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• W3089711134 abstract "Article5 October 2020Open Access Source DataTransparent process Inhibition of fatty acid amide hydrolase prevents pathology in neurovisceral acid sphingomyelinase deficiency by rescuing defective endocannabinoid signaling Adrián Bartoll Adrián Bartoll Centro Biologia Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain Search for more papers by this author Ana Toledano-Zaragoza Ana Toledano-Zaragoza Centro Biologia Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain Search for more papers by this author Josefina Casas Josefina Casas RUBAM, IQAC-CSIC & CIBEREHD, Barcelona, Spain Search for more papers by this author Manuel Guzmán Manuel Guzmán Department of Biochemistry and Molecular Biology, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Instituto Universitario de Investigación Neuroquímica (IUIN), Complutense University, Madrid, Spain Search for more papers by this author Edward H Schuchman Edward H Schuchman Department of Genetics & Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York NY, USA Search for more papers by this author María Dolores Ledesma Corresponding Author María Dolores Ledesma [email protected] orcid.org/0000-0002-5679-6891 Centro Biologia Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain Search for more papers by this author Adrián Bartoll Adrián Bartoll Centro Biologia Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain Search for more papers by this author Ana Toledano-Zaragoza Ana Toledano-Zaragoza Centro Biologia Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain Search for more papers by this author Josefina Casas Josefina Casas RUBAM, IQAC-CSIC & CIBEREHD, Barcelona, Spain Search for more papers by this author Manuel Guzmán Manuel Guzmán Department of Biochemistry and Molecular Biology, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Instituto Universitario de Investigación Neuroquímica (IUIN), Complutense University, Madrid, Spain Search for more papers by this author Edward H Schuchman Edward H Schuchman Department of Genetics & Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York NY, USA Search for more papers by this author María Dolores Ledesma Corresponding Author María Dolores Ledesma [email protected] orcid.org/0000-0002-5679-6891 Centro Biologia Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain Search for more papers by this author Author Information Adrián Bartoll1,‡, Ana Toledano-Zaragoza1,‡, Josefina Casas2, Manuel Guzmán3, Edward H Schuchman4 and María Dolores Ledesma *,1 1Centro Biologia Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain 2RUBAM, IQAC-CSIC & CIBEREHD, Barcelona, Spain 3Department of Biochemistry and Molecular Biology, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Instituto Universitario de Investigación Neuroquímica (IUIN), Complutense University, Madrid, Spain 4Department of Genetics & Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York NY, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +34 911964535; E-mail: [email protected] EMBO Mol Med (2020)12:e11776https://doi.org/10.15252/emmm.201911776 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Acid sphingomyelinase deficiency (ASMD) leads to cellular accumulation of sphingomyelin (SM), neurodegeneration, and early death. Here, we describe the downregulation of the endocannabinoid (eCB) system in neurons of ASM knockout (ASM-KO) mice and a ASMD patient. High SM reduced expression of the eCB receptor CB1 in neuronal processes and induced its accumulation in lysosomes. Activation of CB1 receptor signaling, through inhibition of the eCB-degrading enzyme fatty acid amide hydrolase (FAAH), reduced SM levels in ASM-KO neurons. Oral treatment of ASM-KO mice with a FAAH inhibitor prevented SM buildup; alleviated inflammation, neurodegeneration, and behavioral alterations; and extended lifespan. This treatment showed benefits even after a single administration at advanced disease stages. We also found CB1 receptor downregulation in neurons of a mouse model and a patient of another sphingolipid storage disorder, Niemann–Pick disease type C (NPC). We showed the efficacy of FAAH inhibition to reduce SM and cholesterol levels in NPC patient-derived cells and in the brain of a NPC mouse model. Our findings reveal a pathophysiological crosstalk between neuronal SM and the eCB system and offer a new treatment for ASMD and other sphingolipidoses. Synopsis This study shows the downregulation of the endocannabinoid (eCB) system in neurons of two lysosomal storage disorders: acid sphingomyelinase deficiency (ASMD) and Niemann Pick type C (NPC). Brain pathology in mouse models of these diseases was prevented by eCB pharmacological enhancement. High levels of sphingomyelin (SM) accumulate in ASMD and NPC neurons. High SM levels are responsible for reduced amount of the eCB receptor CB1 and increased localization in lysosomes. Activation of CB1 by treatment with inhibitors of the eCB-degrading enzyme FAAH, reduces SM levels through neutral sphingomyelinase (NSM). CB1 activation prevents ASMD and NPC pathological phenotypes in vitro in cultured mouse and patient cells and in vivo upon oral administration in the respective mouse models The paper explained Problem Acid sphingomyelinase deficiency (ASMD) and Niemann–Pick type C (NPC) are fatal lysosomal storage disorders in which lipids like sphingomyelin accumulate in cells leading to severe neurological involvement and early death. Results Here, we describe the sphingomyelin-induced downregulation of the eCB receptor CB1 in neurons of mouse models and patients of these diseases. Enhancement of the eCB system by oral treatment with inhibitors of the eCB-degrading enzyme fatty acid amide hydrolase (FAAH), reduced lipid storage, prevented neurodegeneration, and increased life span in the mouse models. Impact These results discover a novel therapeutic target and a non-invasive strategy for ASMD and NPC with potential for the treatment of other sphingolipid storage disorders. Introduction The endocannabinoid (eCB) system consists of a family of modulatory lipid messengers together with their specific receptors and metabolic enzymes. This cell signaling system regulates many aspects of neuronal development and function (Maccarrone et al, 2014) and influences synaptic communication in a plethora of physiopathological events, including cognitive and emotional processes (Katona & Freund, 2012; Mechoulam & Parker, 2013). Both anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG), the major eCBs, bind to the G protein-coupled receptors CB1 and CB2, of which CB1 is by large the most abundant in the brain (Pertwee et al, 2010). Concertedly, the enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) contribute to the bulk of AEA and 2-AG degradation, respectively, and are essential for maintaining appropriate levels of both molecules (Di Marzo, 2009; Shin et al, 2019). The neuromodulatory actions of eCBs are believed to rely on their release from a postsynaptic neuron upon stimulation and their subsequent retrograde action on presynaptic terminals, where they activate CB receptors, thus modulating plasma membrane ion conductivity and depressing neurotransmitter release (Castillo et al, 2012). CB1 receptor activation has been long related to inhibitory and excitatory synaptic plasticity underlying learning and memory (Sullivan, 2000; Domenici et al, 2006; Kawamura et al, 2006; Takahashi & Castillo, 2006). Alterations in the brain eCB system have been linked to various psychiatric conditions. For example, impaired AEA degradation underlies attention deficits and hyperactivity disorder (Centonze et al, 2009). It has also been proposed that endogenous CB1 receptor activation serves as a buffer against depression, while its downregulation results in depressive symptoms (Mangieri & Piomelli, 2007; Patel & Hillard, 2009; Fowler, 2015). In addition, the eCB system plays important prohomeostatic, anti-inflammatory, and neuroprotective functions (Sarne & Mechoulam, 2005; van der Stelt & Di Marzo, 2005; Chiarlone et al, 2014; Gomez-Galvez et al, 2016), and its possible involvement in different neurodegenerative diseases (van der Stelt et al, 2005; Blazquez et al, 2011; Aymerich et al, 2018) has stimulated the therapeutic interest of eCB modulation for the treatment of neurological disorders. A series of previous studies point to the membrane environment as a critical factor for the regulation of the eCB system. For example, acute cholesterol depletion increases CB1-dependent signaling in neuronal cells (Bari et al, 2005a,b), and lipid raft-dependent and non-dependent endocytosis might be differentially involved in the transport of AEA and the axonal sorting of CB1 (McFarland & Barker, 2004; Leterrier et al, 2006; Fletcher-Jones et al, 2020). Moreover, eCBs are believed to bind to and activate CB1 receptors by lateral diffusion within the lipid bilayer, rather than reaching the binding site from the extracellular surface (Lynch & Reggio, 2006; Hua et al, 2017). These observations highlight the relevance of membrane lipids to eCB function and the need to understand in further detail their contribution. The infantile, neurovisceral form of acid sphingomyelinase deficiency (ASMD also known as acute neuronopathic ASMD or Niemann–Pick type A) is a fatal lipidosis that leads to hepatosplenomegaly, pulmonary involvement, impaired psychomotor development, and neurodegeneration (McGovern et al, 2017). Disease-causing mutations in the gene encoding ASM (SMPD1), a key enzyme responsible for SM degradation, induce the accumulation of this lipid in all body cells (Schuchman & Desnick, 2017). In neurons from ASM knockout (ASM-KO) mice, high SM levels in the lysosomes and the synaptic and plasma membrane lead to lysosomal permeabilization and autophagy impairment (Gabande-Rodriguez et al, 2014), unpolarized distribution of proteins (Galvan et al, 2008), dendritic spine anomalies (Arroyo et al, 2014), and Ca2+ imbalance (Perez-Canamas et al, 2016). Enzyme replacement therapy (ERT) by intravenous infusion of recombinant human ASM efficiently treats the non-neurological pathology in ASM-KO mice (Miranda et al, 2000) and in ASMD patients (Wasserstein et al, 2015). Two clinical trials (ASCEND and ASCEND-Peds) are currently ongoing (NCT02004691 and NCT02292654, respectively) for ASMD patients that lack neurological involvement (i.e., Niemann–Pick type B or chronic visceral ASMD) (McGovern et al, 2017). However, the inability of the recombinant enzyme to cross the blood–brain barrier and the absence of improvement in the neurological component after ERT in ASM-KO mice (Miranda et al, 2000) indicate that this approach would be inadequate for the neurovisceral forms. In addition to ASM, there are other sphingomyelinases that are active in the mammalian brain and can degrade SM. Indeed, pharmacological enhancement of the neutral sphingomyelinase (NSM) by the glucocorticoid dexamethasone reduced SM levels in synapses of ASM-KO mice and improved their memory and learning capabilities (Arroyo et al, 2014). These findings pointed to the activation of NSM, an enzyme that is not genetically affected in ASMD, as a suitable strategy to diminish high brain SM levels in the disease. It has been shown that cannabinoid-evoked stimulation of CB1 receptors activates NSM through the adaptor protein FAN, thus reducing SM levels in cultured rat astrocytes (Sanchez et al, 2001). Early work in cultured fibroblasts from a severe ASMD patient also showed the capacity of cannabidiol to reduce SM levels (Burstein et al, 1984). We thus hypothesized that enhancing the activity of the eCB system in brain and peripheral organs could be therapeutically beneficial for patients with both the neurovisceral and visceral forms of ASMD. In this study, we provide proof of concept for this hypothesis by showing that (i) elevated SM leads to reduced CB1 expression and altered localization in neurons, (ii) there is a pathological downregulation of the eCB system in neurons of ASM-KO mice and of an infantile ASMD patient, and (iii) inhibitors of the eCB hydrolytic enzyme FAAH exhibit safety and efficacy to treat brain and peripheral pathology in ASM-KO mice. We also extend this concept to another sphingolipid storage disease, NPC, by showing CB1 receptor downregulation in a mouse model and a patient and that FAAH inhibition reduces SM and cholesterol levels in NPC patient-derived cells and in the brain of a NPC mouse model. Results Characterization of CB1 receptor anomalies in the brains of ASM-KO mice and a infantile neurovisceral ASMD patient CB1 and CB2 receptors mediate eCB signaling in the mammalian brain. To determine whether these receptors are altered in ASM-KO mice, we measured their mRNA and protein levels in the brains of age-matched, 4-month-old WT and ASM-KO mice. We focused on the analysis of the cerebellum, which is the most affected brain area in the disease. Real-time quantitative PCR (RT-qPCR) showed no significant differences in the levels of CB2 mRNA (Fig 1A). Protein levels of this receptor were also unchanged as determined by Western blot in cerebellar extracts of ASM-KO mice compared to WT littermates (Fig 1B). In contrast, mRNA levels of CB1 were 30% diminished in the cerebellum (Fig 1C). Similar reductions (40 and 30%, respectively) were also found in other brain areas such as the hippocampus and prefrontal cortex (Appendix Fig S1A). Western blot of cerebellar extracts showed a slight 13% reduction in CB1 protein levels that was not statistically significant (Fig 1D). Similar results were found by Western blot of hippocampal and cortical extracts (Appendix Fig S1B). Slight decreases in CB1 protein levels were detected by immunofluorescence in these two brain areas (Appendix Fig S1C). To monitor the cell type specificity of CB1 protein expression, we performed co-labeling with cellular markers by immunofluorescence in the cerebellum. This analysis indicated a significant 26% reduction in the levels of CB1 protein in the Purkinje neurons, identified by calbindin staining, and a reduced co-localization between CB1 and these cells as indicated by the Mander's coefficient, which reflects the amount of CB1 in this particular cell population with respect to the total CB1 staining in the tissue (Fig 1E). CB1-associated intensity was not significantly changed in astrocytes, identified by GFAP staining. However, the Mander's coefficient between CB1 and astrocytes increased, probably due to the elevated number of these cells in the ASM-KO compared to WT cerebellum (Fig 1F and Appendix Fig S2A). CB1 levels and co-localization did not change in microglia, identified by F4/80 staining, which also increased their number in the ASM-KO cerebellum (Fig 1G and Appendix Fig S2B). These changes in the balance among cell populations in the ASM-KO mouse brains might prevent the detection of the neuronal-specific CB1 reduction in the biochemical experiments. To determine whether the reduction in CB1 protein levels observed in the Purkinje neurons of the ASM-KO mouse also occurred in patients, we gained access to fixed tissue from the cerebellum of a 3-year-old child affected by infantile neurovisceral ASMD and compared CB1 levels in the calbindin-positive cells by immunofluorescence with an age-matched non-affected child. CB1 protein was notably reduced in the Purkinje cells of the ASMD patient (Fig 1H). Reduction of CB1 levels was also observed in neurons (identified by MAP2 staining) of the medium bulb, the other brain area to which we had access in the ASMD patient (Appendix Fig S3). While these observations are not conclusive, since they were made in a single patient, they suggest common CB1 alterations in ASMD-affected humans and mice. Figure 1. eCB alterations in the cerebellum of ASM-KO mice and an infantile, neurovisceral ASMD patient A. Mean ± SEM CB2 mRNA levels in cerebellar extracts of WT and ASM-KO mice (n = 5 mice per group). B. Western blot against CB2 and PSD-95 (used as loading control) and graph showing mean ± SEM CB2 protein levels in cerebellar extracts of WT and ASM-KO mice (n = 6 mice per group). C. Mean ± SEM CB1 mRNA levels in cerebellar extracts of WT and ASM-KO mice (**P = 0.0013, n = 6 mice per group, Student's t-test). D. Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cerebellar extracts of WT and ASM-KO mice (n = 3 mice per group). E–G. Immunofluorescence images against CB1 and the following cellular markers: calbindin for Purkinje cells (E), GFAP for astrocytes (F), and F4/80 for microglia (G) in the cerebellum of WT and ASM-KO mice. DAPI stains cell nuclei. Graphs to the left show mean ± SEM intensity associated with CB1 in the Purkinje cells (shown by white arrows, E), astrocytes (F), and microglia (G) expressed as percentage of the values obtained in WT mice. Graphs to the right show the Mander's coefficient that indicates degree of co-localization between CB1 and the cellular markers for Purkinje cells (E), astrocytes (F), and microglia (G) (E: **PFluorecence intensity = 0.0082, *PMander's Coefficient = 0.0158; F: *PMander's Coefficient = 0.0115; n = 5 mice per group, Student's t-test). Scale bar, 100 μm. H. Immunofluorescence images against CB1 and the Purkinje cell marker calbindin in the cerebellum of age-matched control and ASMD-affected children. Graph shows mean ± SEM intensity associated with CB1 in the Purkinje cells expressed as percentage of the values obtained in the control child (16 and 15 replicates in control and ASMD, respectively). Scale bar, 10 μm. Source data are available online for this figure. Source Data for Figure 1 [emmm201911776-sup-0002-SDataFig1.xlsx] Download figure Download PowerPoint High SM induces CB1 receptor reduction and misdistribution in ASM-KO neurons To further understand the impact of ASM deficiency on CB1 expression in neurons, the cell type in which we observed alterations in the tissue analysis (Fig 1), we analyzed CB1 levels and distribution in cultured primary neurons from WT and ASM-KO mice. CB1 mRNA levels were drastically reduced (95%) in the ASM-KO hippocampal neurons (Fig 2A). This was accompanied by a significant 66% reduction of CB1 protein levels measured by Western blot of neuronal extracts (Fig 2B). The immunofluorescence analysis also indicated a change in the distribution of CB1. Thus, while the receptor was preferentially lost from the neuronal processes (65% reduction), its localization in the lysosomal compartment (as visualized by the lysosomal marker Lamp1) increased in the ASM-KO neurons (Mander's coefficient 0.15 and 0.33 in WT and ASM-KO neurons, respectively) (Fig 2C). A higher co-localization of CB1 with lysosomes was also observed in vivo in the Purkinje cells of the cerebellum of ASM-KO compared to WT mice (Mander's coefficient 0.019 and 0.042 in WT and ASM-KO mice, respectively [Fig 2D]). Figure 2. CB1 alterations in ASM-KO neurons are due to high SM levels A. Mean ± SEM CB1 mRNA levels in cultured hippocampal neurons from WT and ASM-KO mice (****P < 0.0001, n = 5 independent cultures, Student's t-test). B. Western blot against CB1 and GAPDH (used as loading control). Images belong to the same blot but to non-consecutive lanes as indicated by the boxes. Graph showing mean ± SEM CB1 protein levels in cultured neurons from WT and ASM-KO mice (***P = 0.0009, n = 5 independent cultures, Student's t-test). C. Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in cultured neurons from WT and ASM-KO mice. TOPRO stains cell nuclei. Graphs show mean ± SEM intensity associated with CB1 in the neuronal processes expressed in arbitrary units (left) or the Mander's coefficient for the co-localization of CB1 with Lamp1 (right; ****P < 0.0001, n = 3 independent cultures, > 30 neurons per culture, Student's t-test). Scale bar, 10 μm. D. Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in Purkinje cells of the cerebellum from WT and ASM-KO mice. Graph shows mean ± SEM Mander's coefficient indicative of the degree of co-localization of CB1 and Lamp1 (*P = 0.0213, n = 4 mice per group, Student's t-test). Scale bar, 5 μm. E, F. Graphs show mean ± SEM SM levels, expressed as percentage of WT values, in extracts containing the same amount of protein of cultured neurons from WT and ASM-KO mice (E) or from WT mice incubated or not with 40μΜ SM (F). (E: *P = 0.0405; F: **P = 0.0100, n = 2 independent cultures, Student's t-test). G. Mean ± SEM CB1 mRNA levels in cultured neurons from WT mice incubated or not with 40 μM SM (**P = 0.0021, n = 5 independent cultures, Student's t-test). H. Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cultured neurons from WT mice incubated or not with 40 μΜ SM (n = 2 independent cultures, Student's t-test) I. Immunofluorescence images against CB1 and Lamp1 in cultured neurons from WT mice incubated or not with 40 μΜ SM. Graphs show mean ± SEM intensity associated with CB1 in the neuronal processes expressed in arbitrary units (left) or the Mander's coefficient for the co-localization of CB1 with Lamp1 (right; ***P < 0.0001, n = 3 independent cultures, > 30 neurons per culture, Student's t-test). Scale bar, 10 μm. J. Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cultured neurons from WT mice treated with vehicle (veh), with 40 μM SM (SM), with 0.1 μM bafilomycin (BafA1), or with 40 μM SM and 0.1 μM bafilomycin (SM + BafA1). Graph shows mean ± SEM CB1 levels normalized to GAPDH in arbitrary units (n = 2 independent cultures, one-way ANOVA). K. Mean ± SEM SM levels in cultured neurons from ASM-KO mice incubated or not with exogenous SMase (*P = 0.0157, n = 3 independent cultures, Student's t-test). L. Mean ± SEM CB1 mRNA levels in cultured neurons from ASM-KO mice incubated or not with exogenous SMase (**P = 0.0015, n = 5 independent cultures, Student's t-test). M. Western blot against CB1 and GAPDH (used as loading control). Images belong to the same blot but to non-consecutive lanes as indicated by the boxes. Graph showing mean ± SEM CB1 protein levels normalized to GAPDH in extracts from cultured neurons from ASM-KO mice incubated or not with exogenous SMase (n = 5 independent cultures, Student's t-test). N. Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in cultured neurons from ASM-KO mice incubated or not with exogenous SMase. DAPI stains cell nuclei. Graph shows mean ± SEM Mander's coefficient for the co-localization of CB1 with Lamp1 (*P = 0.0189, n = 3 independent cultures, > 30 neurons per culture, Student's t-test). Scale bar, 10 μm. Source data are available online for this figure. Source Data for Figure 2 [emmm201911776-sup-0003-SDataFig2.xlsx] Download figure Download PowerPoint Sphingomyelin accumulation is a key pathological hallmark in ASM-KO neurons, which we confirmed in our hippocampal neuronal cultures (Fig 2E). To determine whether this was responsible for the aforementioned alterations in CB1 expression, cultured WT neurons were incubated with 40 μM SM for 48 h. These conditions increased SM to levels similar to those found in ASM-KO neurons (Fig 2F). SM addition produced a 60% decrease in CB1 mRNA, as measured by RT-qPCR (Fig 2G). CB1 protein levels were reduced by 10% as determined by Western blot, although this value was not statistically significant (Fig 2H). Immunofluorescence analysis in the cultured WT neurons incubated with SM showed CB1 protein reduction from the neuronal processes (by 58%) and increased localization of the receptor in the lysosomes (Mander's coefficient raised from 0.15 to 0.26) (Fig 2I). To determine whether increased co-localization of CB1 in lysosomes could be responsible for its overall low levels due to increased degradation, we measured the amount of the protein in WT neurons incubated or not with 40 μM SM for 48 h in the presence or absence of the lysosomal inhibitor bafilomycin. This drug prevented by 61% the SM-induced CB1 reduction (Fig 2J). In a set of complementary experiments to those of SM addition to WT neurons, we diminished SM levels in cultured ASM-KO neurons by exogenous addition of sphingomyelinase. This treatment, which lowered SM levels by 46% (Fig 2K), resulted in a 55% increase in CB1 mRNA (Fig 2L). CB1 protein levels also increased by 45%, although this value was not statistically significant (Fig 2M). Immunofluorescence analyses showed the decreased co-localization of CB1 and Lamp1 upon sphingomyelinase treatment in the ASM-KO neurons (Mander's coefficient 0.26 in non-treated cells and 0.15 in sphingomyelinase-treated cells; Fig 2N). This treatment induced reductions on the SM amount and CB1 mRNA levels, although not statistically significant, and no evident effect on CB1 protein levels in WT neurons (Appendix Fig S4). Altogether, these data show that reduced expression of CB1 in ASM-KO neurons correlated with elevated SM and increased delivery of CB1 to lysosomes, leading to its degradation. CB1 expression and localization could be recovered in ASM-KO neurons by reducing SM levels. eCB signaling reduces SM levels in cultured ASM-KO neurons The reduced levels of CB1 observed in ASM-KO neurons, especially evident in the neuronal processes, and the reported ability of this receptor to activate NSM and hydrolyze SM in astrocytes (Sanchez et al, 2001) prompted us to assess the therapeutic potential of enhancing eCB action in ASMD. To this end, we first treated cultured ASM-KO neurons with different doses of AEA. This treatment reduced SM levels in a dose-dependent manner (Fig 3A). To determine whether NSM was involved in this effect, we treated ASM-KO neurons with AEA in the presence or absence of the NSM inhibitor GW4869. This compound blocked the effect of AEA on SM levels (Fig 3B). Also consistent with the contribution of NSM, we observed increased levels of this SM-degrading enzyme upon AEA treatment (Fig 3C). This treatment did not have significant effects on CB1 levels as assessed by Western blot (Appendix Fig S5A). Altogether, these observations point to the enhancement of eCB signaling as a potential intervention to increase NSM and reduce SM levels in neurons of ASM-KO mice and ASMD patients. Figure 3. eCB enhancement reduces SM levels in cultured ASM-KO neurons A. Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle or with the indicated concentrations of AEA (***P < 0.0001, n = 3 independent cultures, one-way ANOVA, Bonferroni post hoc). B. Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, the inhibitor of NSM GW, AEA, or the combination of GW and AEA (***P < 0.0001, n = 3 independent cultures, one-way ANOVA, Bonferroni post hoc). C. Western blot against NSM and GAPDH (used as loading control) and graph showing mean ± SEM NSM protein levels in extracts from cultured neurons treated with vehicle or with 50 μM AEA (*P = 0.0230, n = 6 independent cultures, Student's t-test). D. Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, or with JNJ, PF, or URB in the presence or absence of AEA (**PJNJ = 0.0013, *PPF = 0.0143, ***PURB = 0.0002, **PJNJ + AEA = 0.0069, **PPF + AEA = 0.0077, ***PURB + AEA < 0.0001, n = 3 independent cultures, one-way ANOVA, Bonferroni post hoc). E. Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, with PF, or with SR141716 + PF (*PPF = 0.0109, *PSR + PF = 0.0343, n = 3 independent cultures, one-way ANOVA, Bonferroni post hoc). F. Immunofluorescences against the dendritic marker MAP2 of cultured neurons from ASM-KO mice treated with vehicle, AEA, JNJ, PF, or URB. Scale bar, 50 μm. G. Mean ± SEM number of apoptotic cells measured by TUNEL assays in cultured neurons from ASM-KO mice treated with H2O2 (as positive control), vehicle, AEA, JNJ, PF, or URB (n = 3 independent cultures). Source data are available online for this figure. Source Data for Figure 3 [emmm201911776-sup-0004-SDataFig3.xlsx] Download figure Download PowerPoint To boost eCB signaling in disease settings, selective inhibition of eCB degradation is currently being considered as a better therapeutic avenue than direct CB1 receptor agonism since the former avoids the psychoactive side effects of the latter (Di Marzo, 2008; Pertwee, 2012). Thus, we tested the ability of three different FAAH inhibitors (FAAHi) to reduce SM levels in ASM-KO neuronal cultures. Among the different currently available FAAHi, JNJ-1661010 (JNJ), PF-04457845 (PF), and URB-597 (URB) were chosen because they can readily cross the blood–brain barrier and have shown good tolerability in preclinical and clinical studies (Ahn et al, 2009, 2011; D'Souza et al, 2019). All three FAAHi reduced SM levels, an" @default.
• W3089711134 created "2020-10-08" @default.
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• W3089711134 title "Inhibition of fatty acid amide hydrolase prevents pathology in neurovisceral acid sphingomyelinase deficiency by rescuing defective endocannabinoid signaling" @default.
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• W3089711134 doi "https://doi.org/10.15252/emmm.201911776" @default.
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