The mitochondriogenic but not the immunosuppressant effects of mTOR inhibitors prompt neuroprotection and delay disease evolution in a mouse model of progressive multiple sclerosis.

INTRODUCTION: Purportedly, the progression of multiple sclerosis (MS) occurs when neurodegenerative processes due to derangement of axonal bioenergetics take over the autoimmune response. However, a clear picture of the causative interrelationship between autoimmunity and axonal mitochondrial dysfunction in progressive MS (PMS) pathogenesis waits to be provided. METHODS: In the present study, by adopting the NOD mouse model of PMS, we compared the pharmacological effects of the immunosuppressants dexamethasone and fingolimod with those of mTOR inhibitors rapamycin and everolimus that, in addition to immunosuppression, also regulate mitochondrial functioning. Female Non-Obese Diabetic (NOD) mice were immunized with MOG35-55 and treated with drugs to evaluate functional, immune and mitochondrial parameters during disease evolution. RESULTS: We found that dexamethasone and fingolimod did not affect the pattern of progression as well as survival. Conversely, mTOR inhibitors rapamycin and everolimus delayed disease progression and robustly extended survival of immunized mice. The same effects were obtained when treatment was delayed by 30 days after immunization. Remarkably, dexamethasone and fingolimod prompted the same degree of immunosuppression of rapamycin within both spleen and spinal cord of mice. However, only rapamycin prompted mitochondriogenesis by increasing mitochondrial content, and expression of several mitochondrial respiratory complex subunits, thereby preventing mtDNA reduction in the spinal cords of immunized mice. These pharmacodynamic effects were not reproduced in healthy NOD mice, suggesting a disease context-dependent pharmacodynamic effect. DISCUSSION: Data corroborate the key role of mitochondriogenesis to treatment of MS progression, and for the first time disclose the translational potential of mTOR inhibitors in PMS therapy.

Early derangement of axonal mitochondria occurs in a mouse model of progressive but not relapsing-remitting multiple sclerosis.

INTRODUCTION: Derangement of axonal mitochondrial bioenergetics occurs during progressive multiple sclerosis (PMS). However, whether this is a delayed epiphenomenon or an early causative event of disease progression waits to be understood. Answering this question might further our knowledge of mechanisms underlying neurobiology of PMS and related therapy. METHODS: MOG35-55-immunized NOD and PLP139-151-immunized SJL female mice were adopted as models of progressive or relapsing-remitting experimental autoimmune encephalomyelitis (EAE), respectively. Multiple parameters of mitochondrial homeostasis were analyzed in the mouse spinal cord during the early asymptomatic stage, also evaluating the effects of scavenging mitochondrial reactive oxygen species with Mito-TEMPO. RESULTS: Almost identical lumbar spinal cord immune infiltrates consisting of Th1 cells and neutrophils without B and Th17 lymphocytes occurred early upon immunization in both mouse strains. Still, only NOD mice showed axon-restricted dysregulation of mitochondrial homeostasis, with reduced mtDNA contents and increased cristae area. Increased expression of mitochondrial respiratory complex subunits Nd2, Cox1, Atp5d, Sdha also exclusively occurred in lumbar spinal cord of NOD and not SJL mice. Accordingly, in this region genes regulating mitochondrial morphology (Opa1, Mfn1, Mfn2 and Atp5j2) and mitochondriogenesis (Pgc1α, Foxo, Hif-1α and Nrf2) were induced early upon immunization. A reduced extent of mitochondrial derangement occurred in the thoracic spinal cord. Notably, the mitochondrial radical scavenger Mito-TEMPO reduced H2O2 content and prevented both mtDNA depletion and cristae remodeling, having no effects on dysregulation of mitochondrial transcriptome. DISCUSSION: We provide here the first evidence that axonal-restricted derangement of mitochondrial homeostasis already occurs during the asymptomatic state exclusively in a mouse model of PMS. Data further our understanding of mechanisms related to EAE progression, and point to very early axonal mitochondrial dysfunction as central to the neuropathogenesis of MS evolution.

Histone Deacetylase Inhibitors Counteract CGRP Signaling and Pronociceptive Sensitization in a Rat Model of Medication Overuse Headache.

Chronic triptan exposure in rodents recapitulates medication overuse headache (MOH), causing cephalic pain sensitization and trigeminal ganglion overexpression of pronociceptive proteins including CGRP. Because of these transcriptional derangements, as well as the emerging role of epigenetics in chronic pain, in the present study, we evaluated the effects of the histone deacetylase inhibitors (HDACis) panobinostat and givinostat, in rats chronically exposed to eletriptan for 1 month. Both panobinostat and givinostat counteracted overexpression of genes coding for CGRP and its receptor subunit RAMP1, having no effects on CLR and RCP receptor subunits in the trigeminal ganglion (TG) of eletriptan-exposed rats. Within the trigeminal nucleus caudalis (TNc), transcripts for these genes were neither upregulated by eletriptan nor altered by concomitant treatment with panobinostat or givinostat. HDACis counteracted hypersensitivity to capsaicin-induced vasodilatation in the trigeminal territory, as well as photophobic behavior and cephalic allodyniain eletriptan-exposed rats. Eletriptan did not affect CGRP, CLR, and RAMP1 expression in cultured trigeminal ganglia, whereas both inhibitors reduced transcripts for CLR and RAMP-1. The drugs, however, increased luciferase expression driven by CGRP promoter in cultured cells. Our findings provide evidence for a key role of HDACs and epigenetics in MOH pathogenesis, highlighting the therapeutic potential of HDAC inhibition in the prevention of migraine chronification. PERSPECTIVE: The present study highlights a key epigenetic role of HDAC in the rodent model of medication overuse headache, furthering our understanding of the molecular mechanisms responsible for pronociceptive sensitization during headache chronification.

Effects of cluster headache preventatives on mouse hypothalamic transcriptional homeostasis.

OBJECTIVE: To investigate how cluster headache preventatives verapamil, lithium and prednisone affect expression of hypothalamic genes involved in chronobiology. METHODS: C57Bl/6 mice were exposed to daily, oral treatment with verapamil, lithium, prednisone or amitriptyline (as negative control), and transcripts of multiple genes quantified in the anterior, lateral and posterior hypothalamus. RESULTS: Verapamil, lithium or prednisone did not affect expression of clock genes of the anterior hypothalamus (Clock, Bmal1, Cry1/2 and Per1/2). Prednisone altered expression of hypothalamic neuropeptides melanin-concentrating hormone and histidine decarboxylase within the lateral and posterior hypothalamus, respectively. The three preventatives did not affect expression of the neurohypophyseal hormones oxytocin and arginine-vasopressin in the posterior hypothalamus. Conversely, amitriptyline reduced mRNA levels of Clock, oxytocin and arginine-vasopressin. CONCLUSION: Data suggest that cluster headache preventatives act upstream or downstream from the hypothalamus. Our findings provide new insights on hypothalamic homeostasis during cluster headache prophylaxis, as well as neurochemistry underlying cluster headache treatment.

SIRT1-dependent restoration of NAD+ homeostasis after increased extracellular NAD+ exposure.

In the last several years, NAD+ supplementation has emerged as an innovative and safe therapeutic strategy for a wide spectrum of disorders, including diabetes and neuropathy. However, critical questions remain as to how NAD+ and its precursors are taken up by cells, as well as the effects of long-lasting intracellular NAD+ (iNAD+) increases. Here, we investigated the kinetics of iNAD+ levels in different cell types challenged with prolonged exposure to extracellular NAD+ (eNAD+). Surprisingly, we found that after the initial increase, iNAD+ contents decreased back to control levels (iNAD+ resetting). Focusing our attention on HeLa cells, we found that oxygen and ATP consumption occurred with similar temporal kinetics after eNAD+ exposure. Using [3H]NAD+ and [14C]NAD+, we determined that NAD+ resetting was not due to increased dinucleotide extrusion but rather due to reduced uptake of cleaved NAD+ products. Indeed, eNAD+ exposure reduced the expression of the ecto-5'-nucleotidase CD73, the nicotinamide adenine mononucleotide transporter solute carrier family 12 member 8, and the nicotinamide riboside kinase. Interestingly, silencing the NAD+-sensor enzyme sirtuin 1 prevented eNAD+-dependent transcriptional repression of ecto-5'-nucleotidase, solute carrier family 12 member 8, and nicotinamide riboside kinase, as well as iNAD+ resetting. Our findings provide the first evidence for a sirtuin 1-mediated homeostatic response aimed at maintaining physiological iNAD+ levels in conditions of excess eNAD+ availability. These data may be of relevance for therapies designed to support the NAD+ metabolome via extracellular supplementation of the dinucleotide or its precursors.

Treatment with Non-specific HDAC Inhibitors Administered after Disease Onset does not Delay Evolution in a Mouse Model of Progressive Multiple Sclerosis.

Drugs able to efficiently counteract progression of multiple sclerosis (MS) are still an unmet need. Several lines of evidence indicate that histone deacetylase inhibitors (HDACi) are clinically-available epigenetic drugs that might be repurposed for immunosuppression in MS therapy. Here, we studied the effects of HDACi on disease evolution in myelin oligodendrocyte glycoprotein (MOG)-immunized NOD mice, an experimental model of progressive experimental autoimmune encephalomyelitis (PEAE). To obtain data of potential clinical relevance, the HDACi panobinostat, givinostat and entinostat were administered orally adopting a daily treatment protocol after disease onset. We report that the 3 drugs efficiently reduced in vitro lymphocyte proliferation in a dose-dependent manner. Notably, however, none of the drugs delayed evolution of PEAE or reduced lethality in NOD mice. In striking contrast with this, however, the lymphocyte proliferation response to MOG as well as Th1 and Th17 spinal cord infiltrates were significantly lower in animals exposed to the HDACi compared to those receiving vehicle. When put into a clinical context, for the first time data cast doubt on the relevance of HDACi to treatment of progressive MS (PMS). Also, our findings further indicate that, akin to PMS, neuropathogensis of PEAE in NOD mice becomes independent from autoimmunity, thereby corroborating the relevance of this model to experimental PMS research.

Neuroprotective Effects of Thymoquinone by the Modulation of ER Stress and Apoptotic Pathway in In Vitro Model of Excitotoxicity.

Experimental evidence indicates that the activation of ionotropic glutamate receptors plays an important role in neurological disorders' models such as epilepsy, cerebral ischemia and trauma. The glutamate receptor agonist kainic acid (KA) induces seizures and excitotoxic cell death in the CA3 region of the hippocampus. Thymoquinone (TQ) is the most important component of the essential oil obtained from black cumin (Nigella sativa L.) seeds. It has many pharmacological actions including antioxidant, anti-inflammatory, and anti-apoptotic effects. TQ was used in an in vitro experimental model of primary cultures where excitotoxicity was induced. Briefly, rat organotypic hippocampal slices were exposed to 5 µM KA for 24 h. Cell death in the CA3 subregions of slices was quantified by measuring propidium iodide fluorescence. The cross-talk between TQ, ER stress and apoptotic pathways was investigated by Western blot. In untreated slices TQ (10 µM) induced a significant increase on the PSD95 levels and it decreased the excitotoxic injury induced by KA. Additionally, TQ was able to ameliorate the KA-induced increase in unfolded proteins GRP78 and GRP94 expression. Finally, TQ was able to partially rescue the reduction of the KA-induced apoptotic pathway activation. Our results suggest that TQ modulates the processes leading to post-kainate neuronal death in the CA3 hippocampal area.

Dexpramipexole Enhances K+ Currents and Inhibits Cell Excitability in the Rat Hippocampus In Vitro.

Dexpramipexole (DEX) has been described as the first-in-class F1Fo ATP synthase activator able to boost mitochondrial bioenergetics and provide neuroprotection in experimental models of ischemic brain injury. Although DEX failed in a phase III trial in patients with amyotrophic lateral sclerosis, it showed favorable safety and tolerability profiles. Recently, DEX emerged as a Nav1.8 Na+ channel and transient outward K+ (IA) conductance blocker, revealing therefore an unexpected, pleiotypic pharmacodynamic profile. In this study, we performed electrophysiological experiments in vitro aimed to better characterize the impact of DEX on voltage-dependent currents and synaptic transmission in the hippocampus. By means of patch-clamp recordings on isolated hippocampal neurons, we found that DEX increases outward K+ currents evoked by a voltage ramp protocol. This effect is prevented by the non-selective voltage-dependent K+ channel (Kv) blocker TEA and by the selective small-conductance Ca2+-activated K+ (SK) channel blocker apamin. In keeping with this, extracellular field recordings from rat hippocampal slices also demonstrated that the compound inhibits synaptic transmission and CA1 neuron excitability. Overall, these data further our understanding on the pharmacodynamics of DEX and disclose an additional mechanism that could underlie its neuroprotective properties. Also, they identify DEX as a lead to develop new modulators of K+ conductances.

Cuprizone-Dependent De/Remyelination Responses and Functional Correlates in Mouse Strains Adopted to Model Relapsing, Chronic and Progressive Experimental Autoimmune Encephalomyelitis.

NOD mice represent a unique strain that recapitulates some aspects of progressive MS when subjected to experimental autoimmune encephalomyelitis (EAE). It is unknown, however, whether a proneness to demyelination and/or defect in remyelination contribute to disease progression in NOD mice. Answering to this question might help deciphering the molecular and cellular events underpinning disease evolution in progressive MS. Here, we compared the cuprizone-dependent demyelination and remyelination responses, as well as their functional correlates, in NOD, C57BL/6, and SJL mice typically adopted to model progressive, chronic or relapsing EAE. We report that demyelination occurred to a similar extent in the three mice strains, and that in none of them there was evidence of axonal degeneration during prolonged demyelination. Moreover, immunostaining for GFAP+ astrocytes, Iba1+ microglia, and NG2+ oligodendrocyte precursor cells similarly increased in the 3 mouse strains after cuprizone exposure. The mice underwent concomitant and complete remyelination 2 weeks after cuprizone withdrawal. On a functional level, NOD mice showed the earliest reduction of spontaneous motility and full recovery, but no impairment of motor skill. Conversely, C57BL/6 animals showed phasic reduction of both spontaneous motility and motor skill. Lastly, SJL mice presented the most severe neurological impairment with long-lasting reduction of spontaneous motility and motor skill. Overall, data suggest that the unique feature of EAE progression in NOD mice is not due to proneness to demyelination or intrinsic defects in myelin formation. Findings also unravel important functional differences in the response of the three mouse stains to cuprizone that can be harnessed to design and interpret future experiments.

Ethanol neurotoxicity is mediated by changes in expression, surface localization and functional properties of glutamate AMPA receptors.

Modifications in the subunit composition of AMPA receptors (AMPARs) have been linked to the transition from physiological to pathological conditions in a number of contexts, including EtOH-induced neurotoxicity. Previous work from our laboratory showed that EtOH withdrawal causes CA1 pyramidal cell death in organotypic hippocampal slices and changes in the expression of AMPARs. Here, we investigated whether changes in expression and function of AMPARs may be causal for EtOH-induced neurotoxicity. To this aim, we examined the subunit composition, localization and function of AMPARs in hippocampal slices exposed to EtOH by using western blotting, surface expression assay, confocal microscopy and electrophysiology. We found that EtOH withdrawal specifically increases GluA1 protein signal in total homogenates, but not in the post-synaptic density-enriched fraction. This is suggestive of overall increase and redistribution of AMPARs to the extrasynaptic compartment. At functional level, AMPA-induced calcium influx was unexpectedly reduced, whereas AMPA-induced current was enhanced in CA1 pyramidal neurons following EtOH withdrawal, suggesting that increased AMPAR expression may lead to cell death because of elevated excitability, and not for a direct contribution on calcium influx. Finally, the neurotoxicity caused by EtOH withdrawal was attenuated by the non-selective AMPAR antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt as well as by the selective antagonist of GluA2-lacking AMPARs 1-naphthyl acetyl spermine. We conclude that EtOH neurotoxicity involves changes in expression, surface localization and functional properties of AMPARs, and propose GluA2-lacking AMPARs as amenable specific targets for the development of neuroprotective drugs in EtOH-withdrawal syndrome.

ß3-Adrenoceptors as Putative Regulator of Immune Tolerance in Cancer and Pregnancy.

Understanding the mechanisms of immune tolerance is currently one of the most important challenges of scientific research. Pregnancy affects the immune system balance, leading the host to tolerate embryo alloantigens. Previous reports demonstrated that ß-adrenergic receptor (ß-AR) signaling promotes immune tolerance by modulation of NK and Treg, mainly through the activation of ß2-ARs, but recently we have demonstrated that also ß3-ARs induce an immune-tolerant phenotype in mice bearing melanoma. In this report, we demonstrate that ß3-ARs support host immune tolerance in the maternal microenvironment by modulating the same immune cells populations as recently demonstrated in cancer. Considering that ß3-ARs are modulated by oxygen levels, we hypothesize that hypoxia, through the upregulation of ß3-AR, promotes the biological shift toward a tolerant immunophenotype and that this is the same trick that embryo and cancer use to create an aura of immune-tolerance in a competent immune environment. This study confirms the analogies between fetal development and tumor progression and suggests that the expression of ß3-ARs represents one of the strategies to induce fetal and tumor immune tolerance.

3-Iodothyronamine Affects Thermogenic Substrates' Mobilization in Brown Adipocytes.

We investigated the effect of 3-iodothyronamine (T1AM) on thermogenic substrates in brown adipocytes (BAs). BAs isolated from the stromal fraction of rat brown adipose tissue were exposed to an adipogenic medium containing insulin in the absence (M) or in the presence of 20 nM T1AM (M+T1AM) for 6 days. At the end of the treatment, the expression of p-PKA/PKA, p-AKT/AKT, p-AMPK/AMPK, p-CREB/CREB, p-P38/P38, type 1 and 3 beta adrenergic receptors (ß1-ß3AR), GLUT4, type 2 deiodinase (DIO2), and uncoupling protein 1 (UCP-1) were evaluated. The effects of cell conditioning with T1AM on fatty acid mobilization (basal and adrenergic-mediated), glucose uptake (basal and insulin-mediated), and ATP cell content were also analyzed in both cell populations. When compared to cells not exposed, M+T1AM cells showed increased p-PKA/PKA, p-AKT/AKT, p-CREB/CREB, p-P38/P38, and p-AMPK/AMPK, downregulation of DIO2 and ß1AR, and upregulation of glycosylated ß3AR, GLUT4, and adiponectin. At basal conditions, glycerol release was higher for M+T1AM cells than M cells, without any significant differences in basal glucose uptake. Notably, in M+T1AM cells, adrenergic agonists failed to activate PKA and lipolysis and to increase ATP level, but the glucose uptake in response to insulin exposure was more pronounced than in M cells. In conclusion, our results suggest that BAs conditioning with T1AM promote a catabolic condition promising to fight obesity and insulin resistance.

Neuroprotection induced by dexpramipexole delays disease progression in a mouse model of progressive multiple sclerosis.

BACKGROUND AND PURPOSE: Drugs able to counteract progressive multiple sclerosis (MS) represent a largely unmet therapeutic need. Even though the pathogenesis of disease evolution is still obscure, accumulating evidence indicates that mitochondrial dysfunction plays a causative role in neurodegeneration and axonopathy in progressive MS patients. Here, we investigated the effects of dexpramipexole, a compound with a good safety profile in humans and able to sustain mitochondria functioning and energy production, in a mouse model of progressive MS. EXPERIMENTAL APPROACH: Female non-obese diabetic mice were immunized with MOG35-55 . Functional, immune and neuropathological parameters were analysed during disease evolution in animals treated or not with dexpramipexole. The compound's effects on bioenergetics and neuroprotection were also evaluated in vitro. KEY RESULTS: We found that oral treatment with dexpramipexole at a dose consistent with that well tolerated in humans delayed disability progression, extended survival, counteracted reduction of spinal cord mitochondrial DNA content and reduced spinal cord axonal loss of mice. Accordingly, the drug sustained in vitro bioenergetics of mouse optic nerve and dorsal root ganglia and counteracted neurodegeneration of organotypic mouse cortical cultures exposed to the adenosine triphosphate-depleting agents oligomycin or veratridine. Dexpramipexole, however, was unable to affect the adaptive and innate immune responses both in vivo and in vitro. CONCLUSION AND IMPLICATION: The present findings corroborate the hypothesis that neuroprotective agents may be of relevance to counteract MS progression and disclose the translational potential of dexpramipexole to treatment of progressive MS patients as a stand-alone or adjunctive therapy.

Dexpramipexole blocks Nav1.8 sodium channels and provides analgesia in multiple nociceptive and neuropathic pain models.

Selective targeting of sodium channel subtypes Nav1.7, Nav1.8, and Nav1.9, preferentially expressed by peripheral nociceptors, represents a unique opportunity to develop analgesics devoid of central side effects. Several compounds that target Nav1.7 and Nav1.8 with different degrees of selectivity have been developed and are currently being tested in clinical trials for multiple pain indications. Among these chemicals, benzothiazole-like compounds emerged as potent sodium channel blockers. We evaluated the effects of dexpramipexole, a benzothiazole-bearing drug with pleiotypic neuroactive properties and a good safety profile in humans, on sodium conductances of dorsal root ganglia neurons, as well as in multiple nociceptive and neuropathic pain models. Dexpramipexole blocks TTX-resistant sodium conductances in cultured rat dorsal root ganglion neurons with an IC50 of 294.4 nM, suggesting selectivity towards Nav1.8. In keeping with this, dexpramipexole does not affect sodium currents in dorsal root ganglion neurons from Nav1.8 null mice and acquires binding pose predicted to overlap that of the Nav1.8 channel-selective blocker A-8034637. The drug provides analgesia when parenterally, orally, or topically applied in inflammatory and visceral mouse pain models, as well as in mice affected by neuropathic pain induced by oxaliplatin, nerve constriction, or diabetes. Pain reduction in mice occurs at doses consistent with those adopted in clinical trials. The present findings confirm the relevance of selective targeting of peripheral Nav1.8 channels to pain therapy. In light of the excellent tolerability of dexpramipexole in humans, our results support its translational potential for treatment of pain.

Neuroimmunological characterization of a mouse model of primary progressive experimental autoimmune encephalomyelitis and effects of immunosuppressive or neuroprotective strategies on disease evolution.

Progressive multiple sclerosis (PMS) is a devastating disorder sustained by neuroimmune interactions still wait to be identified. Recently, immune-independent, neural bioenergetic derangements have been hypothesized as causative of neurodegeneration in PMS patients. To gather information on the immune and neurodegenerative components during PMS, in the present study we investigated the molecular and cellular events occurring in a Non-obese diabetic (NOD) mouse model of experimental autoimmune encephalomyelitis (EAE). In these mice, we also evaluated the effects of clinically-relevant immunosuppressive (dexamethasone) or bioenergetic drugs (bezafibrate and biotin) on functional, immune and neuropathological parameters. We found that immunized NOD mice progressively accumulated disability and severe neurodegeneration in the spinal cord. Unexpectedly, although CD4 and CD8 lymphocytes but not B or NK cells infiltrate the spinal cord linearly with time, their suppression by different dexamethasone treatment schedules did not affect disease progression. Also, the spreading of the autoimmune response towards additional immunogenic myelin antigen occurred neither in the periphery nor in the CNS of EAE mice. Conversely, we found that altered mitochondrial morphology, reduced contents of mtDNA and decreased transcript levels for respiratory complex subunits occurred at early disease stages and preceded axonal degeneration within spinal cord columns. However, the mitochondria boosting drugs, bezafibrate and biotin, were unable to reduce disability progression. Data suggest that EAE NOD mice recapitulate some features of PMS. Also, by showing that bezafibrate or biotin do not affect progression in NOD mice, our study suggests that this model can be harnessed to anticipate experimental information of relevance to innovative treatments of PMS.

Increased Lacrimal Fluid Level of HMGB1 in Vernal Keratoconjunctivitis.

Purpose: The aim of the present prospective study was to evaluate the lacrimal fluid concentration of HMGB1 in young patients affected by Vernal Keratoconjunctivitis (VKC) compared to a control group of healthy subjects of same age. Methods: Tear fluids was collected in a group of VKC patients and compared to a control group of healthy subjects. HMGB1 concentration was measured using the HMGB1 ELISA II test both in VCK and control subjects. Results: The mean concentration of HMGB1 in tear fluids of 45 VKC patients was 0,977 ± 0,72 ng/ml whereas in the control group was 0,24 ± 0,25 ng/ml and the difference was statistically significant (p = 0,000106) Conclusion: The concentration of HMGB1 in VCK patients was found to be significantly increased, suggesting a possible role of this protein in the inflammatory mechanism of VKC.

ß3-Adrenoreceptors Control Mitochondrial Dormancy in Melanoma and Embryonic Stem Cells.

The early phases of embryonic development and cancer share similar strategies to improve their survival in an inhospitable environment: both proliferate in a hypoxic and catecholamine-rich context, increasing aerobic glycolysis. Recent studies show that ß3-adrenergic receptor (ß3-AR) is involved in tumor progression, playing an important role in metastasis. Among ß-adrenergic receptors, ß3-AR is the last identified member of this family, and it is involved in cancer cell survival and induction of stromal reactivity in the tumor microenvironment. ß3-AR is well known as a strong activator of uncoupling protein 1 (UCP1) in brown fat tissue. Interestingly, ß3-AR is strongly expressed in early embryo development and in many cancer tissues. Induction of uncoupling protein 2 (UCP2) has been related to cancer metabolic switch, leading to accelerated glycolysis and reduced mitochondrial activity. In this study, for the first time, we demonstrate that ß3-AR is able to promote this metabolic shift in both cancer and embryonic stem cells, inducing specific glycolytic cytoplasmic enzymes and a sort of mitochondrial dormancy through the induction of UCP2. The ß3-AR/UCP2 axis induces a strong reduction of mitochondrial activity by reducing ATP synthesis and mitochondrial reactive oxygen species (mtROS) content. These effects are reverted by SR59230A, the specific ß3-AR antagonist, causing an increase in mtROS. The increased level of mtROS is neutralized by a strong antioxidant activity in embryonic stem cells, but not in cancer stem cells, where it causes a dramatic reduction in tumor cell viability. These results lead to the possibility of a selective antitumor therapeutic use of SR59230A. Notably, we demonstrate the presence of ß3-AR within the mitochondrial membrane in both cell lines, leading to the control of mitochondrial dormancy.

Repurposing of dexpramipexole to treatment of neonatal hypoxic/ischemic encephalopathy.

Dexpramipexole (DEX) is a drug with a good safety profile in humans, known for its ability to increase mitochondrial ATP production and prompt neuroprotection in adult rodents subjected to cerebral ischemia. In the present study we evaluated the effect of DEX in rat pups subjected to common carotid artery occlusion plus hypoxia (CCAoH, the classic Rice-Vannucci model). Because of the wide range of infarct size distribution in the CCAoH model, a priori subanalysis based on the effect of DEX on mild/moderate or severe brain injuries was conducted. The subanalysis showed that the drug (3 mg/kg bid i.p, after the hypoxic insult) decreased the infarction size in pups with mild/moderate injuries. Next, we developed a distal middle cerebral artery occlusion plus hypoxia (dMCAoH) model, characterized by an intra-experimental infarct size variability lower than that of the CCAoH model. Post-ischemic treatment with DEX (3 mg/kg bid i.p, after the hypoxic insult) reduced brain infarcts in pups exposed to dMCAoH. For the first time, we show that DEX reduces brain injury in different models of neonatal HIE. In light of the favorable safety profile of DEX in humans, the drug might have a realistic translational potential to treatment of perinatal cerebrovascular disorders.

Dexpramipexole enhances hippocampal synaptic plasticity and memory in the rat.

Even though pharmacological approaches able to counteract age-dependent cognitive impairment have been highly investigated, drugs improving cognition and memory are still an unmet need. It has been hypothesized that sustaining energy dynamics within the aged hippocampus can boost memory storage by sustaining synaptic functioning and long term potentiation (LTP). Dexpramipexole (DEX) is the first-in-class compound able to sustain neuronal bioenergetics by interacting with mitochondrial F1Fo-ATP synthase. In the present study, for the first time we evaluated the effects of DEX on synaptic fatigue, LTP induction, learning and memory retention. We report that DEX improved LTP maintenance in CA1 neurons of acute hippocampal slices from aged but not young rats. However, we found no evidence that DEX counteracted two classic parameters of synaptic fatigue such as fEPSP reduction or the train area during the high frequency stimulation adopted to induce LTP. Interestingly, patch-clamp recordings in rat hippocampal neurons revealed that DEX dose-dependently inhibited (IC50 814 nM) the IA current, a rapidly-inactivating K+ current that negatively regulates neuronal excitability as well as cognition and memory processes. In keeping with this, DEX counteracted both scopolamine-induced spatial memory loss in rats challenged in Morris Water Maze test and memory retention in rats undergoing Novel Object Recognition. Overall, the present study discloses the ability of DEX to boost hippocampal synaptic plasticity, learning and memory. In light of the good safety profile of DEX in humans, our findings may have a realistic translational potential to treatment of cognitive disorders.

Trigeminal ganglion transcriptome analysis in 2 rat models of medication-overuse headache reveals coherent and widespread induction of pronociceptive gene expression patterns.

We attempted to gather information on the pathogenesis of medication-overuse headache, as well as on the neurochemical mechanisms through which symptomatic medication overuse concurs to headache chronification. Transcriptional profiles were therefore evaluated as an index of the homeostasis of the trigeminovascular system in the trigeminal ganglion of female rats exposed for 1 month to daily oral doses of eletriptan or indomethacin. We report that both drug treatments change trigeminal ganglion gene expression to a similar extend. Of note, qualitative transcriptomic analysis shows that eletriptan and indomethacin prompt nearly identical, increased expression of genes coding for proteins involved in migraine pathogenesis and central pain sensitization such as neuropeptides, their cognate receptors, prostanoid, and nitric oxide-synthesizing enzymes, as well as TRP channels. These genes, however, were not affected in thoracic dorsal root ganglia. Of note, lowering of orofacial nociceptive thresholds, as well as forepaw hyperalgesia occurred in both indomethacin- and eletriptan-treated rats. Our study reveals that chronic rat exposure to 2 acute headache medications with completely different mechanisms of action prompts pain sensitization with highly similar induction of pronociceptive genes selectively within the trigeminal ganglion. Data further our understanding of medication-overuse headache pathogenesis and provide hints for specific mechanism-based treatment options.