Evodiamine derivatives improve cognitive abilities in APPswe/PS1ΔE9 transgenic mouse models of Alzheimer's disease

Abstract Background Alzheimer's disease (AD) is a complex neurodegenerative disease. Due to the complexity of its molecular pathogenesis and the interaction of the numerous factors involved, the etiology and pathogenesis of AD have not been fully elucidated. Therefore, effective treatment for AD remains to be developed. Evodiamine, a quinolone alkaloid, has been found to improve learning and memory ability to in the APPswe/PS1△E9 mouse model of dementia. However, the cytotoxicity and physicochemical properties of evodiamine have limited its use in the treatment of AD. Methods Evodiamine and its derivatives were effectively synthesized by EDCI‐mediated condensation at room temperature. These target compounds contained 1 thio‐ and 21 oxo‐evodiamine derivatives with different substituted groups. The cytotoxicity of evodiamine and its derivatives and the neuroprotective effects of the evodiamine derivatives against H2O2‐induced cell loss in SH‐SY5Y cells were investigated using the WST‐8 assay. The Morris water‐maze test was used to detect the effect of evodiamine and its derivatives on improving learning and memory in APPswe/PS1△E9 mice. Results In this study, a series of oxo‐ and thio‐evodiamine derivatives was synthesized. Several derivatives showed lower cytotoxicity and stronger neuroprotective effects than evodiamine and elicited enhanced cognitive improvement, especially in the test of spatial memory in APPswe/PS1△E9 mice. Conclusion Our study provides insights for developing novel evodiamine derivatives for chemical intervention and treatment of AD.


| INTRODUC TI ON
Alzheimer's disease (AD) is a complex neurodegenerative disease that affects more than 35 million people worldwide. Memory loss and cognitive impairment can cause death 3-9 years after diagnosis. 1 The main pathological features of AD are senile plaques formed by amyloid deposition and tangles formed by tau protein hyperphosphorylation. 2 AD is mainly divided into genetic and non-genetic forms. Patients with genetic AD inherit a familial autosomal dominant form of the disease, presenting as early-onset dementia, usually occurring between the ages of 30 and 60. This familial form of AD is caused by mutations in genes such as APP, PSEN1 and PSEN2. 3 These gene mutations lead to the accumulation of amyloid beta protein outside neurons by affecting amyloid beta production, the Aβ-42/ Aβ-40 ratio, or the structure of amyloid beta. 2 More than 99% of AD is non-genetic, and sufferers tend to develop the disease later in life, usually after the age of 65 years. Because of the complexity of its molecular pathogenesis and the interaction of the many factors involved in the disease, the etiology and developmental mechanisms of AD have not yet been fully elucidated. Therefore, an effective treatment for AD remains to be developed. 4 APP swe /PS1 ΔE9 double transgenic mice express two major mutations in the human APP gene, as well as human PS1 mutations knocked-in into the mouse PS1 gene. These mice were bred on a C57BL/6J background. 5 The APP swe transgene encodes a mousehuman hybrid with the mouse sequence in the extracellular and intracellular regions and a human sequence within the Aβ domain with Swedish mutations K594N/M595L. The PS1 ΔE9 transgene encodes the Δexon9 human presenilin-1. This mouse shows spatial memory deficits at 3 months of age and senile plaques in brain tissue at 4.5 months of age. 6 Therefore APP swe /PS1 ΔE9 double transgenic mice develop behavioral phenotypic and pathological features which make them useful as an AD model. Chinese herbal medicine, an important element of Chinese medicine, is becoming increasingly popular among physicians and patients. 7,8 Many medical plant preparations are marketed to the public for various ailments, including Alzheimer's disease. 9 Evodiamine is a quinazolinone alkaloid isolated from the fruit of fructus evodia, which has been used to treat cancer, cardiovascular disease, headaches, abdominal pain, postpartum bleeding, dysentery and amenorrhea. [10][11][12][13] In addition, some studies have found that evodiamine can reduce overexpression of cytokines IL-1β, IL-6, TNF-α, inhibit the inflammatory response and overactivation of glial cells in the brain, and improve the learning and memory ability of APP swe /PS1 ΔE9 dementia model mice. 6 However, the cytotoxicity of evodiamine is relatively high and its physicochemical properties are not suitable for pharmacological use. 14 These disadvantages limited its therapeutic use. In order to discover novel and potent lead compounds for AD treatment, we developed an efficient method for generating quinazolinone alkaloids via carbodiimide-mediated condensation between carbolines with anthranilic acids. 15 This provided potential for increasing the scaffold diversity of evodiamine. In this study, a series of oxo-and thio-evodiamine derivatives was designed and synthesized using this approach and their potential roles for treatment of AD were investigated.

| Animals
APP swe /PS1 ΔE9 double-transgenic mice were maintained on a C57BL/6J genetic background. The mice developed spatial memory deficits at 3 months of age and senile plaques in brain tissue at 4.5 months of age, as previously reported. 16

| Morris water-maze test
The protocol of the Morris water-maze test was modified from the reported methods. 17,18 Briefly, the water maze is a 100 cm diameter pool with a 15 cm diameter escape platform placed 0.5 cm below the water surface. Since C57BL/6J mice are black, we poured an appropriate amount of white paint into the water to make the water white and opaque, and maintained the water temperature at 22-24°C. The mice were placed in the water near the side of the pool. East, west, south, and north positions were randomly selected and each mouse was tested once. We recorded the times taken by the animals to find the underwater platform. In previous training sessions, if this time exceeded 60 seconds, we guided the animal to the platform and let the animal stay on the platform for 10 seconds. The mice were then removed and dried with a towel and placed in a cage. Each mouse was trained 4 times a day, with a 2 hour interval between two training sessions, for 5 consecutive days.
The platform was removed on the sixth day and exploration training lasted for 60 seconds. The animals were placed in the water at the side opposite the original platform quadrant. The duration that animals spent in the quadrant where the platform was originally placed and the number of times they entered the quadrant were recorded, and were used as detection indicators of spatial memory. A video tracking system (Noldus Ltd, Ethovision XT, Holland) was used for detection and analysis. The results were expressed as SD ± SEM.

| Statistical analysis
The data were analysed by two-tailed unpaired t-tests and oneway ANOVA followed by Tukey's post hoc analysis. Data shown are means ± S.D, with P < .05 considered statistically significant.

| Synthesis of evodiamine and its derivatives
As shown in Figure 1, evodiamine and its derivatives were effectively synthesized by EDCI-mediated condensation at room temperature.
These target compounds contained 1 thio-and 21 oxo-evodiamine derivatives with different substituted groups.

F I G U R E 1
The structure and synthesis of evodiamine and its derivatives. Evodiamine and its derivatives were effectively synthesized by EDCI-mediated condensation at room temperature

| Treatment with evodiamine derivatives increased spatial learning and memory in APP swe /PS1 ΔE9 transgenic mice
We used APP swe /PS1 ΔE9 transgenic mice to explore the effect of evodiamine and its derivatives on learning and memory, using the Morris water-maze test. After 4 weeks of treatment, the residence durations in the target quadrant of the mice treated with evodiamine and its derivatives (49, 51, 58 and 60) was significantly higher than that of the untreated APP swe /PS1 ΔE9 transgenic mice group ( Figure 4A,B, n = 7, P < .001), and the effect of the evodiamine derivatives (49, 51, 58 and 60) was stronger than that of evodiamine.

F I G U R E 2
The SH-SY5Y and HepG2 cells viability was assessed by the CCK-8 assay. A, Effect of 5 μg/mL evodiamine and its derivatives on SH-SY5Y cell viability (n = 6, ****P < .0001, ***P < .001, evodiamine derivatives treatment groups vs evodiamine treatment group). B, Effect of 5 μg/mL evodiamine and its derivatives on HepG2 cell viability (n = 6, ****P < .0001, ***P < .001, evodiamine derivatives treatment groups vs evodiamine treatment group) Compared with the APP swe /PS1 ΔE9 group, the number of times that mice crossed the target quadrant increased significantly in the evodiamine and derivatives groups. Among the derivatives groups, the improvements recorded for groups 49 and 60 were more obvious than that of the evodiamine group.

| D ISCUSS I ON
Scaffold diversity has proved to be a powerful strategy for the discovery of novel lead compounds. In this study, a series of oxo-and thio-evodiamine derivatives was synthesized using our reported F I G U R E 3 Effects of evodiamine and its derivatives on H 2 O 2 -induced cytotoxicity in SH-SY5Y cells determined by CCK-8 assay. The SH-SY5Y cells were seeded in 96-well plates (10 000 cells/ well) and cultured for 24 h. Evodiamine and its derivatives (41, 47, 49, 51, 54, 58, 59, 60 and 61) were then added to the wells at doses of 0.5 µg/mL, 0.05 µg/mL and 0.005 µg/mL and the cells were cultured for 3 h. During the brain aging process in female AD patients, mitochondrial respiration decreases and mitochondrial hydrogen peroxide production increases. 24 Over the past few decades, the neuroprotective activity of many natural compounds has been evaluated for its protective effect on H 2 O 2 -induced cell damage in SH-SY5Y and other related neuronal cell lines. [25][26][27][28] Our results showed that evodiamine and its derivatives 49, 51, 54, 58, 59 and 60 displayed increased neuroprotective effects ( Figure 3). Furthermore, using the Morris water-maze test, we showed that treatment with evodiamine and its derivatives for 4 weeks can improve spatial learning and memory in APP swe /PS1 ΔE9 transgenic mice with AD symptoms at 5 months of age ( Figure 4). Compared with evodiamine, our derivatives numbers 49, 51, 58 and 60 significantly improved the spatial memory ability of APP swe /PS1 ΔE9 transgenic mice.
In this study, a series of oxo-and thio-evodiamine derivatives was designed and synthesized using the strategy of scaffold diversification. Some of the derivatives showed lower cytotoxicity, and stronger neuroprotective effects, and elicited significant improvements in spatial memory in the APP swe /PS1 ΔE9 AD mouse model.
This study further explores the use of evodiamine as a treatment for AD, and also provides insights into developing novel and potent evodiamine derivatives for chemical intervention in AD.

AUTH O R CO NTR I B UTI O N S
YY, XP and LZ designed the experiment. YY, XP, SP, CS and SG performed the experiment. SP and YY worked on the statistical analysis of the data. YY, XP and LZ prepared the manuscript. All authors read and approved the final manuscript.