The toxin MPTP generates similar cognitive and locomotor deficits in hTau and tau knock-out mice

Accepted Manuscript Research report The toxin MPTP generates similar cognitive and locomotor deficits in hTau and Tau Knock-Out mice Maud Gratuze, Nicolas Josset, Franck R. Petry, Mathieu Pflieger, Laura Eyoum Jong, Geoffrey Truchetti, Isabelle Poitras, Jacinthe Julien, François Bezeau, Françoise Morin, Pershia Samadi, Francesca Cicchetti, Frédéric Bretzner, Emmanuel Planel PII: DOI: Reference:

S0006-8993(19)30031-9 https://doi.org/10.1016/j.brainres.2019.01.016 BRES 46110

To appear in:

Brain Research

Received Date: Revised Date: Accepted Date:

1 December 2018 7 January 2019 10 January 2019

Please cite this article as: M. Gratuze, N. Josset, F.R. Petry, M. Pflieger, L.E. Jong, G. Truchetti, I. Poitras, J. Julien, F. Bezeau, F. Morin, P. Samadi, F. Cicchetti, F. Bretzner, E. Planel, The toxin MPTP generates similar cognitive and locomotor deficits in hTau and Tau Knock-Out mice, Brain Research (2019), doi: https://doi.org/10.1016/ j.brainres.2019.01.016

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The toxin MPTP generates similar cognitive and locomotor deficits in hTau and Tau Knock-Out mice

Maud Gratuzea,b*, Nicolas Josset a,b, Franck R. Petrya,b, Mathieu Pflieger b, Laura Eyoum Jonga,b, Geoffrey Truchettia,b, Isabelle Poitrasa,b, Jacinthe Julienb, François Bezeaua,b, Françoise Morinb, Pershia Samadia,b, Francesca Cicchettia,b, Frédéric Bretzner a,b, Emmanuel Planela,b,*

a

Université Laval, Faculté de Médecine, Département de Psychiatrie et Neurosciences, Québec, QC,

Canada;

b

Centre de recherche du Centre Hospitalier de l’Université Laval de Québec, Axe Neurosciences, Québec,

QC, Canada;

* Correspondence to: M. Gratuze or E. Planel. E-mail: [email protected] or [email protected].

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Abstract

Parkinson's disease (PD) is characterized by motor deficits, although cognitive disturbances are frequent and have been noted early in the disease. The main pathological characteristics of PD are the loss of dopaminergic neurons and the presence of aggregated α-synuclein in Lewy bodies of surviving cells. Studies have also documented the presence of other proteins within Lewy bodies, particularly tau, a microtubule-associated protein implicated in a wide range of neurodegenerative diseases, including Alzheimer's disease (AD). In AD, tau pathology correlates with cognitive dysfunction, and tau mutations have been reported to lead to dementia associated with parkinsonism. However, the role of tau in PD pathogenesis remains unclear. To address this question, we induced parkinsonism by injecting the toxin 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in hTau mice, a mouse model of tauopathy expressing human tau, and a mouse model knock-out for tau (TKO). We found that although MPTP impaired locomotion (gait analysis) and cognition (Barnes maze), there were no discernable differences between hTau and TKO mice. MPTP also induced a slight but significant increase in tau phosphorylation (Thr205) in the hippocampus of hTau mice, as well as a significant decrease in the soluble and insoluble tau fractions that correlated with the loss of dopaminergic neurons in the brainstem. Overall, our findings suggest that, although MPTP can induce an increase in tau phosphorylation at specific epitopes, tau does not seem to causally contribute to cognitive and locomotor deficits induced by this toxin.

Keywords: Tau - Parkinson's disease – MPTP – Tauopathies – Locomotion - Cognition

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1. Introduction Neurodegenerative diseases share a number of pathological features including cell degeneration accompanied by the accumulation of various pathological peptides or proteins such as -amyloid, tau, synuclein etc. Parkinson’s disease (PD), which is characterized primarily by the loss of dopaminergic neurons within the nigro-striatal pathway, is no exception. The resulting dopamine depletion leads to some of the most prominent motor dysfunctions this condition is known for, which include bradykinesia, rigidity and tremors (Fahn, 2003; Olanow et al., 2009). However, cell loss is not restricted to these structures and, as the disease progresses, other systems are targeted. Notably, the disease is characterized by the presence of Lewy bodies (LB); nuclear inclusions mainly composed of the protein α-synuclein (Baba et al., 1998; Spillantini et al., 1997).

It has recently emerged is that other pathological proteins such as tau, which has been suggested to underlie impairments of cognitive functions in Alzheimer’s disease (AD) (Arriagada et al., 1992; Bretteville and Planel, 2008; Duff and Planel, 2005), may also play a role in PD (Zhang et al., 2018). In addition to LB, neurofibrillary tangles, which are formed of hyperphosphorylated tau, have been observed in PD brains (Bancher et al., 1993; Joachim et al., 1987). In particular, tau is present within filaments of LB and has been shown to co-localize with α-synuclein (Arima et al., 1999; Ishizawa et al., 2003). Remarkably, several genome-wide association studies in PD subjects of European descent have revealed an association between the MAPT locus and PD risk (Edwards et al., 2010; Pankratz et al., 2009; SimonSanchez et al., 2009). Mutations in MAPT, the gene responsible for producing tau proteins, has also been identified in familial frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) (Hutton et al., 1998; Lee et al., 2001). Cognitive impairments are commonly diagnosed in parkinsonian patients (Owen et al., 1997; Watson and Leverenz, 2010) and have been documented to occur from the early stages of disease evolution (Lewis et al., 2003).

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Despite emerging evidence, the role of tau in PD pathogenesis remains unclear. In this study, we investigated whether the administration of the toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), classically used as a model of PD by generating subtle dopaminergic degeneration, altered motor and cognitive behaviours in mice knock-out for Tau (TKO) or mice expressing the human tau protein (hTau). Our study aimed to dissect the impact of MPTP-induced parkinsonism in locomotor and cognitive behavioural aspects in relation to tau or phosphorylated tau levels.

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2 Results 2.1 MPTP affects locomotion independently of Tau expression Gait parameters were quantified during treadmill locomotion at a walking speed of 15 cm/s before and after MPTP injections (Fig. 1). As previously reported (Wang et al., 2012), we found that MPTP injections increased the duty cycle of the stance phase during locomotion and decreased the duration of the swing phase of both tau mutants without changing the step cycle duration (Fig. 1A,B,C), thus supporting that mice spent more time on the ground. The MPTP treatment decreased the percentage of weight support on two limbs but conversely increased that on three limbs (Fig. 1D,E). This suggests that both TKO and hTau mice spent more time on three rather than two limbs, suggesting reduced postural stability during locomotion. Although MPTP altered locomotor parameters and induced a slower locomotor gait, no differences were found between TKO and hTau mice. Taken together, behavioural motor assessment revealed that MPTP injections undermine postural stability during locomotion regardless of the presence of tau.

2.2 MPTP affects memory independently of tau expression Cognitive impairments are common in PD patients and spatial working memory has been specifically described as being altered in individuals diagnosed with the disease (Owen et al., 1997). Spatial working memory can also be measured following MPTP injection, as previously shown in a number of MPTPinduced parkinsonian mice models (Deguil et al., 2010; Ferro et al., 2005; Tanila et al., 1998). We therefore evaluated spatial learning and memory in hTau and TKO mice, with or without MPTP exposure, using a Barnes maze. The animals were subjected to four consecutive trials per day over a period of four days. For each group, latencies to find the escape target decreased over the course of the acquisition training independently of MPTP injections (Fig. 2A,B). On day 5, a probe trial was performed to evaluate short-term memory. Latencies did not differ between MPTP-injected hTau compared to control hTau mice, or between MPTP-injected TKO with respect to control TKO mice (Fig. 2A,B). However, the probe trial conducted at day 12 to assess long-term memory revealed that control hTau and TKO mice visited the target hole more frequently as compared to the MPTP-injected hTau and TKO mice, respectively (Fig. 5

2C-E). However, the mouse genotype (presence or absence of tau) did not influence the number of visits to the target hole (Fig. 2E), suggesting that the alteration of long-term memory in MPTP-injected mice is, again, independent of tau protein levels.

2.3 Tau phosphorylation in MPTP-injected hTau mice We next assessed whether MPTP could promote tau phosphorylation in hTau mice, as tau hyperphosphorylation has previously been reported following MPTP injections (Duka et al., 2006; Duka et al., 2009; Qureshi and Paudel, 2011) as well as in PD patients (Bancher et al., 1993; Joachim et al., 1987). We evaluated tau phosphorylation in the brainstem, hippocampus and cortex of MPTP-injected hTau mice using several anti-phospho-tau antibodies (Table S1). The specificity of the tau antibodies used in this study have already been extensively characterized in our laboratory (Petry et al., 2014b). Western blot analysis revealed no obvious change in tau phosphorylation in either the cortex or hippocampus of MPTP-injected hTau mice (Fig. 3B,C), with the exception of a slight increase in the phosphorylation of tau on the threonine 205 moiety in the hippocampus (Fig 3C3). Total tau and -actin levels remained unchanged in both brain areas (Fig. 3B8-9, C8-9). Brainstem analyses revealed a significant decrease of Tau-1 signal - which recognizes tau non-phosphorylated at Ser195/Ser198/Ser199/Ser202 - in MPTPinjected mice compared to control mice (Fig. 3A7). However, the decrease of tau-1 signal could be explained by the lower level of total tau protein in MPTP-injected mice. Indeed, a significant decrease of total tau levels was measured in the brainstem of MPTP injected mice (Fig. 3A8). Our results show that MPTP treatment has a modest effect on tau phosphorylation at specific epitopes.

2.4 Brainstem tau level and solubility in MPTP-injected hTau mice Given the lower level of total tau in the brainstem of MPTP-hTau mice, we analyzed the insoluble and soluble fractions of tau in the different groups of mice to determine which fractions of tau was specifically affected by this decrease (Fig. 4). Both soluble and insoluble fractions were present at lower levels in MPTP-injected hTau mice compared to control mice. However, the decrease of insoluble tau was greater

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(Fig. 4B: -64.62% compared to control group) than soluble tau (Fig. 4A: -24.09% compared to control group). Additional analyses revealed that the lower level of total tau observed in these mice (Fig. 5A) was directly correlated with the reduction of tyrosine hydroxylase (TH) protein level measured in the brainstem of MPTP-injected mice (Fig. 5B,D), suggesting a link between the loss of dopaminergic neurons in MPTP-injected mice and total tau decrease in this structure. The MPTP treatment lowered levels of both the soluble and insoluble fractions of the tau protein, and this decrease correlated with the loss of brainstem dopaminergic neurons.

2.5 Loss of synaptic integrity in MPTP-injected hTau mice Finally, we tested a number of synaptic markers in the brainstem of MPTP-hTau mice to assess synaptic integrity previously shown to be altered in MPTP mouse models (Shin et al., 2016; Toy et al., 2014). We determined the relative expression of one post-synaptic marker, PSD-95 - anchoring protein localized to the post-synaptic terminal - and two pre-synaptic markers, synaptophysin and SNAP25 - major membrane proteins of presynaptic vesicles. MPTP injections impacted pre-synaptic markers as there was a decrease in SNAP25 and synaptophysin levels within the brainstem of hTau mice compared to control hTau mice (Fig. 6A,B). However, PSD95 remained unchanged regardless of treatment (Fig. 6C), suggesting that MPTP cause retraction of presynaptic terminals but not collapse of spines. The decrease in synaptic markers did not seem to be caused by loss of neurons since levels of the neuronal marker NeuN were similar in MPTP-injected and control mice (Fig. 6C). These results confirm previous findings suggesting that MPTP-induced parkinsonian models can generate synaptic impairments (Shin et al., 2016; Toy et al., 2014).

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3 Discussion We herein report that a classic MPTP regimen inducing subtle dopaminergic degeneration i) alters some aspects of locomotion and memory independently of the expression of the tau protein; ii) does not induce overt tau hyperphosphorylation in the brain of mice that express the human tau protein; iii) leads to lower levels of total tau (both soluble and insoluble fractions) in brainstem of treated hTau mice, which correlates with the loss of TH+ neurons; iv) results in retraction of presynaptic terminals but not collapse of spines in brainstem of treated hTau mice, but without significant neuronal loss. These results suggest that tau disturbances could be a consequence rather than a cause of the parkinsonian-like features induced by the toxin MPTP that we analyzed here.

We investigated the role of tau in PD-related motor behaviour, based on recent data showing that tau may account for motor abnormalities associated with other neurodegenerative diseases such as Huntington's disease (HD) (Gratuze et al., 2016). Indeed, it has been shown that when R6/1 mice, a well-characterized model of HD, are crossed with TKO mice, they exhibit milder motor impairments than R6/1 mice expressing the tau protein (Fernandez-Nogales et al., 2014). These findings suggest that tau may be involved in HD-related motor aspects since suppressing the protein alleviates such features. We first validated that MPTP mice depict locomotion deficits such as duty cycle, postural stability and gait, as previously shown (Wang et al., 2012). However, these alterations occurred in both TKO and hTau mice treated with MPTP, suggesting that tau protein does not causally contribute to PD-like locomotor deficits, at least in the MPTP paradigm we have used in our study. Morris et al. have suggested that tau depletion does not prevent motor deficits in two other mouse models of PD, the 6-OHDA (6-hydroxydopamine)lesioned and the α-synuclein transgenic mouse models (Morris et al., 2011). Indeed, neither the TKO nor the MAPT heterozygotes mice were spared from motor deficits in these PD models. Our results, using the MPTP model of PD in TKO and hTau mice are in agreement with Morris et al. (Morris et al., 2011)

We then examined whether tau contributes to PD-like cognitive deficits in MPTP-treated mice. Specifically, we assessed the role of tau on spatial working memory deficits previously described to be 8

altered in parkinsonian patients (Owen et al., 1997) and in MPTP models (Deguil et al., 2010; Ferro et al., 2005; Tanila et al., 1998). This hypothesis is based on the fact that reduction of endogenous murine tau can prevent spatial memory and learning deficit in mouse models of AD as well as in traumatic brain injury (Cheng et al., 2014; Ittner et al., 2010; Roberson et al., 2007; Roberson et al., 2011). Via the Barnes maze test, we confirmed that MPTP-injected mice exhibit spatial memory deficits. However, spatial memory alterations were seen in both TKO and hTau mice after MPTP injections, suggesting that the tau protein does not modulate PD-like memory deficits in MPTP-treated animals. These results further support the hypothesis of Morris et al. (Morris et al., 2011) suggesting that tau could trigger functional and pathological alterations in neurodegenerative disease such as AD, but not PD.

We further investigated tau phosphorylation within the brainstem, hippocampus and cortex of MPTPinjected hTau mice since tau hyperphosphorylation has previously been reported in the substantia nigra, enthorinal cortex and striatum of PD patients and MPTP models (Bancher et al., 1993; Duka et al., 2006; Duka et al., 2009; Joachim et al., 1987). We found a slight increase in tau phosphorylation on the threonine 205 in the hippocampus but no measurable tau phosphorylation in the cortex and brainstem when compared to control animals. The different models used could explain the discrepancy between our study and previously reported work. In fact, while a number of in vitro studies have reported significant tau hyperphosphorylation after MPTP treatments (Duka et al., 2006; Duka et al., 2009; Qureshi and Paudel, 2011), in vivo studies have reported much more moderate tau hyperphosphorylation after MPTP injections (Duka et al., 2006), as we observed in our study.

We observed, however, a significant decrease of total tau levels in the brainstem of MPTP-injected mice. This decrease was detectable for both soluble and insoluble fractions of tau, although the decrease of insoluble tau was more pronounced when compared to soluble tau. Decrease of soluble tau has already been described in the substantia nigra of PD patients and MPTP-injected mice (Lei et al., 2012), but without detectable changes in insoluble tau since mice used in these study were WTs that normally do not develop tau aggregates. Here, we used hTau mice, a model of tauopathy that develops tangles (Andorfer et 9

al., 2003; Andorfer et al., 2005), which may explain why we were able to observe changes in insoluble tau levels. It has been suggested that the decrease of soluble tau can induce parkinsonism with dementia by impairing APP-mediated iron export (Lei et al., 2012). The lower level of total tau correlated with the reduction of TH seen in the brainstem of MPTP-injected mice, suggesting that total tau decrease is a consequence of the loss of dopaminergic neurons or connections in the MPTP model we used. However, we observed similar levels of NeuN suggesting that tau decrease was independent of neuronal loss, as reported by Lei et al. (Lei et al., 2012). Nevertheless, future histological or immunohistochemical analysis would be necessary to confirm this observation in our model. Dopaminergic terminal loss is observed earlier and is more pronounced than cell body loss in MPTP mouse models (Burke and O'Malley, 2013). Only one day following MPTP treatment, striatal dopamine levels were shown to drop by 60%, whereas no TH-immunoreactive cell bodies were lost in the substantia nigra (Serra et al., 2002). Other studies have also confirmed that MPTP induces significant axonal degeneration (Li et al., 2009; Mijatovic et al., 2011). As tau proteins are mostly localized within axons (Binder et al., 1985; Brion et al., 1988), the substantial degeneration of axons in the MPTP-mouse model may explain the loss of tau in the brainstem of MPTPinjected hTau mice.

Finally, we observed that MPTP injections decreased levels of synaptic markers SNAP25 and synaptophysin but not PSD-95. Other studies have reported decreases in synaptophysin and/or PSD-95 (Shin et al., 2016; Toy et al., 2014). This alteration of presynaptic integrity in the brainstem in our mice could be a consequence of striatal dopamine denervation caused by loss of dopaminergic neurons or connections. Alterations of synaptic integrity in the brainstem of MPTP-injected mice are in accordance with the loss of tau caused by axon degeneration in this model of PD. Retraction of presynaptic terminals in these mice could contribute to altered neurotransmission, often report in PD (Bagetta et al., 2010; Picconi et al., 2012; Plowey and Chu, 2011).

Importantly, MPTP can generate a large spectrum of models depending of species, strains, dosage or delivery (Jackson-Lewis and Przedborski, 2007; Meredith et al., 2008; Meredith and Rademacher, 2011), 10

which makes the comparison of the results obtained between studies difficult. In addition, MPTP toxicity varies between mouse strains; for example, C57BL/6 mice are more sensitive to the toxin than BALB/c mice (Schwarting et al., 1999). Moreover, dopaminergic neurons have been shown to reappear, highlighting the inevitable reversibility on MPTP models. Finally, MPTP-induced acute dopaminergic loss does not really reflect the complex and progressive neuropathologies observed in PD. Taken together, these data show MPTP use to induce Parkinson-like models has some limitations that must be considered when interpreting the results. It would be interesting to evaluate tau involvement in PD pathogenesis in transgenic mice expressing mutant alpha-synuclein and LRRK2 for example. Indeed, transgenic models with -synuclein accumulation seems better to simulate the mechanisms underlying the genetic forms of PD, even though their pathological and behavioral phenotypes are often quite different from the human condition. Both toxin and genetic based models have their advantages and disadvantages. However, using these 2 types of PD models could help to better understand the role of tau in PD.

In conclusion, we have shown that both locomotion and spatial working memory were altered in MPTPinjected mice independent of their tau genotype, suggesting that tau protein does not causally contribute to PD-like locomotor and memory deficits characteristic of this MPTP model. However, it should be acknowledged that the TKO mice might not be an ideal model to make a definite conclusion on these points since they may depict compensatory mechanisms for the loss of tau functions, such as increased MAP1B (Harada et al., 1994). Our observations should be further tested and confirmed in an inducible tau KO mouse model when it becomes available.

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4. Experimental procedure 4.1 Animals Tau knockout (TKO) mice generated by targeted disruption - in which cDNA for enhanced green fluorescent protein (EGFP) was inserted into exon one of MAPT (Tucker et al., 2001) - were used. The hTau mice (Andorfer et al., 2003; Andorfer et al., 2005) were obtained by crossing 8c mice that express a tau transgene derived from a human PAC containing the coding sequence, intronic regions, and regulatory regions of the human gene (Duff et al., 2000), with TKO mice (Tucker et al., 2001). The founders of our hTau and TKO mice colony were derived on a C57BL/6J background (B6.Cg-Mapttm1(EGFP)KltTg(MAPT)8cPdav/J, Jackson Laboratories, Bar Harbor, ME, USA). Seven to nine-month old littermates of either sex were maintained in a temperature-controlled room (~23˚C) with a light/dark cycle of 12/12h, and experiments were performed during the light phase (hTau mice: MPTP n=7 (4M/3F); CTL n=11 (6M/5F); TKO mice: MPTP n=10 (9M/1F); CTL n=9 (8M/1F)). All animals had access to food and water ad libitum. Experimental protocols were approved by the Comité de Protection des Animaux du CHUL and carried out following the guidelines of the Canadian Council on Animal Care.

4.2 MPTP treatment Mice received seven intraperitoneal injections of MPTP (MPTP-HCL; 20 mg/kg free base Sigma Aldrich, St. Louis, MO), dissolved in 0.9% saline and prepared fresh, twice on the first two days at an interval of 12 h, and once daily on subsequent three days (Gibrat et al., 2009; Gibrat et al., 2010). Mice in the control group were administered saline intraperitoneally.

4.3 Locomotor analysis Mice were trained to walk on a single-lane treadmill (LE 8700 Series, Panlab), as previously described (Lemieux et al., 2016). The inner dimensions of the lane were 32 × 5 cm. Mice were subjected to a 20-min acclimation period and subsequently tested on the treadmill at 15cm/s once week prior and 3 weeks following MPTP injections. Steady locomotion for a minimum of 10 contiguous steps was recorded with high-frequency (90 frames/s) cameras (Genie HM640, Dalsa Teledyne). Films were digitized using 12

StreamPix 6.0 (Norpix) and analyzed offline. Videos were analyzed and the timing of all four feet lifts and contacts were manually extracted by using custom-designed software (graciously provided by Drs. S. Rossignol and T. Drew, Université de Montréal). Data were exported and processed with custom-written routines in Matlab (MathWorks) allowing us to analyze multiple gait parameters including phase duration and weight support. The step cycle is a division of locomotion. Each step cycle amounts to all events that occur between the first paw lift and the subsequent paw lift. It is therefore divided in two phases with a swing phase where the paw is off the ground and a stance phase, where the paw is on the ground.

4.4 Cognitive analysis Spatial learning and memory were assessed over a 12-day period using a Barnes maze one week after the last injection of MPTP, as previously described (Hernandez-Rapp et al., 2015). The test consisted of a metallic circular disk with 20 evenly spaced holes around its circumference. One of the holes was the target escape that led to a box beneath the surface. For aversive stimulation, intense light (800 lux) and noise (80 dB) were used. A tracking camera device connected to a computer was positioned over the maze to capture the behavioural experiments. At day 0, the mice were placed in individual cages and brought to the behavioural test room 24 h before the commencement of the test to allow for the mice to acclimatise to their new environment. The next morning, before the first trial, an acclimatization/exploration of 90 s was allowed for each mouse. The learning phase (day 1 to day 4) consisted to 4 × 3-min trials per day. After each trial, the maze's surface was wiped clean with 70% ethanol and dried. To assess short- and long-term memory, two-probe trials were performed on days 5 and 12 respectively; the escape compartment was replaced with a standard hole and the mice were tested during a 90 s period. The latency and the number of visits to the target hole were recorded for the training and probe phases.

4.5 Tissue preparation and protein extraction Mice were sacrificed 15 days after the last MPTP (or saline) injection by decapitation without anesthesia, as anesthesia can lead to hypothermia-induced tau hyperphosphorylation (Planel et al., 2007). Brains were immediately removed and the tissues dissected on ice. The left hemisphere was dissected into 13

hippocampus, cortex, brainstem and cerebellum, frozen on dry ice, and stored at -80˚C until they were processed for Western immunoblot analyses, as previously described (Julien et al., 2012). In this study, the term brainstem is used to define the following areas: brainstem per se (interbrain, midbrain and hindbrain) + cerebral nuclei (striatum and pallidum). Briefly, dissected brain structures were homogenized, without thawing, in 5 times volume/weight of radioimmunoprecipitation assay RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 150 mM NaCl, 0.25% Na-deoxycholate, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, 1 mM PMSF, 10 µl/ml of proteases inhibitors cocktail (P8340, Sigma-Aldrich, St. Louis, MO)), using a mechanical homogenizer (TH, Omni International, Marietta, GA). Samples were then centrifuged for 20 min at 20,000 g at 4˚C. The supernatant was recovered, diluted in sample buffer (NuPAGE LDS; Invitrogen, Carlsbad, CA) containing 5% of 2-β-mercapto-ethanol, 1 mM Na3VO4, 1 mM NaF, 1 mM PMSF, 10 µl/ml of proteases inhibitors cocktail (P8340; Sigma-Aldrich), boiled for 5 min and stored at -20˚C.

4.6 Analysis of aggregates and soluble tau Tau aggregates were extracted following previously published protocols (Julien et al., 2012). This procedure uses 1% sarkosyl and is based on methodologies developed to isolate tau aggregates from brains of AD patients (Greenberg and Davies, 1990). Briefly, the RIPA supernatant was adjusted to 1% sarkosyl (N-lauroylsarcosine), incubated for 30 min at room temperature with constant shaking, and centrifuged at 100,000x g for 1 h at 20˚C. The pellet containing sarkosyl-insoluble aggregated (SP fraction) was resuspended and diluted in Sample buffer (NuPAGE LDS) containing 5% of 2-β-mercaptoethanol, 1 mM Na3VO4, 1 mM NaF, 1 mM PMSF, 10 µl/ml of proteases inhibitors cocktail (P8340, Sigma-Aldrich), boiled for 5 min, and stored at -20˚C.

For heat stable soluble tau, the RIPA supernatant was boiled for 5 min and centrifuged at 20,000x g for 20 min. The supernatant was recovered, diluted in sample buffer (NuPAGE LDS; Invitrogen, Carlsbad, CA) containing 5% of 2-β-mercapto-ethanol, 1 mM Na3VO4, 1 mM NaF, 1 mM PMSF, 10 µl/ml of proteases inhibitors cocktail (P8340; Sigma-Aldrich) and boiled for 5 min (Heat stable fraction). 14

4.7 Western-blot analysis SDS-PAGE and Western blot analysis were performed as previously described (Petry et al., 2014b). All antibodies used in this study are listed in Table S1 and are commercially available except for PHF-1 (Otvos et al., 1994), CP13 (Weaver et al., 2000), MC-6 (Jicha et al., 1997) and CP27 (Duff et al., 2000), which were generously provided by Dr. Peter Davies (Albert Einstein University, New York, NY, USA). Between 5 and 30 µg of brain protein were analyzed, depending on the protein. Brain homogenates were separated on an SDS-10% polyacrylamide gel and transferred onto nitrocellulose membranes (Amersham Biosciences, Pittsburgh, PA). Non-specific binding sites were blocked with 5% non-fat dry milk in Phosphate-buffered saline containing 0.1% Tween 20 (PBS-T) for 1 h at room temperature and subsequently incubated overnight at 4°C with the specific primary antibodies (Table S2). The following day, membranes were washed 3 times in PBS-T and incubated for 1 h at room temperature with the corresponding secondary antibody in 5% non-fat dry milk in PBS-T (Petry et al., 2014a), and the immunoreactive signal intensity was visualized by enhanced chemiluminescence (ECL Plus, GE Healthcare Biosciences, Piscataway, NJ). Immunoreactive bands were visualized using the ImageQuant LAS 4000 imaging system (Fujifilm USA, Valhalla, NY) and densitometric analyses were performed using the Image Gauge analysis software (Fujifilm USA, Valhalla, NY).

4.8 Statistical analysis Statistical analyses were performed with GraphPad Prism software 4.0 (Graphpad Software, La Jolla, CA). For comparisons of 2 groups, statistical analyses were performed using unpaired t-tests. For Figs. 1 and 2, statistical analysis was performed using a two-way ANOVA. Analysis of correlation presented in Fig. 5 was performed using Pearson correlation calculations. *, **, and *** indicated significant differences vs. control groups with p < 0.05, p < 0.01, p < 0.001, respectively. Unless otherwise stated, all data are expressed as means ± SD.

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Competing interests The authors declare no competing interests of any sort.

Acknowledgments We thank Drs. Peter Davies (Albert Einstein University, New York, NY, USA) for the generous gift of antibodies. This work was supported by grants to E.P. from the Alzheimer Society of Canada, the FRQS (Fonds de Recherche du Québec en Santé; 16205, 20048) and the Natural Sciences and Engineering Research Council of Canada (354722). M.G. and F.R.P were recipients of Biomedical Doctoral Awards from the Alzheimer Society of Canada. F.B. had a grant from the Natural Sciences and Engineering Research Council of Canada (RGPIN/418635-2012), and is supported by a FRQS scholarship. F.C. is a recipient of a Research Chair from the FRQS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author contributions M.G., N.J., F.R.P., M.P., G.T., F.B., I.P., J.J. and F.M. performed experiments; M.G., N.J., M.P., G.T. and F.M. analyzed data; M.G., N.J., S.P., E.P., and F.B. interpreted results of experiments; M.G., N.J., and prepared figures; M.G., N.J., F.C., E.P. and F.B. drafted manuscript; E.P., F.B. and S.P. conception and design of research. E.P. is the guarantor of the manuscript presented here and takes full responsibility for the work as a whole, including the study design, access to data, and the decision to submit and publish the manuscript.

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Figure legends Figure 1: Kinematic recording analysis in MPTP-injected TKO and hTau mice. Duration of the step cycle (A), duration of the swing phase (B), and duty cycle (C) during treadmill locomotion at 15cm/s. Percentage of the step cycle duration when the mouse’s body weight is supported on two (D) or three (E) limbs. No differences were found between genotypes before or after MPTP injections. Data are mean ± SD with n=4 hTau and 5 TauKO mice. Statistical analyses were performed using a two-way ANOVA followed by a multi-comparison test. Asterisks indicate significant differences between pre- and postMPTP injection, with * p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 2: Barnes maze analysis in MPTP-injected TKO and hTau mice. Animals were trained during a period of 4 days to learn the location of the target hole. A first probe trial evaluated short-term memory (Day 5). A second probe trial assessed long-term memory (Day 12). During acquisition (Day 1–4), learning was monitored by recording primary latency during the training sessions (A,B). During the first probe trial (Day 5), the target hole was closed. Short-term memory retention was evaluated by measuring the primary latency (A,B). Mice were not trained between Days 6 to 11. During the recall trial, spatial long-term memory was assessed by measuring the primary latency (A,B) and the number of visits in the target hole (C,D,E). Holes −1 and +1 are the closest to the target hole. Data are expressed as mean ± SEM with n=7-11 mice for each condition. Results of statistical analyses are indicated below each graph.

Figure 3: Tau phosphorylation in MPTP-injected hTau mice. Proteins were extracted from brainstem (A), cortex (B), and hippocampus (C), separated by SDS-PAGE, and probed with the following antibodies: 1. AT8, 2. CP13, 3. pT205, 4. PHF1, 5. 12E8, 6. AT100, 7. Tau1, 8. Total tau and 9. -actin (loading control). Two lanes from representative immunoblots are displayed for each condition. Quantifications of phospho-epitopes were performed vs. total tau (ratio of phospho-tau/total tau). Total tau quantification was performed vs. -actin. Results are expressed as percentage of the CTL group. Data are

17

shown as mean ± SD with n=7-11 mice for each condition. Asterisks indicate significant differences, with * p < 0.05 and ** p < 0.01.

Figure 4. Soluble and insoluble Tau in the brainstem of MPTP-injected hTau mice. (A) Soluble total tau: Soluble tau was extracted in heat stable fraction, separated by SDS-PAGE, and probed with total tau antibody. (B) Insoluble total tau: Aggregates of tau protein were extracted with sarkosyl, separated by SDS-PAGE, and probed with total tau antibody. Two lanes from representative immunoblots are displayed for each condition. Results are expressed as percentage of the CTL group. Data are shown as mean ± SD with n=7-11 mice for each condition. Asterisks indicate significant differences, with * p < 0.05 and *** p < 0.001.

Figure 5. Correlation between total tau and tyrosine hydroxylase levels. Proteins were extracted from brainstem, separated by SDS-PAGE, and probed with the following antibodies: (A) Total tau and (B) TH. Two lanes from representative immunoblots are displayed for each condition. Results are expressed as percentage of the CTL group. Data are shown as mean ± SD with n=7-11 mice for each condition. Asterisks indicate significant differences, with * p < 0.05 and ** p < 0.01. C. Correlation between tau and TH level in the brainstem.

Figure 6: Synaptic integrity in MPTP-injected hTau mice. Proteins were extracted from brainstem of hTau mice, separated by SDS-PAGE, and probed with the following antibodies: (A) SNAP25, (B) Synaptophysin, (C) PSD-95, and (D) NeuN (neuronal marker). Two lanes from representative immunoblots are displayed for each condition. Total protein expression was quantified vs. -actin. Data are mean ± SD with n=7-11 mice for each condition. Asterisks indicate significant differences, with * p < 0.05 and ** p < 0.01.

Table S1: Antibodies used in this study.

18

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Tau protein does not contribute to cognitive and locomotor deficits induced by MPTP

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MPTP injections decrease soluble and insoluble tau fractions in hTau mice brain

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Decreased tau correlates with the loss of dopaminergic neurons in the brainstem

24

Figure 1

A.

B.

Step cycle duration

Swing phase duration

0.4

Time (s)

Duration (s)

0.6

0.2

0.0

hTau

C.

TKO

D.

TKO

Two-limb support

% of the step cycle

% of the weight support

Duty Cycle

hTau

TKO

Three-limb support

% of the weight support

E.

hTau

hTau

TKO

hTau

TKO

Figure 2 A.

B.

C.

D.

E.

Figure 3 B. Hippocampus

A. Brainstem 2. CP13

1. AT8

3. pT205

1. AT8

% of controls

3.pT205

1. AT8

150

150

200

150

150

100

100

100

150

100 50

50 0

50 CTL MPTP

0

100

50 CTL MPTP

5. 12E8

0

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CTL MPTP

6. AT100

CTL MPTP

4. PHF1

63kDa

2. CP13

3. pT205

63kDa

150

4. PHF1

% of controls

2. CP13

63kDa

63kDa

0

CTL MPTP

5. 12E8

150

150

150

100

100

100

100

50

50

50

50

0

*

0

CTL MPTP

6. AT100

CTL MPTP

4. PHF1

0

CTL MPTP

5. 12E8

0

150

200

150

150

150

150

150

150

150

100

150

100

100

100

100

100

100

100

50

50

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50

50

50

0

0

0

0

0

0

0

100

50

50

0

CTL MPTP 7. Tau1

0

CTL MPTP 8. Total Tau

CTL MPTP 9. β-actin

CTL MPTP 7. Tau1 48kDa

150

150 100

*

50

50 0

100

CTL MPTP

0

**

CTL MPTP

CTL MPTP 8. Total Tau

CTL MPTP 9. β-actin

63kDa

CTL MPTP 7. Tau1 48kDa

CTL MPTP 8. Total Tau

CTL MPTP 9. β-actin

63kDa

48kDa

150

150

150

150

150

150

150

100

100

100

100

100

100

100

50

50

50

50

50

50

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0

CTL MPTP

CTL MPTP

6. AT100

63kDa

63kDa

63kDa

% of controls

C. Cortex

0

CTL MPTP

0

CTL MPTP

0

CTL MPTP

0

CTL MPTP

0

CTL MPTP

0

CTL MPTP

Figure 4 A. Soluble Tau 63kDa

63kDa

% of controls

B. Insoluble Tau

150

150 100

*

50

50 0

100

CTL MPTP

0

*** CTL MPTP

Figure 5 A. Total Tau

B. TH

63 kDa

63 kDa

% of controls

150 100

150

**

50 0

100

*

50 CTL MPTP

0

CTL MPTP

C. Correlation between total Tau and TH levels

Figure 6 Synaptic markers B. Synaptophysin

% of controls

25kDa

150

100

100

50 0

** CTL MPTP

100kDa

35kDa

150

150

*

50 0

100 50

CTL MPTP

D. NeuN

C. PSD-95

0

48kDa

% of controls

A. SNAP25

Neuronal marker

CTL MPTP

200 150 100

**

50 0

CTL MPTP