Correction: Prophylactic versus Therapeutic Fingolimod: Restoration of Presynaptic Defects in Mice Suffering from Experimental Autoimmune Encephalomyelitis.

[This corrects the article DOI: 10.1371/journal.pone.0170825.].

Fasting-mimicking diet cycles reduce neuroinflammation to attenuate cognitive decline in Alzheimer's models.

The effects of fasting-mimicking diet (FMD) cycles in reducing many aging and disease risk factors indicate it could affect Alzheimer's disease (AD). Here, we show that FMD cycles reduce cognitive decline and AD pathology in E4FAD and 3xTg AD mouse models, with effects superior to those caused by protein restriction cycles. In 3xTg mice, long-term FMD cycles reduce hippocampal Aß load and hyperphosphorylated tau, enhance genesis of neural stem cells, decrease microglia number, and reduce expression of neuroinflammatory genes, including superoxide-generating NADPH oxidase (Nox2). 3xTg mice lacking Nox2 or mice treated with the NADPH oxidase inhibitor apocynin also display improved cognition and reduced microglia activation compared with controls. Clinical data indicate that FMD cycles are feasible and generally safe in a small group of AD patients. These results indicate that FMD cycles delay cognitive decline in AD models in part by reducing neuroinflammation and/or superoxide production in the brain.

Safety and Feasibility of Fasting-Mimicking Diet and Effects on Nutritional Status and Circulating Metabolic and Inflammatory Factors in Cancer Patients Undergoing Active Treatment.

In preclinical studies, fasting was found to potentiate the effects of several anticancer treatments, and early clinical studies indicated that patients may benefit from regimes of modified fasting. However, concerns remain over possible negative impact on the patients' nutritional status. We assessed the feasibility and safety of a 5-day "Fasting-Mimicking Diet" (FMD) as well as its effects on body composition and circulating growth factors, adipokines and cyto/chemokines in cancer patients. In this single-arm, phase I/II clinical trial, patients with solid or hematologic malignancy, low nutritional risk and undergoing active medical treatment received periodic FMD cycles. The body weight, handgrip strength and body composition were monitored throughout the study. Growth factors, adipokines and cyto/chemokines were assessed by ELISA. Ninety patients were enrolled, and FMD was administered every three weeks/once a month with an average of 6.3 FMD cycles/patient. FMD was largely safe with only mild side effects. The patients' weight and handgrip remained stable, the phase angle and fat-free mass increased, while the fat mass decreased. FMD reduced the serum c-peptide, IGF1, IGFBP3 and leptin levels, while increasing IGFBP1, and these modifications persisted for weeks beyond the FMD period. Thus, periodic FMD cycles are feasible and can be safely combined with standard antineoplastic treatments in cancer patients at low nutritional risk. The FMD resulted in reduced fat mass, insulin production and circulating IGF1 and leptin. This trial was registered on Clinicaltrials.gov in July 2018 with the identifier NCT03595540.

SIRT6 enhances oxidative phosphorylation in breast cancer and promotes mammary tumorigenesis in mice.

BACKGROUND: Sirtuin 6 (SIRT6) is a NAD+-dependent deacetylase with key roles in cell metabolism. High SIRT6 expression is associated with adverse prognosis in breast cancer (BC) patients. However, the mechanisms through which SIRT6 exerts its pro-oncogenic effects in BC remain unclear. Here, we sought to define the role of SIRT6 in BC cell metabolism and in mouse polyoma middle T antigen (PyMT)-driven mammary tumors. METHODS: We evaluated the effect of a heterozygous deletion of Sirt6 on tumor latency and survival of mouse mammary tumor virus (MMTV)-PyMT mice. The effect of SIRT6 silencing on human BC cell growth was assessed in MDA-MB-231 xenografts. We also analyzed the effect of Sirt6 heterozygous deletion, of SIRT6 silencing, and of the overexpression of either wild-type (WT) or catalytically inactive (H133Y) SIRT6 on BC cell pyruvate dehydrogenase (PDH) expression and activity and oxidative phosphorylation (OXPHOS), including respiratory complex activity, ATP/AMP ratio, AMPK activation, and intracellular calcium concentration. RESULTS: The heterozygous Sirt6 deletion extended tumor latency and mouse survival in the MMTV-PyMT mouse BC model, while SIRT6 silencing slowed the growth of MDA-MB-231 BC cell xenografts. WT, but not catalytically inactive, SIRT6 enhanced PDH expression and activity, OXPHOS, and ATP/AMP ratio in MDA-MB-231 and MCF7 BC cells. Opposite effects were obtained by SIRT6 silencing, which also blunted the expression of genes encoding for respiratory chain proteins, such as UQCRFS1, COX5B, NDUFB8, and UQCRC2, and increased AMPK activation in BC cells. In addition, SIRT6 overexpression increased, while SIRT6 silencing reduced, intracellular calcium concentration in MDA-MB-231 cells. Consistent with these findings, the heterozygous Sirt6 deletion reduced the expression of OXPHOS-related genes, the activity of respiratory complexes, and the ATP/AMP ratio in tumors isolated from MMTV-PyMT mice. CONCLUSIONS: Via its enzymatic activity, SIRT6 enhances PDH expression and activity, OXPHOS, ATP/AMP ratio, and intracellular calcium concentration, while reducing AMPK activation, in BC cells. Thus, overall, SIRT6 inhibition appears as a viable strategy for preventing or treating BC.

Neuroinflammation in Aged Brain: Impact of the Oral Administration of Ellagic Acid Microdispersion.

The immune system and the central nervous system message each other to preserving central homeostasis. Both systems undergo changes during aging that determine central age-related defects. Ellagic acid (EA) is a natural product which is beneficial in both peripheral and central diseases, including aging. We analyzed the impact of the oral administration of a new oral ellagic acid micro-dispersion (EAm), that largely increased the EA solubility, in young and old mice. Oral EAm did not modify animal weight and behavioral skills in young and old mice, but significantly recovered changes in "ex-vivo, in vitro" parameters in old animals. Cortical noradrenaline exocytosis decreased in aged mice. EAm administration did not modify noradrenaline overflow in young animals, but recovered it in old mice. Furthermore, GFAP staining was increased in the cortex of aged mice, while IBA-1 and CD45 immunopositivities were unchanged when compared to young ones. EAm treatment significantly reduced CD45 signal in both young and old cortical lysates; it diminished GFAP immunopositivity in young mice, but failed to affect IBA-1 expression in both young and old animals. Finally, EAm treatment significantly reduced IL1beta expression in old mice. These results suggest that EAm is beneficial to aging and represents a nutraceutical ingredient for elders.

Simulated microgravity induces nuclear translocation of Bax and BCL-2 in glial cultured C6 cells.

Alterations in the control of apoptotic processes were observed in cells during space flight or under simulated microgravity, the latter obtained with the 3D-Random Positioning Machine (3D-RPM). Usually the proteins Bax and Bcl-2, act as pro- or anti-apoptotic regulators. Here we investigated the effects of simulated microgravity obtained by the 3D-RPM on cell viability, localization and expression of Bax and Bcl-2 in cultures of glial cancerous cells. We observed for the first time a transient cytoplasmic/nuclear translocation of Bax and Bcl-2 triggered by changing gravity vector. Bax translocates into the nucleus after 1 h, is present simultaneously in the cytoplasm after 6 h and comes back to the cytoplasm after 24 h. Bcl-2 translocate into the nucleus only after 6 h and comes back to the cytoplasm after 24 h. Physiological meaning, on the regulation of apoptotic event and possible applicative outcomes of such finding are discussed.

Environmental Training and Synaptic Functions in Young and Old Brain: A Presynaptic Perspective.

BACKGROUND: Aging is an unavoidable, physiological process that reduces the complexity and the plasticity of the synaptic contacts in Central Nervous System (CNS), having profound implications for human well-being. The term "cognitive reserve" refers to central cellular adaptations that augment the resilience of human brain to damage and aging. The term "Cognitive training" indicates the cultural, social and physical stimulations proposed as add-on therapy for the cure of central neurological diseases. "Cognitive training" reinforces the "cognitive reserve" permitting to counteract brain impairments and rejuvenating synaptic complexity. The research has begun investigating the clinical impact of the "cognitive training" in aged people, but additional work is needed to definitively assess its effectiveness. In particular, there is a need to understand, from a preclinical point of view, whether "cognitive training" promotes compensatory effects or, alternatively, if it elicits genuine recovery of neuronal defects. Although the translation from rodent studies to the clinical situation could be difficult, the results from pre-clinical models are of high clinical relevance, since they should allow a better understanding of the effects of environmental interventions in aging-associated chronic derangements in mammals. CONCLUSION: Data in literature and the recent results obtained in our laboratory concerning the impact of environmental stimulation on the presynaptic release of noradrenaline, glutamate and gamma amino butyric acid (GABA) suggest that these neurotransmitters undergo different adaptations during aging and that they are differently tuned by "cognitive training". The impact of "cognitive training" on neurotransmitter exocytosis might account for the cellular events involved in reinforcement of "cognitive reserve" in young and old animals.

5-HT2A-mGlu2/3 receptor complex in rat spinal cord glutamatergic nerve endings: A 5-HT2A to mGlu2/3 signalling to amplify presynaptic mechanism of auto-control of glutamate exocytosis.

Presynaptic mGlu2/3 autoreceptors exist in rat spinal cord nerve terminals as suggested by the finding that LY379268 inhibited the 15 mM KCl-evoked release of [3H]D-aspartate ([3H]D-Asp) in a LY341495-sensitive manner. Spinal cord glutamatergic nerve terminals also possess presynaptic release-regulating 5-HT2A heteroreceptors. Actually, the 15 mM KCl-evoked [3H]D-Asp exocytosis from spinal cord synaptosomes was reduced by the 5-HT2A agonist (±)DOI, an effect reversed by the 5-HT2A antagonists MDL11,939, MDL100907, ketanserin and trazodone (TZD). We investigated whether mGlu2/3 and 5-HT2A receptors colocalize and cross-talk in these terminals and if 5-HT2A ligands modulate the mGlu2/3-mediated control of glutamate exocytosis. Western blot analysis and confocal microscopy highlighted the presence of mGlu2/3 and 5-HT2A receptor proteins in spinal cord VGLUT1 positive synaptosomes, where mGlu2/3 and 5-HT2A receptor immunoreactivities largely colocalize. Furthermore, mGlu2/3 immunoprecipitates from spinal cord synaptosomes were also 5-HT2A immunopositive. Interestingly, the 100 pM LY379268-induced reduction of the 15 mM KCl-evoked [3H]D-Asp overflow as well as its inhibition by 100 nM (±)DOI became undetectable when the two agonists were concomitantly added. Conversely, 5-HT2A antagonists (MDL11,939, MDL100907, ketanserin and TZD) reinforced the release-regulating activity of mGlu2/3 autoreceptors. Increased expression of mGlu2/3 receptor proteins in synaptosomal plasmamembranes paralleled the gain of function of the mGlu2/3 autoreceptors elicited by 5-HT2A antagonists. Based on these results, we propose that in spinal cord glutamatergic terminals i) mGlu2/3 and 5-HT2A receptors colocalize and interact one each other in an antagonist-like manner, ii) 5-HT2A antagonists are indirect positive allosteric modulator of mGlu2/3 autoreceptors controlling glutamate exocytosis.

Genetic inactivation of mGlu5 receptor improves motor coordination in the Grm1crv4 mouse model of SCAR13 ataxia.

Deleterious mutations in the glutamate receptor metabotropic 1 gene (GRM1) cause a recessive form of cerebellar ataxia, SCAR13. GRM1 and GRM5 code for the metabotropic glutamate type 1 (mGlu1) and type 5 (mGlu5) receptors, respectively. Their different expression profiles suggest they could have distinct functional roles. In a previous study, homozygous mice lacking mGlu1 receptors (Grm1crv4/crv4) and exhibiting ataxia presented cerebellar overexpression of mGlu5 receptors, that was proposed to contribute to the mouse phenotype. To test this hypothesis, we here crossed Grm1crv4 and Grm5ko mice to generate double mutants (Grm1crv4/crv4Grm5ko/ko) lacking both mGlu1 and mGlu5 receptors. Double mutants and control mice were analyzed for spontaneous behavior and for motor activity by rotarod and footprint analyses. In the same mice, the release of glutamate from cerebellar nerve endings (synaptosomes) elicited by 12mM KCl or by α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) was also evaluated. Motor coordination resulted improved in double mutants when compared to Grm1crv4/crv4 mice. Furthermore, in in vitro studies, glutamate release elicited by both KCl depolarization and activation of AMPA autoreceptors resulted reduced in Grm1crv4/crv4 mice compared to wild type mice, while it presented normal levels in double mutants. Moreover, we found that Grm1crv4/crv4 mice showed reduced expression of GluA2/3 AMPA receptor subunits in cerebellar synaptosomes, while it resulted restored to wild type level in double mutants. To conclude, blocking of mGlu5 receptor reduced the dysregulation of glutamate transmission and improved motor coordination in the Grm1crv4 mouse model of SCAR13, thus suggesting the possible usefulness of pharmacological therapies based on modulation of mGlu5 receptor activity for the treatment of this type of ataxia.

Immuno-pharmacological characterization of group II metabotropic glutamate receptors controlling glutamate exocytosis in mouse cortex and spinal cord.

BACKGROUND AND PURPOSE: We recently proposed the existence of mGlu3 -preferring autoreceptors in spinal cord terminals and of mGlu2 -preferring autoreceptors in cortical terminals. This study aims to verify our previous conclusions and to extend their pharmacological characterization. EXPERIMENTAL APPROACH: We studied the effect of LY566332, an mGlu2 receptor positive allosteric modulator (PAM), and of LY2389575, a selective mGlu3 receptor negative allosteric (NAM) modulator, on the mGlu2/3 agonist LY379268-mediated inhibition of glutamate exocytosis [measured as KCl-evoked release of preloaded [3 H]-D-aspartate]. The mGlu2 PAM BINA and the mGlu3 NAM ML337, as well as selective antibodies recognizing the N-terminal of the receptor proteins, were used to confirm the pharmacological characterization of the native receptors. KEY RESULTS: Cortical synaptosomes possess LY566332-sensitive autoreceptors that are slightly, although significantly, susceptible to LY2389575. In contrast, LY566332-insensitive and LY2389575-sensitive autoreceptors are present in spinal cord terminals. BINA and ML337 mimicked LY566332 and LY2389575, respectively, in controlling LY379268-mediated inhibition of glutamate exocytosis from both cortical and spinal cord synaptosomes. Incubation of cortical synaptosomes with anti-mGlu2 antibody prevented the LY379268-induced inhibition of glutamate exocytosis, and this response was partially reduced by the anti-mGlu3 antibody. Incubation of spinal cord synaptosomes with the anti-mGlu3 antibody abolished LY379268-mediated reduction of glutamate exocytosis from these terminals, while the anti-mGlu2 antibody was inactive. Western blot analysis and confocal microscopy data were largely consistent with these functional observations. CONCLUSIONS AND IMPLICATIONS: We confirmed that mGlu3 -preferring autoreceptors exist in spinal cord terminals. Differently, cortical glutamatergic terminals possess mGlu2 /mGlu3 heterodimers, whose inhibitory effect is largely mediated by mGlu2 receptors.

Prophylactic versus Therapeutic Fingolimod: Restoration of Presynaptic Defects in Mice Suffering from Experimental Autoimmune Encephalomyelitis.

Fingolimod, the first oral, disease-modifying therapy for MS, has been recently proposed to modulate glutamate transmission in the central nervous system (CNS) of mice suffering from Experimental Autoimmune Encephalomyelitis (EAE) and in MS patients. Our study aims at investigating whether oral fingolimod recovers presynaptic defects that occur at different stages of disease in the CNS of EAE mice. In vivo prophylactic (0.3 mg/kg for 14 days, from the 7th day post immunization, d.p.i, the drug dissolved in the drinking water) fingolimod significantly reduced the clinical symptoms and the anxiety-related behaviour in EAE mice. Spinal cord inflammation, demyelination and glial cell activation are markers of EAE progression. These signs were ameliorated following oral fingolimod administration. Glutamate exocytosis was shown to be impaired in cortical and spinal cord terminals isolated from EAE mice at 21 ± 1 d.p.i., while GABA alteration emerged only at the spinal cord level. Prophylactic fingolimod recovered these presynaptic defects, restoring altered glutamate and GABA release efficiency. The beneficial effect occurred in a dose-dependent, region-specific manner, since lower (0.1-0.03 mg/kg) doses restored, although to a different extent, synaptic defects in cortical but not spinal cord terminals. A delayed reduction of glutamate, but not of GABA, exocytosis was observed in hippocampal terminals of EAE mice at 35 d.p.i. Therapeutic (0.3 mg/kg, from 21 d.p.i. for 14 days) fingolimod restored glutamate exocytosis in the cortex and in the hippocampus of EAE mice at 35 ± 1 d.p.i. but not in the spinal cord, where also GABAergic defects remained unmodified. These results improve our knowledge of the molecular events accounting for the beneficial effects elicited by fingolimod in demyelinating disorders.

CXCR4 and NMDA Receptors Are Functionally Coupled in Rat Hippocampal Noradrenergic and Glutamatergic Nerve Endings.

Previous studies had shown that the HIV-1 capsidic glycoprotein gp120 (strain IIIB) modulates presynaptic release-regulating NMDA receptors on noradrenergic and glutamatergic terminals. This study aims to assess whether the chemokine CXC4 receptors (CXCR4s) has a role in the gp120-mediated effects. The effect of CXCL12, the endogenous ligand at CXCR4, on the NMDA-mediated releasing activity was therefore investigated. Rat hippocampal synaptosomes were preloaded with [3H]noradrenaline ([3H]NA) or [3H]D-aspartate ([3H]D-Asp) and acutely exposed to CXCL12, to NMDA or to both agonists. CXCL12, inactive on its own, facilitated the NMDA-evoked tritium release. The NMDA antagonist MK-801 abolished the NMDA/CXCL12-evoked tritium release of both radiolabelled tracers, while the CXCR4 antagonist AMD 3100 halved it, suggesting that rat hippocampal nerve endings possess presynaptic release-regulating CXCR4 receptors colocalized with NMDA receptors. Accordingly, Western blot analysis confirmed the presence of CXCR4 proteins in synaptosomal plasmamembranes. In both synaptosomal preparations, CXCL12-induced facilitation of NMDA-mediated release was dependent upon PLC-mediated src-induced events leading to mobilization of Ca2+ from intraterminal IP3-sensitive stores Finally, the gp120-induced facilitation of NMDA-mediated release of [3H]NA and [3H]D-Asp was prevented by AMD 3100. We propose that CXCR4s are functionally coupled to NMDA receptors in rat hippocampal noradrenergic and glutamatergic terminals and account for the gp120-induced modulation of the NMDA-mediated central effects. The NMDA/CXCR4 cross-talk could have a role in the neuropsychiatric symptoms often observed in HIV-1 positive patients.

Development and Pharmacological Characterization of Selective Blockers of 2-Arachidonoyl Glycerol Degradation with Efficacy in Rodent Models of Multiple Sclerosis and Pain.

We report the discovery of compound 4a, a potent ß-lactam-based monoacylglycerol lipase (MGL) inhibitor characterized by an irreversible and stereoselective mechanism of action, high membrane permeability, high brain penetration evaluated using a human in vitro blood-brain barrier model, high selectivity in binding and affinity-based proteomic profiling assays, and low in vitro toxicity. Mode-of-action studies demonstrate that 4a, by blocking MGL, increases 2-arachidonoylglycerol and behaves as a cannabinoid (CB1/CB2) receptor indirect agonist. Administration of 4a in mice suffering from experimental autoimmune encephalitis ameliorates the severity of the clinical symptoms in a CB1/CB2-dependent manner. Moreover, 4a produced analgesic effects in a rodent model of acute inflammatory pain, which was antagonized by CB1 and CB2 receptor antagonists/inverse agonists. 4a also relieves the neuropathic hypersensitivity induced by oxaliplatin. Given these evidence, 4a, as MGL selective inhibitor, could represent a valuable lead for the future development of therapeutic options for multiple sclerosis and chronic pain.

Presynaptic, release-regulating mGlu2 -preferring and mGlu3 -preferring autoreceptors in CNS: pharmacological profiles and functional roles in demyelinating disease.

BACKGROUND AND PURPOSE: Presynaptic, release-regulating metabotropic glutamate 2 and 3 (mGlu2/3) autoreceptors exist in the CNS. They represent suitable targets for therapeutic approaches to central diseases that are typified by hyperglutamatergicity. The availability of specific ligands able to differentiate between mGlu2 and mGlu3 subunits allows us to further characterize these autoreceptors. In this study we investigated the pharmacological profile of mGlu2/3 receptors in selected CNS regions and evaluated their functions in mice with experimental autoimmune encephalomyelitis (EAE). EXPERIMENTAL APPROACH: The comparative analysis of presynaptic mGlu2/3 autoreceptors was performed by determining the effect of selective mGlu2/3 receptor agonist(s) and antagonist(s) on the release of [(3)H]-D-aspartate from cortical and spinal cord synaptosomes in superfusion. In EAE mice, mGlu2/3 autoreceptor-mediated release functions were investigated and effects of in vivo LY379268 administration on impaired glutamate release examined ex vivo. KEY RESULTS: Western blot analysis and confocal microscopy confirmed the presence of presynaptic mGlu2/3 receptor proteins. Cortical synaptosomes possessed LY541850-sensitive, NAAG-insensitive autoreceptors having low affinity for LY379268, while LY541850-insensitive, NAAG-sensitive autoreceptors with high affinity for LY379268 existed in spinal cord terminals. In EAE mice, mGlu2/3 autoreceptors completely lost their inhibitory activity in cortical, but not in spinal cord synaptosomes. In vivo LY379268 administration restored the glutamate exocytosis capability in spinal cord but not in cortical terminals in EAE mice. CONCLUSIONS AND IMPLICATIONS: We propose the existence of mGlu2-preferring and mGlu3-preferring autoreceptors in mouse cortex and spinal cord respectively. The mGlu3 -preferring autoreceptors could represent a target for new pharmacological approaches for treating demyelinating diseases.

The impact of long-term exposure to space environment on adult mammalian organisms: a study on mouse thyroid and testis.

Hormonal changes in humans during spaceflight have been demonstrated but the underlying mechanisms are still unknown. To clarify this point thyroid and testis/epididymis, both regulated by anterior pituitary gland, have been analyzed on long-term space-exposed male C57BL/10 mice, either wild type or pleiotrophin transgenic, overexpressing osteoblast stimulating factor-1. Glands were submitted to morphological and functional analysis.In thyroids, volumetric ratios between thyrocytes and colloid were measured. cAMP production in 10(-7)M and 10(-8)M thyrotropin-treated samples was studied. Thyrotropin receptor and caveolin-1 were quantitized by immunoblotting and localized by immunofluorescence. In space-exposed animals, both basal and thyrotropin-stimulated cAMP production were always higher. Also, the structure of thyroid follicles appeared more organized, while thyrotropin receptor and caveolin-1 were overexpressed. Unlike the control samples, in the space samples thyrotropin receptor and caveolin-1 were both observed at the intracellular junctions, suggesting their interaction in specific cell membrane microdomains.In testes, immunofluorescent reaction for 3ß- steroid dehydrogenase was performed and the relative expressions of hormone receptors and interleukin-1ß were quantified by RT-PCR. Epididymal sperm number was counted. In space-exposed animals, the presence of 3ß and 17ß steroid dehydrogenase was reduced. Also, the expression of androgen and follicle stimulating hormone receptors increased while lutenizing hormone receptor levels were not affected. The interleukin 1 ß expression was upregulated. The tubular architecture was altered and the sperm cell number was significantly reduced in spaceflight mouse epididymis (approx. -90% vs. laboratory and ground controls), indicating that the space environment may lead to degenerative changes in seminiferous tubules.Space-induced changes of structure and function of thyroid and testis/epididymis could be responsible for variations of hormone levels in human during space missions. More research, hopefully a reflight of MDS, would be needed to establish whether the space environment acts directly on the peripheral glands or induces changes in the hypotalamus-pituitary-glandular axis.