Journal: Redox Biology

Article Title: YAP1 preserves tubular mitochondrial quality control to mitigate diabetic kidney disease

doi: 10.1016/j.redox.2024.103435

Figure Lengend Snippet: Yes-associated protein 1 (YAP1) and mitochondrial quality control (MQC)-related mediators were disrupted in renal tubule cells from diabetic kidney. ( A, B ) Expression of YAP1 and MQC-related genes in tubulointerstitial compartment of subjects with diabetic kidney disease (DKD) in GSE104954. ( C ) The diagnostic performance of YAP1 and each individual MQC-related genes for DKD. ( D ) The diagnostic performance of a comprehensive predictive model consisted of YAP1 and MQC-related genes for DKD. ( E, F ) Representative Western blots and quantification of p-YAP1, YAP1, MQC-related mediators, and interleukin 6 (IL6) in HK-2 cells exposed to palmitic acid and high glucose (PA + HG). ( G ) Transmission electron microscopy images of mitochondria within HK-2 cell with or without PA + HG treatment. Bar = 200 nm. ( H ) Transcripts of YAP1 , MQC-related mediators, and proinflammatory genes were measured by quantitative real-time polymerase chain reaction. ( I, J ) Representative plots and quantification of flow cytometry analysis for JC-1 staining in HK-2 cells with or without PA + HG treatment. The JC-1 red/green fluorescence ratio was calculated to represent mitochondrial membrane potential. ( K, L ) Representative plots and quantification of flow cytometry analysis for MitoSox Red staining in HK-2 cells with or without PA + HG treatment. The mean fluorescence intensity of MitoSox Red was calculated to represent mitochondrial reactive oxygen species level. Data are expressed as the mean ± SEM. All experiments were repeated at least three times. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. PGC1ɑ, peroxisome proliferator-activated receptor γ coactivator α; TFAM, mitochondrial transcription factor A; TOMM20, translocase of the outer mitochondrial membrane complex subunit 20; PINK1, tensin homolog induced kinase 1; LC3B, microtubule-associated protein 1 light chain 3B; p62, ubiquitin-binding protein sequestosome 1; IL8, interleukin 8; p-YAP1, phosphorylated YAP1.

Article Snippet: Renal tubule cell-specific Yap1 knockout mice (TKO, KspCre / Yap1 flox/flox ) and their control counterparts lacking the KspCre allele (CTL, WT/Yap1 flox/flox ) were bred at the Shanghai Model Organisms Center in China.

Techniques: Control, Expressing, Diagnostic Assay, Western Blot, Transmission Assay, Electron Microscopy, Real-time Polymerase Chain Reaction, Flow Cytometry, Staining, Fluorescence, Membrane, Ubiquitin Proteomics, Binding Assay

Journal: Redox Biology

Article Title: YAP1 preserves tubular mitochondrial quality control to mitigate diabetic kidney disease

doi: 10.1016/j.redox.2024.103435

Figure Lengend Snippet: Hippo signaling pathway was activated in tubule cells under the context of diabetic kidney disease (DKD). ( A ) Representative images of the immunofluorescence staining for yes-associated protein 1 (YAP1) in cultured HK-2 cells with or without palmitic acid and high glucose (PA + HG) treatment. Bar = 50 μm. ( B , C ) Representative Western blots and quantification of YAP1 in cytoplasmic and nuclear fraction of HK-2 cells exposed to PA + HG. ( D ) Representative images of immunohistochemistry staining for YAP1 and p-YAP1 in kidney sections from control donors, early-stage DKD patients, and late-stage DKD patients. Bar = 50 μm. ( E , F ) Quantitative assessment for percentage of YAP1 and p-YAP1 expression (n = 8 for each group). ( G ) Transcripts of genes involved in Hippo signaling pathway were measured by quantitative real-time polymerase chain reaction in cultured HK-2 cells with or without PA + HG treatment. ( H , I ) Representative Western blots and quantification of key molecules involved in Hippo signaling pathway in HK-2 cells exposed to PA + HG. ( J ) Schematic diagram showing the activation of Hippo signaling pathway and the ultimately degraded YAP1 in tubule cells under the context of DKD. Data are expressed as the mean ± SEM. All experiments were repeated at least three times. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. MST1, serine/threonine kinase 4; MST2, serine/threonine kinase 3; p-MST1/2, phosphorylated MST1/2; MOB1, MOB kinase activator 1; p-MOB1, phosphorylated MOB1; LATS1, large tumor suppressor kinase 1; LATS2, large tumor suppressor kinase 2; p-LATS1/2, phosphorylated LATS1/2; TAZ, Tafazzin; TEAD, TEA domain transcription factor; DAPI, 4′,6-diamidino-2-phenylindole; p-YAP1, phosphorylated YAP1.

Article Snippet: Renal tubule cell-specific Yap1 knockout mice (TKO, KspCre / Yap1 flox/flox ) and their control counterparts lacking the KspCre allele (CTL, WT/Yap1 flox/flox ) were bred at the Shanghai Model Organisms Center in China.

Techniques: Immunofluorescence, Staining, Cell Culture, Western Blot, Immunohistochemistry, Control, Expressing, Real-time Polymerase Chain Reaction, Activation Assay

Journal: Redox Biology

Article Title: YAP1 preserves tubular mitochondrial quality control to mitigate diabetic kidney disease

doi: 10.1016/j.redox.2024.103435

Figure Lengend Snippet: Renal tubule cell-specific yes-associated protein 1 (Yap1) deletion aggregated kidney injury via disrupting mitochondrial quality control (MQC) in diabetic kidney disease (DKD) mice. ( A ) The schematic diagram of in vivo experiment. ( B–I ) Body weight, kidney weight/body weight, blood glucose, serum triglyceride, cholesterol, low-density lipoprotein cholesterol (LDL-c), and creatinine and urinary albumin-to-creatinine ratio (UACR) in the indicated mouse groups. ( J ) Representative images of histologic images of Periodic acid-Schiff (PAS) staining, Masson's trichrome (MASSON) staining, as well as immunohistochemistry staining for kidney injury molecule-1 (KIM1) and ɑ-smooth muscle actin (ɑ-SMA) in kidney sections. Black arrows indicated expanded mesangial expansion and tubular atrophy, dilation, or brush border loss. Bar = 50 μm. ( K-M ) Quantitative assessment for tubular injury score, percentage of collagen staining and percentage of KIM1 expression. ( N, O ) Representative Western blots and quantification of p-YAP1, YAP1, MQC-related mediators, and interleukin 6 (IL6) in renal cortex from indicated mouse groups. ( P ) Representative transmission electron microscopy images of mitochondria with abnormal morphology in tubule cells from indicated mouse groups. Damaged mitochondria were observed with matrix swelling and collapsed cristae. An early phase of mitophagy was seen as the formation of double membrane, wrapped around a mitochondrion. The red arrowheads indicate autophagic vacuoles containing damaged mitochondria, or autophagosomes surrounded by a double-membrane with undigested damaged mitochondria inside. Bar = 1 μm. Mt, mitochondrion; Av, autophagic vacuole; N, nucleus. Data are expressed as the mean ± SEM. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 (n = 6 for each group). ND, normal diet; HFD, high-fat diet; STZ, streptozotocin; TKO, tubule cell-specific knockout; Veh, vehicle; PGC1ɑ, peroxisome proliferator-activated receptor γ coactivator α; TFAM, mitochondrial transcription factor A; TOMM20, translocase of the outer mitochondrial membrane complex subunit 20; PINK1, tensin homolog induced kinase 1; p62, ubiquitin-binding protein sequestosome 1; LC3B, microtubule-associated protein 1 light chain 3B; p-YAP1, phosphorylated YAP1.

Article Snippet: Renal tubule cell-specific Yap1 knockout mice (TKO, KspCre / Yap1 flox/flox ) and their control counterparts lacking the KspCre allele (CTL, WT/Yap1 flox/flox ) were bred at the Shanghai Model Organisms Center in China.

Techniques: Control, In Vivo, Staining, Immunohistochemistry, Expressing, Western Blot, Transmission Assay, Electron Microscopy, Membrane, Knock-Out, Ubiquitin Proteomics, Binding Assay

Journal: Redox Biology

Article Title: YAP1 preserves tubular mitochondrial quality control to mitigate diabetic kidney disease

doi: 10.1016/j.redox.2024.103435

Figure Lengend Snippet: Renal tubule cells-specific yes-associated protein 1 (Yap1) deletion boosted CXCL1 secretion and affected macrophage polarization in diabetic kidney. ( A ) Overlapped differentially expressed genes (DEGs) recognized among GSE104954, GSE175759 and GSE30529. ( B ) Gene ontology enrichment analyses of overlapped DEGs. ( C ) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of overlapped DEGs. ( D ) Renal expression of chemokines C-X-C motif chemokine ligand 1 (CXCL1), C–C motif chemokine ligand (CCL19) and CXCL6 in 3 selected GSE datasets (GSE30529, GSE104954, and GSE175759). ( E ) Transcripts of CXCL1 , CCL19 and CXCL6 were measured by quantitative real-time polymerase chain reaction in cultured HK-2 cells with or without palmitic acid and high glucose treatment (PA + HG). ( F ) Representative images of the immunofluorescence staining for CXCL1 in cultured HK-2 cells with or without PA + HG treatment. Bar = 20 μm. ( G ) Representative images of the immunofluorescence staining for CXCL1 in kidney sections from indicated mouse groups. Bar = 15 μm. ( H ) Representative Western blots and quantification of CXCL1 in renal cortex from indicated mouse groups. ( I ) Representative images of the immunofluorescence staining for CD68 and CD206 in kidney sections from indicated mouse groups. Bar = 15 μm. Data are expressed as the mean ± SEM. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 (n = 6 for each group). ND, normal diet; HFD, high-fat diet; STZ, streptozotocin; TKO, tubule cell-specific knockout; Veh, vehicle; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Renal tubule cell-specific Yap1 knockout mice (TKO, KspCre / Yap1 flox/flox ) and their control counterparts lacking the KspCre allele (CTL, WT/Yap1 flox/flox ) were bred at the Shanghai Model Organisms Center in China.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Immunofluorescence, Staining, Western Blot, Knock-Out

Journal: Redox Biology

Article Title: YAP1 preserves tubular mitochondrial quality control to mitigate diabetic kidney disease

doi: 10.1016/j.redox.2024.103435

Figure Lengend Snippet: Yes-associated protein 1 (Yap1) activation counteracted diabetes-induced kidney injury via maintaining mitochondrial quality control (MQC) homeostasis in diabetic kidney. ( A ) The schematic diagram of in vivo experiment. ( B–I ) Body weight, kidney weight/body weight, blood glucose, serum triglyceride, cholesterol, low-density lipoprotein cholesterol (LDL-c), and creatinine and urinary albumin-to-creatinine ratio (UACR) in the indicated mouse groups. ( J ) Representative images of histologic images of Periodic acid-Schiff (PAS) staining, Masson's trichrome (MASSON) staining, as well as immunohistochemistry staining for kidney injury molecule-1 (KIM1) in kidney sections. Black arrows indicated expanded mesangial expansion and tubular atrophy, dilation, or brush border loss. Bar = 50 μm. ( K-M ) Quantitative assessment for tubular injury score, percentage of collagen staining and percentage of KIM1 expression. ( N, O ) Representative Western blots and quantification of p-YAP1, YAP1, MQC-related mediators, and interleukin 6 (IL6) in renal cortex from indicated mouse groups. ( P ) Representative images of the immunofluorescence staining for C-X-C motif chemokine ligand 1 (CXCL1) in kidney sections from indicated mouse groups. Bar = 15 μm. ( Q-R ) Representative Western blots and quantification of CXCL1 in renal cortex from indicated mouse groups. ( S ) Representative transmission electron microscopy images of mitochondria with abnormal morphology in tubule cells from indicated mouse groups. Damaged mitochondria were observed with matrix swelling and collapsed cristae. An early phase of mitophagy was seen as the formation of double membrane, wrapped around a mitochondrion. The red arrowheads indicate autophagic vacuoles containing damaged mitochondria, or autophagosomes surrounded by a double-membrane with undigested damaged mitochondria inside. Bar = 1 μm. Mt, mitochondrion; Av, autophagic vacuole; N, nucleus. Data are expressed as the mean ± SEM. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 (n = 6 for each group). HFD, high-fat diet; STZ, streptozotocin; DKD, diabetic kidney disease; Veh, vehicle; PGC1ɑ, peroxisome proliferator-activated receptor γ coactivator α; TFAM, mitochondrial transcription factor A; TOMM20, translocase of the outer mitochondrial membrane complex subunit 20; PINK1, tensin homolog induced kinase 1; LC3B, microtubule-associated protein 1 light chain 3B; p62, ubiquitin-binding protein sequestosome 1; DAPI, 4′,6-diamidino-2-phenylindole; p-YAP1, phosphorylated YAP1.

Article Snippet: Renal tubule cell-specific Yap1 knockout mice (TKO, KspCre / Yap1 flox/flox ) and their control counterparts lacking the KspCre allele (CTL, WT/Yap1 flox/flox ) were bred at the Shanghai Model Organisms Center in China.

Techniques: Activation Assay, Control, In Vivo, Staining, Immunohistochemistry, Expressing, Western Blot, Immunofluorescence, Transmission Assay, Electron Microscopy, Membrane, Ubiquitin Proteomics, Binding Assay

Journal: Redox Biology

Article Title: YAP1 preserves tubular mitochondrial quality control to mitigate diabetic kidney disease

doi: 10.1016/j.redox.2024.103435

Figure Lengend Snippet: Yes-associated protein 1 (YAP1) maintained mitochondrial quality control (MQC) homeostasis in cultured HK-2 cells. ( A, B ) Representative Western blots and quantification of p-YAP1, YAP1, MQC-related mediators, and interleukin 6 (IL6) in adenovirus (Ad)-infected HK-2 cells with or without palmitic acid and high glucose (PA + HG) treatment. ( C, D ) Representative plots and quantification of flow cytometry analysis for JC-1 staining in Ad-infected HK-2 cells with or without PA + HG treatment. The JC-1 red/green fluorescence ratio was calculated to represent mitochondrial membrane potential. ( E, F ) Representative plots and quantification of flow cytometry analysis for MitoSox Red staining in Ad-infected HK-2 cells with or without PA + HG treatment. The mean fluorescence intensity of MitoSox Red was calculated to represent mitochondrial reactive oxygen species level. ( G ) The relative mitochondrial DNA (mtDNA) copy numbers in Ad-infected HK-2 cells with or without PA + HG treatment were measured by qPCR. ( H ) Representative confocal images of mt-Keima in Ad-infected HK-2 cells with or without PA + HG treatment. Confocal microscopy was analyzed to detect the mt-Keima located in mitochondria (mitochondria at neutral pH, green fluorescence) and the mt-Keima delivered to lysosomes (mitochondria at acidic pH, red fluorescence). The zoom images were magnified from boxed areas in overlay images. Bar = 40 μm. ( I ) Quantification of mitophagy index by mt-Keima imaging. Mitophagy index was determined by analyzing the ratio of red/green fluorescence. ( J ) Real-time measurements of the oxygen consumption rate (OCR) in Ad-infected HK-2 cells with or without PA + HG treatment via Seahorse XF96. After basal OCR was obtained, oligomycin (1.5 μM) was added to obtain ATP-linked OCR. Then, the uncoupler FCCP (1 μM) was added to obtain maximal OCR. Finally, none-mitochondrial OCR was obtained after adding Antimycin A + rotenone (0.5 μM each) to inhibit the electron transport chain. Mitochondrial spare respiratory capacity was calculated by subtracting basal respiration from maximal respiratory capacity. Each point in the lines represents the average measurements of six different wells. ( K ) OCR results from the Seahorse analysis of the mitochondrial respiration parameters: basal respiration, spare respiratory capacity, maximal respiration, and ATP production. ( L ) Mitochondrial respiratory chain complex enzyme activities in Ad-infected HK-2 cells with or without PA + HG treatment. ( M ) ELISA of CXCL1 level for Ad-infected HK-2 cells with or without PA + HG treatment. ( N ) ELISA of CXCL1 level for Ad-infected HK-2 cells with or without mitophagy inhibitor Mdivi-1 treatment. Data are expressed as the mean ± SEM. All experiments were repeated at least three times. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. NC, negative control; PGC1ɑ, peroxisome proliferator-activated receptor γ coactivator α; TFAM, mitochondrial transcription factor A; TOMM20, translocase of the outer mitochondrial membrane complex subunit 20; PINK1, tensin homolog induced kinase 1; p62, ubiquitin-binding protein sequestosome 1; IL6, interleukin 6; CXCL1, C-X-C motif chemokine ligand 1; p-YAP1, phosphorylated YAP1; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; ELISA, enzyme-linked immunosorbent assay.

Article Snippet: Renal tubule cell-specific Yap1 knockout mice (TKO, KspCre / Yap1 flox/flox ) and their control counterparts lacking the KspCre allele (CTL, WT/Yap1 flox/flox ) were bred at the Shanghai Model Organisms Center in China.

Techniques: Control, Cell Culture, Western Blot, Infection, Flow Cytometry, Staining, Fluorescence, Membrane, Confocal Microscopy, Imaging, Enzyme-linked Immunosorbent Assay, Negative Control, Ubiquitin Proteomics, Binding Assay

Journal: Redox Biology

Article Title: YAP1 preserves tubular mitochondrial quality control to mitigate diabetic kidney disease

doi: 10.1016/j.redox.2024.103435

Figure Lengend Snippet: Schematic diagram showing the yes-associated protein 1 (YAP1) as a critical molecular pattern for diabetic kidney disease (DKD). The initiation of DKD within tubule cells triggers the activation of the Hippo signaling pathway, leading to the degradation of the downstream core mediator YAP1. This cascade results in the disruption of YAP1-mediated mitochondrial quality control, characterized by impaired mitobiogenesis and mitophagy. Consequently, there is an elevation in the production of mitochondrial reactive oxygen species (mtROS), a decline in mitochondrial membrane potential (MMP, ΔΨm), mtDNA copy numbers, oxygen consumption rate, and the enzymatic activity of mitochondrial respiratory chain complexes. These molecular alterations stimulate the synthesis of inflammatory cytokines (such as IL6) and chemokines (CXCL1, CXCL6, and CCL19) by tubule cells, instigating the recruitment and activation of macrophages. This intricate process significantly contributes to the pathogenesis of DKD. MST1, serine/threonine kinase 4; MST2, serine/threonine kinase 3; MOB1, MOB kinase activator 1; LATS1, large tumor suppressor kinase 1; LATS2, and large tumor suppressor kinase 2; TAZ, Tafazzin; TEAD, TEA domain transcription factor; PGC1ɑ, peroxisome proliferator-activated receptor γ coactivator α; TFAM, mitochondrial transcription factor A; PINK1, tensin homolog induced kinase 1; LC3B-Ⅱ, microtubule-associated protein 1 light chain 3B-Ⅱ; p62, ubiquitin-binding protein sequestosome 1; IL6, interleukin 6; CXCL1, C-X-C motif chemokine ligand 1; CCL19, C–C motif chemokine ligand; CXCL6, C-X-C motif chemokine ligand 6.

Article Snippet: Renal tubule cell-specific Yap1 knockout mice (TKO, KspCre / Yap1 flox/flox ) and their control counterparts lacking the KspCre allele (CTL, WT/Yap1 flox/flox ) were bred at the Shanghai Model Organisms Center in China.

Techniques: Activation Assay, Disruption, Control, Membrane, Activity Assay, Ubiquitin Proteomics, Binding Assay

Journal: eLife

Article Title: Synapsin E-domain is essential for α-synuclein function

doi: 10.7554/eLife.89687

Figure Lengend Snippet: ( A ) Schematic showing pH-sensitive sensor sypHy and principle of pHluorin experiments to quantitatively evaluate the SV cycle (see main text and methods for more details). ( B ) Elimination of all synapsins block α-syn functionality at synapses. Left : Schematic showing design of pHluorin experiments. WT or synapsin TKO cultured hippocampal neurons were co-transduced at 5 days in vitro (DIV) with h-α-syn:mCherry (or mCherry as control) and sypHy, and imaged at 14–15 DIV. Right : Stimulation-induced sypHy fluorescence traces (300 action potentials at 20 Hz, delivered at t=0 s – for clarity, symbols only mark every other mean ± SEM ΔF/F 0 value in all sypHy traces). Note that while h-α-syn over-expression (orange) attenuated sypHy fluorescence in WT neurons, there was no effect in neurons from mice lacking all synapsins (TKO). All sypHy data quantified in ( C ). ( C ) Quantification of peak ΔF/F 0 sypHy values (bars: mean ± SEM). A total of 12–19 coverslips were analyzed for each condition, from at least three separate cultures (***p=9e-7, ns p=0.45, student’s t-test). ( D ) Domain structure of the five main synapsin isoforms. ( E ) Experimental design to identify the synapsin isoform that reinstated α-syn functionality, Synapsin TKO neurons were co-transduced at 5 DIV with each synapsin isoform, h-α-syn, and sypHy; and imaged at 14–15 DIV. ( F ) SypHy fluorescence traces (mean ± SEM). Note that h-α-syn(orange) attenuates SV recycling only if the neurons are also co-expressing the ‘a’ isoforms – synapsins Ia, IIa, and IIIa (300 action potentials at 20 Hz, delivered at t=0 sec). Data quantified in G. ( G ) Quantification of peak ΔF/F 0 sypHy values (bars: mean ± SEM). 13–26 coverslips from at least three separate cultures were analyzed for each condition (from left to right: ***p=0.0009, ns p=0.62, ***p=0.00005, ns p=0.99, **p=0.004, student’s t test). Figure 1—source data 1. Tabular data and statistical analyses for graphs presented in panels B, C, F, G.

Article Snippet: Synapsin triple knock-out (TKO) mice (RRID: MMRRC_041434-JAX ) were backcrossed onto the C57BL/6 background as described previously ( ; ; ), and C57BL/6JRccHsd mice (RRID: IMSR_ENV:HSD-043 ) served as WT controls.

Techniques: Blocking Assay, Cell Culture, In Vitro, Fluorescence, Over Expression, Expressing

Journal: eLife

Article Title: Synapsin E-domain is essential for α-synuclein function

doi: 10.7554/eLife.89687

Figure Lengend Snippet: ( A ) Data from sypHy experiments where reacidification was blocked by bafilomycin (Baf), allowing isolated evaluation of exocytosis only (also see results). Note that h-α-syn over-expression attenuated synaptic exocytosis in WT neurons (left), while there was no effect in synapsin TKO neurons (middle). Reintroduction of tagBFP:synapsin Ia reinstated the h-α-syn-mediated synaptic attenuation (right). All pHluorin data quantified in ( B ). 9–22 coverslips from at least three independent cultures (***p=1.9e-5 Mann-Whitney test, p=0.22 Student’s t-test, **p=0.008 Student’s t-test). ( C ) A representative trace showing how the fluorescence decay was quantified to evaluate endocytosis in the sypHy experiments (also see Results). ( D–E ) Fluorescence decay analyses of h-α-syn over-expression in WT and synapsin TKO neurons ( D ), as well as in synapsin TKO neurons where each synapsin isoform was reintroduced ( E ). Note that there were no significant differences in any of these groups. All data in this figure are represented as mean +/- SEM. Ten to 26 coverslips from at least three independent cultures (D: p=0.36; E: p=0.85 both Kruskal Wallis ANOVA). Figure 1—figure supplement 1—source data 1. Tabular data and statistical analyses for graphs presented in panels A, B, D, and E.

Article Snippet: Synapsin triple knock-out (TKO) mice (RRID: MMRRC_041434-JAX ) were backcrossed onto the C57BL/6 background as described previously ( ; ; ), and C57BL/6JRccHsd mice (RRID: IMSR_ENV:HSD-043 ) served as WT controls.

Techniques: Isolation, Over Expression, MANN-WHITNEY, Fluorescence

Journal: eLife

Article Title: Synapsin E-domain is essential for α-synuclein function

doi: 10.7554/eLife.89687

Figure Lengend Snippet: ( A ) Workflow for co-immunoprecipitation experiments in neuro2a cells. ( B ) Western blots from co-immunoprecipitation experiments show that the synapsin isoforms Ia, IIa, and IIIa associate more robustly with h-α-syn (top panel), when compared to synapsins Ib and IIb (a non-specific band is marked with an asterisk). ( C ) Quantification of blots in ( B ) n=5, all data presented as mean ± SEM (a vs. b isoform, **p=0.003, ***p=0.0003, Student’s t-test). ( D ) Schematic showing synapsin isoforms and their variable domains. Note that the E-domain is common between synapsins Ia, IIa, and Iia. ( E ) Workflow for pulldown of GST-tagged h-α-syn WT/deletions/scrambled mutations after incubation with mouse brain lysates. Equivalent amounts of immobilized GST α-syn variants were used. ( F ) Schematic showing α-syn regions that were scrambled (amino acids between 96–140 and 96–110). ( G ) Top : Samples from GST-pulldown were analyzed by NuPAGE and immunoblotted with an antibody against synapsin I (top panel). Bottom : Ponceau staining shows equivalent loading of fusion proteins. Note that full-length h-α-syn bound synapsin I from mouse brains (lane 2), while deletion of the h-α-syn C-terminus (amino acids 96–140, lane 3) eliminated this interaction. Lanes 4–7 show that the sequence within amino acids 96–110 of h-α-syn is critical for binding to synapsin I. All western blots are quantified below (n=3). Data presented as mean ± SEM (**p=0.003, **p=0.002, ns p=0.99, ns p=0.98, **p=0.004, **p=0.004, comparing to full-length h-α-syn, one-way ANOVA with Tukey’s posthoc test). Figure 2—source data 1. Tabular data and statistical analyses for graphs shown in panels C and G. Figure 2—source data 2. Full western blots for segments shown in panel B. Figure 2—source data 3. Full western blots for segments shown in panel G.

Article Snippet: Synapsin triple knock-out (TKO) mice (RRID: MMRRC_041434-JAX ) were backcrossed onto the C57BL/6 background as described previously ( ; ; ), and C57BL/6JRccHsd mice (RRID: IMSR_ENV:HSD-043 ) served as WT controls.

Techniques: Immunoprecipitation, Western Blot, Incubation, Staining, Sequencing, Binding Assay

Journal: eLife

Article Title: Synapsin E-domain is essential for α-synuclein function

doi: 10.7554/eLife.89687

Figure Lengend Snippet: ( A ) Schematic showing synapsin Ia scrambled E-domain sequence (synapsin Ia scr-E ). Numbers depict amino acid positions, letters in the inset depict amino-acids. Note that the WT amino acids are randomized in the scrambled mutant. ( B ) Design of sypHy experiments co-expressing synapsin Ia scr-E and h-α-syn in cultured neurons from synapsin TKO mice. ( C ) Stimulation-induced sypHy fluorescence traces (300 action potentials at 20 Hz, delivered at t=0 sec). Note that while h-α-syn attenuated sypHy fluorescence in synapsin TKO neurons expressing synapsin Ia, h-α-syn had no effect in neurons expressing synapsin Ia scr-E . Insets: Quantification of peak ΔF/F 0 sypHy values (bars: mean ± SEM). Ten to 16 coverslips from at least three separate cultures were analyzed for each condition (***p=0.0007, ns p=0.67, one-way ANOVA with Tukey’s posthoc analysis). ( D ) Top : Schematic for co-immunoprecipitation experiments, to test the interaction of h-α-syn with WT synapsin Ia or synapsin Ia scr-E . Neuro2a cells were co-transfected with myc-tagged α-syn and respective YFP-tagged synapsin Ia, and the YFP was immunoprecipitated. Bottom : Note that h-α-syn co-immunoprecipitated with synapsin Ia, but not synapsin Ia scr-E ; quantification of the gels below (n=4, all data are means ± SEM ***p<0.001, Student’s t test – a non-specific band is marked with an asterisk). ( E ) Schematic of experiments to test if the synapsin E-domain is sufficient to enable α-syn functionality in synapsin TKO neurons. Synapsin-E (a 46 amino acid sequence) was fused to the C-terminus of sypHy, so that upon expression in neurons, the E-domain would be present on the cytosolic surface of Svs. ( F ) SypHy fluorescence traces (mean ± SEM). Note that while h-α-syn (orange) was unable to attenuate SV recycling in synapsin TKO neurons (as expected), diminished synaptic responses were seen when the E-domain was present. Insets: Quantification of peak ΔF/F 0 sypHy values (bars: mean ± SEM). Twelve 19 coverslips from at least three separate cultures were analyzed for each condition (ns p=0.89, ***p=2.8e-7, one-way ANOVA with Tukey’s posthoc analysis). Figure 3—source data 1. Tabular data and statistical analyses for graphs shown in panels C, D and F. Figure 3—source data 2. Full western blots for segments shown in panel D.

Article Snippet: Synapsin triple knock-out (TKO) mice (RRID: MMRRC_041434-JAX ) were backcrossed onto the C57BL/6 background as described previously ( ; ; ), and C57BL/6JRccHsd mice (RRID: IMSR_ENV:HSD-043 ) served as WT controls.

Techniques: Sequencing, Mutagenesis, Expressing, Cell Culture, Fluorescence, Immunoprecipitation, Transfection, Western Blot

Journal: eLife

Article Title: Synapsin E-domain is essential for α-synuclein function

doi: 10.7554/eLife.89687

Figure Lengend Snippet: ( A ) Schematic of experiments to evaluate quantitative localization of tagBFP:synapsin Ia and tagBFP:synapsin Ia Scr-E in synapsin TKO neurons (with and without h-α-syn over-expression). Neurons were immunostained for synapsin I (for reliable visualization of the transduced synapsin constructs), as well as for the SV-marker vGlut1 (to confidently identify synapses). ( B ) Representative images showing equivalent immunofluorescence of synapsin Ia and synapsin Ia Scr-E at synapses. Over-expression of h-α-syn did not affect their synaptic fluorescence. ( C ) Quantified synaptic fluorescence data, represented as mean +/- SEM, 15–16 coverslips from at least three independent cultures were analyzed for each condition. ns p=0.52, ns p=0.14, one-way ANOVA. Figure 3—figure supplement 1—source data 1. Tabular data and statistical analyses for graph shown in panel C. Figure 3—figure supplement 1—source data 2. Raw images. Full images of anti-synapsin I and anti-vGlut1 immunofluorescence channels containing the details shown in panel B (marked with white dashed square).

Article Snippet: Synapsin triple knock-out (TKO) mice (RRID: MMRRC_041434-JAX ) were backcrossed onto the C57BL/6 background as described previously ( ; ; ), and C57BL/6JRccHsd mice (RRID: IMSR_ENV:HSD-043 ) served as WT controls.

Techniques: Over Expression, Construct, Marker, Immunofluorescence, Fluorescence

Journal: eLife

Article Title: Synapsin E-domain is essential for α-synuclein function

doi: 10.7554/eLife.89687

Figure Lengend Snippet: ( A ) Schematic of experiments to evaluate synaptic targeting of synapsin E-domain constructs in synapsin TKO neurons. Note that EGFP is tagged to the E-domain in these experiments. ( B ) The EGFP:E-domain construct was diffusely distributed in neurons and not enriched to synapses (marked by immunostaining of VGlut1). A representative image showing that the E-domain construct is not targeted to synapses (green: EGFP:E-domain, magenta: vGlut1). ( C ) Over-expression of synapsin E-domain in the context of excessive α-syn did not have any effect on SV recycling (as determined by sypHy experiments), presumably because the E-domain fails to enrich at synapses. Data shown as mean mean ± SEM, 11–20 coverslips from at least 3 independent cultures were analyzed for each condition (p=0.16, Kruskal-Wallis ANOVA). ( D ) Representative images illustrating synaptic localization of the E-domain tagged to sypHy (green: sypHy:E-domain, magenta: vGlut1). ( E ) Expression of sypHy:E-domain in synapsin TKO neurons enhances the synaptic enrichment of h-α-syn. Synaptic enrichment (see Methods section) of h-α-syn was measured in synapsin TKO neurons expressing either sypHy or sypHy-E-domain. We observed significantly higher enrichment of h-α-syn in the latter. Data shown as mean ± SEM, 23 to 25 coverslips from three independent cultures were analyzed for each condition (**p=0.009, Mann-Whitney U-test). Figure 3—figure supplement 2—source data 1. Tabular data and statistical analyses for graphs shown in panels C and E. Figure 3—figure supplement 2—source data 2. Raw images. Full images of EGFP:E-domain and anti-vGlut1 immunofluorescence channels containing the details shown in panel B, and the full images of sypHy:E-domain and anti-vGlut1 immunofluorescence channels containing the details shown in panel D (marked with white dashed square).

Article Snippet: Synapsin triple knock-out (TKO) mice (RRID: MMRRC_041434-JAX ) were backcrossed onto the C57BL/6 background as described previously ( ; ; ), and C57BL/6JRccHsd mice (RRID: IMSR_ENV:HSD-043 ) served as WT controls.

Techniques: Construct, Immunostaining, Over Expression, Expressing, MANN-WHITNEY, Immunofluorescence

Journal: eLife

Article Title: Synapsin E-domain is essential for α-synuclein function

doi: 10.7554/eLife.89687

Figure Lengend Snippet: ( A ) Representative images from WT or synapsin TKO neurons immunostained with an SV marker (vGlut1); zoomed insets marked by yellow boundaries. Note that the compact clustering of SVs is lost in synapsin-null neurons. ( B ) FWHM as a quantitative means to determine spreading of fluorophores at synapses (also see Results). Note that an increase in FWHM corresponds to a decrease in intensity and increased spreading of fluorescence within a bouton. ( C ) Quantification of synaptic fluorescence in WT and synapsin TKO neurons. Overall intensities are decreased in TKO synapses (left), and FWHM is increased (right), compared to WT synapses; consistent with a spreading of SVs in the synapsin null setting. ( D ) Experimental plan to determine effects of h-α-syn over-expression on the overall distribution of SVs in WT and synapsin TKO neurons. ( E ) FWHM of vGlut1 staining at synapses is augmented by h-α-syn over-expression in WT neurons, but not in neurons from synapsin TKO mice. Reintroduction of synapsins Ia/IIa (but not Ib/IIb) in the setting of h-α-syn over-expression rescues the changes in vGlut1-FWHM ( F ). All data in this figure are represented as mean +/-SEM. Nine to 28 coverslips from at least three independent cultures were analyzed for C, E, and F (C, left: ***p=0.0006, Mann-Whitney U-test; right: see E; E: ***p=4e-8, ns p=0.92, one-way ANOVA with Tukey’s posthoc analysis; F, left: ***p=2.7e-4, ns p=1.0, Kruskal-Wallis ANOVA with Dunn’s posthoc test; F, right: **p=0.001, ns p=0.52, one-way ANOVA with Tukey’s posthoc analysis). Figure 4—source data 1. Raw images. Full images of anti-vGlut1 immunofluorescence channels in WT and synapsin TKO neurons, containing the details shown in panel A (marked with white dashed square). Figure 4—source data 2. Tabular data and statistical analyses for graphs shown in panels C, E, and F.

Article Snippet: Synapsin triple knock-out (TKO) mice (RRID: MMRRC_041434-JAX ) were backcrossed onto the C57BL/6 background as described previously ( ; ; ), and C57BL/6JRccHsd mice (RRID: IMSR_ENV:HSD-043 ) served as WT controls.

Techniques: Marker, Fluorescence, Over Expression, Staining, MANN-WHITNEY, Immunofluorescence

Journal: eLife

Article Title: Synapsin E-domain is essential for α-synuclein function

doi: 10.7554/eLife.89687

Figure Lengend Snippet:

Article Snippet: Synapsin triple knock-out (TKO) mice (RRID: MMRRC_041434-JAX ) were backcrossed onto the C57BL/6 background as described previously ( ; ; ), and C57BL/6JRccHsd mice (RRID: IMSR_ENV:HSD-043 ) served as WT controls.

Techniques: Recombinant, Software, Transduction, Fluorescence, Microscopy

Journal: Brain Communications

Article Title: Immunoproteasome deficiency results in age-dependent development of epilepsy

doi: 10.1093/braincomms/fcae017

Figure Lengend Snippet: Immunoproteasome expression in the brains of young and old mice. ( A ) A western blot analysis of immunoproteasome distribution in the spleen, thymus, liver and brain of 2-month-old female WT mice. A representative western blot is shown (left side). The bar graphs display a relative abundance of low - molecular-weight protein 2 (LMP2) and low-molecular-weight protein 7 (LMP7) normalized to the proteasome subunit α3 ( n = 3–4). The statistical analysis was performed by using one-way ANOVA. Data are presented as mean ± SD, * P = 0.01–0.05, *** P < 0.001. Uncropped blots are shown in . ( B ) A western blot of polyubiquitination in the hippocampi of 2-month-old and 1-year-old female WT mice. A representative western blot is shown (left side), and the bar graphs display a relative polyubiquitin signal normalized to β-actin ( n = 3). The statistical analysis was performed by using an unpaired t -test. Data are presented as mean ± SD, * P = 0.01–0.05. ( C ) A western blot of 2- and 8-month-old female WT and TKO mice displays an increase in the polyubiquitination of TKO hippocampi compared with WT hippocampi. A representative western blot is shown. The bar graphs display polyubiquitination levels normalized to β-actin ( n = 3–4). Statistical significance for ( B ) and ( C ) was analysed by using an unpaired t -test. Data are presented as mean ± SD, * P = 0.01–0.05. Uncropped blots are shown in . ( D) A fluorescence staining of LMP7 (green) and NeuN (magenta, a neuronal marker) in the brains of young and old WT mice. A staining of the brains of old TKO mice is shown as a negative control. ( E ) A fluorescence staining of phospho-tau (AT8 = green, white arrows) in the CA3 region of the brains of WT versus TKO mice. The neuronal nuclear protein (NeuN, magenta) serves as a neuronal marker, and DAPI (blue) stains the nuclei. ( F ) A diaminobenzidine staining of phospho-tau (AT8) in the hippocampi of old WT and TKO mice (CA1).

Article Snippet: The TKO mice were kindly provided by Kenneth Rock (University of Massachusetts Medical School) and Regeneron Pharmaceuticals.

Techniques: Expressing, Western Blot, Molecular Weight, Fluorescence, Staining, Marker, Negative Control

Journal: Brain Communications

Article Title: Immunoproteasome deficiency results in age-dependent development of epilepsy

doi: 10.1093/braincomms/fcae017

Figure Lengend Snippet: Immunoproteasome deficiency increases excitability and the risk of developing epilepsy. ( A ) TKO mice develop epileptic seizures at an age of 5–10 months. Female mice ( n = 141) were scored over 20 months. The graph displays the occurrence of seizures in per cent. Statistical significance was analysed by performing a Mantel–Cox test **** P < 0.0001. ( B–G ) Seizure susceptibility of female TKO mice was tested via an i.p. injection of KA (10 mg/kg). The mice were monitored for 60 min after injection. ( B ) A representative EEG of female WT, LMP7 KO and TKO mice. ( C ) The Racine score was estimated in intervals of 5 min. Statistical significance was analysed by using one-way ANOVA (Turkey’s multiple comparisons test, n = 7 mice/group): WT versus LMP7 KO n.s., WT versus TKO **** P < 0.0001, LMP7 KO versus TKO **** P < 0.0001. ( D ) The number of seizures per 5 min was calculated by counting the occurrence of seizures over 40 min ( n = 7 mice/group). Statistical significance was analysed by using one-way ANOVA: WT versus LMP7 n.s., LMP7 versus TKO n.s., WT versus TKO * P = 0.0122. ( E ) The graph displays the survival of animals ( n = 7 mice/group). Statistical significance was analysed by using the Mantel–Cox test: WT versus TKO **** P < 0.0001, WT versus LMP7 KO n.s., LMP7 KO versus TKO ** P = 0.0031. ( F ) The behavioural score of young female WT and TKO animals after an injection of KA ( n = 4 mice/group). Statistical significance was analysed by performing a paired t -test. *** P = 0.0001. ( G ) The Survival of young and old female TKO mice after an injection of KA ( n = 4–7 mice per group). Statistical significance was analysed by using the Mantel–Cox test ** P = 0.003.

Article Snippet: The TKO mice were kindly provided by Kenneth Rock (University of Massachusetts Medical School) and Regeneron Pharmaceuticals.

Techniques: Injection

Journal: Brain Communications

Article Title: Immunoproteasome deficiency results in age-dependent development of epilepsy

doi: 10.1093/braincomms/fcae017

Figure Lengend Snippet: TKO animals display several neurological disorders apart from recurrent seizures. ( A ) Female TKO animals show increased anxiety in the open field study compared with age-matched WT mice (>1-year-old mice; n = 5 mice/group). The statistical analysis was performed by using an unpaired t -test. ( B ) A gait analysis of >1-year-old female TKO and WT mice ( n = 5 mice/group). The stride length, stride width and toe spread were measured. The SD value of each mouse was calculated. ( C ) Calbidin staining in female WT and TKO mice shows a significant loss of Purkinje cells in TKO animals ( n = 4 mice/group). The number of Purkinje cells per 10 mm was calculated. The statistical analysis was performed by using the unpaired t -test. * P = 0.01–0.05; ** P = 0.001–0.01.

Article Snippet: The TKO mice were kindly provided by Kenneth Rock (University of Massachusetts Medical School) and Regeneron Pharmaceuticals.

Techniques: Staining

Journal: bioRxiv

Article Title: Immunoproteasomal Processing of Isolevuglandin Adducts in Hypertension

doi: 10.1101/2023.04.10.536054

Figure Lengend Snippet: Immunoproteasome triple knockout mice (TKO) and control C57BL/6 mice were treated with angiotensin II. Radiotelemetry was used to measure (A) systolic, (B) diastolic, and (C) mean blood pressure. Blood pressure was analyzed using 2-way ANOVA ( n =5-7). (D) Representative flow cytometry diagrams for CD4 + and CD8 + T cells harvested from Kidney. (E) Quantitation of CD8 + renal T cells. (F) Representation of CD8 + T cells as a percentage of total CD3 + T cells. (G) Quantitation of total CD3 + renal T cells. (H) Quantitation of FRET + DCs harvested from aorta. (I) Representation of FRET + aortic DCs as a percentage of total DCs. (J) Representative flow cytometry plots for FRET in DCs harvested from aorta. Data were analyzed by two-tailed Student’s T-test or Mann Whitney U Test ( n = 6-7).

Article Snippet: Immunoproteasome triple knockout mice (TKO) were obtained from Regeneron Therapeutics.

Techniques: Triple Knockout, Control, Flow Cytometry, Quantitation Assay, Two Tailed Test, MANN-WHITNEY