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

Journal: Bioengineering & Translational Medicine

Article Title: Recent advances in cellular biosensor technology to investigate tau oligomerization

doi: 10.1002/btm2.10231

Figure Lengend Snippet: Schematic representation of tau biosensors based on fluorescence resonance energy transfer (FRET), bimolecular fluorescence complementation (BiFC), or split luciferase complementation (SLC) for monitoring intramolecular and intermolecular tau interactions in living cells. (a) Tau intramolecular FRET biosensor where FRET is observed when there is global folding of wild‐type (WT) monomeric tau. (b) Tau intermolecular FRET biosensors where FRET is observed when tau oligomers or aggregates are formed. WT tau is used for the formation of non‐β‐sheet soluble tau oligomers and tau repeat domain (tauRD) with P301S mutation or truncated tau is used for the formation of β‐sheet insoluble tau aggregates. , , (c) BiFC tau fluorescence turn‐off biosensor where fluorescence is absent when there is tau oligomerization or aggregation. , (d) BiFC/SLC tau fluorescence/luminescence turn‐on biosensor where fluorescence or luminescence is present when tau oligomers or aggregates are formed. , , Tau oligomer is drawn as a dimer for illustration but it can be any species more than a dimer (≥2‐mers)

Article Snippet: In a separate study, the above‐mentioned WT 2N4R tau intermolecular FRET biosensor was adopted and expressed in SH‐SY5Y cells to perform HTS on ChemBridge DIVERSet library of 10,000 compounds to identify 195 small molecules that inhibit tau oligomerization.

Techniques: Fluorescence, Förster Resonance Energy Transfer, Luciferase, Mutagenesis

Journal: Bioengineering & Translational Medicine

Article Title: Recent advances in cellular biosensor technology to investigate tau oligomerization

doi: 10.1002/btm2.10231

Figure Lengend Snippet: Cellular biosensor technologies to study tau oligomerization and aggregation

Article Snippet: In a separate study, the above‐mentioned WT 2N4R tau intermolecular FRET biosensor was adopted and expressed in SH‐SY5Y cells to perform HTS on ChemBridge DIVERSet library of 10,000 compounds to identify 195 small molecules that inhibit tau oligomerization.

Techniques:

Journal: Bioengineering & Translational Medicine

Article Title: Recent advances in cellular biosensor technology to investigate tau oligomerization

doi: 10.1002/btm2.10231

Figure Lengend Snippet: Cellular tau fluorescence resonance energy transfer (FRET) biosensors to examine tau oligomerization and aggregation. (a) Through acceptor photobleaching method, basal FRET is observed in both 0N4R wild‐type (WT) tau (T4) and Δ421‐441 truncated tau (T4C3) CFP/YFP FRET biosensors in the presence of kinase‐dead (Kd) glycogen synthase kinase 3 beta (GSK3β) (absence of GSK3β kinase activity). FRET efficiencies are increased with treatment of active GSK3β. (b) T4 FRET biosensors form soluble tau species or oligomers while T4C3 FRET biosensors form insoluble aggregates in the presence of active GSK3β. (c) Lifetime measurement of the WT 2N4R tau intermolecular GFP/RFP FRET biosensor shows a shorter lifetime in tau‐GFP/RFP (donor‐acceptor) sample as compared to tau‐GFP (donor) sample which exhibits efficient FRET. (d) Thioflavin‐S (ThS) staining of human embryonic kidney 293 (HEK293) cells expressing tau‐RFP (same total DNA concentration as the FRET biosensor) shows a positive signal with treatment of tau preformed fibrils (PFF) as a positive control. In the absence of PFF, there is no ThS signal, indicating formation of nonβ‐sheet soluble oligomers by the WT 2N4R tau FRET biosensor. (e) FRET measurement of tauRD CFP/YFP FRET biosensors with different tau variants expressed in HEK293 cells. PP refers to ΔK280/I227P/I308P mutations, ΔK refers to ΔK280, and LM refers to P301L/V337M mutations. (f) Formation of inclusions are observed in WT, ΔK and LM variants of the tauRD CFP/YFP FRET biosensors, as characterized by positive staining of X‐34 which is an amyloid‐specific dye. Permissions obtained from References , , and

Article Snippet: In a separate study, the above‐mentioned WT 2N4R tau intermolecular FRET biosensor was adopted and expressed in SH‐SY5Y cells to perform HTS on ChemBridge DIVERSet library of 10,000 compounds to identify 195 small molecules that inhibit tau oligomerization.

Techniques: Fluorescence, Förster Resonance Energy Transfer, Activity Assay, Staining, Expressing, Concentration Assay, Positive Control

Journal: Bioengineering & Translational Medicine

Article Title: Recent advances in cellular biosensor technology to investigate tau oligomerization

doi: 10.1002/btm2.10231

Figure Lengend Snippet: Translational applications of fluorescence resonance energy transfer (FRET) and tau‐bimolecular fluorescence complementation (BiFC) biosensors in high‐throughput screening drug discovery. (a) High‐throughput screening of NIH Clinical Collection (NCC) library containing 727 compounds using wild‐type (WT) 2N4R tau intermolecular FRET biosensor expressed in human embryonic kidney 293 (HEK293) cells to obtain a small molecule inhibitor (MK‐886) of tau oligomerization. (b) MK‐886 reduces FRET in both 2N4R WT and P301L tau intermolecular biosensors with EC50 values of 1.40 and 1.84 μM, respectively. (c) High‐throughput screening of 1018 FDA approved compounds using Venus‐based tau‐BiFC fluorescence turn‐on biosensor expressed in HEK293 cells. Tau‐BiFC OFF (+), Tau‐BiFC ON (green), FDA library (gray), methylene blue (MB, blue), LMTM (cyan), and levosimendan (hit compound, yellow) are indicated on the plot. (d) Treatment of levosimendan at different timings along the aggregation cascade of K18‐P301L induced tau aggregation in tau‐BiFC cells. Inhibitory effect of levosimendan is characterized by decreases in the tau‐BiFC responses. Permissions obtained from References and

Article Snippet: In a separate study, the above‐mentioned WT 2N4R tau intermolecular FRET biosensor was adopted and expressed in SH‐SY5Y cells to perform HTS on ChemBridge DIVERSet library of 10,000 compounds to identify 195 small molecules that inhibit tau oligomerization.

Techniques: Fluorescence, Förster Resonance Energy Transfer, High Throughput Screening Assay, Drug discovery