Journal: Nature neuroscience

Article Title: Tau induces PSD95-nNOS uncoupling and neurovascular dysfunction independent of neurodegeneration

doi: 10.1038/s41593-020-0686-7

Figure Lengend Snippet: A . Neocortical CBF, assessed by ASL-MRI, shows no reduction in 2-3-month-old rTg4510 and small reduction in 2-3-month-old PS19 mice, compared to age-matched WT mice. N=10/group; one-way analysis of variance (ANOVA) with Tukey’s test for multiple comparisons. B . Neocortical thickness, assessed bilaterally in T2-weighted MRI images at the level of the somatosensory cortex (−1.22 to −1.70 from bregma), is comparable in PS19, rTg4510, and WT mice. Scale bar=1 mm. N=10/group. C . The number of neurons (NeuN + ) does not differ in PS19, rTg4510, and WT mice. N=5/group (see also ). D-F . No cognitive impairment is observed at the Barnes maze test ( D ), novel object recognition ( E ), or Y-maze test ( F ). N=5/group. G-H . The increases in CBF induced in the whisker barrel cortex by mechanical stimulation of the facial whiskers ( G ; N=5/group; one-way ANOVA with Tukey’s test), but not by neocortical superfusion of acetylcholine ( H ), is markedly attenuated both in PS19 and rTg4510 mice, compared to WT mice. I . Neocortical superfusion of recombinant tau (WT tau or P301L mutant) attenuates the increase in CBF evoked by whisker stimulation in a dose-dependent manner. N=5/group; one-way ANOVA with Tukey’s test between groups at each doses. Data are presented as mean±SEM. See for statistical parameters.

Article Snippet: PSD95 was amplified from C57BL6/J mouse cDNA with primers containing Eco RI (5’- gcacGAATTCatatggactgtctctgtatagtg-3’) and Xho I (5’-gataCTCGAGtcagagtctctctcgggctgg-3’) restriction sites and subsequently cloned into Eco RI/ Xho I-digested pCMV-Myc vector (Clontech). pcDNA3.1-nNOS , pRK5-EGFP-Tau WT and pRK5-EGFP-Tau P301L were kind gifts from Yoichi Osawa (University of Michigan Medical School, Ann Arbor, MI) and Karen Ashe (Addgene plasmid #46904 and #46908; University of Minnesota, Minneapolis, MN), respectively. pcDNA3.1 (Thermo Fisher Scientific) and pEGFP-C3 (Clontech) plasmids were used as empty vector expression controls.

Techniques: Whisker Assay, Recombinant, Mutagenesis

Journal: Nature neuroscience

Article Title: Tau induces PSD95-nNOS uncoupling and neurovascular dysfunction independent of neurodegeneration

doi: 10.1038/s41593-020-0686-7

Figure Lengend Snippet: No changes in locomotor activity are observed in novel object recognition and Y-maze tests of rTg4510, compared to WT mice. N=5 for novel object; N=6 for Y-maze. B . The increases in CBF-LDF induced in the whisker barrel cortex by mechanical stimulation of the facial whiskers were markedly attenuated in PS19 mice also under the isoflurane anesthesia regimen used in ASL-MRI studies. N=5/group; two-tailed unpaired t-test. C . The increases in CBF-LDF produced by the endothelium-dependent (bradykinin or A23187) and -independent (SNAP, adenosine, or hypercapnia) vasodilators are comparable in PS19, rTg4510, and WT mice. N=5/group. D . Recombinant full-length mutant (2N4R, P301L; 5 μM) or WT (2N4R; 5 μM) tau has no effect on CBF response induced by acetylcholine or adenosine. N=5/group. Data are presented as mean±SEM. One-way ANOVA and Tukey’s test.

Article Snippet: PSD95 was amplified from C57BL6/J mouse cDNA with primers containing Eco RI (5’- gcacGAATTCatatggactgtctctgtatagtg-3’) and Xho I (5’-gataCTCGAGtcagagtctctctcgggctgg-3’) restriction sites and subsequently cloned into Eco RI/ Xho I-digested pCMV-Myc vector (Clontech). pcDNA3.1-nNOS , pRK5-EGFP-Tau WT and pRK5-EGFP-Tau P301L were kind gifts from Yoichi Osawa (University of Michigan Medical School, Ann Arbor, MI) and Karen Ashe (Addgene plasmid #46904 and #46908; University of Minnesota, Minneapolis, MN), respectively. pcDNA3.1 (Thermo Fisher Scientific) and pEGFP-C3 (Clontech) plasmids were used as empty vector expression controls.

Techniques: Activity Assay, Whisker Assay, Two Tailed Test, Produced, Recombinant, Mutagenesis

Journal: Nature neuroscience

Article Title: Tau induces PSD95-nNOS uncoupling and neurovascular dysfunction independent of neurodegeneration

doi: 10.1038/s41593-020-0686-7

Figure Lengend Snippet: A . Doxycycline treatment ( tau off ) for 3-4 months reduces neuronal loss in rTg4510 mice, compared to rTg4510 mice fed control diet ( con or tau on ). Treatment with doxycycline ( doxy ) has no effect in WT mice (See for quantification). B . Suppressing tau production reduces AT-8 + tau levels, but not thioflavin-S + neurofibrillary tangles. Arrows indicate co-localization between thioflavin-S + neurofibrillary tangles and AT8 + tau, and asterisks denote strong thioflavin-S + neurofibrillary tangles with faint or no AT8 + tau. Representative pictures from N=5 mice/group. C . Doxycycline treatment reduces human tau P301L mRNA, but has no effect on mouse tau mRNA. N=5 for WT in mouse tau mRNA and for rTg4510 (tau on) in human tau mRNA; N=4 for rTg4510 in mouse tau mRNA; N=6 for rTg4510 (tau off) in human tau mRNA. Images are representative of 3 independent experiments, each including 5 mice/group. Two-tailed unpaired t-test. Data are presented as mean±SEM.

Article Snippet: PSD95 was amplified from C57BL6/J mouse cDNA with primers containing Eco RI (5’- gcacGAATTCatatggactgtctctgtatagtg-3’) and Xho I (5’-gataCTCGAGtcagagtctctctcgggctgg-3’) restriction sites and subsequently cloned into Eco RI/ Xho I-digested pCMV-Myc vector (Clontech). pcDNA3.1-nNOS , pRK5-EGFP-Tau WT and pRK5-EGFP-Tau P301L were kind gifts from Yoichi Osawa (University of Michigan Medical School, Ann Arbor, MI) and Karen Ashe (Addgene plasmid #46904 and #46908; University of Minnesota, Minneapolis, MN), respectively. pcDNA3.1 (Thermo Fisher Scientific) and pEGFP-C3 (Clontech) plasmids were used as empty vector expression controls.

Techniques: Control, Two Tailed Test

Journal: Nature neuroscience

Article Title: Tau induces PSD95-nNOS uncoupling and neurovascular dysfunction independent of neurodegeneration

doi: 10.1038/s41593-020-0686-7

Figure Lengend Snippet: A. NMDA (40 μM) increases NO production in neurons dissociated from 2-3-month-old WT mice, but not in neurons from age-matched PS19 or rTg4510 mice. N=6/group; two-tailed paired t-test. B . NMDA (100 μM)-induced NO production, detected amperometrically, is attenuated in PS19 mice compared to WT. N=5/group; two-tailed unpaired t-test. C . Recombinant tau (rTau; P301L mutant; 400 nM) attenuates NO production induced by NMDA in WT neurons. N=7/group; one-way ANOVA and Tukey’s test. D . Total tau and phosphorylated tau (pTau) are elevated in post-synaptic density (PSD) preparations from 2-3-month-old PS19 (PS) compared to age-matched WT mice. N=12 for WT total Tau, pTau (AT8), and pTau (PHF13); N=13 for PS total tau; N=7 for PS pTau (AT8); N=5 for PS pTau (PHF13); two-tailed unpaired t-test. Data are presented as mean±SEM. E . The association of GluN2B to PSD95, detected by co-immunoprecipitation (IP), is comparable in PS19 and WT mice. N=6/group. Data are presented as mean±SEM. F . Association of PSD95 with nNOS, assessed by IP, is markedly reduced in PS19 mice compared to WT. Data are presented as mean±SEM. N=8/group; two-tailed unpaired t-test. Immunoblots in D, E, and F are cropped; full gel pictures are shown in

Article Snippet: PSD95 was amplified from C57BL6/J mouse cDNA with primers containing Eco RI (5’- gcacGAATTCatatggactgtctctgtatagtg-3’) and Xho I (5’-gataCTCGAGtcagagtctctctcgggctgg-3’) restriction sites and subsequently cloned into Eco RI/ Xho I-digested pCMV-Myc vector (Clontech). pcDNA3.1-nNOS , pRK5-EGFP-Tau WT and pRK5-EGFP-Tau P301L were kind gifts from Yoichi Osawa (University of Michigan Medical School, Ann Arbor, MI) and Karen Ashe (Addgene plasmid #46904 and #46908; University of Minnesota, Minneapolis, MN), respectively. pcDNA3.1 (Thermo Fisher Scientific) and pEGFP-C3 (Clontech) plasmids were used as empty vector expression controls.

Techniques: Two Tailed Test, Recombinant, Mutagenesis, Immunoprecipitation, Western Blot

Journal: Nature neuroscience

Article Title: Tau induces PSD95-nNOS uncoupling and neurovascular dysfunction independent of neurodegeneration

doi: 10.1038/s41593-020-0686-7

Figure Lengend Snippet: A. Exogenously expressed Tau P301L in HEK293T cells is phosphorylated, as shown by sensitivity to phosphatase treatment. N=4/group; two-tailed paired t-test. Data are presented as mean±SEM. B . Co-expression of Tau P301L reduces PSD95 association with nNOS. PSD95 was immunoprecipitated (IP) and associated nNOS was detected. N=10/group; two-tailed unpaired t-test. Data are presented as mean±SEM. C. nNOS does not bind to immunoprecipitated Tau P301L. A representative blot is shown from N=9/group. D . Tau P301L associates with PSD95, which was immunoprecipitated (IP) from HEK293T cell lysates. Data are presented as mean±SEM. A representative image is shown from N=9/group. Immunoblots in are cropped; full gel pictures are shown in

Article Snippet: PSD95 was amplified from C57BL6/J mouse cDNA with primers containing Eco RI (5’- gcacGAATTCatatggactgtctctgtatagtg-3’) and Xho I (5’-gataCTCGAGtcagagtctctctcgggctgg-3’) restriction sites and subsequently cloned into Eco RI/ Xho I-digested pCMV-Myc vector (Clontech). pcDNA3.1-nNOS , pRK5-EGFP-Tau WT and pRK5-EGFP-Tau P301L were kind gifts from Yoichi Osawa (University of Michigan Medical School, Ann Arbor, MI) and Karen Ashe (Addgene plasmid #46904 and #46908; University of Minnesota, Minneapolis, MN), respectively. pcDNA3.1 (Thermo Fisher Scientific) and pEGFP-C3 (Clontech) plasmids were used as empty vector expression controls.

Techniques: Two Tailed Test, Expressing, Immunoprecipitation, Western Blot