Galantamine slows down plaque formation and behavioral decline in the 5XFAD mouse model of Alzheimer's disease

Abstract

The plant alkaloid galantamine is an established symptomatic drug treatment for Alzheimer's disease (AD), providing temporary cognitive and global relief in human patients. In this study, the 5X Familial Alzheimer's Disease (5XFAD) mouse model was used to investigate the effect of chronic galantamine treatment on behavior and amyloid β (Aβ) plaque deposition in the mouse brain. Quantification of plaques in untreated 5XFAD mice showed a gender specific phenotype; the plaque density increased steadily reaching saturation in males after 10 months of age, whereas in females the density further increased until after 14 months of age. Moreover, females consistently displayed a higher plaque density in comparison to males of the same age. Chronic oral treatment with galantamine resulted in improved performance in behavioral tests, such as open field and light-dark avoidance, already at mildly affected stages compared to untreated controls. Treated animals of both sexes showed significantly lower plaque density in the brain, i.e., the entorhinal cortex and hippocampus, gliosis being always positively correlated to plaque load. A high dose treatment with a daily uptake of 26 mg/kg body weight was tolerated well and produced significantly larger positive effects than a lower dose treatment (14 mg/kg body weight) in terms of plaque density and behavior. These results strongly support that galantamine, in addition to improving cognitive and behavioral symptoms in AD, may have disease-modifying and neuroprotective properties, as is indicated by delayed Aβ plaque formation and reduced gliosis.

Figure 1. Brain morphology, amyloid plaque formation, gliosis, and microglial activation in 5XFAD transgenic mice.

Coronal brain sections from 22-week-old wild-type control littermates (A, C, E) and 5XFAD transgenic mice (B, D, F, G, H) were subjected to Nissl staining (A, B) revealing similar brain morphology. Amyloid β immunohistochemistry (C, D) or thioflavin-S staining (E, F) detect numerous amyloid plaques in 5XFAD mice (D, F), whereas wild-type control brains (C, E) are completely devoid of plaques. GFAP immunohistochemistry (activated astrocytes, red in G) or GSA-lectin (activated microglia, red in H) in combination with thioflavin S staining (green in G and H) revealed plaques surrounded by reactive astrocytes and associated with activated microglia in 5XFAD mice. Scale bars: 500 µm (A–F), 250 µm (G, H), and 100 µm (inset in G and H).

Figure 2. Progression of plaque formation with age in 5XFAD transgenic mice.

The plaque density in the hippocampus (HC) and entorhinal cortex (EC) of male and female 5XFAD mice at various ages was determined using thioflavin S staining. In males, plaque density increased in both brain areas and reached saturation at 10 months of age. In female mice, plaque accumulation was faster, reached higher levels, and continued to increase after 14 months of age.

Figure 3. Behavioral analysis of 7-month-old 5XFAD transgenic mice.

5XFAD mice (10 female, 8 male) were analyzed in a series of behavioral tests in comparison to non-transgenic littermates (6 female, 8 male). A significant reduction in body weight (A) of transgenic (white columns) compared to non-transgenic (black columns) mice was observed for both sexes. The grip strength (B) differed between sexes but not between transgenic and non-transgenic mice. Maximum grip strength (black, white columns) and average grip strength (grey, stippled columns) were similar for non-transgenic (black, grey columns) and transgenic mice (white, stippled columns). The rota-rod (C; black control females, grey control males, white transgenic females, dotted transgenic males) did not reveal differences between transgenic and control littermate mice. In the open field (D) transgenic mice (white columns) of both sexes stayed less time in the corners compared to their wild-type littermates (black columns). In the light-dark avoidance paradigm (E, F, G) transgenic mice (white columns) showed less transitions (E), stayed longer time in the light (F), and had a much greater latency to enter the dark compartment (G) both at the first encounter or tested for memory 3 weeks later (grey columns non-transgenic, stippled columns 5XFAD in G). In the O-Maze (H, I) 5XFAD mice (white columns) spend more time (H) and traveled longer distances (I) in the open areas compared to littermate controls (black columns). Fear conditioning (J) did not differ significantly between 5XFAD (white, stippled columns) and control littermate mice (black, grey columns) with respect to freezing in the same context (black, white columns) or when exposed to the tone in a novel environment (grey, stippled columns). Prepulse inhibition of the startle response (K) was not obtained in 5XFAD mice (squares) in contrast to wild-type littermates (diamonds) in both sexes (males open, females filled symbols).

Figure 4. Water consumption, galantamine uptake, and body weight of 5XFAD-transgenic mice treated with galantamine.

Water consumption (A) of 5XFAD transgenic was similar irrespective of added galantamine (black triangles: water (n = 16); diamonds: high dose (n = 16), squares: low dose (n = 8). Galantamine uptake (B) was calculated from the amount of drinking water consumed for the low dose (squares, 12 then 60 mg/l) and the high dose (diamonds, 36 then 120 mg/l). The body weight (C) of non-transgenic control (black) or 5XFAD (white) mice was not significantly influenced by the treatment and the behavioral tests.

Figure 5. Behavioral analysis of 5XFAD-transgenic mice after treatment with galantamine.

In the Open Field (A, B) galantamine treatment restored the preference for the corners (A) and the avoidance of the center (B). Non-transgenic mice receiving water (black columns) showed a significantly higher corner preference (**, p = 0.0045) and avoidance of the center (**, p = 0.0048) compared to untreated 5XFAD transgenic mice (white columns). Treatment of 5XFAD transgenic mice with low dose (stippled columns) or high dose (hatched columns) galantamine increased their corner preference and center avoidance in a dose dependent manner. High dose treated transgenic mice spend significantly (**, p = 0.002) more time in the corners compared to untreated transgenic mice or low dose treated mice (*, p = 0.0451). Non-transgenic mice receiving high dose galantamine (grey columns). In the light-dark avoidance paradigm (C, D), high dose galantamine treatment of 5XFAD transgenic mice (hatched columns) reduced the time in the light (C) to untreated non-transgenic (black columns) control levels. In contrast, the number of transitions (D) is even further reduced by galantamine treatment of 5XFAD transgenic mice (*, p<0.005). The magnitude of the startle response (E) was not affected by galantamine treatment of 5XFAD transgenic mice. During fear conditioning (F, G), galantamine increased the context memory (F) and the tone memory (G) of non-transgenic and 5XFAD transgenic mice similarly.

Figure 6. Quantification of plaque density after galantamine treatment.

The plaque density in the hippocampus (A) of high dose galantamine treated female (black column, n = 22 sections) and male (white column, n = 47) 5XFAD transgenic mice is significantly reduced in comparison to littermate untreated 5XFAD transgenic mice (female grey column, n = 28; male stippled, n = 50) (***, p<0.0001). The reduction after treatment is even stronger for the entorhinal cortex (B) (treated female black column, n = 27, and male white column, n = 59) (untreated female grey column, n = 45, and male stippled column, n = 59). Astrocyte density and plaque density are strongly correlated in 5XFAD transgenic mice (C) (R² = 0.62; ANOVA F_(1,21) = 34.232; p<0.0001).

Figure 7. Plaque density after galantamine treatment.

Thioflavin S staining of representative coronal brain sections from untreated (A, C, E, G) in comparison to chronically high dose galantamine treated (B, D, F, H) 22-week-old 5XFAD transgenic littermate mice. Fewer plaques are detected in treated animals. Males (A–D) show fewer plaques in comparison to females (E–H), both, in the entorhinal cortex (A, B, E, F) and in the hippocampus (C, D, G, H). Scale bars 500 µm.