Elsevier

Neurobiology of Disease

Volume 71, November 2014, Pages 260-269
Neurobiology of Disease

Phosphorylated tau potentiates Aβ-induced mitochondrial damage in mature neurons

https://doi.org/10.1016/j.nbd.2014.08.016Get rights and content

Highlights

  • Tau phosphorylated at S396/S404 is involved in the pathogenesis of Alzheimer's disease.

  • The molecular mechanisms by which tau phosphorylated induce neuronal damage are unknown.

  • Phosphorylated tau enhanced Aβ-induced mitochondrial damage in mature neurons.

  • Phosphorylated tau contributes to neuronal injury by affecting mitochondrial bioenergetics.

Abstract

Tau phosphorylated at the PHF-1 epitope (S396/S404) is likely involved in the pathogenesis of Alzheimer's disease (AD). However, the molecular mechanisms by which tau phosphorylated at these sites negatively impacts neuronal functions are still under scrutiny. Previously, we showed that expression of tau truncated at D421 enhances mitochondrial dysfunction induced by Aβ in cortical neurons. To extend these findings, we expressed tau pseudo-phosphorylated at S396/404 (T42EC) in mature and young cortical neurons and evaluated different aspects of mitochondrial function in response to Aβ. Expression of T42EC did not induce significant changes in mitochondrial morphology, mitochondrial length, or mitochondrial transport, compared to GFP and full-length tau. However, T42EC expression enhanced Aβ-induced mitochondrial membrane potential loss and increased superoxide levels compared to what was observed in mature neurons expressing full-length tau. The same effect was observed in mature neurons that expressed both pseudo-phosphorylated and truncated tau when they were treated with Aβ. Interestingly, the mitochondrial failure induced by Aβ in mature neurons that expressed T42EC, was not observed in young neurons expressing T42EC. These novel findings suggest that phosphorylated tau (PHF-1 epitope) enhances Aβ-induced mitochondrial injury, which contributes to neuronal dysfunction and to the pathogenesis of AD.

Introduction

Tau is a microtubule-associated protein essential for neuronal function (Grundke-Iqbal et al., 1986a, Grundke-Iqbal et al., 1986b). The interest in this protein increased significantly after the realization that tau was the basic component of paired helical filaments (PHFs), which form the neurofibrillary tangles (NFTs) found in the AD brain (Ihara et al., 1986, Kosik et al., 1986). Tau that forms these filaments is abnormally phosphorylated at specific sites and this modification attenuates the ability of tau to stabilize microtubules (Grundke-Iqbal et al., 1986a, Grundke-Iqbal et al., 1986b, Ihara et al., 1986, Kosik et al., 1986). Accumulating evidence suggest that tau likely contributes to the neuronal dysfunction induced by Aβ in AD patients. Indeed, primary neurons from tau knockout mice (tau −/−) are resistant to Aβ-induced cell death (Rapoport et al., 2002). In addition, recent studies have shown that knocking out tau significantly attenuates Aβ-induced behavioral deficits, but does not affect Aβ levels in a mouse overexpressing APP (Roberson et al., 2007), and that Aβ-induced deficits in axonal transport do not occur in neurons from tau (−/−) mice (Vossel et al., 2010). These findings have contributed to a re-evaluation of the role of tau in neurodegeneration, including the exploration of different targets that could be affected.

It is clear that anomalous tau phosphorylation occurs in AD brain and is associated with its pathogenesis (Johnson and Stoothoff, 2004, Pritchard et al., 2011). Phosphorylation at specific sites can significantly increase the tendency of tau to aggregate, and modifications at S396 and S404 appears to be very important in this respect (Alonso et al., 2008). For example, pseudo-phosphorylation of S396 and S404 (PHF-1 epitope) makes tau more fibrillogenic (Reynolds et al., 2007, Alonso et al., 2008). Also, tau phosphorylated at S396 and S404 has been shown to be a major component of PHFs in AD and its presence induces toxicity in neurons by affecting microtubule stability (Busciglio et al., 1995, Reynolds et al., 2007). Pathogenic phosphorylation of tau could be involved in synaptic dysfunction observed in AD (Hwan-Ching Tai et al., 2012). Phospho-tau oligomers phosphorylated mainly at S396/S404 were found accumulated in both presynaptic and postsynaptic terminals in AD brain (Hwan-Ching Tai et al., 2012). Despite the evidence, how and when phosphorylated tau affects the cascade of events that contributes to neuronal dysfunction remain unsolved.

Mitochondrial dysfunction is a prominent player in the pathogenesis of AD, as substantial evidence demonstrates that aberrant mitochondrial metabolism and localization contribute to synaptic dysfunction and loss (Bubber et al., 2005, Quintanilla et al., 2012b, von Bernhardi and Eugenín, 2012). We have demonstrated that the expression of an AD-relevant, truncated form of tau (tau-D421) in neuronal cell lines and primary neurons negatively affects mitochondrial health in response to calcium stress and Aβ treatment (Quintanilla et al., 2009, Quintanilla et al., 2012a). Recently, a few studies began exploring the effects of phosphorylated tau on mitochondrial function, raising awareness about the possible effect of these pathological species on mitochondrial function (Manczak and Reddy, 2012). Since our evidence indicates that truncated tau negatively affects mitochondrial function (Quintanilla et al., 2012a) and phosphorylated tau could participate in mitochondrial impairment in AD (Manczak and Reddy, 2012), we focused on evaluating the effect of pseudo-phosphorylated tau on mitochondrial function and the mechanisms underlying these events.

In this paper we analyzed the effect of phosphorylated tau on mitochondrial function in cortical neurons derived from rat and APP/PS1 mice. In mature neurons, expression of pseudo-phosphorylated tau (GFP-T42EC) did not affect mitochondrial morphology or transport compared to neurons expressing full-length tau (GFP-T4). Further, pseudo-phosphorylated tau enhanced mitochondrial dysfunction and increased superoxide levels in mature cortical neurons exposed to Aβ. Interestingly, mitochondrial impairment induced by pseudo-phosphorylated tau did not occur in young cortical neurons exposed to Aβ. In addition, pathological forms of tau were expressed in cortical neurons obtained from APP/PS1 mice and mitochondrial function was evaluated. The expression of full-length and pseudo-phosphorylated tau did not affect mitochondrial health in young untreated cortical neurons from APP/PS1 transgenic mice. In contrast, expression of a combined construct for pseudo-phosphorylated and truncated tau significantly reduced basal mitochondrial potential levels in the same neuronal cultures.

Altogether these observations indicate that phosphorylated tau contributes to mitochondrial dysfunction in aging neuronal cultures, apparently affecting mitochondrial bioenergetics (mitochondrial membrane potential and oxidative stress). These novel observations which extend previous findings indicate that phosphorylated tau is likely an important factor in the pathogenesis of AD.

Section snippets

Materials

All materials for cell culture and transfection were obtained from Sigma-Aldrich (St. Louis, MO, USA) and Invitrogen (Carlsbad, CA, USA). Tetramethyl rhodamine methyl ester (TMRM), pluronic acid, and dihydroethidium (DHE) were obtained from Molecular Probes (OR, USA). Amyloid peptide1–40 (Aβ1–40) was obtained from Calbiochem (MED Milipore, MA, USA).

Tau constructs

Full-length (T4) and mutant tau constructs: T4-2EC (pseudo-phosphorylated, S396/S404E), T4C3 (truncated at D421) and T4-2EC-C3

Expression of pseudo-phosphorylated tau did not affect mitochondrial morphology in mature neurons

Previously, we showed that expression of caspase-cleaved (truncated at D421) tau induced mitochondrial fragmentation in cortical neurons (Quintanilla et al., 2012a). However, the effects of site-specific phosphorylated tau on mitochondrial function have not been thoroughly studied. Therefore, we analyzed the effects of the expression of pseudo-phosphorylated (tau-T42EC, which is tau with S  E mutations at S396/S404 to mimic phosphorylation) (Ding et al., 2006, Shahani et al., 2006, Yeh et al.,

Discussion

In this study, we analyzed the effects of phosphorylated tau on mitochondrial function in primary neuronal cultures from rat and APP/PS1 mice. Mitochondrial function was evaluated in neurons that expressed phosphorylated tau in combination with truncation, which is a tau modification produced by caspase 3 cleavage, and that induced mitochondrial injury in neuronal cells (Gamblin et al., 2003, Quintanilla et al., 2012a). Our studies indicate that expression of tau pseudo-phosphorylated at

Conclusions

Our observations indicate that phosphorylated tau contributes to mitochondrial dysfunction in aging neuronal cultures, affecting mitochondrial membrane potential and increasing oxidative stress. These novel observations which extend previous findings indicate that phosphorylated tau is likely an important factor in the pathogenesis of AD and that mitochondrial impairment could be an important factor in neuronal dysfunction induced by phosphorylated tau.

The following are the supplementary data

Acknowledgments

This work was supported by FONDECYT #1140968 (RAQ), grant of Vicerrectoría de Investigación, Pontificia Universidad Católica de Chile (VRI, PUC, Chile), P18, (RAQ), FONDECYT # 1090353 (RvB), and by National Institutes of Health (USA), grant NS076789 (GVWJ). The authors declare that they have no competing interests.

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