AD-linked, toxic NH2 human tau affects the quality control of mitochondria in neurons
Introduction
Mitochondria dysfunction and clearance are contribution factors to mammalian aging and to several age-related neurodegenerative disorders named tauopathies, including Alzheimer's Disease (AD) (Batlevi and La Spada, 2011). Compelling evidence shows a causal link between mitochondrial dysfunction and impaired synaptic plasticity in AD pathogenesis at the early preclinical stage (Du et al., 2012, Lee et al., 2012a, Lee et al., 2012b, Lin and Beal, 2006). Early deficits of synaptic mitochondria are indeed detected in mutant APP transgenic mice prior to the extracellular Aβ deposition (Du et al., 2010) and mitochondria dysfunction in the hippocampal CA3 region precedes the synaptic deterioration in Tg2576 (APPswe) mice, another FAD (Familiar Alzheimer's Disease) model (Lee et al., 2012a, Lee et al., 2012b).
Mitochondria exhibit dynamic properties and their homeostasis in a healthy, functional network is a closely interrelated process that involves an intimate crosstalk between organelle quality control and autophagy (Twig et al., 2008). Indeed, as in other cells (Youle and Narendra, 2011), neuronal mitochondria undergo continuous reshaping by paired fission/fusion, transport, biogenesis and selective degradation (mitophagy) which relies on Parkin-mediated priming followed by their sequestration into autophagosomes and retro-trafficking along microtubules into peri-nuclar clusters (mitoaggresomes) (Cai et al., 2012, Sheng et al., 2012, Van Humbeeck et al., 2011, Van Laar and Berman, 2013, Van Laar et al., 2011, Wang et al., 2009, Zhu et al., 2012, Zhu et al., 2013). All these events are closely coordinated and reciprocally interact in an integrated system, constantly monitored by every cell in order to maintain a healthy mitochondrial population and consequent viability. Moreover, because of their high energy demands and very polarized morphology, the proper maintenance of mitochondrial biomass is especially crucial for survival of post-mitotic neurons that continually modulate their size and number according to the variable energy demands and metabolic states in diverse times and/or sub-cellular compartments (Chen and Chan, 2006, Santos et al., 2010, Van Laar and Berman, 2013, Vives-Bauza and Przedborski, 2011). A pathological deregulation of mitochondria turnover adversely impinges on their overall shape, location and metabolic functions so that they are no longer transported along neuritis and provided to highly-ATP utilization sites, such as synaptic terminals (Nunnari and Suomalainen, 2012, Sheng et al., 2012). Consequently, neuronal populations –especially those with poorly myelinated, long, thin axons located in selectively vulnerable AD brain areas– are crucially sensitive to improper mitochondrial dynamics (Verstreken et al., 2005).
To this regard, a growing body of studies actually shows that, along with an altered trafficking and interorganellar communication, changes in the quality control of mitochondria –including quality, number and distribution of their functional population– are causally correlated with the synaptic demise in vulnerable, affected AD neurons (Campello and Scorrano, 2010, Eckert et al., 2011, Karbowski and Neutzner, 2012, Palmer et al., 2011, Reddy, 2011, Reddy et al., 2012, Santos et al., 2010, Sheng et al., 2012). An impaired mitochondrial biogenesis, followed by an ensuing deterioration of their bioenergetics (Rintoul and Reynolds, 2010, Sheng et al., 2012, Young-Collier et al., 2012), and a severe reduction of these metabolically-active organelles from distal processes in correlation with their parallel accumulation into neuronal soma are both early events in AD synaptic failure (Chen and Chan, 2009, Selfridge et al., 2013, Wang et al., 2008a, Wang et al., 2008b, Wang et al., 2009) and, importantly, are detectable prior to any obvious clinical sign (Stokin et al., 2005). Ultrastuctural changes in mitochondria length with a dramatic loss in their inner cristae organization, as well as a drastic reduction in global biomass of these organelles, were early found in three different FAD transgenic mice mainly at synapses, in correlation with their energetic failure and prior to the onset of the memory impairment or of the plaque appearance (Trushina et al., 2012). In AD patients, a morphometric analysis carried out on affected brain regions reports a significant paucity of mitochondria (Baloyannis, 2006) that exhibit evident signs of destruction, including rupture of their inner membrane, lipofuscin-filled vacuoles, and variable mtDNA (mitochondrial DNA) content (Hirai et al., 2001). Functional parameters of mitochondria –such as the respiratory capacity and the antioxidant enzyme activities– are strongly reduced of approximately two-thirds in AD specimens, almost in part due to a loss of their biomass and/or to their impaired biogenesis (Young-Collier et al., 2012). Biochemical unbalance in the expression levels of several mitochondria-shaping proteins (OPA1, Mfn1/2, Drp1, Fis) or alterations in their posttranslational modifications are also found in cellular and animal AD models, as well as in human AD neurons, in association with an excessive fragmentation and mislocalization of these organelles, with neuritic beading and with a loss of dendritic spines (Barsoum et al., 2006, Calkins and Reddy, 2011, Calkins et al., 2011, Cho et al., 2009, Manczak et al., 2011, Tan et al., 2007, Wang et al., 2008a, Wang et al., 2008b, Wang et al., 2009). In neurons from Tg2576 (APPswe) mice, an impaired mitochondrial biogenesis is accompanied with a mislocalization of these BrdU-positive small, defective organelles that reside mainly in the cell soma and cannot be transported to axons and dendrites, hence depriving the nerve terminals of ATP and calcium-buffering ability (Calkins et al., 2011). Finally an impaired autophagic flux, including an increased rate of mitochondrial autophagic removal that leads to selective loss of these organelles –which progressively lessen in number, volume and cytoplasmic density (Moreira et al., 2007a, Moreira et al., 2007b)– has been described as a prominent AD hallmark in vulnerable hippocampus and prefrontal cortex (Baloyannis, 2006, Young-Collier et al., 2012). Altogether, these findings point out that pathogenetic defects in the maintenance of functional mitochondrial biomass are early events participating in the AD synaptic failure and further bolster the notion that it may be possible to alleviate the AD bioenergetic collapse at least in part by interfering with mitochondrial homeostasis (Bonda et al., 2010). However whether excessive mitophagy, owing to an uncontrolled quality control pathway of these organelles, is detrimental, beneficial, or simply an epiphenomenon and whether it acts as an early causal event in AD pathogenesis or a late neuroprotective consequence of other neuropathological insults, remains under intense debate (Batlevi and La Spada, 2011, Cherra and Chu, 2008, Santos et al., 2010, Selfridge et al., 2013, Tolkovsky, 2009).
We have previously reported that a 20–22 kDa NH2-tau fragment (aka NH2htau), mapping between 26 and 230 amino acids of the human tau40, (i) is neurotoxic in primary cultured neurons (Amadoro et al., 2004, Amadoro et al., 2006); (ii) is detected in cellular and animal AD models (Corsetti et al., 2008) and (iii) is also largely enriched in human mitochondria from AD synaptosomes in correlation with the pathological synaptic changes, with the Aβ multimeric species and with the organelle functional impairment (Amadoro et al., 2010). The NH2htau– but not the physiological full-length protein– preferentially interacts with Aβ peptide(s) at human AD synapses and cooperates with it in inhibiting the mitochondrial ANT-1-dependent ADP/ATP exchange (Amadoro et al., 2012). In view of that: (i) a structural and functional impairment of neuronal mitochondria jointly contribute to early synaptic alterations in AD (Baloyannis, 2011, Du et al., 2010, Manczak et al., 2011, Nakamura and Lipton, 2010, Schon and Przedborski, 2011, Selfridge et al., 2013, Trushina et al., 2012, Wang et al., 2009) and (ii) pathological Aβ and tau converge in perishing the mitochondria (Amadoro et al., 2012, Eckert et al., 2011, Quintanilla et al., 2012, Rhein et al., 2009), we sought to determine whether the NH2htau can also affect the distribution, shape and size of the mitochondria. Using both in vivo and in vitro systems, we found out that pathological NH2htau enhances the mitochondria turnover by harmfully affecting their fusion/fission dynamics and their clearance by selective autophagy.
Section snippets
The NH2htau fragment early affects the neuronal mitochondria morphology in vitro, leading to fragmentation and a net decrease of their biomass
As NH2htau specifically interacts with mitochondria in AD (Amadoro et al., 2012), we sought to investigate whether its enhanced intracellular level, as we detected in vivo (Amadoro et al., 2012), influences the mitochondrial network integrity in primary cultured neurons.
To this aim, mature hippocampal primary neurons (DIV 15) were transduced (75% efficiency) by adenovirus-mediated infection with myc-tagged NH2-26–230 tau (myc-NH2htau) and with myc-tagged empty vector (mock) at low/moderate
Discussion
The results we outline here provide evidence that the pathological N-terminal truncation of human tau –which has known to be critically involved in onset and progression of diverse human age-related tauopathies including AD (García-Sierra et al., 2008) and whose level we previously found to be increased in mitochondria from AD brains (Amadoro et al., 2010, Amadoro et al., 2012)– associates to an impairment of mitochondrial quality control in primary hippocampal neurons. By different and
Conclusions
In conclusion, in line with other papers referring that pathological tau per se can affect the mitochondria quality control (Duboff et al., 2012, Manczak and Reddy, 2012, Quintanilla et al., 2009, Quintanilla et al., 2012) as well as the autophagic pathway (Inoue et al., 2012, Pacheco et al., 2009) and that an increased autophagic turnover of mitochondria was detected in affected AD neurons (Young-Collier et al., 2012, Baloyannis, 2006, Moreira et al., 2007a), the present study further
Reagents and antibodies
Bafilomycin A1 (BAF-A1) B1793, chloroquine (CQ) C-6628, and FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) C2759 were from Sigma Aldrich. The following antibodies were used: α-synuclein antibody mouse 610787 BD Transduction Laboratories; β-III tubulin antibody rabbit ab18207 Abcam; kinesin antibody (clone IBII) K1005 Sigma Aldrich; cytochrome C (136F3) rabbit 4280 Cell Signaling Technology; cytochrome C antibody (clone 7H8.2C12) mouse ab13575Abcam; COX IV antibody (clone 1D6E1A8)
Funding
This research was supported by grant from FIRB B81J07000070001 and Fondazione Roma to P.C and by grant from PRIN 2010–2011 (prot. 2010M2JARJ-003) to G.A. The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.
Conflict of interest statement
The authors declare that they have no actual or potential conflicts of interest and that these data are not published elsewhere. In addition all authors approve the study described in this report.
Acknowledgments
We wish to thank Dott. Francesca Natale for her assistance in culturing HeLa cells; Ms G. Baldini and Ms R. Bortul for electron microscopy technical assistance; and Dott. Paolo Fiorenzo, Dott. Mauro Cozzolino and Dott.ssa Flavie Strapazzon for providing several mitochondrial antibodies.
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These authors equally contributed to this work.