No abstract

Melanin concentrating hormone (MCH) is a cyclic neuropeptide present in the hypothalamus of all vertebrates. MCH is implicated in a number of behaviors but direct evidence is lacking. To selectively stimulate the MCH neurons the gene for the light-sensitive cation channel, channelrhodopsin-2, was inserted into the MCH neurons of wild-type mice. Three weeks later MCH neurons were stimulated for 1 min every 5 min for 24 h. A 10 Hz stimulation at the start of the night hastened sleep onset, reduced length of wake bouts by 50%, increased total time in non-REM and REM sleep at night, and increased sleep intensity during the day cycle. Sleep induction at a circadian time when all of the arousal neurons are active indicates that MCH stimulation can powerfully counteract the combined wake-promoting signal of the arousal neurons. This could be potentially useful in treatment of insomnia.

Multiple myeloma (MM) is a plasma cell neoplasm that proceeds through a premalignant state of monoclonal gammopathy of unknown significance; however, the molecular events responsible for myelomagenesis remain uncharacterized. To identify cellular pathways deregulated in MM, we addressed that sumoylation is homologous to ubiquitination and results in the attachment of the ubiquitin-like protein Sumo onto target proteins. Sumoylation was markedly enhanced in MM patient lysates compared with normal plasma cells and expression profiling indicated a relative induction of sumoylation pathway genes. The Sumoconjugating enzyme Ube2I, the Sumoligase PIAS1, and the Sumo-inducer ARF were elevated in MM patient samples and cell lines. Survival correlated with expression because 80% of patients with low UBE2I and PIAS1 were living 6 years after transplantation, whereas only 45% of patients with high expression survived 6 years. UBE2I encodes the sole Sumoconjugating enzyme in mammalian cells and cells transfected with a dominantnegative sumoylation-deficient UBE2I mutant exhibited decreased survival after radiation exposure, impaired adhesion to bone marrow stroma cell and decreased bone marrow stroma cell-induced proliferation. UBE2I confers cells with multiple advantages to promote tumorigenesis and predicts decreased survival when combined with PIAS1. The sumoylation pathway is a novel therapeutic target with implications for existing proteasomal- IntroductionMultiple myeloma (MM) is a neoplasia hallmarked by the clonal expansion of malignant plasma cells (PCs) and the accumulation of a monoclonal immunoglobulin (Ig) or Ig fragment detectable in the serum and/or urine. [1][2][3] MM is the second most commonly diagnosed hematologic malignancy in the Western world, accounts for nearly 20% of all hematologic malignancies, and despite conventional treatment or high-dose therapy with stem cell transplantation is generally considered incurable. 4,5 Recent advances in mechanistic understanding and treatment modalities have extended median survival to exceed 6 years, and 10% of MM patients survive beyond 10 years. [6][7][8] Although high-dose therapy and novel agents that include thalidomide, its analog lenalidomide, and proteasome inhibitors have significantly improved prognosis, patient survival remains highly variable and cannot be accurately predicted with current models in part because the cellular pathways that determine patient response to treatment remain unidentified.In eukaryotes, a highly conserved multienzyme system is used in a sequential process to covalently attach the polypeptide ubiquitin to proteins targeted for degradation. 9,10 Ubiquitination is an essential process that maintains cellular homeostasis through dynamic switches in protein functional states. Ubiquitin-protein conjugates are then degraded in by the ATP-dependent 26S proteasome complex. 11,12 Deregulation of ubiquitination in tumor models has resulted in malignant transformation and tumor progression. 13 In myeloma, proteasomal-dependent catabolis...

A distributed network of neurons regulates wake, non-rapid eye movement sleep (NREM), and rapid eye movement sleep. However, there are also glia in the brain, and there is growing evidence that neurons and astroglia communicate intimately to regulate behavior. To identify the effect of optogenetic stimulation of astrocytes on sleep, the promoter for the astrocyte specific cytoskeletal protein, glial fibrillary acidic protein (GFAP) was employed to direct the expression of channelrhodopsin-2 (ChR2) and the linked reporter gene, enhanced yellow fluorescent protein (EYFP), in astrocytes. rAAV-GFAP-ChR2 (H134R) -EYFP or rAAV-GFAP-EYFP were microinjected (750nl) into the posterior hypothalamus (bilateral) of mice. Three weeks later baseline sleep was recorded (0 Hz) and 24h later optogenetic stimulation applied during the first 6h of the lights-off period. Mice with ChR2 were given 5, 10 or 30Hz stimulation for 6h (10 msec pulses; 1mW; one minute on- 4 minutes off). At least 36h elapsed between the stimulation periods (5, 10, 30Hz) and although 0Hz was always first, the order of the other three stimulation rates were randomized. In mice with ChR2 (n=7), 10 Hz, but not 5 or 30Hz stimulation increased both NREM and REM sleep during the 6hr period of stimulation. Delta power did not increase. In control mice (no ChR2; n=5) 10Hz stimulation had no effect. This study demonstrates that direct stimulation of astrocytes powerfully induces sleep during the active phase of the sleep-wake cycle and underscores the inclusion of astrocytes in network models of sleep-wake regulation.

Cataplexy, a sudden unexpected muscle paralysis, is a debilitating symptom of the neurodegenerative sleep disorder, narcolepsy. During these attacks, the person is paralyzed, but fully conscious and aware of their surroundings. To identify potential neurons that might serve as surrogate orexin neurons to suppress such attacks, the gene for orexin (hypocretin), a peptide lost in most human narcoleptics, was delivered into the brains of the orexin-ataxin-3 transgenic mouse model of human narcolepsy. Three weeks after the recombinant adenoassociated virus (rAAV)-mediated orexin gene transfer, sleep–wake behavior was assessed. rAAV-orexin gene delivery into neurons of the zona incerta (ZI), or the lateral hypothalamus (LH) blocked cataplexy. Orexin gene transfer into the striatum or in the melanin-concentrating hormone neurons in the ZI or LH had no such effect, indicating site specificity. In transgenic mice lacking orexin neurons but given rAAV-orexin, detectable levels of orexin-A were evident in the CSF, indicating release of the peptide from the surrogate neurons. Retrograde tracer studies showed that the amygdala innervates the ZI consistent with evidence that strong emotions trigger cataplexy. In turn, the ZI projects to the locus ceruleus, indicating that the ZI is part of a circuit that stabilizes motor tone. Our results indicate that these neurons might also be recruited to block the muscle paralysis in narcolepsy.

These data indicate that a natural product with antioxidant properties from broccoli has great potential to be used in chemoprevention and treatment of BEAC.

We have previously demonstrated that interleukin-17A (IL-17) producing Th17 cells are significantly elevated in blood and bone marrow (BM) in multiple myeloma (MM) and IL-17A promotes MM cell growth via the expression of IL-17 receptor. In this study, we evaluated anti-human IL-17A human monoclonal antibody (mAb), AIN457 in MM. We observe significant inhibition of MM cell growth by AIN457 both in the presence and absence of BM stromal cells (BMSC). While IL-17A induces IL-6 production, AIN457 significantly down-regulated IL-6 production and MM cell-adhesion in MM-BMSC co-culture. AIN-457 also significantly inhibited osteoclast cell–differentiation. More importantly, in the SCIDhu model of human myeloma administration of AIN-457 weekly for 4 weeks after the first detection of tumor in mice led to a significant inhibition of tumor growth and reduced bone damage compared to isotype control mice. To understand the mechanism of action of anti-IL-17A mAb, we report here, that MM cells express IL-17A. We also observed that IL-17A knock-down inhibited MM cell growth and their ability to induce IL-6 production in co-cultures with BMSC. These pre-clinical observations suggest efficacy of AIN 457 in myeloma and provide the rationale for its clinical evaluation for anti-myeloma effects and for improvement of bone disease.

Neurons containing melanin-concentrating hormone (MCH) are located in the hypothalamus. In mice optogenetic activation of the MCH neurons induces both NREM and REM sleep at night, the normal wake-active period for nocturnal rodents (Konadhode et al., 2013). Here we selectively activate these neurons in rats to test the validity of the sleep network hypothesis in another species. Channelrhodopsin-2 (ChR2) driven by the MCH promoter was selectively expressed by MCH neurons after injection of rAAV-MCHp-ChR2-EYFP into the hypothalamus of Long-Evans rats. An in vitro study confirmed that the optogenetic activation of MCH neurons faithfully triggered action potentials. In the second study, in Long-Evans rats, rAAV-MCH-ChR2, or the control vector, rAAV-MCH-EYFP, were delivered into the hypothalamus. Three weeks later baseline sleep was recorded for 48h without optogenetic stimulation (0 Hz). Subsequently, at the start of the lights-off cycle, the MCH neurons were stimulated at 5, 10, or 30Hz (1 mW at tip; 1 min on – 4 min off) for 24 h. Sleep was recorded during the 24h stimulation period. Optogenetic activation of MCH neurons increased both REM and NREM sleep at night, whereas during the day cycle only REM sleep was increased. Delta power, an indicator of sleep intensity, was also increased. In control rats without ChR2, optogenetic stimulation did not increase sleep or delta power. These results lend further support to the view that sleep-active MCH neurons contribute to drive sleep in mammals.