Age-related changes in tau and autophagy in human brain in the absence of neurodegeneration

Abstract

Tau becomes abnormally hyper-phosphorylated and aggregated in tauopathies like Alzheimers disease (AD). As age is the greatest risk factor for developing AD, it is important to understand how tau protein itself, and the pathways implicated in its turnover, change during aging. We investigated age-related changes in total and phosphorylated tau in brain samples from two cohorts of cognitively normal individuals spanning 19-74 years, without overt neurodegeneration. One cohort utilised resected tissue and the other used post-mortem tissue. Total soluble tau levels declined with age in both cohorts. Phosphorylated tau was undetectable in the post-mortem tissue but was clearly evident in the resected tissue and did not undergo significant age-related change. To ascertain if the decline in soluble tau was correlated with age-related changes in autophagy, three markers of autophagy were tested but only two appeared to increase with age and the third was unchanged. This implies that in individuals who do not develop neurodegeneration, there is an age-related reduction in soluble tau which could potentially be due to age-related changes in autophagy. Thus, to explore how an age-related increase in autophagy might influence tau-mediated dysfunctions in vivo, autophagy was enhanced in a Drosophila model and all age-related tau phenotypes were significantly ameliorated. These data shed light on age-related physiological changes in proteins implicated in AD and highlights the need to study pathways that may be responsible for these changes. It also demonstrates the therapeutic potential of interventions that upregulate turnover of aggregate-prone proteins during aging.

Fig 1. Total tau levels are significantly decreased in the healthy older brains compared to the younger brains.

Representative western blot images for total tau, PHF1 and Actin in post-mortem brains (A i) and quantification of total tau normalized to Actin (A iii). Representative western blot image for NeuN and Actin in post-mortem brains (A ii) and quantification of NeuN normalized to Actin (A iii). Representative western blot images for total tau, PHF1 and Actin in resected tissues (B i) and quantification of total tau normalized to Actin (B ii). Regression plot showing an increased trend of PHF1/Tau signal in the older cohorts compared to the younger group in resected tissue samples (B iii) In post-mortem samples n = 6 (older brains 70–74 years), n = 6 (younger brains 20–33 years), in resected tissue samples n = 13 (older brains 50–70 years and younger brains 19–42 years). ***p<0.0001, *p<0.05 by two-tailed unpaired t test. Data represent mean ± SEM (A iii and B ii). An increased level of PHF1 immunoreactivity was observed in older brains of resected tissues although it was not significant (B iii).

Fig 2. The levels of total α-tubulin and its post-translational modifications are not altered with age in post-mortem brains.

Representative western blot images for acetylated α-tubulin, total α-tubulin and GAPDH in post-mortem brains (A i) and quantification of actetylated α-tubulin relative to total α-tubulin normalized to GAPDH (A ii). Representative western blot images for tyrosinated α-tubulin, total α-tubulin and GAPDH in post-mortem brains (B i) and quantification of tyrosinated α-tubulin relative to total α-Tubulin normalized to GAPDH (B ii). In post-mortem samples n = 6 (older brains) and n = 6 (younger brains). p values are not significant by two-tailed unpaired t test. Data represent mean ± SEM (A ii and B ii).

Fig 3. Autophagy is significantly increased in the healthy older brains compared to the younger brains in post-mortem tissues.

Representative western blot images for p62 and Actin in post-mortem brains (A i) and quantification of p62 normalized to Actin (A ii). Representative western blot images for LC3 and Actin in post-mortem brains (B i) and quantification of LC3-II/LC3-I normalized to Actin (B ii) and LC3-II normalised to Actin (B iii). In post-mortem samples n = 7 (older brains), n = 6 (younger brains). *p<0.05, by two-tailed unpaired t test. Data represent mean ± SEM (A ii and B ii).

Fig 4. Early autophagy gene Beclin 1 is not significantly increased in the older brains compared to the younger brains in post-mortem tissues.

Representative western blot images for Beclin 1 and Actin in post-mortem brains (i) and quantification of Beclin 1 normalized to Actin (ii). In post-mortem samples n = 7 (older brains), n = 6 (younger brains). p values are not significant by two-tailed unpaired t test. Data represent mean ± SEM.

Fig 5. Upregulation of autophagy decreases the age-related accumulation of hyperphosphorylated tau and ameliorates tau-induced locomotor defects in human Tau (0N3R) expressing transgenic flies.

Pan-neuronal expression of human 0N3R tau significantly increases the age-dependent accumulation of hyperphosphorylated tau that is restricted by coexpression of autophagy marker Atg1. Representative western blot images for PHF1, total tau and Actin (A i) and quantification of PHF1 relative to total tau normalized to Actin (A ii) in htau^0N3R and htau^0N3R/Atg1 overexpressing Drosophila models respectively. *p<0.01 by one-way ANOVA with Bonferroni’s correction. Data represent mean ± S.E.M. Expression of 03NR human tau pan-neuronally in adult flies induces locomotor deficits that are rescued by coexpression of Atg1 (B) Comparison of the climbing ability with age over a period of 4 weeks for hTau^0N3R, Atg1 and hTau^0N3R/Atg1 transgenics (n = 50). (2 way ANOVA; *p<0.05, **p<0.001, ****p<0.0001). Error bars are plotted as ± S.E.M. Genotypes: hTau^0N3R = {w; Elav-Geneswitch/UAS-htau^0N3R}, Atg1 = {w; Elav-Geneswitch/+; UAS-Atg1/+}, hTau^0N3R/Atg1 = {w; Elav-Geneswitch /UAS-htau^0N3R; UAS-Atg1/+}, on an OreR background.