Targeting RAGE prevents muscle wasting and prolongs survival in cancer cachexia

Background: Cachexia, a multifactorial syndrome affecting more than 50% of patients with advanced cancer and responsible for ~20% of cancer-associated deaths, is still a poorly understood process without a standard cure available. Skeletal muscle atrophy caused by systemic inflammation is a major clinical feature of cachexia, leading to weight loss, dampening patients' quality of life, and reducing patients' response to anticancer therapy. RAGE (receptor for advanced glycation end-products) is a multiligand receptor of the immunoglobulin superfamily and a mediator of muscle regeneration, inflammation, and cancer. Methods: By using murine models consisting in the injection of colon 26 murine adenocarcinoma (C26-ADK) or Lewis lung carcinoma (LLC) cells in BALB/c and C57BL/6 or Ager−/− (RAGE-null) mice, respectively, we investigated the involvement of RAGE signalling in the main features of cancer cachexia, including the inflammatory state. In vitro experiments were performed using myotubes derived from C2C12 myoblasts or primary myoblasts isolated from C57BL/6 wild type and Ager−/− mice treated with the RAGE ligand, S100B (S100 calcium-binding protein B), TNF (tumor necrosis factor)α±IFN (interferon) γ, and tumour cell- or masses-conditioned media to analyse hallmarks of muscle atrophy. Finally, muscles of wild type and Ager−/− mice were injected with TNFα/IFNγ or S100B in a tumour-free environment. Results: We demonstrate that RAGE is determinant to activate signalling pathways leading to muscle protein degradation in the presence of proinflammatory cytokines and/or tumour-derived cachexia-inducing factors. We identify the RAGE ligand, S100B, as a novel factor able to induce muscle atrophy per se via a p38 MAPK (p38 mitogen-activated protein kinase)/myogenin axis and STAT3 (signal transducer and activator of transcription 3)-dependent MyoD (myoblast determination protein 1) degradation. Lastly, we found that in cancer conditions, an increase in serum levels of tumour-derived S100B and HMGB1 (high mobility group box 1) occurs leading to chronic activation/overexpression of RAGE, which induces hallmarks of cancer cachexia (i.e. muscle wasting, systemic inflammation, and release of tumour-derived pro-cachectic factors). Absence of RAGE in mice translates into reduced serum levels of cachexia-inducing factors, delayed loss of muscle mass and strength, reduced tumour progression, and increased survival. Conclusions: RAGE is a molecular determinant in inducing the hallmarks of cancer cachexia, and molecular targeting of RAGE might represent a therapeutic strategy to prevent or counteract the cachectic syndrome

Enhanced endocannabinoid-mediated modulation of rostromedial tegmental nucleus drive onto dopamine neurons in sardinian alcohol-preferring rats

The progressive predominance of rewarding effects of addictive drugs over their aversive properties likely contributes to the transition from drug use to drug dependence. By inhibiting the activity of DA neurons in the VTA, GABA projections from the rostromedial tegmental nucleus (RMTg) are well suited to shift the balance between drug-induced reward and aversion. Since cannabinoids suppress RMTg inputs to DA cells and CB1 receptors affect alcohol intake in rodents, we hypothesized that the endocannabinoid system, by modulating this pathway, might contribute to alcohol preference. Here we found that RMTg afferents onto VTA DA neurons express CB1 receptors and display a 2-arachidonoylglycerol (2-AG)-dependent form of short-term plasticity, that is, depolarization-induced suppression of inhibition (DSI). Next, we compared rodents with innate opposite alcohol preference, the Sardinian alcohol-preferring (sP) and alcohol-nonpreferring (sNP) rats. We found that DA cells from alcohol-naive sP rats displayed a decreased probability of GABA release and a larger DSI. This difference was due to the rate of 2-AG degradation. In vivo, we found a reduced RMTg-induced inhibition of putative DA neurons in sP rats that negatively correlated with an increased firing. Finally, alcohol failed to enhance RMTg spontaneous activity and to prolong RMTg-induced silencing of putative DA neurons in sP rats. Our results indicate functional modifications of RMTg projections to DA neurons that might impact the reward/aversion balance of alcohol attributes, which may contribute to the innate preference observed in sP rats and to their elevated alcohol intak

Assessment of the olfactory function in Italian patients with type 3 von Willebrand disease caused by a homozygous 253 Kb deletion involving VWF and TMEM16B/ANO2.

Type 3 Von Willebrand disease is an autosomal recessive disease caused by the virtual absence of the von Willebrand factor (VWF). A rare 253 kb gene deletion on chromosome 12, identified only in Italian and German families, involves both the VWF gene and the N-terminus of the neighbouring TMEM16B/ANO2 gene, a member of the family named transmembrane 16 (TMEM16) or anoctamin (ANO). TMEM16B is a calcium-activated chloride channel expressed in the olfactory epithelium. As a patient homozygous for the 253 kb deletion has been reported to have an olfactory impairment possibly related to the partial deletion of TMEM16B, we assessed the olfactory function in other patients using the University of Pennsylvania Smell Identification Test (UPSIT). The average UPSIT score of 4 homozygous patients was significantly lower than that of 5 healthy subjects with similar sex, age and education. However, 4 other members of the same family, 3 heterozygous for the deletion and 1 wild type, had a slightly reduced olfactory function indicating that socio-cultural or other factors were likely to be responsible for the observed difference. These results show that the ability to identify odorants of the homozygous patients for the deletion was not significantly different from that of the other members of the family, showing that the 253 kb deletion does not affect the olfactory performance. As other genes may compensate for the lack of TMEM16B, we identified some predicted functional partners from in silico studies of the protein-protein network of TMEM16B. Calculation of diversity for the corresponding genes for individuals of the 1000 Genomes Project showed that TMEM16B has the highest level of diversity among all genes of the network, indicating that TMEM16B may not be under purifying selection and suggesting that other genes in the network could compensate for its function for olfactory ability

Calcium concentration jumps reveal dynamic ion selectivity of calcium-activated chloride currents in mouse olfactory sensory neurons and TMEM16b-transfected HEK 293T cells.

Ca(2+)-activated Cl(-) channels play relevant roles in several physiological processes, including olfactory transduction, but their molecular identity is still unclear. Recent evidence suggests that members of the transmembrane 16 (TMEM16, also named anoctamin) family form Ca(2+)-activated Cl(-) channels in several cell types. In vertebrate olfactory transduction, TMEM16b/anoctamin2 has been proposed as the major molecular component of Ca(2+)-activated Cl(-) channels. However, a comparison of the functional properties in the whole-cell configuration between the native and the candidate channel has not yet been performed. In this study, we have used the whole-cell voltage-clamp technique to measure functional properties of the native channel in mouse isolated olfactory sensory neurons and compare them with those of mouse TMEM16b/anoctamin2 expressed in HEK 293T cells. We directly activated channels by rapid and reproducible intracellular Ca(2+) concentration jumps obtained from photorelease of caged Ca(2+) and determined extracellular blocking properties and anion selectivity of the channels. We found that the Cl(-) channel blockers niflumic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and DIDS applied at the extracellular side of the membrane caused a similar inhibition of the two currents. Anion selectivity measured exchanging external ions and revealed that, in both types of currents, the reversal potential for some anions was time dependent. Furthermore, we confirmed by immunohistochemistry that TMEM16b/anoctamin2 largely co-localized with adenylyl cyclase III at the surface of the olfactory epithelium. Therefore, we conclude that the measured electrophysiological properties in the whole-cell configuration are largely similar, and further indicate that TMEM16b/anoctamin2 is likely to be a major subunit of the native olfactory Ca(2+)-activated Cl(-) curren

THE ROLE OF ROSTROMEDIAL TEGMENTAL NUCLEUS IN THE REGULATION OF DOPAMINE NEURONS IN SARDINIAN ALCOHOL PREFERRING RATS

Alcoholism is a psychiatric disorder, whose aetiology involves inherited predispositions and environmental factors. Alcohol activates the brain reward circuitry, which stems from the ventral tegmental area (VTA) where dopamine (DA) cells are located. DA neuron spontaneous activity tightly depends on afferent inputs. Among these, those arising from the GABAergic rostromedial tegmental nucleus (RMTg) play a major role in controlling their impulse activity, and in mediating the effects of drugs of abuse on DA cells. In this study we took advantage of significant differences in voluntary alcohol drinking between the selectively bred Sardinian alcohol-preferring (sP) and -nonpreferring (sNP) rat lines and investigated their electrophysiological properties in vivo. Extracellular single unit recordings revealed a difference in baseline DA cell firing activity between sP and sNP rats. Accordingly, the duration of inhibition elicited by electrical stimulation of the RMTg onto DA cells was reduced in sP rats. Consistently, RMTg neurons showed a reduced spontaneous activity in sP rats. When alcohol was systemically administered, we found an increased duration of inhibition from the RMTg on DA cells in sP rats. Given the crucial role played by RMTg cells in modulating DA cell activity, and given that sP and sNP rats are phenotypes of alcohol preference and aversion, respectively, we support the key role of RMTg nucleus in the regulation of the net reward signal encoded by the reward system and suggest its involvement in the individual vulnerability to excessive alcohol drinking

ENDOCANNABINOID-MEDIATED PLASTICITY AT INHIBITORY SYNAPSES ONTO MIDBRAIN DOPAMINE NEURONS AS A POSSIBLE MARKER OF VULNERABILITY TO ADDICTION

Addiction is a psychiatric disorder, whose aetiology involves interaction of inherited predispositions and environmental factors. Both clinical and preclinical findings indicate that there are important genetic variations in vulnerability to drug addiction, and that such differences may be mediated by the same biological mechanisms. Addictive drugs share the properties of being self-administered by laboratory animals, and of activating the brain reward circuitry, which stems from the ventral tegmental area (VTA) where dopamine (DA) cells are located. Endocannabinoids serve as retrograde signaling molecules at many synapses in the brain, including the VTA, and regulate reward seeking by modulating DA signaling. We took advantage of significant differences between pairs of lines of rats selectively bred for their voluntary alcohol preference or aversion, that is Sardinian alcohol-preferring (sP) or nonpreferring (sNP) rat line. We have found that depolarization-induced suppression of inhibition (DSI), a form of endocannabinoid-mediated short term synaptic plasticity, is differently expressed by two discrete sets of inhibitory synapses arising from rostral and caudal afferents onto VTA DA neurons. This phenomenon is selectively mediated by the endocannabinoid 2-arachidonoylglycerol (2-AG), which activates presynaptic type 1-cannabinoid (CB1) receptors. However, the two discrete DSI do not seem to depend upon differences in CB1 number and/or function, but rather on the rate 2-AG is degraded. Thus, 2-AG by differently depressing inhibitory synapses arising from either rostral or caudal afferents might indirectly alter DA neuron functional state, and enhance the responsiveness of the reward pathway to phasic DA. Given that sP rats are vulnerable phenotypes, and that they possess this endocannabinoid-mediated form of short term plasticity, our results suggest that differences in equipment of the endocannabinoid system machinery might control specific sources of vulnerability