Anxiolytic-Like Effects of Lupinus angustifolious Protein Hydrolysates in Alzheimer Model Mice

: Alzheimer’s disease (AD), which is characterized by a devastating and progressive loss of memory, is the principal neurodegenerative disease in the elderly population worldwide. As a con-sequence, AD patients present neuropsychiatric symptoms, such as anxiety, causing sleeping diffi-culty, irritability, agitation, or aggressiveness. Previous studies have demonstrated that a high-fat diet, in addition to exacerbating AD, aggravates anxiety. We have demonstrated that Lupinus angustifolius protein hydrolysates (LPHs) have anti-inflammatory and antioxidant effects, key factors for AD and anxiety. Thus, this study aimed to evaluate the potential effects of LPHs on spatial memory and anxiety of a preclinical model of AD. ApoE − / − mice fed with a western diet were intra-gastrically treated with LPHs (or a vehicle) for 14 weeks. Spatial memory and anxiety were then assessed through the Morris water maze and elevated plus maze, respectively. The results did not show significant differences in spatial memory between groups. However, a significant increase ( p < 0.05) in time in open arms, center time, the number of crossings, and a reduction of anxiety behavior were observed in LPH-treated mice. This is the first study showing that an LPH treatment causes anxiolytic effects, pointing to LPHs as a potential component of future nutritional therapies.


Introduction
Alzheimer´s disease (AD) is a neurodegenerative disorder characterized by quick progressive cognitive dysfunction, loss of memory, and behavior impairment. AD is the world's biggest cause of dementia, affecting more than 40 million people [1]. Besides principal symptoms, AD produces a variety of neuropsychiatric effects, such as anxiety, which generates sleep disturbances, irritability, agitation, or aggressiveness [2]. Although the pathophysiological mechanisms of AD have not yet been fully described, diet westernization is described as one of the main causes of the increase in risk factors of AD development, such as cerebrovascular diseases, diabetes, hypertension, obesity, and  dyslipidemia [3,4]. In this context, previous studies have demonstrated that a high-fat diet, in addition to exacerbating AD, aggravates anxiety [5]. The relationship between diet and AD has generated a search for nutritional treatments that ameliorate the symptoms and consequences of AD. Hence, the beneficial effects of vegetable peptides from soy [6] on improving memory activity and anxiety have been demonstrated.
We have previously demonstrated that Lupinus angustifolius protein hydrolysates (LPHs) reduce inflammation and oxidative stress [7], key risk factors for AD and anxiety. Thus, this study aimed to evaluate the potential effects of LPHs on spatial memory and anxiety of a preclinical model of AD, the ApoE −/− mice.

Animals and Experimental Design
The experimental design is shown in Figure 1. Twelve male ApoE −/− (B6.129P2-Apo-E tm1Unc /J) mice were housed under specific pathogen-free conditions in a room with controlled temperature (22 ± 2 °C), humidity (< 55%), and 12 h light/dark cycle. Animals had free access to water and food. Animals were initially classified in two groups: mice that were fed with a standard diet (SD, n = 4) or a western diet (WD, n = 8) from the Special Diets Production Section of the University of Granada (Granada, Spain). Six-week-old mice from the WD group were randomly divided into two groups and treated intragastrically with LPHs (100 mg/kg) or a vehicle for 14 weeks, respectively. Individual body weight was measured and recorded weekly. Behavioral tests were performed at the Laboratory of Animal Behavior & Neuroscience (Facultad de Psicología, Seville, Spain). Before performing the behavioral tests, animals were conditioned to these installations for a week.

Elevated Plus Maze
Anxiety-like behavior was evaluated using the elevated plus maze (EPM) test at 17 weeks of age, after 12 weeks of consumption of SD, WD, or WD + LPHs. The test was performed as previously described [8]. All of the sessions were recorded using a camera located over the maze. Other anxiety-related behaviors, such as head dips and rears, were considered. Afterwards, the recording was processed using the plugin Animal Tracker for ImageJ software v1.52a and the time spent in the arms and center of the maze were measured.

Morris Water Maze
The Morris water maze (MWM) was performed at 19 weeks to study the processes of spatial memory and learning. Experimental procedures were performed as described by Janseen et al. [9]. All sessions were recorded with a video tracking system overlooking the pool from above. The test started at 11:30 a.m. during the light phase of the light/dark cycle, and nobody stayed in the room during the test. The latency time, distance traveled, and the time spent in each quadrant were analyzed using the plugin Animal Tracker for ImageJ software.

Statistical Analysis
All results were presented as mean ± SEM using GraphPad Prism 7 software (San Diego, CA, USA). Statistical differences were evaluated using the non-parametric Mann-Whitney test, and p < 0.05 was considered statistically significant. The analysis was performed using SPSS ® software v24 (IBM Corporation, Armonk, NY, USA)

LPHs Treatment Does Not Change Body Weight
As shown in Table 1, no differences were observed in the baseline body weight (BBW), final body weight (FBW), and body weight gain (BWG) between groups.  With these results, we can conclude that the effects observed in this study were not related to BWG of mice, which remained unchanged between the three experimental groups.

LPHs Exert an Anxiolytic-Like Effect
Representative images of the tracks of mice in the maze appear in the Figure 2A-C. WD mice spent significantly less time in opened arms and more in closed arms compared to the SD group. This effect was reversed after LPH supplementation ( Figure 2D,E). Moreover, the time spent in the center was significantly lower in WD compared to SD and WD + LPHs ( Figure 2F). Furthermore, other anxiety-related behaviors were evaluated. The number of head dips was significantly lower after WD intake compared to SD, but not in the WD + LPHs group ( Figure 2G). There were no differences in the rears between groups ( Figure 2H). It is well-known that WD accelerates the cognitive deficits in ApoE −/− mice [5]. In the present study, we observed that WD increased anxiety levels significantly. These results are in accordance with previous studies in humans [10] and mice [11]. LPHs, therefore, were shown to minimize the anxiolytic effects caused by the intake of the WD. It has already been shown that bioactive peptides from soy [6] may exert anti-anxiety activity, but, to the best of our knowledge, this is the first time that a Lupine hydrolysate showed anxiolytic-like properties.

LPHs Do Not Alter Spatial Memory
The latency time, which reflects the ability to learn the platform location, is shown in Figure 3B. During the training phase, we did not observe any significant differences between groups. This result revealed that all groups were equally capable of learning the task. The probe phase shows that there were no differences in the time spent in the platform zone ( Figure 3C) among groups. Interestingly, a decreasing trend in the total distance traveled after WD consumption was observed (p < 0.058, compared to SD), which was reverted after LPH treatment (p < 0.0571 compared to WD) ( Figure 3D). Our results revealed no impairment of memory or spatial learning after WD consumption. This fact could be associated with the age of the mice and time of WD consumption [9]. Unlike other peptides [12], LPHs improved neither spatial memory nor learning after a 14-week treatment.

Conclusions
This is the first study showing that an LPH treatment causes anxiolytic effects, pointing to LPHs as a potential component of future nutritional therapies in AD patients with anxiety, a serious side effect of this disease.

Conflicts of Interest:
The authors declare no conflict of interest.