Growth of B16F10 cells is enhanced in DJ-1-deficiency pancreas.

Association between cancer risk and Parkinson's disease is still debated. DJ-1, a Parkinson's disease (PD)-related gene, is encoded by PARK-7 gene and its deficiency causes early-onset PD. In our last studies, it was found that the immunosuppressive microenvironment established in DJ-1 knockout (KO) mice can enhance metastasis of melanoma cells to lungs. Therefore, we wanted to further examine whether there were some niche in other organs of DJ-1-deficiency mouse to facilitate cell growth of tumors. We used in vivo tissue-specific models of tumor growth and in vitro cellular model to verify the hypothesis. We also used protein blot assay, cell-adhesion assay and bioinformatic tools to conduct experiments. In the mouse model of subcutaneous injection, there was no difference on tumor growth between WT and DJ-1 KO mice. Moreover, the results of experimental liver metastasis by intrasplenic injection model showed that there was no difference of nodules number in both mice, but a dramatic enhancement of nodule formation and increased mucin4 levels were found in pancreas of DJ-1 KO mice. In cell cultures, we further found that B16F10 cells indeed tended to adhere well to primary DJ-1-deficiency pancreatic epithelial cells, which had higher protein levels of mucin4. Notably, a human database also showed the inverse relationship in human pancreas between DJ-1 and mucin4, and mucin4 down-regulation can reverse the enhanced cellular adhesion in DJ-1 KO pancreatic epithelial cells. These results indicated that DJ-1 KO pancreatic tissue creating an appropriate microenvironment benefited development of the cancer cells.

CXCL12/CXCR4 Signaling Contributes to the Pathogenesis of Opioid Tolerance: A Translational Study.

BACKGROUND: Long-term opioid therapy for chronic pain may lead to analgesic tolerance, especially when administered intrathecally, thus preventing adequate pain relief. Discovering drug targets to treat opioid tolerance using a mechanism-based approach targeting opioid-induced neuroinflammation provides new therapeutic opportunities. In this study, we provide translational evidence that CXCL12/CXCR4 signaling contributes to the pathogenesis of opioid tolerance. METHODS: The CXCL12 levels in the cerebrospinal fluid of opioid-tolerant patients were compared with those of opioid-naive subjects. For further investigation, a rodent translational study was designed using 2 clinically relevant opioid delivery paradigms: daily intraperitoneal morphine injections and continuous intrathecal morphine infusion. We measured rats' tail flick responses and calculated the percentage of maximum possible effects (%MPE) to demonstrate opioid acute antinociception and the development of analgesic tolerance. The effects of exogenous CXCL12, CXCL12 neutralizing antibody, and receptor antagonist AMD3100 were investigated by intrathecal administration. Data were presented as mean ± SEM. RESULTS: CXCL12 was significantly upregulated in the cerebrospinal fluid of opioid-tolerant patients for 892 ± 34 pg/mL (n = 27) versus 755 ± 33 pg/mL (n = 10) in naive control subjects (P = .03). Furthermore, after 2 and 5 days of intrathecal morphine infusion, rat lumbar spinal cord dorsal horn CXCL12 messenger RNA levels were significantly upregulated by 3.2 ± 0.7 (P = .016) and 3.4 ± 0.3 (P = .003) fold, respectively. Results from the daily intraperitoneal morphine injection experiments revealed that administering an intrathecal infusion of CXCL12 for 24 hours before the first morphine injection did not decrease antinociception efficacy on day 1 but accelerated tolerance after day 2 (%MPE 49.5% vs 88.1%, P = .0003). In the intrathecal morphine coinfusion experiments, CXCL12 accelerated tolerance development (%MPE 9.4% vs 43.4% on day 1, P < .0001), whereas coadministration with CXCL12 neutralizing antibody attenuated tolerance (72.5% vs 43.4% on day 1, P < .0001; 47.6% vs 17.5% on day 2, P < .0001). Coadministration of receptor antagonist AMD 3100 can persistently preserve morphine analgesic effects throughout the study period (27.9% ± 4.1% vs 0.9% ± 1.6% on day 5, P = .03). CONCLUSIONS: The CXCL12/CXCR4 pathway contributes to the pathogenesis of opioid tolerance. Our study indicates that intervening with CXCL12/CXCR4 signaling has therapeutic potential for opioid tolerance.

Growth hormone is increased in the lungs and enhances experimental lung metastasis of melanoma in DJ-1 KO mice.

BACKGROUND: Growth hormone (GH) mainly serves an endocrine function to regulate somatic growth, but also serves an autocrine function in lung growth and pulmonary function. Several recent studies have demonstrated the role of autocrine GH in tumor progression in some organs. However, it is not clear whether excessive secretion of GH in the lungs is related to pulmonary nodule formation. METHODS: Firstly, the lung tissues dissected from mice were used for Western blotting and PCR measurement. Secondly, the cultured cells were used for examining effects of GH on B16F10 murine melanoma cells. Thirdly, male C57BL/6 mice were intravenously injected with B16F10 cells and then subcutaneously injected with recombinant GH twice per week for three weeks. Finally, stably transfected pool of B16F10 cells with knockdown of growth hormone receptor (GHR) was used to be injected into mice. RESULTS: We found that expression of GH was elevated in the lungs of DJ-1 knockout (KO) mice. We also examined the effects of GH on the growth of cultured melanoma cells. The results showed that GH increased proliferation, colony formation, and invasive capacity of B16F10 cells. In addition, GH also increased the expression of matrix metalloproteinases (MMPs) in B16F10 cells. Administration of GH in vivo enhanced lung nodule formation in C57/B6 mice. Increased lung nodule formation in DJ-1 KO mice following intravenous injection of melanoma cells was inhibited by GHR knockdown in B16F10 cells. CONCLUSIONS: These results indicate that up-regulation of GH in the lungs of DJ-1 KO mice may enhance the malignancy of B16F10 cells and nodule formation in pulmonary metastasis of melanoma.

Role of spinal CXCL1 (GROα) in opioid tolerance: a human-to-rodent translational study.

BACKGROUND: The pivotal role of glial activation and up-regulated inflammatory mediators in the opioid tolerance has been confirmed in rodents but not yet in humans. Here, the authors investigated the intraspinal cytokine and chemokine profiles of opioid-tolerant cancer patients; and to determine if up-regulated chemokines could modify opioid tolerance in rats. METHODS: Cerebrospinal fluid samples from opioid-tolerant cancer patients and opioid-naive subjects were compared. The cerebrospinal fluid levels of tumor necrosis factor-alpha, CXCL1, CXCL10, CCL2, and CX3CL1 were assayed. The rat tail flick test was utilized to assess the effects of intrathecal CXCL1 on morphine-induced acute antinociception and analgesic tolerance. RESULTS: CXCL1 level in cerebrospinal fluid was significantly up-regulated in the opioid-tolerant group (n = 30, 18.8 pg/ml vs. 13.2 pg/ml, P = 0.02) and was positively correlated (r = 0.49, P < 0.01) with opioid dosage. In rat experiment, after induction of tolerance by morphine infusion, the spinal cord CXCL1 messenger RNA was up-regulated to 32.5 ± 11.9-fold. Although CXCL1 infusion alone did not affect baseline tail-flick latency, the analgesic efficacy of a single intraperitoneal injection of morphine dropped significantly on day 1 to day 3 after intrathecal infusion of CXCL1. After establishing tolerance by intrathecal continuous infusion of morphine, its development was accelerated by coadministration of CXCL1 and attenuated by coadministration of CXCL1-neutralizing antibody or CXCR2 antagonist. CONCLUSIONS: CXCL1 is up-regulated in both opioid-tolerant patients and rodents. The onset and extent of opioid tolerance was affected by antagonizing intrathecal CXCL1/CXCR2 signaling. Therefore, the CXCL1/CXCR2 signal pathway may be a novel target for the treatment of opioid tolerance.

Upregulation of drug transporter expression by osteopontin in prostate cancer cells.

Multidrug resistance is a major cause of chemotherapy failure. Recent studies indicate that drug resistance can be rapidly induced by some soluble factors, such as cytokines, chemokines, growth factors, and cell adhesion factors in the tumor microenvironment. Osteopontin (OPN), an extracellular matrix protein, has a functional arginine-glycine-aspartic acid (RGD) domain for binding to integrin. Here we found OPN expression to be upregulated by hypoxic condition in PC-3 prostate tumor cells. OPN increased the mRNA and protein expression of p-glycoprotein (P-gp), a subfamily of ATP-binding cassette transporter in a concentration- and time-dependent manner. The increase in P-gp transporter by OPN was mediated by binding to αvß3 integrin. Daunomycin (DUN), a chemotherapeutic agent with autofluorescence, was used to evaluate the pump activity, and OPN increased the drug pumping-out activity. OPN inhibited DUN-induced cell death, which was antagonized by αvß3 monoclonal antibody. Long-term treatment with DUN further enhanced the expression of OPN. Knockdown of endogenous OPN potentiated the DUN-induced apoptosis of PC-3 cells. Furthermore, knockdown of OPN enhanced cell death caused by other drugs, including paclitaxel, doxorubicin, actinomycin-D, and rapamycin, which are also P-gp substrates. The animal studies also showed that OPN knockdown enhanced the cytotoxic action of DUN. These results indicate that OPN is a potential therapeutic target for cancer therapy to reduce drug resistance in sensitive tumors.

Dextromethorphan inhibits osteoclast differentiation by suppressing RANKL-induced nuclear factor-κB activation.

UNLABELLED: Dextromethorphan (DXM), a commonly used antitussive, is a dextrorotatory morphinan. Here, we report that DXM inhibits the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis and bone resorption by abrogating the activation of NF-κB signalling in vitro. Oral administration of DXM ameliorates ovariectomy (OVX)-induced osteoporosis in vivo. INTRODUCTION: DXM was reported to possess anti-inflammatory properties through inhibition of the release of pro-inflammatory factors. However, the potential role and action mechanism of DXM on osteoclasts and osteoblasts remain unclear. In this study, in vitro and in vivo studies were performed to investigate the potential effects of DXM on osteoclastogenesis and OVX-induced bone loss. METHODS: Osteoclastogenesis was examined by the TRAP staining, pit resorption, TNF-α release, and CCR2 and CALCR gene expression. Osteoblast differentiation was analyzed by calcium deposition. Osteogenic and adipogenic genes were measured by real-time PCR. Signaling pathways were explored using Western blot. ICR mice were used in an OVX-induced osteoporosis model. Tibiae were measured by µCT and serum markers were examined with ELISA kits. RESULTS: DXM inhibited RANKL-induced osteoclastogenesis. DXM mainly inhibited osteoclastogenesis via abrogation of IKK-IκBα-NF-κB pathways. However, a higher dosage of DXM antagonized the differentiation of osteoblasts via the inhibition of osteogenic signals and increase of adipogenic signals. Oral administration of DXM (20 mg/kg/day) partially reduced trabecular bone loss in ovariectomized mice. CONCLUSION: DXM inhibits osteoclast differentiation and activity by affecting NF-κB signaling. Therefore, DXM at suitable doses may have new therapeutic applications for the treatment of diseases associated with excessive osteoclastic activity.

A forward loop between glioma and microglia: glioma-derived extracellular matrix-activated microglia secrete IL-18 to enhance the migration of glioma cells.

The mediators and cellular effectors of inflammation are important constituents of the local environment of tumors. In some occasions, oncogenic changes induce an inflammatory microenvironment that promotes the progression of tumors. In gliomas, the presence of microglia may represent tumor-related inflammation and microglia activation, and subsequent inflammatory responses may influence tumor growth and metastasis. Here, we found that C6 glioma--but not primary astrocyte-derived extracellular matrix (ECM) could activate microglia, including primary microglia and BV-2 cell line, and activated microglia-secreted interleukin (IL)-18, a potent inflammatory cytokine of the IL-1 family, to promote C6 migration. In addition, by coating purified ECM components, it was found that secretion of IL-18 by activated microglia was enhanced when microglia encountered with fibronectin and vitronectin. Furthermore, IL-18-induced C6 migration and microfilament disassembly were antagonized by iNOS inhibitor, guanylate cyclase inhibitor, and protein kinase G inhibitor. Taken together, these results indicate that IL-18 secreted by microglia, which was activated by C6 glioma-derived ECM, enhanced migration of C6 glioma through NO/cGMP pathway.

Involvement of 15-lipoxygenase in the inflammatory arthritis.

15-Lipoxygenase (15-LOX) is involved in many pathological processes. The aim of this study is to examine the role of 15-LOX in the matrix metalloproteinase (MMP) expression and inflammatory arthritis. It was found that treatment of 15-LOX downstream product of 15-(S)-HETE (15-S-hydroxyeicosatetraenoic acid) increased the mRNA and protein levels of MMP-2 in rheumatoid arthritis synovial fibroblast (RASF) derived from rheumatoid arthritis patients. The enhancement effect of 15-(S)-HETE was antagonized by the addition of LY294002 (PI3K inhibitor) and PDTC (NF-κB inhibitor). Treatment of 15-(S)-HETE increased the phosphorylation of AKT, nuclear translocation of p65 and the breakdown of IκBα. TNF-α and IL-1ß are the key cytokines involved in arthritis and also increase the activity of MMP-2 in RASF, which was antagonized by pretreatment with 15-LOX inhibitor PD146176 or knockdown of 15-LOX. It was also found that these two cytokines increased the expression of 15-LOX in RASF. Treatment of glucocorticoid but not NSAIDs inhibited 15-(S)-HETE-induced expression of MMP-2. In comparison with wild-type mice, adjuvant-induced arthritis and MMP-2 expression in synovial membrane were markedly inhibited in 15-LOX knockout (KO) mice. These results indicate that 15-LOX plays an important role in the disease progression of arthritis and may be involved in the inflammatory action induced by TNF-α and IL-1ß. 15-LOX is thus a good target for developing drugs in the treatment of inflammatory arthritis.

Development of a Neuroprotective Erythropoietin Modified with a Novel Carrier for the Blood-Brain Barrier.

Extremely high doses of erythropoietin (EPO) has been used for neuroprotection in ischemia-reperfusion brain injury to deliver sufficient amounts of EPO across the blood-brain barrier (BBB); however, harmful outcomes were observed afterward. We aimed to test the ability of HBHAc (heparin-binding haemagglutinin adhesion c), an intracellular delivery peptide for macromolecules, as an EPO carrier across the BBB. The cellular internalization and transcytosis ability of HBHAc-modified EPO (EPO-HBHAc) were evaluated in bEnd.3 cells and in the bEnd.3/CTX TNA2 co-culture BBB model, respectively. Subsequently, the NMDA-induced-toxicity model and ischemia-reperfusion rat model were used to understand the neuronal protective activity of EPO-HBHAc. The biodistribution of EPO-HBHAc was demonstrated in rats by the quantification of EPO-HBHAc in the brain, plasma, and organs by ELISA. Our results demonstrate that EPO-HBHAc exhibited significantly higher cellular internalization in dose- and time-dependent manners and better transcytosis ability than EPO. In addition, the transported EPO-HBHAc in the co-culture transwell system maintained the neuronal protective activity when primary rat cortical neurons underwent NMDA-induced toxicity. The calculated cerebral infarction area of rats treated with EPO-HBHAc was significantly reduced compared to that of rats treated with EPO (29.9 ± 7.0% vs 48.9 ± 7.9%) 24 h after occlusion in 3VO rat experiments. Moreover, the EPO amount in both CSF and damaged cortex from the EPO-HBHAc group was 4.0-fold and 3.0-fold higher than the EPO group, respectively. These results suggest that HBHAc would be a favorable tool for EPO brain delivery and would further extend the clinical applications of EPO in neuroprotection.

Overexpression of heme oxygenase-1 protects dopaminergic neurons against 1-methyl-4-phenylpyridinium-induced neurotoxicity.

Heme oxygenase-1 (HO-1) is up-regulated in response to oxidative stress and catalyzes the degradation of pro-oxidant heme to carbon monoxide (CO), iron, and bilirubin. Intense HO-1 immunostaining in the Parkinsonian brain is demonstrated, indicating that HO-1 may be involved in the pathogenesis of Parkinsonism. We here locally injected adenovirus containing human HO-1 gene (Ad-HO-1) into rat substantia nigra concomitantly with 1-methyl-4-phenylpyridinium (MPP(+)). Seven days after injection of MPP(+) and Ad-HO-1, the brain was isolated for immunostaining and for measurement of dopamine content and inflammatory cytokines. It was found that overexpression of HO-1 significantly increased the survival rate of dopaminergic neurons; reduced the production of tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in substantia nigra; antagonized the reduction of striatal dopamine content induced by MPP(+); and also up-regulated brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) expression in substantia nigra. Apomorphine-induced rotation after MPP(+) treatment was also inhibited by Ad-HO-1. On the other hand, inhibition of HO enzymatic activity by zinc protoporphyrin-IX facilitated the MPP(+)-induced rotatory behavior and enhanced the reduction of dopamine content. HO-1 overexpression also protected dopaminergic neurons against MPP(+)-induced neurotoxicity in midbrain neuron-glia cocultures. Overexpression of HO-1 increased the expression of BDNF and GDNF in astrocytes and BDNF in neurons. Our results indicate that HO-1 induction exerts neuroprotection both in vitro and in vivo. Pharmacological or genetic approaches targeting HO-1 may represent a promising and novel therapeutic strategy in treating Parkinsonism.

Cytokine MIF Enhances Blood-Brain Barrier Permeability: Impact for Therapy in Ischemic Stroke.

Ischemic stroke is a devastating disease with limited therapeutic options. It is very urgent to find a new target for drug development. Here we found that the blood level of MIF in ischemic stroke patients is upregulated. To figure out the pathological role of MIF in ischemic stroke, both in vitro and in vivo studies were conducted. For in vitro studies, primary cortical neuron cultures and adult rat brain endothelial cells (ARBECs) were subjected to oxygen-glucose deprivation (OGD)/reoxygenation. Middle cerebral artery occlusion (MCAo) rodent models were used for in vivo studies. The results show that MIF exerts no direct neuronal toxicity in primary culture but disrupts tight junction in ARBECs. Furthermore, administration of MIF following MCAo shows the deleterious influence on stroke-induced injury by destroying the tight junction of blood-brain barrier and increasing the infarct size. In contrast, administration of MIF antagonist ISO-1 has the profound neuroprotective effect. Our results demonstrate that MIF might be a good drug target for the therapy of stroke.

Impairment of social behaviors in Arhgef10 knockout mice.

Background: Impaired social interaction is one of the essential features of autism spectrum disorder (ASD). Our previous copy number variation (CNV) study discovered a novel deleted region associated with ASD. One of the genes included in the deleted region is ARHGEF10. A missense mutation of ARHGEF10 has been reported to be one of the contributing factors in several diseases of the central nervous system. However, the relationship between the loss of ARHGEF10 and the clinical symptoms of ASD is unclear. Methods: We generated Arhgef10 knockout mice as a model of ASD and characterized the social behavior and the biochemical changes in the brains of the knockout mice. Results: Compared with their wild-type littermates, the Arhgef10-depleted mice showed social interaction impairment, hyperactivity, and decreased depression-like and anxiety-like behavior. Behavioral measures of learning in the Morris water maze were not affected by Arhgef10 deficiency. Moreover, neurotransmitters including serotonin, norepinephrine, and dopamine were significantly increased in different brain regions of the Arhgef10 knockout mice. In addition, monoamine oxidase A (MAO-A) decreased in several brain regions. Conclusions: These results suggest that ARHGEF10 is a candidate risk gene for ASD and that the Arhgef10 knockout model could be a tool for studying the mechanisms of neurotransmission in ASD. Trial registration: Animal studies were approved by the Institutional Animal Care and Use Committee of National Taiwan University (IACUC 20150023). Registered 1 August 2015.

Quantitative evaluation of focused ultrasound with a contrast agent on blood-brain barrier disruption.

The use of an ultrasound contrast agent (UCA) with focused ultrasound sonication has the potential to disrupt the blood-brain barrier (BBB) noninvasively and reversibly at target locations. This study investigated the effects of UCA dose and ultrasound pressure on BBB disruption. Sonications were applied at 1 MHz with a burst length of 10 ms, a 1% duty cycle and a repetition frequency of 1 Hz. The duration of the sonication was 30 s. In in vivo experiments, 16 male Wistar rats were sonicated in the presence of UCA at four doses (0, 30, 60 and 90 microL/kg). BBB integrity was evaluated by injecting Evans blue (EB) into the femoral vein of anesthetized rats. The relationship between UCA dose and the region of EB extravasation was evaluated at ultrasound pressures of 0.9 and 1.2 MPa. The BBB disruption, as quantified by the amount of EB extravasation, was significantly greater in rats injected with UCA at a dose of 60 or 90 microL/kg than at a dose of 0 or 30 microL/kg. The amount of EB extravasation increased monotonically with the quantity of UCA injected into the femoral vein before sonication. Furthermore, the BBB disruption could be enhanced in the focal region relative to the surrounding region with a higher dose of UCA (60 or 90 microL/kg). This study demonstrates that BBB disruption can be both increased and localized to the focal region by injecting an appropriate quantity of UCA before performing focused ultrasound sonications.

Pulsed-wave low-dose ultrasound hyperthermia selectively enhances nanodrug delivery and improves antitumor efficacy for brain metastasis of breast cancer.

The clinical application of chemotherapeutics for brain tumors remains a challenge due to limitation of blood-brain barrier/blood-tumor barrier (BBB/BTB). In this study, we investigated the effects of low-dose focused ultrasound hyperthermia (UH) on the delivery and therapeutic efficacy of pegylated liposomal doxorubicin (PLD) for brain metastasis of breast cancer. Murine breast cancer cells (4T1-luc2) expressing firefly luciferase were implanted into mouse striatum as a brain tumor model. The mice were intravenously injected with PLD with/without transcranial pulsed-wave/continuous-wave UH (pUH/cUH) treatment on day-6 after tumor implantation. pUH (frequency: 500kHz, PRF: 1000Hz, duty cycle: 50%) was conducted under equal acoustic power (2.2-Watt) and sonication duration (10-min) as cUH. The amounts of doxorubicin accumulated in the normal brain and tumor tissues were measured with fluorometry. The tumor growth responses for the control, pUH, PLD, PLD+cUH, and PLD+pUH groups were evaluated with IVIS. The PLD distribution and cell apoptosis were assessed with immunofluorescence staining. The results showed that pUH significantly enhanced the PLD delivery into brain tumors and the tumor growth was further inhibited by PLD+pUH without damaging the sonicated normal brain tissues. This indicates that low-dose transcranial pUH is a promising method to selectively enhance nanodrug delivery and improve the brain tumor treatment.

Inhibition of osteoporosis by the αvß3 integrin antagonist of rhodostomin variants.

Integrins are heterodimeric cell surface receptors that mediate cell-cell and cell-matrix interaction. The vitronectin and osteopontin receptor αvß3 integrin has increased expression levels and is implicated in the adhesion, activation, and migration of osteoclasts on the bone surface as well as osteoclast polarization. αvß3 integrin plays an important role in osteoclast differentiation and resorption. In addition, Arg-Gly-Asp (RGD)-containing peptides, small molecular inhibitors, and antibodies to αvß3 integrin have been shown to inhibit bone resorption in vitro and in vivo. Here we examined the effects of a disintegrin HSA-ARLDDL a genetically modified mutant of rhodostomin conjugated with human serum albumin, which is highly selective of αvß3, on RANKL-induced osteoclastogenesis and ovariectomy (OVX)-induced osteoporosis. In RANKL-induced osteoclastogenesis, HSA-ARLDDL significantly inhibited osteoclast formation, and IC50 was at nM range. Post-treatment HSA-ARLDDL also inhibits osteoclast formation. Furthermore, weekly administration of HSA-ARLDDL significantly inhibits the increase in serum bone resorption marker levels and decrease in cancellous bone loss in tibia and femur induced by OVX. On the other hand, HSA-ARLDDL did not affect the differentiation and calcium deposition of osteoblasts. These results indicate that the highly selective and long-acting αvß3 integrin antagonists could be developed as effective drugs for postmenopausal osteoporosis.

Hypoxia-induced iNOS expression in microglia is regulated by the PI3-kinase/Akt/mTOR signaling pathway and activation of hypoxia inducible factor-1alpha.

Exposure to hypoxia induced microglia activation and animal studies have shown that neuronal cell death is correlated with microglial activation following cerebral ischemia. Thus, it is likely that toxic inflammatory mediators produced by activated microglia under hypoxic conditions may exacerbate neuronal injury following cerebral ischemia. The hypoxia-inducible factor-1 (HIF-1) is primarily involved in the sensing and adapting of cells to changes in the O(2) level, which is regulated by many physiological functions. However, the role of HIF-1 in microglia activation under hypoxia has not yet been defined. In the current work, we investigate the signaling pathways of HIF-1alpha involved in the regulation of hypoxia-induced overexpression of inducible NO synthase (iNOS) in microglia. Exposure of primary rat microglial cultures as well as established microglial cell line BV-2 to hypoxia induced the expression of iNOS, indicating that hypoxia could lead to the inflammatory activation of microglia. iNOS induction was accompanied with NO production. Moreover, the molecular analysis of these events indicated that iNOS expression was regulated by the phosphatidylinositol 3-kinase (PI3-kinase)/AKT/ mammalian target of rapamycin (mTOR) signaling pathway and activation of hypoxia inducible factor-1alpha (HIF-1alpha). Thus, during cerebral ischemia, hypoxia may not only directly damage neurons, but also promote neuronal injury indirectly via microglia activation. In this study, we demonstrated that hypoxia induced iNOS expression by regulation of HIF-1alpha in microglia.

Microglia-Derived Cytokines/Chemokines Are Involved in the Enhancement of LPS-Induced Loss of Nigrostriatal Dopaminergic Neurons in DJ-1 Knockout Mice.

Mutation of DJ-1 (PARK7) has been linked to the development of early-onset Parkinson's disease (PD). However, the underlying molecular mechanism is still unclear. This study is aimed to compare the sensitivity of nigrostriatal dopaminergic neurons to lipopolysaccharide (LPS) challenge between DJ-1 knockout (KO) and wild-type (WT) mice, and explore the underlying cellular and molecular mechanisms. Our results found that the basal levels of interferon (IFN)-γ (the hub cytokine) and interferon-inducible T-cell alpha chemoattractant (I-TAC) (a downstream mediator) were elevated in the substantia nigra of DJ-1 KO mice and in microglia cells with DJ-1 deficiency, and the release of cytokine/chemokine was greatly enhanced following LPS administration in the DJ-1 deficient conditions. In addition, direct intranigral LPS challenge caused a greater loss of nigrostriatal dopaminergic neurons and striatal dopamine content in DJ-1 KO mice than in WT mice. Furthermore, the sensitization of microglia cells to LPS challenge to release IFN-γ and I-TAC was via the enhancement of NF-κB signaling, which was antagonized by NF-κB inhibitors. LPS-induced increase in neuronal death in the neuron-glia co-culture was enhanced by DJ-1 deficiency in microglia, which was antagonized by the neutralizing antibodies against IFN-γ or I-TAC. These results indicate that DJ-1 deficiency sensitizes microglia cells to release IFN-γ and I-TAC and causes inflammatory damage to dopaminergic neurons. The interaction between the genetic defect (i.e. DJ-1) and inflammatory factors (e.g. LPS) may contribute to the development of PD.

Local immunosuppressive microenvironment enhances migration of melanoma cells to lungs in DJ-1 knockout mice.

DJ-1 is an oncoprotein that promotes survival of cancer cells through anti-apoptosis. However, DJ-1 also plays a role in regulating IL-1ß expression, and whether inflammatory microenvironment built by dysregulated DJ-1 affects cancer progression is still unclear. This study thus aimed to compare the metastatic abilities of melanoma cells in wild-type (WT) and DJ-1 knockout (KO) mice, and to check whether inflammatory microenvironment built in DJ-1 KO mice plays a role in migration of cancer cells to lungs. First, B16F10 melanoma cells (at 6 × 10(4)) were injected into the femoral vein of mice, and formation of lung nodules, levels of lung IL-1ß and serum cytokines, and accumulation of myeloid-derived suppressor cells (MDSCs) were compared between WT and DJ-1 KO mice. Second, the cancer-bearing mice were treated with an interleukin-1 beta (IL-1ß) neutralizing antibody to see whether IL-1ß is involved in the cancer migration. Finally, cultured RAW 264.7 macrophage and B16F10 melanoma cells were respectively treated with DJ-1 shRNA and recombinant IL-1ß to explore underlying molecular mechanisms. Our results showed that IL-1ß enhanced survival and colony formation of cultured melanoma cells, and that IL-1ß levels were elevated both in DJ-1 KO mice and in cultured macrophage cells with DJ-1 knockdown. The elevated IL-1ß correlated with higher accumulation of immunosuppressive MDSCs and formation of melanoma module in the lung of DJ-1 KO mice, and both can be decreased by treating mice with IL-1ß neutralizing antibodies. Taken together, these results indicate that immunosuppressive tissue microenvironment built in DJ-1 KO mice can enhance lung migration of cancer, and IL-1ß plays an important role in promoting the cancer migration.

LC3 overexpression reduces Aß neurotoxicity through increasing α7nAchR expression and autophagic activity in neurons and mice.

Autophagy is an intracellular degradation pathway with dynamic interactions for eliminating damaged organelles and protein aggregates by lysosomal digestion. The EGFP-conjugated microtubule-associated protein 1 light chain 3 (EGFP-LC3) serves to monitor autophagic process. Extracellular ß-amyloid peptide accumulation is reported as a major cause in Alzheimer's disease (AD) pathogenesis; large numbers of autophagic vacuoles accumulate in patients' brains. We previously demonstrated that extracellular Aß (eAß) induces strong autophagic response and α7nAChR acts as a carrier to bind with eAß; which further inhibits Aß-induced neurotoxicity via autophagic degradation. In the present study, we overexpressed LC3 in both neuroblastoma cells (SH-SY5Y/pEGFP-LC3) and mice (TgEGFP-LC3) to assess the effect of LC3 overexpression on Aß neurotoxicity. SH-SY5Y/pEGFP-LC3 cells and primary cortical neuron cultures derived from E17 (embryonic day 17) TgEGFP-LC3 mice showed not only better resistance against Aß neurotoxicity but also higher α7nAChR expression and autophagic activity than control. Administration of α-bungarotoxin (α-BTX) to block α7nAChR antagonized the neuroprotective action of SH-SY5Y/pECGF-LC3 cells, suggesting that eAß binding with α7nAChR is an important step in Aß detoxification. LC3 overexpression thus exerts neuroprotection through increasing α7nAChR expression for eAß binding and further enhancing autophagic activity for Aß clearance in vitro and in vivo.