The CD200R1 microglial inhibitory receptor as a therapeutic target in the MPTP model of Parkinson's disease.

BACKGROUND: It is suggested that neuroinflammation, in which activated microglial cells play a relevant role, contributes to the development of Parkinson's disease (PD). Consequently, the modulation of microglial activation is a potential therapeutic target to be taken into account to act against the dopaminergic neurodegeneration occurring in this neurological disorder. Several soluble and membrane-associated inhibitory mechanisms contribute to maintaining microglial cells in a quiescent/surveillant phenotype in physiological conditions. However, the presence of activated microglial cells in the brain in PD patients suggests that these mechanisms have been somehow overloaded. We focused our interest on one of the membrane-associated mechanisms, the CD200-CD200R1 ligand-receptor pair. METHODS: The acute MPTP experimental mouse model of PD was used to study the temporal pattern of mRNA expression of CD200 and CD200R1 in the context of MPTP-induced dopaminergic neurodegeneration and neuroinflammation. Dopaminergic damage was assessed by tyrosine hydroxylase (TH) immunoreactivity, and neuroinflammation was evaluated by the mRNA expression of inflammatory markers and IBA1 and GFAP immunohistochemistry. The effect of the modulation of the CD200-CD200R1 system on MPTP-induced damage was determined by using a CD200R1 agonist or CD200 KO mice. RESULTS: MPTP administration resulted in a progressive decrease in TH-positive fibres in the striatum and TH-positive neurons in the substantia nigra pars compacta, which were accompanied by transient astrogliosis, microgliosis and expression of pro- and anti-inflammatory markers. CD200 mRNA levels rapidly decreased in the ventral midbrain after MPTP treatment, while a transient decrease of CD200R1 mRNA expression was repeatedly observed in this brain area at earlier and later phases. By contrast, a transient increase in CD200R1 expression was observed in striatum. The administration of a CD200R1 agonist resulted in the inhibition of MPTP-induced dopaminergic neurodegeneration, while microglial cells showed signs of earlier activation in CD200-deficient mice. CONCLUSIONS: Collectively, these findings provide evidence for a correlation between CD200-CD200R1 alterations, glial activation and neuronal loss. CD200R1 stimulation reduces MPTP-induced loss of dopaminergic neurons, and CD200 deficiency results in earlier microglial activation, suggesting that the potentiation of CD200R1 signalling is a possible approach to controlling neuroinflammation and neuronal death in PD.

Sleep modulates haematopoiesis and protects against atherosclerosis.

Sleep is integral to life1. Although insufficient or disrupted sleep increases the risk of multiple pathological conditions, including cardiovascular disease2, we know little about the cellular and molecular mechanisms by which sleep maintains cardiovascular health. Here we report that sleep regulates haematopoiesis and protects against atherosclerosis in mice. We show that mice subjected to sleep fragmentation produce more Ly-6Chigh monocytes, develop larger atherosclerotic lesions and produce less hypocretin-a stimulatory and wake-promoting neuropeptide-in the lateral hypothalamus. Hypocretin controls myelopoiesis by restricting the production of CSF1 by hypocretin-receptor-expressing pre-neutrophils in the bone marrow. Whereas hypocretin-null and haematopoietic hypocretin-receptor-null mice develop monocytosis and accelerated atherosclerosis, sleep-fragmented mice with either haematopoietic CSF1 deficiency or hypocretin supplementation have reduced numbers of circulating monocytes and smaller atherosclerotic lesions. Together, these results identify a neuro-immune axis that links sleep to haematopoiesis and atherosclerosis.

Bidirectional and context-dependent changes in theta and gamma oscillatory brain activity in noradrenergic cell-specific Hypocretin/Orexin receptor 1-KO mice.

Noradrenaline (NA) and hypocretins/orexins (HCRT), and their receptors, dynamically modulate the circuits that configure behavioral states, and their associated oscillatory activities. Salient stimuli activate spiking of locus coeruleus noradrenergic (NALC) cells, inducing NA release and brain-wide noradrenergic signalling, thus resetting network activity, and mediating an orienting response. Hypothalamic HCRT neurons provide one of the densest input to NALC cells. To functionally address the HCRT-to-NA connection, we selectively disrupted the Hcrtr1 gene in NA neurons, and analyzed resulting (Hcrtr1Dbh-CKO) mice', and their control littermates' electrocortical response in several contexts of enhanced arousal. Under enforced wakefulness (EW), or after cage change (CC), Hcrtr1Dbh-CKO mice exhibited a weakened ability to lower infra-θ frequencies (1-7 Hz), and mount a robust, narrow-bandwidth, high-frequency θ rhythm (~8.5 Hz). A fast-γ (55-80 Hz) response, whose dynamics closely parallelled θ, also diminished, while ß/slow-γ activity (15-45 Hz) increased. Furthermore, EW-associated locomotion was lower. Surprisingly, nestbuilding-associated wakefulness, inversely, featured enhanced θ and fast-γ activities. Thus HCRT-to-NA signalling may fine-tune arousal, up in alarming conditions, and down during self-motivated, goal-driven behaviors. Lastly, slow-wave-sleep following EW and CC, but not nestbuilding, was severely deficient in slow-δ waves (0.75-2.25 Hz), suggesting that HCRT-to-NA signalling regulates the slow-δ rebound characterizing sleep after stress-associated arousal.

Importance of CD200 expression by tumor or host cells to regulation of immunotherapy in a mouse breast cancer model.

Cell-surface CD200 expression by mouse EMT6 breast tumor cells increased primary tumor growth and metastasis to the draining lymph nodes (DLN) in normal (WT) BALB/c female recipients, while lack of CD200R1 expression in a CD200R1-/- host negated this effect. Silencing CD200 expression in EMT6siCD200 tumor cells also reduced their ability to grow and metastasize in WT animals. The cellular mechanisms responsible for these effects have not been studied in detail. We report characterization of tumor infiltrating (TILs) and draining lymph node (DLN) cells in WT and CD200-/- BALB/c mice, receiving WT tumor cells, or EMT6 lacking CD200 expression (EMT6siCD200 cells). Our data show an important correlation with augmented CD8+ cytotoxic T cells and resistance to tumor growth in mice lacking exposure (on either host cells or tumor) to the immunoregulatory molecule CD200. Confirmation of the importance of such CD8+ cells came from monitoring tumor growth and characterization of the TILs and DLN cells in WT mice challenged with EMT6 and EMT6siCD200 tumors and treated with CD8 and CD4 depleting antibodies. Finally, we have assessed the mechanisms(s) whereby addition of metformin as an augmenting chemotherapeutic agent in CD200-/- animals given EMT6 tumors and treated with a previously established immunotherapy regime can increase host resistance. Our data support the hypothesis that increased autophagy in the presence of metformin increases CD8+ responses and tumor resistance, an effect attenuated by the autophagy inhibitor verteporfin.

A comparison of serum miRNAs influencing metastatic growth of EMT6 vs 4THM tumor cells in wild-type and CD200R1KO mice.

PURPOSE: We investigated whether miRNAs in exosomes from EMT6 or 4THM tumor-bearing mice played a role in regulating inflammatory cytokine expression and/or metastasis in WT mice injected with EMT6 and/or 4THM tumor cells. METHODS: EMT6 tumors in BALB/c CD200R1KO mice resolve following surgical resection of localized tumor and immunization with irradiated EMT6 cells along with CpG as adjuvant. Wild-type (WT) animals treated in the same fashion develop pulmonary and liver metastases within 20 days of surgery. DLNs from CD200R1KO mice contain no tumor cells detectable at limiting dilution. In contrast, 4THM tumor cells injected into CD200R1KO show increased metastasis compared with WT mice. Transfer of serum exosomes from 4THM tumor-bearing mice to WT animals increased metastasis of EMT6 tumors, an effect attenuated by anti-IL-6 antibody. We compared miRNA expression in exosomes from the serum of 4THM/EMT6 WT or CD200R1KO tumor-bearing mice, and the effects of antagomirs to miRNAs on tumor growth. RESULTS: Complex changes in miRNA expression were observed in the isolated exosomes. Some miRNAs, including miR155, have been reported to potentiate inflammatory responses and augment inflammatory cytokine expression. Expression of miR155 increased in exosomes from 4THM relative to EMT6 tumor bearers, and antagomirs to miR155 attenuated tumor growth and metastasis, and improved survival, following infusion into WT mice. Antagomirs to the miR205 family were thought to affect metastasis by targeting epithelial-to-mesenchymal transition (EMT), increased growth and metastasis in both 4THM and EMT6 tumor-bearing mice, and decreased survival, with some modulation of inflammatory cytokine production. CONCLUSIONS: Multiple pathways are implicated in differential metastasis of EMT6/4THM, and targeting these may have clinical utility in human breast cancer.

Involvement of the orexin/hypocretin system in the pharmacological effects induced by Δ(9) -tetrahydrocannabinol.

BACKGROUND AND PURPOSE: Anatomical, biochemical and pharmacological evidence suggest the existence of a crosstalk between the orexinergic and endocannabinoid systems. While the orexin receptor 1 (OX1 receptor) modulates the reinforcing properties of cannabinoids, the participation of orexins in the acute pharmacological effects of Δ(9) -tetrahydrocannabinol (THC) remains unexplored. EXPERIMENTAL APPROACH: We assessed the possible role of orexins in THC-induced hypolocomotion, hypothermia, antinociception, anxiolytic- and anxiogenic-like effects and memory impairment. Selective OX1 and OX2 receptor antagonists and OX1 knockout (KO) mice as well as prepro-orexin (PPO) KO mice were used as pharmacological and genetic approaches. CB1 receptor levels in control and PPO KO mice were evaluated by immunoblot analysis. The expression of c-Fos after THC treatment was analysed in several brain areas in wild-type mice and in mice lacking the PPO gene. KEY RESULTS: The hypothermia, supraspinal antinociception and anxiolytic-like effects induced by THC were modulated by orexins through OX2 receptor signalling. OX1 receptors did not seem to be involved in these THC responses. No differences in CB1 receptor levels were found between wild-type and PPO KO mice. THC-induced increase in c-Fos expression was reduced in the central amygdala, medial preoptic area and lateral septum in these mutant mice. CONCLUSIONS AND IMPLICATIONS: Our results provide new findings to further clarify the interaction between orexins and cannabinoids. OX1 and OX2 receptors are differently implicated in the pharmacological effects of cannabinoids.

Comparison of immunity in mice cured of primary/metastatic growth of EMT6 or 4THM breast cancer by chemotherapy or immunotherapy.

PURPOSE: We have compared cure from local/metastatic tumor growth in BALB/c mice receiving EMT6 or the poorly immunogenic, highly metastatic 4THM, breast cancer cells following manipulation of immunosuppressive CD200:CD200R interactions or conventional chemotherapy. METHODS: We reported previously that EMT6 tumors are cured in CD200R1KO mice following surgical resection and immunization with irradiated EMT6 cells and CpG oligodeoxynucleotide (CpG), while wild-type (WT) animals developed pulmonary and liver metastases within 30 days of surgery. We report growth and metastasis of both EMT6 and a highly metastatic 4THM tumor in WT mice receiving iv infusions of Fab anti-CD200R1 along with CpG/tumor cell immunization. Metastasis was followed both macroscopically (lung/liver nodules) and microscopically by cloning tumor cells at limiting dilution in vitro from draining lymph nodes (DLN) harvested at surgery. We compared these results with local/metastatic tumor growth in mice receiving 4 courses of combination treatment with anti-VEGF and paclitaxel. RESULTS: In WT mice receiving Fab anti-CD200R, no tumor cells are detectable following immunotherapy, and CD4+ cells produced increased TNFα/IL-2/IFNγ on stimulation with EMT6 in vitro. No long-term cure was seen following surgery/immunotherapy of 4THM, with both microscopic (tumors in DLN at limiting dilution) and macroscopic metastases present within 14 d of surgery. Chemotherapy attenuated growth/metastases in 4THM tumor-bearers and produced a decline in lung/liver metastases, with no detectable DLN metastases in EMT6 tumor-bearing mice-these latter mice nevertheless showed no significantly increased cytokine production after restimulation with EMT6 in vitro. EMT6 mice receiving immunotherapy were resistant to subsequent re-challenge with EMT6 tumor cells, but not those receiving curative chemotherapy. Anti-CD4 treatment caused tumor recurrence after immunotherapy, but produced no apparent effect in either EMT6 or 4THM tumor bearers after chemotherapy treatment. CONCLUSION: Immunotherapy, but not chemotherapy, enhances CD4+ immunity and affords long-term control of breast cancer growth and resistance to new tumor foci.

Acute suppressive and long-term phase modulation actions of orexin on the mammalian circadian clock.

Circadian and homeostatic neural circuits organize the temporal architecture of physiology and behavior, but knowledge of their interactions is imperfect. For example, neurons containing the neuropeptide orexin homeostatically control arousal and appetitive states, while neurons in the suprachiasmatic nuclei (SCN) function as the brain's master circadian clock. The SCN regulates orexin neurons so that they are much more active during the circadian night than the circadian day, but it is unclear whether the orexin neurons reciprocally regulate the SCN clock. Here we show both orexinergic innervation and expression of genes encoding orexin receptors (OX1 and OX2) in the mouse SCN, with OX1 being upregulated at dusk. Remarkably, we find through in vitro physiological recordings that orexin predominantly suppresses mouse SCN Period1 (Per1)-EGFP-expressing clock cells. The mechanisms underpinning these suppressions vary across the circadian cycle, from presynaptic modulation of inhibitory GABAergic signaling during the day to directly activating leak K(+) currents at night. Orexin also augments the SCN clock-resetting effects of neuropeptide Y (NPY), another neurochemical correlate of arousal, and potentiates NPY's inhibition of SCN Per1-EGFP cells. These results build on emerging literature that challenge the widely held view that orexin signaling is exclusively excitatory and suggest new mechanisms for avoiding conflicts between circadian clock signals and homeostatic cues in the brain.

Synthesis and evaluation of carbon-linked analogs of dual orexin receptor antagonist filorexant.

Analogs of the dual orexin receptor antagonist filorexant were prepared. Replacement of the ether linkage proved highly sensitive toward modification with an acetylene linkage providing compounds with the best in vitro and in vivo potency profiles.

Anti-CD200R2, anti-IL-9, anti-IL-35, or anti-TGF-ß abolishes increased graft survival and Treg induction induced in cromolyn-treated CD200R1KO.CD200tg mice.

BACKGROUND: Rejection is associated with early degranulation (≥80%) of graft-infiltrating CD200R1 receptor-positive mast cells (MCs). Survival is increased, and MC degranulation is decreased, in CD200 mice but not in CD200R1KO mice. CD200 engagement of CD200R2 (not present on MCs) alters dendritic cell differentiation and enhances induction of Foxp3 regulatory T cells (Tregs). We investigated whether attenuation of MC degranulation by sodium cromoglycate allowed CD200 to increase survival in CD200R1KO mice. METHODS: C57BL/6 control, CD200R1KO, CD200, or CD200R1KO.CD200 mice received BALB/c grafts with or without treatment with cromoglycate. Survival was monitored daily from day 10, with mixed lymphocyte culture responses measured on day 14 or 21 and graft immunohistology performed on day 14. RESULTS: Decreased MC degranulation and increased graft Foxp3 Treg infiltration/survival occurred in CD200 mice and in CD200-treated control mice or CD200R1KO.CD200 mice receiving cromoglycate. Neutralizing anti-CD200 or anti-CD200R1/R2 monoclonal antibody caused graft rejection, as did anti-interleukin (IL)-9, anti-IL-35, or anti-transforming growth factor-ß antibodies, with the latter also decreasing graft-infiltrating Tregs. CONCLUSION: These data imply a coordinated effect of MCs and Tregs on increased graft survival induced by CD200, with a critical role for IL-9, IL-35, and transforming growth factor-ß in the development/function of Tregs.