neurobiology of disease - core · 2016. 5. 26. · g university of salerno, department of...

Neurobiology of Disease 62 (2014) 489–507

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Neurobiology of Disease

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AD-linked, toxic NH2 human tau affects the quality control ofmitochondria in neurons

G. Amadoro a,b,⁎,1, V. Corsetti b,1, F. Florenzano b,d, A. Atlante c, M.T. Ciotti d, M.P. Mongiardi d, R. Bussani e,V. Nicolin f, S.L. Nori g, M. Campanella b,h, P. Calissano b

a Institute of Translational Pharmacology (IFT), CNR, Via Fosso del Cavaliere 100-00133, Rome, Italyb European Brain Research Institute (EBRI), Via del Fosso di Fiorano 64-65-00143, Rome, Italyc Insitute of Biomembrane and Bioenergetic (IBBE), CNR, Via Amendola 165/A-70126, Bari, Italyd Institute of Cellular Biology and Neurobiology (IBCN), CNR, IRCSS Fondazione Santa Lucia, Via del Fosso di Fiorano 64-65-00143, Rome, Italye UCO Anatomy and Pathological Histology, Hospital of Cattinara, Strada di Fiume 447-34149, Trieste Italyf University of Trieste, Clinical Department of Medical, Surgical and Health Science-section of Human Morphology, Via Manzoni 16-34138, Trieste, Italyg University of Salerno, Department of Pharmaceutical and Biomedical Sciences (FARMABIOMED), NANOMATES, Via Ponte don Melillo 1-85084, Fisciano (SA), Italyh Department of Comparative Biomedical Sciences, The Royal Veterinary College, and Consortium for Mitochondrial Research, University College London, Royal College Street,NW1 0TU, United Kingdom

Abbreviations: NH2htau, NH2-human tau fragment oNucleotide Translocator-1; AD, Alzheimer's Disease; APP,amyloid beta; PD, Parkinson's disease; α-syn, α-synuclDisease; MOI, multiplicity of infection; mtDNA, mitochonCOX, cytochrome c oxidase; BAF-A1, bafilomycin A1; CQ, canide 4-(trifluoromethoxy)phenylhydrazone); MT, micro⁎ Corresponding author at: Institute of Translational Pha

del Cavaliere 100-00133-Rome, Italy.E-mail address: [email protected] (G. Amadoro)Available online on ScienceDirect (www.sciencedire

1 These authors equally contributed to this work.

0969-9961/$ – see front matter © 2013 Published by Elsehttp://dx.doi.org/10.1016/j.nbd.2013.10.018

a b s t r a c t
a r t i c l e i n f o
Article history:Received 15 May 2013Revised 10 September 2013Accepted 16 October 2013Available online 24 October 2013

Keywords:NH2-tau fragmentMitochondrial dynamicsAutophagySynapse(s)NeurodegenerationAlzheimer's Disease (AD)

Functional as well as structural alterations in mitochondria size, shape and distribution are precipitating,early events in progression of Alzheimer's Disease (AD). We reported that a 20–22 kDa NH2-tau fragment(aka NH2htau), mapping between 26 and 230 amino acids of the longest human tau isoform, is detectedin cellular and animal AD models and is neurotoxic in hippocampal neurons. The NH2htau –but not thephysiological full-length protein– interacts with Aβ at human AD synapses and cooperates with it ininhibiting the mitochondrial ANT-1-dependent ADP/ATP exchange. Here we show that the NH2htau alsoadversely affects the interplay between the mitochondria dynamics and their selective autophagic clear-ance. Fragmentation and perinuclear mislocalization of mitochondria with smaller size and density areearly found in dying NH2htau-expressing neurons. The specific effect of NH2htau on quality control ofmitochondria is accompanied by (i) net reduction in their mass in correlation with a general Parkin-mediated remodeling of membrane proteome; (ii) their extensive association with LC3 and LAMP1 autoph-agic markers; (iii) bioenergetic deficits and (iv) in vitro synaptic pathology. These results suggest that NH2htaucan compromise the mitochondrial biology thereby contributing to AD synaptic deficits not only by ANT-1inactivation but also, indirectly, by impairing the quality control mechanism of these organelles.

© 2013 Published by Elsevier Inc.

Introduction

Mitochondria dysfunction and clearance are contribution factors tomammalian aging and to several age-related neurodegenerative disor-ders named tauopathies, including Alzheimer's Disease (AD) (Batleviand La Spada, 2011). Compelling evidence shows a causal link betweenmitochondrial dysfunction and impaired synaptic plasticity in AD

f 20–22 kDa; ANT-1, Adenineamyloid precursor protein; Aβ,ein; FAD, Familiar Alzheimer'sdrial DNA; CS, citrate synthase;hloroquine; FCCP, (carbonyl cy-tubules.rmacology (IFT), CNR, Via Fosso

.ct.com).

vier Inc.

pathogenesis at the early preclinical stage (Du et al., 2012; Lee et al.,2012b; Lin and Beal, 2006). Early deficits of synaptic mitochondriaare indeed detected in mutant APP transgenic mice prior to the ex-tracellular Aβ deposition (Du et al., 2010) and mitochondria dys-function in the hippocampal CA3 region precedes the synapticdeterioration in Tg2576 (APPswe) mice, another FAD (FamiliarAlzheimer's Disease) model (Lee et al., 2012a,b).

Mitochondria exhibit dynamic properties and their homeostasis in ahealthy, functional network is a closely interrelated process that involvesan 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 sequestrationinto autophagosomes and retro-trafficking along microtubules into peri-nuclar clusters (mitoaggresomes) (Cai et al., 2012; Sheng et al., 2012; VanHumbeeck et al., 2011; Van Laar and Berman, 2013; Van Laar et al., 2011;Wang et al., 2009; Zhu et al., 2012, 2013). All these events are closely

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490 G. Amadoro et al. / Neurobiology of Disease 62 (2014) 489–507

coordinated and reciprocally interact in an integrated system, con-stantly monitored by every cell in order to maintain a healthy mito-chondrial population and consequent viability. Moreover, becauseof their high energy demands and very polarized morphology, theproper maintenance of mitochondrial biomass is especially crucialfor survival of post-mitotic neurons that continually modulatetheir size and number according to the variable energy demandsand metabolic states in diverse times and/or sub-cellular compart-ments (Chen and Chan, 2006; Santos et al., 2010; Van Laar andBerman, 2013; Vives-Bauza and Przedborski, 2011). A pathologicalderegulation of mitochondria turnover adversely impinges on theiroverall shape, location and metabolic functions so that they are no lon-ger transported along neuritis and provided to highly-ATP utilizationsites, such as synaptic terminals (Nunnari and Suomalainen, 2012;Sheng et al., 2012). Consequently, neuronal populations –especiallythose with poorly myelinated, long, thin axons located in selectivelyvulnerable AD brain areas– are crucially sensitive to improper mito-chondrial dynamics (Verstreken et al., 2005).

To this regard, a growing body of studies actually shows that,alongwith an altered trafficking and interorganellar communication,changes in the quality control of mitochondria –including quality,number and distribution of their functional population– are causallycorrelated with the synaptic demise in vulnerable, affected AD neu-rons (Campello and Scorrano, 2010; Eckert et al., 2011; Karbowskiand Neutzner, 2012; Palmer et al., 2011; Reddy, 2011; Reddy et al.,2012; Santos et al., 2010; Sheng et al., 2012). An impaired mitochon-drial biogenesis, followed by an ensuing deterioration of their bioen-ergetics (Rintoul and Reynolds, 2010; Sheng et al., 2012; Young-Collieret al., 2012), and a severe reduction of thesemetabolically-active organ-elles from distal processes in correlation with their parallel accumula-tion into neuronal soma are both early events in AD synaptic failure(Chen and Chan, 2009; Selfridge et al., 2013; Wang et al., 2008a,b,2009) and, importantly, are detectable prior to any obvious clinicalsign (Stokin et al., 2005). Ultrastuctural changes inmitochondria lengthwith a dramatic loss in their inner cristae organization, aswell as a dras-tic reduction in global biomass of these organelles, were early found inthree different FAD transgenic mice mainly at synapses, in correlationwith their energetic failure and prior to the onset of the memoryimpairment or of the plaque appearance (Trushina et al., 2012). In ADpatients, a morphometric analysis carried out on affected brain regionsreports a significant paucity of mitochondria (Baloyannis, 2006) thatexhibit evident signs of destruction, including rupture of their innermembrane, lipofuscin-filled vacuoles, and variable mtDNA (mitochon-drial DNA) content (Hirai et al., 2001). Functional parameters of mito-chondria –such as the respiratory capacity and the antioxidantenzyme activities– are strongly reduced of approximately two-thirdsin AD specimens, almost in part due to a loss of their biomass and/orto their impaired biogenesis (Young-Collier et al., 2012). Biochemicalunbalance in the expression levels of several mitochondria-shapingproteins (OPA1, Mfn1/2, Drp1, Fis) or alterations in their posttransla-tional modifications are also found in cellular and animal AD models,as well as in human AD neurons, in association with an excessive frag-mentation and mislocalization of these organelles, with neuritic bead-ing and with a loss of dendritic spines (Barsoum et al., 2006; Calkinsand Reddy, 2011, 2011; Cho et al., 2009; Manczak et al., 2011; Tanet al., 2007; Wang et al., 2008a,b, 2009). In neurons from Tg2576(APPswe) mice, an impaired mitochondrial biogenesis is accompaniedwith amislocalization of these BrdU-positive small, defective organellesthat reside mainly in the cell soma and cannot be transported to axonsand dendrites, hence depriving the nerve terminals of ATP and calcium-buffering ability (Calkins et al., 2011). Finally an impaired autophagicflux, including an increased rate of mitochondrial autophagic removalthat leads to selective loss of these organelles –which progressivelylessen in number, volume and cytoplasmic density (Moreira et al.,2007a,b)– has been described as a prominentADhallmark in vulnerablehippocampus and prefrontal cortex (Baloyannis, 2006; Young-Collier

et al., 2012). Altogether, these findings point out that pathogeneticdefects in the maintenance of functional mitochondrial biomass areearly events participating in the AD synaptic failure and further bolsterthe notion that it may be possible to alleviate the AD bioenergeticcollapse at least in part by interfering with mitochondrial homeostasis(Bonda et al., 2010). However whether excessive mitophagy, owing toan uncontrolled quality control pathway of these organelles, is detri-mental, beneficial, or simply an epiphenomenon and whether it actsas an early causal event in AD pathogenesis or a late neuroprotectiveconsequence of other neuropathological insults, remains under intensedebate (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 thehuman tau40, (i) is neurotoxic in primary cultured neurons (Amadoroet al., 2004, 2006); (ii) is detected in cellular and animal AD models(Corsetti et al., 2008) and (iii) is also largely enriched in human mito-chondria from AD synaptosomes in correlation with the pathologicalsynaptic changes, with the Aβ multimeric species and with the organ-elle functional impairment (Amadoro et al., 2010). The NH2htau– butnot the physiological full-length protein– preferentially interacts withAβ peptide(s) at human AD synapses and cooperates with it ininhibiting the mitochondrial ANT-1-dependent ADP/ATP exchange(Amadoro et al., 2012). In view of that: (i) a structural and functionalimpairment of neuronal mitochondria jointly contribute to early synap-tic 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 NH2htaucan also affect the distribution, shape and size of the mitochondria.Using both in vivo and in vitro systems, we found out that patholog-ical NH2htau enhances the mitochondria turnover by harmfullyaffecting their fusion/fission dynamics and their clearance by selec-tive autophagy.

Results

TheNH2htau fragment early affects the neuronalmitochondriamorphologyin vitro, leading to fragmentation and a net decrease of their biomass

AsNH2htau specifically interactswithmitochondria in AD (Amadoroet al., 2012), we sought to investigatewhether its enhanced intracellularlevel, as we detected in vivo (Amadoro et al., 2012), influences themito-chondrial network integrity in primary cultured neurons.

To this aim, mature hippocampal primary neurons (DIV 15) weretransduced (75% efficiency) by adenovirus-mediated infection withmyc-tagged NH2-26–230 tau (myc-NH2htau) and with myc-taggedempty vector (mock) at low/moderate multiplicity (MOI 50). In agree-ment with our previous investigations (Amadoro et al., 2004, 2006),the specific expression of exogenous myc-NH2htau in cultured neuronswas confirmed to be correlated with a significant reduction of neuronalsurvival. As assessed byMTT assay and intact nuclei count (seeMaterialsand methods), after 12–16 h post-infection at MOI 50 only 60% of cellssurvived while no significant difference in viability was found upontransduction with mock or unrelated LacZ-protein (not shown).

The morphological profile of mitochondria was next examined byultrastructural analysis of Transmission Electron Microscopy (TEM). Incontrol neurons themajority of mitochondria appeared healthy in elon-gated or tubular shape, along with a well-preserved nuclear envelopewhich surrounded a disperse chromatin (Fig. 1A). As previously report-ed (Frezza et al., 2006), matrices were dark and uniform and the cristaewere homogeneously distributed and highly interconnected, showingregular intercristal cross-sectional distances. On the contrary, NH2htau-expressing neurons revealed heterogeneous mitochondrial alterations

491G. Amadoro et al. / Neurobiology of Disease 62 (2014) 489–507

in a similar pattern to that found in vivo in AD brains (Wang et al., 2008a,2009). In detail, several of these organelles appeared shorter, globularand less elongated and sometimes cloudy (Fig. 1A, arrow heads), justresembling the in vitro deleterious changes due to the neuronal expres-sion of mitochondria-targeted, toxic, intracellular Aβ (Cha et al., 2012).Numerous mitochondria appeared roundish and pale with patchymatrices, sometimes swollen and suffering from the total disap-pearance of their inner cristae (Fig. 1A, arrows), mimicking the ADpathological changes found in vivo (Baloyannis, 2006; Hirai et al.,2001; Moreira et al., 2007a,b). We also detected a dramatic and ab-errant remodeling in inner membrane cristae revealing enlarged,dilated electron-transparent space (Fig. 1A, curved arrows), a mito-chondrial morphology which is evident during the OPA-1/DRP-1mediated fragmentation of these organelles (Frezza et al., 2006;Jahani-Asl et al., 2011; Knott et al., 2008) and upon the Parkin-mediated reshaping/degradation in their outer membrane (Yoshiiet al., 2011). Moreover mitochondria, along with an increasedcircularity and a decreased connectivity (see below) which areboth indicators of a reduction in length of these organelles due totheir excessive fragmentation (Dagda et al., 2009, 2011; Xie andChung, 2012a,b), showed disorganized cristae (Fig. 1A asterisks)with an irregular and interrupted intermembrane space. We alsoobserved discontinuous, fractured outer membranes (Fig. 1A, boldarrows) which were similar to that visualized during the impairedturnover of these organelles promoted by the exogenous expressionof α-synuclein (Choubey et al., 2011; Nakamura et al., 2011), anotherneuronal protein which also bindsmitochondria and sometimes coexistsin several FADpedigrees at synaptic terminalswith hyperphosphorylatedtau (Muntané et al., 2008). In addition, ultrastructural analysis revealedthat NH2htau-transduced cultures also contained cytosolic vesicleswhich appeared to be early autophagosomes enclosing dark mitochon-dria (Fig. 1A, upper inset), alongwith late/degradative vesicles containingamorphous, totally and/or partially digested material which are morecomparable to autophagolysomes and/or lysosomes (Fig. 1A, lowerinset; Supplemental Fig. S1). It is also noteworthy that, although theoverall number of mitochondria significantly decreasedwith the increas-ing MOI in our in vitro neuronal model, we found between control andNH2htau-transduced cultures –even at lowest dosage– a drastic diminu-tion in density of these organelles, asmeasured bymass (see below), dueto a smaller average size and/or to complete loss in inner structures.

Next, in order to determine whether the changes of mitochondriaintegrity (size and shape) as we detected in the NH2htau-expressingneurons were specific –i.e. associated to changes of mitochondrial pro-teome and not of other subcellular compartments– we analyzed thewhole extracts from cultures infected at increasing MOI (25–50–100)by reacting immunoblots with antibodies against several structural orsoluble mitochondrial proteins located to outer membrane (VDAC1,Tomm20) (Figs. 1C–D), inner membrane (COXIV, Timm 23, ATP syn-thase β-subunit) (Figs. 1E, F, G), intermembrane space (cytC) (Fig. 1H)or to matrix (MnSOD-II, mtHSP60) (Figs. 1I–J). β-III Tubulin was usedas internal loading control (Fig. 1K). Although with different kineticsand with a relatively parallel pattern discernible during the explicitinduction of mitophagy (Chan et al., 2011; Geisler et al., 2010; Liuet al., 2012; Narendra et al., 2008), the forced expression of myc-NH2htau –but not of the mock vector used at high MOI (MOI 100)(Fig. 1B)– induced at 12 h a decline in the mitochondrial mass even atlowest-toxicity dosage (MOI 25), as already previously revealed by elec-tron micrographs (Fig. 1A). Concomitantly, the increasing intracellularlevel of NH2htau reduced in a dose-dependent manner a large numberofmitochondrialmarkers, thus lessening the content of these organellesper cell over controls (Chan et al., 2011; Liu et al., 2012; Van Humbeecket al., 2011;Wang et al., 2011). At 12h post-infection, such decrease didnot involve mtHSP60 (Fig. 1J) and F1β (not shown) whose delayeddecline –as previously reported (Chan et al., 2011; Wang et al., 2011)–occurs only at later times and more slowly after mitochondrial stress.Actually, in line with other reports (Chan et al., 2011; Wang et al.,

2011), a more pronounced degradation of all the above mentionedmitochondrial proteins (including HSP60 and F1β) was observed inour in vitro system at prolonged times of incubation (18–24h). Densito-metric quantification of results is shown in Fig. 1L, by normalizingvalues on the β-III tubulin signal to establish the relative abundance ofmitochondrial proteins per overall cell protein content. Importantly,no difference was contextually found in the expression levels of non-mitochondrial markers such as for endoplasmic reticulum (calnexin)(Fig. 1M), for Golgi complex (α-58K) (Fig. 1N), and for peroxisomes(catalase) (Fig. 1O) that remarkably use the same machinery offission as the mitochondria (Koch et al., 2010), or for axonal trans-port motors (kinesin/dynein) that are involved in the organelledelivery (Figs. 1P-Q), pointing out a selective process which drivesthe mitochondria removal in these neuronal cultures. Moreover,when expressed at similar low level (MOI 50) of toxic NH2htau frag-ment, the full-length human myc-tau 1–441 (htau40) did not affectthe neuronal survival (Amadoro et al., 2006) and did not cause themitochondrial content decline (see below, Fig. 5), further strength-ening that the event was also specific and not simply an artifact ofoverexpression of myc-tagged tau proteins. Likewise, significantchange neither in viability (Amadoro et al., 2006) nor in mitochon-drial mass (not shown) was found upon adenovirus-mediated trans-duction of primary neuronal cultures with unrelated LacZ-protein.

To ascertain whether the almost general decrease in the amount ofmitochondrial proteins detected by Western blotting was due to anactual decrease in mitochondrial mass, the quantity of mitochondriawas further estimated in these neuronal cultures transduced at MOI50 by two different methodologies. One was spectrophotometric mea-surement of the activity of citrate synthase (CS), amitochondrialmatrixenzyme of the Krebs cycle, which remains highly constant in theseorganelles and is considered to be a reliablemarker of their intracellularcontent (Barrientos, 2002; Choubey et al., 2011; Pallotti and Lenaz,2001; Rustin et al., 1994; Zeviani et al., 1991). The other method wasthe estimation of their DNA amount (mtDNA), using etoposide aspositive internal control, by real-time PCR on mitochondrial NADH-ubiquinone oxidoreductase chain 2 (mtND2) gene (expressed asmtDNA/nuclear DNA ratio by normalizing to that of hTERT genomicDNA) (Fu et al., 2008). As shown (Fig. 1R), a significant decrease ofabout 50% in CS activity (normalized by the total homogenate pro-teins content) was found in NH2htau-infected hippocampal cultureswhen compared to mock-infected control neurons. Consistently andin agreement with its specific effect on decline of mitochondrial pro-teins observed by our previous Western blotting (Figs. 1C–K), theNH2htau fragment –but not full-length human tau protein expressedat similar level– induced a substantial parallel decrease of about 35–40% in the mtDNA copy number (Fig. 1S) in human SY5Y, a neuronalcell-line undergoing apoptosis with endogenous production of theNH2htau fragment (Corsetti et al., 2008) and properly used to assessthe mitochondrial DNA content in comparison with post-mitoticneurons provided (Fu et al., 2008).

Collectively these findings suggest that the moderate and early ex-pression of NH2htau fragment in neurons is sufficient (i) to evokechanges in mitochondria morphology by inducing shorter organellesthat undergo enlargement, cristae disorganization and complete lossof inner structures and (ii) to cause a frank and specific decrease of mi-tochondriamass, bothmirroring the pathogenetic in vivo changes foundin AD brains (Baloyannis, 2006; Moreira et al., 2007a,b; Wang et al.,2008a, 2009; Young-Collier et al., 2012).

Defective mitochondria are redistributed in soma and reduced in distalneuritis in NH2htau-expressing neurons

Pathological changes in mitochondria morphology/integrity cannegatively impact on their proper transport along neuritis (Nunnariand Suomalainen, 2012; Sheng et al., 2012; Xie and Chung, 2012). Like-wise, the Pink 1/Parkin pathway directly participates in regulating the


distribution of damaged mitochondria during mitophagy by arrestingtheir anterograde movements along axons (Cai et al., 2012; Kane andYoule, 2011a,b; Liu et al., 2012; Wang et al., 2011).

In viewof thesefindings, we set out to examine the subcellular local-ization of mitochondria in NH2htau-transduced neurons turning to twodifferent –but complementary– experimental approaches. We firstexplored the mitochondrial network with MitoTracker CMxRos dye(mitochondrial membrane potential-dependent) (Cho et al., 2012;Trushina et al., 2012) because (i) the morphology of these organellesremains intact after the cell fixation procedure (Zhu et al., 2011; Zhuet al., 2012) and (ii) the accumulation of this functional probe into thelipid environment of mitochondria is driven in live cells by their mem-brane potential, so that its fluorescence intensity indirectly also indi-cates the energetic state of these organelles. As shown in Figs. 2A–B, at12 h post-infection (MOI 50) we found that in control neurons most

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mitochondriawere energized, respiration-competent and appeared as avery thin, interconnected, tubular network that homogeneously cov-ered the soma and neuronal processes. On the contrary, the moderateexpression of NH2htau in neuronal cultures caused an evident and netdecrease in proportion of these polarized, healthy organelles –that isconsistent with our previous TEM analysis (Fig. 1A)– along with a dras-tic alteration in their transport/subcellular localization (Figs. 2A–B,arrow heads). In detail, dying neurons exhibited “trafficking jams”of normal as well as more globular degenerating mitochondria thatappeared unevenly distributed along the neuritis (bold arrows),thus resembling the “beads-on-string” distribution pattern (Caiet al., 2012; Trushina et al., 2012; Vande Velde et al., 2011). In addi-tion, as typically discernible during Parkin-mediated selectivemitophagy in neurons (Cai et al., 2012; Van Humbeeck et al., 2011),a large part of these organelles were clustered around the nucleus,


so that they residedmainly in the soma and could not be transportedto axons and dendrites hence depriving the distal nerve terminals ofATP and calcium-buffering capacity (Calkins et al., 2011). This intracellu-lar pattern was very similar to that visualized in NH2htau-infected cul-tures after immunocytochemistry with GRP75/mortalin/PBP74/mthsp70(glucose-regulated protein 75), another non-functional (i.e. membranepotential-independent) mitochondrial marker that labels the wholepopulation of these organelles (Fig. 2C). Furthermore this segregationwas also reminiscent of our in vivo studies carried out on degeneratingAD neurons, whose mitochondria are perinuclear and express a high en-dogenous level of NH2htau fragment (Amadoro et al., 2012).

Next we quantified the changes inmitochondria distribution detect-ed in control and NH2htau-expressing cultures, by measuring thesubcellular occupancy of these organelles in somatic and in neuriticcompartments. Morphometric analysis of the MitoTracker red fluo-rescence intensity shows that these organelles (i.e. energized mito-chondria) diminished in neuronal processes (−140%) (Fig. 2E) in alarger extent than in the soma (−60%) (Fig. 2D) of NH2htau-expressingcultures if compared to controls (Student's t test *pb0.05). Consistently,the percentage of cellular area occupied by MitoTracker red-positivemitochondria shows greater decrease in neuronal processes (−400%)(Fig. 2G) than in soma (−220%) (Fig. 2F) of NH2htau-expressing neu-rons if compared to controls (Student's t test *pb 0.05). Similar results(not shown) were obtained by quantification of BrdU-labeled mito-chondria with newly-synthesized mtDNA (Calkins et al., 2011).

Further, as the reduction in mitochondria interconnectivity and theincrease in their circularity are typically used to evaluate fragmentationof the mitochondrial network and as mitophagy indexes (Dagda et al.,2009), we performed a morphological quantification of these twoparameters onGRP75-positive organelles. Evaluation of interconnec-tivity (area/perimeter ratio) (Fig. 2H) and elongation (major/minoraxis ratio) (Fig. 2I) on GRP75-positive mitochondria shows a signif-icant reduction (−871% and −393% respectively) in NH2htau-expressing cultures when compared to controls (Student's t test**p b0.01).

These findings point up that NH2htau fragment is sufficient to evokedrastic in vitro changes not only inmorphology but also inmitochondrialocalization, leading to a peri-nuclear accumulation of these defectiveorganelles and in parallel to their uneven coverage in distal neuronalprocesses, as evident during the explicit induction of selectivemitophagy in cultured neurons (Cai et al., 2012) or as detectable inAD brains (Amadoro et al., 2012; Wang et al., 2009).

Fig. 1. NH2htau-expressing neurons exhibit early alterations in morphology of mitochondria wmature hippocampal primary neurons (DIV 15) adenovirally transduced at MOI 50 with myc-tand examined at 12–16h post-infection. The greatmajority ofmitochondria in the control groupcristae. On the contrary, organelles in myc-NH2htau-expressing neurons (myc-NH2htau pane(curved arrows) or disorganized (asterisks) inner membrane cristae. Mitochondria “discontinuof the inner cristae (arrows) were also evident in NH2htau-expressing neurons. Ultrastructuralthe initial stages of mito-autophagosome digestion and localized around an altered nuclear envrial (lower inset), jointly indicating the complete and efficient autophagic removal of organellWestern blotting analysis (n = 5) on total protein extracts from hippocampal primary neuroand with mock control (MOI 100) and examined at 12h post-infection. Immunoblots and relattochondrial proteins located to outer membrane (VDAC1, Tomm20) (C–D), inner membrane (matrix (MnSOD-II, mtHSP60) (I–J) are shown. The dose-dependent expression of myc-tagged Ntometric analysis is shown (L) bynormalizing the immunoreactivity protein intensity on theβ-Iproteins. Values are means of five independent experiments and statistically significant diff***p b 0.0001 versus mock). (M to S) Western blotting analysis (n= 5) on total protein extract(25–50–100) with myc-NH2htau and with mock control (MOI 100) and examined at 12 h posmarkers such as for endoplasmic reticulum (calnexin) (M), for Golgi complex (α-58K) (N), foand dynein p150 glued (P–Q, respectively) or for two autophagic markers Microtubule-Associatively). (T) Estimation of mitochondrial mass carried out at 12 h on rat hippocampal neurons iactivity of citrate synthase expressed as nanomoles per minute per milligram of protein. Valuecalculated by unpaired-two tailed Student's t test (***p b 0.0001 versus mock). (U) Quantitattransduced human SH-SY5Y neuroblastoma cells, by measuring the content of a specific mitochscriptase (hTERT) genomic DNA was used to normalize and 10 μM etoposide (not shown) wastically significant differences were calculated by unpaired-two tailed Student's t test (*p b 0.05

Parkin-driven changes in the levels of mitochondria reshaping proteinsOPA1 and Mfn1/2 also occur in NH2htau-expressing neurons

The E3 ubiquitin ligase Parkin plays a critical role in regulating thequality control of mitochondria in neurons (Karbowski and Youle,2011; Sheng et al., 2012; Vives-Bauza and Przedborski, 2011; Vives-Bauza et al., 2010; Ziviani and Whitworth, 2010), by intersecting thedynamics of these organelles and then by stimulating their selectiveautophagic degradation (Cai et al., 2012; Joselin et al., 2012; Tanakaet al., 2010; Van Humbeeck et al., 2011). As well as for fragmentationand for arrest of motility, it has been reported that Parkin can facilitatethe selective clearance of mitochondria by promoting the rupture,ubiquitination and proteasome-dependent degradation (Ashrafi andSchwarz, 2012) in several membrane-bound proteins of these organ-elles —such as GTPases Optic Atrophy 1 (OPA1) (Chan et al., 2011;Yoshii et al., 2011) and Mitofusin 1/2 (Mfn1/2) (Glauser et al., 2011;Ziviani and Whitworth, 2010).

In view of these considerations, we moved about ascertainingwhether the NH2htau-induced mitochondrial loss was associated to aParkin-dependent targeting/remodeling of mitochondrial membraneproteome and/or to changes in the expression of reshaping proteinswith nonrespiratory functions but which more directly control theintegrity of these organelles in neurons (Trushina et al., 2012; Wanget al., 2009). To this aim, the pattern of several key proteins for mito-chondrial fission (i.e. Drp1) and fusion (i.e. OPA1, Mfn1/2) along withthe upstream Pink 1/Parkin pathway (Glauser et al., 2011; Springeret al., 2011; Wang et al., 2011; Yu et al., 2011) was also analyzed inour in vitro neuronal model. By Western blotting analysis on totalprotein extracts, we found that the graded increase of myc-NH2htauintracellular level was accompanied at 12 h post-infection in primaryhippocampal cultures by a modulation in processing/stability towardsfull-length high molecular weight form (63 kDa) of Pink 1 (Fig. 3A), aSer/Thr kinase known to cooperate with Parkin in promoting the selec-tive removal of mitochondria in neurons (Joselin et al., 2012; Wanget al., 2011) during the explicit induction of mitophagy (Ashrafi andSchwarz, 2012). Remarkably, the myc-tagged NH2htau increased thePink 1 66/55 kDa ratio at mitochondria (Fig. 3A) by favoring thesteady-state accumulation of 66-kDa full-length form over the 53 kDalower one, the latter is localized in the inner membrane fraction andrepresents a mature form (i.e. avoided of the mitochondrial targetingsequence) that does not recruit Parkin (Jin et al., 2010). On the contrary,inmock-infected neurons, the 66-kDa full-length Pink 1 –that is instead

ith selective and net removal of these organelles. (A) Electron microscopy images (TEM) ofagged NH2-26–230 tau (i.e.myc-NH2htau) and with myc-tagged control vector (i.e.mock)(mockpanel) had a typical filamentous-elongatednetworked patternwithwell-organizedls) showed mainly round-shaped, fragmented morphology (arrow heads) with enlargedous”with fractured outer membranes (bold arrows) or “ghost”with a total disappearanceanalysis by TEM also demonstrated electron-dense mitochondria (upper inset), resemblingelopes, along with larger electron-translucent autolysosomes with partially digested mate-es. The right upper panels show enlarged views of the boxed area. Scale bar 1 μm. (B to L)ns (DIV 15) adenovirally transduced at increasing MOI (25–50–100) with myc-NH2htauive densitometric graphs revealing an extensive dose-dependent proteolysis of several mi-COXIV, Timm 23, ATP synthase β-subunit) (E, F, G), intermembrane space (cytC) (H) or toH2htau was checked (B) and β-III tubulin was used as internal loading control (K). Densi-

II tubulin signal to establish the relative abundance ofmitochondrial proteins per overall cellerences were calculated by unpaired-two tailed Student's t test (*p b 0.05; **p b 0.001;s from hippocampal primary neurons (DIV 15) adenovirally transduced at increasing MOIt-infection. Immunoblots were probed with specific antibodies against non-mitochondrialr peroxisomes (catalase) (O), for two axonal transport motors Kinesin High Chain (KHC)ted Protein 1A/1B-light chain 3 (LC3) and NBR1 (neighbor of BRCA1 gene 1) (R–S, respec-nfected with of myc-tagged vectors (MOI 50) by spectrophotometric measurement of thes are means of five independent experiments and statistically significant differences wereive real-time PCR determination of mitochondrial DNA (mtND2DNA) at 12 h on MOI 50-ondrial gene NADH-ubiquinone oxidoreductase chain 2. Human-Telomerase Reverse Tran-included as internal control. Values are means of two independent experiments and statis-versusmock).

Fig. 2. Fragmented and defective mitochondria accumulate in the soma and in the proximal dendritic regions in NH2htau-expressing neurons. (A to C) The sub-cellular localization ofmi-tochondria was assessed at 12 h post-infection (MOI 50) in mock- and myc-NH2htau-expressing hippocampal cultures by live-staining (red channel) with MitoTracker CMxRos (mem-brane potential-dependent) (A–B) and by immunofluorescence (green channel) with GRP75 (glucose-regulated protein 75) (C), two mitochondrial markers that label the polarizedand whole organelle population respectively. Nuclei (blue channel) were stainedwith DAPI. A DIC (Differential Interference Contrast) light transmitted channel was acquired to visualizeneuritic structures (B). Note that fewpolarizedmitochondria aremainly clustered in somatodendritic regions of NH2htau-expressing neuronswhile amore uniformnetwork of energized,respiration-competent and interconnected organelles is clearly detectable in healthy, control cultures. (D–E) Estimation of theMitoTrackermean pixelfluorescence intensity in cell bodies(D) and in neuritis (E) between control (white column) and NH2htau-infected (black column) cultures. Values are means of three independent experiments and statistically significantdifferenceswere calculated by unpaired-two tailed Student's t test (*pb0.05 versusmock). (F–G) Estimation of the proportion of cellular area occupied byMitoTracker-positivemitochon-dria (ratio between the area occupied by the fluorescence and the area sampled) in cell bodies (F) and in neuronal processes (G) between control (white column) and NH2htau-infected(black column) (unpaired-two tailed Student's t test *p b 0.05). (H–I) Quantification, on GRP75-positive mitochondria, of interconnectivity (area/perimeter ratio; H) and elongation(major/minor axis ratio; I) between control (white column) and NH2htau-infected (black column) cultures. Values are means of three independent experiments and statistically signif-icant differences were calculated by paired-two tailed Student's t test (**p b 0.01 versus mock). Scale bar: MitoTracker=20 μm; DAPI-GRP75=15 μm.


specifically located on the outer mitochondrial membrane where it canrecruit Parkin (Jin et al., 2010; Narendra et al., 2010)– was only faintlydetected. Furthermore, a significant depletion in immunoreactivity oflong OPA1 isoform (Fig. 3B) and Mfn1 and Mfn2 (Figs. 3C–D) was alsodetected already after the moderate myc-NH2htau-overexpression(MOI 25), suggesting that the low-toxicity level of intracellular NH2htauis sufficient to perturbate the mitochondria integrity at least in part bypromoting a non-fusing state of these organelles, as previously de-scribed to occur in vivo in affected AD neurons (Wang et al., 2009).Notably, as in mitophagy induced by Parkin-mediated remodeling ofouter membrane proteome (Chan et al., 2011), the graded intracellularexpression of NH2htau destabilized in a dose-dependent way the longOPA1 isoform (Fig. 3B) which disappeared more rapidly than the shortone that, on the contrary, is more resistant to degradation (Song, et al.,2007). The progressive decline of the OPA1 steady-state level detectedin NH2htau-expressing neurons with the increasing MOI not only sup-ported our previous ultrastructural findings (Fig. 1A and SupplementalFig. S1), showing that a much larger number of small and roundedmitochondria was found in these cultures than in control ones, butalso aligned with the fact that an up-regulation of OPA1 expressionlevel in neurons is per se sufficient to restoremitochondrialmorphology(Jahani-Asl et al., 2011) while a depletion of its immunoreactivity islinked to a diminished fusion capacity of these organelles (Twig et al.,2008). Moreover, consistently with others (Tanaka et al., 2010) and ina similar way of mutant A53T α-synuclein that also binds to mitochon-dria and promotes their fission through a Drp-1 independent pathway(Nakamura et al., 2011; Xie and Chung, 2012), no significant changewas found in the relative total level –or in its Ser616 phoshorylation

(not shown)– of Drp1 (dynamin-related protein 1), a mitochondrial-fission GTPase known to promote fragmentation of these organelles inAD (Wang et al., 2009). Densitometric quantification of results is shown(Fig. 3F), obtained by normalizing on mtHSP60 intensity (Fig. 3E) toestablish the relative abundance per mitochondrion.

Next we checked whether the Parkin translocation to mitochondria–which triggers mitophagy in primary cortical neurons in response to avariety of mitochondrial damaging agents (Joselin et al., 2012)– also oc-curred in our 12h-infected cultures. As shown by biochemical fraction-ation of cytosolic/mitochondrial proteins (Figs. 3G–H–I–L), not only didwe confirm that the largest part of overexpressed myc-NH2htau(MOI 50) was localized at mitochondria in tau-transduced neurons(Amadoro et al., 2010; Corsetti et al., 2008), but also we found outthat its distribution to these organelles was paralleled by that of endog-enous Parkin, which was indeed recovered mainly in mitochondria-enriched fractions and nearly excluded from corresponding cytosoliccompartments. Conversely, we did not find any significant recruitmentof endogenous Parkin at mitochondria in mock-cultures at 12 h post-infection even after treatment for 5 h with 10 μM FCCP (carbonyl cya-nide 4-(trifluoromethoxy)phenylhydrazone), a protonophore which isknown to lead to fragmentation of these organelles and to their selec-tive removal by autophagy in primary neurons only after prolongedtimes (18–24h) of incubation (Cai et al., 2012).

Altogether, these findings suggest that the inverse correlationdetected in NH2htau-expressing neurons between the recruitment ofendogenous Parkin to mitochondria and the downregulation in the ex-pression levels of their reshapingproteinsMfn1,Mfn2, OPA1 –justmim-icking the parallel changes occurring in AD neurons (Wang et al.,

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Fig. 3.Mitochondrial Parkin translocation and perturbations inmitochondria dynamics also occur in NH2htau-expressing neurons. (A to F) Representative blots (A–E) ofWestern blottinganalysis (n=3) on total protein extracts fromhippocampal primary neurons (DIV 15) adenovirally transduced at increasingMOI (25–50–100)withmyc-NH2htau andwithmock control(MOI 100) and examined at 12 h post-infection. The expression pattern of several key proteins controlling mitochondria turnover was assessed by probing immunoblots with specificantibodies against Pink 1 (A), and two pro-fusion proteins OPA1 (B) and Mfn1 and Mfn2 (C–D). Densitometric quantification of results was obtained (F) by normalizing onmtHSP60 in-tensity (E) to establish the relative proteins abundance permitochondrion. Values aremeans of three independent experiments and statistically significant differences were calculated byunpaired-two tailed Student's t test (***pb0.0001 versusmock). (G to L) Representative blot ofWestern blotting analysis (n=2) on equal proteins amount of cytosolic andmitochondrialfractions (40 μg) from hippocampal primary neurons (DIV 15) adenovirally transduced for 12h with myc-NH2htau (MOI 50) and with mock control (MOI 100)—in the absence or in thepresence of 10 μM FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) added to cultures for the last 5 h. The co-distribution of overexpressed myc-NH2htau and endogenousParkin atmitochondria in infectedneuronswas ascertained byprobingwithmyc-tag antibody (H) andwithmouse Parkin (clone Park8) (G). The efficiency and thepurity ofmitochondrialpreparationwere checkedbyprobing fractionatedprotein fractionswith several specific antibodies reactingwith cytoplasmatic (copper/zinc–Cu/Zn–SODI) (L) andmitochondrialmarkers(mtHSP60) (I).


2009)–may account for the generation of more roundish, ruptured anddisorganized organelles relative to the controls (Fig. 1A) and for the fa-cilitation of mitophagy flux (Ashrafi and Schwarz, 2012; Glauser et al.,2011; Joselin et al., 2012).

Mitochondrial autophagy is increased in NH2 human tau-expressingneurons

Next,we ascertainedwhether theNH2htau-induced abnormal clear-ance ofmitochondria that leads in neurons to reduction of their biomassoccurred via the autophagy–lysosomal pathway, as previously sug-gested in Figs. 1R–S. The autophagic nature of this event was tested byconfocal analysis on MOI 50-infected hippocampal cultures at 12 h

after double immunofluorescence with cytC (green) and LC3 (red),two markers used routinely to detect activation of mitophagy in neu-rons (Cai et al., 2012; Van Humbeeck et al., 2011). CytC is a dynamicmi-tochondrial marker localized to intermembrane space of theseorganelles and its cytosolic release is a very late event occurring duringexcitotoxic death (Cheung et al., 2005; Cregan et al., 2002), as previous-ly reported to occur after the exogenous expression of NH2htau in neu-rons (Amadoro et al., 2006). LC3 (Microtubule-Associated Protein 1A/1B-light chain 3) is a known autophagic indicator that localizes in itslipidated form (LC3II) to autophagosomal membranes (Klionsky et al.,2008, 2012). As shown in Fig. 4A, NH2htau-expressing neurons wereendowed with a large number of LC3-stained puncta colocalizing withcytC-positive fragmented mitochondria that –as we have previously

image of Fig.�3


detected (Figs. 2A–B)– were clearly accumulated in somatodendriticcompartment (mito-aggresomes). No overlapping labeled dots (yel-low) were found in mock-infected neurons that showed a healthy andeven distributed cytC-positive mitochondrial network along with a dis-perse and nearly undetectable LC3 staining in the cytoplasm. It is note-worthy that under physiological conditions, axons and dendrites arealmost devoid of autophagic vacuoles and lysosomes, since neuronsare particularly vulnerable to developing autophagic stress (Chu,2006). In healthy neurons autophagosomes are rarely observed, likelydue to their high basal autophagic rates compared to other cell typesso that the autophagic material is rapidly degraded (Yue et al., 2009),and LC3 exists under normal condition predominantly in its solubleLC3-I form which shows a faint and diffuse staining (Wang et al.,2006). Consistently,morphometric analysis of autophagic vesicles num-ber showed a 43% increase in the number of LC3-positive mitophagicbodies (tau-infected versus mock-infected: Student's t test *p b 0.05)and their relative diameter similarly raised to 52% (tau-infected versusmock-infected: Student's t test *p b 0.05). These data put forward thatthe expression of NH2htau-truncated formwas sufficient to significantlyincrease the autophagic neuronal phenotype, alike another COOH-derived (Asp 421) caspase-cleaved tau fragment (Dolan and Johnson,2010). In addition, the number of cytC-positive fragmented mitochon-dria that were also labeled by LC3 enhanced to 74% (tau-infected versusmock-infected: Student's t test **pb0.01), thus confirming that theseorganelles were mainly degraded via the autophagy–lysosomalpathway in NH2htau-expressing neurons. Remarkably, the possibleactivation of non selective-autophagy was ruled out by colocalizationanalysis of cytC/LC3 double staining performed with the Pearson'scorrelation coefficient (Zinchuk and Grossenbacher-Zinchuk, 2009)that estimated an average value (Rr) of 0.89 ± 0.02 in NH2htau-infected cultures and of 0.35 ± 0.27 in controls (Student's t test**pb0.01), indicating a high and low degree of codistribution respec-tively. Of note the Manders' coefficients showed a medium-high de-gree of overlap between cytC and LC3 signals in mock-infectedcultures (LC3/cytC = 0.73 ± 0.02; cytC/LC3 = 0.68 ± 0.07) whichsignificantly (Student's t test **p b 0.01) increased nearly to 100% (LC3/cytC=0.98± 0.01; cytC/LC3=0.97±0.01) of overlap in NH2htau-infected. Notably these findings suggested that in mock-conditionsthere were basal co-expression levels of LC3/cytC markers thatwere not correlated –as indicated by the low Pearson's coefficient–which, on the contrary, appeared to increase and correlate afterNH2htau-infection. Importantly, the NH2htau overexpression alsoproduced a dramatic increase in dotted labeling of lysosome markerLAMP-1 that –consistent with the preferential subcellular localiza-tion of mature and acid lysosomes in neurons (Cai et al., 2012)–was detected mainly in the perinuclear area and partially colocalizedwith Tomm20-positive roundish mitochondria (Fig. 4A′), therebysuggesting that a proportion of defective mitochondria were effi-ciently removed by lysosomes. On the contrary, in mock-infectedneurons, the LAMP-1 staining appeared as small puncta that didnot colocalize with Tomm20-positive elongated mitochondria. Thecomplete and preferential clearance of these organelles occurringalong the autophagic flux in NH2htau-expressing neurons was alsodemonstrated by ultrastructural analysis by electron microscopywhich revealed the presence of perinuclear electron-dense mitochon-dria (Fig. 1A, upper inset), just resembling the initial stages of mito-autophagosome digestion/maturation (Viscomi et al., 2012). Early/degradative, doublemembrane-limited vesicles containing cytoplasmicmaterial and/or organelles at various stages of degradation were alsofound along with more mature “empty” late/degradative vesicles(Fig. 1A, lower inset; Supplemental Fig. S1). These vesicular profiles(Boland et al., 2008) indicated that the fusion of autophagosomeswith late endosomal and lysosomal compartments was efficient in ourin vitroneuronalmodel. In linewith thesefindings,we evaluated the co-efficient of variation (CV = SD/mean) for LC3 fluorescence intensitypresent in the neuronal bodies which has previously been used as an

index of the compartmentalization/diffusion state of a protein in the cy-tosol and it is considered a reliable measure of the endogenous level ofautophagic activity (Campanella et al., 2009; Klionsky et al., 2012). Thefluorescence CV (Fig. 4A′) was of 0.69 for LC3 in the NH2htau-expressing neurons, demonstrating a net shift towards amore compart-mentalized cytosolic state of this autophagic marker when compared tothe corresponding lower and roughly similar values estimated in themock ones (0.45 for LC3). For LAMP1, due to the variability in the sizeand number of positive lysosomial organelles, which could affect theevaluation of the fluorescence intensity (Falcón-Pérez et al., 2005) anadditional analytical indexwas also carried out. To estimate the concen-tration of LAMP1 staining, the size and the mean fluorescence intensityof the fluorescent structures were taken into account. Again, in theNH2htau-expressing neurons a significant 71% increase in respect of thecontrol ones was observed (Fig. 4A′). The findings that in NH2htau-expressing the analytical values were significantly (Student's t test*pb0.05) higher when compared to control ones for both early and lateautophagicmarkers supported the notion that a removal ofmitochondriaoccurred up to thefinal steps in these cultures, according to the increasedexpression level of exogenous NH2htau.

As known, during autophagy, soluble proLC3 is proteolysed togenerate its cytosolic form (LC3-I) which is conjugated to phospha-tidylethanolamine to form LC3–phosphatidylethanolamine conju-gate (LC3-II) that, eventually, is recruited to autophagosomalmembranes. Therefore the mobility shift of LC3-II –which exhibitsa faster migration by SDS-PAGE– as well as the increased conversionratio LC3II/LC3I is frequently used to assess the induction of autoph-agy in neurons (Kabeya et al., 2000; Klionsky et al., 2008, 2012;Viscomi et al., 2012). Moreover, an increased autophagic flux leadsto an augmented elimination of p62/SQTM-1 (Sequestosome-1)and NBR1 (neighbor of BRCA1 gene 1) (Bjørkøy et al., 2005; Kirkinet al., 2009; Komatsu et al., 2007; Viscomi et al., 2012), two proteinsthat interact with LC3-II and thus become incorporated into theautophagosomes and subsequently degraded in lysosomes (Yue et al.,2009). Consistently with our morphological observations, the autopha-gy activation upon overexpression ofmyc-NH2htauwas further validat-ed by the fact that the diminution in level of p62/SQTM-1 and NBR1immunoreactivity was contextually correlated in these neurons by aninverse elevation in LC3-II/LC3-I ratio (Figs. 4B–C–D), as shown byWestern blotting on total protein extracts and relative densitometricanalysis (Fig. 4F). Notably, no change or even a modest and transientincrease in the amount of p62/SQTM-1 has also been described inconditions where there is an augmented autophagic flux (Klionskyet al., 2012). Finally, in agreement with others (Chu et al., 2007; Zhuet al., 2007) reporting that mitophagy can also occur independently ofBeclin-1, no significant difference (not shown) was found in level ofthis key pro-autophagic protein whose decline was associated withaging and AD pathogenesis (Pickford et al., 2008). Although an inversecorrelation between the increase in LC3-II immunoreactivity and thedecrease in p62/SQTM-1 and in NBR1 is largely considered a reliablemethod to monitor the endogenous autophagic flux (Klionsky et al.,2008, 2012), we decided to measure it by an alternative and otherwiseexact experimental procedure, such as by inferring the LC3-II turnoverin the presence and in the absence of agents that eventually neutralizethe pH lysosomal degradation (Klionsky et al., 2008, 2012). To thisaim, we quantified by Western blotting analysis and relative densito-metric analysis (Figs. 4H–K), the difference in the intensity level ofLC3-II in the presence and in the absence of bafilomycin A1 (BAF-A1,50 nM) and chloroquine (CQ 25 μM), by comparing NH2htau-expressing neurons to control ones. BAF-A1 is a cell-permeable, selec-tive inhibitor of vacuolar-type (v-type) H+ATPase while CQ is anotheralkalizing reagent –such as NH4Cl– used to raise the lysosomal pHwhich leads to inhibition of both fusion of autophagosome withlysosome and lysosomal protein degradation. Under basal conditionsNH2htau-expressing neurons showed, relative to control ones, an in-creased LC3-II level which –when the lysosomal degradation was

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Fig. 4. The NH2htau fragment accelerates the autophagic flux of mitochondria (mitophagy). (A–A′) In A: Confocal microscopy analysis of double immunofluorescence for cytC (green channel)and LC3 (red channel), carried out on primary hippocampal cultures at 12 h post-infection (MOI 50) with mock- and myc-NH2htau vectors. Nuclei were stained with DAPI (blue channel).LC3immunofluorescence is very faint in mock-treated group while numerous labeled LC3 stained vesicles –which were also intensely positive (colocalized) for cytC– were observed in myc-NH2htau neurons. Morphometric graphs show the diameters (expressed in μm) and the number of autophagic vesicles (LC3 positive) between control- (white column) and NH2htau-infected neurons (black column). Plot of the spatial profile (white line in the LC3/cytC merged image) of pixel intensity for cytC (green line) and LC3 (red line) indicates the net increase of im-munoreactivity in correspondence of three double-labeled vesicles. Values aremeans of three independent experiments and statistically significant differenceswere calculated by unpaired-twotailed Student's t test (*p b 0.05 versusmock). In A′: Double immunofluorescence analysis with lysosome marker LAMP-1 (green channel) and mitochondrial marker Tomm20 (red channel),carried out on primary hippocampal cultures at 12h post-infection (MOI 50) withmock- andmyc-NH2htau vectors. Some fragmented TOM20-positive mitochondria (red channel) andmanyLAMP1-positive vesicles (green channel) were colocalized (yellow dots) and accumulated in the somatic compartment of NH2htau-expressing cultures. In mock-expressing cultures, LAMP1showeda faint immunoreactivity and ahealthy anduniformly distributedTOM20-positivemitochondria networkwas evident. Scale bar: 10μm.Histograms show the analysis of LC3 autophagicactivity performedbyevaluation of the coefficient of variation (CV=SD/mean) andof immunofluorescence intensity alone or dividedby the area for LAMP-1 (pixel intensity andpixel intensity/μm2; seeMaterials andmethods) between control- (white column) and NH2htau-infected neurons (black column). CVwas evaluated on cell bodieswhile the fluorescence intensity representsthemean pixel intensity only in the vesicles. Values aremeans of three independent experiments and statistically significant differences were calculated by unpaired-two tailed Student's t test(*p b 0.05; **p b 0.01 versus mock). (B to F) The modulation in expression levels of p62/SQTM-1 (B), NBR1 (C) and LC3-II/LC3-I ratio (D), three key proteins involved in autophagosomesformation, induced by neuronal transduction (MOI 25–50) with myc-NH2htau were evaluated by biochemical Western blotting on total protein extracts and relative densitometric analysis(F). Densitometric quantification of immunoreactivity levels was calculated by normalizing the values on the β-III tubulin intensity (E). Values are means of three independent experimentsand statistically significant differenceswere calculated by unpaired-two tailed Student's t test (***pb0.0001 versusmock). (G to L)Western blotting analysis and relative densitometric analysis(H–K), showing the difference in the intensity level of LC3-II in the absence and in the presence of bafilomycin A1 (BAF-A1, 50nM) and chloroquine (CQ 25 μM) for the last 6 h, by comparingNH2htau-expressing neurons to control ones. Assessment of autophagic flux calculated as the ratio of LC3II densitometric value of BAFA1- or CQ-treated samples over the corresponding un-treated samples. Values are means of three independent experiments and statistically significant differences were calculated by unpaired-two tailed Student's t test (**p b 0.01 versus mockormyc-NH2htau; ***pb 0.0001 versusmock). Representative immunoblot (n=3) for endogenous COXIVwas also shown (I) and relative densitometric analysis (L) was calculated by normal-izing the values on the β-III tubulin intensity (J). Values aremeans of three independent experiments and statistically significant differences were calculated by unpaired-two tailed Student's ttest (***p b 0.0001 versusmock; **p b 0.01 versusmyc-NH2htau).



inhibited with BAF-A1 or CQ– was further potentiated (Fig. 4H lane 5versus 4 comparedwith lane 2 versus1; Fig. 4H lane 6 versus4 comparedwith lane 3 versus 1). The fact that the higher basal level in LC3II immu-noreactivity was enhanced after lysosomal inhibition in NH2htau-expressing neurons much more than in control clearly demonstratedthat an accelerated turnover of protein cargoes via autophagy occurredin these cells (Fig. 4K). Importantly, an upregulation of COXIV immuno-reactivity was also found in NH2htau-expressing cultures after inhibitortreatment (Figs. 4I–L), supporting the notion that their elevated rate ofautophagic flux was associated to a selective removal of mitochondria.Collectively, these results point out that the increased intracellularlevel of NH2htau fragment –as we found in vivo in AD mitochondria(Amadoro et al., 2012)– is associated to selectivemitochondrial autoph-agic clearance in neurons.

Mitochondrial quality control changes in NH2htau-expressing neurons incorrelation with the in vitro synaptic pathology

Themorphological aswell as functional impairment ofmitochondriais crucial in synaptic demise occurring during the AD onset (Lee et al.,2012b,; Du et al., 2010). A potential consequence of an unbalance of mi-tochondria dynamics in neurons is indeed the failure to provide thesehealthy organelles to sites of high-energy consumption, such as synap-tic terminals, thus depriving of ATP and rendering themmore suscepti-ble to reactive oxygen species (ROS) (Li et al., 2004). In addition,mitochondria buffer calcium ions and its inefficient handling can alsocontribute to synaptic dysfunction and subsequently to “dying-back” neurodegeneration (Calkins and Reddy, 2011; Nunnari andSuomalainen, 2012; Reddy and Beal, 2005; Sheng and Cai, 2012). To in-vestigate whether the morphological abnormalities of mitochondria(i.e. reduced size and density) and their accumulation in thesomatoneuritic regions were accompanied to concomitant modifica-tions in synaptic domains, we assessed the expression level and distri-bution of several specific pre- and post-synaptic markers in NH2htau-transduced neurons, by Western blotting and immunocytochemistryat 12 h post-infection. Consistently with that found in vivo in AD neu-rons (Wang et al., 2009), the immunoreactivity of α-synuclein(Fig. 5A), synaptosome-associated protein of 25 kDa (SNAP-25)(Fig. 5B), synapsin I (Fig. 5C), synapthophysin (Fig. 5D) and PSD95(Fig. 5E) significantly decreased with the increasing MOI (25–50–100). Densitometric quantification of immunoreactivity level wasshown (Fig. 5G), by normalizing the values on the β-III tubulinintensity (Fig. 5F). Accordingly, confocal microscopy showed thatNH2htau-infected cultures presented fewer and less immunoreac-tive synaptic puncta if compared with control, mock-infected neu-rons (Figs. 5H–I). Morphometric analysis (Figs. 5H–I) revealed asignificant decline in the fluorescent intensity and in the number ofsynapsin-labeled (tau-infected versus mock-infected: 97% in intensityand 88% in number; Student's t test *p b 0.05, **p b 0.01) and ofsynapthophysin-positive buttons (tau-infected versus mock-infected:98% in intensity and 299% in number; Student's t test **p b 0.01). Re-markably and in agreement with our estimation of mtDNA content inneuronal cultures (Fig. 1S) the full-length human myc-tau 1–441(htau40) (Tolkovsky, 2009) –when expressed at similar low level(MOI 50) of toxic NH2htau fragment (Fig. 5J)– did not alter the Pink-1stability (Fig. 5K) and did not decrease the expression of p62/SQTM-1(Fig. 5L) nor of OPA-1, VDAC1, cytC, α-synuclein and SNAP-25 (Figs.5M–N–O–P–Q), as shown byWestern blotting analysis on total proteinextracts. These findings further corroborate the specific and potenteffect of neurotoxic NH2-truncated tau because the physiological formof human tau was ineffective in perturbating the expression level ofproteins entailing with the mitochondrial autophagy (Figs. 5K–L–M–

N–O) as well as with the synapse stability (Figs. 5P–Q).Altogether, these findings suggest that the dysfunction in quality

control of mitochondria induced by a specific enhanced level ofNH2htau fragment in cultured neurons is associated with pathological

synaptic alterations, as found in pathological AD conditions (Wanget al., 2009).

Impairment in COX activity and increased ROS production also occur inNH2htau-expressing neurons in correlation with pathological changes ofmitochondria dynamics

Changes in mitochondrial morphology can affect the mitochondrialmetabolism (Nunnari and Suomalainen, 2012; Sheng et al., 2012;Trushina et al., 2012; Van Laar and Berman, 2013) and mitochondriafunctions were reduced in AD brains almost in part owing to a loss oftheir mass (Young-Collier et al., 2012) as well as in three FAD mousemodels (Trushina et al., 2012). Elongatedmitochondria display a highercristae density and then an enhanced efficiency of ATP synthase, whichsustains a more intense metabolic intracellular activity (Gomes et al.,2011). In view of these findingswe quantified the variation of the respi-ratory capacity in adenovirally-transduced cultures (MOI 50) by mea-suring at 14 h post-infection a mitochondrial functional parameter,such as cytochrome c oxidase (COX) activity which is commonly usedas an index of oxidative ability (Choubey et al., 2011; Wredenberget al., 2002). Fig. 6A reports the values found in total cell homogenates(for details see Materials and methods) –obtained from mock– andNH2htau-tranduced neurons, respectively. As shown, a significant de-crease of 33%, in COX activity (normalized by the total homogenate pro-teins content), was found inNH2htau-infected cultureswhen comparedto control ones. Importantly, considering the mtDNA-derived CS(Fig. 1R) as reliable marker of mitochondrial content, the ratio COX/CSactivity in NH2htau-expressing neurons (MOI 50) was quite differentfrom that of control ones (compare Fig. 6A to Fig. 1R), regardless ofthe reduction in expression level of COXIV subunit protein (Fig. 1E).

Furthermore, a regulatory role for reactive oxygen species (ROS) ofmitochondrial origin as signaling molecules in selective autophagy hasbeen described (Scherz-Shouval and Elazar, 2007). Excessive oxidativestress is inseparably linked to mitochondrial dysfunction in tautransgenic mice and in flies (David et al., 2005; Dias-Santagataet al., 2007), as these organelles are both generators of and targetsfor ROS (Lee et al., 2012a). Furthermore, one of the most importantnon-enzymatic antioxidant defenses in neurons is the glutathione(GSH) system which is in part localized in the mitochondrial matrix,where it plays a key cellular role in counteracting the detrimentaleffects elicited by an increased ROS production (Dasuri et al.,2012). Therefore, as the glutathione disulfide (GSSG)/reduced gluta-thione (GSH) ratio (GSH/GSSG ratio) is routinely used as an indicatorof the intracellular redox state levels, we decided to compare it betweenmock- versus NH2htau-expressing cultures (MOI 50, 14 h) in order toinvestigate whether an overproduction of ROS also occurred in theseneurons. As shown in Fig. 6B, a dramatic increase of 255% (normalizedby the total homogenate protein content) in GSSG/GSH ratio wasfound in NH2htau-infected cultures in contrast to control ones, furthersuggesting that their mitochondria were largely defective being sourceof deleterious ROS.

Consistently with obvious ultrastructural changes (Fig. 1A) and in asimilar way of other pathogenetic proteins that are also localized tomitochondria in different neurodegenerative human diseases, such asParkinson's Disease (PD)-associated A53Tα-synuclein (Choubey et al.,2011; Nakamura et al., 2011) and DJ1 (Wang et al., 2012) and ALS-linked mutant SODG93A (Magrané et al., 2012), these results demon-strate that the NH2htau fragment impinges on themitochondrial oxida-tive capacity and the antioxidant defense in neurons also by affectingthe quality control of mitochondria.

Discussion

The results we outline here provide evidence that the pathologicalN-terminal truncation of human tau –which has known to be criticallyinvolved in onset and progression of diverse human age-related

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Fig. 5. Physiological full-length tau is ineffective in perturbating the expression level of proteins entailingwith themitochondrial autophagy as well aswith the synapses stability. (A to G)Representative blots (A–G) of Western blotting analysis (n=3) on total protein extracts from hippocampal primary neurons (DIV 15) adenovirally transduced at increasing MOI(25–50–100) with myc-NH2htau and withmock control (MOI 100) and examined at 12h post-infection. The intracellular levels of NH2htau down-regulates in a dose-dependentmanner (MOI 25–50–100) the immunoreactivity of pre-synaptic α-synuclein (A), synaptosome-associated protein of 25 kDa (SNAP-25) (B), synapsin I (C), synapthophysin (D)and post-synaptic PSD95 (E). Densitometric quantification of immunoreactivity level was shown (G), by normalizing the values on the β-III tubulin intensity (F). Values aremeans of three independent experiments and statistically significant differences were calculated by unpaired-two tailed Student's t test (*p b 0.05; ***p b 0.0001 versusmock). (H–I) Representative images at 12 h post-infection (MOI 50) of synaptic structures labeled with synapsin (H) and synapthophysin (I) (red channel). Nuclei were stainedwith DAPI (blue channel). A DIC (Differential Interference Contrast) light transmitted channel was acquired to visualize neuritic structures. Synapsin- and synapthophysin-labeled punctashow amarked decrease of relative fluorescence intensity and number inmyc-NH2htau-expressing hippocampal neurons compared to controls. Morphometric graphs confirm as signif-icant the detected differences in staining intensity and number of synapsin- and synapthophysin-positive dots between control (white column) and NH2htau-infected (black column)cultures. Values are means of three independent experiments and statistically significant differences were calculated by unpaired-two tailed Student's t test (*p b 0.05; **p b 0.01 versusmock). Scale bar: 10μm. (J to R) Representative blots (J–Q)ofWestern blotting analysis (n=4) carried out on total protein extracts fromhippocampal primary neurons (DIV15) thatwereadenovirus-mediated infected for 12 h with the full-length human myc-tau 1–441 (myc-htau) and with truncated myc-NH2htau (J) at similar low level (MOI 50). Control neurons were in-fectedwithmock-vector aMOI 100 and collected at 12h asmyc-tagged-expressing cultures. The activation ofmitophagic pathway and synapses abundancewas compared in infected cultures,by assessing the expression pattern of Pink-1 (K), p62/SQSTM1 (L), OPA-1 (M), VDAC1 (N), cytochrome c (O), α-synuclein (P), SNAP-25 (Q). β-III tubulin (R) was used ad loading control.Values are means of three independent experiments and statistically significant differences were calculated by unpaired-two tailed Student's t test (*p b 0.05; ***p b 0.0001 versusmock).


tauopathies including AD (García-Sierra et al., 2008) and whose levelwe previously found to be increased in mitochondria from AD brains(Amadoro et al., 2010, 2012)– associates to an impairment of

mitochondrial quality control in primary hippocampal neurons. By dif-ferent and complementary experimental approaches, we report thatthe toxic human NH2-truncated tau strongly affects the turnover of

Fig. 6. Impairment in COX activity and increased GSSG/GSH ratio are associated to alter-ation of mitochondria turnover in NH2htau-expressing neurons. (A) Spectrophotometricdetermination of mitochondria COX activity, measured by monitoring the decrease inabsorbance during the cyanide-sensitive oxidation of reduced form cytochrome cred(10 μM), in the presence of 3 μM rotenone plus 0.8 μM antimycin A, and normalized bythe total homogenate proteins content, in total cell homogenates from mock-and NH2htau-tranduced neurons (MOI 50) at 14 h post-infection (for detail seeMaterials and methods). Absorbance decrease was recorded with a Jasco doublebeam/double-wavelength spectrophotometer UV-550 at 548 nm–540 nm. Valuesare means of five independent experiments and statistically significant differences werecalculated byunpaired-two tailed Student's t test (***pb0.0001 versusmock). (B) Spectro-photometric determination of the glutathione disulfide (GSSG)/Glutathione (GSH) ratio,which measures the ROS-dependent-thiol oxidation state, in total cell homogenatesfrom mock- and NH2htau-tranduced neurons (MOI 50) at 14 h post-infection (for detailsee Materials and methods). Values are means of five independent experiments and sta-tistically significant differences were calculated by unpaired-two tailed Student's t test(***p b 0.0001 versus mock).


neuronal mitochondria since we found out an inverse correlation be-tween the accumulation of endogenous Parkin at these organelles andsevere perturbations in their turnover. Importantly, the early changesof mitochondrial dynamics occur in dying neuronal cultures in concom-itance to bioenergetic deficits and synaptic damages. Taken togetherand consistently with other papers reporting that unbalancedmitophagy could be extremely deleterious in post-mitotic neurons(Cheung and Ip, 2011; Wong et al., 2011a,b) and that pathological taucan be per se a potent and direct upstream regulator of autophagy inneurons (Inoue et al., 2012; Pacheco et al., 2009), our data suggestthat this pathogenetic form of NH2htau –in addition to its direct inhibi-tory action on ANT-1 translocator (Amadoro et al., 2012; Bobba et al.,2013)– may also affect in primary neurons the mitochondrial qualitycontrol which, in turn, may critically impinge on their synapses.

Gain of function of tau protein and mitochondria quality control inneurodegeneration

A potential role for abnormalities of mitochondria quality control intauopathies has been long put forward but the mechanisms by whichthe over-expression of wild-type –as well as of mutant tau– influencesthe size, shape and distribution of these organelles in neurons (Dixitet al., 2008; Gilley et al., 2012; Stamer et al., 2002; Stoothoff et al.,2009) and consequently the synapses (Chee et al., 2005; Thies andMandelkow, 2007) are still not finally known. We cannot completelyrule out that the proposed mechanisms (Dubey et al., 2008; Ittneret al., 2009; Kanaan et al., 2011, 2012; Magnani et al., 2007) may con-tribute to mislocalization of mitochondria into soma induced by the ex-pression of MT-free NH2htau in neurons, but it is all the same possiblethat this pathogenetic form of NH2-truncated tau can also influencethe distribution of these organelles by interfering with their dynamics,likely through anomalous interactions with Parkin. We do favor thispossibility because Mfn2, a specific substrate of Parkin ubiquitination(Gegg et al., 2010; Tanaka et al., 2010), is required for the movementof mitochondria in neurons (Misko et al., 2010) and the activation ofPink-1/Parkin pathway prevents the anterograde movement of theseorganelles in order to quarantine them prior to autophagic clearance(Kane and Youle, 2011; Liu et al., 2012; Wang et al., 2011). Besides, al-though authors report that soluble P301L human tau adversely affectsthe mitochondria distribution in rTg4510 mice in the absence of con-comitant alterations in size of these organelles (Kopeikina et al.,2011), our in vitro data are more in line with the in vivo AD conditionsin which a dramatic loss of inner cristae organization and of total massof mitochondria at synapses was retrieved in three different pre-symptomatic FAD mice (Trushina et al., 2012) as well as in humandiseased specimens (Baloyannis, 2006; Chan, 2006; Hirai et al., 2001),due almost in part to an increased autophagic removal (Moreira et al.,2007a,b). Nevertheless, although mutant R406W tau was recentlyreported to be able to induce an anomalous elongation of mitochondriain flies and in mouse neurons (Duboff et al., 2012), our in vitro study isstrengthened by the finding that the expression of another truncatedtau form (COOH-Asp-421) –which is more toxic to immortalized corti-cal neurons when compared with a physiological full length human tauisoform (htau40) in a similar way of the present NH2htau (Fig. 5)–causes fragmentation and impairment of mitochondria (Quintanillaet al., 2009, 2012). Accordingly, in our in vitro model, we also foundthat the exogenous NH2htau drastically compromises the integrity ofthese organelles that appeared more roundish, disorganized beingavoided of inner structures (Fig. 1A) and respiration-defective(Figs. 2A, 6), just mirroring their excessive fragmentation describedto occur in post-mitotic neurons after APP overexpression or expo-sure to oligomeric Aβ (Manczak et al., 2011; Wang et al., 2008b).Finally, another recent work supports the notion that pathogenetictau can perturbate the quality control of neuronal mitochondriain vivo showing that AD-like phosphorylated tau (AT8) physically in-teracts in AD brains with the mitochondrial Drp1 and that its fission-

linked GTPase activity was significantly elevated in APP, APP/PS1 and3XTg AD mice (Manczak and Reddy, 2012).

Mitophagy in neurons: a question of balance

Compelling evidence has revealed that –although the normal elimi-nation of defectivemitochondria is beneficial playing a homeostatic andneuroprotective role in neurons– it can cause “autophagic stress” if it isexcessive, inadequate or incomplete (Cheung and Ip, 2011;Wong et al.,2011a,b). In fact, if mitophagic rate cannot be counterbalanced by an


equivalent functional reserve of these organelles or if it exceeds the re-generative capacity of cellular protein synthesis, postmitotic neurons –especially those suffering or aged– are no longer able to counteract itand consequently the excessive autophagic demand inevitably committhem to die (Wong et al., 2011a). On the other hand, limiting theextreme autophagic/mitophagy response can allow neurons to success-fully repair/regenerate a sufficient pool of functional mitochondria andthen to survive (Zhu et al., 2012). In the present paper we show thatthe toxic NH2htau-derived fragment triggers a marked depletion of alarge amount ofmitochondria in primary hippocampal neurons contrib-uting thus to diminish their capacity for aerobic respiration, as yet de-scribed for another protein involved in AD neurodegeneration such asα-synuclein which also affects the activity of ANT-1 translocator(Choubey et al., 2011; Nakamura et al., 2011; Xie and Chung, 2012). Inline with our in vitro data (Figs. 1, 2, 4, 6), Chakrabarti et al. (2009)observed that a robust and indiscriminate upregulation of autophagytakes place in Purkinje cell degeneration (PCD) transgenicmice, leadingto selective removal ofmitochondria in vivowhich is causally associatedwith a severe neurodegenerative phenotype. Likewise, the overex-pression of mutant PD-associated A53Tα-synuclein leads in primarycortical neurons to a massive mitochondrial destruction, and then tobioenergetic deficits, which are both partially suppressed by overex-pression of wtMfn2 as well as of dominant negative Drp1K38A or bysilencing in several autophagy-related genes, such as beclin 1 andATG12 (Choubey et al., 2011). Moreover, even though we do notdetect changes in beclin 1 levels in NH2htau-expressing neurons, asreported by Choubey et al. (2011), a beclin-independent mitophagyhas been also described in neurons when acutely exposed to theparkinsonian neurotoxin 1-methyl-4-phenylpyridinium (MPP+)(Zhu et al., 2007). However, whether the in vitro genetic and/orpharmacological inhibition of autophagy could offer a significantneuroprotection against the NH2htau-induced neuronal death andwhether the relocalization of Parkin to mitochondria is causallyinvolved in NH2htau-induced selective clearance of these organelles,likely by a direct or indirect stable interaction with it, are all ques-tions that at the moment are under our ongoing investigations. Tothis regard, it's worthmentioning that recent studies have demonstrat-ed that the knocking-down of Parkin by shRNA-encoding plasmids issufficient per se to suppress the excessive mitophagy –and then toprovide partial neuroprotection– in A53Tsynuclein-expressing dopami-nergic neurons (Choubey et al., 2011). The direct interaction betweenendogenous Parkin and autophagic protein Ambra 1 (activating mole-cule in Beclin 1-regulated autophagy) enhances in neurons the dynam-ics and efficiency of Parkin-dependent clearance of mitochondria (VanHumbeeck et al., 2011). Moreover, Parkin-null cortical neuronal/glialcultures aremore resistant thanwild-type ones to toxicity of oligomericAβ 1–42 (Solano et al., 2012) that, as well as the endogenous NH2htau(Amadoro et al., 2010, 2012), targets and impairs mitochondria(Manczak et al., 2011). Interestingly, our preliminary in vitro resultsseem to be in line to these findings indicating that the viability aswell as the mitochondrial morphology and content are not affectedupon NH2htau overexpression in HeLa cells (Supplemental Fig. S2),a human cervical cancer line which has little or no endogenousParkin expression (Denison et al., 2003; Pawlyk et al., 2003; VanHumbeeck et al., 2011).

It is also worth mentioning that increasing evidence implicates thecytosolic ubiquitin/proteasome system (UPS) as part of the quality con-trol of mitochondria (Chan et al., 2011; Yoshii et al., 2011). Moreover, ifthe proteasomal degradation actually promotes or inhibits the Parkin-dependent mitophagy is still an unanswered question, especially inview of the fact that post-mitotic primary neurons are more differentfrom cell-lines (Chan et al., 2011; Yoshii et al., 2011). Therefore, we donot exclude the possibility that in our in vitro system Parkin could regu-late themitochondrial turnover in both an autophagy-dependent and aproteasome-dependentmanner but further experimentswill be neededto clarify this important point.

Finally, there are no doubts on the interplay between the bioener-getic and mitochondrial dynamics to be uniquely and specifically regu-lated in post-mitotic neurons (Cai et al., 2012; Van Laar et al., 2011).Interestingly, to this regard it has been clearly demonstrated that(i) the recruitment of Parkin tomitochondria is very slow in neurons,unlike other cells, as it occurs only after 18–24 h CCCP treatmentwhile it is barely detectable at 12 h of depolarization (Cai et al.,2012) –as in our in vitro experimental conditions– and (ii) therapid loss of ATP after a global mitochondrial insult may prevent inneurons the full-scale Parkin-associated mitophagy, in comparisonto other non-neuronal glycolytic cells (Van Laar and Berman, 2013;Van Laar et al., 2011).

Conclusions

In conclusion, in line with other papers referring that pathologicaltau per se can affect the mitochondria quality control (Duboff et al.,2012; Manczak and Reddy, 2012; Quintanilla et al., 2009, 2012) aswell as the autophagic pathway (Inoue et al., 2012; Pacheco et al.,2009) and that an increased autophagic turnover of mitochondria wasdetected in affected AD neurons (Young-Collier et al., 2012; Baloyannis,2006; Moreira et al., 2007a,b), the present study further highlights thecritical role ofmitochondrial turnover control in ADpathogenesis show-ing theNH2-truncation of human tau protein –whose levelwe previous-ly found to be increased in mitochondria from AD brains (Amadoroet al., 2010, 2012)– as a potential mediator in affecting it.

Materials and methods

Reagents and antibodies

Bafilomycin A1 (BAF-A1) B1793, chloroquine (CQ) C-6628, and FCCP(carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) C2759 werefrom Sigma Aldrich. The following antibodies were used: α-synucleinantibody mouse 610787 BD Transduction Laboratories; β-III tubulinantibody rabbit ab18207 Abcam; kinesin antibody (clone IBII)K1005 Sigma Aldrich; cytochrome C (136F3) rabbit 4280 Cell Signal-ing Technology; cytochrome C antibody (clone 7H8.2C12) mouseab13575Abcam; COX IV antibody (clone 1D6E1A8) mouse ab14705Abcam; p150 (Glued) antibody mouse 610473 BD TransductionLaboratories; LAMP1 antibody (clone LY1C6) mouse VAM-EN001Stressgen; LC3antibody rabbit PD014 MBL; LC3 antibody mouse(clone 5F10) ALX-803-080 Enzo Life Sciences; Mfn2 antibody rabbitM6444 Sigma Aldrich; c-Myc (9E10) antibody mouse sc-40 SantaCruz Biotechnology; c-Myc (A14) antibody rabbit sc-789 SantaCruz Biotechnology; OPA1 antibody mouse 612606 BD TransductionLaboratories; Parkin antibody rabbit ab15954 Abcam; Parkin anti-body mouse (clone Park8) P6248 Sigma-Aldrich; Pink-1 antibodyrabbit P0051 Sigma-Aldrich; Pink-1 antibody rabbit NB100-493Novus Biologicals; PSD95 antibody rabbit 2507 Cell SignalingTechnology; SNAP 25 (synaptosome associated protein of 25 kDa,C-terminal-specific) rabbit SL3730 Biomol International; synapsin Iantibody rabbit AB1543P Millipore Corporation; synaptophysinantibody mouse VAM-SV011 Stressgen Biotechnologies; SOD II(MnSOD, mitochondrial superoxide dismutase) rabbit SOD-110DStressgen Biotechnologies; mtHSP70 (Mitochondrial Heat ShockProtein 70) mouse MA3-028 Affinity Bioreagents; COXIV subunit Imouse A6403 Molecular Probes; SQSTM1/p62 antibody rabbit5114 Cell signaling; NBR1 antibody rabbit 5202 Cell signaling;Tomm20 antibody rabbit (FL-145) sc-11415 Santa Cruz Biotech-nology; ATPB antibody (3D5) mouse ab14730 Abcam; Drp1 anti-body rabbit NB11055288 Novus Biological; Timm23 antibodymouse 611222 BD transduction Laboratories; Mfn1 Antibodymouse NBP1-71775 Novus Biologicals; anti-VDAC/Porin antibodyab34726 Abcam; beclin-1 antibody mouse 3738 Cell Signaling;phospho-DRP1 (Ser616) antibody 3455 Cell Signaling; anti-MFN2


(AB2) antibody rabbit AV42420 Sigma; Grp75/mortalin/PBP74/mthsp70 antibody rabbit 2816 Cell Signaling.

Cell culture and treatments

SH-SY5Y human neuroblastoma cells (American Type culture Col-lection, Rockville, MD, U.S.A.) were grown in DMEM/F12 medium(Invitrogen, Gibco BRL 31331-028) supplemented with 10% fetal calfserum (Invitrogen, Gibco BRL 10108-157), 100 U/ml of penicillin and100 μg/ml of streptomycin (Invitrogen, Gibco BRL 15140-122) andmaintained at 37 °C in a saturated humidity atmosphere containing95% air and 5% CO2. Naive cells at 20% confluency were differentiated,as we previously reported (Corsetti et al., 2008), by incubation for 5–6 days in DMEM/F12 medium containing N2 (Invitrogen, Gibco BRL17502-048), dbcAMP 1mM (Sigma-Aldrich, Oakville, Ontario, Canada-D-0627) and NGF 100 ng/ml. For all treatments, the media with addi-tives were changed after 1 and 3 days. Hippocampal neurons wereprepared from embryonic day 17–18 (E17/E18) embryos from timedpregnant Wistar rats (Charles River), as we previously reported(Amadoro et al., 2006, 2010). In detail, the hippocampus was dissectedout inHanks' balanced salt solution bufferedwithHEPES anddissociatedvia trypsin/EDTA treatment. Cells were plated at 1×106 cells on 3.5-cmdishes pre-coated with poly-DL-lysine. After 2 days of culturing inneurobasal medium with B-27 supplement and glutamax, cytosinearabinofuranoside was added to reduce glial proliferation. Half of themedium was changed every 3–4 days and mature neuronal cultureswere infected at 15days in vitro, as reported in Amadoro et al. (2006).

Assessment of neuronal viability

Cell viability was quantified by counting the number of intact nuclei,after lysing in detergent-containing solution (Soto and Sonnenschein,1985; Volontè et al., 1994) and by the MTT tetrazolium salt assay(Manthorpe et al., 1986).

Immunofluorescence

Following treatment, neuronal cultureswerewashed twicewith PBSand fixed in PBS containing 4% (w/v) paraformaldehyde for 15min atroom temperature. Cells were washed with PBS 1× pH7.4 and perme-abilized with 0.1% (v/v) Triton X-100/PBS pH7.4 for 4min at room tem-perature. Coverslips were saturated with 2% BSA and 10% FCS in PBS 1×for 3h followed by incubation overnight at 4°C in a humidified chamberwith primary antibody. Unbound antibody was removed by threewashes with PBS 1×. Bound antibody was detected by incubationwith donkey anti-rabbit Alexa 488 (1:500) and donkey anti-mouse rho-damine-conjugated (1:500) secondary antibodies (Invitrogen) at roomtemperature for 30 min. Nuclei were stained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma, St. Louis, MO, U.S.A.)1:1000 in PBS for 10min. Controls were performed either by omittingthe primary antibody. For staining of mitochondria, MitoTracker redCMxRos (M7512 Molecular Probes, Eugene, OR, USA) was added at aconcentration of 500nM and then incubated for 30min before fixationas standard procedures.

Isolation of mitochondria

The mitochondrial extraction was performed using mitochondrialextraction kit (Qiagen 37612), as previously described (Amadoroet al., 2012). The purity of mitochondrial preparation was checked byprobing fractionated protein extracts with several specific antibod-ies reacting with mitochondrial markers, as previously described(Amadoro et al., 2012).

Protein cellular lysate preparation

Total proteins were extracted by scraping the cells in an SDS-reducing sample buffer or lysis in ice-cold RIPA buffer (50 mM Tris–HCl pH8, 150mMNaCl, 1% Triton, 2mMEDTA, 0.1% SDS) plus proteasesinhibitor cocktail (Sigma Aldrich, P8340) and phosphatase inhibitorcocktail (Sigma Aldrich, P5726/P2850) and centrifuged at 4 °C for15min at 1000× g. The supernatant was then collected and boiled for5 min. Proteins concentration was determined using the Bio-Rad RCDC protein assay kit.

Western blot analysis and densitometry

Equal amounts of protein were subjected to SDS-PAGE 7.5–15%linear gradient or Bis–Tris gel 4–12% (NuPage, Invitrogen). Afterelectroblotting onto a nitrocellulose membrane (Hybond-C AmershamBiosciences, Piscataway, NJ) the filters were blocked in TBS containing5% non-fat dried milk for 1 h at room temperature or overnight at4 °C. Proteins were visualized using appropriate primary antibodies.All primary antibodies were diluted in TBS and incubated with thenitrocellulose blot overnight at 4 °C. Incubation with secondary peroxi-dase coupled anti-mouse, anti-rabbit or anti-goat antibodies was per-formed by using the ECL system (Amersham, Arlington Heights, IL,U.S.A.) Final figures were assembled by using Adobe Photoshop 6 andAdobe Illustrator 10 and quantitative analysis of acquired images wasperformed by using ImageJ (http://imagej.nih.gov/ij/).

Confocal microscopy and image analysis

Slideswere examined under a confocal laser scanningmicroscope(Leica SP5, Leica Microsystems, Wetzlar, Germany) equipped with 4laser lines: violet diode emitting at 405 nm, argon emitting at488nm, helium/neon emitting at 543nm, and helium/neon emittingat 633 nm. In addition, a transmitted light detector for DifferentialInterference Contrast (DIC; Nomarski) imaging was available. Confo-cal acquisition setting was identical between control and experi-mental cases. For production of figures, brightness and contrast ofimages were adjusted and final figures were assembled by usingAdobe Photoshop 6 and Adobe Illustrator 10. Image analysis wasperformed by using Imaris Suite 7.4® (Bitplane A.G., Zurich,Switzerland) or ImageJ 1.4 (imagej.nih.gov/ij/) softwares on fivedifferent images derived from each experimental group. Image anal-ysis was performed under visual control to determine thresholdsthat subtracts background noise and takes into account neuronaland mitochondrial structures. During image processing, the imageswere compared with the original raw data to make sure that nostructures were introduced that were not seen in the original dataseries or that structures present in the original data series were notremoved. To determine fluorescent signal colocalization betweendifferent channels a median filter was applied to the images, to re-duce the background noise, and the degree of channels overlap wascharacterized by Manders' and Pearson's coefficients. To evaluatethe cell bodies and neuritic areas, their relative fluorescence intensi-ty, the mitochondria diameters and relative fluorescence intensity,two different masks were drawn on these two cellular domains byusing the light transmitted channel to visualize cell bodies andneurites. After which the DAPI and light transmitted channels weredeleted and measures were performed by identifying mitochondriaas previously described (Colaço et al., 2012). Evaluation of synapsefluorescence intensity, diameters, numbers and of autophagic vesiclefluorescence intensity, diameters, numbers was performed on the allimage field (130×130μm). Analysis of the neuritic network was per-formed by using the filament tracer module and by considering theaverage immunofluorescent area and the average neuritic length.To evaluate mitochondria fragmentation indexes images were proc-essed using ImageJ software and plugins. Using a custom-written

http://imagej.nih.gov/ij/


ImageJ macro, processed images were converted to binary (black andwhite) images by auto-thresholding, and mitochondrial particleswere analyzed for length (Major axis), width (minor axis), andarea:perimeter ratio as previously described (Dagda et al., 2009).Analysis of autophagic activity was performed by evaluation of thecoefficient of variation (CV; =SD/mean) for LC3 immunofluores-cence intensity in the neuronal cell bodies as previously described(Campanella et al., 2009; Klionsky et al., 2012). Briefly, with Imarissoftware a mask was drawn on the cell bodies, as described above,and the mean and SD were compared. Cells with increased levels ofautophagic activity, greater protein compartmentalization, greaternumber of autophagosomes or autolysosomes in their cytosol,show a CV higher. Conversely, in cells where autophagy is not occur-ring, LC3 immunofluorescence is uniformly distributed throughoutthe cytosol and the CV is lower. Vesicle immunofluorescence intensi-ty was evaluated by filtering the vesicles through thresholding basedon surface segmentation and background subtraction operated byapplying a local contrast algorithm. This procedure is able to detectfoci-like structures within a cell. LAMP1 immunofluorescence evalu-ation was performed only on positive vesicles-structures whichdisplayed a high variability in number, size and shape as previouslydescribed (Falcón-Pérez et al., 2005). To take into account LAMP1structure variability we adapted from this latter paper an analyticalstrategy by considering the mean immunofluorescence intensity di-vided by the area of positive structures per cell.

Citrate synthase and cytochrome c oxidase assay

Citrate synthase (CS) and cytochrome c oxidase (COX) activitieswere measured in cell homogenates. CS is a mitochondrial matrix en-zyme which is part of the tricarboxylic acid cycle and whose activity isfrequently used as an index of the mitochondrial content (Fernández-Vizarra et al., 2011; Kirby et al., 2007). COX activity was chosen as thereference for the OXPHOS system activity, as this enzyme (complexIV) constitutes the last step in the respiratory chain (RC) and it is,most probably, limiting its electron flux (Kunz et al., 2000; Mazatet al., 2001). CS and COX activities weremeasured by spectrophotomet-ric standard methods. Briefly, measurements of CS activity were per-formed using about 20–40 μg protein of total cell homogenate in afinal volume of 2ml. The increase of the absorbance at 412nm (extinc-tion coefficient of 13.6 mM−1 cm−1) due to the formation of a yellowcomplex of free CoA with DTNB was measured at 20 °C (Srere, 1968).The CoA is formed in the reaction of acetyl-CoA with oxalacetate toform citrate, catalyzed by CS. Absorbance change was recorded with aJasco double beam/double-wavelength spectrophotometer UV-550and the rate of CoA formation, obtained as tangent to the initial partsof the progress curves, was expressed as nmolmin−1mg−1 cell protein.Complex IV, i.e. COX, was measured by recording the decrease inabsorbance during the cyanide-sensitive oxidation of 10 μMferrocytochrome c (reduced with substoichiometric concentrationsof potassium ascorbate) at 548–540 nm, in the presence of 3 μMrotenone and 0.8 μM antimycin A (Atlante et al., 2008). Absorbancechange was recorded with a Jasco double beam/double-wavelengthspectrophotometer UV-550 and the rate of ferrocytochrome c oxida-tion, obtained as tangent to the initial parts of the progress curves,was expressed as nmol min−1mg−1 cell protein.

Glutathione disulfide/reduced glutathione ratio measurement

Glutathione (GSH) or glutathione disulfide (GSSG) was assayed incell homogenate, according to Atlante et al. (2003). Briefly, GSH in thepresence of methylglyoxal (2mM) and glyoxalase I (6 e.u.) was specif-ically converted into S-lactoyl-GSH which could be monitored directlyat 240nm;GSSG amountwas assayed in the same cuvette bymeasuringthe stoichiometric conversion of NADPH (10μM) spectrophotometrical-ly at 340nm in the presence of glutathione reductase (1 e.u.).

Transmission Electron Microscopy (TEM)

Cells were grown on coverslips in the same conditions previouslyused for optical microscopy. The coverslip was removed from the cellslayer by immersion in liquid nitrogen after resin polymerization atwarm temperature (60 °C). After a brief rinse in PBS, cells attached toa glass surface were fixed with 2.5% glutaraldehyde in 0.1M phosphatebuffer, pH 7.3, for 10min at room temperature and then for 20min at4 °C. Cells were then rinsed in 0.1M phosphate buffer, pH7.3, postfixedwith 1% osmium tetroxide in the same buffer for 1 h at 4 °C thendehydrated in ascending alcohols, and finally treated with propyleneoxide. After embedding in Araldite (Electron Microscopy Sciences, Ft.Washington, PA), ultrathin section was cut with a Reichert OMultrami-crotome. Sections were stained with uranyl and lead citrate, and thenexamined with a Transmission Electron Microscope Jeol model 100S(JEOL, Peabody, MA) operated at 80 kV. Images were contrasted byusing the ImageJ plugin CLAHE (Contrast Limited Adaptive HistogramEqualization) filter which enhances the contrast of grayscale images.Figures were prepared by using Adobe Illustrator 10.

Mitochondrial DNA content

Mitochondrial DNA content (mtDNA) was measured as described inFu et al. (2008). Briefly, neuronal cell line SH-SY5Y cells were infected asdescribed in the text. After 12h post-infection, cells were washed twicewith PBS and DNA was prepared by lysing cells in 1mM EDTA, 10mMTris, pH 8.0, 1% SDS, followed by denaturation at 95 °C for 20min anda 25-fold dilution in water. Four microliters of the diluted cell lysateswas used for Real Time PCR. Mitochondrial DNA was quantified bymeasuring content of a specific mitochondrial gene NADH-ubiquinoneoxidoreductase chain 2 (mtND2). Human-Telomerase Reverse Tran-scriptase (hTERT) DNA was used as the internal control. The quantita-tive Real Time PCR reaction was carried out in a total volume of 20 μlusing Sensimix SYBR-Green (Bioline) following the manufacturer'sinstructions. The gene specific primers utilized were: MT ND2 left:5′-ctaccgcattcctactactcaactt-3′; MT ND2 right: 5′-ggtggatggaattaagggtgt-3′; hTERT left: 5′-ccctcctttgccttccac-3′; and hTERT right: 5′-agctcccagggtccttctc-3′. Sequence-specific amplification was detected onthe ABI Prism 7900 sequence detection system (Applied Biosystems).

Statistical analysis

Experiments were carried out in triplicates and repeated at leastthree times. Data were expressed as means± SD (n= 4). Statisticallysignificant differenceswere calculated by unpaired-two tailed Student'st test (*p b 0.05; **p b 0.01; ***p b 0.0001) as indicted in the figurelegends. For Western blot analysis and immunofluorescence studiesshown were representative of at least three separate experiments.Relative differenceswere expressed as percentage change andwere cal-culated as (Exp−CTRL)/Exp×100.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nbd.2013.10.018.

Funding

This research was supported by grant from FIRB B81J07000070001and 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 de-sign, data collection and analysis, decision to publish or preparation ofthe manuscript.

Conflict of interest statement

The authors declare that they have no actual or potential conflicts ofinterest and that these data are not published elsewhere. In addition allauthors approve the study described in this report.




Acknowledgments

Wewish to thank Dott. Francesca Natale for her assistance in cultur-ing HeLa cells; Ms G. Baldini and Ms R. Bortul for electron microscopytechnical assistance; and Dott. Paolo Fiorenzo, Dott. Mauro Cozzolinoand Dott.ssa Flavie Strapazzon for providing several mitochondrialantibodies.

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