Modulation of p25 and inflammatory pathways by fisetin maintains cognitive function in Alzheimer's disease transgenic mice

Abstract

Alzheimer's disease (AD) is the most common type of dementia. It is the only one of the top ten causes of death in the USA for which prevention strategies have not been developed. Although AD has traditionally been associated with the deposition of amyloid β plaques and tau tangles, it is becoming increasingly clear that it involves disruptions in multiple cellular systems. Therefore, it is unlikely that hitting a single target will result in significant benefits to patients with AD. An alternative approach is to identify molecules that have multiple biological activities that are relevant to the disease. Fisetin is a small, orally active molecule which can act on many of the target pathways implicated in AD. We show here that oral administration of fisetin to APPswe/PS1dE9 double transgenic AD mice from 3 to 12 months of age prevents the development of learning and memory deficits. This correlates with an increase in ERK phosphorylation along with a decrease in protein carbonylation, a marker of oxidative stress. Importantly, fisetin also reduces the levels of the cyclin-dependent kinase 5 (Cdk5) activator p35 cleavage product, p25, in both control and AD brains. Elevated levels of p25 relative to p35 cause dysregulation of Cdk5 activity leading to neuroinflammation and neurodegeneration. These fisetin-dependent changes correlate with additional anti-inflammatory effects, including alterations in global eicosanoid synthesis, and the maintenance of markers of synaptic function in the AD mice. Together, these results suggest that fisetin may provide a new approach to the treatment of AD.

Keywords: ERK; astrogliosis; eicosanoid; lipoxygenase; oxidative stress; prostaglandin.

Figure 1

Fisetin maintains learning and memory in AD mice but does not affect plaque number. (A) Performance of the mice during the acquisition phase of the MWM test. Nine-month-old AD mice failed to learn the task over the 5-day testing period, whereas fisetin-treated AD mice learned to locate the submerged platform. (B) Performance of the mice during the memory phase of the MWM test. AD mice did not spend a greater than random percentage of their time in the quadrant where the submerged platform was previously located, whereas fisetin-treated AD mice spent greater than random amounts of time in the platform quadrant. (C) Performance of 12-month-old mice during the 2-day WM test. Although all the mice learned to find the visible platform on day 1, the AD mice did not remember where the platform was located when it was submerged on day 2, whereas fisetin-treated AD mice did remember. (D) 12-month-old AD mice spend more time in the open arm of the elevated plus maze, an indicator of disinhibition, as compared with fisetin-treated AD mice. Data represent means ± SD, n = 9–12 per group. Analysis by one-way anova followed by Tukey-Kramer multiple comparisons post hoc test. *P < 0.05 and **P < 0.01 relative to control non-Tg mice. (E) 30 micrometer thick coronal sections from 12-month-old AD mice were stained with antibody 6E10, and plaque counts in the hippocampus were quantified using Image J software. The average plaque counts for each mouse group is expressed as the number of plaques ± SD (n = 6 per group; unpaired t-test). Aβ_1-40 and Aβ_1-42 levels were measured in the insoluble (100 000 g pellet) (F) and RIPA soluble (G) hippocampal fractions by ELISA in untreated AD mice and AD mice fed fisetin. The results are expressed as mean ± SD, n = 6–8 per group. Analysis by unpaired t-test. *P < 0.05. AD, Alzheimer’s disease; MWM, Morris water maze.

Figure 2

Fisetin reduces oxidative stress, increases ERK activation, and maintains p35. (A) Fisetin reduces protein carbonylation, a marker of oxidative stress, in hippocampal extracts of non-Tg and AD mice. (B) Fisetin increases ERK activation in non-Tg and AD mice. (C) Fisetin reduces the cleavage of the Cdk5 activator p35 to pro-neurodegeneration p25. (D) Fisetin does not alter Cdk5 levels. (E) The phosphorylation of the p35 substrate PSD95, which correlates with synapse efficacy, is reduced in AD mice and maintained by fisetin. (F) Fisetin increases the synaptic spine marker drebrin in AD mice. Representative Western blots are shown. Data represent means ± SD, n = 9–12 per group. Analysis by one-way anova followed by Tukey-Kramer multiple comparisons post hoc test. *P < 0.05, **P < 0.01 and ***P < 0.001 relative to control non-Tg mice or AD mice. AD, Alzheimer’s disease; PSD95, postsynaptic density protein 95; Cdk5, cyclin-dependent kinase 5.

Figure 3

Fisetin reduces astrocytic reactivity in the brains of AD mice. (A) Representative images of GFAP immunostaining in the hippocampus. The graphs show quantification of (B) the number of astrocytes per mm², (C) the average astrocyte area (μm²), and (D) the intensity of GFAP staining in relation to non-Tg mice. Scale bar: 10 μm. Data represent means ± SEM, n = 3 per group (4–5 slices per brain). Analysis by one-way anova followed by Tukey-Kramer multiple comparisons post hoc test. *P < 0.05 and **P < 0.01 relative to control non-Tg mice or AD mice. GFAP, glial fibrillary acidic protein.

Figure 4

Fisetin reduces the protein expression of markers of inflammation in the AD mice. (A) GFAP, (B), PLA2c, (C), iNOS (D), Cox1 (E) Cox2, (F) 5-LOX and (G) 12-LOX. Representative Western blots are shown. Data represent means ± SD, n = 9–12 per group. Analysis by one-way anova followed by Tukey-Kramer multiple comparisons post hoc test. *P < 0.05 and ***P < 0.001 relative to control non-Tg mice or AD mice. AD, Alzheimer’s disease; GFAP, glial fibrillary acidic protein; iNOS, inducible nitric oxide synthase; Cox1, cyclooxygenase 1.

Figure 5

Fisetin modulates the metabolism of AA and DHA eicosanoid derivatives in AD mice. Data represent means ± SEM, n = 6 per group. Analysis by one-way anova followed by Newman–Keuls post hoc test. *P < 0.05 and **P < 0.01 relative to control non-Tg mice or AD mice. AD, Alzheimer’s disease.

Figure 6

Summary of the protective mode of action of fisetin in the AD mice. AD, Alzheimer’s disease.