Synaptic mitochondria isolated from mice expressing non-mutant human tau isoforms exhibit age-associated bioenergetic alterations
To explore whether alterations in mitochondrial bioenergetics are involved in tauopathy pathogenesis, non-synaptic and synaptic mitochondria were isolated from htau transgenic and WT mice at 5- and 8-months of age and oxygen consumption rates (OCR) driven by complex II were measured utilizing the coupling assay [44]. Non-synaptic mitochondria from htau mice did not show alterations in respiration compared to WT mice at either age examined (Fig. 1A). Synaptic mitochondria isolated from htau mice at 8- (but not 5-) months of age exhibit a significant increase in the rate of complex II driven state 2 (basal), state 3 (ADP-stimulated), and state 3u (maximum uncoupled) respiration compared to those from age-matched WT mice (Fig. 1B). Tau pathology has been described in the brain of htau mice at 8-months [38], thus, mitochondria from the synapse are particularly sensitive to pathologic tau accumulation, and exhibit functional changes reported during aging [45].
Quantitative mitochondrial proteomics reveals accumulation of tau in association with synaptic mitochondria
To explore the molecular mechanisms underlying altered synaptic mitochondrial respiration in the absence of respiratory changes in non-synaptic mitochondria, we investigated the influence of human tau expression in mice on brain mitochondria protein expression profiles using the quantitative mass spectrometry-based technique SWATH-MS [52]. Synaptic and non-synaptic mitochondria were isolated from 5- and 8-month-old htau transgenic and WT mice, and the proteome was analyzed. In total, 1,578 proteins were identified and the complete list of these proteins with quantitative values is provided in Additional File 1: Supplementary Table S1. SWATH sample replicates exhibited a high degree of correlation, suggesting there was minimal sample-to-sample variablity within groups (Additional File 2: Supplemental Figs. 1–4). To uncover which proteins were differentially expressed (DE; FDR q < 0.05) in mitochondria isolated from htau as compared to age-matched WT mice, we used a Bayesian regularized t-test analysis and multiple testing corrections, which revealed 20 (5-months) and 12 (8-months) DE proteins in non-synaptic mitochondria samples, and 54 (5-months) and 31 (8-months) DE proteins in synaptic mitochondria samples (Additional File 1: Supplementary Table S2A-D). As shown in Fig. 2A, only 1 protein (Dephospho-CoA kinase domain containing protein (Dcakd)) was found to be DE in both non-synaptic and synaptic mitochondria isolated from 5-month old htau mice (as compared to age-matched WT mice), whereas 2 proteins (Mapt (tau) and tubulin alpha-4 chain (Tuba4a)) were found to be DE in synaptic mitochondria isolated from 5- and 8-month old htau mice (as compared to age-matched WT mice). Dcakd was found to be elevated in isolated non-synaptic and synaptic mitochondria from htau mice as compared to WT mice at 5-months (Fig. 2B). However, at 8-months, the levels of Dcakd are similar in isolated mitochondria between htau and WT mice. While Mapt (tau) levels are increased in isolated synaptic mitochondria from htau mice as compared to WT mice at both ages (Fig. 2C), Tuba4a is decreased at 5-months and increased at 8-months (Fig. 2D). Of note, Mapt (tau) levels are increased in isolated mitochondria from 8- as compared to 5-month-old htau mice, highlighting an age-dependent increase.
To uncover protein-protein interaction networks and perform enrichment analysis to gain insight into functional associations of the mitochondrial protein changes, the lists of DE proteins in htau versus age-matched WT mice for each mitochondrial population (non-synaptic and synaptic mitochondria) were uploaded to the STRING database. Only interactions which were of high confidence (minimum required interaction score of 0.07) were used to generate the interaction networks using the MCL clustering method (network clusters are denoted by node color). This analysis identified 8 interacting proteins organized in 4 networks for the 5-month non-synaptic mitochondria DE proteins (Fig. 3A), 2 proteins organized in 1 network for the 8-month non-synaptic mitochondria DE proteins (Fig. 3B), 29 interacting proteins organized in 7 networks with 9 distinct network clusters for the 5-month synaptic mitochondria DE proteins (Fig. 3C), and 13 interacting proteins organized in 3 networks for the 8-month synaptic mitochondria DE proteins (Fig. 3D). While the networks for the non-synaptic mitochondria DE proteins were composed of single interactions between 2 proteins, the networks for the synaptic mitochondria DE proteins were more complex, evidenced by the increased numbers of nodes and edges. Functional enrichments in the STRING networks revealed gene ontology (GO) biological process (BP) terms that were altered according to our proteomics results (Additional File 1: Supplementary Table S3A-C). While no BP terms were identified for the 5-month non-synaptic mitochondria, one term was found for the DE proteins in non-synaptic mitochondria from 8-month htau compared to WT mice were “positive regulation of hydrolase activity” (FDR = 0.0078). In contrast, several BP terms (131 at 5-months and 9 at 8-months) were significantly enriched based on the DE proteins in synaptic mitochondria from htau as compared to WT mice, with the top two terms “synaptic vesicle docking” and “glycerol-3-phosphate metabolic process” at 5-months, and “microtubule-based process” and “cytoskeleton organization” at 8-months. Of note, Mapt (tau) was a node in the STRING networks for synaptic mitochondria DE proteins at both ages studied.
Human tau expression alters expression of electron transport chain components
To determine whether proteomic changes affecting the electron transport chain (ETC) and oxidative phosphorylation (OxPhos) correlate with the observed respiratory alterations, we assessed the expression of the ETC component proteins as well as regulators of complex assembly and function (Fig. 4). We used the web served based MEV to cluster log2-fold expression data comparing synaptic and non-synaptic data between htau and WT mice at 5- and 8-months of age (Fig. 4A). Clustering indicated that expression changes in ETC components were most similar between synaptic and non-synaptic mitochondria a 5-months. Furthermore, changes 8-month synaptic mitochdonria were the most divergent between genotypes, a finding that correlates well with the changes we observed in bioeneregetics of 8-month synaptic mitochondria. Interestingly, the age-dependent alterations of mitochondrial respiratory complex subunit expression in synaptic mitochondria from WT mice were similar to changes observed in htau transgenic mice consistent with the observation of similar synaptic mitochondrial functional alterations with age in htau transgenic and WT mice. However, despite the predominant conservation of age-dependent changes in the synaptic mitochondrial ETC subunits and regulator proteins in htau mice, differences exist which may contribute to the functional differences between htau and WT mice at 8-months of age. Therefore, we focused on the synaptic mitochondrial proteomic changes between htau and WT mice at 8-months of age to gain mechanistic insight into the contribution of tau pathology to alterations in synaptic mitochondrial function. Upon examination of the differences in the expression of the ETC subunits between 8-month-old htau and WT mice it becomes apparent that several of the mitochondrial DNA (mtDNA) encoded subunits (Mtnd4 (log2 = -1.43), Mtnd5 (log2 = 1.10), and Mtco1 (log2 = -0.176)) exhibit altered expression (Additional File 1: Supplementary Table 1, Fig. 4A (red row names)). Although not reaching significance in our proteomic analysis due to the stringency of our statistical analysis, lower Mtco1 levels were confirmed via immunoblotting (Fig. 4B). Similar to Mtco1, our proteomic analysis revealed a slight reduction in Uqcrc2 expression in synaptic mitochondria from 8-month-old htau compared to WT mice (log2 = -0.256), which was confirmed by immunobot quanitfication (Supplementary Table 1; Fig. 4). A similar observation was made for Atp5a1 (log2 = -0.44). Since htau mice exhibit hyperphosphorylation and aggregation of tau within the span from 5- to 8-months of age [38], we next sought to characterize tau associated with synaptic mitochondria as our functional and proteomic results suggested this popoulation of mitochondria was the most divergent.
Accumulation of pathologic tau in association with synaptic mitochondria
Phosphorylation of tau at certain serine and threonine residues serves as a biochemical marker for pathologic alterations in AD and fronto-temporal dementia (FTD) [2, 10, 26, 36, 38]. We therefore sought to determine whether alterations in synaptic mitochondrial bioenergetics were associated with this hallmark of disease progression. We used immunoblotting to determine if tau was present in purified synaptic mitochondrial isolates (Fig. 5). In agreeance with our SWATH analysis (Fig. 2) we observed a significant increase in the amount of total tau protein present in htau mice at both 5- and 8-months of age compared with WT mice, which was more pronounced at 8 compared to 5 months of age (Fig. 5A). We also detected CP13 [8, 9, 53] and PHF1 [54, 55] positive immunoreactivity in isolated synaptic mitochondria, which specifically detect abnormal phosphorylated residues S202 (Fig. 5B) and S396/404 (Fig. 5C), respectively. In synaptic mitochondria we observed low but comparable levels of CP13 immunoreactivity between 5-month-old WT and htau mice, however, at 8-months we detected a significant increase of CP13 signal in synaptic mitochondria from htau mice (Fig. 5B). In contrast to CP13, we detected a significant increase in PHF1 signal at both 5- and 8-months in synaptic mitochondria from htau mice as compared to WT, and this exaggerated at 8-months (Fig. 5C). In addition to pathologic hyperphosphorylation, tau also changes its conformation during disease progression [56], which finally culminates in aggregation and formation of tangles. These early conformational changes can be monitored by probing with the antibody MC1, which is specific for a pathologic conformation of tau as it recognizes neurofibrillary tangles (NFTs) [56]. In synaptic mitochondria from 5-month-old htau mice there was a trending albeit insignificant increase in MC1 signal that achieved significance at 8-months (Fig. 5D). Interestingly, a significant increase in the detection of PFH1 tau was observed at both 5- and 8-months timepoints, suggesting increased phosphorylation at S396/404 may be an earlier event in the progression of AD and related tauopathies (Fig. 5C).
Tau localizes to the mitchondrial outer membrane
Other studies have reported the association of tau protein with mitochondria in a number of systems [11, 13, 28, 36, 57–59]. This association was determined in some cases to be the result of tau incorporation into the outer mitochondrial membrane, yet in others the consequence of protein-protein interactions with outer mitochondrial membrane proteins, one such protein is voltage dependent anion channel 1 (VDAC1) [60–63]. With this in mind we sought to determine the mechanism by which tau was co-purifying with synaptic mitochondria in our isolations. To address this, we used sodium carbonate extraction, an established method to differentiate integral mitochondrial membrane proteins from membrane associated mitochondrial proteins. In this assay integral membrane proteins fractionate into an insoluble (pellet; “P”) fraction, while peripheral membrane (i.e. membrane associated) proteins segregate into the soluble (“S”) fraction. Synaptic mitochondria isolated from 8-month-old htau mice were extracted with sodium carbonate and the resulting fractions were probed by western blot (Fig. 6). As control we probed for the OxPhos panel (Fig. 6A, red) as well as heat-shock protein 60 (HSP60) (Fig. 6A, green). With the exception of ATP synthase F1 subunit alpha (Atp5a1), we primarily detected the proteins of the OxPhos panel in the pellet as expected since most are integral membrane proteins, also as expected we observed the majority of HSP60 in the soluble fraction. Finally, we observed tau protein segregating almost entirely into the soluble fraction (Fig. 6B). Our results suggest that tau associated with synaptic mitochondria is not inserted into the mitochondrial membrane, but likely associated via protein-protein interaction.
To further support our sodium carbonate extraction results, we employed a protease digest assay to determine the rate at which tau was degraded compared to two mitochondrial proteins. We reasoned that if tau was associated with mitochondria via protein-protein interactions it would be degraded at lower concetrations of protease than either integral membrane or mitochondrial matrix proteins. Isolated synaptic and non-synpatic mitochondria from approximately 8-month-old htau mice were digested by trypsin and immunoblotted for tau, VDAC, and succinate dehydrogenase complex subunit A (SDHA) (Fig. 6C). We observed a reduced percentage of remaining full length tau protein at lower trypsin concentration than either SDHA or VDAC, suggesting mitochondrially associated tau is more accessible to proteolytic action. Taken together with our sodium carbonate extraction results, this suggests that tau is present on the mitochondrial outer membrane and this interaction is most likely mediated by protein-protein interactions.