Journal: Acta Neuropathologica Communications

Article Title: Tau modulation through AAV9 therapy augments Akt/Erk survival signalling in glaucoma mitigating the retinal degenerative phenotype

doi: 10.1186/s40478-024-01804-0

Figure Lengend Snippet: Modulation of Tau expression in C57BL/6 mice retinas A Scheme of AAV plasmid vector containing GFP, mTau, scrambled shRNA, and mTau-shRNAmir sequences. mTau (overexpression) and mTau-shRNAmir (knockdown, KD) sequences were cloned in AAV9 viral vector plasmid fused to the ampicillin-resistance gene. For mTau protein expression, a T2A self-cleaving peptide sequence was used. B Schematic depicting the experimental design and AAV administration experimental timeline. C Immunofluorescence images of retinal sections from control, AAV-GFP, AAV-mTau, AAV-Scramble and AAV-mTau KD groups showing GFP (green, FITC), tau (Green, Alexa Fluor 488), and ptau (Ser199/Ser202) (Red, Cy3). NeuN (red-Cy3) and nucleus (blue, DAPI) (representative images, Scale bar = 50 μm, arrows indicate the changes in the expressions; Antibody concentrations: GFP (1:1000), tau (1:800), ptau (Ser199/Ser202) (1:800), NeuN (1:1000)). D Quantification of GFP relative fluorescence intensity (RFI) percentage (**** P < 0.0001, n = 5). E Quantification of Tau RFI percentage (**** P < 0.0001, n = 5) F Quantification of pTau (Ser199/Ser202) RFI percentage (**** P < 0.0001, n = 5). Statistical significance was assessed by employing One-way ANOVA analysis with Tukey’s multiple comparison test

Article Snippet: For the over expression of the Tau, murine mutant Tau (P232S) cDNA (NCBI ref: BC014748) under the transcriptional control of the cytomegalovirus chicken β-actin (CAG2) hybrid promotor was inserted into the adeno-associated virus serotype 9 (AAV9) vector with the enhanced green fluorescence protein reporter (eGFP) and 2A linker in between GFP and the mTau sequence (AAV9-CAG2-eGFP-2A-mTau (P232S)-WPRE or AAV9- Tau) (Vector Biolabs, USA).

Techniques: Expressing, Plasmid Preparation, shRNA, Over Expression, Knockdown, Clone Assay, Sequencing, Immunofluorescence, Control, Fluorescence, Comparison

Journal: Acta Neuropathologica Communications

Article Title: Tau modulation through AAV9 therapy augments Akt/Erk survival signalling in glaucoma mitigating the retinal degenerative phenotype

doi: 10.1186/s40478-024-01804-0

Figure Lengend Snippet: Electrophysiological and structural alterations in C57BL/6 mice retinas subjected to AAV-tau overexpression and AAV-tau knockdown under normal IOP conditions. A Positive scotopic threshold responses (pSTRs) of the control, AAV-GFP, and AAV-mTau injected mice. B Quantification of amplitudes indicates a significant decrease in pSTR amplitudes in AAV9-Tau overexpressing mice (n = 10, P < 0.0001 t-test). C pSTRs of the control, AAV-scrambled, and AAV-tau shRNA expressing mice and D their quantification indicates a significant decrease of pSTR amplitudes in AAV9-KD mice retinas (n = 10, **** P < 0.0001, t-test). No significant difference in pSTR amplitudes was observed between the control and AAV9-GFP or AAV9-scramble-shRNA sequence-expressing eyes. E H and E staining of retinal sections shows GCL cell density loss in Tau overexpression and Tau KD retinas compared to the controls, arrows indicate the changes in cell densities (representative images, Scale bar = 50 μm). F Quantification of cell density in the GCL from the retinal H and E-stained images (**** P < 0.0001, One-way ANOVA analysis with Tukey’s multiple comparison test, n = 5 for each group)

Article Snippet: For the over expression of the Tau, murine mutant Tau (P232S) cDNA (NCBI ref: BC014748) under the transcriptional control of the cytomegalovirus chicken β-actin (CAG2) hybrid promotor was inserted into the adeno-associated virus serotype 9 (AAV9) vector with the enhanced green fluorescence protein reporter (eGFP) and 2A linker in between GFP and the mTau sequence (AAV9-CAG2-eGFP-2A-mTau (P232S)-WPRE or AAV9- Tau) (Vector Biolabs, USA).

Techniques: Over Expression, Knockdown, Control, Injection, shRNA, Expressing, Sequencing, Staining, Comparison

Journal: Acta Neuropathologica Communications

Article Title: Tau modulation through AAV9 therapy augments Akt/Erk survival signalling in glaucoma mitigating the retinal degenerative phenotype

doi: 10.1186/s40478-024-01804-0

Figure Lengend Snippet: Tau overexpressing mice retinas shows exacerbated degenerative changes in the inner retina in high-IOP glaucoma condition. A Chronic elevation of IOP in C57BL/6 mice eyes was induced by intracameral microbead injections in control, AAV-GFP and AAV-mTau administered mice for 2 months. B Positive scotopic threshold response (pSTR) traces from control, glaucoma, glaucoma + AAV-GFP, and glaucoma + AAV-mTau overexpression mice retinas, as indicated and C their pSTR amplitudes quantification indicated a significant decline of the pSTR in the glaucoma AAV9-tau overexpression animals. (n = 10, *** P < 0.001 **** P < 0.0001) D Western blot analysis of Tau, pTau S199/202 and pTau S404 levels in control and glaucomatous retina. β-actin was used as a loading control E Fold change of Tau, pTau S199/202 and pTau. S404 showed a significant increase in retinas in high IOP conditions (*** P < 0.001, n = 3 each group, t-test). F H and E staining of retinal sections of control, glaucoma, glaucoma + AAV9-GFP, and glaucoma + AAV9-mTau mice (representative images, arrows indicate the changes in cell densities, Scale bar, 50 μm). G Quantification of the cell density in GCL indicated a significant decrease in the number of cells in glaucoma and AAV-tau overexpressed retinas compared to the controls (n = 5, **** P < 0.0001, One-way ANOVA analysis with Tukey’s multiple comparison test)

Article Snippet: For the over expression of the Tau, murine mutant Tau (P232S) cDNA (NCBI ref: BC014748) under the transcriptional control of the cytomegalovirus chicken β-actin (CAG2) hybrid promotor was inserted into the adeno-associated virus serotype 9 (AAV9) vector with the enhanced green fluorescence protein reporter (eGFP) and 2A linker in between GFP and the mTau sequence (AAV9-CAG2-eGFP-2A-mTau (P232S)-WPRE or AAV9- Tau) (Vector Biolabs, USA).

Techniques: Control, Over Expression, Western Blot, Staining, Comparison

Journal: Acta Neuropathologica Communications

Article Title: Tau modulation through AAV9 therapy augments Akt/Erk survival signalling in glaucoma mitigating the retinal degenerative phenotype

doi: 10.1186/s40478-024-01804-0

Figure Lengend Snippet: Silencing of Tau expression using AAV protects the inner retinal function and laminar structure in glaucomatous eyes. A Chronic elevation of IOP in C57BL/6 mice eyes was induced by intracameral microbead injections for 2 months. B Positive scotopic threshold response (pSTR) traces from control, glaucoma, glaucoma + scrambled and glaucoma + Tau KD mice retinas, as indicated. C Quantification of the pSTR amplitudes from the glaucoma AAV9-Tau KD mice eyes compared to glaucoma scrambled sequence expressing controls indicated significant protection against pSTR amplitudes loss in the glaucoma-AAV-Tau KD animals. (n = 10, *** P < 0.001, One-way ANOVA analysis with Tukey’s multiple comparison test). D H and E staining of retinal sections of the control, glaucoma, glaucoma + AAV9-scramble, and glaucoma + AAV9-tau KD mice eyes. (representative images, arrows indicate the changes in cell densities, Scale bar, 50 μm). E Quantification of the cell density in GCL indicated significant protection against cell loss in glaucoma AAV9-tau KD-subjected retinas compared to the microbead-injected control eyes. (n = 5, *** P < 0.001, **** P < 0.0001, One-way ANOVA analysis with Tukey’s multiple comparison test)

Article Snippet: For the over expression of the Tau, murine mutant Tau (P232S) cDNA (NCBI ref: BC014748) under the transcriptional control of the cytomegalovirus chicken β-actin (CAG2) hybrid promotor was inserted into the adeno-associated virus serotype 9 (AAV9) vector with the enhanced green fluorescence protein reporter (eGFP) and 2A linker in between GFP and the mTau sequence (AAV9-CAG2-eGFP-2A-mTau (P232S)-WPRE or AAV9- Tau) (Vector Biolabs, USA).

Techniques: Expressing, Control, Sequencing, Comparison, Staining, Injection

Journal: Acta Neuropathologica Communications

Article Title: Tau modulation through AAV9 therapy augments Akt/Erk survival signalling in glaucoma mitigating the retinal degenerative phenotype

doi: 10.1186/s40478-024-01804-0

Figure Lengend Snippet: Western blot (WB) analysis of retinal endoplasmic reticulum (ER) stress markers changes in glaucoma and Tau modulation conditions. A WBs of control and glaucoma C57BL/6 mice retinal lysates probed with GRP78(1:1000), CHOP(1:1000), P-PERK(1:1000), and β-actin (1:5000) antibodies and B-D their respective relative band intensities quantified as the fold change using β-actin as loading control (n = 3 **** P < 0.0001, t-test). E WB of retinal lysates of the control, glaucoma, AAV-GFP, AAV-mTau (overexpression), AAV-GFP + glaucoma, and AAV-mTau + glaucoma mice were probed with GRP-78, CHOP, P-PERK, and actin antibodies, and F-H their respective relative band intensities quantified as the fold change using β-actin as loading control (n = 3, ** P < 0.01, *** P < 0.001, **** P < 0.0001, One-way ANOVA analysis with Tukey’s multiple comparison test,). I WB of retinal lysates from control, glaucoma, AAV9-scramble, AAV-mTau KD, AAV9-scramble + glaucoma, AAV-mTau KD + glaucoma mice were probed with GRP 78, CHOP, P-PERK, and actin antibodies, and (J-L) their respective band intensities quantified as fold change using β-actin as a loading control (*** P < 0.001, **** P < 0.0001, One-way ANOVA analysis with Tukey’s multiple comparison test, n = 3 per group)

Article Snippet: For the over expression of the Tau, murine mutant Tau (P232S) cDNA (NCBI ref: BC014748) under the transcriptional control of the cytomegalovirus chicken β-actin (CAG2) hybrid promotor was inserted into the adeno-associated virus serotype 9 (AAV9) vector with the enhanced green fluorescence protein reporter (eGFP) and 2A linker in between GFP and the mTau sequence (AAV9-CAG2-eGFP-2A-mTau (P232S)-WPRE or AAV9- Tau) (Vector Biolabs, USA).

Techniques: Western Blot, Control, Over Expression, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest

doi: 10.1073/pnas.1707316114

Figure Lengend Snippet: Screening for the most potent mitogens in immature hCO. (A) Validation of proliferation induction in neonatal rat cardiomyocytes. Representative immunostaining of mitotic (pH3+) cardiomyocytes (α-actinin+) after all treatments after 48 h of culture. (B) Quantification of mitotic (pH3+) cardiomyocytes (α-actinin+) revealed that CHIR99021, miR-199a, miR-590, and Ad-YAP(S112A) were capable of inducing proliferation in neonatal rat cardiomyocytes; n = 4 replicates per group. The 14,339 cardiomyocytes were manually counted. (C) Schematic outlining protocol for directed differentiation of hESCs into cardiac cells (15 d) and formation and exercise of hCO in the Heart-Dyno (6 d). The tissues were then stimulated with mitogens for 2 d before analysis. (D) Whole-tissue imaging after transfection of a Cy3-labeled siRNA shows efficient transfection of small RNAs throughout the hCO in the Heart-Dyno. (E) qPCR shows increased expression of miR-199a or miR-590 after transfection vs. a scramble control; n = 5–6 from two experiments. (F) Staining of GFP-infected hCOs after 48 h of AAV6-GFP treatment. (Scale bar, 20 µm.) (G) Quantification of GFP+ hPSC-CMs (α-actinin+) after 48 h of AAV6-GFP treatment; n = 4. (H) Quantification of mitotic (pH3+) hPSC-CMs (α-actinin+) after all treatments after 48 h of treatment; n = 8–21 from two to three experiments. The 35,690 hPSC-CMs were manually counted for this analysis. (I) Analysis of force of contraction reveals that CHIR99021 decreases force; however, constitutively active β-catenin does not; n = 11–29 from two to three experiments. (J) Representative immunostaining of mitotic (pH3+) hPSC-CMs (α-actinin+) after 48-h treatment with CHIR99021. (Scale bar, 20 μm.) (K) Representative immunostaining of mitotic (pH3+) hPSC-CMs (α-actinin+) after 48-h treatment with CHIR99021, revealing that proliferating hPSC-CMs are located throughout the hCO. Data are mean ± SEM. (Scale bar, 200 μm.) *P < 0.05; **P < 0.01; and ***P < 0.001, using t test (E) or ANOVA with Tukey’s posttest (H and I). CHIR, CHIR99021.

Article Snippet: Control AAV6-MCS or AAV6-GFP (Vector Biolabs) controls were used at the same titers in these experiments.

Techniques: Immunostaining, Imaging, Transfection, Labeling, Expressing, Staining, Infection

Journal: Neurobiology of Pain

Article Title: Nrxn3 reduces myofascial nociceptive pain

doi: 10.1016/j.ynpai.2025.100197

Figure Lengend Snippet: Nrxn3 shRNA reduced Nrxn3 expression The central amygdala was infused with adeno-associated virus containing either a control (scrambled sequence) shRNA or a Nrxn3 shRNA expression construct. After four weeks the masseter tendon was ligated bilaterally, or a sham surgery was performed and the animals sacrificed three weeks after ligature. Central amygdala tissue was isolated by biopsy punches of frozen sections and the quantity of Nrxn3 was quantitated by ELISA. Significant differences of (P < 0.05) between the groups are indicated by “*”. Each point on the graph is from a different animal. Values are the means ± SEM for 8 animals per treatment group.

Article Snippet: A separate group of rats was infused with 1 μl of 1X10 13 TU/mL AAV1 containing a scrambled shRNA sequence (AAV1-GFP-U6-shRNA, Vector Biolabs).

Techniques: shRNA, Expressing, Virus, Control, Sequencing, Construct, Isolation, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Mitotic regulators and the SHP2-MAPK pathway promote IR endocytosis and feedback regulation of insulin signaling

doi: 10.1038/s41467-019-09318-3

Figure Lengend Snippet: IRS1/2 are required for insulin-activated IR endocytosis. a HepG2 cells stably expressing IR-GFP WT were transfected with the indicated siRNAs or siRNA-resistant Myc-IRS1, serum starved, treated without or with 100 nM insulin for 5 min, and stained with anti-GFP antibodies. b Quantification of the ratios of PM and IC IR-GFP signals of cells in a (siLUC, n = 26; siLUC + Ins, n = 42; si-IRS1, n = 41; si-IRS1, n = 26; si-IRS2, n = 20; si-IRS2 + Ins, n = 10; si-IRS1/2, n = 48, si-IRS1/2 + Ins, n = 66; si-IRS1/2 + IRS1, n = 14; si-IRS1/2 + IRS1 + Ins, n = 14; mean ± s.d.; **** p < 0.0001; two-tailed unpaired t test). c Domains and YXXΦ motifs of human IRS1 and mouse IRS2. PH, pleckstrin homology domain; PTB, phosphotyrosine-binding domain. AP2M1- and SHP2-binding regions are indicated. YXXΦ motifs and phosphotyrosine sites of IR for SHP2 binding are shown as blue and red bars, respectively. The MAPK phosphorylation sites are labeled as green letters in the sequences. d 293FT cells stably expressing IR-GFP WT were transfected with the indicated siRNAs or siRNA-resistant Myc-IRS1, serum starved, treated without or with 100 nM insulin for 5 min, and stained with anti-GFP (IR; green), anti-Myc (IRS1; red), and DAPI (blue) (3YA, the IRS1 Y612A/Y632A/Y662A triple mutant; 3YF, the Y612F/Y632F/Y662F triple mutant; 3SA, the S616A/S636A/S666A triple mutant; 3SD, the S616D/S636D/S666D triple mutant; 2YA, the Y1179A/Y1229A double mutant). Scale bar, 5 µm. e Quantification of the ratios of PM and IC IR-GFP signals of cells in d (siLUC, n = 49; siLUC + Ins, n = 87; si-IRS1/2, n = 46; si-IRS1/2 + Ins, n = 87; si-IRS1/2 + WT, n = 65; si-IRS1/2 + WT + Ins, n = 112; si-IRS1/2 + 3YA, n = 54; si-IRS1/2 + 3YA + Ins, n = 63, si-IRS1/2 + 3SA, n = 40; si-IRS1/2 + 3SA + Ins, n = 66; si-IRS1/2 + 3 SD, n = 42; si-IRS1/2 + 3 SD + Ins, n = 63; si-IRS1/2 + 3YF, n = 48; si-IRS1/2 + 3YF + Ins, n = 58; si-IRS1/2 + Y612A, n = 48; si-IRS1/2 + Y612A, n = 58; si-IRS1/2 + 1–864, n = 23; si-IRS1/2 + 1–864 + Ins, n = 35; si-IRS1/2 + 2YA, n = 28, si-IRS1/2 + 2YA + Ins, n = 42; mean ± s.d.; **** p < 0.0001; two-tailed unpaired t test). f 293FT cells were serum starved and treated without or with 100 nM insulin for 5 min. Total cell lysate (TCL), anti-IRS1 IP, and IgG IP were blotted with anti-IRS1 and anti-AP2B1 antibodies. g Serum-starved primary hepatocytes were treated with DMSO or 10 µM SHP099 for 2 h and treated with 100 nM insulin for 5 min. TCL, anti-IRS1 IP were blotted with anti-IRS1 and anti-AP2B1 antibodies. Source data are provided as a Source Data file

Article Snippet: After 1 day, the cells were serum starved for 14 h and treated with DMSO or SHP099 for 2 h. AAV encoding SHP2 shRNA (AAV8-GFP-U6-mPTPN11-shRNA) or control shRNA (AAV-GFP-U6-scrmb-shRNA) were generated at Vector Biolabs.

Techniques: Stable Transfection, Expressing, Transfection, Staining, Two Tailed Test, Binding Assay, Labeling, Mutagenesis

Journal: Nature Communications

Article Title: Mitotic regulators and the SHP2-MAPK pathway promote IR endocytosis and feedback regulation of insulin signaling

doi: 10.1038/s41467-019-09318-3

Figure Lengend Snippet: Mitotic regulators and SHP2 promote feedback inhibition of IR. a Insulin signaling in the liver from mice fed HFD for 5 weeks. The mice were administered vehicle or SHP099 for 5 days, fasted overnight, and administered vehicle or SHP099 once more. At 2 h after the last administration, the mice were injected with or without 1 U insulin via inferior vena cava. The livers were collected at the indicated time points. Lysates were prepared from these tissues and subjected to quantitative immunoblotting with the indicated antibodies. b Quantification of the blots in a . Mean ± range; n = 2 independent experiments. c Targeting feedback regulation of IR endocytosis for diabetes treatment. Left panel depicts the feedback regulation of IR endocytosis by ERK1/2 and SHP2 during unperturbed insulin signaling. Right panel illustrates the mechanism by which SHP2 inhibitor (SHP099) or shRNA blocks growth-promoting IR signaling and IR endocytosis, and prolongs insulin signaling through the PI3K-AKT pathway, which controls metabolism. Source data are provided as a Source Data file

Article Snippet: After 1 day, the cells were serum starved for 14 h and treated with DMSO or SHP099 for 2 h. AAV encoding SHP2 shRNA (AAV8-GFP-U6-mPTPN11-shRNA) or control shRNA (AAV-GFP-U6-scrmb-shRNA) were generated at Vector Biolabs.

Techniques: Inhibition, Injection, Western Blot, shRNA