Carolina Doerrier

Extrapineal melatonin: analysis of its subcellular distribution and daily fluctuations

Abstract:  We studied the subcellular levels of melatonin in cerebral cortex and liver of rats under several conditions. The results show that melatonin levels in the cell membrane, cytosol, nucleus, and mitochondrion vary over a 24‐hr cycle, although these variations do not exhibit circadian rhythms. The cell membrane has the highest concentration of melatonin followed by mitochondria, nucleus, and cytosol. Pinealectomy significantly increased the content of melatonin in all subcellular compartments, whereas luzindole treatment had little effect on melatonin levels. Administration of 10 mg/kg bw melatonin to sham‐pinealectomized, pinealectomized, or continuous light‐exposed rats increased the content of melatonin in all subcellular compartments. Melatonin in doses ranging from 40 to 200 mg/kg bw increased in a dose‐dependent manner the accumulation of melatonin on cell membrane and cytosol, although the accumulations were 10 times greater in the former than in the latter. Melatonin levels in the nucleus and mitochondria reached saturation with a dose of 40 mg/kg bw; higher doses of injected melatonin did not further cause additional accumulation of melatonin in these organelles. The results suggest some control of extrapineal accumulation or extrapineal production of melatonin and support the existence of regulatory mechanisms in cellular organelles, which prevent the intracellular equilibration of the indolamine. Seemingly, different concentrations of melatonin can be maintained in different subcellular compartments. The data also seem to support a requirement of high doses of melatonin to obtain therapeutic effects. Together, these results add information that assists in explaining the physiology and pharmacology of melatonin.

Disruption of the NF-κB/NLRP3 connection by melatonin requires retinoid-related orphan receptor-α and blocks the septic response in mice

We determined the NF‐κB‐ and NOD‐like receptor (NLR)P3‐dependent molecular mechanisms involved in sepsis and evaluated the role of retinoid‐related orphan receptor (ROR)‐α in melatonins anti‐inflammatory actions. Western blot, RT‐PCR, ELISA, and spectrophotometric analysis revealed that NF‐κB and NLRP3 closely interact, leading to proinflammatory and pro‐oxidant status in heart tissue of septic C57BL/6J mice. Moreover, mitochondrial oxygen consumption was reduced by 80% in septic mice. In vivo and in vitro analysis showed that melatonin administration blunts NF‐κB transcriptional activity through a sirtuinl‐dependent NF‐κB deacetylation in septic mice. Melatonin also decreased NF‐κB‐dependent proinflammatory response and restored redox balance and mitochondrial homeostasis, thus inhibiting the NLRP3 inflammasome. In an important finding, the inhibition of NF‐κB by melatonin, but not that of NLRP3, was blunted in RORαsg/sg mice, indicating that functional RORα transcription factor is necessary for the initiation of the innate immune response against inflammation. Our results are evidence of the NF‐κB/NLRP3 connection during sepsis and identify NLRP3 as a novel molecular target for melatonin. The multiple molecular targets of melatonin in this study explain its potent anti‐inflammatory efficacy against systemic innate immune activation and herald a promising therapeutic application for melatonin in the treatment of sepsis.—García, J. A., Volt, H., Venegas, C., Doerrier, C., Escames, G., López., L. C., Acuña‐Castroviejo, D. Disruption of the NF‐κB/NLRP3 connection by melatonin requires retinoid‐related orphan receptor‐α and blocks the septic response in mice. FASEB J. 29, 3863‐3875 (2015). www.fasebj.org

Melatonin blunts the mitochondrial/NLRP3 connection and protects against radiation-induced oral mucositis

Mucositis is a common and distressing side effect of chemotherapy or radiotherapy that has potentially severe consequences, and no treatment is available. The purpose of this study was to analyze the molecular pathways involved in the development of oral mucositis and to evaluate whether melatonin can prevent this pathology. The tongue of male Wistar rats was subjected to irradiation (X‐ray YXLON Y.Tu 320‐D03 irradiator; the animals received a dose of 7.5 Gy/day for 5 days). Rats were treated with 45 mg/day melatonin or vehicle for 21 days postirradiation, either by local application into their mouths (melatonin gel) or by subcutaneous injection. A connection between reactive oxygen species, generating mitochondria and the NLRP3 (NLR‐related protein 3 nucleotide‐binding domain leucine‐rich repeat containing receptor‐related protein 3) inflammasome, has been reported in mucositis. Here, we show that mitochondrial oxidative stress, bioenergetic impairment and subsequent NLRP3 inflammasome activation are involved in the development of oral mucositis after irradiation and that melatonin synthesized in the rat tongue is depleted after irradiation. The application of melatonin gel restores physiological melatonin levels in the tongue and prevents mucosal disruption and ulcer formation. Melatonin gel protects the mitochondria from radiation damage and blunts the NF‐κB/NLRP3 inflammasome signaling activation in the tongue. Our results suggest new molecular pathways involved in radiotherapy‐induced mucositis that are inhibited by topical melatonin application, suggesting a potential preventive therapy for mucositis in patients with cancer.

The beneficial effects of melatonin against heart mitochondrial impairment during sepsis: inhibition of iNOS and preservation of nNOS.

While it is accepted that the high production of nitric oxide (NO˙) by the inducible nitric oxide synthase (iNOS) impairs cardiac mitochondrial function during sepsis, the role of neuronal nitric oxide synthase (nNOS) may be protective. During sepsis, there is a significantly increase in the expression and activity of mitochondrial iNOS (i‐mtNOS), which parallels the changes in cytosolic iNOS. The existence of a constitutive NOS form (c‐mtNOS) in heart mitochondria has been also described, but its role in the heart failure during sepsis remains unclear. Herein, we analyzed the changes in mitochondrial oxidative stress and bioenergetics in wild‐type and nNOS‐deficient mice during sepsis, and the role of melatonin, a known antioxidant, in these changes. Sepsis was induced by cecal ligation and puncture, and heart mitochondria were analyzed for NOS expression and activity, nitrites, lipid peroxidation, glutathione and glutathione redox enzymes, oxidized proteins, and respiratory chain activity in vehicle‐ and melatonin‐treated mice. Our data show that sepsis produced a similar induction of iNOS/i‐mtNOS and comparable inhibition of the respiratory chain activity in wild‐type and in nNOS‐deficient mice. Sepsis also increased mitochondrial oxidative/nitrosative stress to a similar extent in both mice strains. Melatonin administration inhibited iNOS/i‐mtNOS induction, restored mitochondrial homeostasis in septic mice, and preserved the activity of nNOS/c‐mtNOS. The effects of melatonin were unrelated to the presence or the absence of nNOS. Our observations show a lack of effect of nNOS on heart bioenergetic impairment during sepsis and further support the beneficial actions of melatonin in sepsis.

Dysfunctional Coq9 protein causes predominant encephalomyopathy associated with CoQ deficiency

Coenzyme Q10 (CoQ(10)) or ubiquinone is a well-known component of the mitochondrial respiratory chain. In humans, CoQ(10) deficiency causes a mitochondrial syndrome with an unexplained variability in the clinical presentations. To try to understand this heterogeneity in the clinical phenotypes, we have generated a Coq9 Knockin (R239X) mouse model. The lack of a functional Coq9 protein in homozygous Coq9 mutant (Coq9(X/X)) mice causes a severe reduction in the Coq7 protein and, as consequence, a widespread CoQ deficiency and accumulation of demethoxyubiquinone. The deficit in CoQ induces a brain-specific impairment of mitochondrial bioenergetics performance, a reduction in respiratory control ratio, ATP levels and ATP/ADP ratio and specific loss of respiratory complex I. These effects lead to neuronal death and demyelinization with severe vacuolization and astrogliosis in the brain of Coq9(X/X) mice that consequently die between 3 and 6 months of age. These results suggest that the instability of mitochondrial complex I in the brain, as a primary event, triggers the development of mitochondrial encephalomyopathy associated with CoQ deficiency.

Same molecule but different expression: aging and sepsis trigger NLRP3 inflammasome activation, a target of melatonin

The connection between the innate immune system, clock genes, and mitochondrial bioenergetics was analyzed during aging and sepsis in mouse heart. Our results suggest that the sole NF‐κB activation does not explain the inflammatory process underlying aging; the former also triggers the NLRP3 inflammasome that enhances caspase‐1‐dependent maturation of IL‐1β. In this way, aged mice enter into a vicious cycle as IL‐1β further activates the NF‐κB/NLRP3 inflammasome link. The origin of NF‐κB activation was related to the age‐dependent Bmal1/Clock/RORα/Rev‐Erbα loop disruption, which lowers NAD+ levels, reducing the SIRT1 deacetylase ability to inactivate NF‐κB. Consequently, NF‐κB binding to DNA increases, raising the formation of proinflammatory mediators and inducing mitochondrial impairment. The cycle is then closed with the subsequent NLRP3 inflammasome activation. This paired contribution of the innate immune pathways serves as a catalyst to magnify the response to sepsis in aged compared with young mice. Melatonin administration blunted the septic response, reducing inflammation and oxidative stress, and enhancing mitochondrial function at the levels of nonseptic aged mice, but it did not counteract the age‐related inflammation. Together, our results suggest that, although with different strengths, chronoinflammaging constitutes the biochemical substrate of aging and sepsis, and identifies the NLRP3 inflammasome as a new molecular target for melatonin, providing a rationale for its use in NLRP3‐dependent diseases.

Analysis of the daily changes of melatonin receptors in the rat liver

Melatonin membrane (MT1 and MT2) and nuclear (RORα) receptors have been identified in several mammalian tissues, including the liver. The mechanisms regulating hepatic melatonin receptors are yet unknown. This study investigated whether these receptors exhibit daily changes and the effects of melatonin on their levels. Our results show that mRNAs for MT1/MT2 receptors exhibit circadian rhythms that were followed by rhythms in their respective protein levels; the acrophases for the two rhythms were reached at 04:00 and 05:00 hr, respectively. Pinealectomy blunted the rhythms in both mRNAs and protein levels. In contrast, mRNA and protein levels of nuclear receptor RORα increased significantly after pinealectomy. The cycles of the latter receptor also exhibited circadian rhythms which peaked at 03:00 and 03:45 hr, respectively. Melatonin administration (10–200 mg/kg) increased in a dose‐dependent manner the protein content of MT1/MT2 receptors, with no effects on RORα. Lunzindole treatment, however, did not affect melatonin receptor expression or content of either the membrane or nuclear receptors. Together with previously published findings which demonstrated the intracellular distribution of melatonin in rat liver, the current results support the conclusion that the circadian rhythms of MT1/MT2 and RORα receptors are under the control of the serum and intracellular melatonin levels. Moreover, the induction of MT1/MT2 receptors after the administration of high doses of melatonin further suggests that the therapeutic value of melatonin may not be restricted to only low doses of the indoleamine.

Melatonin rescues zebrafish embryos from the parkinsonian phenotype restoring the parkin/PINK1/DJ-1/MUL1 network.

Multiple studies reporting mitochondrial impairment in Parkinsons disease (PD) involve knockout or knockdown models to inhibit the expression of mitochondrial‐related genes, including parkin, PINK1, and DJ‐1 ones. Melatonin has significant neuroprotective properties, which have been related to its ability to boost mitochondrial bioenergetics. The meaning and molecular targets of melatonin in PD are yet unclear. Zebrafish are an outstanding model of PD because they are vertebrates, their dopaminergic system is comparable to the nigrostriatal system of humans, and their brains express the same genes as mammals. The exposure of 24 hpf zebrafish embryos to MPTP leads to a significant inhibition of the mitochondrial complex I and the induction of sncga gene, responsible for enhancing γ‐synuclein accumulation, which is related to mitochondrial dysfunction. Moreover, MPTP inhibited the parkin/PINK1/DJ‐1 expression, impeding the normal function of the parkin/PINK1/DJ‐1/MUL1 network to remove the damaged mitochondria. This situation remains over time, and removing MPTP from the treatment did not stop the neurodegenerative process. On the contrary, mitochondria become worse during the next 2 days without MPTP, and the embryos developed a severe motor impairment that cannot be rescued because the mitochondrial‐related gene expression remained inhibited. Melatonin, added together with MPTP or added once MPTP was removed, prevented and recovered, respectively, the parkinsonian phenotype once it was established, restoring gene expression and normal function of the parkin/PINK1/DJ‐1/MUL1 loop and also the normal motor activity of the embryos. The results show, for the first time, that melatonin restores brain function in zebrafish suffering with Parkinson‐like disease.

Melatonin treatment counteracts the hyperoxidative status in erythrocytes of patients suffering from Duchenne muscular dystrophy.

OBJECTIVES To analyze whether the antioxidant melatonin could reduce the hyperoxidative status in the blood of patients with Duchennes muscular dystrophy. DESIGN AND METHODS Ten patients aged 12.8±0.9 years were treated with melatonin (60mg at 21:00h plus 10mg at 09:00h) for 9 months, and erythrocyte markers of oxidative stress were determined at 3, 6, and 9 months of treatment. Healthy age- and sex-matched subjects served as controls. RESULTS Prior to treatment, the patients had higher glutathione disulfide/glutathione ratio and higher glutathione transferase and superoxide dismutase activities, and lower glutathione reductase activity than controls. After 3 months of melatonin treatment, the hyperoxidative status of these patients was counteracted, being reduced to the normal redox state between 3 and 9 months. CONCLUSION These results, together with the reduction in the inflammatory process and in muscle injury recently reported in the same patients, support the efficacy of melatonin therapy in DMD patients.

Ubiquinol-10 ameliorates mitochondrial encephalopathy associated with CoQ deficiency

Coenzyme Q10 (CoQ10) deficiency (MIM 607426) causes a mitochondrial syndrome with variability in the clinical presentations. Patients with CoQ10 deficiency show inconsistent responses to oral ubiquinone-10 supplementation, with the highest percentage of unsuccessful results in patients with neurological symptoms (encephalopathy, cerebellar ataxia or multisystemic disease). Failure in the ubiquinone-10 treatment may be the result of its poor absorption and bioavailability, which may be improved by using different pharmacological formulations. In a mouse model (Coq9(X/X)) of mitochondrial encephalopathy due to CoQ deficiency, we have evaluated oral supplementation with water-soluble formulations of reduced (ubiquinol-10) and oxidized (ubiquinone-10) forms of CoQ10. Our results show that CoQ10 was increased in all tissues after supplementation with ubiquinone-10 or ubiquinol-10, with the tissue levels of CoQ10 with ubiquinol-10 being higher than with ubiquinone-10. Moreover, only ubiquinol-10 was able to increase the levels of CoQ10 in mitochondria from cerebrum of Coq9(X/X) mice. Consequently, ubiquinol-10 was more efficient than ubiquinone-10 in increasing the animal body weight and CoQ-dependent respiratory chain complex activities, and reducing the vacuolization, astrogliosis and oxidative damage in diencephalon, septum-striatum and, to a lesser extent, in brainstem. These results suggest that water-soluble formulations of ubiquinol-10 may improve the efficacy of CoQ10 therapy in primary and secondary CoQ10 deficiencies, other mitochondrial diseases and neurodegenerative diseases.