Inhibition of Src kinase activity attenuates amyloid associated microgliosis in a murine model of Alzheimer’s disease

Anti-Aβ, clones 6E10 and 4G8 were from Covance (Emeryville, CA, USA). The anti-Lyn antibody, anti-Src, anti-α-tubulin antibodies, FITC and Texas Red conjugated secondary antibodies, and horseradish peroxidase conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse TNF-α Elisa kit was obtained from R&D Systems (Minneapolis, MN, USA). Anti-phosphotyrosine (4G10) antibody was from EMD Millipore (Billerica, MA, USA) and anti-pLyn (Tyr 396) antibody was purchased from Abcam (Cambridge, MA, USA). Anti-Iba1 antibody was from Wako Chemicals USA, Inc (Richmond, VA, USA). Elite Vectastain ABC avidin and biotin and alkaline phosphatase kits, biotinylated anti-rabbit, anti-mouse, and anti-rat antibodies and the Vector VIP and Vector Blue chromogen kits were from Vector Laboratories Inc. (Burlingame, CA, USA). Anti-CD68 was obtained from Serotec (Raleigh, NC, USA). Anti-PSD95 and anti-pSrc (Tyr416) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-APP antibody was purchased from Invitrogen (Camarillo, CA, USA). Synaptophysin and βIII tubulin antibodies were purchased from Chemicon International, Inc (Temecula, CA, USA). The non-receptor tyrosine kinase inhibitor, dasatinib, was obtained from LC Laboratories (Woburn, MA, USA). The transgenic mouse line, strain 005864 B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J and wild type mouse line, C57BL/6 were obtained from the Jackson Laboratory (Bar Habor, ME, USA).

All animal use was approved by the University of North Dakota Institutional Animal Care and Use Committee (UND IACUC). Mice were provided food and water ad libitum and housed in a 12 h light:dark cycle. The investigation conforms to the National Research Council of the National Academies Guide for the Care and Use of Laboratory Animals (8^th edition).

Immortalized murine microglial BV2 cells were obtained from Dr. Gary E. Landreth, Cleveland, OH, USA. The cells were maintained at 3 × 10⁶ cells/dish in 100-mm dishes in DMEM/F12 (Gibco RBL, Rockville, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (U.S. Biotechnologies Inc., Parkerford, PA, USA), 5% horse serum (Equitech-Bio, Inc., Kerrville, TX, USA), penicillin G (100 units/ml), streptomycin (100 mg/ml), and L-glutamine (2 mM) and incubated at 37°C in a humidified atmosphere containing 5% CO₂ and 95% air.

Microglia were derived, as described previously [53], from the brains of postnatal day 1 to 3 C57BL/6 J mice. Briefly, meninges-free cortices were removed, trypsinized and triturated in microglia media (DMEM/F12 media containing L-Glutamine (Invitrogen, Carlsbad, CA, USA) and 20% heat inactivated FBS) and placed in T-75 flasks. The media in the flasks was replaced completely after 24 h and partially after 7 days with fresh media. The cells were ready to harvest and count after 14 days.

Aβ 1–42 fibrils (American Peptide, Sunnyvale, CA, USA) were prepared according to an established protocol [54]. Microglia were stimulated by removing them from growth media into serum-free DMEM/F12 media containing Aβ fibrils. To assess dasatinib effects, BV2 or microglia were pretreated with the drug for 30 minutes before Aβ stimulations. For Western blot analyses, five- minute Aβ stimulations were performed and cells were lysed using radioimmunoprecipitation assay buffer (RIPA) (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM Na3VO4 10 mM NaF, 1 mM EDTA, 1 mM EGTA, 0.2 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 0.1% SDS, and 0.5% deoxycholate) with protease inhibitors (AEBSF 104 mM, Aprotinin 0.08 mM, Leupeptin 2.1 mM, Bestatin 3.6 mM, Pepstatin A 1.5 mM, E-64 mM). Protein concentrations were determined using the Bradford method [55]. For ELISA and toxicity analyses, cells were stimulated with Aβ for 24 hours. To assess the level of proinflammatory cytokine, TNF-α, secreted after 24 h stimulation of microglia with Aβ fibril, the media from the cells was collected and analyzed using a commercially available ELISA kits (R & D Systems) according to the manufacturer’s protocol. Cell viability was assessed by the MTT reduction assay. After 24-hour stimulation of microglia, media was removed for ELISA and the cells were incubated with 3[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide (MTT, 100 μg/mL) for 4 hours. The media was aspirated and the reduced formazan precipitate was dissolved in isopropanol. Absorbance values were read at 560/650 nm via plate reader and averaged +/− SEM.

Lysates from cell stimulation experiments were diluted into sample buffer and separated via 10% SDS-PAGE, transferred to polyvinylidene difluoride membrane and Western blotted using anti-pSrc (Tyr416), anti-pLyn (Tyr396), Src (loading control) and Lyn (loading control) and enhanced chemiluminescence for detection (GE Healthcare, Piscataway, NJ, USA). pSrc/pLyn optical densities (O.D.) from visualized Western blots were normalized to their respective loading controls (Src/Lyn) and averaged from five independent experiments.

Brains from different aged APP/PS1 mice were collected for longitudinal analyses. 2-, 4-, 6- and 12-month old transgenic mice (n = 5 to 6) along with their age-matched C57BL/six wild type controls were euthanized and perfused with PBS-CaCl₂. Brains were rapidly dissected and divided into left and right hemispheres, with right hemispheres fixed in 4% paraformaldehyde for sectioning. The left hemispheres were further dissected into different brain regions to obtain hippocampus, temporal cortex, frontal cortex and cerebellum, and flash frozen using liquid N₂. The frozen tissue was lysed using RIPA with protease inhibitors and used for Western blot analysis.

Dasatinib was infused subcutaneously into female APP/PS1 mouse at 13 months of age. Dasatinib was delivered via mini-osmotic pumps (model 1004, 0.25 μL/hour delivery rate, Alzet, Cupertino, CA, USA). Pumps delivered either vehicle (DMSO/Hepes) (n = 6) or dasatinib (500 ng/kg/day) (n = 7) for 28 days. At the end of the infusion period, mice were euthanized, brains perfused with PBS-CaCl₂ and rapidly collected. Control untreated APP/PS1 animals were collected at a comparable age of completion, 14 months. The right hemispheres were collected for fixing in 4% paraformaldehyde and the left hemispheres were flash frozen in liquid nitrogen for biochemical analysis.

The paraformaldehyde fixed right hemispheres for different age mice or from dasatinib treated and control mice were cut using a freezing microtome. Briefly, paraformaldehyde fixed tissue was embedded in a 15% gelatin (in 0.1 M phosphate buffer) matrix and immersed in a 4% paraformaldehyde solution for two days to harden the gelatin matrix. The blocks were then cryoprotected through three cycles of 30% sucrose for three to four days each. The blocks were then flash frozen using dry ice/isomethylpentane, and serial 40 μm sections were cut using a freezing microtome. Serial sections were used for immunostaining with anti- pTyr (4G10) antibody at a dilution of 1:1,000, anti-Aβ (4G8) and anti-CD68 at a dilution of 1:500 to 1:1,000, anti-pSrc as 1:250, anti-Iba1 at 1:1,000, and anti-GFAP antibody at a dilution of 1:1,000, followed by incubation with biotinylated secondary antibodies (1:2,000 dilution) (Vector Laboratories Inc.) and avidin/biotin solution (Vector ABC kit). The immunoreactivity was observed using Vector VIP as chromogen. For pSrc/CD68 double staining, FITC and Texas Red conjugated secondary antibodies were used. For phosphotyrosine/CD68 or Iba1 double-labeling, the sections were first immunostained with anti-phosphotyrosine antibody 4G10 using Vector VIP as the chromogen. The tissue was then incubated in 0.2 N HCl to strip off antibodies then the tissue was double-labeled with either CD68 or Iba1 using Vector Blue as the chromogen. The slides were dehydrated and cover slipped using VectaMount (Vector Laboratories, Inc.) following a standard dehydrating procedure through a series of ethanol solutions and Histo-Clear (National Diagnostics, Atlanta, GA, USA). Images were taken using an upright Leica DM1000 microscope and Leica DF320 digital camera system (Leica Microsystems Inc., Buffalo Grove, IL, USA). Figures were made using Adobe Photoshop 7.0 software (Adobe Systems, San Jose, CA). For quantitation purposes, 1.25X images were taken from three consecutive serial sections, (960 μm apart) throughout the hippocampal region. Optical densities from the temporal cortex or CA1 regions from the same serial sections were measured using Adobe Photoshop software. All sections were immunostained simultaneously to minimize variability and background values in an unstained area of tissue for each section were set to zero using the curve tool before quantifying optical density values. The optical density of the entire temporal cortex region/CA1 region from a representative section was selected via marquee and the same size marquee was applied to all sections per condition to allow comparison of optical densities (O.D.) independent of area changes. The values for each section were averaged (three sections/brain, five to seven brains per condition) and plotted for Aβ, immunoreactivity for dasatinib infusion animals, pSrc immunoreactivities of dastinib treated mice, and Aβ and CD68 immunoreactivities for longitudinal study animals. For quantitating phospho-tyrosine immunostaining from different aged APP/PS1 and wild type mice, the serial sections were viewed under a microscope and the number of 4G10 positive plaques were viewed from the entire CA1 and temporal cortex regions for all the animals in each condition. The numbers of plaques were averaged (three sections/brain, five to seven brains per condition) and plotted. To insure reliability in comparison, only immunostains that were processed together were quantitatively compared to minimize any variability in staining processing from day-to-day. This allows an accurate assessment of relative comparisons within parallel processed conditions and samples although not necessarily a reflection of absolute values.

Hippocampus and temporal cortex were removed from flash frozen brains of treated mice, lysed, sonicated in RIPA buffer and quantitated using the Bradford method [55]. The lysates were resolved using a custom-built 28-well comb and 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes for Western blotting using anti-pTyr (4G10), anti-APP, anti-Aβ (6E10), anti-GFAP, anti-TNF-α and anti-CD68 antibodies with α-tubulin as their loading control, anti-pSrc (Tyr416), anti-pLyn (Tyr396) antibodies with anti-Src and anti-Lyn as their respective loading controls and anti-PSD95 and anti-synaptophysin with βIII-tubulin as the loading control. Antibody binding was detected using enhanced chemiluminescence for detection. Western blots were quantified using Adobe Photoshop software. Optical density (O.D.) of bands were normalized against their respective loading controls and averaged (+/−SEM).

T-maze analysis was performed as described by Wenk, 1998 [56]. Briefly, upon completion of the in vivo infusion period, control and treated mice were placed into the starting arm, and the door was raised to allow animals to walk down the stem and choose an arm. Once the mice entered an arm with all four feet, they were returned to the starting arm and the door was closed. After 30 sec, the door was opened and the mice were allowed to choose an arm again. The process was repeated for nine trials with a 30 sec interval between each trial. The choice of arms was noted each time and the number of alternations between trials for each mouse was averaged and plotted.

Data are presented as mean +/− standard deviation. Values statistically different from controls were determined using one-way ANOVA or Student t-test when appropriate. The Tukey-Kramer multiple comparisons post-hoc test was used to determine P-values.

Based upon our prior work demonstrating that both fibrillar and oligomeric Aβ stimulate increased non-receptor tyrosine kinase activity in microglia [38], we determined whether a brain penetrant tyrosine kinase inhibitor, dasatinib, could attenuate activity of Src kinase in vitro. In order to validate the Src-kinase inhibitory ability of dasatinib, the mouse microglia cell line, BV2, was treated with varying concentrations of the drug. Dasatinib is a small molecule ATP-competitive inhibitor of Bcr-Abl and Src kinase with IC50s for the isolated kinases of 0.55 and 3.0 nM, respectively [57,58]. FDA-approved for use in cases of chronic phase Philadelphia chromosome-positive chronic myelogenous leukemia (CP-CML), dasatinib (Sprycel, Bristol-Myers Squibb, Princeton, NJ, USA) has been shown to cross the blood-brain barrier [59]. Although dasatinib will certainly attenuate c-Abl activity, based upon limited demonstration of c-Abl in microglia [60] and our prior work demonstrating robust Aβ-stimulated increase in Src family kinase activity [38,41], we expected a major target of dasatinib inhibition to be the Src family of non-receptor tyrosine kinases. As expected, active, phosphorylated Src levels significantly decreased with increasing dasatinib concentrations from 1nM to up 1 μM compared to vehicle treated BV2 cells (Figure 1). For subsequent treatments of microglial cells in vitro, a dose of 100 nM dasatinib was chosen to obtain an optimum effect of Aβ-dependent tyrosine kinase inhibition in primary cultures.

In order to determine whether dasatinib was able to reduce an Aβ stimulated increase in active tyrosine kinase levels, primary microglia cultures were treated with Aβ fibril with or without dasatinib. Microglial cells were pretreated with 100 nM dasatinib followed by a five-minute stimulation by 10 μM Aβ_f to detect changes in the activated, phosphorylated forms of the specific non-receptor tyrosine kinases, Src and Lyn. As expected, Aβ fibrils stimulated an increase in active phospho-Src levels compared to vehicle treated cells (Figure 1). The Aβ-dependent increase in phospho-Src levels was significantly attenuated when cells where stimulated in the presence of dasatinib (Figure 1). Although Aβ fibrils did not significantly increase the levels of active, phosphorylated Lyn kinase at the concentrations used, dasatinib was still able to significantly reduce levels of active phospho-Lyn indicating that the drug was not specific to any particular Src family member (Figure 1). In addition, the data demonstrated that the Aβ stimulated increase in microglial active Src kinase levels could be inhibited using a clinically relevant non-receptor tyrosine kinase Src/Abl inhibitor, dasatinib.

In order to determine whether the Aβ-stimulated change in active Src levels was required for changes in phenotype, changes in secretion of the pro-inflammatory cytokine, TNF-α, were quantified from the primary microglia cultures in the presence or absence of dasatinib. Fibrillar Aβ stimulated a significant increase in TNF-α secretion compared to control or vehicle treated microglial cells (Figure 2). This increase in TNF-α secretion was attenuated by 100nM pretreatment of dasatinib with no effect of cellular viability (Figure 2). The data demonstrated that inhibition of Src family kinase activity was sufficient to prevent microglia from acquiring a reactive secretory phenotype upon Aβ fibril stimulation in vitro.

To determine an appropriate age in vivo for examining increased tyrosine kinase activity and Aβ-associated microgliosis, a transgenic APP/PS1 mouse model of AD was used. Varying age brains of APP/PS1 mice were compared to age-matched controls to validate the use of this transgenic mouse model for our study. The APP/PS1 mice were collected at 2, 4, 6 and 12 months of age along with their age-matched wild type mice (C57BL/6) and brains were sectioned for histology. Immunostaining demonstrated a significant increase in APP/PS1 temporal cortex CD68 immunoreactivity in 6- and 12-month old mice compared to earlier 2- and 4-month APP/PS1 age groups and their age-matched C57BL/6 controls (Figure 3). This increase correlated precisely with increased plaque-associated Aβ immunoreactivity in the 6- and 12-month old APP/PS1 mice (Figure 3). Analyses of hippocampi from all groups showed similar results to the temporal cortex (data not shown). These data supported the notion that microglial activation in AD and its mouse models may be a consequence of Aβ fibril interaction. However, to validate an involvement of tyrosine kinase activity in microglial phenotype changes during disease, APP/PS1 brains were next immunostained with anti-phosphotyrosine antibody as an indirect method to assess overall tyrosine kinase activity changes. Immunostaining mouse brains with anti-phosphotyrosine antibody, 4G10, demonstrated positive microglial-like as well as vascular immunoreactivity (Figure 4). Microglial-like immunoreactivity increased in intensity demonstrating transition from ramified to a more compact, plaque-clustered morphology with increasing age in APP/PS1 mice compared to controls (Figure 4). Counting plaque-associated, 4G10 positive staining as putative microglia demonstrated a trend of age-associated increase in APP/PS1 temporal cortex staining with a significant increase by 4 months of age with significantly higher immunoreactivity at 6 and 12 months (Figure 4). Similar trends were observed in the hippocampus (data not shown). To validate that phosphotyrosine immunoreactivity partially co-localized to microglia, sections were double-labeled with both antiphosphotyrosine antibody and anti-CD68 or anti-Iba1 antibodies. Double-labeling demonstrated that a portion of the phosphotyrosine immunoreactivity demonstrated clear co-localization with either microglial marker, CD68 or Iba1 (Figure 4). These data confirmed that by 12 months of age, this particular transgenic line had robust phosphotyrosine microglial reactivity in association with Aβ plaques providing us with the appropriate time point for use.

Based upon the temporal profiling of microgliosis we next determined whether inhibition of tyrosine kinase activity could attenuate the microglial reactivity in this line. Based upon our in vitro data we hypothesized that dasatinib treatment would attenuate the Aβ-associated increase in tyrosine kinase activity, particularly Src, and subsequent inflammatory changes in the mice. In order to answer this question, 13 month old APP/PS1 female mice were subcutaneously infused with dasatinib or vehicle for 28 days. Dasatinib infusion significantly decreased protein phosphotyrosine levels in the hippocampus but not the temporal cortex compared to vehicle infused control animals (Figure 5). More importantly, dasatinib infusion significantly decreased levels of active, phospho-Src in both temporal cortex and hippocampus compared to control mice (Figure 5). However, dasatinib infusion had no effect on the active, phosphorylated levels of the related Src family member, Lyn kinase, demonstrating some specificity of the drug (Figure 5). These data demonstrated that a subcutaneous route of dasatinib delivery was able to inhibit levels of active Src in the brains of the APP/PS1 mice.

Based upon the encouraging findings that dasatinib was able to exert brain effects and decrease active Src levels, we expected that dasatinib infusion should also attenuate microgliosis and TNFα secretion as observed in our in vitro findings. Dasatinib infusion significantly decreased TNFα levels in both the hippocampus and temporal cortex correlating with the decrease in active Src levels and demonstrating a clear brain anti-inflammatory effect of the drug (Figure 6). Moreover, levels of CD68, the reactive microglial marker protein, were significantly decreased in the hippocampus of dasatinib infused brains but not the temporal cortex. These data demonstrated that dasatinib inhibition of brain active Src levels correlates with a significant anti-inflammatory, microglial-inhibitory effect in APP/PS1 mice. Interestingly, these effects appeared most robust in the hippocampus compared to the temporal cortex (Figure 6).

To examine the breadth of changes that might result from dasatinib infusion we also determined whether adverse effects on neurons, astrocytes, or Aβ deposition resulted from drug treatment in addition to the anti-inflammatory, microglial-inhibitory changes. However, dasatinib administration had no effect on either total APP levels or Aβ levels in the hippocampus or temporal cortex of treated mice compared to vehicle controls (Figure 7). In addition, dasatinib treatments did not increase levels of the reactive astrocyte marker protein, GFAP, in the infused animals compared to vehicle control mice (Figure 7). Finally, dasatinib treatment did not have any significant effects on levels of either presynaptic, synaptophysin, or post-synaptic, PSD 95, proteins in either the temporal cortex or hippocampus compared to vehicle treated mice (Figure 7). These data demonstrated that the Src inhibitory, anti-inflammatory, microglia-inhibitory effects of dasatinib treatment did not correlate with any robust adverse effects such as increased astrogliosis, neuron or synaptic loss, and increased Aβ deposition.

To provide qualitative assessments along with the quantitative Western blot analyses, select immunohistochemical studies were also performed from the treated animals. As expected, protein phosphotyrosine immunoreactivity in dasatinib infused mouse brains appeared dramatically less than vehicle treated mice in both the hippocampus and temporal cortex (Figure 8). A similar trend of dasatinib decreased immunoreactivity was observed for active phospho-Src versus vehicle controls (Figure 8). Similar to phosphotyrosine, pSrc immunoreactivity also localized to vasculature as well as microglial-like immunoreactivity (Figure 8). Double-labelling verified that a portion of the pSrc immunoreactivity co-localized to microglia (Figure 8). Because the majority of pSrc immunoreactivity was observed in plaque-associated microglial-like cells we suggest that the significant decreases in active Src and phosphotyrosine levels observed by Western blot analyses were a reflection of microglial changes due to the drug. However, certainly a portion of both phosphotyrosine and pSrc level changes detected by the Western blot analyses is due to contributions from cells other than microglia. Also as predicted, levels of CD68 reactive microglial immunoreactivity in dasatinib infused mice was noticeably decreased compared to vehicle treated mice in both the hippocampus and temporal cortex (Figure 8). Consistent with the Western blot analysis, GFAP immunoreactivity for reactive astrocytes was not visually different between dasatinib and vehicle infused mice in either temporal cortex or hippocampus (Figure 8). The qualitative immunostaining findings corroborated the quantitative Western blot analyses and suggest that the significant decreases in pSrc levels were largely due to decreased microglial immunoreactivity, our intended target for the drug.

One possible consequence of attenuated tyrosine kinase activity in microglia could be inhibition of phagocytic ability. It is well known, for instance, that tyrosine kinase activity can affect macrophage phagocytic ability [61]‐[63]. Therefore, inhibition of Src or related kinase activity might attenuate any ability microglia have to clear Aβ deposits in the brain producing an unwanted side-effect of increased plaque load. Based upon the Western blot analysis, dasatinib treatment had no effect on altering APP or Aβ levels in the brains of mice (Figure 8). However, to better assess whether the drug had any effects on insoluble fibrillar deposits in the brain, quantitative immunostaining of Aβ plaques was performed. Dasatinib treatment had no effect on plaque load in treated mice compared to vehicle controls in either the hippocampus or temporal cortex (Figure 9). These data demonstrate that the microglial-inhibitory effects of dasatinib do not result in unwanted effects of increasing Aβ deposition or accumulation.

Although there was no effect of dasatinib treatment on Aβ deposition, the clear and somewhat specific effect of Src inhibition correlating with anti-inflammatory, microglial-inhibitory effects provided an opportunity to assess whether dasatinib could also provide cognitive improvement to the mice. After treatments, spontaneous alternations from dasatinib treated, vehicle treated and control animals were quantified during T maze analyses. This particular model of spatial memory was selected based upon its sensitivity of detection of cognitive deficit in AD mouse models [64] and its preference for detecting changes in hippocampal-based performance differences [65,66] since our most significant effects of drug were observed in the hippocampus. Dasatinib infused animals demonstrated improved cognitive performance represented by a significant increase in spontaneous alternations compared to vehicle treated mice (Figure 10). These data validated that subcutaneous administration of the Src/Abl tyrosine kinase inhibitor, dasatinib, is able to provide cognitive enhancing effects to APP/PS1 mice while attenuating microglial activation and proinflammatory cytokine, TNFα, levels in the brain.