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Multi-target Action of the Novel anti-Alzheimer Compound CHF5074: In Vivo Study of Long Term Treatment in Tg2576 Mice

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Multi-target Action of the Novel anti-Alzheimer Compound CHF5074: In Vivo Study of Long Term Treatment in Tg2576 Mice

Sandra Sivilia et al. BMC Neurosci.

Abstract

Background: Alzheimer disease is a multifactorial disorder characterized by the progressive deterioration of neuronal networks. The pathological hallmarks includes extracellular amyloid plaques and intraneuronal neurofibrillary tangles, but the primary cause is only partially understood. Thus, there is growing interest in developing agents that might target multiple mechanisms leading to neuronal degeneration. CHF5074 is a nonsteroidal anti-inflammatory derivative that has been shown to behave as a γ-secretase modulator in vitro and to inhibit plaque deposition and to reverse memory deficit in vivo in transgenic mouse models of Alzheimer's disease (AD). In the present study, the effects of a long-term (13-month) treatment with CHF5074 on indicators of brain functionality and neurodegeneration in transgenic AD mice (Tg2576) have been assessed and compared with those induced by a prototypical γ-secretase inhibitor (DAPT).

Results: To this end, plaque-free, 6-month-old Tg2576 mice and wild-type littermates were fed with a diet containing CHF5074 (125 and 375 ppm/day), DAPT (375 ppm/day) or vehicle for 13 months. The measured indicators included object recognition memory, amyloid burden, brain oligomeric and plasma Aβ levels, intraneuronal Aβ, dendritic spine density/morphology, neuronal cyclin A positivity and activated microglia. Tg2576 mice fed with standard diet displayed an impairment of recognition memory. This deficit was completely reverted by the higher dose of CHF5074, while no effects were observed in DAPT-treated mice. Similarly, amyloid plaque burden, microglia activation and aberrant cell cycle events were significantly affected by CHF5074, but not DAPT, treatment. Both CHF5074 and DAPT reduced intraneuronal Aβ content, also increasing Aβ40 and Aβ42 plasma levels.

Conclusions: This comparative analysis revealed a profoundly diverse range of clinically relevant effects differentiating the multifunctional anti-inflammatory derivative CHF5074 from the γ-secretase inhibitor DAPT and highlighted unique mechanisms and potential targets that may be crucial for neuroprotection in mouse models of AD.

Figures

Figure 1
Figure 1
Novel object recognition memory in the different treatment groups. Bars represent the average (± SEM) of the recognition index in the novel object recognition task. Vehicle-treated Tg2576 mice showed a borderline significant impairment of recognition compared to control non-transgenic mice treated with vehicle (p = 0.052). CHF5074-treatment (375 ppm) completely reversed memory impairment in Tg2576 mice (p = 0.031). DAPT was ineffective both in Tg2576 and wild-type animals (wild type not shown). The number of animals included in each group is indicated under the corresponding bar. *p < 0.05 and # p = 0.052 vs vehicle-treated Tg2576 mice.
Figure 2
Figure 2
Effect of CHF5074 and DAPT treatments on Aβ-related, cellular and molecular parameters. Representative images of amyloid plaques stained with the 6E10 monoclonal antibody in the cerebral cortex (A, C, E) and in the hippocampus (B, D, F) of untreated (vehicle, A, B) and CHF5074-treated Tg2576 mice (C-F) are shown in the figure. CHF5074 dose and animal code number (#) are indicated in each panel. Quantification of plaque immunostaining in both brain areas of untreated (“vehicle”), CHF5074-treated and DAPT-treated animals (indicated by the different bar colours) is shown in panel G. Plasma Aβ40 and Aβ42 levels in the same groups of untreated, CHF5074- and DAPT-treated animals are reported in panel H. The levels of monomeric Aβ and of different Aβ oligomeric species detected with the 6E10 antibody in low-detergent (0.1% Triton-X100, 0.01% Nonidet-P40) and high-detergent (3% SDS, 0.5% Triton X-100, 1% deoxycholate) brain extracts are shown in panels I and J, respectively. Data refer to the quantification of the indicated Tg2576-specific immunoreactive bands (i.e., polypeptides not present in brain extracts from wild-type littermates) in untreated Tg2576 mice and in Tg2576 mice treated with CHF5074 or DAPT (same number of animals/group as in panel G) by near-infrared fluorescence (NIRF; arbitrary units). Data in panels G-J are expressed as mean ± SEM; **p < 0.01 or *p < 0.05 vs vehicle-treated Tg2576 mice; the number of animals in each group is indicated below the corresponding bar (see Additional file 2: Figure S1 for a representative example of gel fractionation and immunoblot analysis).
Figure 3
Figure 3
Effect of CHF5074 and DAPT treatments on 6E10 intraneuronal immunoreactivity. A-C. Representative images of 6E10-immunostaining of intraneuronal 6E10-immunoreactivity in cerebral cortex sections of vehicle- (A), CHF5074 125 ppm- (B), CHF5074 375 ppm- (C) and DAPT 375 ppm-treated Tg2576 mice (D); # indicates the animal code. E. Quantification of intraneuronal 6E10-immunoreactivity in the different treatment groups. The number of animals included in each group (N) is indicated under the corresponding bar, data are expressed as mean ± SEM, *p < 0.05 and **p < 0.01 for CHF5074 treated vs untreated Tg2576 mice.
Figure 4
Figure 4
Effect of CHF5074 and DAPT treatments on microglia activation. A-D. Representative images of Iba1-immunostaining of activated microglia in hippocampus sections from wild-type (A) and Tg2576 vehicle- (B) and CHF5074-(C, D)-treated animals; # indicates the animal code. E. Quantification of Iba1-immunostaining of activated microglia in the different treatment groups. CHF5074, but not DAPT, reduces the level of microglial activation observed in Tg2576 mice. **p < 0.01 vs vehicle-treated Tg2576 mice.
Figure 5
Figure 5
Effect of CHF5074 and DAPT treatments on dendritic spine density and length in hippocampal pyramidal neurons. Low-power mag of CA1 hippocampal cortex (A) visualized by the Golgi-Cox technique; B-E: high-power mag (100x, 1.35NA) of spine morphology in hippocampal pyramidal neurons of wild-type (B), Tg2576 vehicle- (C), Tg2576 CHF5074 125 ppm- (D), CHF5074 375 ppm- (E) and DAPT 375 ppm- (F) treated mice. G. Quantification of the data, including those related to DAPT treatment, expressed as mean (± SEM) spine density and length of pyramidal neurons from the cerebral cortex of animals belonging to different experimental groups; the number of animals utilized for this analysis is indicated below the bars corresponding to each treatment group. *p < 0.05; **p < 0.01. Bars: A, 200 μm; B, 5 μm.
Figure 6
Figure 6
Effect of CHF5074 and DAPT treatments on neuronal cell-cycle functionality. A-F. Representative images of cyclin A immunoreactivity (red staining, B and E) in NeuN-positive cells (green staining, C and F) and of the resulting merge images (A, D) in Tg2576-mice treated with vehicle (A-C) or with CHF5074 375 ppm (D-F); # indicates the animal code. G. Quantification of cyclin-A positive cells versus total NeuN-positive cells in the different treatment groups. While approximately 50% of neurons express Cyclin A in untreated (“vehicle”) Tg2576 mice, the percentage of Cyclin A/NeuN-positive neurons is reduced to less than 20% in CHF5074-treated, but not DAPT-treated, animals. * p < 0.05 and ***p < 0.001 vs vehicle-treated Tg2576 mice; c p < 0.001 vs CHF5074 125 and 375 ppm-treated Tg2576 mice.

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