Memory Improvement by Yokukansankachimpihange and Atractylenolide III in the Olfactory Bulbectomized Mice

Alzheimer’s Disease (AD) shows cognitive dysfunction as core symptoms and Behavioral and Psychological Symptoms of Dementia (BPSD). Since acetylcholine nerve system derived from septum is collapsed in the AD patients, we have used Olfactory Bulbectomized (OBX) mice whose cholinergic system is largely impaired in the septum. Recently, Yokukansankachimpihange (YKH), a traditional Japanese Kampo medicine has used for BPSD in addition to improve cognitive dysfunction in AD patients. However the essential components for cognition and BPSD improvement and their mechanism are largely unknown. In present study, we found that Atractylenolide III (Aen-III), one of the components of YKH, improved cognitive deficits and depression in the OBX mice. OBX mice were orally administered with Aen-III (1.0 and 3.0 mg/kg) and YKH extracts daily for 18 days. Like YKH extracts, the Aen-III treatments ameliorated cognitive deficits and depression-like behavior observed in OBX mice. Importantly, Aen-III administration significantly restored the decreases in Ca2+/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and phosphorylation of Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) and cyclic AMP response element binding protein (CREB). The restoration of CaMKII and CaMKIV signaling is closely related to the increased BDNF levels. Furthermore, ATP reduction in OBX mice was rescued by Aen-III (3.0 mg/kg) and YKH (1000 mg/kg) treatment. In summary, Aen-III as a component of YKH ameliorates cognitive dysfunctions and depression via restoring CaMKII and CaMKIV signaling.


Introduction
Alzheimer's Disease (AD) is a progressive neurodegenerative disorder that causes cognitive impairment as core symptoms and depression and aggressive-like behaviors as Behavioral and Psychological Symptoms of Dementia (BPSD) [1].In AD patients, the downregulation of acetylcholine neurons is the causative pathology in the Central Nervous System (CNS), which is confirmed by improvement of clinical symptoms by treatments of acetylcholinesterase inhibitors, such as donepezil and rivastigmine [2].It has been reported that AD patients have theolfactory system impairment [3]- [6].The olfactory bulb is innervated by acetylcholine nerve from the septum, and Olfactory Bulbectomy (OBX) causes the retrograde nerve injury of the septal cholinergic neurons [7].Therefore, the acetylcholine nerve projections to cerebral cortex and hippocampus from the septum are also impaired, thereby causing cognitive impairment [8] [9] and depression [10].We have used the OBX mice as AD model to verify the improvement of cognition and BPSD by AD drugs [11] [12].
Yokukansankachimpihange (YKH), traditional Japanese herbal medicine (also Kampo medicine), is used traditionally for treatment of neurasthenia, hysteria, insomnia, menopausal neurosis, and pediatric epilepsy [13].YKH is consists of Atractylodis rhizome, Japanese angelica root, Bupleurum root, Citrus unshiu peel, Cnidium rhizome, Glycyrrhiza, Poriasclerotium, Pinellia tuber, and Uncaria hook.Recently Yokukansan also has a clinical efficacy against BPSD in Alzheimer's disease patients [14] and in cerebrovascular dementia model rat [15].Likewise, YKH improves the memory dysfunction a core symptom of AD in this rat [16].Recently, atractylenolide III (Aen-III), component of Byaku-jutsu (Atractylodis rhizome), has been reported to have effect of improving learning and memory dysfunction following chronic high-dose homocysteine administration in rat [17].Aen-III exerts the effect by enhancing phosphorylation of the PKC and inhibiting the ROS levels and caspase-3 activity, suggesting that Aen-III may have therapeutic potential to the cognitive dysfunction.
Therefore, we here investigated whether oral administration of Aen-III improves the cognitive impairment and depression in OBX mice.We also clarify the mechanisms underlying cognitive improvement in OBX.

Animal
Adult male DDY mice of 8 weeks were purchased from Nippon SLC (Hamamatsu, Japan) and housed in cages under the conditions of constant temperature (23˚C ± 1˚C) and humidity (55% ± 5%) with a 12-h light/dark cycle (light; 09:00-21:00 h), and could freely access to food and water.All experimental procedures using animals were approved by the Committee on Animal Experiments at Tohoku University.

Operation and Drug Treatment Diagram
OBX mice were prepared as described previously [7] [8].After a two-week recovery period, drugs were administered once a day for 18 days, and then mice were subjected to behavioral tests and subsequently dissected as shown in Figure 1.To obtain YKH powder, Atractylodis rhizome 4 g, Japanese angelica root 3 g, Bupleurum root 2 g, Citrus unshiu peel 3 g, Cnidiumrhizome 3 g, Glycyrrhiza 1.5 g, Poria sclerotium 4 g, Pinellia tuber 5 g and Uncaria hook 3 g were mixed and extracted with hot water, and then spray-dried to obtain 5 g of YKH Figure 1.Experimental protocol.Two weeks after OBX operation, drugs were administered once a day for 18 days.From day 28, mice were subjected to behavioral tests.At day 32, finishing behavioral tests, mice were sacrificed and the hippocampal CA1 region was dissected for Western blotting.powder extract.Aen-III (Wako, Osaka, Japan) was dissolved in Corn oil (Wako, Osaka, Japan) and administered orally at two doses (1.0 and 3.0 mg/kg in a volume of 0.5 mL/100g body weight).YKH was suspended in 0.5% carboxymethyl cellulose (CMC) and administered orally at 1000 mg/kg in a volume of 0.5 mL/100g body weights.Administration of YKH at 1000 mg/kg is equal to 130 µg/kg of Aen-III.Sham-operated mice were treated with the same volume of suspended solution.

Y-Maze Task
Short-term spatial reference memory and attentions were evaluated by the spontaneous alternation behavior in a Y-maze as described [18].The apparatus consisted of three identical arms (50 cm long, 16 cm wide, 32 cm high) made from black plexiglas.Each mouse was placed on the edge of one arm and allowed to move freely through the maze during an 8-min session.An alternation was defined as entries into all three arms on consecutive choices.The number of maximum alternations was defined as the total number of arms entries minus two, and the percentage of alternation was calculated as actual alternation/maximum alternations ×100.In addition, the total number of arms entries during the session was also determined as an indicator of locomotion activity.

Novel Object Recognition Task
Long-term and cognitive memory was evaluated by novel object recognition task.Mice were individually habituated an open-field box (35 cm × 25 cm × 35 cm) for 2 consecutive days.The experimenter scoring behavior was blinded to the treatment.The novel task was assessed as described [18].In the acquisition phase, two objects of the same material ware placed in the test chamber and mice were allowed to explore them for 10 min.After 24 h, one of the objects was replaced by novel object and mice were allowed to explore for 5 min.The exploratory behavior was analyzed.After each session, objects were thoroughly cleaned with 75% ethanol to prevent odor recognition.Exploration of an object was defined as rearing on the object or sniffing it at a distance of less than 1 cm, touching it with the nose, or both.Discrimination of spatial novelty was assessed by comparing the difference between exploratory contacts to familiar and novel objects and the total number of contacts, which made it possible to adjust for difference in total exploratory contacts.

Step-Through Passive Avoidance Task
The step-through passive avoidance task is a paradigm to evaluate long-term memory was assessed as described [18].Training and retention trials of the passive avoidance task were conducted in a box consisting of dark (25 × 25 × 25 cm) and light (14 × 10 × 25 cm) compartments and a floor of stainless steel rods.Floor rods in the dark compartment were connected to an electronic stimulator (Nihon Kohden, Tokyo, Japan).Mice were habituated to the apparatus the day before passive avoidance acquisition.In the training trial, mice were placed in the light compartment.Entering the dark compartment, the door was closed and an electric shock (0.3 mA, 2 sec) was delivered to grid floor and mice were removed from the apparatus 30 s later.In the testing trial, 24 h later, mice were placed in the light compartment and step-through latency was recorded until 300 sec had elapsed to assess retention level.

Tail Suspension Task
In the tail suspension task, duration of immobility time was measured according to the method described by Steru et al. [19].In this test, mice were suspended 50 cm above the floor via attachment to a tail hanger with adhesive tape.Immobility time was recorded during a 10 min.Mice were considered immobile only when they hung passively and remained completely motionless.

ATP Assay
Hippocampal regions were dissected out and rapidly frozen in liquid nitrogen and stored at −80˚C until using.ATP contents in the hippocampal extracts were measures using the ATP assay kit (Toyo Ink, Tokyo, Japan) according to the manufacturer's protocol.In brief, frozen hippocampal samples were homogenized in the homogenate buffer (0.25 M sucrose, 10 mM HEPES-NaOH, pH 7.4), and centrifuged at 1000 g for 10 min at 4˚C.The supernatant was collected and mixed with the extraction buffer at room temperature, and 30 min later, luciferin buffer was added to each sample.Oxyluciferin was detected using a luminometer (Gene Light 55, Microtec, Funabashi, Japan).

Statistical Evaluation
All values in graphs are expressed as mean ± SEM.Significant differences among groups were determined using one-way analysis of variance (ANOVA) followed by Dunnett's test for all experiments.p < 0.05 represented a statistically significant difference.

YKH and Aen-III Administration Ameliorates Cognitive Deficits and Depression-Like Behavior in OBX Mice
We first asked whether YKH and Aen-III administration improve the memory dysfunction and depression observed in OBX mice.In Y-mazetask, the percentage of alternations significantly decreased in vehicle-treated OBX mice compared with sham-operated mice (sham control: 73.9% ± 2.0%, n = 5; OBX: 48.9% ± 4.9%, n = 7) without changes in the total number of arm entries (Figure 2 We also performed novel object recognition task to assess the cognitive function [21].In a training session, there were no significant differences in discrimination index among the groups (Figure 2(c)).After 24 h retention interval between trial and test sessions, the vehicle-treated OBX mice failed to discriminate between the familiar and novel objects (Figure 2(d)).Under the same condition, the decreased discrimination index in OBX mice was significantly restored by administration Aen-III (1.0, 3.0 mg/kg) and YKH (1000 mg/kg) (Figure 2(d)).
These results show that YKH and Aen-III have the equal efficacy to improve the cognitive deficits and depression-like behavior observed in OBXmice.

Discussion
The improvement of memory and BPSD-like behaviors with yokukansan (YKS) has been reported in OBX mice [27] and amyloid precursor protein transgenic mice [28].However, the essential components of YKS and its mechanism to elucidate the improvement have not been defined.Since Aen-III, a component of Byaku-jutsu  (Atractylodis rhizome), has been reported to have effect of improving learning and memory dysfunction following chronic high-dose homocysteine administration in rat [16], we focused effects of Aen-III to elucidate the mechanism underling memory improvement in the present study.In addition, we tried to get proof-of-concept to improve memory by YKH, another Kampo medicine in animal studies, because it now popular to treat BPSDlike behaviors in human.
As expected, like YKH, Aen-III ameliorated memory deficits and depression behaviors observed in OBX mice.In addition, the previous study focused on PKC activation by Aen-III to reduce the oxidative stress and apoptosis [16].We here investigated CaMKII and CaMKIV/CREB signaling to elucidate the mechanisms underling improvement of memory and depression behaviors.CaMKII autophosphorylation is essential for longterm potentiation (LTP) induction in the hippocampal CA1 region [29] and memory formation [30].Indeed, CaMKII autophosphorylation significantly decreased and LTP was impaired in OBX mice [11].As expected, Aen-III or YKH treatment significantly ameliorated the reduced CaMKII autophosphorylation in hippocampal CA1 region.Additionally, CaMKIV and CREB phosphorylation decreased in OBX mice were also significantly restored by Aen-III or YKH treatment.Furthermore, CaMKIV/CREB downstream target, BDNF has been known to mediate the antidepressant effects of selective serotonin reuptake inhibitors (SSRI) [31]- [34].We previously observed the reduction of phosphorylated CaMKIV, CREB and BDNF levels in OBX mice [11].In addition, since the hippocampal LTP is impaired in mice lacking the BDNF gene, BDNF is closely involved in the hippocampus-mediated learning and memory [35] [36].In addition, BDNF treatment of the hippocampal slices of BDNF knockout mice rescues deficits in LTP and synaptic transmission [37].In AD patients, BDNF expression is severely decreased in the hippocampus and cortex [38] [39].Taken together, BDNF is considered to be important in the pathogenesis of AD.In this context, the observation that improvement of CaMKIV-CREB-BDNF signaling with YKH and Aen-III is critical for not only improvement of memory but also rescue of depression behaviors in AD patients.Furthermore, YKH and Aen-III restored the ATP levels in OBX mouse hippocampus.ATP is key regulator for energy production in the brain.YKH and Aen-III are likely to have potency of brain metabolism enhancement.

Conclusion
Taken together, this study showed the first evidence for the improvement effects of Aen-III in cognitive deficits as core symptoms of AD and depression as BPSD in AD model mice.Aen-III ameliorated behavioral dysfunction through restoring the decreased CaMKII autophosphorylation and CaMKIV-CREB-BDNF signaling.Moreover, Aen-III has ability to enhance the ATP levels.Thus, our study shows the potential of Aen-III for AD therapy.

Figure 2 .
Figure 2. Administration of YKH and Aen-III improves cognitive deficits and depression-like behavior in OBX mice.(a) In a Y-maze task, there were no differences in total number of arms entries among all groups; (b) Alternation in a Y-maze task was measured 14 days after OBX operation; (c) The discrimination index (%) was measured 15 days after OBX operation in a trial session; (d) The discrimination index (%) in a test session was determined 16 days after OBX operation, 24 h after trial session; (e) Latency time in step-through passive avoidance task was measured 16, 17 days after OBX operation; (f) Immobility time in tail suspension task was measured at 18 days after OBX operation.Each bar represents the mean ± S.E.M. ( * p < 0.05, ** p < 0.01 vs. sham-operated mice, # p < 0.05, ## p < 0.01 vs. OBX mice).

Figure 4 .
Figure 4. Quantitative analysis of ATP concentration in the hippocampus.The decreased ATP levels were restored by YKH and Aen-III administration in OBX mice.Data are showed as percent of sham-operated mice.Each bar represents the mean ± S.E.M. ( ** p < 0.01 vs. sham-operated mice, # p < 0.05, ## p < 0.01 vs. OBX mice).