosis contributes to the loss of vision associated with age-related macular degeneration (AMD). Retinal pigment epithelial (RPE) cells play a key role in the pathogenesis of AMD including the fibrotic reaction. We examined the role of retinoic acid receptor-γ (RAR-γ) in the epithelial-mesenchymal transition (EMT) and other fibrosis-related processes in mouse RPE cells cultured in a type I collagen gel. Transforming growth factor-β2 (TGF-β2)-induced collagen gel contraction mediated by the RPE cells was inhibited by the RAR-γ agonist R667 in a concentration- and time-dependent manner. Expression of the mesenchymal markers α-smooth muscle actin and fibronectin, the release of interleukin-6, and the phosphorylation of paxillin, mitogen-activated protein kinases (ERK, p38, and JNK), Smad2, and AKT induced by TGF-β2 were also suppressed by the RAR-γ agonist. Furthermore, gelatin zymography and immunoblot analysis revealed that the TGF-β2-induced release of matrix metalloproteinase (MMP)-2, MMP-3, MMP-8, and MMP-9 from RPE cells was inhibited by R667, and the MMP inhibitor GM6001 attenuated TGF-β2-induced RPE cell contraction. Finally, immunohistofluorescence analysis with antibodies to glial fibrillary acidic protein showed that R667 inhibited the development of subretinal fibrosis in a mouse model in vivo. Our results thus suggest that RAR-γ agonists may prove effective for the treatment of subretinal fibrosis associated with AMD.RAR-γ agonist R667 suppressed collagen gel contraction mediated by RPE cells. Epithelial-mesenchymal transition (EMT) in RPE cells was inhibited by RAR-γ agonist R667. RAR-γ agonist R667 inhibited fibrosis-related processes in RPE cells. RAR-γ agonists may attenuate AMD-associated fibrosis.

Lactoylglutathione lyase (GLO1), a ubiquitously expressed methylglyoxal (MG) detoxification enzyme, is implicated in the progression of various human malignant diseases. However, the role of GLO1 in the development or progression of murine fibrosarcoma is still unclear. We performed proteomic analysis to identify differences in the intracellular proteins of the regressive tumor cell line QR-32 and the inflammatory cell-promoting progressive tumor cell line QRsP-11 of murine fibrosarcoma by 2DE combined with MS. Seven upregulated proteins were identified in QRsP-11 compared to QR-32 cells, namely, GLO1, annexin A1, adenylate kinase isoenzyme 1, transcription factor BTF3, myosin light polypeptide 6, low molecular weight phosphotyrosine protein phosphatase and nucleoside diphosphate kinase B. Heat shock protein beta-1 (HspB1), a methylglyoxal-adducted protein, is concomitantly over-expressed in QRsP-11 as compared to QR-32 cells. We also found out that GLO1 is translocated into the nucleus to a higher extent in QRsP-11 compared to QR-32 cells, which can be reversed by using a MEK inhibitor (U0126). Moreover, U0126 and GLO1 siRNA can inhibit cell proliferation and migration in QRsP-11 cells. Our data suggest that overexpression and nuclear translocation of GLO1 might be associated with tumor progression in murine fibrosarcoma.

Three clones with differential metastatic potential were established from the parental SN12C human renal cell carcinoma (HRCC). We previously reported that in the two high metastatic SN12C clones, two isoforms of ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH‑L1) showed decreased expression by using two-dimensional electrophoresis (2‑DE) covering a pH range (pH 3.0‑10.0) followed by liquid chromatography‑tandem mass spectrometry. However, in the case of the low metastatic clone, the spot volume for UCH‑L1 was almost the same as for the parental SN12C. In the present study, we found one protein spot which was correlated with the metastatic potential of SN12C clones by using 2‑DE over a narrow pH range (pH 4.0‑7.0). The protein glyoxalase 1 (GLO1) appeared to be directly proportional to the metastatic potential of the SN12C clones. GLO1 was the only protein which consistently varied according to the metastatic potentials of SN12C clones. GLO1 was increased in high metastatic cell lines by western blot analysis. These findings suggest that GLO1 is associated with the metastatic potential of SN12C HRCC clones. We expanded our experimental range to include clones of scirrhous gastric cancer cell lines (OCUM‑2M, OCUM‑2D and OCUM‑2MLN) and similar results were obtained, thereby further strengthening our original findings.

Children with neurofibromatosis type 1 (NF1) frequently have cognitive and behavioral deficits. Some of these deficits have been successfully modeled in Nf1 genetically-engineered mice that develop optic gliomas (Nf1 OPG mice). In the current study, we show that abnormal motivational influences affect the behavior of Nf1 OPG mice, particularly with regard to their response to novel environmental stimuli. For example, Nf1 OPG mice made fewer spontaneous alternations in a Y-maze and fewer arm entries relative to WT controls. However, analysis of normalized alternation data demonstrated that these differences were not due to a spatial working memory deficit. Other reported behavioral results (e.g., open-field test, below) suggest that differential responses to novelty and/or other motivational influences may be more important determinants of these kinds of behavior than simple differences in locomotor activity/spontaneous movements. Importantly, normal long-term depression was observed in hippocampal slices from Nf1 OPG mice. Results from elevated plus maze testing showed that differences in exploratory activity between Nf1 OPG and WT control mice may be dependent on the environmental context (e.g., threatening or non-threatening) under which exploration is being measured. Nf1 OPG mice also exhibited decreased exploratory hole poking in a novel holeboard and showed abnormal olfactory preferences, although L-dopa (50 mg/kg) administration resolved the abnormal olfactory preference behaviors. Nf1 OPG mice displayed an attenuated response to a novel open field in terms of decreased ambulatory activity and rearing but only during the first 10 min of the session. Importantly, Nf1 OPG mice demonstrated investigative rearing deficits with regard to a novel hanging object suspended on one side of the field which were not rescued by L-dopa administration. Collectively, our results provide new data important for evaluating therapeutic treatments aimed at ameliorating NF1-associated cognitive/behavioral deficits.

Benzodiazepines (BDZs) enhance GABAAreceptor inhibition by direct actions on central BDZ receptors (CBRs). Although some BDZs also bind mitochondrial receptors [translocator protein (18 kDa) (TSPO)] and promote the synthesis of GABA-enhancing neurosteroids, the role of neurosteroids in the clinical effects of BDZs is unknown. In rat hippocampal slices, we compared midazolam, an anesthetic BDZ, with clonazepam, an anticonvulsant/anxiolytic BDZ that activates CBRs selectively. Midazolam, but not clonazepam, increased neurosteroid levels in CA1 pyramidal neurons without changing TSPO immunostaining. Midazolam, but not clonazepam, also augmented a form of spike inhibition after stimulation adjacent to the pyramidal cell layer and inhibited induction of long-term potentiation. These effects were prevented by finasteride, an inhibitor of neurosteroid synthesis, or 17PA [17-phenyl-(3α,5α)-androst-16-en-3-ol], a blocker of neurosteroid effects on GABAAreceptors. Moreover, the synaptic effects were mimicked by a combination of clonazepam with FGIN (2-[2-(4-fluorophenyl)-1H-indol-3-yl]-N,N-dihexylacetamide), a selective TSPO agonist, or a combination of clonazepam with exogenous allopregnanolone. Consistent with thesein vitroresults, finasteride abolished the effects of midazolam on contextual fear learning when administrated 1 d before midazolam injection. Thus, dual activation of CBRs and TSPO appears to result in unique actions of clinically important BDZs. Furthermore, endogenous neurosteroids are shown to be important regulators of pyramidal neuron function and synaptic plasticity.

Oseltamivir (Tamiflu), a neuraminidase inhibitor, is widely used for treatment of influenza. Because abnormal behaviors have been observed in some Japanese teenagers following oseltamivir use, its safety has been questioned. Oseltamivir is known to alter neuronal function and behavior in animals, particularly when administered in combination with ethanol. Based on this, it has been hypothesized that interactions of oseltamivir with other drugs may result in altered CNS excitability in this study. It has been found that injection of ephedrine and caffeine overcame inactivity induced by oseltamivir and ethanol but did not alter changes in novelty seeking behavior in a Y-maze test. In ex-vivo hippocampal slices, oseltamivir carboxylate (OTC), an active form of oseltamivir, alters excitability in the absence of ethanol. In slices pretreated with OTC, long-term depression (LTD), a form of synaptic plasticity that is correlated with Y-maze performance was not altered if caffeine or ephedrine was administered individually. However, LTD could not be induced in slices pretreated with OTC if caffeine and ephedrine were administered simultaneously. These observations suggest that combination of oseltamivir with other neurostimulants may alter synaptic plasticity and this may contribute to behavioral changes associated with the drug.

Concerns have been raised about whether operating microscopes and endoillumination used during ophthalmic surgeries contribute to retinal damage. Despite the recognition that ascorbic acid (vitamin C) helps to protect the eye from light and the abundance of vitamin C in the retina, artificial aqueous humors used during surgery only contain the antioxidant glutathione. To test whether inclusion of antioxidants other than glutathione in surgical solutions might help to preserve retinal integrity, we studied the effects of vitamin C on acute toxicity in isolated rat retinas. Male Sprague-Dawley rats (PND 30 ± 2) were sacrificed for retinal isolation. In the presence or absence of vitamin C (1 or 3 mM), retinas were exposed to 302 nm ultraviolet B (UVB) light for 1 hour and were incubated for a total of 5 hours at 30°C. Retinal damage was assessed by morphological examination and biochemical assay measuring the amount of lactate dehydrogenase (LDH) released from injured cells. In control retinas, LDH release was significantly increased after UVB exposure. The presence of 1 mM vitamin C in the incubation media significantly reduced LDH release during the post-incubation period following UV exposure. No difference was found between 1 mM and 3 mM vitamin C. Microscopic examination revealed that disorganization in the outer nuclear layer after UVB exposure was markedly attenuated by administration of 1 mM vitamin C. One mM vitamin C, a concentration found in the anterior chamber in humans, but not glutathione, prevented phototoxic injury following UV exposure. Although vitamin C itself cannot be used in intraocular irrigating solutions because of adverse interactions with iron released during bleeding, inclusion of antioxidants equivalent to vitamin C should be considered to help protect the retina from intraoperative light toxicity.

Poly (ADP-ribose) polymerase-1 (PARP-1) and autophagy play increasingly important roles in DNA damage repair and cell death. Gemcitabine (GEM) remains the first-line chemotherapeutic drug for pancreatic cancer (PC). However, little is known about the relationship between PARP-1 expression and autophagy in response to GEM. Here we demonstrate that GEM induces DNA-damage response and degradation of mono-ADP ribosylated PARP-1 through the autophagy pathway in PC cells, which is rescued by inhibiting autophagy. Hypoxia and serum starvation inhibit autophagic activity due to abrogated GEM-induced mono-ADP-ribosylated PARP-1 degradation. Activation of extracellular regulated protein kinases (ERK) induced by serum starvation shows differences in intracellular localization as well as modulation of autophagy and PARP-1 degradation in GEM-sensitive KLM1 and -resistant KLM1-R cells. Our study has revealed a novel role of autophagy in PARP-1 degradation in response to GEM, and the different impacts of MEK/ERK signaling pathway on autophagy between GEMsensitive and -resistant PC cells., peer-reviewed