Journal: The Journal of Biological Chemistry

Article Title: Lipid droplet targeting of the lipase coactivator ABHD5 and the fatty liver disease-causing variant PNPLA3 I148M is required to promote liver steatosis

doi: 10.1016/j.jbc.2025.108186

Figure Lengend Snippet: Mutations in C terminus of PNPLA3 I148M prevent localization to lipid droplets, the interaction with ABHD5, and LD accumulation . A , schematic of human PNPLA3. B , fluorescent imaging of COS-7 cells transfected with EYFP-tagged PNPLA3 WT or mutants. Cells were treated with 0.2 mM oleic acid overnight. Images are representative of results seen in three consecutive experiments. C , fluorescent imaging and FRET analysis shows EYFP-tagged PNPLA3 and ECFP-tagged ABHD5 localization and complementation in COS-7. Cells were treated with 0.2 mM oleic acid overnight. Images are representative of results seen in three consecutive experiments. D , Gluc protein complementation (PC) assay of HEK293A cells. Data are from five trials with four technical replicates per trial. Statistics calculated using Brown–Forsythe and Welch ANOVA test with Dunnett’s T3 multiple comparisons, ∗ p < 0.05. E , Gluc protein complementation (PC) assay of HEK293A cells. Data show that a 3 h 0.2 mM oleic acid treatment significantly increases PNPLA3 and ABHD5 interaction in both WT and I148M, but causes no significant changes in the mutants. Data are from three trials with four technical replicates per trial. Statistics calculated using two-way ANOVA with Sidak’s multiple comparisons, ∗ p < 0.05. F , graph showing blinded quantification of transfected cells with visible lipid droplets after fluorescent imaging of COS-7 cells transfected with EYFP-tagged PNPLA3 I148M or mutants, mCherry-tagged ABHD5, ECFP-tagged PNPLA2 WT or ATGL S47A , and PLIN5. Data are from three independent trials with six observations each. Statistics calculated using a One-way ANOVA with Sidak’s multiple comparisons, ∗ p < 0.05. The scale bar represents 10 μm. ABHD5, α/β hydrolase domain-containing protein 5; PNPLA3, patatin-like phospholipase domain-containing 3.

Article Snippet: AAV vectors for the expression of EGFP pENN.AAV.TBG.PI.EGFP, human PNPLA3 (hPNPLA3) pENN.AAV.TBG.PI.hPNPLA3 and human PNPLA3 I148M pENN.AAV.TBG.PI.hPNPLA3 I148M were produced as AAV8 serotype and obtained from Vector BioLabs.

Techniques: Imaging, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Lipid droplet targeting of the lipase coactivator ABHD5 and the fatty liver disease-causing variant PNPLA3 I148M is required to promote liver steatosis

doi: 10.1016/j.jbc.2025.108186

Figure Lengend Snippet: Lipid droplet/membrane targeting of ABHD5 is required to facilitate PNPLA3–ABHD5 interactions. A , fluorescent imaging and FRET analysis of COS-7 cells transfected with YFP-tagged mPNPLA3 (WT or I148M variant) or YFP-tagged G0S2, and ECFP-tagged mABHD5 (WT or R116N variant). Images are representative of results seen in four consecutive experiments. The scale bar represents 10 μm. B , Gluc protein complementation assay of HEK293A cells transfected with GlucN-tagged mPNPLA3 WT or mPNPLA3 I148M and GlucC-tagged mABHD5 WT or mABHD5 R116N. Data show that cytosolic ABHD5 significantly reduces PNPLA3 and ABHD5 complementation. Data represent averages of three trials with four technical replicates per trial. Statistics calculated using Brown–Forsythe and Welch ANOVA test with Dunnett’s T3 multiple comparisons, ∗∗∗∗ p < 0.001. ABHD5, α/β hydrolase domain-containing protein 5; PNPLA3, patatin-like phospholipase domain-containing 3.

Article Snippet: AAV vectors for the expression of EGFP pENN.AAV.TBG.PI.EGFP, human PNPLA3 (hPNPLA3) pENN.AAV.TBG.PI.hPNPLA3 and human PNPLA3 I148M pENN.AAV.TBG.PI.hPNPLA3 I148M were produced as AAV8 serotype and obtained from Vector BioLabs.

Techniques: Membrane, Imaging, Transfection, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Lipid droplet targeting of the lipase coactivator ABHD5 and the fatty liver disease-causing variant PNPLA3 I148M is required to promote liver steatosis

doi: 10.1016/j.jbc.2025.108186

Figure Lengend Snippet: Modeling interactions between PNPLA3 and LDs. A , the contact fraction between each PNPLA3 residue and various lipid components (POPC, DOPE, SAPI, and TAG). B , the conformation of PNPLA3 bound to the LD, with PNPLA3, phospholipids, and TAG depicted in green , cyan , and gray , respectively. The three regions (aa 80–88, 147–155, and 200–225), which form interactions with the LD, are highlighted in red . The C terminal of aa 369 to 428 and 433 to 481 is shown in blue . The helix of aa 345 to 368 and aa 370 to 373 is shown in orange and magenta , respectively. C , GaMD snapshot of WT PNPLA3 bound to the LD, I148, the 370 QRLV 373 motif, and the phospholipid interacting with R371, colored in magenta , purple , and cyan , respectively. DOPE, dioleoylphosphatidylethanolamine; LD, lipid droplet; POPC, palmitoyl-2-oleoyl-glycero-3-phosphocholine; PNPLA3, patatin-like phospholipase domain-containing 3; SAPI, 1-stearoyl-2-arachidonoyl-phosphatidylinositol; TAG, triacylglycerol.

Article Snippet: AAV vectors for the expression of EGFP pENN.AAV.TBG.PI.EGFP, human PNPLA3 (hPNPLA3) pENN.AAV.TBG.PI.hPNPLA3 and human PNPLA3 I148M pENN.AAV.TBG.PI.hPNPLA3 I148M were produced as AAV8 serotype and obtained from Vector BioLabs.

Techniques: Residue

Journal: The Journal of Biological Chemistry

Article Title: Lipid droplet targeting of the lipase coactivator ABHD5 and the fatty liver disease-causing variant PNPLA3 I148M is required to promote liver steatosis

doi: 10.1016/j.jbc.2025.108186

Figure Lengend Snippet: Fluorescence cross-correlation spectroscopy of ABHD5 with WT PNPLA3 and PNPLA3 I148M. FCCS was performed to analyze the codiffusion of ABHD5 and PNPLA3. The mean and SEM of the autocorrelations and cross-correlations for ( A ) ABHD5 and WT PNPLA3 and ( B ) ABHD5 and PNPLA3 I148M are shown. Larger amplitude correlations, greater similarity between correlation amplitudes, and faster cluster diffusion in the presence of the variant versus WT are significant. C , a greater fraction of ABHD5 and the variant were codiffusing than ABHD5 and WT PNPLA3 ( p = 0.00002). D , the multicolor clusters of ABHD5 and PNPLA3 demonstrated a slower diffusion time with WT PNPLA3 than the variant ( p = 0.002). C and D , noise within each collected scan and uncertainties of the fits resulted in a wide distribution of individual scan fit results ( symbols ). Boxplots show the median and extent of the data ( lines ). ABHD5, α/β hydrolase domain-containing protein 5; PNPLA, patatin-like phospholipase domain.

Article Snippet: AAV vectors for the expression of EGFP pENN.AAV.TBG.PI.EGFP, human PNPLA3 (hPNPLA3) pENN.AAV.TBG.PI.hPNPLA3 and human PNPLA3 I148M pENN.AAV.TBG.PI.hPNPLA3 I148M were produced as AAV8 serotype and obtained from Vector BioLabs.

Techniques: Fluorescence, Spectroscopy, Diffusion-based Assay, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Lipid droplet targeting of the lipase coactivator ABHD5 and the fatty liver disease-causing variant PNPLA3 I148M is required to promote liver steatosis

doi: 10.1016/j.jbc.2025.108186

Figure Lengend Snippet: ABHD5 interacts with multiple PNPLA family members. A , schematic of PNPLA4 fusions. B , fluorescent imaging of U2OS cells transfected with YFP-tagged PNPLA4, PNPLA3 WT, PNPLA3-PNPLA4 fusion, or PNPLA3-PNPLA4 fusion AAEE mutant to show lipid droplet targeting. Cells were treated with 0.2 mM oleic acid overnight and stained with Mitotracker Red and Lipi-Blue. Images are representative of results seen in three technical replicates of three experiments. C , fluorescent imaging and FRET analysis of COS-7 cells transfected with YFP-tagged PNPLA4, PNPLA3 WT, or PNPLA3 mutant and ECFP-tagged ABHD5 R229N. Cells were treated with 0.2 mM oleic acid overnight and stained with LipidTOX Neutral Red. Images are representative of results seen in three technical replicates of three consecutive experiments. D , Gluc protein complementation assay of HEK293A cells transfected with various PNPLA family proteins tagged with GlucN and ABHD5-GlucC. Cells were treated with 0.2 mM oleic acid in 10% BSA or 10% BSA alone overnight. Data show that oleic acid treatment significantly alters the interaction of ABHD5 with PNPLA3 WT, PNPLA3 I148M, and PNPLA5. Data represent averages of three trials with four technical replicates per trial. Statistics calculated using two-way ANOVA with Sidak’s multiple comparison test, ∗ p < 0.05. The scale bar represents 10 μm. ABHD5, α/β hydrolase domain-containing protein 5; BSA, bovine serum albumin; PNPLA, patatin-like phospholipase domain.

Article Snippet: AAV vectors for the expression of EGFP pENN.AAV.TBG.PI.EGFP, human PNPLA3 (hPNPLA3) pENN.AAV.TBG.PI.hPNPLA3 and human PNPLA3 I148M pENN.AAV.TBG.PI.hPNPLA3 I148M were produced as AAV8 serotype and obtained from Vector BioLabs.

Techniques: Imaging, Transfection, Mutagenesis, Staining, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Lipid droplet targeting of the lipase coactivator ABHD5 and the fatty liver disease-causing variant PNPLA3 I148M is required to promote liver steatosis

doi: 10.1016/j.jbc.2025.108186

Figure Lengend Snippet: PNPLA3 I148M targeting to LDs is required to promote liver steatosis. A , liver TAGs were quantified from PNPLA3-KO mice with liver expression of GFP, WT PNPLA3 (WT), PNPLA3 I148M (I148M), or PNPLA3 I148M AAEE (AAEE). B , representative Oil Red O staining of frozen liver sections from mice in A . C , livers from mice in A were fractionated into cytosol, mitochondria/membrane (Mito/MB), and lipid droplets (LDs) and the fractions were blotted for hPNPLA3, mPLIN2, mMek1/2, mAIF, and mABHD5. Fractionation data are representative from three independent experiments. ∗ p < 0.05 as determined by one-way ANOVA with Sidak’s multiple comparison test. The scale bar represents 200 μm. PNPLA, patatin-like phospholipase domain; TAG, triacylglycerol.

Article Snippet: AAV vectors for the expression of EGFP pENN.AAV.TBG.PI.EGFP, human PNPLA3 (hPNPLA3) pENN.AAV.TBG.PI.hPNPLA3 and human PNPLA3 I148M pENN.AAV.TBG.PI.hPNPLA3 I148M were produced as AAV8 serotype and obtained from Vector BioLabs.

Techniques: Expressing, Staining, Membrane, Fractionation, Comparison

Journal: bioRxiv

Article Title: Adolescent Alcohol Exposure Disrupts Astrocyte-Synaptic Structural And Functional Coupling In The Male Dorsal Hippocampus

doi: 10.1101/2025.10.14.682478

Figure Lengend Snippet: a. Experimental design. Schematic representation of intracranial injection of the astrocyte-specific Ca 2+ sensor (AAV5.GfaABC1D.cyto.GCaMP6f) into the dHipp on PND 26. Beginning at PND 30, animals received AIE or H 2 O intermittently through PND 45. Brains were collected on PND 72, and acute hippocampal slices were prepared for physiology. A bipolar stimulating electrode was placed in CA1 region of the Schaffer Collaterals to induce neuronal stimulation with simultaneous recording of astrocyte-specific Ca 2+ signaling within the CA1 dHipp. b. Representative images demonstrating confirmation of GCaMP6f (green) expression and specificity for astrocytes (GFAP, blue). Scale bar = 125 µm, 2 µm. c. Representative images of astrocyte Ca 2+ signaling before and after Schaffer Collateral stimulation following H 2 O and AIE, in adulthood. Scale bar = 10 µm. d Quantification of GCaMP6f signal shown as ΔF/F ratio. Data are presented as violin plots representing the distribution of individual data points. Analysis: unpaired t-tests. n = 6-10 astrocytes/animal, 6 animals/treatment group.

Article Snippet: HEK 293T/17 cells (cat #CRL-11268; ATCC, Manassas, VA) were grown to ∼90% confluency in 150 mm tissue-culture treated dishes (cat # 229650; CELLTREAT, Pepperell, MA) and co-transfected with 68.6 ug pAd-DeltaF (Addgene #112867; RRID: Addgene_112867; gift from James M. Wilson), 137 ug pUCmini-iCAP- PHP.eB (Addgene # 103005; RRID: Addgene_103005 ) and 34.4 ug PhP.eB::AAV- GfaABC1D-GRAB_Ado1.0 (Addgene # 153288; RRID:Addgene_153288 ) or AAV gfaABC1D Lck-GFP (Addgene # 105598; RRID:Addgene_105598 ).

Techniques: Injection, Expressing

Journal: bioRxiv

Article Title: Adolescent Alcohol Exposure Disrupts Astrocyte-Synaptic Structural And Functional Coupling In The Male Dorsal Hippocampus

doi: 10.1101/2025.10.14.682478

Figure Lengend Snippet: a. Schematic representation of intracranial injection of the astrocyte-specific extracellular glutamate release sensor (AAV5.GFAP.iGluSnFr.WPRE.SV40) in the dHipp on PND 26. Beginning at PND 30, animals received AIE or H 2 O intermittently through PND 45. Brains were collected on PND 72 and prepared for acute slice physiology. A bipolar stimulating electrode was placed in CA1 region of the Schaffer Collaterals to induce neuronal stimulation with simultaneous recording of astrocyte-membrane specific glutamate signaling within the CA1 dHipp. b. Representative trace of AIE and control extracellular glutamate release following single neuronal stimulation. c. Representative images demonstrating confirmation of iGluSnFR (green) expression and specificity for astrocytes (GFAP, blue) within the dHipp. Scale bar =125 µm, 2 µm. d. Representative images of iGluSnFR in astrocytes before and after neuronal stimulation. Scale bar = 10 µm. e. Quantification of ΔF/F ratio prior to neuronal stimulation. f-g . Quantification of ΔF/F ratio and signal decay rate following neuronal stimulation. h. Schematic representation of intracranial injection of the astrocyte membrane-specific adeno-associated virus (AAV5.gfaABC1D.Lck.GFP) in the dHipp on PND 26. i. Representative images of astrocyte surface rendering (Lck-GFP, green), mGluR2/3 (magenta), and mGluR5 (blue) in the adult dHipp following AIE. Scale bar= 20 µm, 5 µm. j-m . Quantification of membrane-bound glutamate receptor and transporter expression. Data are presented as violin plots representing the distribution of individual data points. Analysis: unpaired t-tests, n = 6-10 astrocytes/animal, 6-8 x 80 µm image stacks/slide, 2 slices/animal, 6 animals/treatment group.

Article Snippet: HEK 293T/17 cells (cat #CRL-11268; ATCC, Manassas, VA) were grown to ∼90% confluency in 150 mm tissue-culture treated dishes (cat # 229650; CELLTREAT, Pepperell, MA) and co-transfected with 68.6 ug pAd-DeltaF (Addgene #112867; RRID: Addgene_112867; gift from James M. Wilson), 137 ug pUCmini-iCAP- PHP.eB (Addgene # 103005; RRID: Addgene_103005 ) and 34.4 ug PhP.eB::AAV- GfaABC1D-GRAB_Ado1.0 (Addgene # 153288; RRID:Addgene_153288 ) or AAV gfaABC1D Lck-GFP (Addgene # 105598; RRID:Addgene_105598 ).

Techniques: Injection, Membrane, Control, Expressing, Virus

Journal: bioRxiv

Article Title: Adolescent Alcohol Exposure Disrupts Astrocyte-Synaptic Structural And Functional Coupling In The Male Dorsal Hippocampus

doi: 10.1101/2025.10.14.682478

Figure Lengend Snippet: a. Left, Schematic representation of intracranial injection of the astrocyte membrane-specific adeno-associated virus (AAV5.gfaABC1D.Lck.GFP) in the dHipp on PND 26. Beginning at PND 30, animals received AIE or H 2 O intermittently through PND 45. Brains were collected on PND 72 and prepared for acute slice physiology. Right, Representative image of astrocyte-specific AAV (Lck-GFP, green) and astrocyte excitation of GFP in the CA1 dHipp. b. A bipolar stimulating electrode was placed in CA1 region of the Schaffer Collaterals to induce neuronal stimulation and measure changes in astrocyte membrane potential following exogenous glutamate release (glutamate puff; 100 µM) in the CA1 dHipp. c. Representative trace of membrane potential following 100 uM glutamate puff for 300 ms. d. Quantification of astrocyte peak current amplitude. Analysis: unpaired t-tests, n= 6-10 astrocytes/animal, 5 animals/treatment group.

Article Snippet: HEK 293T/17 cells (cat #CRL-11268; ATCC, Manassas, VA) were grown to ∼90% confluency in 150 mm tissue-culture treated dishes (cat # 229650; CELLTREAT, Pepperell, MA) and co-transfected with 68.6 ug pAd-DeltaF (Addgene #112867; RRID: Addgene_112867; gift from James M. Wilson), 137 ug pUCmini-iCAP- PHP.eB (Addgene # 103005; RRID: Addgene_103005 ) and 34.4 ug PhP.eB::AAV- GfaABC1D-GRAB_Ado1.0 (Addgene # 153288; RRID:Addgene_153288 ) or AAV gfaABC1D Lck-GFP (Addgene # 105598; RRID:Addgene_105598 ).

Techniques: Injection, Membrane, Virus

Journal: bioRxiv

Article Title: Adolescent Alcohol Exposure Disrupts Astrocyte-Synaptic Structural And Functional Coupling In The Male Dorsal Hippocampus

doi: 10.1101/2025.10.14.682478

Figure Lengend Snippet: a. Schematic representation of intracranial injection of the astrocyte membrane-specific adeno-associated virus (AAV5.GFAP.hM3D(Gq).mCherry + AAVPHP.eB.GfaABC1D.GRAB_Ado1) and a fiber implant in the dHipp on PND 26. Beginning at PND 30, animals received AIE or H 2 O intermittently through PND 45. Following 26 day forced abstinence period, animals received saline or clozapine-N-oxide (CNO, 2 mg/kg, i.p.) to activate astrocytes. After 30 minutes, animals were then assessed for cognitive performance in a contextual fear conditioning paradigm along with real-time glutamate or adenosine availability measurements. b. Representative images of the dHipp, hM3DGq (magenta), GRAB_Ado1.0 (green), GFAP (blue), and colocalization of GRAB_Ado1.0+hM3DGq confirming astrocyte specificity of AAV and fiber photometry representative trace across day 1 of fear acquisition. c. Quantification of the adenosine ΔF/F ratio before and after foot shock: Pre-FS: (# vs. &; p= 0.0499; $ vs. @; p= 0.0103) Post FS1-3: ($ vs. @; p= 0.0103; & vs. @; p= 0.0041). d. Quantification of ΔF/F ratio collapsed across the session. e. Scatterplot and trendlines comparing adenosine ΔF/F with freezing behavior (H 2 O+SAL, β= 0.007, SE = 0.003, p = 0.015, R 2 =0.486; AIE+SAL, β=-0.013, SE = 0.004, p < 0.001, R 2 =0.268; H 2 O+CNO, β= -0.005, SE = 0.004, p = 0.012 , R 2 =-0.029; AIE+CNO, β = -0.004, SE = 0.003, p = 0.120, R 2 =-0.044). f. Schematic representation of intracranial injection of the astrocyte membrane-specific adeno-associated virus (AAV5.GFAP.hM3D(Gq).mCherry + AAV5.hSyn.iGluSnFr.WPRE.SV40) g. Representative images of the dHipp, hM3DGq (magenta), iGluSnFr (green), GFAP (blue), and localization of iGluSnFr and hM3DGq within a dHipp astrocyte and neuron. h. Quantification of glutamate availability (ΔF/F ratio) before and after foot shock (&; p=0.0166 ). i. Quantification of the glutamate availability (ΔF/F ratio) collapsed across session ( all p’s=0.4147-0.9998 ). j. Scatterplot and trendlines comparing glutamate availability ΔF/F and freezing behavior (H 2 O+SAL, β= -0.008, SE = 0.0024, p = 0.001, R 2 =0.319; H 2 O+CNO, β= 0.005, SE = 0.004, p = 0.229, R 2 =0.034; AIE+SAL, β =0.008, SE = 0.004, p = 0.047, R 2 =0.015; AIE+CNO, β =0.006, SE = 0.225, p = 0.066, R 2 =0.020). Analysis: Repeated measures Two-way ANOVA and Linear Regression Analysis, n = 6 animals/treatment group. ( p < 0.05* p < 0.001**)

Article Snippet: HEK 293T/17 cells (cat #CRL-11268; ATCC, Manassas, VA) were grown to ∼90% confluency in 150 mm tissue-culture treated dishes (cat # 229650; CELLTREAT, Pepperell, MA) and co-transfected with 68.6 ug pAd-DeltaF (Addgene #112867; RRID: Addgene_112867; gift from James M. Wilson), 137 ug pUCmini-iCAP- PHP.eB (Addgene # 103005; RRID: Addgene_103005 ) and 34.4 ug PhP.eB::AAV- GfaABC1D-GRAB_Ado1.0 (Addgene # 153288; RRID:Addgene_153288 ) or AAV gfaABC1D Lck-GFP (Addgene # 105598; RRID:Addgene_105598 ).

Techniques: Injection, Membrane, Virus, Saline

Journal: Cell reports

Article Title: Hepatocyte membrane potential regulates serum insulin and insulin sensitivity by altering hepatic GABA release

doi: 10.1016/j.celrep.2021.109298

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: TBG driven PSEM89S ligand depolarizing channel, cre-independent virus , Penn Vector Core , AAV8.TBG PI.PSAM(Y115F,L141F)a75HT3-HC.IRES.GFP.WPRE.bGH (RRID: Addgene_32477).

Techniques: Virus, Plasmid Preparation, Control, Recombinant, Glucose Oxidase Assay, Enzyme-linked Immunosorbent Assay, Picogreen Assay, RNA Sequencing, Software