Journal: Neuroscience

Article Title: Cre-inducible Adeno Associated Virus-mediated Expression of P301L Mutant Tau Causes Motor Deficits and Neuronal Degeneration in the Substantia Nigra

doi: 10.1016/j.neuroscience.2019.10.001

Figure Lengend Snippet: iAAV-mediated delivery of transgenes in mouse substantia nigra. (A) In vitro validation of Cre-dependent transgenes expression in HEK293 cells by western blot. Co-transfection of AAV-FLEX-WT tau (positive control) and AAV-FLEX-P301L tau plasmid with Cre expression plasmid showed a robust expression of human tau using monoclonal HT7 antibody. No tau gene expression was observed when Cre was replaced with ΔCre plasmid, which expresses inactive Cre recombinase. (B) Unilateral injection of saline, AAV2/6-FLEX-GFP, or AAV2/6-FLEX-P301L tau in the SN of 20-week-old TH-Cre mice. The injection site (coordinates: −3.10 mm AP, −1.12 mm ML, −4.34 mm DV) is indicated by a red asterisk, where the substantia nigra is located. (C) Experimental paradigm. Mice underwent surgery at postnatal week 20 and behavioral tests were performed from week 26 to week 30 as shown. (D) Expression of Cre-dependent GFP in tyrosine hydroxylase promoter Cre-driver mice. The TH expressing dopaminergic neurons in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA), rather than nondopaminergic neurons in the substantia nigra pars reticulata (SNr), can express GFP fluorescence. (E) Expression of Cre-dependent tau in tyrosine hydroxylase promoter Cre-driver mice. Targeted human P301L tau was specifically immunostained with HT7 antibody in TH cells of the substantia nigra (indicated by white arrowheads). Scale bars: A, 500 μm; C, 200 μm (left) and 100 μm (right); D, 100 μm and 20 μm (magnification).

Article Snippet: Of them, AAV-FLEX-GFP and AAV-FLEX-P301L tau plasmids were packaged by SignaGen Laboratories (SignaGen Laboratories, USA) to create recombinant pseudotyped AAV2/6 virus, while AAV-FLEX-WT tau was only used for HEK293 cells transfection in vitro .

Techniques: In Vitro, Expressing, Western Blot, Cotransfection, Positive Control, Plasmid Preparation, Injection, Fluorescence

Journal: Neuroscience

Article Title: Cre-inducible Adeno Associated Virus-mediated Expression of P301L Mutant Tau Causes Motor Deficits and Neuronal Degeneration in the Substantia Nigra

doi: 10.1016/j.neuroscience.2019.10.001

Figure Lengend Snippet: P301L tau transfer causes significant loss of TH neurons and degeneration of SN. (A) Immunohistochemistry of tyrosine hydroxylase and transgenes (GFP or P301L tau) in the substantia nigra. TH was preserved in both the ipsilateral and contralateral sides in the GFP-injected mice, whereas P301L tau expression obliterated TH on the ipsilateral side of injection. (B-C) Comparison of total TH positive cells between contralateral and ipsilateral side in SN for AAV-FLEX-GFP (B) and AAV-FLEX-P301L tau (C) group. Tau transfer reduced 50% of TH neurons in the ipsilateral side relative to contralateral side. (D) Number of transduced cells by AAV-FLEX-GFP or AAV-FLEX-P301L tau vector. The number of cells expressing GFP or tau are comparable, suggesting the similar transduction efficiency in each group. (E) Percentage of TH+ GFP+ cells in total GFP+ cells and TH+ HT7+ cells in total HT7+ cells, respectively. Fewer tau positive cells are simultaneously expressing TH, revealing the cytotoxicity of tau in dopaminergic neurons. (C-E) N = 4 animals per group for quantification. (F) Immunohistochemistry of astrocyte stained by GFAP or microglia stained by Iba1 with transgenes (GFP or P301L tau) in the substantia nigra. No transfected cells were co-localized with astrocyte or microglia in the GFP-injected side, whereas some of HT7+ cells were co-localized with Iba1+ microglia (indicated by white arrows), suggesting the engulfment of tau-induced degenerative neurons by microglia. (G) Tau pathology was detected with phosphorylated tau antibodies (AT8 and CP13, indicated by white arrows) in the substantia nigra of Tau-injected ipsilateral side. Scale bars: A, 200 μm; F, 100 μm; G, 100 μm and 20 μm (4 × Enlarged). Data are presented by box and whisker plots which show the median and the 25th to 75th percentiles; ****p < 0.0001, n.s., no significance, using Wilcoxon matched pairs test (B-C) or using Mann–Whitney U test (D-E).

Article Snippet: Of them, AAV-FLEX-GFP and AAV-FLEX-P301L tau plasmids were packaged by SignaGen Laboratories (SignaGen Laboratories, USA) to create recombinant pseudotyped AAV2/6 virus, while AAV-FLEX-WT tau was only used for HEK293 cells transfection in vitro .

Techniques: Immunohistochemistry, Injection, Expressing, Plasmid Preparation, Transduction, Staining, Transfection, Whisker Assay, MANN-WHITNEY