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In Alzheimer’s disease (AD), deposition of pathological tau and amyloid-β (Aβ) is thought to drive synaptic loss and cognitive decline. Major questions in the field center around possi-ble connections between the two pathologies and what molecular mechanisms can impact tau pathology progression. We aimed to determine, if different proteins reported to modify tau pathology could modify tau phosphorylation in vitro and tau spreading in vivo. Two kinases, Pyk2 (Ptk2b) and Fyn, are part of a signaling cascade activated by Aß oli-gomers binding to the cellular prion protein receptor (PrPC) and have also been reported to interact with GSK3ß, one of the best characterized tau kinases. We hypothesized that Aß might induce tau phosphorylation through PrpC-mGluR5-Fyn-Pyk2-GSK3ß signaling. We first studied the interactions of the three kinases and tau in different in vitro model systems, in-cluding HEK-293T over-expression system and iPSC-derived neurons. In HEK-293T cells, Pyk2 and Fyn worked in synergy to increase GSK3ß phosphorylation and subsequent tau phosphor-ylation. Furthermore, GSK3ß co-immunoprecipitated with Pyk2, and Fyn kinase inhibition reduced Fyn’s proximity to tau as well as reduced tau spreading in mouse cortical neuron cultures seeded with human tau. Thus, initial data supported a role for the three kinases in contributing to tau hyperphosphorylation and spreading. Contrary to these results, GSK3ß phosphorylation remained unchanged in human iPSC-derived neurons upon inhibition of Pyk2 and Fyn kinases. Surprisingly, tau phosphorylation on some epitopes was even increased up-on Pyk2 inhibition. In addition, iPSC-derived neurons were incubated either short- or long-term with synthetic Aßo. Upon long-term treatment, neurons showed decreases in synaptic density as expected. In contrast to expectations, short-term treatment with sAßo did not ac-tivate Pyk2 and Fyn kinases and showed no effect on GSK3ß, tau or other downstream tar-gets. To study modulation of tau spreading by different factors, misfolded tau aggregates ex-tracted from human AD brains were injected into WT and transgenic mice to drive templat-ed spreading of tau pathology. We assessed the impact of Aβ co-pathology, of deleting loci known to modify AD risk (Ptk2b, Grn, and Tmem106b) and of pharmacological intervention with a Fyn kinase inhibitor on tau spreading after injection of AD tau extracts. The density and spreading of tau inclusions triggered by human tau seed were unaltered in the hippo-campus and cortex of APPswe/PSEN1ΔE9 transgenic and AppNL-F/NL-F knock-in mice. In mice with human tau sequence replacing mouse tau, template matching enhanced neuritic tau burden. Human AD brain tau-enriched preparations contained aggregated Aβ, and the Aβ co-injection caused a redistribution of Aβ aggregates in mutant AD model mice. The injection-induced Aβ phenotype was spatially distinct from tau accumulation and could be ameliorat-ed by depleting Aβ from tau extracts. These data suggest that Aβ and tau pathologies prop-agate by largely independent mechanisms after their initial formation. Altering the activity of the Fyn and Pyk2 kinases involved in Aβ-oligomer–induced signaling, or deleting expres-sion of the progranulin and TMEM106B lysosomal proteins, did not alter the somatic tau in-clusion burden or spreading. However, mouse aging had a prominent effect to increase the accumulation of neuritic tau after injection of human AD tau seeds into WT mice. Overall, these results suggest that observations from over-expression non-neuronal model studies of Pyk2 and Fyn acting on GSK3ß and tau do not translate faithfully into neu-ronal model systems. Further, these studies refined our knowledge of factors (in-)capable of modulating tau spreading and lend evidence to the hypothesis of a more complex role of tau phosphorylation regulation by Fyn and Pyk2 in different model systems.

Additional file 1.

Additional file 1: Figure S1. Reduced Fyn Activation in Mice Fed AZD0530-Containing Food. (A) Representative blots using anti-pY416, Fyn, and ß-actin antibodies of the RIPA-soluble fraction of the hippocampus of WT mice treated with Vehicle or AZD0530 for 9 months. (B) Quantification of pY416-immunoreactive bands from the immunoblot from A by densitometric analysis. The bands indicated by an arrow in A were quantified. The band intensity was normalized to that of ß-actin and then normalized to the mean of the Vehicle-treated WT group. Data are represented as mean ± SEM. n = 6 /group. *p < 0.05; t-test. (C) Quantification of Fyn-immunoreactive bands from the immunoblot in A by densitometric analysis. The band intensity was normalized to that of ß-actin and then normalized to the mean of the Vehicletreated WT group. Data are represented as mean ± SEM. n = 6 /group. t-test. Figure S2. Behavioral Tests of PS19 or WT Mice Treated with AZD0530 or Vehicle (A) Morris water maze distance traveled for forward and reverse swims in 8-month-old PS19 and WT mice after 6 months of treatment. Pathlength is measured as the total distance traveled (in cm) before the mouse reaches the submerged platform. Data are mean ± SEM. n = 11-15 /group. One-way ANOVA. (B) Morris water maze latency to target for forward and reverse swims in 8-month-old PS19 and WT mice after 6 months of treatment. The latency was measured as the time for the mouse to find a submerged platform in a forward and a reverse swim after a platform relocation. Data are mean ± SEM. n = 11-15 /group. One-way ANOVA. (C) Morris water maze visible platform trial after reverse swim. Latency is measured as the average amount of time the mouse takes to reach the flagged platform in an average of 12 trials or until the latency has plateaued for 3 trials, whichever comes first. Data are mean ± SEM. n = 11-15 /group, each dot from one mouse. *p < 0.05, One-way ANOVA with Holm-Sidak’s multiple comparisons test. (D) Rotarod trials in 8-month-old PS19 and WT mice of the prophylactic cohort. Latency to fall is measured as the time it takes to fall from the rotating, accelerating rod. Each data represents the average of 5 trials for one mouse. Data are mean ± SEM. n = 18-19 /group. One-way ANOVA. Figure S3. Low Magnification Survey of Gliosis in PS19 Mice Unaffected by AZD0530. (A) Representative images of Iba1 immunostaining in the hippocampus in 9-month-old PS19 and WT mice after 7 months of treatment. Scale bar, 100 μm. (B) Quantification of Iba1-positive area (%) in the hippocampus in 9-month-old PS19 and WT mice collected after 7 months of treatment. Data are mean ± SEM. n = 7-10 /group. One-way ANOVA. (C) Representative images of immunofluorescent staining GFAP in the hippocampus in 9-month-old PS19 and WT mice after 7 months of treatment. Scale bar, 100 μm. (D) Quantification of GFAP-positive area (%) in the hippocampus in 9-month-old PS19 and WT mice collected after 7 months of treatment. Data are mean ± SEM. n = 7-10 /group. One-way ANOVA. Figure S4. Minimal Tissue Damage or Neuronal Loss after rmTBI/Stress. (A) Representative cresyl violet stained images of Sham Vehicle-treated (Sham) and Injured Vehicletreated (Injured) of the cortex and hippocampal regions containing the injury site collected more than 3 months after injury. (B) Representative NeuN stained images of Sham Vehicle-treated (Sham) and Injured Vehicletreated (Injured) coronal sections of cerebral cortex within 0.5-1 mm medial to the site of injury, using 20X magnification. Scale bar, 20 μm. (C) Representative NeuN stained images of sham vehicle treated (Sham) group and injured vehicle treated (Injured) coronal sections from the CA1 region of the hippocampus within 1 mm of the site of injury, using 20X magnification. Scale bar, 20 μm. Figure S5. Fyn Inhibitor Treatment of Chronic rmTBI/Stress Mice. (A) Timeline for a second cohort of mice that underwent a similar 14 days of chronic variable stress (CVS) plus closed head injury (CHI) or Sham CVS & CHI paradigm, and then starting on Day 121 were treated with either AZD0530 (5 mg/kg/d) or Vehicle for 10 weeks. The mice subsequently underwent Morris water maze testing at 11 months of age. (B) Latency to reach a hidden platform in reverse Morris water maze for 11-month-old WT mice from Sham Vehicle-treated (SV), Injured Vehicle-treated (IV), and Injured AZD0530-treated (IA) groups. Latency is measured as the time it takes for the mouse to reach the hidden platform. Both Injured groups exhibited longer latency to the hidden platform compared to the Sham group, but the two Injured groups were not significantly different from one another. Data are mean ± SEM. n = 8-26 /group. Twoway ANOVA, ****p < 0.0001; Tukey’s multiple comparisons test. (C) Morris water maze probe trial performed 24 hours after training trials in B for 11-month-old WT mice from SV, IV, and IA groups. Neither mice from IV nor IA groups demonstrated preference towards the target quadrant. Dashed line indicates random chance performance of 25% in the target quadrant. Data are mean ± SEM. n = 8-26 /group, each dot is one mouse. Two-tailed Wilcoxon signed rank test for non-Gaussian distribution versus random chance: SV, ***p = 0.0001; IV and IA, n.s., p > 0.05 . One way ANOVA, Tukey’s multiple comparisons test: SV vs IV, ****p < 0.0001; IV vs IA, n.s., p > 0.05; SV vs IA, ****p < 0.0001. Figure S6. Minimal Microgliosis and Astrogliosis Months after rmTBI/Stress Unaffected by Fyn Inhibitor. (A) Representative images of immunofluorescent staining Iba1 of cortical sections in the same region as Fig. 5f,h in 7.5-month-old WT mice from Sham Vehicle-treated (SV), Injured Vehicle-treated (IV), and Injured AZD0530-treated (IA) groups. Scale bar, 20 μm. (B) Quantification of Iba1-positive area (%) in 7.5-month-old WT mice from SV, IV, and IA groups. Data are mean ± SEM. n = 5 / group, each dot is one mouse. One way ANOVA with Dunnett’s multiple comparisons test, (C) Representative images of immunofluorescent staining GFAP of cortical sections in the same region as in Fig. 5f, h in 7.5-month-old WT mice from SV, IV, and IA groups. Scale bar, 20 μm. (D) Quantification of GFAP-positive area (%) in 7.5-month-old WT mice from SV, IV, and IA groups. Data are mean ± SEM. n = 5 / group, each dot is one mouse. One way ANOVA with Tukey’s multiple comparison test. Table S1. Mouse Cohorts