Brain targeting of 9c,11t-Conjugated Linoleic Acid, a natural calpain inhibitor, preserves memory and reduces Aβ and P25 accumulation in 5XFAD mice

Abstract

Deregulation of Cyclin-dependent kinase 5 (CDK5) by binding to the activated calpain product p25, is associated with the onset of neurodegenerative diseases, such as Alzheimer's disease (AD). Conjugated Linoleic Acid (CLA), a calpain inhibitor, is a metabolite of Punicic Acid (PA), the main component of Pomegranate seed oil (PSO). We have shown recently that long-term administration of Nano-PSO, a nanodroplet formulation of PSO, delays mitochondrial damage and disease advance in a mouse model of genetic Creutzfeldt Jacob disease (CJD). In this project, we first demonstrated that treatment of mice with Nano-PSO, but not with natural PSO, results in the accumulation of CLA in their brains. Next, we tested the cognitive, biochemical and pathological effects of long-term administration of Nano-PSO to 5XFAD mice, modeling for Alzheimer's disease. We show that Nano-PSO treatment prevented age-related cognitive deterioration and mitochondrial oxidative damage in 5XFAD mice. Also, brains of the Nano-PSO treated mice presented reduced accumulation of Aβ and of p25, a calpain product, and increased expression of COX IV-1, a key mitochondrial enzyme. We conclude that administration of Nano-PSO results in the brain targeting of CLA, and suggest that this treatment may prevent/delay the onset of neurodegenerative diseases, such as AD and CJD.

Conflict of interest statement

R.G. is one of the founders of Granalix Biotechnologies LTD, a company producing Granagard, a commercial version of Nano-PSO. O.B., K.N., K.F., Y.U. declare no potential conflict of interest.

Figures

Figure 1

Brain targeting of CLA following administration of Nano-PSO to WT mice. C57B mice were treated either with PSO or with Nano-PSO as described in the methods and subsequently lipids were extracted from brains and livers for detection of PA or CLA. (a) PA and CLA after a single dose of PSO or Nano-PSO administration (b) PA and CLA after continuous administration of PSO and Nano-PSO. Unpaired t-test: *p < 0.05; ***p < 0.001.

Figure 2

Nano-PSO administration to both 5XFAD and WT mice improved their performance on the T-maze test. 5XFAD and WT mice treated with or without Nano-PSO were subjected to the T-Maze test at 7 months of age as described in the methods. (a) cartoon of the experiment (b) Number of entries to the new arm on day 2. Statistical analysis were performed by planned contrast test of numbers of entries to unfamiliar arm reveal significant difference between the groups [f(3,38) = 14.009, p < 0.001)].

Figure 3

Effect of Nano-PSO treatment on long-term non-associative spatial memory in the open field habituation task. 5XFAD and WT mice treated with or without Nano-PSO were subjected to the open field habituation test at 7 months of age as described in the methods. (a) Cartoon of the experiment (b) Difference in movement duration between day 1 and day 2. One-way Anova (Tukey post hoc analysis) present significant difference between Tg untreated group (n = 12) and Tg treated group (***p < 0.001; n = 18).

Figure 4

Recognition of a novel object is improved by Nano-PSO treatment of 5XFAD mice. Nano-PSO treated and untreated 5XFAD and WT mice were subjected to the novel object recognition task at 10 months of age as described in the methods. (a) Cartoon of the experiment. (b) Percentage of time spent in boundary of new object in day 2. Statistical analysis by simple contrast test was performed and showed a trend toward a difference between the groups ([f (3,39) = 0.49 p = 0.077].

Figure 5

Nano-PSO treatment restored mitochondrial COX IV-1 expression in brains of 5XFAD mice. (a) Paraffin embedded brain sections of untreated 5XFAD mice (at 3, 5 and 10 months of age), treated Tg (10 months) as well as untreated and treated WT (10 months) mice were stained with α COX IV-1 mAb (red) and counterstained with dapi (blue). The magnification of cortex area presented in this figure is (x20). (b) Ten months old 5XFAD mice, treated (I) and untreated (II) with Nano-PSO, were double-stained with the 6E10 antibody (green) and α COX IV-1 mAb(red) and presented at a magnification of x60. Magnification of single cells (x100) presented for treated (III) and untreated (IV) 5XFAD brains. Cortical neurons from 10 mounts WT brain also stained with 6E10 and COX IV-1, presented in picture V (x100).

Figure 6

Amyloid plaque burden is decreased in brains of Nano-PSO treated 5XFAD mice. (a) Sagittal serial brains sections of - 5XFAD untreated mice in ages of 3, 5, 7 and 10 months and 10 months old Nano-PSO treated mice were immunostained with α-Aβ antibody (green; 6E10 mAb) (b) quantitative assessment of Aβ plaques burden per hemisphere of 10 months 5XFAD mice, treated (green) or untreated (red) with Nano-PSO, (four section of each brain; n = 5 per group: unpaired t-test; **p = 0.01; ***p < 0.001). (c) Enlargement of Aβ plaques from 10 m old untreated 5XFAD brain -and Nano-PSO treated 5XFAD brain. (d) Quantitative assessment of Thioflavin S dense-core plaques per hemisphere of treated and untreated 10 months 5Xfad mice (four section of each brain; n = 5 per group; unpaired t-test; ***p < 0.001). (e) Sagittal section of 10 months 5Xfad mice, treated and untreated, were stain with Thioflavin S, to label dense-core plaques in cortex (x20).

Figure 7

Decreased levels of Aβ in brains of 5XFAD mice treated with Nano-PSO. Brain homogenates of WT, untreated and treated 5XFAD transgenic mice were immunoblotted with the anti Aβ 6E10mAb. Figure (a) shows levels of APP and Aβ oligomers. Figure (b) shows Aβ monomers in brains. Beta-actin served as loading control (lower panel of b). In some cases, blots have been cropped and increased in exposure; full length original blots are presented in Supplementary Fig. 1. Figure (c) represent quantitative analysis of immunoreactive bands. The bars represent the relative levels of Aβ compared with beta actin and are expressed as percentage of the 3m WT value. Unpaired t-test (***p = 0.006).

Figure 8

Nano-PSO treatment reduced p25 levels in neurodegenerative brains. (a) Brain homogenates of 5XFAD and TgMHu2ME199K mice treated and untreated with Nano-PSO as well as brain homogenates from WT mice of different ages were immunoblotted with a p35/ p25 antibody. Blots have been cropped for conciseness; full length original blots are presented in Supplementary Fig. 2. (b) Quantitative analysis of immunoreactive bands of p25. The bars are expressed as percentage of the 3 m WT value. Unpaired t-test revealed significant difference between the treated and untreated 5XFAD mice (**p < 0.05) and treated and untreated TgMHu2ME199K mice (p < 0.02).