Cammarota, Mariarosaria (2022) "Study of the remyelination and neuroprotective potential of novel pharmacological compounds targeting the Na+/Ca+2 exchanger NCX3 in oligodendrocytes or the endocannabinoid and melatoninergic systems in brain explants". [Tesi di dottorato]


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Item Type: Tesi di dottorato
Resource language: English
Title: "Study of the remyelination and neuroprotective potential of novel pharmacological compounds targeting the Na+/Ca+2 exchanger NCX3 in oligodendrocytes or the endocannabinoid and melatoninergic systems in brain explants".
Date: 9 February 2022
Number of Pages: 123
Institution: Università degli Studi di Napoli Federico II
Department: Neuroscienze e Scienze Riproduttive ed Odontostomatologiche
Dottorato: Neuroscienze
Ciclo di dottorato: 34
Coordinatore del Corso di dottorato:
Boscia, FrancescaUNSPECIFIED
Date: 9 February 2022
Number of Pages: 123
Keywords: sodium calcium exchanger NCX3,endocannabinoid and melatoninergic system
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/14 - Farmacologia
Date Deposited: 14 Feb 2022 13:11
Last Modified: 28 Feb 2024 14:19

Collection description

Neurodegeneration, neuroinflammation, and failure of regenerative processes contribute to the pathogenesis of several neurodegenerative disorders including multiple sclerosis (MS). Indeed, MS treatment are primarily immunomodulatory, but no neuroprotective or remyelinating therapies are available to prevent the accumulation of neurological deficit and progression of the disease. Currently, a significant effort is underway to develop molecules with the potential to halt the neurodegenerative and demyelination processes, modulate the microglia/macrophage response to neuroinflammation and stimulate myelin repair, particularly for the treatment of progressive MS forms, for which current therapies are relatively ineffective. In the present research project, we investigated the neuroprotective and remyelinating potential of two distinct pharmacological treatments modulating ionic or lipid homeostasis and contributing to distinct signaling pathways important for oligodendrocyte differentiation and neuroprotection. The Na+/Ca2+ exchanger NCX3 is an important regulator of sodium and calcium homeostasis in oligodendrocyte lineage, and a recent work performed by our research group demonstrated that [Ca2+]i transients mediated by NCX3 play an important role during oligodendrocyte differentiation and myelin formation. The observation that any change in NCX activity dynamically affect Na+ and Ca2+ fluxes and that Na+ and Ca2+ gradients tightly control the kinetics and directionality of NCX operation indicates that more studies are required to understand the pharmacological effects of selective NCX3 blockers in oligodendroglia and neuronal cells under demyelinating conditions. These studies are fundamental to develop and identify novel neurorepair strategies targeting NCX exchangers. In the first part of the thesis, we investigated, by means of biochemical, morphological and functional analyses, the pharmacological effects of the NCX3 inhibitor, the 5–amino‐N‐butyl‐2–(4–ethoxyphenoxy)-benzamide hydrochloride (BED), on NCXs expression and activity, as well as intracellular [Na+]i and [Ca2+]i levels, during treatment and following drug washout both in human MO3.13 oligodendrocytes and rat primary oligodendrocyte precursor cells (OPCs). BED exposure antagonized NCX activity, induced OPCs proliferation and [Na+]i accumulation. By contrast, 2 days of BED washout after 4 days of treatment significantly upregulated low molecular weight NCX3 proteins, reversed NCX activity, and increased intracellular [Ca2+]i . This BED-free effect was accompanied by an upregulation of NCX3 expression in oligodendrocyte processes and accelerated expression of myelin markers in rat primary oligodendrocytes. Collectively, these findings show that the pharmacological inhibition of the NCX3 exchanger with BED blocker maybe followed by a rebound increase in NCX3 expression and reversal activity that accelerate myelin sheet formation in oligodendrocytes. In addition, they indicate that a particular attention should be paid to the use of NCX inhibitors for possible rebound effects and suggest that further studies will be necessary to investigate whether selective pharmacological modulation of NCX3 exchanger may be exploited to benefit demyelination and remyelination in demyelinating diseases. Another interesting attractive target for MS treatment is the endocannabinoid system (EC) which consists to a complex network of endocannabinoid ligands, cannabinoid receptors and enzymes responsible for synthesizing and degrading endocannabinoids (ECs). Enhancing endogenously-released cannabinoid ligands through the inhibition of the degrading fatty acid amide hydrolase (FAAH) enzyme in the brain may provide therapeutic effects more safely and effectively than administering drugs directly acting at the cannabinoid receptors. FAAH inhibition prevents the breakdown of anandamide (AEA), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), producing potentially therapeutic effects, some through cannabinoid receptors and some through peroxisome proliferator-activated receptors (PPAR). Currently, FAAH inhibitors are under clinical investigation for the treatment of Tourette Syndrome (NCT02134080), and chronic pain. Furthermore, a large number of studies highlight the beneficial role of melatonin in neuroinflammatory disorders. Melatonin, a neurohormone produced by the pineal gland, regulates the sleep wake-cycle and exerts potent antioxidant, anti-inflammatory, immunomodulatory, and neuroprotective effects. In experimental models of MS, melatonin prevented demyelination, axonal injury and stimulated OPCs differentiation and remyelination. It is currently used as dietary supplement for the short-term treatment of insomnia but its use has also been proposed as add-on therapy in several neuroinflammatory conditions. In the second part of the thesis, we investigated the neuroprotective effects of a novel pharmacological compound, UCM-1341, a bivalent ligand with FAAH inhibitory activity and melatonin receptors agonism. To this aim, we set up models of excitoxic and neuroinflammatory damage in rat hippocampal explant cultures to study the time – and dose-dependent effects of compounds on cell death. Our expression studies showed that FAAH protein levels early increased in CA1 hippocampal neurons and astrocytes after the neuroinflammatory insult. At later time points, the increased FAAH immunosignal persisted in astrocytes, but not in neurons, and emerged in microglia, particularly in that one surrounding damaged cells. By using an antibody recognizing melatonin receptors we found that MT1 levels persistently increased after the insult while MT2 proteins decreased after a transient upregulation. Our neuroprotection studies showed that while the damage occurring in the CA1 and the less vulnerable CA3 region in slices exposed to NMDA concentrations was not greatly affected by UCM-1341, the bivalent ligand exerted a marked dose-dependent neuroprotection against LPS+IFN--induced neuroinflammatory damage and attenuated axonal demyelination. More interestingly, we showed that cotreatment of slices with the reference compounds melatonin or URB-597 exerted a greater neuroprotection against LPS+IFN-exposure, if compared to single compounds applications. This effect was comparable to that observed with UCM-1341, thus indicating that the reference compounds exerted a synergistic neuroprotective actions against LPS+IFN--induced inflammatory damage. Finally, by performing biochemical and quantitative colocalization analyses we showed that UCM-1341 had also protective effects on astrocyte and microglia cells and polarized microglia/macrophages to a foamy phenotype expressing the anti-inflammatory M2 marker CD206. Collectively, our findings demonstrated that the combined modulation of EC and melatoninergic signaling exerted potent neuroprotective effects against the neuroinflammatory damage in hippocampal explant cultures. The modulation effects of UCM-1341 on microglia activities might contribute to the synergistic beneficial effects against the inflammatory injury. Nevertheless, further studies will be required to understand whether enhancing the EC and melatoninergic tone with UCM-1341 may represent a novel neuroprotective strategy to treat neuroinflammatory conditions.


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