Animals
Tg PS1/APPsw, Tg APPsw, Tg Tau P301S and wild-type mice were generated and maintained in a C57BL/6 genetic background as previously described [28]. All mice were maintained under specific pathogen free conditions in ventilated racks in the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) accredited vivarium of the Roskamp Institute. All experiments involving mice were reviewed and approved by the Institutional Animal Care and Use Committee of the Roskamp Institute before implementation and were conducted in compliance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Tissue processing
All mice were humanely euthanatized and their brains were collected and fixed in 4% paraformaldehyde (PFA) for 48 h. The method of euthanasia used follow the AVMA (American Veterinary Medical Association) guidelines for the euthanasia of animals. Briefly, mice were rendered unconscious through inhalation of 5% isoflurane in oxygen using a vaporizer and a gas chamber. While under anesthesia, after verifying the absence of reflexes, mice were euthanatized by exsanguination (blood was withdrawn from cardiac puncture).
Subsequently, the hemispheres were processed in a Sakura Tissue-Tek VIP (Leica Biosystems Inc., IL, USA) vacuum infiltration processor. Brains were then embedded in paraffin with the Sakura Tissue-Tek (Leica Biosystems Inc., IL, USA) and stored at 4 °C for 2 days for subsequent cutting with a Leica RM2235 microtome (Leica Biosystems Inc., IL, USA). All brains were cut at a thickness of 12 μm. Sagittal slices were mounted on glass slides and dried for 48 h at 37 °C for subsequent immunofluorescence staining and confocal imaging.
Immunofluorescence
Paraffin sections were washed in two baths of histoclear (National Diagnostics, USA) and progressively rehydrated with ethanol gradients and phosphate buffered saline (PBS, Sigma Aldrich, MO, USA). Brain sections were subjected to antigen retrieval for 7 min in citric acid buffer (pH 6) at 100 °C. All sections were treated with 0.05% Sudan Black in 70% ethanol to quench autofluorescence. Sections were then blocked in PBS containing 10% donkey serum (Abcam, MA, USA) for 1 h. Sections were incubated in PBS containing 1% donkey serum and the respective panel of primary antibodies overnight at 4 °C. The following antibodies were used: CP13 (anti(α)-phospho-tau (pTau) S202, 1:200, Dr. Peter Davies’ Lab), MC1 (α-conformational tau, 1:200, Dr. Peter Davies’ Lab), TOC1 (1:200, Dr. Lester Binder’s Lab), PHF-1 (α-pTau S396/404, 1:200, Dr. Peter Davies’ Lab), 9G3 (α-pTau Y18, 1:200, MediMabs Inc., QC, Canada), DA9 (α-total-tau (tTau), 1:200, Dr. Peter Davies’ Lab), α-BACE1 (1:200 Cell Signaling, MA, USA), α-sAPPβ with Swedish mutation (1:100 Immuno-Biological Laboratories Co, Ltd., Japan), α-Iba1 (1:300, Abcam, MA, USA), α-GFAP (1:5000, Aves Labs, OR, USA), α-pSyk (Y525/526, 1:200, Cell Signaling, MA, USA). In addition to the α-pSyk (Y525/526, 1:200, Cell Signaling, MA, USA), we used the α-pSyk (Y525/526, 1:100, Abgent, CA, USA) and obtained similar results. After three washing steps in PBS for 5 min, sections were incubated in a solution containing PBS, 1% donkey serum and the respective panel of secondary antibodies for 1 h in the dark at room temperature in a humidified chamber. The following secondary antibodies were used: donkey α-rabbit, α-goat, α-mouse conjugated to Alexa 488, 568 and 647, respectively (1:500, Life technologies). After three washing steps in PBS for 5 min, sections were mounted in Fluoroshield with or without DAPI (Sigma Aldrich, MO, USA). All images were acquired using the confocal microscope LSM 800 (Carl Zeiss AG, Germany), the ZEN Blue 2.1 (Carl Zeiss AG, Germany) software and a 20× or 63× objective. The acquisition settings were kept the same for all genotypes within the same experiment.
For qualitative analysis of the pSyk burden in Tg PS1/APPsw and Tg APPsw mice compared to age-matched WT littermates (n = 6 for each genotype, equal amount of male and female), 116 ± 13.5 (avg. ± SEM) weeks of age were stained and analyzed as described above (Fig. 1).
For qualitative analysis of the pSyk burden in Tg Tau P301S mice compared to WT littermates, hippocampi and cortices of 16 male and female mice ranging from 8 to 56 weeks of age were stained and analyzed as described above.
For the quantitative analysis of the pSyk burden (Fig. 3), 140 randomly-selected microscopic fields of four non-consecutive brain slices (containing the hippocampus) from six animals per genotype (equal number of male and female) were acquired. The area covered with the pSyk immunopositive staining was quantified with Fiji [34] in microscopic fields containing Aβ plaques as well as in microscopic fields not containing Aβ deposits. The PS1/APPsw, APPsw and WT mice of the younger cohort were on average 45 ± 0.3 (avg. ± SEM) weeks old. The average age of the mice of the older cohort was 116 ± 13.5 weeks (±SEM). The pSyk burden of the transgenic mice was normalized to the level of pSyk burden quantified in wild-type littermates of the respective age-group. As a negative control, primary antibodies were omitted to determine background and autofluorescence (not shown).
For the quantitative analysis of the colocalization of pSyk and different tau epitopes (Fig. 8) between 400 and 570 cortical fields (50,000 μm2 per field) from four male Tg Tau P301S animals (average age 47 ± 3.1 (SEM) weeks) were analyzed for each tau epitope. To quantify the percentage of the immunopositive neurons a total of 2546 microscopic fields and 21,800 neurons were counted using the Zen Blue 2.1 software (Carl Zeiss AG, Germany).
The fluorescence intensities (Figs. 9, 10, 11, 12 and 13) of 30 to 40 neurons immunopositive for pSyk, pTau or both (colocalized) were determined for each tau epitope (total of 90 neurons per epitope) using Zen Blue 2.1 (Carl Zeiss AG, Germany). The male Tg Tau P301S mice (n = 4) used for quantification were on average 47 ± 3.1 weeks old (avg. ± SEM).
In addition, the different immunostainings mentioned above were performed on paraffin-embedded tissue sections (10 μm, dorsolateral frontal cortex) from a 67-year-old, male patient with AD (Braak VI) and a 102-year-old, male non-demented control that were provided by Dr. Ann McKee (Boston University, MA, USA). Institutional review board approval for brain donation was obtained through the Boston University Alzheimer’s Disease Center (BUADC, Boston, MA, USA).
Cell culture
SH-SY5Y cells were purchased from American Type Culture Collection (VA, USA). SH-SY5Y cells were grown in DMEM/F12 medium (Thermo Fisher Scientific, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, MA, USA), GlutaMAX and 1% penicillin/streptomycin/fungizone.
Generation of Syk overexpressing SH-SY5Y cells
A human cDNA ORF Clone of the human SYK gene (NM_003177, transcript variant 1) was purchased from OriGene Technologies (MD, USA). The cDNA fragment encoding human SYK was amplified by PCR using PfuUltra II Fusion HS DNA polymerase (Agilent Genomics, CA, USA) and subcloned into the p3xFLAG-Myc-CMV™-26 Expression Vector (Sigma-Aldrich, MO, USA) to generate the pCMV-SYK-Flag plasmid. The entire reading frame of the plasmid was confirmed by DNA sequencing. SH-SY5Y cells were maintained in advanced DMEM/F-12 medium supplemented with 10% fetal bovine serum, 1% GlutaMAX, 1% penicillin/streptomycin (Thermo Fisher Scientific, MA, USA) and incubated in a humidified 5% CO2 atmosphere at 37 °C. For stable transfection, SH-SY5Y cells were grown in 6-wells cell culture plates until reaching 70-80% confluence and transfected with 3 μg of empty pCMV vector (control cells) or pCMV-SYK-Flag plasmids per well using lipofectamine 2000 (Thermo Fisher Scientific, MA, USA). After 48 h, the medium surrounding transfected cells was replaced with fresh medium containing 0.2 mg/ml of G418 for selection. After 14 days of selection, G418 resistant cells were trypsinized and expanded. The expression efficiency of SYK was analyzed by Western blot using antibodies against SYK (4D10 Syk antibody, Santa Cruz Biotechnology, TX, USA) and the Flag tag (Sigma-Aldrich, MO, USA).
Immunoblotting
SH-SY5Y cells were cultured in 24-well-plates for 24 h and subsequently lysed with mammalian protein extraction reagent (MPER, Thermo Fisher Scientific, MA, USA) containing Halt protease & phosphatase single use inhibitor/EDTA (Thermo Fisher Scientific, MA, USA) and 1 mM PMSF. Proteins of cell lysates were separated by 10% tris-glycine-SDS-PAGE using 1 mm Criterion TGX gels (Bio-Rad Laboratories, CA, USA) and electro-transferred onto 0.2 μm PVDF membranes (Bio-Rad Laboratories, CA, USA). Membranes were blocked in TBS containing 5% non-fat dried milk for 1 h and were hybridized with the primary antibody (αSyk (4D10, 1:1000, Santa Cruz, TX, USA), αpTau S396/404 (PHF-1, 1:1000, Dr. Peter Davies’ Lab), αtTau (DA9, 1:1000, Dr. Peter Davies’ Lab), αpTau Y18 (9G3, 1:1000, MediMabs Inc., QC, Canada,) overnight at 4 °C. Subsequently, the membranes were incubated for 1 h in HRP-conjugated αmouse secondary antibody (1:1000, Cell Signaling, MA, USA). Western blots were visualized using chemiluminescence (Super Signal West Femto Maxium Sensitity Substrate, Thermo Fisher Scientific, MA, USA). Signals were quantified using ChemiDoc XRS (Bio-Rad Laboratories, CA, USA) and densitometric analyses were performed using Quantity One (Bio-Rad Laboratories, CA, USA) image analysis software.
Statistical analyses
The data were analyzed and plotted with GraphPad Prism (GraphPad Software, Inc., CA, USA). The Shapiro-Wilk test for normality was used to test for Gaussian distribution. Statistical significance was determined by either Kruskal-Wallis followed by Dunn’s post-hoc test or the non-parametric Mann–Whitney test. All data are presented as mean ± the standard error of the mean (SEM) and p < 0.05 was considered significant.