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The role that astrocytes play in central nervous system (CNS) myelination is poorly understood. We investigated the contribution of astrocyte-derived factors to myelination and revealed a substantial overlap in the secretomes of human and rat astrocytes. Using in vitro myelinating co-cultures of primary retinal ganglion cells and cortical oligodendrocyte precursor cells, we discovered that factors secreted by resting astrocytes, but not reactive astrocytes, facilitated myelination. Soluble brevican emerged as a new enhancer of developmental myelination in vivo, CNS and its absence was linked to remyelination deficits following an immune-mediated damage in an EAE mouse model. The observed reduction of brevican expression in reactive astrocytes and human MS lesions suggested a potential link to the compromised remyelination characteristic of neurodegenerative diseases. Our findings suggested brevican's role in myelination may be mediated through interactions with binding partners such as contactin-1 and tenascin-R. Proteomic analysis of resting versus reactive astrocytes highlighted a shift in protein expression profiles, pinpointing candidates that either facilitate or impede CNS repair, suggesting that depending on their reactivity state, astrocytes play a dual role during myelination.
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The endocannabinoid system (ECS) is critically involved in the pathophysiology of Multiple Sclerosis (MS), a neuroinflammatory and neurodegenerative disease of the central nervous system (CNS). Over the past decade, researchers have extensively studied the neuroprotective and anti-inflammatory effects of the ECS. Inhibiting the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) has emerged as a promising strategy to mitigate brain damage in MS. In this study, we investigated the effects of a novel reversible MAGL inhibitor (MAGLi 432) on C57/BL6 female mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. We assessed its implications on motor disability, neuroinflammation, and synaptic dysfunction. Systemic in vivo treatment with MAGLi 432 resulted in a less severe EAE disease, accompanied by increased 2-AG levels and decreased levels of arachidonic acid (AA) and prostaglandins (PGs) in the brain. Additionally, MAGLi 432 reduced both astrogliosis and microgliosis, as evidenced by decreased microglia/macrophage density and a less reactive morphology. Flow cytometry analysis further revealed fewer infiltrating CD45+ and CD3+ cells in the brains of MAGLi 432-treated EAE mice. Finally, MAGLi treatment counteracted the striatal synaptic hyperexcitability promoted by EAE neuroinflammation. In conclusion, MAGL inhibition significantly ameliorated EAE clinical disability and striatal inflammatory synaptopathy through potent anti-inflammatory effects. These findings provide new mechanistic insights into the neuroprotective role of the ECS during neuroinflammation and highlight the therapeutic potential of MAGLi-based drugs in mitigating MS-related inflammatory and neurodegenerative brain damage.
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Ácidos Araquidónicos , Encefalomielitis Autoinmune Experimental , Endocannabinoides , Glicéridos , Ratones Endogámicos C57BL , Animales , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Glicéridos/metabolismo , Ratones , Endocannabinoides/metabolismo , Ácidos Araquidónicos/farmacología , Ácidos Araquidónicos/metabolismo , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/metabolismo , Sinapsis/efectos de los fármacos , Sinapsis/patología , Sinapsis/metabolismo , Microglía/efectos de los fármacos , Microglía/metabolismo , Monoacilglicerol Lipasas/antagonistas & inhibidores , Monoacilglicerol Lipasas/metabolismoRESUMEN
This study aimed to evaluate (R)-[18F]YH134 as a novel PET tracer for imaging monoacylglycerol lipase (MAGL). Considering the ubiquitous expression of MAGL throughout the whole body, the impact of various MAGL inhibitors on (R)-[18F]YH134 brain uptake and its application in brain-periphery crosstalk were explored. Methods: MAGL knockout and wild-type mice were used to evaluate (R)-[18F]YH134 in in vitro autoradiography and PET experiments. To explore the impact of peripheral MAGL occupancy on (R)-[18F]YH134 brain uptake, PET kinetics with an arterial input function were studied in male Wistar rats under baseline and blocking conditions. Results: In in vitro autoradiography, (R)-[18F]YH134 revealed a heterogeneous distribution pattern with high binding to MAGL-rich brain regions in wild-type mouse brain slices, whereas the radioactive signal was negligible in MAGL knockout mouse brain slices. The in vivo brain PET images of (R)-[18F]YH134 in wild-type and MAGL knockout mice demonstrated its high specificity and selectivity in mouse brain. A Logan plot with plasma input function was applied to estimate the distribution volume (V T) of (R)-[18F]YH134. V T was significantly reduced by a brain-penetrant MAGL inhibitor but was unchanged by a peripherally restricted MAGL inhibitor. The MAGL target occupancy in the periphery was estimated using (R)-[18F]YH134 PET imaging data from the brain. Conclusion: (R)-[18F]YH134 is a highly specific and selective PET tracer with favorable kinetic properties for imaging MAGL in rodent brain. Our results showed that blocking of the peripheral target influences brain uptake but not the V T of (R)-[18F]YH134. (R)-[18F]YH134 can be used for estimating the dose of MAGL inhibitor at half-maximal peripheral target occupancy.
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Monoacilglicerol Lipasas , Neuroimagen , Ratas , Ratones , Masculino , Animales , Monoacilglicerol Lipasas/metabolismo , Ratas Wistar , Neuroimagen/métodos , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Tomografía de Emisión de Positrones/métodos , Ratones Noqueados , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/químicaRESUMEN
Monoacylglycerol lipase (MAGL) regulates endocannabinoid 2-arachidonoylglycerol (2-AG) and eicosanoid signalling. MAGL inhibition provides therapeutic opportunities but clinical potential is limited by central nervous system (CNS)-mediated side effects. Here, we report the discovery of LEI-515, a peripherally restricted, reversible MAGL inhibitor, using high throughput screening and a medicinal chemistry programme. LEI-515 increased 2-AG levels in peripheral organs, but not mouse brain. LEI-515 attenuated liver necrosis, oxidative stress and inflammation in a CCl4-induced acute liver injury model. LEI-515 suppressed chemotherapy-induced neuropathic nociception in mice without inducing cardinal signs of CB1 activation. Antinociceptive efficacy of LEI-515 was blocked by CB2, but not CB1, antagonists. The CB1 antagonist rimonabant precipitated signs of physical dependence in mice treated chronically with a global MAGL inhibitor (JZL184), and an orthosteric cannabinoid agonist (WIN55,212-2), but not with LEI-515. Our data support targeting peripheral MAGL as a promising therapeutic strategy for developing safe and effective anti-inflammatory and analgesic agents.
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Monoacilglicerol Lipasas , Monoglicéridos , Animales , Ratones , Rimonabant , Endocannabinoides , Analgésicos/farmacología , Receptor Cannabinoide CB1 , Ratones Endogámicos C57BLRESUMEN
Despite its essential role in the (patho)physiology of several diseases, CB2R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB2R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB2R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB2R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB2R based drugs.
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Microglia, the innate immune cells of the brain, are essential for maintaining homeostasis by their ramified, highly motile processes and for orchestrating the immune response to pathological stimuli. They are implicated in several neurodegenerative diseases like Alzheimer's and Parkinson's disease. One commonality of these diseases is their strong correlation with aging as the highest risk factor and studying age-related alterations in microglia physiology and associated signaling mechanism is indispensable for a better understanding of age-related pathomechanisms. CD22 has been identified as a modifier of microglia phagocytosis in a recent study, but not much is known about the function of CD22 in microglia. Here we show that CD22 surface levels are upregulated in aged versus adult microglia. Furthermore, in the amyloid mouse model PS2APP, Aß-containing microglia also exhibit increased CD22 signal. To assess the impact of CD22 blockage on microglia morphology and dynamics, we have established a protocol to image microglia process motility in acutely prepared brain slices from CX3CR1-GFP reporter mice. We observed a significant reduction of microglial ramification and surveillance capacity in brain slices from aged versus adult mice. The age-related decrease in surveillance can be restored by antibody-mediated CD22 blockage in aged mice, whereas surveillance in adult mice is not affected by CD22 inhibition. Moreover to complement the results obtained in mice, we show that human iPSC-derived macrophages exhibit an increased phagocytic capacity upon CD22 blockage. Downstream analysis of antibody-mediated CD22 inhibition revealed an influence on BMP and TGFß associated gene networks. Our results demonstrate CD22 as a broad age-associated modulator of microglia functionality with potential implications for neurodegenerative disorders.
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Envejecimiento/fisiología , Encéfalo/citología , Microglía/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Lectina 2 Similar a Ig de Unión al Ácido Siálico/antagonistas & inhibidores , Envejecimiento/efectos de los fármacos , Envejecimiento/genética , Animales , Encéfalo/efectos de los fármacos , Encéfalo/fisiología , Recuento de Células , Modelos Animales de Enfermedad , Humanos , Macrófagos/metabolismo , Masculino , Ratones , Microglía/citología , Fagocitosis/genética , Lectina 2 Similar a Ig de Unión al Ácido Siálico/genética , Lectina 2 Similar a Ig de Unión al Ácido Siálico/metabolismo , Transducción de SeñalRESUMEN
Pharmacological modulation of cannabinoid type 2 receptor (CB2R) holds promise for the treatment of numerous conditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this receptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CB2R signaling, especially in cell-type and tissue-dependent contexts. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CB2R fluorescent probes, used successfully across applications, species, and cell types. These include flow cytometry of endogenously expressing cells, real-time confocal microscopy of mouse splenocytes and human macrophages, as well as FRET-based kinetic and equilibrium binding assays. High CB2R specificity was demonstrated by competition experiments in living cells expressing CB2R at native levels. The probes were effectively applied to FACS analysis of microglial cells derived from a mouse model relevant to Alzheimer's disease.
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Enfermedad de Alzheimer/metabolismo , Colorantes Fluorescentes/química , Microglía/metabolismo , Receptor Cannabinoide CB2/análisis , Animales , Células CHO , Cricetulus , Modelos Animales de Enfermedad , Citometría de Flujo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Ligandos , Ratones , Simulación del Acoplamiento Molecular , Sondas Moleculares/química , Imagen Óptica , Sensibilidad y Especificidad , Transducción de SeñalRESUMEN
Individual reports suggest that the central nervous system (CNS) contains multiple immune cell types with diverse roles in tissue homeostasis, immune defense, and neurological diseases. It has been challenging to map leukocytes across the entire brain, and in particular in pathology, where phenotypic changes and influx of blood-derived cells prevent a clear distinction between reactive leukocyte populations. Here, we applied high-dimensional single-cell mass and fluorescence cytometry, in parallel with genetic fate mapping systems, to identify, locate, and characterize multiple distinct immune populations within the mammalian CNS. Using this approach, we revealed that microglia, several subsets of border-associated macrophages and dendritic cells coexist in the CNS at steady state and exhibit disease-specific transformations in the immune microenvironment during aging and in models of Alzheimer's disease and multiple sclerosis. Together, these data and the described framework provide a resource for the study of disease mechanisms, potential biomarkers, and therapeutic targets in CNS disease.