Journal: Human Molecular Genetics

Article Title: Trans -splicing correction of tau isoform imbalance in a mouse model of tau mis-splicing

doi: 10.1093/hmg/ddt108

Figure Lengend Snippet: Correction of tau mis-splicing in cultured cortical neurons from htau mouse after lentivirus-mediated PTM delivery. Cultured cortical neurons from htau mice were transduced at DIV3 with LV-TauPTM6 at the indicated m.o.i., or with a control LV at m.o.i. = 10. Total RNA was extracted 7 days after transduction and reverse-transcribed. ( A ) PCR analysis of reverse-transcribed RNA from transduced neurons. Top panel: a 580 bp PCR product was detected with 9F-FLAGR primers only in neurons transduced with LV-TauPTM6 at m.o.i. = 5 and 10. Middle panel: 3R and 4R tau isoforms were detected with primers 9F and 13R. GAPDH mRNA was used for normalization (bottom panel). No RT: PCR with no RT using RNA from cells transduced with LV-TauPTM6 at m.o.i. = 10. ( B ) Details of the sequence of the 9F-FLAGR PCR product showing a correct splice junction between exons 9 and 10 and the presence of the FLAG epitope sequence at the 3′ end of the trans -spliced product. ( C ) Quantitative analysis of 4R/3R tau RNA ratios. 4R/3R ratios are expressed as mean ± SEM ( ** P < 0.01, n = 3). The percentage of isoform conversion is indicated above the respective bars. ( D ) Individual values of 4R and 3R tau RNA content. Values are expressed in arbitrary units as mean ± SEM (* P < 0.05, ** P < 0.025, n = 2).

Article Snippet: Htau transgenic mice, in a C57BL/6 background, were obtained from Jackson Laboratories (Bar Harbour, Maine, USA; B6.Cg-Mapttm1 (EGFP) Klt Tg(MAPT)8cPdav/J.

Techniques: Cell Culture, Control, Transduction, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Sequencing, FLAG-tag

Journal: Human Molecular Genetics

Article Title: Trans -splicing correction of tau isoform imbalance in a mouse model of tau mis-splicing

doi: 10.1093/hmg/ddt108

Figure Lengend Snippet: Translation of trans -spliced RNA into a chimeric protein. ( A ) Diagram of the strategy used for the detection of chimeric proteins by immunoprecipitation. An immobilized mouse monoclonal FLAG antibody was used to immunoprecipitate chimeric proteins. Immunoprecipitated proteins were analyzed by western blotting with N-terminal or C-terminal tau antibodies. The N-terminal part of the protein (grey box) corresponds to the part encoded by endogenous RNA (exons 1–9) and the C-terminal part (white box) corresponds to the part encoded by the PTM (exons 10–13 and FLAG epitope). ( B–D ) Immunoprecipitation analysis of FLAG-containing proteins from transduced cells. SH-SY5Y cells ( B ) or cortical neurons from htau mice at DIV3 ( C and D ) were transduced with LV-TauPTM6 at the indicated m.o.i. or with a control lentivirus (m.o.i. = 10). Transduced cells were lysed 14–16 days later and proteins immunoprecipitated with a FLAG antibody were analyzed by western blotting with an N-terminal tau antibody, a GAPDH antibody ( B and C ) or a C-terminal tau antibody ( D ) (input: 5 μl of cell lysate before immunoprecipitation). All samples had similar levels of tau before immunoprecipitation. No GAPDH immunoreactivity was detected in immunoprecipitates, ruling out non-specific precipitation by the FLAG antibody or by the affinity gel.

Article Snippet: Htau transgenic mice, in a C57BL/6 background, were obtained from Jackson Laboratories (Bar Harbour, Maine, USA; B6.Cg-Mapttm1 (EGFP) Klt Tg(MAPT)8cPdav/J.

Techniques: Immunoprecipitation, Western Blot, FLAG-tag, Transduction, Control

Journal: Human Molecular Genetics

Article Title: Trans -splicing correction of tau isoform imbalance in a mouse model of tau mis-splicing

doi: 10.1093/hmg/ddt108

Figure Lengend Snippet: Correction of tau mis-splicing in vivo in adult htau mouse brain. ( A ) Map of LV Syn -TauPTM6/LV Ub -TauPTM6 designed for TauPTM6 delivery in vivo . The viral backbone is the same as for LV-TauPTM6 (Fig. ). TauPTM6 is under the control of the synapsin promoter or of the ubiquitin promoter and is followed by the WPRE sequence (TSD: trans -splicing domain; F: FLAG epitope sequence). LV Syn -TauPTM6 or LV Ub -TauPTM6 was stereotaxically injected into the prefrontal cortex of 12–14-week-old htau mice; control mice were uninjected or injected with an LV expressing a PTM lacking the trans -splicing domain (LV-PTMΔTSD). RNA and protein were analyzed 8 months after injection. ( B ). RT–PCR analysis with the 9.1F-FLAGR primer combination. In addition to a 360 bp product, demonstrating PTM expression, a 550 bp trans -spliced product was detected after injection with LV Syn -TauPTM6 or LV Ub -TauPTM6. ( C ) RT-PCR analysis with the 9F-13R primer combination, demonstrating an increase in 4R tau mRNA after injection with LV Syn -TauPTM6. ( D ) Immunoprecipitation analysis of FLAG-containing proteins using N- or C- terminal tau antibodies (Input: 5 μl of brain homogenate before immunoprecipitation). An increased signal was detected with both antibodies after injection with LV Syn -TauPTM6.

Article Snippet: Htau transgenic mice, in a C57BL/6 background, were obtained from Jackson Laboratories (Bar Harbour, Maine, USA; B6.Cg-Mapttm1 (EGFP) Klt Tg(MAPT)8cPdav/J.

Techniques: In Vivo, Control, Ubiquitin Proteomics, Sequencing, FLAG-tag, Injection, Expressing, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation

Journal: Acta Neuropathologica Communications

Article Title: P53 aggregation, interactions with tau, and impaired DNA damage response in Alzheimer’s disease

doi: 10.1186/s40478-020-01012-6

Figure Lengend Snippet: p53 oligomers detected in aged tau mouse models, but not in aged control mice. a Representative immunofluorescent confocal images of cortex of 14-month-old htau, 5-month-old P301L, and 9-month-old C57BL/6 mice. Brain tissue probed with anti-p53 (red) and anti-I11 (green; oligomer-specific) in merge panel. b Magnified ROI from merged panel in A. (htau ROI 1 and 2) Peri-nuclear colocalization between p53 and I11 is shown with other oligomers in the vicinity. (P301L ROI 1 and 2) Peri-nuclear, circular colocalization between p53 and I11 is shown with other oligomers in the vicinity. (C57BL/6 ROI 1) No colocalization between p53 and I11 is detected in C57BL/6 mice. Keyence Microscope 60X magnification. Scale bar = 50 μm. c Graph depicting immunofluorescent intensity for I11 oligomer ( n = 2 in technical triplicate) and ( d ) p53 intensities from htau and C57BL/6 mice ( n = 2 in technical triplicate). Keyence Microscope 60X magnification. Scale bar = 50 μm. e Western blot of aged C57BL/6, htau, P301L, and Tau KO mice probed with anti-p53. f Densitometry of p53 bands shows a significant increase in p53 in htau mice at multiple bands compared to C57BL/6 and Tau KO mice ( n = 3). g Western blot of same mice probed with anti-MDM2 (90 kD). h Densitometry of MDM2 from G shows significant increase in MDM2 in htau compared to all other mouse models. P301L also show significantly more MDM2 compared to control and Tau KO mice. ( n = 3 in technical duplicate)

Article Snippet: C57BL/6 J (The Jackson Laboratory #000664), Tg2576/Tau P301L (P301L) (Taconic #2469), Tau knockout (KO) (The Jackson Laboratory #007251), and hemizygous human tau htau mice (The Jackson laboratory stock #005491) [ ] were bred at UTMB.

Techniques: Control, Microscopy, Western Blot

Journal: Biomolecules

Article Title: A Combination of Heavy Metals and Intracellular Pathway Modulators Induces Alzheimer Disease-like Pathologies in Organotypic Brain Slices

doi: 10.3390/biom14020165

Figure Lengend Snippet: Spreading of human amyloid-beta (hAβ42) and P301S aggregated tau (aggTau) in postnatal wild-type and transgenic organotypic brain slices. Brain slices were prepared from postnatal day 8–10 wild-type (WT) C57BL6 mice or transgenic (TG) amyloid precursor protein _Swedish–Dutch–Iowa (APP_SDI) mice. Collagen hydrogels with hAβ42, P301S aggTau, or a mix of both was loaded after 1 week in culture. Collagen hydrogels with an empty load were included as negative controls. Slices were cultured for 8 weeks, fixed and analyzed by immunohistochemistry for Aβ using the antibody clone 6E10 or for tau using the antibodies Tau5 or AT8. Images were taken at the 20× magnification with a field size of 523 × 392 µm, which were quantified using ImageJ (Version 5.5.0). ( A ) Quantification of the number of Aβ+ cells in the ventral areas of WT and TG APP_SDI slices. ( B ) Quantification of the Aβ+ fiber density. ( C ) Quantification of the number of Tau5+ cells in the ventral areas of WT and TG APP_SDI slices. ( D ) Quantification of the Tau5+ fiber density. The corresponding fiber densities were calculated by counting the number of times fibers crossed a predefined grid on the same image used to analyze the number of Aβ+ or Tau5+ cells. Note that no mature Aβ plaques or tau neurofibrillary tangles were observed in these postnatal slices. Values are given as mean ± SEM for each group and the values in parentheses represent the number of analyzed animals. The dots represent individual raw data values. Statistical analyses were performed using a one-way ANOVA with a Fisher’s LSD post-hoc test. * p < 0.05, *** p < 0.001 signifies comparisons against the respective (−) groups. § p < 0.05, §§§ p < 0.001 signifies comparisons between hAβ42 + P301S aggTau and hAβ42 groups. # p < 0.05 signifies comparisons between hAβ42+ P301S aggTau and P301S aggTau groups.

Article Snippet: The fixed coronal brain cryosections of transgenic mice expressing human tau (hTau) were commercially obtained from QPS Austria GmbH (Grambach, Austria).

Techniques: Transgenic Assay, Cell Culture, Immunohistochemistry

Journal: Biomolecules

Article Title: A Combination of Heavy Metals and Intracellular Pathway Modulators Induces Alzheimer Disease-like Pathologies in Organotypic Brain Slices

doi: 10.3390/biom14020165

Figure Lengend Snippet: Characterization of amyloid-beta (Aβ) plaques and tau neurofibrillary tangles (NFT) in post-mortem human and transgenic mouse slices. Tissue sections from the temporal lobe of a human Alzheimer’s disease (AD) patient ( A – D ) were probed for the appearance of Aβ plaques and tau NFT. Following the deparaffinization and antigen retrieval protocols, the slices were subject to immunohistochemistry. ( A ) The lack of a primary antibody (w/o) served as a negative control with background staining. ( B ) An Aβ plaque was detected by using the Aβ antibody clone 6E10 with a dense plaque core and the surrounding corona. ( C ) Tau containing NFTs were visualized in human tissue sections with a densely stained cytoplasm and unstained nuclei. ( D ) A representative image displayed the pyramid-shaped tau NFT (green; AlexaFluor-488), Aβ plaques (red; Thiazine Red dye) and nuclei (blue; DAPI). ( E ) Slices from transgenic (TG) amyloid precursor protein _Swedish–Dutch–Iowa (APP_SDI) mice ( F – H ) exhibited no staining without a primary antibody (w/o). ( F ) Slices from TG APP_SDI mice displayed numerous Aβ plaque structures. ( G ) Slices from a TG mouse model expressing the human tau protein were commercially obtained and probed for tau NFTs, which could be detected with intracellular staining and unstained nuclei. ( H ) A representative image from the human tau-expressing TG mice showed strong staining in neuronal cells detected by AT8 antibody (green; AlexaFluor-488) with nuclear staining (blue; DAPI). Scale bar in G = 50 µm in ( A – C ); 27 µm in ( D ); 100 µm in ( E – G ); 27 µm in ( H ).

Article Snippet: The fixed coronal brain cryosections of transgenic mice expressing human tau (hTau) were commercially obtained from QPS Austria GmbH (Grambach, Austria).

Techniques: Transgenic Assay, Immunohistochemistry, Negative Control, Staining, Expressing

Journal: Biomolecules

Article Title: A Combination of Heavy Metals and Intracellular Pathway Modulators Induces Alzheimer Disease-like Pathologies in Organotypic Brain Slices

doi: 10.3390/biom14020165

Figure Lengend Snippet: Culturing of adult slices from wild-type (WT) and transgenic (TG) animals. ( A ) Composite image of a half-brain adult slice shows AT8+ immunoreactivity after 9 weeks of culture with the magnified image showing representative AT8+ immunoreactivity in the ventral areas. ( B ) Acute fresh slices from 3 different adult WT animals (day 0) were collected and frozen immediately. Conversely, adult WT slices were cultured for 9 weeks and then compared via Western blotting. The acute slices display a strong signal for neurofilament and GFAP, whereas cultured slices show a severely reduced signal indicating the general decline of viability post-culture. ( C ) Adult slices from TG amyloid precursor protein _Swedish–Dutch–Iowa (APP_SDI) mice were incubated with collagen hydrogels containing human amyloid-beta 42 (hAβ42) or P301S aggregated tau (aggTau) for 9 weeks. A significantly increased number of AT8+ cells were quantified in the ventral areas between hAβ42- or P301S aggTau-loaded hydrogels and the empty hydrogels (minus group). The number of AT8+ cells were counted from the ventral regions and the values are reported as mean ± SEM. ( D ) Adult WT slices were supplemented with either scopolamine, wortmannin, MHY1485, lead and cadmium (50, 10, 50, 100, 100 nM final concentration, respectively) or normal media after 4 weeks of culture and then further cultured for 4 weeks. Slices were fixed and immunostained with the Aβ clone 6E10 or AT8 antibodies. No significant differences were found in the groups treated with the combination treatment and normal slice media. Images were quantified using ImageJ. Raw optical density measurements were inverted by correcting for slice background for each image such that 0 represents white and 255 represents black. Values are generated from an average of two images from left and right hemispheres per animal and values in parentheses indicate the number of analyzed animals. Statistical analyses were performed using a student’s t -test with equal variance, where p values < 0.05 represent significance versus the Minus (−) group (* p < 0.05, ** p < 0.01).

Article Snippet: The fixed coronal brain cryosections of transgenic mice expressing human tau (hTau) were commercially obtained from QPS Austria GmbH (Grambach, Austria).

Techniques: Transgenic Assay, Cell Culture, Western Blot, Incubation, Concentration Assay, Generated

Journal: International Journal of Alzheimer's Disease

Article Title: Drosophila Models of Tauopathies: What Have We Learned?

doi: 10.1155/2012/970980

Figure Lengend Snippet: Tau constructs available in Drosophila.

Article Snippet: The Lu lab showed that the 0N4R hTau R406W/S2A construct is not phosphorylated at some Sgg sites and is refractory to toxic enhancement by Sgg overexpression [ ], while the Jackson lab found that 2N4R hTau S2A , although less toxic, was still phosphorylated at Sgg sites when Sgg is overexpressed [ ].

Techniques: Construct, Mutagenesis

Journal: International Journal of Alzheimer's Disease

Article Title: Alpha 1-Antichymotrypsin, an Inflammatory Protein Overexpressed in the Brains of Patients with Alzheimer's Disease, Induces Tau Hyperphosphorylation through c-Jun N-Terminal Kinase Activation

doi: 10.1155/2013/606083

Figure Lengend Snippet: Tau hyperphosphorylation in ACT expressing mice: cortex and hippocampal regions from normal nontransgenic (mTau), ACT expressing (ACT/mTau), human tau expressing (hTau), and both ACT and hTau expressing (ACT/mTau/hTau) mice were analyzed for changes in phosphorylation of tau at PHF-1 (a), p-Ser202 (b), pThr231 (c) specific epitopes, and total tau (d) using the corresponding P-tau or the TG5 total tau antibody by western blot and quantified using the Image J image analysis software. The data shows significant increase in P-tau levels in ACT and hTau as well as the double transgenic ACT/hTau mice (* P < 0.05). Only P-Ser202 tau showed significant change in double transgenic mice compared to mice expressing hTau alone ( # P < 0.05).

Article Snippet: The human tau (hTau) mice [ ] were purchased from Jackson laboratories.

Techniques: Expressing, Phospho-proteomics, Western Blot, Software, Transgenic Assay