Tau protein aggregation is associated with cellular senescence in the brain

Abstract

Tau protein accumulation is the most common pathology among degenerative brain diseases, including Alzheimer's disease (AD), progressive supranuclear palsy (PSP), traumatic brain injury (TBI), and over twenty others. Tau-containing neurofibrillary tangle (NFT) accumulation is the closest correlate with cognitive decline and cell loss (Arriagada, Growdon, Hedley-Whyte, & Hyman, ), yet mechanisms mediating tau toxicity are poorly understood. NFT formation does not induce apoptosis (de Calignon, Spires-Jones, Pitstick, Carlson, & Hyman, 2009), which suggests that secondary mechanisms are driving toxicity. Transcriptomic analyses of NFT-containing neurons microdissected from postmortem AD brain revealed an expression profile consistent with cellular senescence. This complex stress response induces aberrant cell cycle activity, adaptations to maintain survival, cellular remodeling, and metabolic dysfunction. Using four AD transgenic mouse models, we found that NFTs, but not Aβ plaques, display a senescence-like phenotype. Cdkn2a transcript level, a hallmark measure of senescence, directly correlated with brain atrophy and NFT burden in mice. This relationship extended to postmortem brain tissue from humans with PSP to indicate a phenomenon common to tau toxicity. Tau transgenic mice with late-stage pathology were treated with senolytics to remove senescent cells. Despite the advanced age and disease progression, MRI brain imaging and histopathological analyses indicated a reduction in total NFT density, neuron loss, and ventricular enlargement. Collectively, these findings indicate a strong association between the presence of NFTs and cellular senescence in the brain, which contributes to neurodegeneration. Given the prevalence of tau protein deposition among neurodegenerative diseases, these findings have broad implications for understanding, and potentially treating, dozens of brain diseases.

Keywords: Alzheimer’s disease; aging; cellular senescence; neurodegeneration; senolytic; tau.

Figure 1

Neurofibrillary tangles were associated with cellular senescence‐associated gene pathways in human Alzheimer’s disease neurons and tau transgenic mouse brains. (a) Pathways and predicted upstream regulators identified by ingenuity pathway analyses (IPA, QIAGEN) as significantly enriched in Alzheimer’s disease patient‐derived neurons with neurofibrillary tangles compared to non‐tangle‐containing neurons; z‐score plotted on x‐axis and (p‐value) indicated in bar graph. Cellular functions and (b) predicted upstream regulators employed by neurofibrillary tangle‐containing neurons derived from Alzheimer’s disease patient are shown. (c) Predicted upstream regulators of gene transcription in tau_NFT mice after the onset of neurofibrillary tangles (~6 months old vs. ~2 months old); z‐score plotted on x‐axis and (p‐value) indicated in bar graph. (d‐e) Representative immunoblot generated by capillary electrophoresis on chromatin‐bound fractions from mouse forebrain homogenate probed with anti‐γ‐H2ax antibody. (e) Densitometric normalization of γ‐H2ax to total protein content (CTL: n = 3; tau_WT n = 4; tau_NFT: n = 5; ANOVA, p = 0.0056. Mice aged 16 to 18 months old). (f–g) Quantitative gene expression on RNA isolated from CTL (open bar, n = 3), tau_WT (closed bar, n = 3), and tau_NFT (red bar, n = 4) mouse forebrain targeting (f): Cdkn2a, p = 0.0066, and (g) Cdkn1a, p = 0.0207. Gene expression was analyzed by one‐way ANOVA Tukey’s multiple comparison post hoc. Data are graphically represented as mean ± SEM

Figure 2

Neurofibrillary tangles were associated with upregulation of SASP gene expression and NFκB activation. (a) Quantitative gene expression on RNA isolated from CTL (open bar, n = 3), tau_WT (closed bar, n = 3), and tau_NFT (red bar, n = 4) mouse forebrain targeting SASP‐associated genes Il1b, p = 0.0025; (b) Cxcl1, p = 0.0040; (c) Tnfa, p = 0.0114; and (d) Tlr4, p = 0.0144. (d) Immunoblot generated by capillary electrophoresis on subcellular fractionated mouse forebrain homogenate probed with anti‐NFκB p65 antibody. Total cellular p65 (top blot) and nuclear‐localized p65 protein levels (bottom blot) were (e) normalized to total protein content. Total p65, p = 0.0758; nuclear p65, p = 0.0223. CTL: open bar, n = 3; tau_WT: closed bar, n = 4; tau_NFT: red bar, n = 5. In all experiments, mice were aged 16–18 months old; both males and females were included. Significance was determined by one‐way ANOVA Tukey’s multiple comparison post hoc. Data are graphically represented as mean ± SEM

Figure 3

Brain regions with neurofibrillary tangles displayed altered cellular respiration. (a–c) Representative respirometric traces from cortical and (d–f) hippocampal tissues using the SUIT protocol to measure oxygen consumption (top gray traces: CTL; black middle traces: tau_WT; bottom red traces: tau_NFT). (g) Tissue mass‐specific respiration analyses in cortical and (h) hippocampal tissue. Two‐way ANOVA Tukey’s multiple comparison post hoc: **p < 0.005. (i) Biochemical analyses of citrate synthase (CS) activity to assess total mitochondrial content in the cortex and hippocampus (n = 5/group). Experimental mice were aged 16–18 months old with n = 6/group; both males and females were included. (j) Total oxygen consumption and (k) Cdkn2a gene expression were measured in the cerebellum, a brain region devoid of NFTs. n = 3/group. Data are graphically represented as mean ± SEM. ETF_L (fat oxidation in the absence of ADP [state 2]), ETF_P (fat oxidation coupled to ATP production), CI_P (complex I activity linked to ATP production [state 3]), CI + CII_P (complex I and complex II linked respiration [state 3]), CI + CII_E (complex I and complex II linked respiration uncoupled [maximum respiration]), and CII_E (complex II activity uncoupled). Data are graphically represented as mean ± SEM

Figure 4

Senescence‐associated Cdkn2a was significantly upregulated in mouse and human brains with neurofibrillary tangles and tracked with total tangle deposition and brain atrophy. (a) Genetically ablating endogenous mouse tau to significantly reduce neurofibrillary tangle load resulted in a concomitant 60% reduction in Cdk2na expression (two‐tailed t test: p = 0.0041; n = 3/group) and (b) significant reduction in brain atrophy (two‐tailed t test, p = 0.0143; n = 3/group). (c) Tracking Cdkn2a expression in tau_WT mice revealed a significant age‐dependent increase (one‐way ANOVA: p = 0.0043; n = 3/group for tau_WT and n = 4 tau_P301L). In contrast to significantly lower expression than tau_NFT mice at 16 months old (p = 0.0075), Dunnett's multiple comparison test indicated that at 22 months of age, tau_WT mouse Cdkn2a expression was no longer statistically lower than tau_NFT mice (p = 0.0577) and by 28–30 months they were are statistically the same (p = 0.999). (d) Immunofluorescence and Bielschowsky silver staining revealed neurofibrillary tangles in 18‐month‐old tau_WT mouse hippocampal CA1 (NeuN, neuron, green; PHF1: phosphorylated tau, red; DAPI, blue, nuclei). (e) qPCR analyses of RNA extracted from 3xTgAD mice with Aβ plaques were compared to tau_NFT set at y = 1. 3xTgAD Cdkn2a expression was no different than age‐matched C57BL/6 mice (two‐tailed t test, p = 0.1081; n = 3 WT, n = 6 3xTgAD; n = 4 tau_NFT). Both mouse cohorts expressed significantly less Cdkn2a than tau_NFT mice (one‐way ANOVA: p < 0.0001). (f) Cdkn2a expression level was significantly correlated with brain atrophy (R ² = 0.5615, p < 0.0001; n = 43). (g) qPCR analyses of RNA extracted from brains from control older adult humans (n = 10; ave. age = 85.70 years) and age‐matched progressive supranuclear palsy (n = 14; ave. age = 83.86 years) indicated a 57% upregulation of CDKN2A with progressive supranuclear palsy diagnosis (unpaired t test, p = 0.0415) that (h) positively correlated with neurofibrillary tangle deposition in the parietal lobe (ANOVA, p = 0.0008; Kendall’s Tau rank correlation p = 0.059). (i) Immunoblot generated by capillary electrophoresis on cortical brain homogenate from control and progressive supranuclear palsy human brains probed with total tau antibody, HT7. The individual with the highest CDKN2A expression (panel g) displayed high molecular weight tau, lane 9*. Data are graphically represented as error bars, mean ± SEM

Figure 5

Senolytic treatment reduced neurofibrillary tangle burden, ventricular enlargement, and neurodegeneration in 23‐month‐old tau transgenic mice. (a) Representative brain images analyzed for neurofibrillary tangles in tau transgenic mice treated with either vehicle or dasatinib and quercetin (DQ). (phosphorylated tau, PHF1, red; total tyrosine phosphorylation, pTyr, yellow; and DAPI nuclei; blue. Scale bar = 10 µm). (b) Neurofibrillary tangle counts from n = 3 mice/group sampled from 12 cortical images/mouse and analyzed with unpaired two‐tailed t test, ****p < 0.0001. (c) DQ significantly reduced hippocampal Tlr4, p = 0.0459, and Cxcl1, p = 0.0142, gene expression as measured by qPCR. Vehicle‐treated (open symbols, n = 7) and DQ‐treated (red closed symbols, n = 5); data analyzed by unpaired two‐tailed t test. (d) Representative brain images from anatomical T2‐weighted MRI. (e) Quantification of ventricle volume analyzed by one‐way ANOVA, p = 0.0010; Holm–Sikak's post hoc: ***p = 0.0007, *p = 0.05. (f) Immunoblot generated by capillary electrophoresis on forebrain homogenates (n = 6/group) with antibodies against neuronal proteins NeuN, synaptophysin (synapto.), and PSD95 normalized to total protein. (g) Neuronal protein expression was normalized to total protein and analyzed by unpaired two‐tailed t test, NeuN: *p = 0.0432, (h) synaptophysin (synapto): *p = 0.0416 and (i) PSD95: *p = 0.0398. n = 6/group. Data represented as mean ± SEM