Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Filters applied. Clear all
. 2015 Jun;77(6):953-71.
doi: 10.1002/ana.24394. Epub 2015 Mar 21.

Fyn Inhibition Rescues Established Memory and Synapse Loss in Alzheimer Mice

Affiliations
Free PMC article

Fyn Inhibition Rescues Established Memory and Synapse Loss in Alzheimer Mice

Adam C Kaufman et al. Ann Neurol. .
Free PMC article

Abstract

Objective: Currently no effective disease-modifying agents exist for the treatment of Alzheimer disease (AD). The Fyn tyrosine kinase is implicated in AD pathology triggered by amyloid-ß oligomers (Aßo) and propagated by Tau. Thus, Fyn inhibition may prevent or delay disease progression. Here, we sought to repurpose the Src family kinase inhibitor oncology compound, AZD0530, for AD.

Methods: The pharmacokinetics and distribution of AZD0530 were evaluated in mice. Inhibition of Aßo signaling to Fyn, Pyk2, and Glu receptors by AZD0530 was tested by brain slice assays. After AZD0530 or vehicle treatment of wild-type and AD transgenic mice, memory was assessed by Morris water maze and novel object recognition. For these cohorts, amyloid precursor protein (APP) metabolism, synaptic markers (SV2 and PSD-95), and targets of Fyn (Pyk2 and Tau) were studied by immunohistochemistry and by immunoblotting.

Results: AZD0530 potently inhibits Fyn and prevents both Aßo-induced Fyn signaling and downstream phosphorylation of the AD risk gene product Pyk2, and of NR2B Glu receptors in brain slices. After 4 weeks of treatment, AZD0530 dosing of APP/PS1 transgenic mice fully rescues spatial memory deficits and synaptic depletion, without altering APP or Aß metabolism. AZD0530 treatment also reduces microglial activation in APP/PS1 mice, and rescues Tau phosphorylation and deposition abnormalities in APP/PS1/Tau transgenic mice. There is no evidence of AZD0530 chronic toxicity.

Interpretation: Targeting Fyn can reverse memory deficits found in AD mouse models, and rescue synapse density loss characteristic of the disease. Thus, AZD0530 is a promising candidate to test as a potential therapy for AD.

Figures

Figure 1
Figure 1. AZD0530 potently inhibits Fyn activity and indirectly blocks Pyk2
(A) The enzyme kinetics of the interaction between FynB and AZD0530 shown as ATP varies indicate a competitive model of inhibition. The best fit of the data indicates that the Ki is 8.3 nM ± 3.4 nM. (B) Immunoblot analysis of RIPA-soluble extracts from brain slices incubated with or without Aβo and with or without AZD0530 for the indicated antigens for 30 min. (C) Densitometric analysis of the immunoblot from B showing that Aβo treatment induces significantly increased Src family kinase phosphorylation, which can be significantly reduced by incubating with AZD0530. Data are mean ± SEM of 8-9 brain slices per condition. (** p<0.01, *** p<0.001, one-way ANOVA with post-hoc LSD comparisons as noted). (D) Densitometric analysis of the immunoblot from B showing that Aβo treatment also induces significantly increased Pyk2 phosphorylation, which can also be significantly reduced by AZD0530. Data are mean ± SEM of 10-11 brain slices per condition. (*** p<0.001, **** p<0.0001, one-way ANOVA with Tukey’s multiple comparisons as noted). (E) Densitometric analysis of the immunoblot from B showing that Aβo treatment induces significantly increased NR2B phosphorylation, which is reduced by AZD0530. Data are mean ± SEM of 3 brain slices per condition. (* p<0.05, *** p<0.001, one-way ANOVA with Tukey’s multiple comparisons as noted) (F) The LanthaScreen binding assay shows that AZD0530 minimally inhibits Pyk2. The IC50 of this interaction is greater than 250 μM.
Figure 2
Figure 2. AZD0530 effectively crosses the blood brain barrier and has a long half life within the brain
(A) AZD0530 can be found in plasma, brain, and CSF of mice that received 6 oral doses given over a 3 day time period. Higher doses correlated with higher levels in all compartments. The data points for plasma and brain are the mean ± SEM of 5 mice. The data points for CSF are single values for samples pooled from 5 mice. (B) AZD0530 levels within the brain fall over time with an estimated half-life of 16 hours. Each data point is the mean ± SEM of 5 mice that received 5 mg/kg/d for 3 days prior to sacrifice. (C) Mice treated with 5 mg/kg/d for 3 days achieve comparable trough levels in CSF to that of humans treated with 125 mg/d for one month. Data are mean ± SEM of the calculated CSF value of 5 mice derived from their brain levels, and the actual CSF value of 5 humans.
Figure 3
Figure 3. Pyk2 levels are returned to WT levels by treatment with AZD0530
(A) Immunoblot analysis of RIPA-soluble lysates from brain hemispheres from mice treated with AZD0530 or vehicle for two weeks stained with an anti Pyk2 antibody and an anti p-Pyk2 antibody. Genotypes and treatment status are indicated above each lane and each lane is from a separate mouse. (B) Densitometric analysis of immunoblot experiment normalized to actin levels from A. Data are mean ± SEM n=3-6 mice per group. There was a significant elevation of the ratio of p-Pyk2 to Pyk2 in the vehicle treated APP/PS1 mice in the hippocampus compared to all other treatment groups and the ratio in the AZD0530 treated APP/PS1 mice normalized (ANOVA with Tukey post-hoc testing performed ***p<0.001). (C-D) Densitometric analysis of immunoblot experiments normalized to actin levels from the same mice used for A however looking in the cortex and the cerebellum. The pattern seen in the cortex matches what was seen in the hippocampus in that there is an elevated ratio of of p-Pyk2 to Pyk2 in the vehicle treated APP/PS1 mice which is returns to the WT baseline in the AZD0530 treated APP/PS1 mice (ANOVA with Tukey post-hoc testing performed *p<0.05, **p<0.01). In the cerebellum, no group of mice showed any elevated ratio (ANOVA p>0.05). (E) WT and APP/PS1 mice at 11 months of age were treated with Vehicle or 5 mg/kg/d AZD0530 for 7 weeks and then the brain was collected for analysis. Immunofluorescence staining of p-Pyk2 in the CA1 regions of the hippocampus is illustrated. Scale bar, 25 μm. (F-G) The intensity of pPyk2 in the cell body layer of the CA3 and CA1 hippocampus was measured. * p<0.05, ** p<0.01, *** p<0.001, one-way ANOVA with post-hoc Tukey comparisons as noted. Data are mean ± SEM from n=6 mice per group.
Figure 4
Figure 4. AZD0530 reverses learning and memory deficits in APP/PS1 mouse model after 3-5 weeks treatment
(A) Spatial learning is plotted as the latency for a cohort of 12 month old mice take to find a hidden platform after receiving treatment for 1-2 weeks prior to testing. Mean ± SEM n=17-26 mice per groups. Performance differed over the last 12 trials by genotype but not treatment status (two way RM-ANOVA for APP/PS1 ***p<0.001; for short pretreatment with AZD0530 p>0.05). Tukey post hoc analysis indicated that both APP/PS1 groups differed from the C57BL/6 group (***p<0.001) while short pre-treatment with AZD0530 of APP/PS1 did not cause the mice to perform differently than the vehicle treated APP/PS1 group (p>0.05). (B) Performance during a 60 second probe trial, completed 24 hours after the training in the Morris water maze is completed, is measured by percent time spent in the quadrant where the platform was located previously. The dashed line indicates random chance at 25%. Mean ± SEM n=9-14 mice per group. Target quadrant differed by genotype but not by treatment status (two way ANOVA for APP/PS1 ***p<0.001; for short pretreatment with AZD0530 p>0.05). By Fisher’s LSD post-hoc pairwise comparisons vehicle treated APP/PS1 and AZD0530 treated APP/PS1 did not differ from each other significantly (p>0.05) while both were significantly different from vehicle treated C57BL/6 (*p<0.05 and **p<0.01 respectively). (C) Spatial learning is plotted as the latency a cohort of 13 month old mice take to find a hidden platform after receiving treatment for 3-5 weeks prior to testing. Mean ± SEM n=17-27 mice per group. Performance differed over the last 12 trials by genotype as well as treatment status (two way RM-ANOVA for APP/PS1 ***p<0.001; for long pretreatment with AZD0530 *p<0.05). There was an interaction between genotype and treatment. (two-way ANOVA , APP/PS1 × AZD0530 p<0.05). The vehicle treated APP/PS1 group significantly differed (**p<0.01, ***p<0.001) from the other groups in Fisher’s LSD post hoc pairwise comparisons while all other comparisons were not different (p>0.05). For the indicated trial blocks, the vehicle treated APP/PS1 group differed from the other groups (*p<0.05, **p<0.01, ***p<0.001) while none of the other groups differed from each other. (D) A 60 second probe trial was performed 24 hours after training in mice that received 3-5 weeks of pretreatment prior to testing. Random chance is 25%. Mean ± SEM n=9-14 mice for each group. A one-way ANOVA indicated that differences existed between the groups (***p<0.001). By Fisher’s LSD post hoc, pairwise comparisons the untreated APP/PS1 group differed from the others (*p<0.05, ***p<0.001), whereas none of the other groups differed from each other (p>0.05). (E) Spatial learning was testing in a cohort of mice that was treated for 3-5 weeks with a lower dose of AZD0530 and plotted as the latency to finding a hidden platform. Mean ± SEM n=8-13 mice per groups. Performance among the groups differed over the last 12 trials of the training (one way RM-ANOVA, ***p<0.01). Tukey post-hoc pairwise comparisons indicate that APP/PS1 treated with the lower dose of AZD0530 performed significantly worse than both higher dose treated APP/PS1 group and the vehicle treated WT group (**p<0.01) while the two latter groups performed similarly (p>0.05). (F) Object recognition learning is plotted as the time a 13 month cohort of mice pre-treated for 6 weeks spent interacting with a novel object compared to time spent interacting with a familiar object. Mean ± SEM n=6-10 mice per group. Vehicle treated APP/PS1 mice showed no preference for a novel object over a familiar object (two-tailed Student’s t-test p>0.05) while the other groups all showed a distinct preference for the novel object (two-tailed Student’s t-test **p<0.01 ***p<0.001).
Figure 5
Figure 5. APP and Aβ levels are not altered by treatment with AZD0530
(A) Immunoblot analysis of TBS-soluble lysates from brain hemispheres stained with anti-Aβ antibody 6E10. Genotypes and treatment status are indicated above each lane and each lane is from a separate mouse. The molecular weight of the standard is shown to the left of the blot. (B) Densitometric analysis of immunoblot experiment normalized to βIII-tubulin levels from A. Data are mean ± SEM n=4 mice per group. There was no difference after treatment between the two APP/PS1 groups (two-tailed Student’s t-test, p>0.05). (C) Treatment with AZD0530 did not alter total Aβ monomer levels as detected by an Aβ ELISA (Tukey post hoc comparison testing, p>0.05). (D) Immunofluorescent detection of Aβ in 13 month APP/PS1 mice treated with vehicle or AZD0530 in the hippocampus using a confocal microscope and a 4X objective lens. Scale bar, 100 μm. (E) Fractional analysis of the immunoreactive area stained for Aβ with antibody 2454. There was no difference between the two groups (two-tailed Student’s t-test p>0.05).
Figure 6
Figure 6. Microgliosis is reduced in APP/PS1 mice after 7 weeks of treatment with AZD0530
(A) The cortex of the indicated groups was stained with Iba1 and imaged with a confocal microscope with a 20X objective. Scale bar, 50 μm. (B) Fractional area of immunoreactivity for Iba1 in the cortex of the groups indicated in A. A one-way ANOVA with Tukey post hoc comparisons show that the vehicle treated APP/PS1 group differs from all groups including the AZD0530 treated APP/PS1 group (*p<0.05, ***p<0.001). The AZD0530 treated APP/PS1 also differed from the two C57Bl6 groups (*p<0.05). Mean ± SEM 5 mg/kg/d AZD0531 APP/PS1 n=6 mice, Vehicle APP/PS1 n=9 mice, 5 mg/kg/d AZD0530 WT n=5 mice, Vehicle WT n=5 mice. (C) The cortex of the indicated groups was stained with GFAP and imaged with a confocal microscope with a 20X objective. Scale bar, 50 μm. (D) Fractional area of immunoreactivity for GFAP in the cortex of the groups indicated in C. A one-way ANOVA with Tukey post hoc comparisons show that the vehicle treated APP/PS1 differs from both C57Bl6 groups (**p<0.01, ***p<0.001) while not differing significantly from the AZD050 treated APP/PS1 group (p>0.05). Mean ± SEM 5 mg/kg/d AZD0531 APP/PS1 n=6 mice, Vehicle APP/PS1 n=9 mice, 5 mg/kg/d AZD0530 WT n=4 mice, Vehicle WT n=4 mice.
Figure 7
Figure 7. Synaptic markers recover to WT levels after 7 weeks of treatment with AZD0530
(A) The dentate gyrus of the hippocampus of the indicated groups was stained with PSD-95 and imaged with a confocal microscope with a 60X objective. (B) Fractional area of immunoreactive puncta for PSD-95 in the dentate gyrus of the groups indicated in A. A one-way ANOVA with Fisher’s LSD post hoc pairwise comparisons show that the vehicle treated APP/PS1 group differs from the other groups including the AZD0530 treated APP/PS1 group (*p<0.05) while no other group exhibited differences from each other (p>0.05). Mean ± SEM n=5-11 mice per group. (C) Fractional area of immunoreactive puncta for SV2a in the dentate gyrus of the hippocampus imaged with a 60x objective. A one-way ANOVA with Fisher’s LSD post hoc pairwise comparisons show that the vehicle treated APP/PS1 differs from the remaining groups (*p<0.05). Mean ± SEM n=5-11 mice per group.
Figure 8
Figure 8. AZD0530 decreases total Tau and phosphorylated Tau in RIPA-soluble and –insoluble fractions
(A) 3xTg-AD mice (11 months of age) received vehicle or 5 mg/kg/d AZD0530 divided in b.i.d. doses by oral gavage for 40 days. After removal of blood by perfusion with cold PBS for 5 min, the cortex and hippocampus from one hemisphere of each mouse were homogenized and centrifuged in TBS and then the insoluble material was extracted with RIPA buffer (n=5-7 per group; age 13 months by the end of gavage). Human Tau, total Tau, and pTau (pS199/S202 and pS396) with the size of P301L Tau (*, asterisk on the right of the blot) were measured. (B, C) AZD0530 did not alter tau levels in TBS-soluble fractions. However, AZD0530 decreased total Tau by 74%, pTau pS199/S202 by 61%, and pS396 by 53% compared to control 3xTg in RIPA extracts of TBS-insoluble material (ANOVA, Tukey’s multiple comparisons test, *p< 0.05, ***p < 0.001, ****p < 0.0001; Error bars show SEM). (D) In RIPA-insoluble fractions, insoluble human Tau and total Tau were also decreased by AZD0530 treatment, by 56% and 41%, respectively (For human Tau, by t-test, **p < 0.01; for total Tau, ANOVA, Tukey’s multiple comparisons test, ***p < 0.001; Error bars show SEM). (E, F) Immunostaining of pTau (pS199/S202) and PHF-tau (AT180) in CA1 of hippocampus showed that the pTau-positive area was decreased by AZD0530 treatment (t-test, **p < 0.01, ***p < 0.001; Error bars show SEM).
Figure 9
Figure 9. No Toxic Effect of Chronic AZD0530 in Dogs
Dogs were treated for 273 days with 0. 0.5, 2 or 5 mg/kg/d of AZD0530. (A) The body weight of the male dogs as a function of age is reported. (B-E) Hematological and coagulation parameters are reported for 8 dogs at each dose level prior to and after 9 months of AZD0530. All data are mean ± sem. There were small changes in various parameters as the dogs aged, but there was no statistically significant difference between AZD0530 groups and vehicle by one-way ANOVA with Tukey post hoc correction.

Similar articles

See all similar articles

Cited by 78 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback