Author’s Accepted Manuscript

Genetic ablation of tau improves mitochondrial

function and cognitive abilities in the hippocampus

Claudia Jara, Alejandra Aránguiz, Waldo Cerpa,

Cheril Tapia-Rojas, Rodrigo A. Quintanilla

www.elsevier.com/locate/redox

PII: S2213-2317(18)30469-5
DOI: https://doi.org/10.1016/j.redox.2018.07.010
Reference: REDOX956
To appear in: Redox Biology
Received date: 1 June 2018
Revised date: 13 July 2018
Accepted date: 18 July 2018
Cite this article as: Claudia Jara, Alejandra Aránguiz, Waldo Cerpa, Cheril
Tapia-Rojas and Rodrigo A. Quintanilla, Genetic ablation of tau improves
mitochondrial function and cognitive abilities in the hippocampus, Redox
Biology, https://doi.org/10.1016/j.redox.2018.07.010
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 Genetic ablation of tau improves mitochondrial function and cognitive

abilities in the hippocampus.

Claudia Jara1, Alejandra Aránguiz1, Waldo Cerpa3, Cheril Tapia-Rojas*2, Rodrigo A.

Quintanilla*1

1
Laboratory of Neurodegenerative Diseases, Universidad Autónoma de Chile, Chile
2
Laboratory of Neurobiology of Aging, Centro de Biología Celular y Biomedicina

(CEBICEM), Universidad San Sebastián, Chile

3
Laboratorio de Función y Patología Neuronal, Departamento de Biología Celular y

Molecular, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile,

8331150 Santiago, Chile.

*
Address for correspondence: Laboratory of Neurodegenerative Diseases, CIB, Universidad

Autónoma de Chile, El Llano Subercaseaux 2801, San Miguel, Santiago, Chile.

[email protected]

*
Laboratory of Neurobiology of Aging, CEBICEM, Universidad San Sebastián, Carmen

Sylva 2444, Providencia, Santiago, Chile. [email protected]

Abstract

Tau is a key protein for microtubule stability; however, post-translationally modified tau

contributes to neurodegenerative diseases by forming tau aggregates in the neurons.

Previous reports from our group and others have shown that pathological forms of tau are

toxic and impair mitochondrial function, whereas tau deletion is neuroprotective. However,

the effects of tau ablation on brain structure and function in young mice have not been fully

elucidated. Therefore, the aim of this study was to investigate the implications of tau

ablation on the mitochondrial function and cognitive abilities of a litter of young mice (3

months old). Our results showed that tau deletion had positive effects on hippocampal cells

by decreasing oxidative damage, favoring a mitochondrial pro-fusion state, and inhibiting

mitochondrial permeability transition pore (mPTP) formation by reducing cyclophilin D

(Cyp-D) protein. More importantly, tau deletion increased ATP production and improved

the recognition memory and attentive capacity of juvenile mice. Therefore, the absence of

tau enhanced brain function by improving mitochondrial health, which supplied more

energy to the synapses. Thus, our work opens the possibility that preventing negative tau

modifications could enhance brain function through the improvement of mitochondrial

health.
Graphical Abstract:

Abbreviations

WT: wild-type; KO: knockout; tau-/-: homozygous tau KO; mPTP: Mitochondrial

permeability transition pore; AD: Alzheimer’s disease; GR: Glucocorticoid Receptor; HO:

heme oxygenase; GCS: γ-glutamine cysteine synthase; ROS: reactive oxygen species;

Mfn1: Mitofusin 1; Mfn2: Mitofusin 2; OPA1: optic atrophy 1; Drp1: Dynamin-related

protein 1; Fis1: Fission 1; Cyp-D: cyclophilin D; VDAC: voltage-dependent anion

channels; ANT: adenine nucleotide translocase; (PGC)-1α: Peroxisome proliferator-

activated receptor-gamma coactivator; SYP: synaptophisin; VAMP: Vesicle-associated

membrane protein; PSD95: Post synaptic density 95; NR2B: N-methyl D-aspartate receptor

subtype 2B; NOR: Novel Object Recognition; MWM: Morris Water Maze;

Keywords: tau, mitochondria, hippocampus, memory, learning,

1. Introduction

Tau is a protein that associates with microtubules and is found prominently in the axons

of neurons (Guo et al., 2017). In physiological conditions tau is involved in neuronal

morphogenesis, especially promoting axonal growth, stabilization, elongation, and

neuronal polarity (Guo et al., 2017). In addition, tau promotes vesicular transport and is

important for synaptic function (Avila et al., 2016). These and other functions of tau are

modulated by post-translational modifications in specific residues (Avila et al., 2016;

Sotiropoulos et al., 2017).

Abnormal modifications of tau are involved in a number of neurodegenerative diseases,

known as tauopathies, which are characterized by the formation of pathological deposits

of tau (Mandelkow and Mandelkow, 2012; Mudher et al., 2017). Hyper-phosphorylated

or cleaved forms of tau are the principal components of neurofibrillary tangles, one of

the neuropathological hallmarks of Alzheimer’s disease (AD) (Wang et al., 2009b).

Pathological forms of tau generate serious alterations in neuronal activity, affecting

synaptic transmission and learning and memory processes, which finally leads to
neurodegeneration (Zuo et al., 2016). Moreover, previous in vitro and in vivo studies

have shown that pathological forms of tau are likely to form tau aggregates and these

events appear to play an important role in early synaptic dysfunction and memory

deficits (Ittner et al., 2010; Lasagna-Reeves et al., 2011).

Genetic deletion of tau could be protective (Rapoport et al., 2002; Wegmann et al.,

2015). Studies in a mouse model of AD have shown that ablation of tau expression

prevents neurotoxicity induced by the Amyloid-β peptide and improves cognitive

damage (Pallo and Johnson, 2015; Quintanilla et al., 2014; Rapoport et al., 2002).

Similarly, tau deletion protects against the effects of stress on neuronal structure and

working memory (Lopes et al., 2017). However, other reports suggest that the absence of

tau could have a negative effect on normal brain function (Kimura et al., 2013).

In neurons, mitochondria are critical for synaptic function (Ly and Verstreken, 2006;

Son et al., 2012; Vos et al., 2010). Mitochondria are highly dynamic organelles that are

necessary to supply energy to the synapse (Ly and Verstreken, 2006). Also, they are

involved in Ca2+ regulation, playing a pivotal role in cytoprotection and oxidative

damage reduction (Dawson et al., 2001). Pathological forms of tau can impair

mitochondrial function, including mitochondrial morphology, transport, and

bioenergetics (Guo et al., 2017; Lasagna-Reeves et al., 2011). Interestingly, we found

that the expression of pathological tau species, in particular truncated tau, induces

mitochondrial fragmentation and bioenergetics failure in immortalized cortical neurons

and primary neuronal cultures (Perez et al., 2017b; Quintanilla et al., 2009). Similarly,

phosphorylated tau induces mitochondrial fragmentation and affects the bioenergetics

function of mature neurons (Cheng and Bai, 2018; Quintanilla et al., 2014). Thus, the
absence of tau protein in neural cells could prevent the effects on mitochondrial structure

and function produced by post-translationally-modified tau.

Considering that limited research has used tau-deficient mouse models and the role of

tau on the regulation of mitochondrial function and the resulting implications on cellular

and cognitive processes are not entirely clear, a study examining the impact of tau

ablation will contribute to the understanding of the physiological function of tau protein

in vivo. The present study was conducted in litters of young mice (3 months old) to

investigate the effects of tau reduction in hippocampal tissue, to identify the implications

of tau on mitochondrial function and behavior during youth. We used a well-known

homozygous tau knockout (KO) (tau-/-) mouse line (B6.129X1-Mapttm1Hnd/J, Stock No:

007251, The Jackson Laboratory) (Dawson et al., 2001), which is phenotypically normal

and reproductively viable (Dawson et al., 2001; Morris et al., 2013). Our findings

suggest that tau ablation improves mitochondrial function through the regulation of

mitochondrial structure and dynamics, indicated by increased levels of proteins involved

in fusion processes and decreased expression of proteins that forms the mitochondrial

permeability transition pore (mPTP). In addition, tau -/- KO mice presented a significant

reduction in oxidative damage, accompanied by the activation of Nrf-2 signaling and

PGC-1α in the hippocampus. These effects generated an improvement in the

mitochondrial bioenergetics state, indicated by increased ATP production in the

hippocampus. Finally, we observed that tau deletion improved cognitive abilities,

including exploration capacity against a stimulus, recognition memory, social behavior,

and most significantly, attention. In conclusion, tau ablation in mice resulted in

enhanced mitochondrial function in the hippocampus and improved cognitive capacities.

2. Materials and Methods

2.1 Animals.

Three month old wild-type (WT) and homozygous tau KO (tau-/-) mice were obtained

from The Jackson Laboratory (strain name: B6.129-Mapttm1Hnd/J Bar Harbor, ME,

Stock no 007251). Tau -/- mice are viable and successful reproducible, and have a

normal life-span reaching 20 month-old (Dawson et al., 2001; Tang et al., 2018). The

animals were handled according to the guidelines of the National Institute of Health

(NIH, Baltimore, MD). Animals were maintained at the Bioterio Central de la

Universidad Autónoma under a sanitary barrier and in closed colonies. All mice were

housed up and grouped to four per cage and keeping them to a monitored room

temperature of 23ºC. Animals were kept on a 12-h light/dark cycle and were given ad

libitum access to food and water according to Jackson lab recommendations (The

Jackson Laboratory, USA). Experimental procedures were approved by the Bioethical

and Biosafety Committee of the Universidad Autónoma de Chile. A total of 8 WT and

7 tau-/- mice were used to perform cognitive test. Also, for biochemical studies, we

used an n=4 of different animals for the WT group and n=5 for tau -/- group. For

mitochondrial isolation and analysis of function, we used an n=5 of different animals

for each group.

2.2 Reagents and antibodies.

The primary antibodies used were: rabbit anti-β-tubulin (sc-9104, Santa Cruz

Biotechnology, Inc., 1:2000), mouse anti-Total OXPHOS Human WB Antibody

Cocktail (ab110411, Abcam, Inc. dilution 1:2000), mouse anti-COX IV (4D11-B3-E8)

(11967S, Cell Signaling, 1:1000), mouse anti-β-actin (sc-47778, Santa Cruz

 Biotechnology, Inc., 1:1000), rabbit anti-Opa1 (PA1-16991, Thermo Scientific,

1:1000), rabbit anti-Cyclophilin D (PA3-022, Thermo Scientific, 1:1000), rabbit anti-

Fis1 (sc-98900, Santa Cruz Biotechnology, Inc., 1:500), rabbit anti-Mfn1 (H-65) (sc-

50330, Santa Cruz Biotechnology, Inc. 1:1000), rabbit anti-Mfn2 (H-68) (sc-50331,

Santa Cruz Biotechnology, Inc., 1:1000), mouse anti-VDAC1 (B-6) (sc-390996, Santa

Cruz Biotechnology, Inc., 1:1000), rabbit anti-phospho-DRP1 (Ser616) (4494, Cell

Signaling, 1:1000), mouse anti-DRP1 (C-5) (sc-271583, Santa Cruz Biotechnology,

Inc., 1:1000), rabbit anti-nitrotyrosine (n-tyr) (141682-AP, US Biological Life

Sciences, 1:500), and goat anti-4HNE (H6275-02, US Biological, Life Sciences,

1:1000). Anti-Total OXPHOS Human WB Antibody Cocktail has been previously used

with mice samples, but is necessary add anti-COX IV mouse to the correct detection,

because OXPHOS Human Cocktail not detect this mouse mitochondrial complex (Chin

et al., 2014). The fluorescent dyes used were MitoTracker™ Red CM-H2Xros (Catalog

number: M7513, Thermo Fisher Scientific), MitoTracker™ Green FM (Catalog

number: M7514, Thermo Fisher Scientific), CM-H2DCFDA (Catalog number: C6827,

Thermo Fisher Scientific), and VECTASHIELD Antifade Mounting Medium with

DAPI (Catalog number: H1200, Vector Laboratories, Inc).

2.3 Behavioral tests

All behavioral tests were monitored using an automatic tracking system (Any-MAZE

Behavioral software).
2.3.1 Morris water maze (MWM) tests

The MWM task was performed as previously described (Tapia-Rojas and Inestrosa,

2018). Briefly, the mice were trained in a 1.2-m-diameter circular pool (opaque

water, 50 cm deep) filled with 19-21 °C water. A submerged 9-cm platform (1 cm

below the surface of the water, invisible to the animal) was used for training, with a

maximum trial duration of 60 s. The mice remained on the platform for 10 s at the

end of each trial. Each animal was trained to locate the platform. The test was

performed with 3 trials per day and for each trial, the latency time required to reach

the platform and the time spent in each quadrant was measured. After testing, the

mouse was gently removed from the maze and returned to its cage.

2.3.2 Memory flexibility (MF)

MF tests were performed as previously described (Tapia-Rojas et al., 2016; Tapia-

Rojas et al., 2015). Briefly, a circular white pool was prepared with non-toxic white

paint and a hidden platform (diameter: 9 cm) in 4 quadrants. The animals were pre-

trained in this pool for 60 s (s) 1 day before the actual testing began. The water

temperature was kept between 18 and 20 °C. To acclimate the animals to the room

and the swimming strategy, the platform was removed from the pool. Then, animals

were subjected to testing for 4 consecutive days with a maximum of 15 trials per

day. Every day, the platform position in a quadrant was changed. Testing stopped

when the animal reached the platform on 3 consecutive trials with an average of 20

s or less. Data are presented as the number of trials after which the animals met this

criteria.
2.3.3 Open field (OF) tests

OF tests were performed 2 days after the MWM test as previously described (Tapia-Rojas

et al., 2015). The animals were individually placed at the center of a 72 x 72 x 32 cm white

acrylic box and were allowed to move freely within it for 10 min. For all behavioral tests,

data were gathered and analyzed with a video tracking system (HVS Imagen, UK). The

room temperature was maintained at 20 °C.

2.3.3 Novel object recognition (NOR) tests

NOR tests were performed in a 38 x 38 x 32 cm white acrylic box one day after the

OF test as previously described (Tapia-Rojas et al., 2015; Vargas et al., 2014). The

animals were habituated to the box without any objects present for 2 consecutive

days. For testing, each animal was placed at the center of the box which contained 2

identical objects (old objects) for 10 min. Then, the box and objects were cleaned

with a 50% methanol solution. Two hours later, the animal was exposed to an old

object and a new object of a different shape and color than the old object. The box

and objects were cleaned again and the next animal was tested. The recognition

index was calculated as the time spent exploring the new object divided by the time

spent exploring both objects.

2.3.4 Social Interaction Test

For this task, a protocol previously described was used (Tapia-Rojas et al., 2018).

Briefly, mice were habituated for 10 min in a 3-chamber box, with each chamber

measuring 20 x 40 x 22 cm. Then, an object and an unknown mouse were placed

inside individual lateral chambers (the same distance from the wall) and presented
 to the experimental mouse. Experimental mice started at the center chamber and

were allowed to explore freely for 10 min. Finally, the object was replaced with

another unknown mice. The experimental mouse was allowed to explore freely for

an additional 10 min. Data were collected using a video tracking system coupled to

the Honestech TVR 2.5 program and analyzed off-line using the ANY-MAZE

software.

2.3.5 Object-based Attention Test

For this task, a protocol previously described was used (Alkam et al., 2011).

Briefly, the test was performed in a rectangular apparatus containing 2 chambers,

the exploring chamber (40 x 40 x 22 cm) and the test chamber (40 x 20 x 22 cm).

First, during the habituation phase, each mouse was subjected to a 10-min

familiarization session, where they explored both empty chambers. Later, in the

acquisition phase, mice were subjected to a 3-min session where they explored five

objects (1, 2, 3, 4 and 5) separated within the chamber. Finally, a retention phase

was performed; immediately following the acquisition phase (<15 s), an old object

(used in the acquisition phase) was placed in its original position and a sixth novel

object was placed in the test chamber. The mice explored both objects for 3 min. A

recognition index of the retention session was calculated as the ratio (T6×100)/(T2

+ T6), where T2 and T6 are the times spent at objects 2 and 6, respectively.

2.4 Total RNA extraction

Total RNA was isolated from 100 mg of tissue using TRIzol® reagent (Invitrogen Life

Technologies, USA) following the manufacturer’s instructions. The residual DNA was
removed with RNase free-DNase I, Amplification Grade (Invitrogen). RNA yield and

purity were determined by TECAN (Infinite 200 PRO series).

2.5 Reverse transcription cDNA synthesis

One μg of total RNA was subjected to reverse transcription using the ImProm-II Reverse

Transcription System (Promega) following the manufacturer's protocol. cDNA was stored

at −20 °C for future use. For qPCR analysis, each cDNA sample was diluted 10 times with

nuclease-free water.

2.6 Real-time PCR

The real-time PCR reaction was performed in triplicate in the LightCycler® 96 System

(Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany) using KAPA

SYBR FAST qPCR Master Mix (2X) in a final volume of 10 μl. Amplification conditions

consisted of an initial denaturation at 95 °C for 10 min followed by amplification of 40

cycles (95 °C for 15 s, 60 °C for 20 s, and 72 °C for 20 s). A melting curve analysis was

performed immediately after amplification from 55 to 95 °C. Values were normalized to

18S expression levels using the ΔCT method.

Gene Forward primer Reverse primer

18S 5'-GCCGCTAGAGGTGAAATTCTTGGA-3' 5'-ATCGCCAGTCGGCATCGTTTAT-3'
Vdac1 5'-CGGCCACACATGATCACAGA-3' 5'-ACCAGTCTCGGGGTCTTCTT-3'
Cyp- D 5'-AGGAGATAGCCCCAGGAGAT-3' 5'-TTGCATACACGGCCTTCTCTT-3'
ANT 5'-CCACCCAGGCTCTCAACTTT-3' 5'-AAGCACAAGGATGTAGCCCC-3'
mT32-t138 (tau) 5'-GTCCTCGCCTTCTGTCGATTATC-3' 5'-GCTGTGGGGGAGACTCTTTTAAG-3'
NRF2 5'-ACCCGAAGCACGCTGAAGGC-3' 5'-GTCACTGAACCCAGGCGGTGG-3'
GR1 5'-GGGGTGACGAGGTGGAGTA-3' 5'-GATCTGGCTCTCGTGAGGAA-3'
GCS 5'-GGGGTGACGAGGTGGAGTA-3' 5'-GTTGGGGTTTGTCCTCTCCC-3'
HO1 5'-CACAGCACTATGTAAAGCGTCT-3' 5'-TGTGCAATCTTCTTCAGGACC-3'
2.7 Immunoblotting

The hippocampus of WT and tau -/- mice were dissected on ice and immediately processed

as previously described (Tapia-Rojas et al., 2018; Tapia-Rojas and Inestrosa, 2018).

Briefly, the hippocampal tissue were homogenized in RIPA buffer (10 mM Tris-Cl, pH 7.4,

5 mM EDTA, 1% NP-40, 1% sodium deoxycholate, and 1% SDS) supplemented with a

protease inhibitor mixture and phosphatase inhibitors (25 mM NaF, 100 mM Na3VO4, and

30 μM Na4P2O7) using a Potter homogenizer and then sequentially passed through

syringes of different calibers. The protein samples were centrifuged twice at 14,000 rpm for

15 min at 4 °C. The protein concentrations were determined using the BCA Protein Assay

Kit (Pierce). The samples were resolved by SDS-PAGE, followed by immunoblotting on

PVDF membranes.

2.8 Hippocampal slices and staining with mitochondrial fluorescent dyes

Coronal 20 µm slices of unfixed tissue were obtained from the brain of WT and tau -/-

mice. Slices were mounted on slides and incubated with both MitoTraker Green FM, a

marker of mitochondrial mass (Perez et al., 2017a), and MitoTraker Red CM-H2Xros, an

indicator of mitochondrial membrane potential (Giordano et al., 2014), in Krebs-Ringer-

Hepes-bicarbonate (KRH) buffer for 45 min at 37 °C. After incubation, slices were washed

3 times for 5 min in PBS and mounted with DAPI fluorescent mounting media. Images

were acquired with a high-resolution fluorescence microscope (Leica 6000x) and analyzed

using Image J software.

2.9 Isolation of hippocampal mitochondria

Hippocampal mitochondria were isolated as previously described (Karadayian et al., 2015).

Briefly, n=5 animals for group (wild type and tau -/-) were euthanized and the hippocampus

was rapidly removed and suspended in MSH buffer (230 mM mannitol, 70 mM sucrose, 5

mM Hepes, pH 7.4) supplemented with 1 mM EDTA and protease inhibitor cocktail.

Homogenates were centrifuged at 600 g for 10 min at 4 ºC to discard nuclei and cell debris.

The supernatant was centrifuged at 8000 g for 10 min; the new mitochondrial pellet was

washed twice in MSH without EDTA. Protein concentration was determined using a

standard BCA kit.

2.10 Estimation of mitochondrial complexes activity

Mitochondrial complex activity I and III were estimated through the indirect evaluation of

the Reactive Oxygen Species (ROS) production in hippocampal mitochondrial enriched

preparations (Brown et al., 2006; Karadayian et al., 2015). Mitochondrial ROS production

was measured using 25µM DCF (485 nm, 530 nm) in the Biotek Synergy HT plate reader

as previously described (Brown et al., 2006). Isolated mitochondria (25 µg of protein) were

added to 100µl of KCl respiration buffer with 5mM pyruvate and 2.5mM malate as

oxidative substrates at 37 °C. ROS production was calculated as the maximum DCF

fluorescence following 2h of incubation, expressed in arbitrary fluorescence units.

2.11 Measurement of ATP concentration

ATP concentration was measured in the hippocampal lysate and in the supernatant of

isolated mitochondria after the incubation with oxidative substrates, using a

luciferin/luciferase bioluminescence assay kit (ATP determination kit #A22066, Molecular

Probes, Invitrogen) as previously described (Tapia-Rojas et al., 2018). For ATP

determinations in isolated mitochondria, we used Trifluoromethoxy carbonylcyanide

phenylhydrazone (FCCP), as an uncoupler of the oxidative phosphorylation in

mitochondria (Benz and McLaughlin, 1983). The amount of ATP in each sample was

calculated from standard curves and normalized to the total protein concentration.

2.12 Statistical analysis

The data are expressed as the mean ± standard error of the mean (SEM), with the number of

experiments indicated in the corresponding figures. All samples included in these studies

were analyzed to normality distribution using the Kolmogorov-Smirnov test. Later, the

obtained data were analyzed using Student’s t-test with Dunnett’s post hoc test or, if

analyzing more than 2 groups, ANOVA followed by Bonferroni’s post hoc test. P<0.05

was considered statistically significant. All statistical analyses were performed using Prism

software (GraphPad Software Inc.). No statistical methods were used to predetermine the

sample size.

3. Results

3.1 Tau ablation reduced oxidative damage in the hippocampus

To determine if the absence of tau has a significant effect on hippocampal cells, we used

litters of 3 months old male WT or tau KO (tau -/-) mice. The hippocampus of WT and KO

mice were dissected and total protein or mRNA was extracted. We performed a PCR

analysis to identify tau mRNA (Figure 1A) and a western blot assay to detect the tau

protein (Figure 1B). In both results, tau was not present in the hippocampus of tau -/- mice,

confirming its ablation in KO mice. Later, we evaluated oxidative stress-mediated damage

in the hippocampus of WT and tau -/- mice (Figure 1). We determined the levels of

oxidized proteins by western blot using an anti-4HNE antibody that recognizes sulfhydryl

or histidine and lysine groups of proteins to form stable HNE-protein adducts (Pecorelli et

al., 2013) (Figure 1C), and anti-nitrotyrosine antibody, a nitric oxide production marker that

detect proteins containing nitrotyrosine (Figure 1D). Our results showed that the levels of

oxidative damage were significantly reduced in the hippocampus of tau -/- mice, as

indicated by a decrease in the levels of lipid peroxidation products and nitrotyrosinated

proteins (Figure 1C and 1D). A potential signaling pathway that is activated under

oxidative conditions is the Nrf-2 transcriptional pathway (Nguyen et al., 2009), which

controls the expression of some antioxidants, thus detoxifying enzymes and improving

mitochondrial function (Dinkova-Kostova and Abramov, 2015). Interestingly, tau -/-

animals presented a significant decrease in the total levels of Nrf-2 protein (Figure 1E), as

indicated by the densitometry analyses (Figure 1F). However, we also found that in the

hippocampus of tau -/- animals, there are increased Nrf2 mRNA levels (Figure 1G) as well

as increased expression of downstream genes of Nrf-2-dependent signaling, including the

Glucocorticoid Receptor (GR) (Figure 1H), heme oxygenase (HO) (Figure 1I), and γ-

glutamine cysteine synthase (GCS) (Figure 1J). Altogether, our results indicate that tau

ablation reduces cellular damage induced by oxidative stress in the hippocampus, possibly

by increasing the activation and transcription of Nrf-2 signaling genes which finally could

lead to Nrf-2 protein degradation.

3.2 The absence of tau positively regulates mitochondrial dynamics in the

hippocampus.

Since the mitochondria is the main producer of reactive oxygen species (ROS) in the cell

(Turrens, 2003) and considering that the Nrf2 pathway also regulates mitochondrial

function (Dinkova-Kostova and Abramov, 2015), we next investigated whether the absence

of tau modifies the expression of proteins that are fundamental for mitochondrial structure

and function. Mitochondria are dynamic organelles that normally undergo fusion/fission

processes to maintain mitochondrial function and optimize bioenergetics capabilities

(Detmer and Chan, 2007; Ni et al., 2015; Westermann, 2012). Fusion events are controlled

by the dynamin-related GTPases, Mitofusins (Mfn1 and Mfn2) and optic atrophy 1

(OPA1), which stimulate the fusion of outer and inner mitochondrial membranes,

respectively (Detmer and Chan, 2007; van der Bliek et al., 2013). When we measured these

protein levels, we observed that Mfn2 was increased, whereas Mfn1 and Opa1 showed no

significant changes in tau -/- animals (Figure 2A). In contrast, fission is mediated by

Dynamin-related protein 1 (Drp1), which is recruited to the outer membrane to constrict

mitochondria and induce its division (Anand et al., 2014; Otera et al., 2013), a process that

is mainly stimulated by Drp1 phosphorylation at Ser616 (Wang et al., 2009a). Additionally,

Drp1 interacts with other mitochondrial receptor proteins, such as Fission 1 (Fis1), to

complete this process (Chan, 2006; Liesa et al., 2009; Westermann, 2012). Our results

show that Fis1 decreased its expression while simultaneously, total Drp1 or its

phosphorylated form at Ser616, remained unaffected (Figure 2B). Therefore, the absence of

tau protein in the hippocampus increases the expression of a fusion protein and decreases
expression of a fission protein, suggesting that a pro-fusion state could be beneficial to

mitochondrial function.

In addition, we evaluated the main protein components of the mPTP, a mega protein

channel formed in the inner membrane of the mitochondria in response to mitochondrial

calcium overload that permits the release of small molecules into the cytoplasm (Halestrap,

2009; Perez and Quintanilla, 2017). The opening of the mPTP promotes uncoupling of the

respiratory chain, blocks ATP production, and can ultimately lead to apoptosis (Halestrap,

2009). mPTPs are composed of cyclophilin D (Cyp-D), voltage-dependent anion channel

(VDAC), and adenine nucleotide translocase (ANT) proteins (Gutierrez-Aguilar and

Baines, 2015). The levels of these three proteins were measured in the hippocampi of WT

and tau -/- mice (Figure 2C). Interestingly, we observed that the levels of Cyp-D and ANT

proteins, but not VDAC, were significantly reduced in tau KO mice. Surprising, mRNA

levels of Cyp-D and ANT were increased in tau -/- animals (Figure 2D-F). Since that

mRNA and protein levels correlation in mPTP components is unknown, we only could

suggest possible explanations to this phenomenon. One possibility is that increased mRNA

levels act a compensatory mechanism to the reduced protein levels, due that when

comparing protein and mRNA levels during dynamic processes, there is a delay between

transcription and translation events in the cell (Liu et al., 2016). Other possibility is that

mRNA and protein levels no shown positive correlation as consequence of differences in

the regulation of the steps in the synthesis of a protein or post-transcriptional defect (Liu et

al., 2016; Sarro et al., 2010). Despite this results, our observations indicate that tau deletion

promotes changes in the proteins that could favoring a pro-mitochondrial fusion state and

reduce Cyp-D expression, this last event could be related with a reduction in the mPTP
activation in tau -/- mice (Du and Yan, 2010; Gutierrez-Aguilar and Baines, 2015; Schinzel

et al., 2005).

3.3 Tau deletion increase hippocampal PGC1α levels

Peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α is a member of a

family of transcription coactivators that plays a key role in regulating cellular energy

metabolism (Liang and Ward, 2006). PGC-1α promotes mitochondrial biogenesis by

regulating both carbohydrate and lipid metabolism and modulating mitochondrial function

(St-Pierre et al., 2006; Wareski et al., 2009). In contrast, the absence of PGC-1α induces

mitochondrial dysfunction and neurodegeneration (Cui et al., 2006). Therefore, we

investigated the effect of tau depletion on PGC-1α protein levels and observed that tau -/-

mice have increased levels of this protein compared to WT mice (1.000 ± 0.03204 WT;

1.176 ± 0.06912 tau -/-) in approximately a 18% (Figure 3A, B). To corroborate whether

increased PGC-1α protein levels results in increased mitochondrial biogenesis, we

incubated unfixed hippocampal slices of WT and tau KO animals with the mitochondrial

dye MitoTracker Green, a cell-permeable probe that passively diffuses across the plasma

membrane and binds to lipids in the mitochondria (Elmore et al., 2004). Our analysis

revealed similar mitochondrial mass between WT and tau -/- mice (Figure 3C) in all the

analyzed regions in the hippocampus, including the dentate gyrus (DG) (Figure 3D), CA1

(Figure 3E), and CA3 (Figure 3F), indicating that tau deletion may not affect mitochondrial

mass. Therefore, our observations suggest a more active state of PGC-1α in tau -/- mice,

which could be possibly regulating mitochondrial function without modulating

mitochondrial mass in the hippocampus. This is further supported by reports which

highlight that PGC1α can modify the composition and function of individual mitochondria

independent of mitochondrial biogenesis (Austin and St-Pierre, 2012).

3.4 Mitochondrial function is improved in the hippocampus of tau -/- mice

To determine how tau proteins affect mitochondrial bioenergetics, we first measured

mitochondrial membrane potential using the fluorescent dye MitoTracker Red CMXH2Ros

(Perez et al., 2017a; Poot et al., 1996). This dye specifically detects functional

mitochondria, with the florescence intensity proportional to mitochondrial membrane

potential (Perez et al., 2017a; Poot et al., 1996). Figure 4A shows that unfixed slices from

the hippocampus of WT and tau -/- mice incubated with MitoTracker Red CMXH2Ros

presented with similar mitochondrial potential levels in the DG, CA1, and CA3 regions of

the hippocampus (Fig. 4A). Later, we evaluated the levels of protein involved in oxidative

phosphorylation using an antibody cocktail (OHPHOS) that contains all 5 mitochondrial

complexes (Chin et al., 2014; Hao et al., 2018). We observed that tau -/- mice present with

significantly reduced levels of V, III, IV, and I complexes in the hippocampus compared to

WT animals (Figure 4B). Finally, we measured the bioenergetics capacity of the

mitochondria by determining total ATP levels using a luminescence assay (see methods).

Using this assay, we determined that tau -/- mice have significantly higher ATP levels

compared to WT mice (Figure 4D). Altogether, our results suggest that despite OXPHOS

protein are decreased in the tau -/- mice, the oxidative phosphorylation of these animals is

more active, producing higher ATP levels. To validate this possibility, we isolated

hippocampal mitochondria from WT and tau -/- mice. These mitochondria were exposed to
malate and pyruvate substrates during two hours to 37°C, in presence of CM-H2CDFA dye,

to measure the ROS production and thus, indirectly the activity of mitochondrial complexes

I and III (Brown et al., 2006). Later, the supernatant was used to detect the ATP

concentration produced. Figure 4E indicate that tau -/- mitochondria produce increased

levels of ROS compared with mitochondria isolated of WT mice, suggesting a higher

activity of the mitochondrial respiratory chain. Furthermore, Figure 4F demonstrate that tau

-/- mitochondria produce significantly more ATP levels, effect that is reduced by the

presence of FCCP (a mitochondrial uncoupler) in both experimental groups. Interestingly,

the increased ROS production in tau -/- mitochondria could be beneficial to hippocampal

cells; due that although very high concentrations of mitochondrial ROS (mROS) are

detrimental to cell, normal levels of mROS are necessary to the cell homeostasis and act as

a signal to activate pathways of response to stress (Raimundo, 2014; Sena and Chandel,

2012). This could explain the activation of Nrf-2 and PGC-1α pathways in tau -/- mice (St-

Pierre et al., 2006) and the resulting decreased oxidative damage in tau -/- mice (Figure 1).

Therefore, our results strongly suggest that tau KO mice have an enhanced mitochondrial

bioenergetics.

3.5 Genetically reducing tau modifies the expression of synaptic proteins in

hippocampal tissue

Considering that mitochondria are fundamental for correct synaptic function (Cheng et al.,

2010; Hollenbeck, 2005), we measured the levels of different synaptic proteins by western

blot (Supplementary Figure 1). As pre-synaptic markers, we evaluated the levels of

synaptophisin (SYP) and vesicle-associated membrane protein (VAMP). We detected that

VAMP protein levels are significantly decreased in tau -/- mice, compared to WT
hippocampi (Supplementary Figure 1A). In contrast, we observed that tau KO mice had

increased expression of the post-synaptic markers Post synaptic density 95 (PSD95) and N-

methyl D-aspartate receptor subtype 2B (NR2B) in the hippocampi. Specifically, these

mice had significantly higher expression of PSD95 compared to WT mice (Supplementary

Figure 1B). Altogether, these results indicate that tau deletion induces changes in synaptic

protein levels, mainly by increasing a key scaffold protein necessary to maintain the correct

function of receptors in the post-synaptic cells (Beique and Andrade, 2003; Chen et al.,

2011).

3.6 Tau reduction enhances exploratory capacity and recognition memory

In order to investigate whether tau deletion influences the learning process and

hippocampus-dependent memory, we performed the Novel Object Recognition (NOR) task

(Broadbent et al., 2010). To carry out this test, we first exposed the animals to a habituation

phase, in which each animal explored the test chamber for 10 min on 2 consecutive days

(Tapia-Rojas et al., 2015). On the test day, mice were subjected to the familiarization

phase. In this stage, each animal had 10 min to explore the chamber, which contained 2

identical objects (Figure 5A). The differences in the behavior of WT mice compared to tau

-/- mice are reported in the heat map (Figure 5B) and the paths traveled (Figure 5C). We

observed that all tau - /- mice (n=7) explored more time the objects than WT mice, as

indicated by the amount of time that the animal’s head spent in object zones 1 (Figure 5D)

and 2 (Figure 5E), as well as by the number of times the animal’s head entered into object

zones 1 (Figure 5F) and 2 (Figure 5G). This is further supported by the amount of time that

the animals spent exploring the objects, as tau -/- animals explored both objects for a

significantly longer amount of time than WT mice (Figure 5H). These results suggest that
tau -/- mice have more explorative behaviors than WT mice. After 2 h, the recognition

phase (Testing phase) was performed. In this phase, for 5 min, animals explored an old

object as well as a novel object that replaced one of the former objects (Figure 5I). Our

results showed that 2 subpopulations within the litter of tau -/- mice exists. These

subpopulations differ in their behaviors toward the novel object, which also differs from the

conduct of WT mice (Figure 5J). Five tau KO animals spent more time exploring the novel

object compared to WT mice (Figure 5J, 5K and 5L), while 2 tau -/- animals had the

opposite behavior, in which they explored the old object longer than the other tau KO mice

and spent a similar amount of time exploring the novel object as WT mice (Figure 5J and

5L). Interestingly, knocking out tau improved recognition memory for the majority of the

animals, but also encouraged them to spend more time exploring objects. This could

explain why the recognition index was not significantly different between the mice (Figure

5M), as this is calculated as the time that mice spent exploring the new object versus the

time spent exploring both objects.

To determine whether the observed differences tau -/- animal behavior could be associated,

at least in part, with stress and/or anxiety, processes related to cortex-hippocampus circuit,

we developed the open field test (Supplementary Figure 2) (Seibenhener and Wooten,

2015). We found that the same animal subpopulations observed in the NOR test were

detected in this task, as indicated by the path traveled and heat map (Supplementary Figure

2A-C). As observed earlier, both subpopulations of tau -/- animals had different behaviors

from those of WT mice. The majority of tau KO animals (n=5) presented with reduced total

distance travelled (Supplementary Figure 2D) and average speed (Supplementary Figure

2E), but increased episodes and freezing time (Supplementary Figure 2F and 2G
respectively). Simultaneously, they spent more time and had more immobile episodes

(Supplementary Figure 2H-2K), and entered/spent less time in the center of the apparatus

(Supplementary Figure 2L-2N). These observations indicate that the main behavior

associated with tau -/- mice is reduced movement when the field is empty, possibly due to

more anxiety. However, this in contrast to the 2 tau -/- mice which presented with the

opposite behavior. Altogether, these results strongly suggest that tau ablation increases the

stress and/or anxiety in an empty field, but in the presence of objects, promotes the animal

to become more mobile and explorative and have a higher recognition memory. It is

important to highlight that several individuals within the litter of brothers had behaviors

that varied from the common population. This could explain the variability of results

observed in the literature.

Additionally, we evaluated spatial memory, other type of hippocampal memory (Bird and

Burgess, 2008). We performed the classic Morris Water Maze (MWM) protocol, but

observed no significant differences on any of the test days (Supplementary Figure 3A-3D).

The next day after the MWM, a probe in absence of the platform was developed. We found

that WT and tau -/- animals spent similar times and had similar entries onto the platform

area (Supplementary Figure 3F-3H). Ten days after the initial MWM test, the escape

latency was measured to evaluate long term memory and no significant differences were

observed (Supplementary Figure 3I). Finally, a memory flexibility test was performed.

There were no observed differences in the number of trials required to reach the criterion

(Supplementary Figure 3J) nor differences in swimming speed (Supplementary Figure 3K).

Therefore, the absence of tau does not modify spatial memory of young animals and only

has positive effects on recognition memory.

3.7 Tau -/- mice have similar social abilities to WT mice

Next, we investigated the effects of tau deletion on social abilities in mice, a behavior

mediated by hippocampal and cortical circuits (Bicks et al., 2015; Montagrin et al., 2017).

For this, a social interaction test was performed (Yang et al., 2011). Before phase 1 of the

task, for 10 min, the animals were exposed to an empty chamber divided into 3

compartments. During the first phase, the animals had 10 min to explore the chamber

containing an unknown mouse in a compartment and an object in the other compartment

(Figure 6A). Figure 6B shows a representative track and the heat maps of the WT and tau -

/- groups. In these representations, one can observe how the mice had an increased

preference for the unknown mouse. This was more evident when we analyzed the number

of entries or the time that the mice spent in the compartment of the object versus the

unknown mouse (Figure 6C and 6D respectively). More importantly, we observed no

significant differences when analyzing the number of entries of the animal’s head in the

area around the unknown mouse or object (Figure 6E), however, the time that the animal’s

head spend in the area around the unknown mouse is higher than the object (Figure 6F).

Additionally, tau -/- mice spent significantly more time exploring the object compared to

WT mice (Figure 6F). Therefore, these results indicate that both WT and tau -/- mice

showed a preference for a similar individual over an object, however, tau KO animals also

spent more time exploring the object, reinforcing the idea that they are more explorative

animals.

Immediately following the first stage, the object was replaced by an unknown mouse and

then the animals explored the chamber for 10 min (phase 2, Figure 6G). Representative

tracks and heat maps of WT and tau -/- animals showed that both experimental groups had
a preference for the novel mouse versus the old mouse (Figure 6H). We found that both

WT and tau -/- mice entered a novel mouse zone more times compared to an old mouse

zone (Figure 6I), but tau KO mice remained in this zone for more time compared to the WT

group (Figure 6J). These results demonstrate that when faced with a new individual, tau -/-

mice presented with similar behavior to WT mice, suggesting that tau ablation does not

modified social abilities in mice.

3.8 Absence of tau protein optimizes attention capacity

Finally, we performed a recognition-based attention test to determine whether tau deletion

favors attentive behavior, a complex process mediated by the prefrontal cortex and its

communication with the hippocampus (Alkam et al., 2011). For this task, 5 different

objects were placed in the chamber in different positions as indicated in Figure 7A. Then,

the animals explored the chamber containing the objects for 3 min. The representative

travel path and heat map of each group are presented in Figure 7B. Figure 7C shows the

amount of time that the animal’s head spent in the area of each object. With the exception

of object 4, the WT and tau KO animals spent similar amounts of time exploring each

object as well as the total time exploring all objects (Figure 7D). Interestingly, tau -/- mice

travelled a longer distance (Figure 7E) and had a higher mean speed (Figure 7F),

suggesting that they were more interested in the objects compared to WT mice. Next, in the

attention test phase (15 s later) the chamber size was reduced by half, where an old object

remained in the chamber (object 2) and a new object was added (object 6). The

representative scheme of this phase is shown in Figure 7G. The animals were allowed to

explore this chamber for 3 min. Figure 7H shows representative tracks and heat maps of

WT and tau -/- groups. In these images, it is evident that tau -/- mice prefer the novel
object, however, the distance traveled (Figure 7I) and the speed of exploration (Figure 7J)

is similar in both groups. Statistical analysis revealed that tau -/- mice entered the novel

object zone more often (Figure 7K) and spent more time exploring it (Figure 7L). This is

more evident when we analyzed the recognition index (Figure 7M). Altogether, these

results indicate that tau ablation improves the attention capacity of young animals.

In conclusion, our study demonstrates that tau deletion induces relevant improvements in

mitochondrial health and this could ultimately result in increased cognitive abilities

associated with recognition memory and attention.

4. Discussion

In the present study, we performed a complete analysis to study the role of tau on

mitochondrial function in the hippocampus of young mice, as well as on cognitive abilities,

including recognition and spatial memory, social capacity, and attention. Surprisingly, our

results indicate that tau ablation reduces oxidative damage of hippocampal cells and

activates the pathways necessary to protect mitochondrial health, such as Nrf-2 (Dinkova-

Kostova and Abramov, 2015) and PGC-1α pathways. In particular, we observed increased

levels of proteins involved in the dynamics of mitochondrial fusion processes and at the

same time, fission proteins were reduced, suggesting a pro-fusion state that could be

beneficial to mitochondrial function (Chen et al., 2007). Interestingly, these effects were

also accompanied by a reduction in the expression of an important mPTP component, Cyp-

D, and increased ATP production in the hippocampal tissue of juvenile tau -/- mice.

Finally, we analyzed the behavior of a litter of young male animals and observed that tau -/-

mice have more exploratory behavior with concomitant improved recognition memory and
attention capabilities. Thus, our results indicate that the absence of tau protein has

beneficial effects on brain function in the youth, increasing the cognitive capacity of mice

by a mechanism implicated in improvement of mitochondrial function.

Limited production of oxidative stress is a consequence of metabolic activity, in which

catabolic processing in the mitochondria forms reactive ROS (Murphy, 2009). The main

producers of ROS in cells are the mitochondria, however, peroxisomes and the endoplasmic

reticulum also contribute to the redox state (Lismont et al., 2015). Diverse reports

demonstrate that mROS have physiological roles maintain the cell homeostasis and serve

such as signaling molecules that modulate the function of pathways adaptive to stress

(Raimundo, 2014; Sena and Chandel, 2012). In contrast, higher levels of ROS are toxic to

neurons, as they damage crucial biomolecules in the cell, such as DNA, RNA, proteins, and

membrane lipids (Therond, 2006). Pathological forms of tau, including phosphorylated tau,

have been associated with increased oxidative stress; however, the increased production of

ROS could also promote tau hyperphosphorylation (Alavi Naini and Soussi-Yanicostas,

2015). This suggests that there is a positive feedback loop between oxidative stress and

phosphorylated tau protein that could result in neurodegeneration (Alavi Naini and Soussi-

Yanicostas, 2015). Therefore, preventing the occurrence of this vicious cycle could protect

neurons. In fact, in this study, we demonstrate that tau ablation reduces oxidative damage in

the hippocampus of young tau KO mice compared to WT mice, as indicated by a reduction

in the levels of nitrotyrosinated proteins and proteins containing 4-hydroxynonenal adducts.

Thus, these findings confirm the relationship between tau protein and oxidative balance, in

which the presence of tau favors an oxidative state. A signaling pathway that could

contribute to the reduced redox state is the Nrf-2 pathway. This signaling pathway is

activated in response to ROS by stimulating the transcription of antioxidant genes and

promoting the reestablishment of mitochondrial function (Narasimhan et al., 2011; Nguyen

et al., 2009). Here, we reported that tau deletion favors the activation of the Nrf-2 pathway

and induces the transcription of its target genes, including GR, HO, and GCS (Figure 1).

Interestingly, we also observed decreased levels of Nrf2 protein in the hippocampus of tau -

/- mice. Apparently the differences between Nrf2 mRNA and protein levels in tau -/- mice

are contradictory, but could be explained considering that Nrf2 is a highly unstable protein

(t½ ∼ 15 min), which translocate directly to the nucleus following its synthesis and induce

the transactivation of its genes (Nguyen et al., 2009; Stewart et al., 2003). Immediately

after, its ubiquitylated and targeted for degradation by a mechanism Keap1-dependent in

the nucleus. In contrast, under cellular stress condition, Nrf2 is stabilized by reducing its

access to Keap1 (Nguyen et al., 2009; Stewart et al., 2003), and for this reason WT mice

may have more Nrf2 protein, considering that present more oxidative damage compared

with tau -/- animals.

In the mitochondria, ROS are formed constantly by oxidative phosphorylation as a

consequence of ATP production. The reduced oxidative damage in the hippocampus of tau

-/- mice could be responsible, in part, for the improved mitochondrial function. To evaluate

this possibility, we first measured the levels of proteins that are important for mitochondrial

structure and function. Generally, mitochondria undergo fission in the presence of toxic

agents, which could result in neuronal death (Cheung et al., 2007; Cho et al., 2009). In

contrast, mitochondrial fusion is thought to be a rescue mechanism against damage and

could prevent apoptosis (Chen and Chan, 2010; Chen et al., 2007). Interestingly, we found

that the absence of tau protein in the hippocampus increases Mfn2 levels, a protein

involved in mitochondrial fusion, with a simultaneous reduction in Fis1 levels, a pro-fission

protein. These results strongly suggest a mitochondrial pro-fusion state that could promote

the health of mitochondria in the hippocampus (Mouli et al., 2009). The formation and

opening of the mPTP, which allows the release of mitochondrial components that can result

in mitochondrial swelling and cell death (Mnatsakanyan et al., 2016; Perez and Quintanilla,

2017), is also indicative of mitochondrial damage. VDAC, ANT, and Cyp-D are among the

proteins that compose the mPTP. Studies reveal that Cyp-D is necessary for mPTP opening

and interestingly, we observed that tau -/- mice had reduced levels of this protein,

suggesting that in the absence of tau protein, mitochondria are less prone to activate the

mPTP. Additional studies investigating mitochondrial swelling in the presence of higher

calcium concentrations are needed to evaluate this hypothesis.

PGC-1α is a protein that modulates the genes involved in energy metabolism. It is the main

regulator of mitochondrial biogenesis, and it was recently demonstrated that it regulates

mitochondrial remodeling by changing its intrinsic properties and promotes ROS

elimination by inducing the transcription of antioxidant enzymes (Austin and St-Pierre,

2012). Therefore, increased expression of PGC-1α improve the respiratory capacity of

individual mitochondria and remove mROS produced, minimizing the impact of ROS on

cell physiology (Austin and St-Pierre, 2012; St-Pierre et al., 2006). We measured the levels

of PGC-1α protein in the hippocampus of both experimental groups and our results

revealed that tau ablation significantly increased the expression of this protein, however, we

did not detect alterations in general mitochondrial mass. Therefore, we suggest that PGC-

1α is activated in tau -/- mice and could be removing ROS and elevating anti-oxidative

metabolism, thus contributing to oxidative homeostasis, as indicated in Figure 1. Also,

PGC-1 could be control cellular mitochondrial respiration without alter the mitochondria
mass, enhancing the respiratory capacity of individual mitochondria. To validate that

increased levels of PGC-1α are involved in mitochondrial metabolism in tau -/- mice, in

future studies we could measure the levels of threonine-177 and serine-538 phosphorylation

in PGC-1α-dependent AMPK (Austin and St-Pierre, 2012; Jager et al., 2007). In contrast,

to evaluate the participation of PGC-1α in mitochondrial biogenesis we may detect the

levels of Arginine methylation-mediated PRMT1 (Austin and St-Pierre, 2012; Teyssier et

al., 2005).

Increased mitochondrial metabolism is associated with enhanced bioenergetics function,

which could result in higher concentrations of ATP in the cell (Lemire et al., 2008; Owen

and Sunram-Lea, 2011). We found that Nrf-2 and PGC-1α signaling pathways are activated

in tau -/- mice, and both pathways could contribute to improved ATP production. In fact,

Nrf-2 target genes are increased, as indicated by increased mRNA levels of GR, HO, and

GCS, as shown in Figure 1. The observed reduction in the protein expression of several

mitochondrial complexes in the absence of mitochondrial potential changes suggest that tau

-/- mice could have increased mitochondrial respiratory chain activity, resulting in a higher

concentration of ATP in hippocampal cells. This possibility was demonstrated isolating

hippocampal mitochondria of WT and tau -/- mice, where we observed increased ATP

production in tau -/- mitochondria. On the other hand, it is interesting that WT mice

presented with similar mitochondrial membrane potential as tau -/- animals, considering

that they have significantly reduced ATP production. Interestingly, it has been described on

several reports that the high oxidative damage is formed predominantly from complex I,

which results in deficient ATP production in conditions of high mitochondrial membrane

potential (proton-motive force) (Murphy, 2009). In fact, these changes could be occurring
in the hippocampus of WT mice, as we observed increased oxidative damage accompanied

by normal mitochondrial membrane potential and reduced ATP production.

Mitochondrial function is fundamental for communication between neurons. We detected

improved mitochondrial function in tau -/- mice; and we observed reduced levels of the pre-

synaptic protein VAMP while simultaneously, PSD95 was significantly increased in tau

KO animals. These changes may be important in synaptic processes, since synaptic strength

can be regulated by PSD-95 (Chen et al., 2015; Chen et al., 2011; Fitzgerald et al., 2015).

Finally, we performed a battery of cognitive tests and reported that the absence of tau

protein in the hippocampus of young mice improves exploratory capacity, recognition

memory, and attention capabilities compared to WT mice. However, other abilities,

including spatial memory and social abilities, were not affected. These last results are

consistent with previous studies which show no significant differences in motor and spatial

learning in the MWM during the first 5 days using 6 month-old tau KO mice (Ahmed,

2014). Interestingly, our behavioral results could be explained, in part, by enhanced

mitochondrial bioenergetics function of tau -/- mice, since mitochondrial dysfunction can

affect cognition, including impaired attention, executive function, and hippocampus-

dependent memory (Finsterer, 2012; Picard and McEwen, 2014).

Attention is a complex process. Part of the prefrontal cortex known as the inferior frontal

junction controls visual processing areas that are tuned to recognize a specific category of

objects; this is a process known as object-based attention which involves focusing on what

is happening in a particular location (Baldauf and Desimone, 2014; Bichot et al., 2015).

This process has a high energetic demand (Verma et al., 2016). Interestingly, chronic stress

results in tau-mediated atrophy of dendrites and spine loss in the prefrontal cortex, an effect

that was protected against in tau KO animals (Lopes et al., 2017), supporting the idea that
tau could affect attention processes in mice. More importantly, tau deletion reduces

alterations to mitochondrial transport, oxidative phosphorylation, and the synaptic

localization of mitochondria in the prefrontal cortex after stress (Lopes et al., 2017; Vossel

et al., 2015). Therefore, our findings provide evidence that tau deletion improves the

cognitive abilities of mice by optimizing mitochondrial structure and function in the

hippocampus.

Our study is very important, because despite that were performed with a reduced number of

animals, the variability between each experimental group is minimal, evidencing the

differences of WT mice with the tau -/- group; mainly in the attentive capacity and in the

mitochondrial bioenergetics function. We used a limited number of animals, because our

focus was evaluated changes in the hippocampus of litters of brother mice in both WT and

tau -/- animals; however, we think that these results are reproducible and representative of

the hippocampal structure and function of all WT and tau -/- animals. Our work also

proposes that preventing negative tau modifications, including tau phosphorylation and tau

cleavage, could enhance mitochondrial and brain function. Additionally, the improvement

in the mitochondrial bioenergetics function and its adaptive response to stress, propose that

ablation of tau could protect the hippocampus of physiological changes such as normal

aging and of pathological conditions such as neurodegenerative diseases.

Disclosure statement

The authors confirm that there are no conflicts of interest.

Acknowledgments

This work was supported by grants FONDECYT N°. 1170441 to RQ; FONDECYT

N°11170546 and CONICYT PAI N°77170091 to CTR; Anillo ACT1411 to WC and RQ;

and a pre-doctoral fellowship from the Universidad Autónoma of Chile to CJ.

Figure 1. Tau ablation reduces oxidative stress in the hippocampus. (A) Representative

detection of the levels of tau mRNA in the hippocampus of WT and tau KO (-/-) mice by

RT-PCR. (B) Representative western blot of tau protein in hippocampal lysates from WT

and tau KO (-/-) mice; tau was measured using a specific tau antibody. Western blot of

hippocampal lysates and densitometric analyses of (C) 4HNE and (D) n-Tyr. (E) Western

blot of hippocampal lysates of Nrf2 and (F) its densitometric analyses. (G) Relative mRNA

expression of Nfr2 and (H-J) its downstream mRNA of target genes. *p < 0.05, **p < 0.01;

data are presented as the mean ± SE. n=4 different animals for WT group and n=5 different

animals for tau -/- group.

Figure 2. Effects of tau deletion on the levels of proteins involved in mitochondrial

dynamics and structure. (A) Representative western blot of hippocampal lysates and

densitometric analysis of proteins involved in mitochondrial fusion, such as Mnf1, Mnf2,

and Opa1. (B) Representative western blot of hippocampal lysates and densitometric

analysis of proteins involved in mitochondrial fission, including phospho-Drp1, total Drp1,

and Fis1. (C) Western blot of hippocampal lysates and densitometric analysis of proteins
that form the mitochondrial permeability transition pore (mPTP), such as Cyp-D, VDAC,

and ANT. (D-F) Relative mRNA expression of Cyp-D, VDAC, and ANT in the

hippocampus of WT and tau -/- animals. *p < 0.05, **p < 0.01; data are presented as the

mean ± SE. n=4 different animals for WT group and n=5 different animals for tau -/- group.

Figure 3. Tau -/- animals have increased levels of PGC-1α without affecting

mitochondrial mass. (A) Representative western blot of hippocampal lysates and (B)

densitometric analyses of PGC-1α. (C) Representative images of unfixed hippocampal

slices from WT and tau -/- mice stained with MitoGreen at 10×. (D-F) Quantitative analysis

for MitoGreen dye in DG, CA1, and CA3 hippocampal regions. *p < 0.05, **p < 0.01; data

are presented as the mean ± SE. n=4 different animals for WT group and n=5 different

animals for tau -/- group.

Figure 4. Absence of tau improves the bioenergetics capacity of hippocampal

mitochondria. (A) Representative images of unfixed hippocampal slices stained with

MitoTracker Red dye sensitive to mitochondrial membrane potential at 10× and its

quantitative analysis for DG, CA1, and CA3. (B) Representative western blot and (C)

densitometry analyses for mitochondrial OXPHOS complexes I-V levels. (D) Whole

hippocampal extracts from lysates of WT and tau KO -/- mice. ATP levels were expressed

as pmol ATP/µg of total protein extract (E) ROS production of isolated mitochondria

measured by the fluorescent dye CM-H2DCFDA after exposition to oxidative substrates, to

evaluate indirectly the mitochondrial activity of I and III complexes. (F) Isolated

mitochondria of WT and tau KO -/- mice were exposed to malate and pyruvate substrates

and then, the ATP levels were measured, expressed as ATP concentration (% of WT mice
mitochondria). *p < 0.05, **p < 0.01; data are presented as the mean ± SE. n=4 different

animals for WT group and n=5 different animals for tau -/- group in western blot analysis.

n=5 different animals for WT and tau -/- groups to mitochondrial studies.

Figure 5. Recognition memory is improved in tau -/- mice. (A) Schematic representation

of the novel object recognition (NOR) test. Mice are allowed to explore two identical

objects for 10 minutes (familiarization phase). (B) Heat maps show mean data for an entire

test group. (C) Representative tracks of WT and tau KO (-/-) mice in the NOR test

familiarization phase. Familiarization phase; the time the animal’s head spent exploring (D)

object zone 1 and (E) object zone 2. Familiarization phase; the number of head entries in

(F) object zone 1 and (G) object zone 2. (H) Familiarization phase; time animals spent

exploring both objects 1 and 2. (I) Schematic representation of the NOR testing phase,

which occurred 2 hours after the end of the familiarization phase with a familiar object and

a novel object. (J) Heat maps for the mean data for an entire test group. (K) Representative

tracks of WT and tau (-/-) mice in the NOR testing phase. (L) Testing phase; the

exploration time in the old and novel object zone. (M) Recognition Index, indicated as the

time spent exploring the novel object divided by the time spent exploring both objects. *p <

0.05, **p < 0.01; data are presented as the mean ± SE. n=8 different animals for WT and

n=7 different animals for tau -/-.

Figure 6. Social interaction is similar between WT and tau -/- mice. (A) Mice are

allowed to explore a mouse and an object for 10 minutes (Phase 1) as indicated in the

scheme. (B) Representative tracks of WT and tau (-/-) mice and the heat map of the Social

Interaction test of the entire experimental group. (C) Graphical representation of head
entries per zone, (D) time the animal’s head spent per zone, (E) head entries per area

around the mouse and object, and (F) time the animal’s head spent in each area around the

mouse or object. (G) Phase 2; mice explored a familiar mouse and a novel mouse as

indicated in the scheme. (H) Representative tracks of WT and tau (-/-) mice and the heat

map of each animal group in the Social Interaction test phase 2. (I) Graphical representation

of head entries per zone, (D) time the animal’s head spent per zone, (E) head entries per

area around the old and novel mice, and (F) time the animal’s head spent in each area

around the old or novel mouse. *p < 0.05, **p < 0.01; data are presented as the mean ± SE.

n=8 different animals for WT and n=7 different animals for tau -/-.

Figure 7. Tau ablation enhances the attention of mice. (A) Representative scheme of

the object-based attention test. Mice were exposed to five objects (1-5) for 3 min

(exploration phase). (B) Representative track and heat maps showing mean data for each

entire experimental group. (C) Graphical representation of the time that the animals spent

exploring all the objects. (D) Quantification of the total time exploring all the objects. (E)

Graphical representation of distance travelled (m) and (F) mean speed (m/s) in the

exploration phase. (G) Representative scheme of the testing phase after a 15 s interval; the

mice were exposed to one old object (2) and a novel object (6) for 3 min. (H)

Representative track and heat maps showing mean data for an entire group in the testing

phase. (I) Graphical representation of distance travelled (m) and (J) mean speed (m/s) in the

testing phase. (K) Graphical representation of the head entries per zone and (L) the amount

of time the head spent per zone. (M) Recognition index, indicated as the time exploring the

novel object divided by the time exploring both objects. *p < 0.05, **p < 0.01; data are
presented as the mean ± SE. n=8 different animals for WT and n=7 different animals for tau

-/-.

Supplementary Figure 1. Tau-/- mice show changes in synaptic proteins. (A)

Representative western blot of hippocampal lysates and densitometry analyses of pre-

synaptic proteins Synaptophysin (SYP) and VAMP. (B) Representative western blots and

densitometric analyses of the post-synaptic proteins PSD95 and NR2B. *p < 0.05, **p <

0.01; data are presented as the mean ± SE. n=4 different animals for WT group and n=5

different animals for tau -/- group.

Supplementary Figure 2. Tau KO mice have different behavior in the Open Field

compared to WT mice. (A-C) Representative tracks and heat map show the mean data for

the entire group in the Open Field test. Graphical representation of the (D) total distance

travelled (m), (E) average speed (m/s), (F) total freezing episode, (G) total time freezing,

(H) total time mobile, (I) total time immobile, (J) total mobile episode, and (K) total

immobile episode. (L) Representative tracks to evaluate the (M) number of entries and (L)

time in the center zone of the field. *p < 0.05, **p < 0.01; data are presented as the mean ±

SE. n=8 different animals for WT and n=7 different animals for tau -/-.

Supplementary Figure 3. WT and tau -/- mice have similar spatial memory in the

Morris water maze test. (A) Escape latency to find the hidden platform in the Morris

Water Maze test. Graphical representation of the escape latency (s) on (B) day 4 and (C)

day 8. (D-E) Representative tracks of the swimming path to find the hidden platform.

Quantification of (F) time (s) in the platform area and (G) number of entries in the platform
area in the probe stage. (H) Heat maps of the probe day show the mean data for the entire

test group. (I) Graphical representation of escape latency (s) 10 days after the MWM test.

(J) Number of trials to reach the criterion in the memory flexibility test. (K) Mean speed

(m/s) in the memory flexibility test. *p < 0.05, **p < 0.01; data are presented as the mean ±

SE. n=8 different animals for WT and n=7 different animals for tau -/-.
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Highlights

 Tau deletion reduce oxidative damage in the hippocampus of juvenile mice.

 Genetic tau reduction improves mitochondrial bioenergetics in the hippocampus of

young animals.

 The absence of tau enhances the attentive capacity of young mice