Journal: International Journal of Molecular Sciences

Article Title: Endocannabinoid Enhancement via MAGL Inhibition in CDKL5 Deficiency: Selective Cellular Benefits and Domain-Specific Functional Effects in Adult Cdkl5 KO Mice

doi: 10.3390/ijms27062773

Figure Lengend Snippet: Experimental design and behavioral assessment following 6-week JZL184 treatment. ( A ) Experimental timeline showing the intermittent JZL184 treatment regimen (10 mg/kg Low dose [LD] or 20 mg/kg High dose [HD], i.p., three times per week as indicated by the triangles) administered for 6 weeks starting at 5 months of age. Behavioral testing was conducted during the final two weeks of treatment. ( B ) Body weight in grams measured at the end of the treatment period of vehicle-treated wild-type (+/Y n = 12), vehicle-treated Cdkl5 KO (−/Y n = 13) and JZL184-treated Cdkl5 KO mice (−/Y JZL LD n = 7; −/Y JZL HD n = 7). ( C ) Autistic-like features in treated Cdkl5 −/Y mice. Marble-burying performance, expressed as the number of marbles buried after 30 min in the same groups described in ( B ). ( D ) Total hindlimb clasping time measured during a 2-min tail suspension test in vehicle-treated Cdkl5 +/Y ( n = 12) and −/Y mice ( n = 12) as well as in JZL184-treated Cdkl5 −/Y mice receiving either a low dose (LD, n = 7) or a high dose (HD, n = 7). ( E ) Rotarod performance expressed as the frequency of passive rotations in vehicle-treated wild-type (+/Y n = 11), vehicle-treated Cdkl5 KO (−/Y n = 13) and JZL184-treated Cdkl5 KO mice (−/Y JZL LD n = 6; −/Y JZL HD n = 7). ( F ) Locomotor activity assessed in a 20-min open field test. The graph shows the average locomotion velocity in vehicle-treated wild-type (+/Y n = 12), vehicle-treated Cdkl5 KO (−/Y n = 13) and JZL184-treated Cdkl5 KO mice (−/Y JZL LD n = 6; −/Y JZL HD n = 7). ( G , H ) Time-binned analysis of locomotor velocity ( G ) and total distance traveled ( H ) during the 20-min open field session in mice as in ( F ). ( I ) Anxiety-like behavior was assessed based on the number of vertical jumps performed by mice during the open field test. ( J ) Cue-dependent freezing, expressed as the percentage of freezing time during fear conditioning, in vehicle-treated Cdkl5 +/Y ( n = 12) and −/Y ( n = 12) mice and in Cdkl5 −/Y mice treated with a high dose of JZL184 (HD, n = 7). Values are represented as means ± SEM. Statistical analyses were performed using one-way ANOVA ( B – F , I ) or two-way ANOVA ( G , H , J ), followed by Fisher’s LSD post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Starting at five months of age (adult stage), mice were treated intraperitoneally (i.p.) with either vehicle (5% DMSO, 40% PEG300, 5% Tween-80, and 50% ddH 2 O) or the monoacylglycerol lipase (MAGL) inhibitor JZL184 (10 or 20 mg/kg; Selleckchem, Munich, Germany) formulated in the same vehicle.

Techniques: Suspension, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Endocannabinoid Enhancement via MAGL Inhibition in CDKL5 Deficiency: Selective Cellular Benefits and Domain-Specific Functional Effects in Adult Cdkl5 KO Mice

doi: 10.3390/ijms27062773

Figure Lengend Snippet: Biochemical assessment of MAGL inhibition and downstream signaling. ( A ) Proposed model of the effects of 6-week JZL184-mediated MAGL inhibition on endocannabinoid signaling and downstream biological outcomes in Cdkl5 KO mice. Inhibition of MAGL increases 2-AG levels, thereby enhancing CB1R activation and promoting AKT phosphorylation. ( B ) Mass spectrometry-based biochemical analyses of plasma 2-AG levels were performed at the end of the 6-week treatment paradigm in vehicle-treated Cdkl5 +/Y ( n = 12) and −/Y ( n = 13) mice, as well as in JZL184-treated Cdkl5 −/Y mice receiving either a low dose (LD, n = 7) or a high dose (HD, n = 6). ( C , D ) Western blot analysis of MAGL, CB1R, CB2R, phospho-AKT (Ser473), total AKT, GAPDH and Vinculin protein expression in hippocampal extracts from mice as described in ( B ). Representative immunoblots from two animals per group are shown in panel ( C ). Histograms in ( D ) show quantification of phospho-AKT (Ser473) normalized to total AKT, total AKT and MAGL normalized to GAPDH, and CB1R and CB2R levels normalized to Vinculin. The data are shown as the mean ± SEM and are expressed as a percentage relative to the controls. Statistical significance was assessed using one-way ANOVA followed by Fisher’s LSD post hoc test. * p < 0.05, ** p < 0.01 and *** p < 0.001. Abbreviations: MAGL = Monoacylglycerol Lipase, 2-AG = 2-arachidonoylglycerol, AA = Arachidonic Acid, CB1R = Cannabinoid Receptor Type 1.

Article Snippet: Starting at five months of age (adult stage), mice were treated intraperitoneally (i.p.) with either vehicle (5% DMSO, 40% PEG300, 5% Tween-80, and 50% ddH 2 O) or the monoacylglycerol lipase (MAGL) inhibitor JZL184 (10 or 20 mg/kg; Selleckchem, Munich, Germany) formulated in the same vehicle.

Techniques: Inhibition, Activation Assay, Phospho-proteomics, Mass Spectrometry, Clinical Proteomics, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Endocannabinoid Enhancement via MAGL Inhibition in CDKL5 Deficiency: Selective Cellular Benefits and Domain-Specific Functional Effects in Adult Cdkl5 KO Mice

doi: 10.3390/ijms27062773

Figure Lengend Snippet: Analysis of dendritic spine morphology and hippocampal neuronal density. ( A ) Total dendritic spine density measured on Golgi-stained basal dendrites of pyramidal neurons located in layers II/III of the primary somatosensory cortex (S1) from vehicle-treated wild-type mice ( n = 6), vehicle-treated Cdkl5 KO mice ( n = 6), and Cdkl5 KO mice treated with JZL184 at the low dose (LD, 10 mg/kg n = 6) or high dose (HD, 20 mg/kg n = 6). ( B ) Quantification of dendritic spine morphology showing the relative proportion of immature (filopodia-like and thin) and mature (mushroom- and cup-shaped) spines expressed as a percentage of the total number of protrusions in mice as described in panel ( A ). ( C ) Representative Golgi-stained images of basal dendrites from cortical pyramidal neurons for each experimental group. Blue arrows indicate mature spines, whereas red arrows indicate immature spines. ( D ) Quantification of DAPI-positive nuclei in the CA1 pyramidal layer of the hippocampus from vehicle-treated Cdkl5 +/Y ( n = 6) and −/Y ( n = 6) mice, and JZL184-treated Cdkl5 −/Y mice receiving either a low dose (LD, n = 4) or a high dose (HD, n = 4). ( E ) Representative DAPI-stained hippocampal sections from one mouse per group. Data are expressed as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Fisher’s LSD post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Starting at five months of age (adult stage), mice were treated intraperitoneally (i.p.) with either vehicle (5% DMSO, 40% PEG300, 5% Tween-80, and 50% ddH 2 O) or the monoacylglycerol lipase (MAGL) inhibitor JZL184 (10 or 20 mg/kg; Selleckchem, Munich, Germany) formulated in the same vehicle.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Endocannabinoid Enhancement via MAGL Inhibition in CDKL5 Deficiency: Selective Cellular Benefits and Domain-Specific Functional Effects in Adult Cdkl5 KO Mice

doi: 10.3390/ijms27062773

Figure Lengend Snippet: Assessment of microglial activation following six-week JZL184 treatment. ( A ) Schematic overview illustrating the proposed impact of sustained MAGL inhibition by JZL184 on endocannabinoid signaling and downstream cellular responses. Elevated 2-AG levels may engage CB2 receptor–mediated signaling in microglia, thereby contributing to the attenuation of neuroinflammatory responses. ( B ) Mean cell body size of AIF-1-positive cells in the primary somatosensory cortex (S1) (on the left) and in hippocampal sections (in the middle) from vehicle-treated wild-type (+/Y n = 6), vehicle-treated Cdkl5 KO (−/Y n = 6) and JZL184-treated Cdkl5 KO mice (−/Y JZL LD n = 4; −/Y JZL HD n = 4). Histogram on the right shows microglial cell density in S1. ( C ) Representative immunofluorescence images of AIF-1–positive microglia in the primary somatosensory cortex (S1) across experimental groups. ( D ) Western blot analysis and densitometric quantification of AIF-1 protein levels in cortical tissue extracts from vehicle-treated wild-type (+/Y n = 11), vehicle-treated Cdkl5 KO (−/Y n = 14) and JZL184-treated Cdkl5 KO mice (−/Y JZL LD n = 7; −/Y JZL HD n = 7). Histogram shows the quantification of AIF-1 levels normalized to GAPDH. The data are shown as the mean ± SEM and are expressed as a percentage relative to the controls. Statistical analyses were performed using one-way ANOVA followed by Fisher’s LSD post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Starting at five months of age (adult stage), mice were treated intraperitoneally (i.p.) with either vehicle (5% DMSO, 40% PEG300, 5% Tween-80, and 50% ddH 2 O) or the monoacylglycerol lipase (MAGL) inhibitor JZL184 (10 or 20 mg/kg; Selleckchem, Munich, Germany) formulated in the same vehicle.

Techniques: Activation Assay, Inhibition, Immunofluorescence, Western Blot