Journal: Molecular Medicine

Article Title: Progressive endocannabinoid system dysregulation in autosomal dominant polycystic kidney disease

doi: 10.1186/s10020-026-01457-w

Figure Lengend Snippet: ECS components are progressively dysregulated in human ADPKD kidney tissue. a Microarray analysis ( GSE7869 ) of human kidney tissue shows stepwise increases in CNR1 transcript from healthy cortex to minimally cystic (PKDm) and fully cystic (PKD) ADPKD tissue, with corresponding reductions in AEA-metabolizing enzymes NAPEPLD and FAAH . b Single-nucleus RNA-sequencing (snRNA-seq) analysis of human ADPKD kidneys ( n = 8) versus healthy controls ( n = 5) demonstrate consistent CNR1 upregulation and marked downregulation of NAPEPLD and FAAH , while 2-AG-metabolizing enzymes remain largely unchanged. c snRNA-seq analysis of diabetic kidney disease (DKD; n = 5 patients; controls n = 6) reveals minimal alterations in CNR1 and ECS-metabolizing enzymes. d Gene expression analysis by qPCR confirms CNR1 upregulation in human ADPKD kidney tissue ( n = 17) versus non-cystic nephrectomy controls ( n = 5), with concurrent changes in ECS enzyme transcription. e-i eCB quantification by liquid chromatography-tandem mass spectrometry (LC–MS/MS) reveals significant depletion of tissue anandamide (AEA); e , N -oleoylethanolamine (OEA); f , 2-arachidonoylglycerol (2-AG); h , and arachidonic acid (AA); i , while N -palmitoylethanolamine (PEA); g remains unchanged. j-l Western blot analysis shows substantial inter-individual variability in CB 1 R protein levels without significant difference between ADPKD ( n = 6) and control kidneys ( n = 4), but significant reductions in DAGLα/β, NAPEPLD, MGLL and FAAH protein. Western blots were normalized to total proteins. Data represent mean ± SEM. Statistics of control versus PKD represented by * and control versus PKDm by #. Statistical significance assessed by Mann–Whitney U test or unpaired t -test: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Sections were stained with rabbit anti-CB 1 R antibody (1:200, 10006590, Cayman) followed by a goat anti-rabbit HRP conjugate (ImmPRESSTM, Vector laboratories).

Techniques: Microarray, RNA Sequencing, Gene Expression, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Western Blot, Control, MANN-WHITNEY

Journal: Molecular Medicine

Article Title: Progressive endocannabinoid system dysregulation in autosomal dominant polycystic kidney disease

doi: 10.1186/s10020-026-01457-w

Figure Lengend Snippet: Temporal endocannabinoid system dysregulation during Pkd1 RC/RC disease progression. a-g Quantitative PCR analysis of ECS-related genes across disease stages (9 and 12 months) showing progressive upregulation of Cnr1 a and Cnr2 b transcripts, with corresponding changes in ECS metabolic enzymes: Dagla c , Daglb d , Napepld e , Mgll f , and Faah g . h-n Western blot analysis and quantification of ECS proteins across disease stages, demonstrating sustained CB 1 R protein elevation at 9 and 12 months h, i , stable 2-AG-related enzyme proteins (DAGLα, DAGLβ, MGLL; h, j, k, m ), and stage-specific changes in AEA-related enzymes including elevated NAPEPLD at 12 months ( h, l ) and reduced FAAH at 9 months ( h, n ). Data represent mean ± SEM from WT ( n = 5) and Pkd1 RC/RC ( n = 6–7) per group per timepoint. Statistical analysis: unpaired t -test comparing Pkd1 RC/RC to age-matched WT. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus WT

Article Snippet: Sections were stained with rabbit anti-CB 1 R antibody (1:200, 10006590, Cayman) followed by a goat anti-rabbit HRP conjugate (ImmPRESSTM, Vector laboratories).

Techniques: Biomarker Discovery, Real-time Polymerase Chain Reaction, Western Blot

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet:

Article Snippet: The rabbit polyclonal anti-GABA A α1 antibody (catalog #: PPS022) was purchased from R&D systems (Minneapolis, MN).

Techniques: Transfection, Construct, Control, Recombinant, Plasmid Preparation, Cloning, Software

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Changes of C3d/S100A10 levels, and pan-reactive and A1/A2 specific gene expression in reactive astrocytes after treatment with microglia-conditioned media (MCM) and P2Y 1 R-ANT. (a), (b). Representative images (a) and quantitative data (b) of western blots showing protein levels of GFAP, C3d, S100A10, and CSPG in non-OGD astrocytes, OGD astrocytes, OGD astrocytes treated with MCM, OGD astrocytes treated with P2Y 1 R-ANT, and OGD astrocytes treated with P2Y 1 R-ANT and MCM. β-actin was used as an internal control. * P < 0.05 vs. non-OGD astrocyte, # P < 0.05 vs. OGD astrocytes. (c). Venn diagram of mRNAs with upregulated (fold change ≥ 1.5) and downregulated (fold change ≤ 0.67) expressions in OGD astrocytes, OGD astrocytes treated with P2Y 1 R-ANT (1 mM), and OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM. (d). Heatmap of the entire mRNA expression in OGD astrocytes, OGD astrocytes treated with P2Y 1 R-ANT (1 mM), and OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM. (e). Heatmaps comparing the mean expression of pan-reactive, A1-specific, and A2 specific genes in OGD astrocytes; OGD astrocytes treated with P2Y 1 R-ANT (1 mM); and OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM. N = 4/group. Values are the mean ± SD. GFAP = glial fibrillary acidic protein, CSPG = chondroitin sulfate proteoglycans, OGD = oxygen–glucose deprivation, P2Y 1 R-ANT = P2Y 1 receptor antagonist, MCM = microglial conditioned medium

Article Snippet: The primary antibodies used in this study were goat polyclonal anti-GFAP (1:40,000; Abcam), goat polyclonal anti-C3d (a marker for A1 astrocytes) (1:2,000, R&D Systems), rabbit polyclonal anti-S100A10 (1:2,000, Proteintech), mouse monoclonal anti-CSPG (1:100,000, Abcam), mouse monoclonal anti-phosphorylated neurofilament heavy chain (pNFH) (1:500; BioLegend), rabbit polyclonal anti-P2Y 1 R-ANT (1:200; Alomone Labs), rabbit polyclonal anti-caspase-3 (1:500, Abcam), and rabbit monoclonal anti-actin (1:10,000, Abcam).

Techniques: Expressing, Western Blot, Control

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Change in inflammatory gene expression and pathway analysis. (a). Top 20 significant canonical pathways of the core analysis in IPA of most highly expressed genes in OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM relative to OGD astrocytes. Blue bars: negative z-score; orange bars: positive z-score; gray bars: no activity pattern available; white bars: activity of zero. (b), (c). Heatmap of mRNA-related Neuroinflammation Signaling expression, and quantitative analysis of representative mRNA-related Neuroinflammation Signaling in OGD astrocytes, OGD astrocytes treated with P2Y 1 R-ANT (1 mM), and OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM. * P < 0.05 vs. OGD astrocyte, # P < 0.05 vs. OGD astrocytes treated with P2Y 1 R-ANT. (d). Signaling pathway predicted by analyzing changes using IPA software in mRNA expression in the OGD astrocytes treated with P2Y 1 R-ANT and MCM relative to OGD astrocytes, or OGD astrocytes treated with P2Y 1 R-ANT. The functional networks were generated via IPA (QIAGEN Inc., https://www.qiagenbio-informatics.com/products/ingenuity-pathway-analysis ). N = 4/group. Values are the mean ± SD. IPA = Ingenuity Pathway Analysis, P2Y 1 R-ANT = P2Y 1 receptor antagonist, MCM = microglial conditioned medium, MAPK = mitogen-activated protein kinase, NF-κB = nuclear factor-κβ, TNF-α = tumor necrosis factor, IL-1β = interleukin-1β, NOX = nitrogen oxides, CASP8 = Caspase 8

Article Snippet: The primary antibodies used in this study were goat polyclonal anti-GFAP (1:40,000; Abcam), goat polyclonal anti-C3d (a marker for A1 astrocytes) (1:2,000, R&D Systems), rabbit polyclonal anti-S100A10 (1:2,000, Proteintech), mouse monoclonal anti-CSPG (1:100,000, Abcam), mouse monoclonal anti-phosphorylated neurofilament heavy chain (pNFH) (1:500; BioLegend), rabbit polyclonal anti-P2Y 1 R-ANT (1:200; Alomone Labs), rabbit polyclonal anti-caspase-3 (1:500, Abcam), and rabbit monoclonal anti-actin (1:10,000, Abcam).

Techniques: Expressing, Activity Assay, Software, Functional Assay, Generated

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Significance of AEVs derived from anti-inflammatory astrocytes to the peri-infarct area after MCAO. (a). The experimental scheme of isolating AEVs from OGD astrocytes treated with MCM and P2Y 1 R-ANT and their application for rats subjected to MCAO and cultured cortical neurons. (b), (c). Modified neurological severity score ( b ) and latency to fall off the rotarod at 56 days after MCAO ( c ) in PBS treatment, treatment with AEVs derived from OGD astrocytes (100 µg), and OGD astrocytes treated with MCM and P2Y 1 R-ANT (100 µg) in rats subjected to MCAO. N = 6–7/group. Values are the mean ± SD. (d), (e). Double immunofluorescent confocal images and quantitative data of the peri-infarct area at 56 days after MCAO with intracerebral administration of PBS, 100 µg AEVs derived from OGD astrocytes, and 100 µg AEVs derived from OGD astrocytes treated with MCM and P2Y 1 R-ANT, showing C3d + area (green) ( d ) and S100A10 + area (green) ( e ), and co-localized with GFAP + area (yellow). Merge ratio of C3d /GFAP ( d ), and S100A10/GFAP ( e ). N = 5/group (three sections per rat, and total of 15 samples in each group). Values are the mean ± SD. * P < 0.05 vs. PBS-treated rats, # P < 0.05 vs. rats treated with AEVs derived from OGD astrocytes. Scale bar = 100 μm AEVs = astrocytic extracellular vesicles, MCAO = middle cerebral artery occlusion, P2Y 1 R-ANT = P2Y 1 receptor antagonist, MCM = microglia-conditioned medium, GFAP = glial fibrillary acidic protein, OGD = oxygen–glucose deprivation

Article Snippet: The primary antibodies used in this study were goat polyclonal anti-GFAP (1:40,000; Abcam), goat polyclonal anti-C3d (a marker for A1 astrocytes) (1:2,000, R&D Systems), rabbit polyclonal anti-S100A10 (1:2,000, Proteintech), mouse monoclonal anti-CSPG (1:100,000, Abcam), mouse monoclonal anti-phosphorylated neurofilament heavy chain (pNFH) (1:500; BioLegend), rabbit polyclonal anti-P2Y 1 R-ANT (1:200; Alomone Labs), rabbit polyclonal anti-caspase-3 (1:500, Abcam), and rabbit monoclonal anti-actin (1:10,000, Abcam).

Techniques: Derivative Assay, Cell Culture, Modification

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Expression of microRNAs (miRNAs) in AEVs and inflammatory regulation in peri-infarct glial scars. (a). Heatmap of miRNA profiles on AEVs derived from OGD astrocytes and OGD astrocytes treated with P2Y 1 R-ANT and MCM. (b). Quantitative analysis of representative miRNAs related to ‘Inflammatory Response’ in AEVs derived from OGD astrocytes treated with P2Y 1 R-ANT and MCM, relative to AEVs derived from OGD astrocytes. N = 4/group. Values are the mean ± SD. * P < 0.05 vs. AEVs derived from OGD astrocytes. (c), (d). Double immunofluorescent confocal images and quantitative data of the peri-infarct area 56 days after MCAO with intracerebral administration of PBS, 100 µg AEVs derived from OGD astrocytes, and 100 µg AEVs derived from OGD astrocytes treated with MCM and P2Y 1 R-ANT, showing TNFα + area (green) ( c ) and NF-κB + area (green) ( d ), and co-localized with GFAP + area (yellow). N = 5/group (three sections per rat, and total of 15 samples in each group). Values are the mean ± SD. * P < 0.05 vs. PBS-treated rats. Scale bar = 100 μm AEVs = astrocytic extracellular vesicles, P2Y 1 R-ANT = P2Y 1 receptor antagonist

Article Snippet: The primary antibodies used in this study were goat polyclonal anti-GFAP (1:40,000; Abcam), goat polyclonal anti-C3d (a marker for A1 astrocytes) (1:2,000, R&D Systems), rabbit polyclonal anti-S100A10 (1:2,000, Proteintech), mouse monoclonal anti-CSPG (1:100,000, Abcam), mouse monoclonal anti-phosphorylated neurofilament heavy chain (pNFH) (1:500; BioLegend), rabbit polyclonal anti-P2Y 1 R-ANT (1:200; Alomone Labs), rabbit polyclonal anti-caspase-3 (1:500, Abcam), and rabbit monoclonal anti-actin (1:10,000, Abcam).

Techniques: Expressing, Derivative Assay

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Axonal outgrowth after AEV treatment and hindering by TNF-α (a), (b). Double immunofluorescent confocal images and quantitative data of the peri-infarct area 56 days after MCAO with intracerebral administration of PBS, 100 µg AEVs derived from OGD astrocytes, and 100 µg AEVs derived from OGD astrocytes treated with MCM and P2Y 1 R-ANT, showing pNFH + axons (green) ( a ) and MAP2 cells + (green) ( b ), with GFAP. N = 5/group (three sections per rat, and total of 15 samples in each group). Values are the mean ± SD. Scale bar = 100 μm. (c). Representative time-lapse microscopic images and quantitative data of primary cortical neurons in a microfluidic chamber showing axonal elongation (distance from yellow arrow to red arrow) in OGD neurons, OGD neurons treated with 1 ng/µl of TNF-α, and OGD neurons treated with 10 ng/µl of TNF-α. N = 3/group. Values are the mean ± SD. Scale bar = 20 μm. Quantitative data of axonal elongation per 30 min prior to 96 h after OGD. * P < 0.05, OGD neurons treated with 1 ng/µL of TNF-α vs. OGD neurons; # P < 0.05, OGD neurons treated with 10 ng/µL of TNF-α vs. OGD neurons; $ P < 0.05, OGD neurons treated with 10 ng/µL of TNF-α vs. OGD neurons treated with 1 ng/µL of TNF-α. AEVs = astrocytic extracellular vesicles, MCAO = middle cerebral artery occlusion, OGD = oxygen–glucose deprivation, P2Y 1 R-ANT = P2Y 1 receptor antagonist, MCM = microglia-conditioned medium, GFAP = glial fibrillary acidic protein, pNFH = phosphorylated neurofilament heavy chain

Article Snippet: The primary antibodies used in this study were goat polyclonal anti-GFAP (1:40,000; Abcam), goat polyclonal anti-C3d (a marker for A1 astrocytes) (1:2,000, R&D Systems), rabbit polyclonal anti-S100A10 (1:2,000, Proteintech), mouse monoclonal anti-CSPG (1:100,000, Abcam), mouse monoclonal anti-phosphorylated neurofilament heavy chain (pNFH) (1:500; BioLegend), rabbit polyclonal anti-P2Y 1 R-ANT (1:200; Alomone Labs), rabbit polyclonal anti-caspase-3 (1:500, Abcam), and rabbit monoclonal anti-actin (1:10,000, Abcam).

Techniques: Derivative Assay

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Diagram depicting the findings of the present study. Microglia and inhibition of P2Y 1 R regulate reactive astrocytes by transforming C3d/S100A10 expression and suppressing neuroinflammation. AEVs derived from reactive astrocytes with anti-inflammatory properties possessing miR-146a-5p regulate glial scars by suppressing NF-κB and TNF-α, which is permissive for axonal outgrowth and improves stroke recovery

Article Snippet: The primary antibodies used in this study were goat polyclonal anti-GFAP (1:40,000; Abcam), goat polyclonal anti-C3d (a marker for A1 astrocytes) (1:2,000, R&D Systems), rabbit polyclonal anti-S100A10 (1:2,000, Proteintech), mouse monoclonal anti-CSPG (1:100,000, Abcam), mouse monoclonal anti-phosphorylated neurofilament heavy chain (pNFH) (1:500; BioLegend), rabbit polyclonal anti-P2Y 1 R-ANT (1:200; Alomone Labs), rabbit polyclonal anti-caspase-3 (1:500, Abcam), and rabbit monoclonal anti-actin (1:10,000, Abcam).

Techniques: Inhibition, Expressing, Derivative Assay