Effects of Chronic Masitinib Treatment in APPswe/PSEN1dE9 Transgenic Mice Modeling Alzheimer’s Disease

Animals and treatments

Transgenic APPswe/PSEN1dE9 mice co-express human APP and PS1 genes with AD familial mutations on a C57BL/6J background [10]. APPswe/PSEN1dE9 (TgAD) mice as well as wild-type (WT) littermates were purchased from Jackson Laboratory and bred in-house.

For the present study, 12-month-old TgAD and WT male mice were treated with masitinib (AB1010, AB Science) at 75 mg/kg/day or vehicle (distilled water) in a single dose by oral gavage for 2.5 months, 6 days/week (WT-Vehicle, n = 10; WT-masitinib, n = 10; TgAD-Vehicle, n = 8; TgAD-masitinib, n = 9).

Mast cell-deficient APPswe/PSEN1dE9 mice were produced by mating APPswe/PSEN1dE9 males with female mice harboring a c-Kit receptor mutation (homozygous Wsh line 13) [13] at the Centre de Recherche en Cancérologie (Marseille, France). Ten-month-old male (M) and female (F) mice were used (WT, n = 6 M + 4 F; Wsh, n = 5 M + 4 F; TgAD, n = 5 M + 5 F; TgAD/Wsh, n = 7 M + 8 F).

Animals were held in accordance with the French animal welfare act and the EU legislation (council directive 2010/63/EU). The present research project was positively evaluated and authorized by the regional ethics committee (Project N°04311.03) and all protocols were carried out according to ARRIVE guidelines.

Cognitive evaluation

Learning and memory of mice were evaluated using the Morris water maze task as described in the Supplementary Methods.

Histology

All mice were sacrificed at 10 or 14 months of age. They were transcardially perfused with phosphate buffered saline following deep anesthesia with sodium pentobarbital. Their brains were collected and cut by half. The right hemisphere was immediately frozen in liquid nitrogen and then transferred at 80°C before subsequent biochemical analysis. The left hemisphere was fixed by immersion in 4% phosphate buffered paraformaldehyde at 4°C for 48 h and then cryoprotected in 2% DMSO –20% glycerol PBS at 4°C, and finally sliced on a freezing microtome (serial 40μm thick sections).

Amyloid load assessment was performed following Congo red staining according to Puchtler’s method [14]. For each APPswe/PSEN1dE9 mouse a whole series of coronal sections spanning the entire rostro-caudal extent of the brain were stained and subsequently analyzed.

Immunohistochemistry was performed on other batches of serial sections using the standard ABC (Avidin-Biotin Complex) detection method with diaminobenzidine as chromogen. Used antibodies were a polyclonal anti-Iba1 antibody (Wako, 1 : 3000), a polyclonal anti-GFAP antibody (Dako,1 : 5000) or a monoclonal anti-synaptophysin antibody SY38 (Millipore, 1 : 1000) that were incubated overnight at 4°C.

All histological sections were scanned with a NanoZoomer 2.0-RS slide scanner (Hamamatsu Photonics, pixel size 0.25 and image analysis was performed using different methods (Supplementary Material).

Biochemical analysis

Aβ peptides, IL-1β, and chemokines dosages were performed according to protocols described in the Supplementary Methods.

Statistical analyses

All data were expressed as mean±SEM and analyzed with student t-tests or ANOVA using GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA) or Statistica 10–13 (StatSoft, Inc., Tulsa, OK, USA) software packages. Statistical significance was defined as p < 0.05. In ANOVA designs, multiple comparisons relied on post-hoc Bonferroni tests. All neuropathological and biochemical measures were normalized to reference groups. This allowed to take into account variability between discrepant factors (e.g., brain region analyzed) to better assess the effect of main factors of interest (i.e., treatment and genotype).

Cognitive function

We first addressed the effects of masitinib treatment on the cognitive performance of TgAD mice trained in the Morris water maze task.

The analysis of navigation strategies (Fig. 1A) underlined that all but the TgAD-Vehicle group succeeded to develop spatial navigation strategies with continuous training. As a noticeable observation, TgAD mice treated with masitinib reached levels of performance very close to those obtained by WT mice. ANOVA confirmed these observations underlining a significant effect of the group factor (F(3,32) = 4.3; p < 0.025). Post-hoc comparisons indicated that TgAD-Vehicle mice performed a reduced number of spatial trials in comparison to WT-Vehicle mice (p < 0.025) while WT and TgAD mice treated with masitinib performed equally (p > 0.99). Besides, TgAD-masitinib mice performed better than TgAD-Vehicle mice (p < 0.05).

OPEN IN VIEWER

Fig. 1

Masitinib mitigates spatial learning deficits in TgAD mice. Analyzed data of the Morris water maze task during the spatial learning phase and during the memory probe test. A) Number of spatial trials performed during the training phase. B) Total distance travelled during training. C) Percent time spent in the target quadrant during the memory probe test. All values are expressed as mean±SEM. *p < 0.05, **p < 0.01.

The learning accuracy, assessed by the distance travelled in the maze, paralleled navigation strategy data (Fig. 1B). ANOVA results indicated an effect of the group factor (F(3,32) = 7.023; p < 0.001). Post-hoc tests showed that TgAD mice were strongly impaired in comparison to WT mice in the vehicle condition (p < 0.001) but not following treatment with masitinib (p > 0.99). TgAD-masitinib mice performed indeed better than TgAD-Vehicle mice as they displayed lower travelled distance (p < 0.05).

During the probe test (Fig. 1C) WT mice showed a clear preference for the target quadrant in both vehicle and masitinib drug conditions indicating memory for the platform location (t-tests to compare percentage of time in the target quadrant to 25% chance level, p < 0.05 for both WT groups). On the contrary, transgenic mice did not perform above the chance level (all p-values >0.7). A two-way ANOVA confirmed a genotype effect on memory retention performance with TgAD mice being globally impaired as compared to WT mice (F(1, 31) = 6.30, p < 0.025) with no significant effect observed for the treatment factor or treatment×genotype interaction (Fs < 1).

Brain amyloidosis and neuroinflammation

Next, we explored if the beneficial effect of masitinib on the cognitive deficits of TgAD mice were associated with a decrease of Aβ accumulation and brain inflammation. In 14-month-old TgAD mice, that display numerous aggregated amyloid deposits, no differences between vehicle- and masitinib-treated mice were observed in terms of Aβ brain concentrations, regardless of peptide isoform (A, A) or conformation (soluble, insoluble) (all ts(15)<0.61; ps > 0.55; see Fig. 2A). Also amyloid loads assessed from brain sections stained with Congo red did not show any differences between TgAD-Vehicle and TgAD-masitinib mice in frontal and hippocampal areas (all ts(15)<0.25; ps > 0.81; Fig. 2B). As anticipated, both transgenic groups showed highly increased IL-1β concentrations in comparison with WT mice confirming a neuroinflammatory background in TgAD mice (WT-Vehicle versus TgAD-Vehicle: t(16) = 5.20; p < 0.0001; WT-Vehicle versus TgAD-masitinib: t(17) = 5.00; p < 0.0001; Fig.2C). Analysis did not however reveal any difference between the two TgAD groups (t(15) = 0.70; ns), indicating no effect of masitinib treatment on the proinflammatory IL-1β cytokine levels. In addition, brain levels of different chemokines (CCL-2, CCL-3, CCL-4) were found to be comparable in the two transgenic groups (All ts(15)<2.117; ps > 0.051; Supplementary Figure 1B).

OPEN IN VIEWER

Fig. 2

Masitinib does not impact brain amyloidosis and inflammation in TgAD mice. A) Biochemically-assessed brain Aβ (soluble and insoluble) concentrations in TgAD mice. B) Histologically-assessed amyloid loads of TgAD mice in the frontal cortex and hippocampus. C) Brain (whole hemisphere) dosage of IL-1β in WT and TgAD mice. D) IBA1 loads of TgAD mice in the frontal cortex and hippocampus. Data expressed as mean±SEM. ***p < 0.0001.

The two TgAD groups under vehicle or masitinib treatment showed comparable IBA1 loads in brain tissue underlining an overall constant number of microglial cells (comparison of IBA1 loads in TgAD-Vehicle versus TgAD-masitinib groups, in frontal and hippocampal areas: all ts(10)<1.44; ps > 0.18; Fig.2D). Similarly, no difference in GFAP loads were observed in the two transgenic groups (All ts(10)<0.54; ps > 0.84) indicating equivalent levels of astrocytocis (Supplementary Figure 1A).

Synaptic integrity

To evaluate possible correlates of pro-cognitive effects of masitinib treatment in TgAD mice, we assessed the drug’s effect on synaptic markers (Fig.3A). As expected, a strong decrease in synaptophysin immunoreactivity was detected in TgAD-Vehicle mice when compared with WT-Vehicle animals (t(15) = 4.4; p < 0.0005), confirming synaptic anomalies in this transgenic line. Chronic treatment with masitinib significantly increased by 20% synaptophysin immunoreactivity in TgAD mice (comparison Tg-Vehicle versus TgAD-masitinib: t(15) = 2.92; p < 0.01). No difference were observed between WT-Vehicle versus TgAD-masitinib mice (t(16) = 2.11; ns) indicating recovery of synaptic integrity in treated Tg mice.

To assess the hypothesis that masitinib protective effect on synapse is related to MCs inhibition, we generated a new model of APPswe/PSEN1dE9 mice in which c-Kit expression was knocked-down leading to a complete depletion of MC populations. This allowed us to investigate the consequences of mast cell deficiency in TgAD mice. Paralleling observations in 14-month-old mice (see above), a lower but still significant decrease in synaptophysin immunoreactivity was evidenced in younger (10-month-old) TgAD mice as compared with WT littermates (t(17) = 2.111; p < 0.05; see Fig. 3B). Mast cell deficiency in TgAD mice promoted an increase of synaptophysin immunoreactivity (comparison TgAD versus TgAD/Wsh: t(22) = 2.80; p < 0.025), mimicking the effects of pharmacological treatment with masitinib and allowing a full recovery of synaptic integrity in this genotype (comparison TgAD/Wsh versus WT: t(23) = 0.56; ns).

OPEN IN VIEWER

Fig. 3

Masitinib and mast cell-deficiency induce a recovery of synaptic markers in TgAD mice. A) Relative optical density of synaptophysin immunoreactivity in the hippocampus of WT and TgAD mice treated with Vehicle or masitinib (left part) and representative microphotographs illustrating synaptophysin immunoreactivity levels in the four studied groups (right part). B) Relative optical density of synaptophysin immunoreactivity in the hippocampus of WT and TgAD mice with or without mast cells depletion induced by the Wsh mutation (left part) and representative microphotographs illustrating synaptophysin immunoreactivity levels in the four studied groups (right part). Data expressed as mean±SEM.***p < 0.0001, **p < 0.01, *p < 0.05.