Tau Oligomers in Traumatic Brain Injury

Traumatic brain injury (TBI) is a serious problem affecting millions of people in the United States alone. Multiple concussions or even a single moderate-severe TBI can also predispose individuals to develop a pathologically distinct form of tauopathyrelated dementia at an early age. No effective treatments are currently available for TBI or TBI-related dementia; moreover, only recently has insight been gained regarding the mechanisms behind their connection. Here, we used antibodies to detect oligomeric and phosphorylated tau proteins in a nontransgenic rodent model of parasagittal fluid percussion injury. Oligomeric and phosphorylated tau proteins were detected 4 and 24 hours and 2 weeks post-TBI in injured, but not sham control rats. These findings suggest that diagnostic tools and therapeutics that target only toxic forms of tau may provide earlier detection and safe, more effective treatments for tauopathies associated with repetitive neurotrauma. Traumatic brain injury (TBI) is often thought of as an acute event resulting from a motor vehicle accident, blast injury, fall, or gunshot wound to the head. In reality, however, the effects from even a mild TBI can be long lasting and negatively impact quality of life. Demonstration of neuronal and glial changes months after TBI (1,2), in combination with evidence of seizures (3,4), endocrine disorders (5,6) and circadian rhythm disruption (7), have led to a paradigm shift in the http://www.jbc.org/cgi/doi/10.1074/jbc.M113.472746 The latest version is at JBC Papers in Press. Published on April 30, 2013 as Manuscript M113.472746 Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on N ovem er 7, 2017 hp://w w w .jb.org/ D ow nladed from Tau Oligomers in Traumatic Brain Injury 2 field and now TBI is acknowledged as a chronic disease, rather than a single event (8,9). Repetitive neurotrauma, believed to contribute to the development of chronic traumatic encephalopathy (CTE) in professional athletes, victims of domestic abuse, and combat veterans, can lead to the development of a progressive form of dementia reminiscent of early onset Alzheimer’s disease (AD) ((10); and reviewed in (11)). Neurofibrillary tangles (NFT) may play a role in the pathophysiology of CTE, but little is known about the mechanisms underlying their formation. Hyperphosphorylation contributes to NFT formation, and until recently it has generally been accepted that NFT trigger processes that lead to neuronal cell death. However, NFT accumulation alone may be insufficient to cause cell death, as neuronal loss and cognitive deficits precede NFT formation (12-15). Tau oligomers precede the formation of NFT and contribute to learning and memory deficits and neuronal cell death (16-19). In support of this idea, we demonstrated that oligomeric tau causes neurotoxicity in vivo (20) and that increased oligomeric tau species are present in post mortem brain samples from AD patients as compared to healthy controls (21). Moreover, we recently injected tau oligomers (isolated from AD brains) into wild-type mice; these oligomers disrupt memory and propagate abnormal tau conformation of endogenous tau after prolonged incubation (22). Previous studies of tau in brain, serum, or cerebrospinal fluid following TBI have only examined total native tau species (23,24). Previously, with our antibody (T22) that specifically recognizes oligomeric tau (21,22,25), we detected tau oligomers in both the PBS and sarkosyl soluble fractions and showed data supporting that tau oligomers as both intracellular and extracellular deposits (21). Extracellular tau is being investigated in recent reports because of the new phenomena of the spreading of tau pathology (26-28), in non-transgenic tauopathy model extracellular tau spreads by multiple mechanisms (29), and release of tau from healthy neurons under stimulation conditions similar to normal neuronal activity (30) and we believe this may play an important role in the development of tauopathies following TBI. Here, we used antibodies against oligomeric and hyperphosphorylated tau in a non-transgenic rodent model of parasagittal fluid percussion injury. We detected oligomeric and phosphorylated tau proteins as early as 4 hrs post TBI. In our model of fluid percussion TBI, we have detected neuronal cell death as early as 4 hrs after TBI and do not normally see more than one or two injured neurons in the hippocampus of sham-injured animals (31,32). Tau oligomers may well be a valuable diagnostic biomarker and therapeutic target for TBI. Strategies designed to prevent tau aggregation and eliminate these oligomeric toxic forms of tau specifically, while leaving the functional tau protein intact and available for microtubule formation (19,33-35), and could lead to more effective treatments for tauopathies, especially ones induced by repetitive neurotrauma. EXPERIMENIAL PROCEDURES Animals-This research was conducted in a facility approved by the American Association for the Accreditation of Laboratory Animal Care (AAALAC) and all experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch. Male Sprague–Dawley (Charles Rivers, Wilmington, MA) rats (400-500 grams) were anesthetized (4% isoflurane), incubated, mechanically ventilated with 1.5% isoflurane in O2:air (20:80) using a volume ventilator (NEMI Scientific: New England Medical Instruments, Medway, MA), and prepared for moderate or sham parasagittal fluid percussion injury (FPI) as previously described (36,37). Rectal and temporalis muscle temperatures were monitored using telethermometers (Physitemp Instruments, Clifton, NJ), and temperatures were maintained within a range of 37.5 ± 0.5 °C using an overhead lamp and a thermostatically controlled water blanket (Gaymar, Orchard Park, NY). Rats were placed in a stereotaxic apparatus, a midline incision of the skin was performed, and the skull was exposed. With the use of a Michele trephine, a craniotomy was performed 1 mm lateral (right) to the sagittal suture, midway between the lambda and bregma. The bone chip was removed, leaving the dura intact. A modified 20-gauge needle hub was secured in place over the by gest on N ovem er 7, 2017 hp://w w w .jb.org/ D ow nladed from Tau Oligomers in Traumatic Brain Injury 3 exposed dura with cyanoacrylic adhesive and cemented into place with hygienic dental acrylic. Parasagittal fluid percussion injury (FPI)-TBI was administered by means of an FPI device (38) consisting of a fluid-filled Plexiglas cylinder 60 cm long and 4.5 cm in diameter, one end of which was connected to a hollow metal cylinder housing a pressure transducer (Statham PA856-100, Data Instruments, Acton, MA), with the other end closed by a Plexiglas piston mounted on O rings. The transducer housing was connected to the rat by a plenum tube at the craniotomy site. Each TBI was induced by dropping a 4.8 kg steel pendulum that struck the piston. The height of the pendulum determined the intensity of the injury. The fluid pressure pulse was recorded on an oscilloscope triggered photoelectrically by the descent of the pendulum. Four or 24 hrs after TBI or sham injury (n = 6 at each time point), rats were reanesthetized, transcardially perfused with chilled saline to remove blood, and then brains were collected and immediately frozen. ELISAFor ELISA, plates were coated with 10 μL of the brain extract using 0.1 M sodium bicarbonate, pH 9.6, as coating buffer, followed by overnight incubation at 4°C, washing three times with Tris-buffered saline (TBST) with low Tween (0.01%), then blocked for 3 hrs at room temperature with 10% non fat dry milk in TBST. The plates were then washed with TBST. Antibodies used include anti-tau oligomer antibody T22 (1:500), Tau1 (clone PC1C6/MAB3420 1:1000; Millipore, Billerica, MA), GAPDH (1:1000; Abcam, Cambridge, MA) AT8, and AT180 (1:1000; ThermoScientific, Waltham, MA). All antibodies were diluted in 5% nonfat milk in TBST and were added and allowed to react for 1 hr at room temperature. The plates were then washed three times with TBST and 100 μL of horseradish peroxidase-conjugated antirabbit IgG (1:3000; GE Healthcare Life Sciences, Pittsburgh, PA) was added, followed by incubation for 1 hr at room temperature. Finally, plates were washed three times with TBST and developed with 3,3',5,5',-tetramethylbenzidine (TMB-1 component substrate) from DAKO (Carpinteria, CA). The reaction was stopped with 100 μl 1M HCl and samples were read at 450 nm using a POLARstar OMEGA plate reader (BMG Labtechnologies, Melbourne, Australia). Western blot analysisTissue samples were homogenized in 5% ice-cold protease inhibitor. 16 μL of each of the brain extracts were mixed with 4 μL of 4X sample buffer (without boiling) and were run on 4-12% Bis-Tris SDS-PAGE gels and subsequently transferred onto nitrocellulose membranes. After blocking overnight at 4°C with 10% nonfat milk, membranes were probed for 1 hr at room temperature with T22 antibody (1:200), Tau-1 (1:1000; Millipore, Billerica, MA), GAPDH antibody (1:1000; Abcam, Cambridge, MA), AT8, or AT180 antibodies (1:1000; ThermoScientific, Waltham, MA) diluted in 5% nonfat dried milk. For detection horseradish peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG (1:3000; GE Healthcare, UK) and ECL plus (GE Healthcare) were used. Western blot densitometry-Analysis was performed using Labworks 4.5 software (UVP Inc., Upland, CA). For protein quantification, the densitometry of each band in the Western blot was normalized with GAPDH. All densitometry results represent the mean and standard deviations (n = 6). Isolation and characterization of tau oligomers derived tau speciesImmunoprecipitation (IP) experiments were performed as previously described (21,39). Briefly, tosyl-activated magnetic Dynabeads (Dynal Biotech, Lafayette Hill, PA) were coated with 20 μg of T22 antibody (1.0 mg/ml) diluted in 0.1 M borate, pH 9.5, overnight at 37°C. Beads were washed (0.2 M Tris, 0.1% bovine serum albumin, pH 8.5) and then incubated with either the 24-hrs TBI or sham brain homogenate with rotation at room temperature for 1 hr. Beads were then washed three times with PBS and eluted using 0.1 M glycine, pH 2.8. The pH of each eluted fraction was adjusted using 1 M Tris pH 8.0. Fractions were pooled and concentrated 5x using Amicon Ultra Centrifugal Filter units with 30 kDa cut-off (Millipore)(21). Characterization of tau oligomers isolated by IP was performed by various methods as previously described (21,40). Size-exclusion chromatography (SEC) analysis was performed using an LC-6AD Shimadsu highperformance liquid chromatography (HPLC) system fitted with a Superdex 200-10/300 GL column (GE Healthcare). 6 μl of isolated sample was adjusted to 50 μl total volume with filtered 1X PBS for injecting into the column. Running buffer by gest on N ovem er 7, 2017 hp://w w w .jb.org/ D ow nladed from Tau Oligomers in Traumatic Brain Injury 4 used 1X PBS, flow rate 0.8 ml/min. The approximate molecular weight of the peaks was estimated using gel filtration standard (Bio-Rad, CA) was used for calibrations (21,40). various methods as previously described (40). The morphology of tau oligomers isolated by IP was assessed as previously described by Atomic Force Microscopy (AFM) using a non-contact tapping method (ScanAsyst-air) with a Multimode 8 AFM machine (Veeco, CA) (21,40). Immunofluorescence-Frozen sections were fixed with chilled acetone for 10 mins at room temperature. After blocking in normal goat serum for 1 hr, sections were incubated overnight with T22 (1:300). The next day, the sections were washed in PBS three times for 10 mins each and then incubated with goat anti-rabbit IgG Alexa 568 (1:350; Invitrogen, Grand Island, NY) for 1 hr. The sections were then washed three times for 10 mins each in PBS before incubation overnight with mouse Tau-1 (1:200; Millipore). The next day the sections were washed in PBS three times for 10 minutes each prior to incubation with goat anti-mouse IgG Alexafluor 488 (1:350; Invitrogen) for 1 hr. The sections were then washed three times for 10 minutes with PBS. Sections were incubated with DAPI (1:3000 in PBS) for 5 mins, washed and mounted in Fluoromount-G mounting medium (Southern Biotech, Birmingham, AL). The sections were examined using a Bio-Rad Radiance 2100 confocal system mounted on a Nikon Eclipse E800 microscope equipped with a CoolSnap-FX monochrome CCD camera (Photometrics, Tucson, AZ) using standard Nikon FITC, Texas Red and DAPI filters set for Alexafluor 488, 568 and DAPI, respectively. Peroxidase immunohistochemistry-Peroxidase immunohistochemistry was performed on frozen sections. In brief, sections (5 μm) were fixed with chilled acetone for 10 mins, at room temperature and blocked for 1 hr with 5% horse serum in PBS. The following antibodies were used for immunostaining: T22 (1:300) and mouse AT8 and AT180 (1:100; ThermoScientific). Primary antibodies were incubated overnight at 4°C and detected with biotinylated goat anti-mouse IgG (Vectastin ABC kit; Vector Laboratories, Burlingame, CA) or biotinylated goat anti-rabbit IgG (Vectastin ABC kit; Vector Laboratories) and visualized using peroxidase substrate kit DAB (Vector Laboratories) according to the manufacturer’s recommendations. Sections were counterstained with hematoxylin (Vector Laboratories) for nuclear staining. Brightfield images were acquired using a Nikon Eclipse 800 microscope equipped with a Nikon DXM1200 color CCD camera (Nikon Instruments Inc, Melville, NY). Statistical Analyses-Data were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni’s correction for multiple comparisons using GraphPad prism 5 and OriginPro 8.0 software. Data in text and figures are represented as mean  SD.