Sodium selenate reduces hyperphosphorylated tau and improves outcomes after traumatic brain injury

Hyperphosphorylation of tau is implicated in traumatic brain injury (TBI) pathology. Shultz et al. show that TBI reduces protein phosphatase 2A (PP2A) activity and increases tau phosphorylation in rats, with similar findings in human post-mortem tissue. Sodium selenate, a PP2A activator, prevents TBI-induced abnormalities in rats and improves behavioural outcomes.

Hyperphosphorylation of tau is implicated in traumatic brain injury (TBI) pathology. Shultz et al. show that TBI reduces protein phosphatase 2A (PP2A) activity and increases tau phosphorylation in rats, with similar findings in human post-mortem tissue. Sodium selenate, a PP2A activator, prevents TBI-induced abnormalities in rats and improves behavioural outcomes.

Abstract

Traumatic brain injury is a common and serious neurodegenerative condition that lacks a pharmaceutical intervention to improve long-term outcome. Hyperphosphorylated tau is implicated in some of the consequences of traumatic brain injury and is a potential pharmacological target. Protein phosphatase 2A is a heterotrimeric protein that regulates key signalling pathways, and protein phosphatase 2A heterotrimers consisting of the PR55 B-subunit represent the major tau phosphatase in the brain. Here we investigated whether traumatic brain injury in rats and humans would induce changes in protein phosphatase 2A and phosphorylated tau, and whether treatment with sodium selenate—a potent PR55 activator—would reduce phosphorylated tau and improve traumatic brain injury outcomes in rats. Ninety young adult male Long-Evans rats were administered either a fluid percussion injury or sham-injury. A proportion of rats were killed at 2, 24, and 72 h post-injury to assess acute changes in protein phosphatase 2A and tau. Other rats were given either sodium selenate or saline-vehicle treatment that was continuously administered via subcutaneous osmotic pump for 12 weeks. Serial magnetic resonance imaging was acquired prior to, and at 1, 4, and 12 weeks post-injury to assess evolving structural brain damage and axonal injury. Behavioural impairments were assessed at 12 weeks post-injury. The results showed that traumatic brain injury in rats acutely reduced PR55 expression and protein phosphatase 2A activity, and increased the expression of phosphorylated tau and the ratio of phosphorylated tau to total tau. Similar findings were seen in post-mortem brain samples from acute human traumatic brain injury patients, although many did not reach statistical significance. Continuous sodium selenate treatment for 12 weeks after sham or fluid percussion injury in rats increased protein phosphatase 2A activity and PR55 expression, and reduced the ratio of phosphorylated tau to total tau, attenuated brain damage, and improved behavioural outcomes in rats given a fluid percussion injury. Notably, total tau levels were decreased in rats 12 weeks after fluid percussion injury, and several other factors, including the use of anaesthetic, the length of recovery time, and that some brain injury and behavioural dysfunction still occurred in rats treated with sodium selenate must be considered in the interpretation of this study. However, taken together these data suggest protein phosphatase 2A and hyperphosphorylated tau may be involved in the neurodegenerative cascade of traumatic brain injury, and support the potential use of sodium selenate as a novel traumatic brain injury therapy.

Introduction

Traumatic brain injury (TBI) is a leading cause of death and morbidity worldwide and there is currently no pharmaceutical intervention that has been shown to improve long-term outcome (Blennow et al., 2012). This may be due to the complex cascade of secondary pathophysiological injury mechanisms that occur in the TBI disease process (Blennow et al., 2012). However, the delayed and progressive nature of these secondary injury processes provides an opportunity for a pharmacological intervention that reduces neurodegeneration and thereby improves outcome in patients with TBI (Blennow et al., 2012).

The hyperphosphorylation of tau has been implicated in the pathogenesis of a number of neurodegenerative diseases (Grundke-Iqbal et al., 1986; Ballatore et al., 2007; Morris et al., 2011), and has been identified as a component of secondary injury in TBI (Thom et al., 2011; Blennow et al., 2012; Johnson et al., 2012; McKee et al., 2013). Tau is a microtubule-associated phospho-protein that is important in the stabilization of microtubules, axonal transport, and neuronal health (Ballatore et al., 2007; Morris et al., 2011; Blennow et al., 2012). When tau is hyperphosphorylated it dissociates from microtubules, resulting in microtubule destabilization and consequent loss of function, and may form neurotoxic neurofibrillary tangles (Alonso et al., 1994, 1996; Ballatore et al., 2007; Li et al., 2007; Morris et al., 2011). Thus, for tau to maintain its proper physiological functions it must possess the ability to attain a dephosphorylated state (Ballatore et al., 2007; Morris et al., 2011; Blennow et al., 2012). Protein phosphatase 2A (PP2A) is a heterotrimeric protein that regulates key signalling pathways in the brain, and is composed of a core A-subunit, a catalytic C-subunit (PP2Ac), and a regulatory B-subunit (Iqbal et al., 2009; Zheng et al., 2014). Of particular relevance to the dephosphorylation of tau, PP2A heterotrimers consisting of the PR55 regulatory B-subunit (PP2A/PR55) represent the major tau phosphatase in the brain (Wang et al., 1996, 2007; Liu et al., 2005; Xu et al., 2008; Iqbal et al., 2009; Shi, 2009). Furthermore, the downregulation of PP2A/PR55 levels and/or a decrease in PP2A activity has been reported after experimental brain insults (Chen et al., 2010; Koh, 2013), and promotes the hyperphosphorylation of tau (Sontag et al., 2008; Bolognin et al., 2012). Therefore, a pharmacological compound that increases PP2A/PR55 may reduce hyperphosphorylated tau and be beneficial to TBI outcome.

Sodium selenate is a potent PP2A/PR55 activator (Corcoran et al., 2010b; Zheng et al., 2014). Previous findings from our laboratory, and others, demonstrate that treatment with sodium selenate activates PP2A/PR55 (Corcoran et al., 2010b; Zheng et al., 2014), decreases hyperphosphorylated tau (Corcoran et al., 2010b; van Eersel et al., 2010; Jones et al., 2012), and improves outcome in animal tauopathy models (Corcoran et al., 2010b; van Eersel et al., 2010; Jones et al., 2012). Given these findings, in this study we used the rat lateral fluid percussion injury (FPI) model to characterize the influence of experimental TBI on PP2A and hyperphosphorylated tau, examined whether similar changes to PP2A and hyperphosphorylated tau occur in humans after TBI, and assessed sodium selenate treatment as a novel TBI therapy in rats. We herein report that FPI decreased PP2A/PR55 protein expression by 2 h post-FPI and PP2A activity by 24 h post-FPI. These changes in PP2A were followed by a hyperphosphorylation of tau, which began at 72 h post-FPI and was still present at 12 weeks post-FPI. Similar acute effects on PR55/PP2A and tau were found in post-mortem brain samples collected from human TBI patients. Furthermore, a 12-week sodium selenate treatment regimen in rats significantly increased PP2A/PR55, and reduced hyperphosphorylated tau, brain damage, and behavioural impairments post-FPI compared to saline-vehicle treatment. These findings demonstrate that a decrease in PP2A/PR55, and the concomitant hyperphosphorylation of tau, occur in the neurodegenerative aftermath of TBI, and that pharmacologically targeting PP2A/PR55 with sodium selenate may be a novel therapeutic approach to reduce the hyperphosphorylation of tau and improve outcome following TBI.

Materials and methods

Subjects

Ninety Long-Evans hooded rats obtained from Monash animal research services (Melbourne, Australia) were 12 weeks of age and weighed 250–300 g at the time of injury. Rats were housed individually under a 12 h light/dark cycle, and given access to food and water ad libitum for the duration of the experiment. All experimental procedures were approved by the Melbourne Health Animal Ethics Committees (AEC#1112173).

Experimental groups

To assess the acute effects of FPI or sham-injury on PP2A and tau, 30 rats were randomly assigned to either FPI (n = 15) or sham-injury (n = 15) groups. These rats were sacrificed at 2, 24, and 72 h post-injury (n = 5/group/time point). To assess the potential therapeutic effects of sodium selenate treatment (1 mg/kg/day; Corcoran et al., 2010b; Jones et al., 2012) on TBI outcome, the remaining 50 rats were randomly assigned to one of four experimental conditions: sham-injury + saline-vehicle treatment (SHAM + VEH; n = 12), sham-injury + sodium selenate treatment (SHAM + SS; n = 12), FPI + saline-vehicle treatment (FPI + VEH; n = 13), or FPI + sodium selenate treatment (FPI + SS; n = 13). The assigned treatment began immediately post-injury, and was delivered continuously via subcutaneous osmotic pump (Model 2006, Alzet) for the entire duration of the study.

Surgery and FPI

FPI and sham-injury procedures were based on a standard protocol as previously described and used by our group (Thompson et al., 2005; Jones et al., 2008; Bao et al., 2012). Briefly, rats were placed in a sealed Plexiglas box for 3 min into which 4% isoflurane and 2 l/min oxygen flow was introduced for anaesthesia. Anaesthesia was maintained throughout surgery using a nose cone with a standard stereotaxic device and 2% isoflurane + 500 ml/min oxygen flow. Under aseptic conditions rats underwent a craniotomy (5 mm diameter) centred −3.0 mm posterior and 4.0 mm lateral of bregma to expose the intact dura mater of the brain. A hollow plastic injury cap was sealed over the craniotomy with cyanoacrylate and dental cement. For the rats assigned to receive saline-vehicle or sodium selenate treatment via subcutaneous osmotic pump, a horizontal incision was made between the shoulder blades to allow for the rapid insertion of the pump immediately post-injury. After a total anaesthetic time of 50 min, the rat was then removed from anaesthesia and attached to the injury device via the head cap. At the first response of hind-limb withdrawal, rats received a FPI pulse to the brain with a force of 3 atmospheres. Upon resumption of spontaneous breathing the head cap was removed, the treatment pump was inserted, and the incisions were sutured. Sham-injury rats underwent the same procedures with the exception that the fluid pulse was not administered. Six weeks post-injury, rats were anaesthetized for 10 min, the initial treatment pump was removed, and a replacement treatment pump was implanted as described above. Rats were then given an additional recovery time of 6 weeks, for a total recovery time of 12 weeks. The follow-up time post-TBI of 12 weeks was chosen for the sodium selenate treatment experiment as we have previously found that the progressive neurodegeneration and behavioural impairments following FPI plateau at 3 months, and do not significantly differ from those observed at 6 months post-FPI (Jones et al., 2008; Liu et al., 2010). Furthermore, previous studies have reported that abnormalities in phosphorylated tau are present by 3 months post-FPI (Hoshino et al., 1998).

Acute injury severity

Apnoea, unconsciousness, and self-righting reflex times were monitored in all rats immediately after injury as indicators of acute injury severity (Gurkoff et al., 2006; Shultz et al., 2011, 2012a). Apnoea was the time from injury to spontaneous breathing. Loss of consciousness was the time from injury to a hind-limb withdrawal response to a toe pinch. Self-righting reflex was the time from injury to the return of an upright position. For rats that were given either FPI or sham-injuries and sacrificed at 2, 24, or 72 h post-injury, FPI worsened apnoea [F(1,24) = 67.429, P < 0.001], unconsciousness [F(1,24) = 736.609, P < 0.001], and self-righting reflex times [F(1,24) = 300.698, P < 0.001] compared to sham-injured rats, and there were no significant differences between the 2, 24, and 72 h groups (Table 1). For rats that were given either FPI or sham-injury and treated with either sodium selenate or saline-vehicle, FPI worsened apnoea [F(1,51) = 222.842, P < 0.001], unconsciousness [F(1,51) = 428.672, P < 0.001], and self-righting reflex times [F(1,51 = 279.852, P < 0.001] compared to sham-injury regardless of the assigned treatment group (Table 1).

Table 1.

Acute injury severity

Western blot studies	2 h		24 h		72 h
	Sham	FPI	Sham	FPI	Sham	FPI
Apnoea (s)	0	44.0a	0	46.4a	0	52.4a
Unconsciousness (s)	0	335.2a	0	333.6a	0	343.0a
Self-righting (s)	109.2	538.4a	123.6	555.6a	109.8	558.5a

Long-term treatment study	SHAM + VEH	SHAM + SS	FPI + VEH	FPI + SS
Apnoea (s)	0	0	42.8a	41.4a
Unconsciousness (s)	0	0	338.9a	364.6a
Self-righting (s)	152.4	160.9	553.1a	573.4a

Top: Injury severity outcomes for acute western blot studies. FPI results in worsened acute injury severity outcomes as indicated by longer apnoea, unconsciousness, and self-righting reflex times compared to the sham-injured group. There were no significant differences between the 2, 24 and 72 h groups.

Bottom: Acute injury severity measures for long-term treatment study. Regardless of the assigned treatment, FPI resulted in worsened acute injury severity outcomes as indicated by increased apnoea, unconsciousness, and self-righting reflex times compared to the sham-injured group.

^aFPI significantly greater than sham-injured groups, P < 0.001. See ‘Results’ section for additional statistical details.

Serial MRI

MRI scanning was performed at baseline and 1, 4 and 12 weeks post-injury using a 4.7 T Bruker Avance III scanner with 30 cm horizontal bore and fitted with a BGA12S2 actively shielded gradient set capable of 440 mT/m and actively decoupled volume transmit and 4-channel surface receive coils. Anaesthetized rats were positioned supinely on a cradle with stereotactic fixation and a nose cone positioned over the rat’s snout to maintain anaesthesia. Body temperature was maintained throughout the experiment with a hot water circulation system built into the cradle.

The scanning protocol consisted of a 3-plane localizer sequence followed by multi-slice axial, coronal and sagittal scout images to accurately determine the position of the rat brain. A T₂-weighted image was acquired using a 2D RARE (Rapid Acquisition with Relaxation Enhancement) sequence (Hennig et al., 1986) with the following imaging parameters: recovery time = 10 000 ms, RARE factor = 8, effective echo time = 36 ms, field of view = 28.8 × 28.8 mm², matrix size = 192 × 192, number of slices = 80, slice thickness = 150 μm and number of repetitions = 2.

HARDI (high angular resolution diffusion weighted imaging) was performed using a 2D echo planar, spin-echo sequence (Stejskal and Tanner, 1965) with the following imaging parameters: recovery time = 9000 ms, echo time = 37 ms, field of view = 38.4 × 38.4 mm², matrix size = 128 × 128, number of slices = 36 and slice thickness = 300 μm. Diffusion weighting was performed with diffusion duration (δ) = 5 ms and diffusion gradient separation (Δ) = 14 ms in 126 non-collinear directions with 10 non-diffusion images. Two diffusion data sets were acquired, the first with b-value = 1200 s/mm² and the second with b-value = 3000 s/mm².

MRI analysis

All volumetric analysis procedures followed those previously described (Bouilleret et al., 2009; Liu et al., 2010; Shultz et al., 2013b, 2014b). Briefly, T₂-weighted MRI volumes of selected brain regions were quantified with manually drawn regions of interest using FSL (Analysis Group, Oxford, UK). A total of eight regions of interest, including the cortex, hippocampus, corpus callosum, and lateral ventricles from each hemisphere, were used in the analysis (Bouilleret et al., 2009; Liu et al., 2010; Shultz et al., 2013b, 2014b). Regions of interest were drawn on consecutive axial MRI slices by an investigator blinded to experimental conditions. Only slices containing hippocampus were analysed. Template images were generated for each cohort at each time point using ANTS (Advanced Normalization Tools Software) that derives an optimal template based on symmetric normalization unbiased by either shape or appearance (Kim et al., 2008; Avants et al., 2010). Fractional anisotropy measures were calculated using FSL’s FDT software using the lower b-value (1200 s/mm²) HARDI data sets. Region of interest masks were then transformed into the diffusion image space using ANTS and mean fractional anisotropy measures were calculated for each animal using MATLAB (The MathWorks). Fibre-tracking was performed using the MRtrix software package (Farquharson et al., 2013; Tournier et al., 2013) using high b-value (3000 s/mm²) HARDI data sets. Probabilistic whole brain fibre-tracking was performed using orientations sampled from the constrained spherical deconvolution and seeded at random within the whole brain mask (Farquharson et al., 2013). Both the total numbers of tracks passing through, and the mean track densities per voxel, were calculated for the corpus callosum masks.

Behavioural tests

Behavioural testing was performed at 12 weeks post-injury, was conducted by an experimenter blinded to experimental conditions, and was completed over five consecutive days. On Day 1 of testing, anxiety-like behaviour was assessed using the elevated-plus maze as previously described (Jones et al., 2008; Bao et al., 2012). Briefly, rats were placed in the centre of the elevated-plus maze facing an open arm and allowed to explore the maze freely for 5 min. Behaviours were recorded using an overhead camera, and the number of entries into, and amount of time spent in, open or closed arms were quantified by Ethovision Tracking Software (Noldus, Netherlands). As time spent in the open arm is decreased in rats that exhibit greater anxiety-related behaviours, a percentage score was calculated for the time spent in the open arm (Walf and Frye, 2007; Saucier et al., 2008). The number of entries into the closed arm of the maze was also calculated as a measure of locomotion (Walf and Frye, 2007; Saucier et al., 2008).

On Day 1 of behavioural testing, after completion of the elevated-plus maze, locomotion and anxiety-like behaviour was assessed using an open field as previously described (Jones et al., 2008; Bao et al., 2012; Shultz et al., 2013a). Rats were placed in the centre of a circular open field arena (100-cm diameter) enclosed by walls 20-cm high, and allowed to freely explore for 10 min. Behaviour in the open field was recorded by an overhead camera, and Ethovision Tracking Software (Noldus) quantified the total distance travelled, as well as the number of entries and time spent in the centre area (66-cm diameter) of the arena.

As previously described (Bao et al., 2012; Shultz et al., 2012b, 2013a), to assess cognition water maze testing was conducted in a circular pool (150-cm diameter) on Days 2 (acquisition session) and 3 (reversal session) of behavioural testing. Briefly, an escape platform was hidden 2 cm below the water surface in the centre of the south-east quadrant of the pool during the acquisition session. The acquisition session consisted of 10 trials. A trial began by placing the rat in the pool adjacent to, and facing, the pool wall, and ended when the rat standing on the hidden platform. Each trial began at one of four pool wall start locations (North, South, East, or West) according to a pseudo-random schedule of start locations that prevented repeated sequential starts from the same location. The reversal session of the water maze was completed on Day 3 of behavioural testing, 24 h after acquisition. Reversal procedures were identical to those for the acquisition session except that the hidden platform was now located in the opposite north-west quadrant of the pool. Behaviour was analysed by Ethovision Tracking Software. Search time and direct and circle swims were used as measures of spatial place memory (Morris, 1989; Whishaw and Jarrard, 1995; Bao et al., 2012). Swim speed (cm/s) was used as a measure of motor ability (Bao et al., 2012).

The beam task was used to assess sensorimotor function (Kolb and Whishaw, 1985; Bao et al., 2012). Beam training occurred on Day 2 of behavioural testing, after the acquisition session of the water maze. During the training session rats were given five trials to traverse a 100 cm long beam with a width of 4 cm, and a further five trials to traverse a 100 cm long beam with a width of 2 cm. Beam testing occurred on Day 3 of behavioural testing, after water maze reversal, and consisted of 10 trials on the 2 cm wide, 100 cm long beam. Each trial began with the rat being placed at one end of the beam and ended when the animal successfully traversed a distance of 100 cm. A maximum of 60 s was allowed for each trial. If the rat fell off the beam it was given a time of 60 s. Each trial was recorded by a camera, and traverse time and the number of slips and falls were used as measures of sensorimotor function.

As previously described, the forced swim test is commonly used in rodents to measure depression-like behaviour (Porsolt et al., 1977; Jones et al., 2008; Shultz et al., 2012a). The forced swim apparatus is a clear perspex cylinder (diameter = 30 cm, height = 40 cm) filled to a depth of 30 cm with water at 25°C. On Day 4 of behavioural testing, rats underwent forced swim training. Training consisted of the rats being placed in the forced swim apparatus for 15 min. On Day 5 of behavioural testing the rats underwent forced swim testing, where each rat was placed in the apparatus for 5 min. Behaviour was recorded by a video camera from a horizontal angle, and the test session was later scored by an individual blinded to experimental conditions to calculate: (i) the time each rat spent immobile (the primary outcome), defined as making only those movements necessary to keep its head above water; (ii) the time spent escaping, defined as vigorous vertical movement with all four limbs; and (iii) the time spent swimming. Only behaviours that persisted longer than 2 s were scored.

Rat western blotting

Depending on experimental assignment, rats were either sacrificed at 2 h, 24 h, 72 h, or 12 weeks post-injury with a lethal dose of pentobarbital (100 mg/kg). The brains were rapidly removed, the cortex from each hemisphere was dissected, and the tissue was rapidly frozen in liquid nitrogen and stored at −80°C. The frozen tissue was grinded on dry ice and solved in RIPA buffer [40 mM Tris, pH 7.5, 150 mM NaCl, 0.1% sodium dodecyl sulphate (SDS), 1% glycerol, 1% Triton™ X-100, 2 mM ethylene diamine tetraacetic acid (EDTA) and 0.5% NP-40] with protease inhibitors cocktail and phosphatase inhibitors cocktail. The homogenate was composed of 1 mg of tissue/ml RIPA buffer. The extracts were centrifuged at 12 000g for 15 min at 4°C and the supernatant was used for western blotting. After the protein concentration of supernatant was determined, the supernatant was mixed [5:1 (v/v) ratio] with sample buffer containing 300 mM Tris-HCl (pH 6.8), 30% 2-mercaptoethanol, 12% SDS, 0.005% bromophenol blue, and 20% glycerol. This mixture was boiled for 10 min at 95°C, then centrifuged briefly, and the supernatant was stored at −20°C for western blot analysis. Protein (40 μg) was loaded for each well; the proteins in samples were separated with SDS-polyacrylamide gel electrophoresis (SDS-PAGE, 12%), and the bands of proteins were electroblotted onto polyvinyl difluoride (PVDF) membranes. The blots on PVDF were developed with anti-pS198 (1:1000 dilution; 0.447 μg/ml IgG; Abcam), anti-pS262 (1:1000 dilution; 1 μg/ml IgG; Abcam); anti-Tau-5 (1:1000 dilution; 0.5 μg/ml IgG; Millipore), anti-PR55 (1:1000 dilution; 0.5 μg/ml IgG; Millipore), anti-PP2Ac (1:1000 dilution; 0.5μg/ml IgG; Millipore), and anti-GAPDH (1:5000 dilution; 0.05 μg/ml IgG; Cell Signaling) antibodies, and visualized by enhanced chemiluminescent substrate kit and exposure to x-films. Hyperphosphorylated tau was determined as the amount of phosphorylated tau (pS198 and pS262) relative to total tau (Tau-5; Liang et al., 2009; Jones et al., 2012). The mean intensity of the blots was quantified using ImageJ software (National Institutes of Health, USA). All western blot procedures were conducted by an experimenter blinded to experimental conditions.

PP2A activity assays

PP2A activity in samples of injured rat cortex were measured with a PP2A immunoprecipitation phosphatase assay. The frozen tissues were ground on dry ice, and dissolved in 20 mM imidazole-HCl, pH 7.0 with protease inhibitor cocktail (Begum and Ragolia, 1996). The lysates were then centrifuged at 12 000g for 15 min at 4°C, and the supernatants (100 µg of total protein) were used to assay phosphatase activity. PP2A was immunoprecipitated by anti-PP2Ac, and the background was pulled down by mouse IgG in parallel samples. These immune complexes were pulled down by protein A agarose beads. The PP2A of these immune complexes were incubated with threonine phosphopeptide for 15 min at 30°C to release free phosphate, which was assayed with malachite green phosphate detection solution. The PP2A activities were calculated as pmol released free phosphate/min/mg protein and expressed as relative to the PP2A activity in sham-injured rats.

Patients and sample collection: human post-mortem brain tissue

All procedures were conducted in accordance with the Australian National Health and Medical Research Council’s National Statement on Ethical Conduct in Human Research, the Victorian Human Tissue Act 1982, the National Code of Ethical Autopsy Practice, and the Victorian Government Policies and Practices in Relation to Post Mortem. Ethics approval was obtained from the Monash University Human Research Ethics Committee.

TBI brain samples from seven individuals aged 39 to 76 years (mean 63.1 years) were obtained from the Australian Brain Bank Network. The causes of injury included motor vehicle accident and falls. Patients had a survival time between 8 and 122 h (mean 49.4 h). The post-mortem interval varied between 40 and 114 h (mean 80.1 h). Frontal cortical tissue was sampled. Control brain samples of nine individuals, aged between 48 and 78 years (mean 66.5 years), with no history of brain trauma or other neurologic or psychiatric disorder and no significant neuropathology (as assessed by a neuropathologist) were also obtained from the Australian Brain Bank Network. The post-mortem interval varied between 24 and 71 h (mean 46.2 h). Clinical information and epidemiological details of all patients are included in Table 2.

Table 2.

Human TBI and control details

	Primary cause of death	Age	Survival time (h)	PMI (h)
Non-TBI case no.
1	Asthma	59	–	30
2	Myocardial infarct	75	–	50
3	Cardiac failure	48	–	50
4	IHD	63	–	68
5	IHD	64	–	24
6	Aortic aneurysm	75	–	46
7	IHD	69	–	71
8	Myocardial fibrosis	63	–	30
9	IHD	78	–	46
TBI case no.
1	TBI (fall)	75	10	89
2	TBI (fall)	38	122	101
3	TBI (fall)	64	8	61
4	TBI (MVA)	56	8	65
5	TBI (MVA)	70	76	114
6	TBI (MVA)	73	29	91
7	TBI (fall)	61	93	40

Clinical and epidemiological details of TBI and non-TBI patients used for Fig. 1.

IHD = ischaemic heart disease; PMI = post-mortem interval; MVA = motor vehicle accident.

Human sample preparation and western blotting

Fresh frozen brain cortex samples were homogenized at 150 mg wet weight per ml of PBS + 1% Triton™ X-100 + protease and phosphatase inhibitors. Lysates were stored at −80°C until further analysis. Samples were boiled in SDS-loading buffer with dithiothreitol, subjected to SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% bovine serum albumin or 5% skim milk prepared in 1× TBS plus 0.05% Tween-20, and then probed overnight with primary antibodies [anti-pS198 (1:2000), anti-pS262 (1:1000), anti-tau HT7 (1:5000), anti-PR55 (1:1000), and anti-GAPDH (1:5000)] followed by the appropriate HRP-conjugated secondary antibody. Signals were revealed by chemiluminescence (Pierce ECL Western Blotting Substrate, ThermoScientific), and quantified as described above for the rat samples. For analysis of tau blots, only bands between ∼45 to 69 kDa were included (Goedert et al., 1989).

Statistical analyses

Independent samples t-tests were used to analyse the acute western blots comparing the sham versus FPI rat groups, and the TBI versus non-TBI human groups. A two-way repeated measures ANOVA was used to analyse imaging measures and water maze search times related to the sodium selenate treatment experiments. All other measures were analysed using two-way ANOVAs. Bonferroni post hoc comparisons were carried out when appropriate. All analyses were performed using SPSS 21.0 software (IBM) with statistical significance set at P < 0.05.

Results

TBI induces the hyperphosphorylation of tau, and reduces PP2A/PR55 expression and PP2A activity

As shown in Fig. 1, FPI in rats induced the increased phosphorylation of tau, as indicated by the ratio of phosphorylated tau (pS198, Fig. 1A; pS262, Fig. 1B) to total tau (Tau-5) and the increased expression of pS198 (Fig. 1C) in the injured cortex, that reached statistical significance by 72 h post-injury compared to sham-injured rats [pS198/total tau: t(1,8) = 2.884, P < 0.05, Fig. 1A; pS262/total tau: t(1,8) = 2.659, P < 0.05, Fig. 1B; pS198/GAPDH: t(1,8) = 2.483, P < 0.05; Fig. 1C]. Furthermore, rats given a FPI had significantly reduced expression of PR55, and this effect preceded the increase in the phosphorylation of tau, reaching statistical significance at 2 h [t(1,8) = 3.609, P < 0.01], 24 h [t(1,8) = 3.968, P < 0.005], and 72 h [t(1,8) = 2.988, P < 0.05] post-injury compared to sham-injured rats (Fig. 1F). PP2A activity was also found to be significantly reduced after TBI, reaching statistical significance at 24 h [t(1,7) = 3.018, P < 0.05, Fig. 1D] and 72 h post-injury [t(1,7) = 2.805, P < 0.05, Fig. 1G]. Expression of pS262, total tau, and PP2Ac relative to GAPDH did not significantly differ between groups at 2, 24 or 72 h post-injury (P > 0.05; Fig. 1).

Figure 1.

Experimental TBI increases tau phosphorylation and reduces PP2A/PR55 B-subunit expression and PP2A activity in rats. Western blot analysis found the hyperphosphorylation of tau by 72 h post-FPI, as indicated by the increased ratio of phosphorylated tau (pS198, A; pS262, B) to total tau (tau-5), and the increased expression of pS198 relative to GAPDH (D) in the injured cortex. This was preceded by an acute reduction of PR55 expression at 2 h, 24 h, and 72 h post-FPI (F), and an acute reduction of PP2A activity at 24 h and 72 h post-FPI (G). Expression of pS262 (D), total tau (E), and PP2Ac (H) did not significantly differ between groups. *Significant difference between FPI and sham injury, P < 0.05.

As shown in Fig. 2, western blot analysis was also conducted on post-mortem brain samples collected from human TBI patients and non-TBI control brain samples (Table 2). The TBI group had a significant increase in phosphorylation of tau, as indicated by the ratio of pS262 to total tau [t(1,14) = 2.183, P < 0.05, Fig. 2C] and an increased expression of pS262 relative to GAPDH [t(1,14 = 2.480, P < 0.05, Fig. 2F]. While analyses of pS198 and PP2A/PR55 did not reach statistical significance (P > 0.05), there were trends suggesting a decrease in PP2A/PR55 expression and the hyperphosphorylation of pS198.

Figure 2.

Western blot analysis of tau phosphorylation and PP2A/PR55 B-subunit expression in human TBI brain tissue. (A and B) Western blots from brain tissue collected from human TBI patients and non-TBI controls (see Table 2 for patient details). Human TBI patients had hyperphosphorylated tau, as indicated by significant group differences on the measures of pS262 to total tau (HT7) ratio (D) and expression of pS262 (G). There were also non-significant trends suggesting increased expression of pS198 (C and F) and decreased expression of PR55 (E) in human TBI tissue. (H) Total tau (HT7) expression. *Significant difference between TBI and non-TBI, P < 0.05.

Sodium selenate treatment increases PP2A/PR55 expression and PP2A activity, and mitigates the increase in the phosphorylation of tau post-TBI

Rats given a FPI and treated with saline-vehicle had hyperphosphorylated tau as indicated by a significant increase in the ratio of pS198 to total tau [injury × treatment interaction: F(1,16) = 8.660, P < 0.01; FPI + VEH > all other groups, all P < 0.05] 12 weeks post-injury, whereas rats given FPI and treated with sodium selenate did not significantly differ from sham-injured rats (Fig. 3A). Rats treated with sodium selenate had increased expression of PP2A/PR55 B-subunit compared to those treated with saline-vehicle, irrespective of injury [F(1,16) = 4.591, P < 0.05; Fig. 3F]. Rats treated with sodium selenate also had increased PP2A activity compared to those treated with saline-vehicle, irrespective of injury [F(1,12) = 6.409, P < 0.05; Fig. 3G]. Rats given a FPI had significantly less total tau than sham-injured rats [F(1,16) = 5.482, P < 0.05; Fig. 3E]. The ratio of pS262 to total tau, and the expression of pS198, pS262, and PP2Ac relative to GAPDH did not significantly differ between groups (P > 0.05; Fig. 3).

Figure 3.

Sodium selenate treatment reduces hyperphosphorylated tau after TBI, and increases PP2A/PR55. (A) Western blot analysis indicated rats given a FPI and treated with saline-vehicle (VEH) had hyperphosphorylated tau as indicated by an increased phosphorylated tau (pS198) to total tau (tau-5) ratio in the injured cortex, whereas rats given and FPI and treated with sodium selenate did not differ from sham-injured groups. A similar trend was observed in the ratio of pS262 to total tau (B), and there was a significant reduction of total tau in rats given an FPI regardless of treatment (E). Continuous treatment with sodium selenate for 12 weeks resulted in significant increases in PR55 expression (F) and PP2A activity (G). Expression of pS198 (C), pS262 (D), and PP2Ac (H) did not differ significantly between groups. ***Significantly greater than all other groups, P < 0.05. *FPI groups significantly less than sham groups, P < 0.05. ^#Sodium selenate significantly greater than VEH treated groups, P < 0.05. SS = sodium selenate.

Sodium selenate treatment reduces progressive atrophy of brain structures post-TBI

Serial MRI at baseline, 1, 4 and 12 weeks post-injury was used to assess progressive brain atrophy after TBI. Rats given a FPI and treated with sodium selenate had significantly more volume in the ipsilateral cortex [injury × treatment interaction: F(1,31) = 4.303, P < 0.05; FPI + VEH < FPI + SS < both sham-injured groups, all P < 0.05; Fig. 4B] and ipsilateral corpus callosum [injury × treatment interaction: F(1,31) = 7.237, P < 0.05; FPI + VEH < all other groups, all P < 0.05; Fig. 4C] than FPI rats treated with saline-vehicle. However, all rats given a FPI, regardless of treatment, did experience some degree of volume loss in the ipsilateral hippocampus [F(1,31) = 4.937, P < 0.05; Fig. 4D] and the ipsilateral and contralateral cortices [F(1,31) = 9.393, P < 0.005], as well as increased ipsilateral [injury × time interaction: F(2,62) = 16.492, P < 0.001; Fig. 4E] and contralateral [injury × time interaction: F(2,62) = 8.567, P < 0.001] ventricle volumes, compared to sham-injured rats.

Figure 4.

Sodium selenate treatment reduces progressive brain damage after TBI. (A) Template T₂-weighted images incorporating the MRI data from each rat in each group at 1 week, 4 weeks, and 12 weeks post-injury. Volumetric analysis indicated that continuous treatment with sodium selenate for 12 weeks post-FPI significantly reduced the damage to the ipsilateral cortex (B) and corpus callosum (C) relative to the rats given a FPI and treated with saline-vehicle (VEH). Rats given a FPI, regardless of sodium selenate or VEH treatment, had damage in the ipsilateral hippocampus (D), and lateral ventricle (E) relative to rats given a sham-injury. *FPI groups significantly different than sham groups, P < 0.05. **Significantly less than both sham groups, P < 0.05. ***Significantly less than all other groups, P < 0.05. See ‘Results’ section for additional statistical details. SS = sodium selenate; w = week.

Sodium selenate treatment attenuates corpus callosum damage post-TBI

In addition to the volumetric analysis, serial DWI was used to assess the integrity of the corpus callosum, a white matter structure commonly affected by TBI, at baseline, 1, 4 and 12 weeks post-injury. Fractional anisotropy in the corpus callosum of rats given a FPI and treated with sodium selenate did not significantly differ from sham-injured groups, whereas rats given a FPI and treated with saline-vehicle had a significant decrease in fractional anisotropy in the ipsilateral [injury × treatment interaction: F(1,31) = 5.425, P < 0.05; FPI + VEH < all other groups, P < 0.05; Fig. 5C] and contralateral corpus callosum [injury × treatment interaction: F(1,31) = 4.836, P < 0.05; FPI + VEH < all other groups, P < 0.05; Fig. 5D]. DWI-based tractography outcomes further supported that sodium selenate treatment after FPI preserved corpus callosum integrity. Rats given a FPI and treated with sodium selenate had an increased number of tracts in the corpus callosum compared to rats given a FPI and treated with saline-vehicle [injury × treatment interaction: F(1,31) = 5.148, P < 0.05; FPI + VEH < all other groups, P < 0.005; Fig. 5E]. Consistent with tract number findings, rats given a FPI and treated with sodium selenate also had increased tract density in the corpus callosum compared to rats given a FPI and treated with saline-vehicle [injury × treatment interaction: F(1,31) = 4.967, P < 0.05; FPI + VEH < all other groups, P < 0.01; Fig. 5F].

Figure 5.

Sodium selenate treatment reduces injury to the corpus callosum after TBI. (A) Representative fractional anisotropy maps at 12 weeks post-injury. Analysis of mean fractional anisotropy in the ipsilateral (B) and contralateral (C) corpus callosum found that rats given a FPI and treated with saline-vehicle (VEH) had decreased fractional anisotropy, whereas rats given a FPI and treated with sodium selenate for 12 weeks did not differ from sham-injured groups. (D) Template whole-brain tractography images at 12 weeks post-injury. (E) Rats given a FPI and treated with VEH had significantly fewer tracks (E) and decreased track density (F) in the corpus callosum, whereas rats given a FPI and treated with sodium selenate for 12 weeks did not differ from sham-injured groups. ***Significantly less than all other groups, P < 0.05. SS = sodium selenate; w = week.

Sodium selenate treatment reduces cognitive impairments post-TBI

Rats were tested in the water maze to assess cognitive function 12 weeks after injury. During the acquisition session of the water maze, rats given a FPI and treated with sodium selenate displayed significantly faster search times than their vehicle-treated counterparts [injury × treatment interaction: F(1,51) = 7.127, P < 0.01; FPI + VEH > all other groups, P < 0.001; Fig. 6A]. Direct and circle swim data during acquisition were consistent with search time findings as rats given a FPI and treated with sodium selenate displayed significantly more direct and circle swims compared to the FPI rats treated with saline-vehicle [injury × treatment interaction: F(1,51) = 4.812, P < 0.05; FPI + VEH < all other groups, all P < 0.01; Fig. 6B].

Figure 6.

Sodium selenate treatment reduces cognitive, anxiety, and motor impairments after TBI. Rats given a FPI and treated with saline-vehicle (VEH) had longer search times (A) and fewer direct and circle swims (B) during acquisition of the water maze, whereas rats given a FPI and treated with sodium selenate did not differ from sham-injured groups. (C) Rats given a FPI and treated with saline-vehicle had longer search times during reversal of the water maze, whereas rats given a FPI and treated with sodium selenate did not differ from sham-injured groups. (D) Rats given a FPI, regardless of sodium selenate or saline-vehicle treatment, displayed fewer direct and circle swims during reversal of the water maze. (E) Rats given a FPI and treated with saline-vehicle (VEH) spent significantly less time in the open arm of the elevated-plus maze, whereas rats given a FPI and treated with sodium selenate did not differ from sham-injured groups. (F) There were no group differences on the number of closed arm entries in the elevated-plus maze, suggesting that locomotion was not a confounding factor. Rats given a FPI exhibited more slips and falls (G) and longer traverse times (H) on the beam task. However, rats given a FPI and treated with sodium selenate had significantly fewer slips and falls than rats given a FPI and treated with saline-vehicle (G). *FPI groups significantly different than sham groups, P < 0.05. **Significantly more than both sham groups, P < 0.05. ***Significantly different than all other groups, P < 0.05.

One day after the water maze acquisition session, rats underwent a water maze reversal session where the location of the escape platform was changed. During the reversal session, post-TBI treatment with sodium selenate was again found to have beneficial effects on cognition as rats given a FPI and treated with sodium selenate exhibited significantly faster search times than FPI rats treated with saline-vehicle [injury × treatment interaction: F(1,51) = 4.794, P < 0.05; FPI + VEH > all other groups, all P < 0.005; Fig. 6C]. However, regardless of treatment, FPI rats had fewer direct and circle swims than sham-injured rats [F(1,51) = 16.379, P < 0.001], indicating that all FPI rats experienced some degree of cognitive impairment during reversal testing (P < 0.001; Fig. 6D). There were no significant effects on the measure of swim speed during water maze acquisition or reversal (P > 0.05, data not shown), suggesting that motor abnormalities were not a confounding factor.

Sodium selenate treatment reduces anxiety-like behaviour post-TBI

Rats were also tested in the elevated-plus maze to assess anxiety-like behaviour. Rats given a FPI and treated with sodium selenate spent significantly more time in the open arm of the elevated-plus maze, indicating less anxiety-like behaviour, compared to FPI rats treated with saline-vehicle [injury × treatment interaction: F(1, 51) = 4.389, P < 0.05; FPI + VEH < all other groups, all P < 0.05; Fig. 6E]. There were no significant effects on the measure of closed arm entries (P > 0.05, Fig. 6F), suggesting that motor abnormalities were not a confounding factor.

Sodium selenate treatment reduces sensorimotor impairments post-TBI

Rats were tested on the beam task to assess sensorimotor function 12 weeks after injury. Rats given a FPI and treated with sodium selenate displayed improved sensorimotor outcome compared to FPI rats treated with saline-vehicle as indicated by fewer slips and falls [injury × treatment interaction: F(1,51) = 5.573, P < 0.05; FPI + VEH > FPI + SS > both sham groups, all P < 0.05; Fig. 6G]. However, all rats given a FPI, regardless of treatment, did displayed some degree of sensorimotor impairment, as indicated by an increased number of slips and falls (Fig. 6G) and longer traverse times [F(1,51) = 12.980, P < 0.001; Fig. 6H] than sham-injured rats. There were no significant effects on any of the open field or forced swim measures (all P > 0.05, data not shown).

Discussion

TBI is a complex and progressive neurodegenerative condition without a proven pharmaceutical intervention to improve long-term outcome (Blennow et al., 2012). Hyperphosphorylated tau has been implicated in the pathogenesis of TBI and other related neurodegenerative disorders (Thom et al., 2011; Blennow et al., 2012; Johnson et al., 2012; McKee et al., 2013; Zheng et al., 2014). PP2A is a phosphatase that regulates key signalling pathways in the brain (Janssens and Goris, 2001; Janssens et al., 2008), and the PR55 regulatory B-subunit is essential for PP2A to dephosphorylate tau (Janssens et al., 2008; Xu et al., 2008; Shi, 2009; Zheng et al., 2014). Here we investigated whether PP2A and tau are affected by TBI. We found that experimental TBI in rats results in the increased phosphorylation of tau, which is consistent with previous rodent (Hoshino et al., 1998; Chen et al., 2010) and human findings (Thom et al., 2011; Johnson et al., 2012; McKee et al., 2013). We also found that experimental TBI induced decreases in PP2A activity and PP2A/PR55 expression, and that these changes preceded the hyperphosphorylation of tau. Hyperphosphorylated tau and decreased PR55 expression were also observed in brain tissue from human patients acutely after TBI. While many of the human findings did not reach statistical significance, they are supportive of the relevance of the findings in the rat model considering the highly heterogeneous nature of human TBI, the variability in post-TBI time points, and the relatively old age of both the TBI and non-TBI patients, all of which may affect PR55 and hyperphosphorylated tau. Taken together, our acute TBI findings are consistent with those from previous studies reporting a downregulation of PR55 and/or PP2A after brain insults including compression (Chen et al., 2010), ischaemic (Koh, 2013), and excitotoxic injuries (Liang et al., 2009), and support a close relationship between PP2A/PR55 and the hyperphosphorylation of tau (Sontag et al., 2008; Xu et al., 2008; Iqbal et al., 2009; Liang et al., 2009; Shi, 2009; Chen et al., 2010; Corcoran et al., 2010b; van Eersel et al., 2010; Bolognin et al., 2012; Zheng et al., 2014).

The molecular mechanisms underlying these TBI-induced changes to PP2A/PR55 and tau are complex and require further investigation. Given the onset of these changes in the acute phase of TBI, it is possible that an early pathogenic event in the injury cascade may initiate the effect on PP2A/PR55 and subsequently phosphorylated tau. For example, glutamate-mediated excitotoxicity begins in the immediate seconds following TBI (Blennow et al., 2012), has been reported to induce hyperphosphorylated tau and decrease PP2A (Liang et al., 2009), and also occurs in neurodegenerative conditions (e.g. Alzheimer’s disease, epilepsy) where similar tau and PP2A changes have been reported (Gong et al., 1993, 1995, 2000; Liang et al., 2009). Brain injury has also been shown to result in a number of abnormalities in the biosynthesis, degradation, and post-translational modification of proteins. Of particular relevance to the potential mechanisms underlying these TBI-induced changes to tau and PP2A, it has also been found that TBI decreases global histone H3 methylation (Gao et al., 2006), suggesting that TBI may induce post-translational modifications leading to relevant expression or functional consequences. Methylation of PP2Ac on Leu³⁰⁹ is necessary for PR55 binding with PP2Ac, and the consequent removal of phosphate residues from phosphorylated tau (Nunbhakdi-Craig et al., 2007; Xu et al., 2008). Notably, the methylation of PP2Ac and PR55 is decreased in neurodegenerative conditions such as Alzheimer disease (Sontag et al., 2007; Zhou et al., 2008), and the demethylation of PP2Ac induces the hyperphosphorylation of tau and a net loss of PR55 (Sontag et al., 2008; Bottiglieri et al. 2012; Yao et al., 2012). Furthermore, alterations to the ubiquitin-proteasome, autophagy-lysosome, and/or calpain-mediated systems, which are all important regulators of protein degradation, are other pathways altered after experimental TBI which may play a mechanistic role in the findings of hyperphosphorylated tau and decreased PR55 expression and PP2A activity after TBI (Yao et al., 2007; Clark et al., 2008; Liu et al., 2008; Saatman et al., 2010; Luo et al., 2011; Oberg et al., 2012; Lee et al., 2013; Sun et al., 2013).

We next examined whether a clinically relevant 12-week treatment with sodium selenate, a PP2A/PR55 activator (Corcoran et al., 2010b; Zheng et al., 2014), would reduce hyperphosphorylated tau and improve outcome in rats given a TBI. It was found that the sodium selenate treatment improved cognitive, sensorimotor, and anxiety-related outcomes following a FPI. We also used serial MRI, a clinically relevant and translatable approach (Shultz et al., 2013b, 2014a), to identify that sodium selenate treatment attenuated the FPI-induced progressive brain atrophy to the cortex and corpus callosum. Advanced serial DWI and tractography outcomes also demonstrated that sodium selenate treatment preserved the integrity of the corpus callosum after TBI. Lastly, post-mortem analyses on brain tissue found that sodium selenate treatment increased PP2A activity and PP2A PR55 B-subunit expression, and that rats given a FPI and treated with sodium selenate had decreased hyperphosphorylated tau compared to their vehicle-treated counterparts. These findings that sodium selenate treatment increased PP2A activity and PP2A/PR55 expression, and decreased the hyperphosphorylation of tau, are consistent with those reported in other experimental tauopathy models (Corcoran et al., 2010b; van Eersel et al., 2010; Jones et al., 2012). While the exact molecular mechanisms underlying the upregulation of PR55/PP2A by sodium selenate remain to determined, previous studies have found that these effects do not occur with other selenium-based compounds (Corcoran et al., 2010b), and that PP2A is necessary for sodium selenate to dephosphorylate tau (van Eersel et al., 2010).

In addition to the questions that remain surrounding molecular mechanisms, there are other factors and limitations that should be considered when interpreting the findings of this study. Despite reductions in cognitive, emotional, and sensorimotor impairments, and less damage to the cortex and corpus callosum in rats given an FPI and treated with sodium selenate, there was still evidence of significant damage to the cortex, corpus callosum and hippocampus, and cognitive and sensorimotor impairments in these rats relative to sham-injury. These effects could be due to the brain damage that is caused by the primary injury at the moment of TBI impact, which is considered to be largely irreversible (Blennow et al., 2012; Xiong et al., 2013). Additionally, a number of other secondary injury pathways are activated in the aftermath of TBI, including neuroinflammation, oxidative stress, and apoptosis (Blennow et al., 2012; Xiong et al., 2013), that could have contributed the brain damage observed in the FPI rats treated with sodium selenate. Our finding of decreased total tau in rats 12 weeks post-FPI should also be noted as it is a contributing factor to the finding of increased phosphorylated tau/total tau ratio in FPI rats treated with vehicle. As described above, TBI may result in abnormalities to a number of protein synthesis and degradation pathways. Of relevance to tau, TBI can result in sustained calpain activation (Saatman et al., 2010), and calpain is a mediator of tau degradation (Xie and Johnson, 1998). Therefore, our findings of decreased total tau in rats given FPI, regardless of sodium selenate or vehicle treatment, may be related to calpain activation, though further studies examining this relationship are required. A potential limitation in the study was that the FPI model requires the use of isoflurane anaesthetic, and isoflurane may alter tau metabolism (Planel et al., 2007; Dong et al., 2012). However, all rats received the same duration of anaesthetic regardless of injury type, all rats were near consciousness at the time of injury, and all rats had their body temperatures maintained throughout anaesthetic exposure. As such, any effects isoflurane may have had on tau were minimized and consistent across both sham and FPI injured rats. Also, rats were not perfused prior to tissue collection, which may affect outcomes due to the inclusion of circulating proteins and/or decapitation ischaemia. However, this was a factor that was consistent amongst all experimental groups. Another potential limitation was the use of a 12-week post-injury recovery time to study the long-term effects of TBI and sodium selenate treatment. Although this 12-week recovery time was based on our previous findings that behavioural and pathological changes after FPI plateau at 12 weeks post-injury and do not differ from those observed at 6 months post-injury (Jones et al., 2008; Liu et al., 2010), future studies could employ more chronic recovery periods where the observed behavioural deficits, tauopathy and associated neurodegeneration may continue to evolve (Hoshino et al., 1998).

Despite these considerations, the current findings indicate that TBI induces abnormalities to PP2A and tau, that sodium selenate treatment is effective in improving experimental TBI outcomes while not resulting in any overt negative effects, and support the notion that targeting PP2A/PR55 and hyperphosphorylated tau with sodium selenate could be a novel therapeutic approach for TBI. Importantly, sodium selenate is highly water soluble and readily crosses the blood–brain barrier, and a sodium selenate treatment regimen similar to the one used here has already been demonstrated to be safe in a 6-month phase I trial in patients with prostate cancer (Corcoran et al., 2010a), and is also currently being assessed in a phase II study for Alzheimer’s disease (Zheng et al., 2014). Considering the lack of an effective pharmacological intervention for TBI patients, sodium selenate has the potential to be rapidly translated into clinical TBI trials.

Conclusions

Experimental TBI in rats induces decreased expression of PP2A/PR55 by 2 h post-injury, decreased PP2A activity by 24 h post-injury, and the hyperphosphorylation of tau by 72 h post-injury. Similar changes in the hyperphosphorylation of tau and expression of PP2A/PR55 were also observed in post-mortem brain tissue collected from human TBI patients. Continuous sodium selenate treatment for 12 weeks increased PP2A/PR55 levels and PP2A activity in rats, and sodium selenate treatment reduced hyperphosphorylated tau, neurodegeneration, and behavioural impairments in rats post-TBI. Taken together, these data suggest an important role for PP2A/PR55 in the hyperphosphorylation of tau and neurodegenerative aftermath of TBI, and support sodium selenate as a clinically translatable treatment to potentially improve outcomes after TBI.

Glossary

Abbreviations

FPI

lateral fluid percussion injury

PP2A

protein phosphatase 2A

TBI

traumatic brain injury

Funding

This study was funded by grants to T.O., C.H., N.J., M.S. and S.S. from the National Health and Medical Research Council (NHMRC #1006077 and #1062653), the Victorian Transport Accident Commission (Victorian Neurotrauma Initiative Grant #DNP13), the Alzheimer’s Australia Dementia Research Fund, the Royal Melbourne Hospital Neuroscience Foundation, and the Canadian Institute of Health Research. Human brain tissues were received from the Victorian Brain Bank Network, supported by the Mental Health Research Institute, The Alfred, Victorian Forensic Institute of Medicine, The University of Melbourne and funded by NHMRC, Helen Macpherson Smith Trust, Parkinson’s Victoria and Perpetual Philanthropic Services.