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Tau induces PSD95–neuronal NOS uncoupling and neurovascular dysfunction independent of neurodegeneration

Abstract

Cerebrovascular abnormalities have emerged as a preclinical manifestation of Alzheimer’s disease and frontotemporal dementia, diseases characterized by the accumulation of hyperphosphorylated forms of the microtubule-associated protein tau. However, it is unclear whether tau contributes to these neurovascular alterations independent of neurodegeneration. We report that mice expressing mutated tau exhibit a selective suppression of neural activity-induced cerebral blood flow increases that precedes tau pathology and cognitive impairment. This dysfunction is attributable to a reduced vasodilatation of intracerebral arterioles and is reversible by reducing tau production. Mechanistically, the failure of neurovascular coupling involves a tau-induced dissociation of neuronal nitric oxide synthase (nNOS) from postsynaptic density 95 (PSD95) and a reduced production of the potent vasodilator nitric oxide during glutamatergic synaptic activity. These data identify glutamatergic signaling dysfunction and nitric oxide deficiency as yet-undescribed early manifestations of tau pathobiology, independent of neurodegeneration, and provide a mechanism for the neurovascular alterations observed in the preclinical stages of tauopathies.

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Fig. 1: Neurovascular coupling is selectively disrupted in tau mice before tau pathology and cognitive impairment.
Fig. 2: Suppression of neurovascular coupling in tau mice results from impaired vasodilatation of intracerebral arterioles.
Fig. 3: Spontaneous and evoked neural activity and NMDA-induced Ca2+ increase are not altered in PS19 mice.
Fig. 4: Suppressing tau production rescues neurovascular coupling and prevents cortical atrophy and cognitive deficits in older rTg4510 mice.
Fig. 5: The NMDA-dependent component of functional hyperemia is selectively suppressed in tau mice.
Fig. 6: The NO-dependent component of functional hyperemia is attenuated in tau mice.
Fig. 7: Suppression of NMDAR-induced neuronal NO production and dissociation of nNOS from PSD95 in tau mice.
Fig. 8: Tau disrupts the PSD95–nNOS association by binding to PSD95.

Data availability

All the data supporting the conclusions of the current study are presented in the figures. If necessary, the data that support the findings of this study are available from the corresponding authors upon reasonable request. There are no restrictions on data availability. Source data are provided with this paper.

Code availability

No code was used for the study.

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Acknowledgements

This work was supported by NIH grants R01-NS37853 (to C.I.), R01-NS097805 (to L.P.) and R01-NS109588 (to K.H.), the Japan Heart Foundation/Bayer Research Grant Abroad (to Y.H.), The Uehara Memorial Foundation Research Fellowship (to Y.H.), the Japan Society for the Promotion of Science Overseas Research Fellowship (to Y.H.), and American Heart Association Postdoctoral Fellowship 20POST35120063 (to S.J.A.). We thank C. B. Schaffer for helpful suggestions and editing. Support from the Feil Family Foundation is gratefully acknowledged.

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Authors

Contributions

L.P., K.H. and Y.H. conducted the experiments and performed the data analyses. S.J.A. and A.A. conducted the 2PM and LSI experiments. G.W. performed the NO production and Ca2+ imaging experiments. J.S. and K.U. contributed to the histology experiments. I.B. and V.P. contributed to the immunoprecipitation and western blotting experiments. D.E. and D.A. provided WT rTau. L.P., K.H., P.Z., J.A. and C.I. supervised the research. L.P., K.H. and C.I. provided funding. L.P. and C.I. wrote the manuscript.

Corresponding authors

Correspondence to Laibaik Park or Costantino Iadecola.

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C.I. serves on the advisory board of Broadview Ventures. The other authors have no conflicts to declare.

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Extended data

Extended Data Fig. 1 Resting CBF and thickness in PS19, rTg4510, and WT mice at 2-3 months of age.

Resting CBF and thickness were bilaterally assessed by ASL- and T2-MRI, respectively, in neocortex, entorhinal cortex, and hippocampus at the bregma level from -1.22 to -1.70 mm. a, b, A small reduction in entorhinal (A) and hippocampal (B) CBF is found in PS19 mice and not in rTg4510 mice compared to age-matched WT mice, while entorhinal cortex thickness (A) and hippocampal volume (B) are comparable in both strains. N = 10/group; one-way analysis of variance (ANOVA) with Tukey’s test for multiple comparisons. c, d, Resting CBF (C) and thickness (D) in the neocortex, entorhinal cortex, and hippocampus are comparable in rTg4510 mice, expressing both tau (Tau+) and transactivator (TA+), and their littermates (WT; Tau+ only; TA+ only). Scale bar=1 mm; N = 5/group; one-way ANOVA with Tukey’s test. Data are presented as mean ± SEM. See Source Data 7 for statistical parameters.

Source data

Extended Data Fig. 2 No neurofibrillary tangles are observed in 2-3-month-old PS19, rTg4510, and WT mice.

Neurofibrillary tangles, assessed by the thioflavin-S stain, are not observed in the somatosensory cortex of 2-3-month-old WT (a–c), PS19 (d), and rTg4510 (g) mice, but phosphorylated tau (AT-8) is observed in PS19 (e, f) and rTg4510 (h, i). Images are representative of 3 independent experiments, each including 5 mice/group.

Extended Data Fig. 3 Microvessels, microglia/macrophages, and astrocytes in the neocortex of 2-3-month-old tau mice.

Neocortical cluster of differentiation (CD) 31 (CD31+) microvessels (a; N = 5/group) and Iba1+ microglia/macrophages (b; N = 5/group) are comparable in PS19, rTg4510, and WT mice at the age of 2-3 months, but enhanced astrogliosis (GFAP+ cells) is found in rTg4510 mice, compared to PS19 and WT mice (c; N = 5/group). One-way ANOVA and Tukey’s test. Data are presented as mean ± SEM. See Source Data 8 for statistical parameters.

Source data

Extended Data Fig. 4 No neuronal loss occurs in PS19 and rTg4510 mice, but mislocalized phosphorylated tau is observed in somatodendritic compartments.

The number of neurons (NeuN+) is comparable in PS19, rTg4510, and WT mice (a, see quantification in Fig. 1c). As anticipated, in PS19 and rTg4510 mice phosphorylated tau (AT-8+) is co-localized with neurons (NeuN+) (A) and axons (myelin basic protein, MBP+) (b). Phosphorylated tau is also observed in dendritic spines (MAP2+) (c), indicating displacement of tau to somatodendritic compartments. Images are representative of 3 independent experiments, each including 5 mice/group.

Extended Data Fig. 5 Locomotor activity and neurovascular response in 2-3-month-PS19 and rTg4510 mice.

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.

Extended Data Fig. 6 Suppressing tau production with doxycycline reduces P301L tau expression and prevents neuronal loss, tau accumulation in 7-8-month-old rTg4510 mice.

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 Fig. 4c 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.

Extended Data Fig. 7 Suppressing tau production with doxycycline prevents cognitive deficits in 7-8-month-old rTg4510 mice.

a, Suppressing tau production with doxycycline (tau off or doxy), compared with control diet (tau on or con), prevents the CBF reduction, assessed by ASL-MRI. N = 7/group; two-way ANOVA with Tukey’s test. b, c, Suppressing tau production prevents cognitive deficits, assessed by novel object recognition (b; N = 10 for WT tau on & off and rTg4510 tau on; N = 9 for rTg4510 tau off) and Y-maze test (c; N = 10/group), but has no effect on locomotor activity, as reflected by distance traveled (b) or number of arm entries (c). Locomotor activity of rTg4510 mice in the novel object test seems more variable (b). Data are presented as mean ± SEM. Two-way ANOVA with Tukey’s test. d, The CBF increase induced by neocortical superfusion of the NO donor SNAP or adenosine is not altered in 7-8 month-old rTg4510 mice. N = 5/group; two-way ANOVA with Tukey’s test. Data are presented as mean ± SEM. See Source Data 9 for statistical parameters.

Source data

Extended Data Fig. 8 Aquaporin-4 immunoreactivity and astrogliosis are unaffected by suppressing tau production with doxycycline in 7-8-month-old rTg4510 mice.

a, AQP-4 immunoreactivity, which labels astrocytic end-feet, was not disrupted in rTg4510 mice with or without doxycycline. N = 5/group; two-way ANOVA with Tukey’s test; p = 0.2420 between WT con & WT doxy, p = 0.9985 between WT con & rTg4510 tau on, p = 0.8585 between WT con & rTg4510 tau off, p = 0.1881 between WT doxy & rTg4510 tau on, p = 0.6502 between WT doxy & rTg4510 tau off, and p = 0.7804 between rTg4510 tau on & off. Data are presented as mean ± SEM. b, The astrogliosis (GFAP+ cells) observed in rTg4510 mice was not reduced with tau suppression. Data are presented as mean ± SEM. N = 5/group; two-way ANOVA with Tukey’s test. See Source Data 10 for statistical parameters.

Source data

Extended Data Fig. 9 MK-801 and TTX effect on CBF, L-NNA effect on NMDA-induced NO production and expression of NMDA receptor subunits in 2-3-month-old rTg4510 mice.

a, MK-801 and/or TTX have no effect on resting CBF or CBF response produced by neocortical superfusion of acetylcholine or adenosine. N = 5/group. Data are presented as mean ± SEM. b, NMDAR subunits mRNA levels are comparable in WT and rTg4510 mice. N = 5/group. Data are presented as mean ± SEM. c, Treatment with the NOS inhibitor L-NNA prevents NMDA-induced NO production in isolated cortical neurons from WT mice. Data are presented as mean ± SEM. N = 6/group; one-way ANOVA with Tukey’s test. See Source Data 11 for statistical parameters.

Source data

Extended Data Fig. 10 NMDAR-related proteins in PS19 and rTg4510 mice, effects of WT tau on nNOS-PSD95 coupling, and putative mechanisms of the effect of tau on neurovascular function.

a–d, Levels and kinase activity of NMDAR-related proteins are not altered in 2-3 month-old PS19 and rTg4510 mice. a, Protein levels of nNOS, GluN2B and PSD95 are unaltered in synaptosomal preparations of PS19 (PS) compared to WT mice. N = 3/group. Data are presented as mean ± SEM. b, Protein levels of nNOS, GluN2B and PSD95 in PS19 (PS) and WT mice are comparable. The presynaptic marker synaptophysin (SYP) and MEK1/2 were used as membrane (MEM) and cytosolic (CYT) markers, respectively. N = 7/group. Data are presented as mean ± SEM. c, As in PS19 mice, nNOS, GluN2B and PSD95 protein levels are unchanged in PSD preparations of rTg4510 (Tg) mice compared to WT mice. N = 4/group. Data are presented as mean ± SEM. d, CaMKIIα level and activity quantified with reference to GAPDH, synaptophysin (SYP), or PSD95 associated with cytoplasm (CYT), membrane (MEM), and/or PSD are not altered in PS19 mice, compared to WT. N = 3/group. Data are presented as mean ± SEM. Immunoblots in a-d are cropped; full gel pictures are shown in Source Data 12. e-g, WT tau impairs binding of nNOS to PSD95 through association with PSD95. e, WT tau over-expressed in HEK293T cells is susceptible to phosphatase treatment, and thus hyperphosphorylated. N = 3/group; unpaired t-test. f, WT tau co-expression disrupts binding of nNOS to precipitated PSD95. N = 8/group; two-tailed unpaired t-test. g, Exogenously expressed WT tau interacts with PSD95 in HEK293T cells. A representative blot from N = 3/group is shown. Data are presented as mean ± SEM. Units for markers on the immunoblots in a-g are kDa. Immunoblots in e-g are cropped; full gel pictures are shown in Source Data 12. h, Putative mechanism by which tau induces a deficit in neuronal NO and neurovascular dysfunction: pathogenic tau (p-tau) binds to PSD95 and prevents its association with nNOS (1) and the resulting suppression in the NO production evoked by glutamatergic synaptic activity (2) dampens the NO dependent component of the increase in CBF produced by activation (3).

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Supplementary Information

Supplementary Tables 1 and 2: primary antibody list and PCR primers.

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Park, L., Hochrainer, K., Hattori, Y. et al. Tau induces PSD95–neuronal NOS uncoupling and neurovascular dysfunction independent of neurodegeneration. Nat Neurosci 23, 1079–1089 (2020). https://doi.org/10.1038/s41593-020-0686-7

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