Genetic background modifies neurodegeneration and neuroinflammation driven by misfolded human tau protein in rat model of tauopathy: implication for immunomodulatory approach to Alzheimer's disease

SHR 72 transgenic rats (referred to as SHR TG) were generated to over-express truncated tau protein under the control of the mouse Thy-1 promoter, as described previously [19]. The WKY 72 transgenic line (WKY TG) was developed by back-crossing the SHR 72 line to the WKY genetic background. This study was performed on the F5 generation.

Rats were born and bred in our animal facility, and housed in standard laboratory conditions in plastic cages (555 × 345 × 195 mm, 5 rats per cage) in a temperature and humidity-controlled environment with a 12/12 hour light/dark cycle and with food and water available ad libitum. Efforts were made to minimize the number of animals utilized. All experiments were performed in accordance to the Slovak and European Community Guidelines, with the approval of the Institute's Ethical Committee and State Veterinary and Food Administration of the Slovak Republic.

Systolic blood pressure was measured using indirect tail-cuff method (LE 5002 Storage pressure meter, BIOSEB) on the tail arteries of 5 animals of each transgenic and non-transgenic group at 6 months of age.

Four-month-old animals, SHR TG males (N = 4) and WKY TG males (N = 4) were used for quantitative western blot analysis. Frozen brain stems of the studied rats were homogenized in 10 volumes of ice-cold extraction buffer [in mm: Tris, 20, pH 7.4; NaCl, 150; ethylenediaminetetraacetic acid (EDTA), 1; dithiothreitol, 1; 0.5% Triton X-100; Na₃VO₄, 1; NaF, 20; supplemented with protease inhibitors Complete^® without EDTA (Roche Diagnostics GmbH, Mannheim, Germany) using an OMNI TH tissue homogenizer (OMNI International, Marietta, GA, USA)]. After incubation on ice for 5 min the homogenates were cleared by centrifugation at 20 000 g for 20 min at 4°C. The supernatants were collected and the total protein concentration was determined using the BioRad protein Assay (BioRad Laboratories GmbH, Munich, Germany). The supernatants were then mixed with equal volume of 2 × sodium dodecyl sulphate (SDS) sample loading buffer [23] and heated at 95°C for 5 min. Ten micrograms of total protein was separated by electrophoresis in 12% SDS-polyacrylamide gels and then transferred to nitrocellulose membrane in 10 mm N-cyclohexyl-3-aminopropanesulphonic acid (CAPS; pH 12). After the transfer, the membranes were stained with Ponceau S to verify the uniform transfer of the proteins and then incubated with cell culture supernatant (1:5) of Hybridoma-producing pan-tau monoclonal antibody DC25 recognizing residues 347-354 (Axon Neuroscience, Vienna, Austria), followed by polyclonal goat anti-mouse IgG, horseradish peroxidase-conjugated (1:10 000; Dako, Glostrup, Denmark). The blots were developed using Super- Signal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL, USA) and detected with LAS3000 imaging system (FUJI Photo Film Co., Ltd, Tokyo, Japan). The band intensities were quantified using aida (Advanced Image Data Analyser software; Raytest, Straubenhardt, Germany).

To demonstrate the mature neurofibrillary pathology in neurons, Gallyas silver iodide staining was performed [24]. Sections were examined with an Olympus BX51 microscope and microphotographs were created with Olympus camera DP-50.

This method was used for the identification of the cell surface markers sensitive to paraformaldehyde fixation. Rats (5 animals per group, transgenic rats in the terminal stage of phenotype with age-matched non-transgenic controls) were deeply anaesthetised with ketamine-xylasine and perfused intracardially with phosphate-buffered saline (PBS) for 2 min using a peristaltic pump. After perfusion, the brainstem was removed and embedded without fixation in cryostat embedding medium (Leica) in aluminium foil vessel, and frozen above the surface of liquid nitrogen until the medium solidified. The sample was left for 1 hour on dry ice, and then stored at -70°C until used. 10-μm-thick sections were cut on a cryomicrotome (Leica CM 1850), affixed onto poly-L-lysine-coated slides, and left to dry at room temperature for 1 hour. Sections were fixed for 10 min in a 80% acetone:20% ethanol solution. Immunostaining was performed using the standard avidin-biotin-peroxidase method (Vectastain ABC kit, Vector Laboratories, USA). After 20 min in 1% H₂O₂ and 1 hour blocking in 5% bovine serum albumin (Sigma), sections were incubated with primary antibodies OX-42 (CD11b/CD18) and W3/25 (anti-CD4) (Serotec, UK) overnight at 4°C. After washing, the sections were incubated 1 hour in biotinylated secondary antibody (Vectastain ABC kit). The reaction product was visualized using avidin-biotin and Vector VIP as chromogen (Vector).

This method was used to prepare material for immunohistochemistry of markers tolerant to paraformaldehyde fixation as ED-1 (anti-CD68, Serotec), DC11 or MN423 (Axon Neuroscience) and for stereological analysis. Rats were deeply anaesthetised with ketamine-xylasine and perfused intracardially using a peristaltic pump: for 1 min with phosphate-buffered saline (PBS) and for 7 min with 4% paraformaldehyde in PBS, pH 7.2 (4% PFA). The brain was post-fixed overnight in 4% PFA, cryoprotected with 15% and 25% sucrose solutions (subsequently overnight), frozen in 2-methylbutane (30 seconds at -42°C) and transferred to dry ice. Sagittal brainstem sections (40 μm thick) were cut on a Leica CM1850 cryomicrotome.

For stereological analysis, every 13th section was included in the series (section sampling fraction = 1/13, total 17-19 sections per animal). Tissue sections were incubated with primary antibodies AT8 (Pierce Endogen), OX-6 (anti-MHCII, Serotec), anti-Iba1 (WAKO, USA), anti-NeuN antibody (Chemicon, USA) or anti- GFAP (Dako, Belgium) overnight at 4°C. Sections were immunostained using the standard avidin-biotin-peroxidase method (Vectastain ABC kit) with VIP as chromogen and 5 minute counterstaining of cell nuclei by methyl green (Vector).

For stereological quantification, male rats of transgenic lines SHR TG (N = 5) and WKY TG (N = 5), in the terminal stage of transgenic phenotype development, were used along with their non-transgenic littermates as age-matched controls (SHR crl, N = 4 and WKY crl, N = 6). The numbers of Iba1-positive microglia, MHCII-positive microglia, neurofibrillary tangles, neurons and astrocytes were quantified by stereology in brainstem (pons and medulla oblongata). This region of interest was chosen because it is highly affected by the neurofibrillary pathology in the brain of these tau transgenic rats.

The study was accomplished using an optical fractionator approach on a modified light microscope (Olympus BX51) equipped with a computer-based stereological system (StereoInvestigator; MicroBrightField, Williston, VT, USA). The region of interest was selected at low magnification (objective 4 × UPlanFI), and counting was performed at high magnification using an objective with a high numerical aperture (60 × oil immersion objective, Olympus, UPlanFI, NA = 1.25) and oil condenser (Olympus, UAAC, PlanApo, NA = 1.40). Parameters of the stereological analysis (disector base, sampling grid) were chosen according to density of the particular structure (e.g. NFT, microglia) in the tissue - sparse NFTs required a denser grid than did frequent microglia to ensure precise analysis with appropriately low coefficient of error. Detailed parameters of the stereological analysis are described in Table 1.

All data sets are expressed as mean ± standard error of the mean (SEM). Results were examined using an unpaired Student's t-test (with Welch's correction in case of uneven variances) and two-way ANOVA using Prism GraphPad Software Version 4.03 (Graph Pad Software, Inc., USA). Differences were considered to be statistically significant if p < 0.05.

The SHR TG line was back-crossed to the WKY genetic background. WKY TG rats in the F5 generation showed typical WKY habitus (Figure 1A, C) characterized by elongated skull shape and increased body size and body weight compared to SHR rats (Figure 1B, D). At 24 weeks, the mean body weight was 425 g ± 18 in WKY TG rats, 501 g ± 14 in WKY controls, 295 g ± 8 in SHR TG rats and 337 g ± 7 in SHR non-transgenic littermates. While SHR TG and SHR control rats suffered chronic hypertension, WKY TG remained normotensive as did WKY control rats (Figure 1E). Transgene expression, measured as human misfolded tau to rat endogenous tau ratio, was equivalent in both transgenic lines (Figure 1F) (Student's t-test with Welch's correction, t = 0.81, df = 3, p = 0.48).

Argyrophilic neurofibrillary tangles (NFTs) and axonal degeneration were prominent in the brainstem and spinal cord of transgenic SHR and WKY rats. Neurofibrillary pathology was detected by Gallyas silver staining (Figure 2A, B) and immunolabeled with monoclonal antibody AT8 that recognizes tau protein phosphorylated at Ser202 and Thr205 (Figure 2C-F). Immunostaining with antibodies that label conformationally modified tau (DC11, MN423) did not reveal significant differences between transgenic rat lines (data not shown). Despite similar transgene expression levels in the brainstems of both lines, stereological analysis of terminal-staged transgenic rats revealed a significantly lower number of AT8-positive NFTs in the brainstem of WKY TG line than in SHR TG (Student's t-test, t = 2.67, df = 8, p < 0.05) (Figure 2G, H). The difference was approximately 1,6-fold.

In brain areas harbouring neurofibrillary pathology and axonopathy, extensive inflammation mediated by reactive microglia was observed. In both transgenic lines, immunohistochemical staining showed activated morphology of microglia (thick shortened processes, macrophage-like morphology, forming of cell clusters) and increased microglial expression of activation markers: CD4, CD11b/CD18 and CD68 (Figure 3). Stereological quantification of cells stained by the general microglial marker Iba1 was performed in both transgenic lines and age-matched controls (Figure 4). The terminal stage of the transgenic phenotype was characterized by a two-fold increase in Iba1-positive microglia compared to control WKY or SHR rats (Figure 4G). Two way ANOVA showed that the transgenic factor creates approximately 74.5% of variability in microglial counts (F = 73.08, p < 0.0001), while the factor of genetic background demonstrates only a non-significant trend towards increased number of Iba1-positive microglia in SHR TG rats and control rats compared to WKY TG and WKY controls (F = 4.198, p = 0.057). The results were not significantly influenced by the interaction of examined factors (F = 2.06, p = 0.17). In the stereological quantification of the Iba1-positive cells, phagocytic morphology (Figure 4E, F - highlighted by arrows) was likewise considered. Macrophage-like structures were frequently observed in transgenic brainstems, but occasionally seen in control rats. The average ratio of phagocytic morphology observed among Iba1-positive cells was 8.9% in WKY TG rats, 14.5% in SHR TG, 0.8% in WKY controls and 1.5% in SHR control rats. Phagocytic morphology followed a similar but more pronounced pattern as stereological quantification of the number of the Iba1-positive microglia, and showed significant differences between WKY TG and SHR TG rats (Figure 4H). In two-way ANOVA analysis, the transgenic factor accounted for 69% of variability in the number of macrophage-like structures (F = 106.7, p < 0.0001). However, the factor of genetic background was responsible for 9.85% of the variability (F = 15.13, p = 0.001). Interaction (F = 12.17, p = 0.003) also influenced the result significantly, indicating that genetic background did not have the same effect on the number of macrophage-like structures in transgenic and control rats.

Brainstems of transgenic rats were stained for MHC class II antigens - RT1B (Figure 5.). Quantification of non-transgenic rats was not undertaken due to lack of MHCII-positive cells in several control brainstems. A striking difference in the number of MHCII-immunoreactive microglia (Student's t-test, t = 8.2, df = 8, p < 0.0001) was detected between the genetic backgrounds, showing different types of microglial activation. In WKY TG, 23.2% of total (Iba1-positive) brainstem microglia expressed MHCII, while in SHR TG rats, only 1.6% of total microglia were MHCII-positive, respectively.

Besides microglial activation, the reaction of astrocytes was observed and quantified (Figure 6). Independently from the genetic background, an elevated number of GFAP-positive astrocytes were counted in both lines of transgenic rats (SHR TG and WKY TG) in comparison with non-transgenic age-matched controls. Two-way ANOVA revealed a significant role of the transgenic factor impacting 71.53% of variability (F = 71.53, p < 0.0001), while genetic background (F = 0.1, p = 0.80) or interaction (F = 0.71, p = 0.51) did not influence the number of GFAP-positive astrocytes.