Mice. Male C57BL/6J mice, referred to as WT mice, were purchased from Envigo (Holland). Male B6.129S4(Cg)-Mapttm1(EGFP)Klt/J, on a pure C57BL/6J background, referred to as tau knock-out (KO) mice, were from Jackson Laboratory (#029219, USA) [21]. Male JNPL3 mice expressing 0N4R human tau with the P301L mutation (P301L) were obtained from Taconic Biosciences (New York, USA) (Tau- Model 2508). Controls were non-transgenic (Non-Tg) male mice with the same mixed C57BL/6, DBA/2, SW genetic background as P301L mice. Mice were housed in a specific pathogen-free animal room at a constant temperature of 21 ± 1 °C, humidity 60 ± 5%, with a 12 h light/dark cycle, and ad libitum access to food and water.
Procedures involving animals and their care were conducted in conformity with the institutional guidelines at the Istituto di Ricerche Farmacologiche Mario Negri IRCCS in compliance with national (D.lgs 26/2014; Authorization n. 19/2008-A issued March 6, 2008 by Ministry of Health) and international laws and policies (EEC Council Directive 2010/63/UE; the NIH Guide for the Care and Use of Laboratory Animals, 2011 edition). They were reviewed and approved by the Mario Negri Institute Animal Care and Use Committee that includes ad hoc members for ethical issues, and by the Italian Ministry of Health (Decreto no. D/07/2013-B and 301/2017-PR). Animal facilities meet international standards and are regularly checked by a certified veterinarian who is responsible for health monitoring, animal welfare supervision, experimental protocols and review of procedures.
Mouse model of TBI. The controlled cortical impact brain injury mouse model used in this study replicates both the mechanical forces and the main secondary injury processes observed in severe TBI patients with brain contusion and gives clinically-relevant behavioral and histopathological outcomes [14, 22]. WT and tau KO mice were anesthetized by isoflurane inhalation (induction 3%; maintenance 1.5%) in an N2O/O2 (70%/30%) mixture and placed in a stereotaxic frame. Rectal temperature was maintained at 37 °C. Mice were then subjected to craniectomy followed by induction of controlled cortical impact brain injury as previously described [23]. Briefly, the injury was induced using a 3-mm rigid impactor driven by a pneumatic piston rigidly mounted at an angle of 20° from the vertical plane and applied to the exposed dura mater, between bregma and lambda, over the left parietotemporal cortex (antero-posteriority: -2.5 mm, laterality: -2.5 mm), at an impactor velocity of 5 m/s and deformation depth 1 mm, resulting in a severe level of injury [24]. The craniotomy was then covered with a cranioplasty and the scalp sutured. Sham mice received identical anesthesia and surgery without brain injury. Mice were allocated to surgery by a list randomizer (http://www.random.org/list). All behavioral evaluations and magnetic resonance imaging (MRI) analysis were done blinded to injury status and genetic background.
Sensorimotor function. TBI-induced sensorimotor deficits were assessed on WT and tau KO mice by the Neuroscore and Simple Neuroassessment of Asymmetric Impairment (SNAP) tests 4 months post-TBI. Neuroscore. Mice are scored from 4 (normal) to 0 (severely impaired) for each of the following indices: 1) forelimb function while walking on the grid and flexion function response when suspended by the tail; 2) hindlimb function while walking on the grid and extension function when suspended by the tail; and 3) resistance to lateral right and left pulsion. The maximum score per mouse is 12 [23, 25]. SNAP. The test evaluates eight neurological parameters including vision, proprioception, motor strength and posture. Score ranges from 0 (normal) to 5 (severely impaired) for each test. The scores from each of the eight tests are summed to give the total SNAP score. A neurologically intact animal would be expected to have a SNAP score of 0. The higher the score the more the asymmetric deficits [26].
Magnetic resonance imaging. Imaging studies were done four months after TBI. Images were acquired on a 7T Bruker Biospec (Ettlingen, Germany) running ParaVision 6.01, equipped with a quadrature cryogenic surface coil as transmitter and receiver. Mice were anesthetized (induction 3–4%, maintenance 1.5-2% in an air/O2 -70%/30% mixture) and body temperature was maintained at 37 °C. Acquisitions: coronal, 2D, T2-weighted RARE sequences with FOV 1.5 × 1.5 cm, matrix 150 × 150, 37 slices 300 µm thick, repetition time TR 5500 ms, echo time E = 66 ms, RARE factor of 8 and number of averages N = 12. Contusion volume was computed using the ITK-SNAP software.
Recombinant tau. Recombinant tau WT (TauWT) and P301L (TauP301L) were expressed in E. coli and purified by exchange chromatography followed by size-exclusion chromatography. The purification protocol was adapted from Rossi et al. [27]. Aggregation was induced by incubating the proteins with heparin (Mr 6000–20000, Selleckchem), in a tau:heparin ratio of 4:1 (w/w) in 10 mM phosphate buffered saline (PBS), pH 7.4, for 24 h at 37 °C with shacking. To assess aggregation, the samples were examined in a detergent insolubility assay adapted from Drisaldi et al. [28] and the proteins in the supernatant and pellet were analyzed by Western blot using the anti-tau rabbit polyclonal antibody DAKO (Agilent Technologies Italy SpA, Milan, Italy) (1:10000 dilution). Anti-rabbit IgG peroxidase conjugate (1:20000, Sigma) was used as secondary antibody.
Proteinase-K digestion. Brain homogenates (30 µg proteins) from the ipsi pericontusional area of 3 months post-TBI and sham-injured mice, Non-Tg and P301L were incubated for 1 h at 37 °C with 10 µg/mL proteinase-K (PK) or the same volume of water. After incubation, 2 µg of digested proteins were suspended in 4X SDS (sodium dodecyl sulfate) loading buffer, boiled for 5 min and analyzed in 10% SDS-PAGE, followed by Comassie Blue R250 staining for 30 min.
C. elegans studies. Bristol N2 nematodes were obtained from the Caenorhabditis elegans Genetic Center (CGC, University of Minnesota, Minneapolis, MN, USA) and propagated at 20 °C on solid Nematode Growth Medium (NGM) seeded with E. coli OP50 (CGC) for food. We used the bleaching technique to prepare age-synchronized animals [29]. C. elegans at the first larval stage were then transferred to fresh NGM plates and grown at 20 °C.
At L3-L4 larval stage nematodes were collected with M9 buffer, centrifuged, and washed twice with 10 mM PBS, pH 7.4, to eliminate bacteria. Worms were incubated for 2 h at room temperature with orbital shaking, in the absence of E. coli, with homogenates from ipsi or contralateral (contra) areas from WT or tau KO TBI mice, the equivalent area of either WT or tau KO sham mice, or brain homogenates from Non-Tg and P301L mice (30–60 µg protein/100 worms/100 µL in 10 mM PBS, pH 7.4). TauWT or TauP301L (30 µg protein/100 worms/100 µL) were administered to worms in 10 mM PBS, pH 7.4. Worms incubated with brain homogenates of naïve mice (30 µg protein/100 worms/100 µL) or 10 mM PBS, pH 7.4 (100 worms/100 µL) were used as negative controls. Worms were then plated onto NGM plates seeded with OP50 E. coli, grown at 20 °C and transferred every day to new NGM plates seeded with E. coli to avoid overlapping generations.
The locomotor activity and the pharyngeal function of nematodes were scored before (time 0) and 2, 4, and 7 days after the treatment [30, 31]. To measure the locomotor activity, worms were picked up, transferred into a well of a 96-well ELISA plate containing 100 µL of ddH2O and their motility was evaluated by counting the number of left–right movements in 1 minute (body bends/min). The pharyngeal pumping rate was measured by counting the number of times the terminal bulb of the pharynx contracted in 1minute (pumps/min).
Brain homogenates (30 µg proteins) from the pericontusional area of 12 months post-TBI and sham-injured mice, Non-Tg and P301L were incubated for 1 h at 37 °C with 10 µg/mL PK or the same volume of water. The homogenates were then given to C. elegans (30 µg proteins/100 worms/100 µL) as described above. Control worms were treated with 100 µL of water ± 10 µg/mL PK. Worms were then plated onto NGM plates seeded with OP50 E. coli, grown at 20 °C and transferred every day for six days to new NGM plates seeded with E. coli. Locomotor activity was rated on the seventh day after treatment.
In some experiments, N2 nematodes were fed for 2 h with the different brain homogenates (30 µg proteins/100 worms/100 µL) previously incubated or not for 30 min at room temperature with 5–50 ng/µL anti-mouse tau monoclonal antibody T46 (Thermo Scientific) or anti-human tau monoclonal antibody SP70 (Rockland, Limerick, PA, USA). Antibodies alone were employed as controls. Worms were also treated in the same conditions with brain homogenates incubated for 30 min at room temperature with 10 ng/µL of T46 antibody inactivated by incubation at 100 °C for 10 min (inactivated T46) or the corresponding volume of 10 mM PBS, pH 7.4. Worms were then plated onto NGM plates seeded with OP50 E. coli, grown at 20 °C and transferred every day for six days to new NGM plates seeded with E. coli. Locomotor activity was rated on the seventh day. All behavioral evaluations were done blinded.
Sensitivity to aldicarb and levamisole. The sensitivity of worms to aldicarb and levamisole was evaluated four days after treatment with brain homogenates. Nematodes (50 worms/plate) on NGM agar plates with OP50 E. coli, were treated with 0.5 mM aldicarb (Sigma Aldrich) or 1 mM levamisole (Sigma Aldrich). The worms were prodded on the nose after 15 min and 30-min intervals for the next 4 h to determine when paralysis was complete [32].
Lifespan. For lifespan experiments, L4 worms were fed for 2 h with the different brain homogenates, described above. Nematodes were then plated onto NGM plates seeded with OP50 E. coli and kept at 20 °C. To avoid overlapping generations, live worms were transferred daily to fresh NGM plates seeded with OP50 E. coli without fluorodeoxyuridine until they were all dead. Nematodes were scored as dead when there was no touch-provoked movement. The first day of adulthood is day 1 in survival curves.
Statistical analysis. The data were analyzed using GraphPad Prism 8.0 software (CA, USA) by Student’s t-test, one-way or two-way ANOVA and Bonferroni’s or Tukey’s post hoc test. The median survivals were determined using the same software. A p value < 0.05 was considered significant.