The Cannabinoids, CBDA and THCA, Rescue Memory Deficits and Reduce Amyloid-Beta and Tau Pathology in an Alzheimer’s Disease-like Mouse Model

Most studies related to hemp are focused on Cannabidiol (CBD) and Tetrahydrocannabinol (THC); however, up to 120 types of phytocannabinoids are present in hemp. Hemp leaves contain large amounts of Cannabidiolic acid (CBDA) and Tetrahydrocannabinolic acid (THCA), which are acidic variants of CBD and THC and account for the largest proportion of CBDA. In recent studies, CBDA exhibited anti-hyperalgesia and anti-inflammatory effects. THCA also showed anti-inflammatory and neuroprotective effects that may be beneficial for treating neurodegenerative diseases. CBDA and THCA can penetrate the blood–brain barrier (BBB) and affect the central nervous system. The purpose of this study was to determine whether CBDA and THCA ameliorate Alzheimer’s disease (AD)-like features in vitro and in vivo. The effect of CBDA and THCA was evaluated in the Aβ1–42-treated mouse model. We observed that Aβ1–42-treated mice had more hippocampal Aβ and p-tau levels, pathological markers of AD, and loss of cognitive function compared with PBS-treated mice. However, CBDA- and THCA-treated mice showed decreased hippocampal Aβ and p-tau and superior cognitive function compared with Aβ1–42-treated mice. In addition, CBDA and THCA lowered Aβ and p-tau levels, alleviated calcium dyshomeostasis, and exhibited neuroprotective effects in primary neurons. Our results suggest that CBDA and THCA have anti-AD effects and mitigate memory loss and resilience to increased hippocampal Ca2+, Aβ, and p-tau levels. Together, CBDA and THCA may be useful therapeutic agents for treating AD.


Introduction
Alzheimer's disease (AD) is an age-related neurodegenerative disease accompanied by memory and cognitive deficits [1]. It has been more than 100 years since AD was first reported. However, there is currently no treatment for AD. The causes of AD are unclear, although several hypotheses include the amyloid-beta hypothesis and the highly phosphorylated tau hypothesis [2,3]. The accumulation of amyloid-beta and hyper-phosphorylated tau cause neuronal cell death, synaptic collapse, and neuro-inflammation, which are hallmark symptoms of AD. This results in a decrease in memory and cognitive function, leading to severe dementia [4].

CBDA and THCA Treatment Ameliorates Learning and Memory Loss in Aβ 1-42 -Treated Mice
To determine the effect of CBDA and THCA on the pathogenesis of AD, the hippocampus of the mice was unilaterally infused with Aβ 1-42 (3 µg/mouse) or PBS. Two days after injection, CBDA (6 µmol/mouse) or THCA (12 µmol/mouse) was similarly injected into the hippocampus of Aβ 1-42 -treated mice to determine the effect of CBDA and THCA on learning and memory. We conducted Morris water maze and object location tests to evaluate spatial learning ability and novel object recognition tests to assess the ability to recognize new objects (Figure 3). The experimental schedule for the behavioral tests is summarized in Figure 3A. Two-way ANOVA analysis of mean escape latency (i.e., the time required to locate the escape platform) in the Morris water maze test revealed statistically significant differences between the three groups (interaction (p = 0.047), Treatment (p < 0.0001), Time (p < 0.0001)). Aβ 1-42 -treated mice learned the location of the submerged platform more slowly compared with PBS-treated mice during training sessions and showed less improvement throughout training. However, mice treated with CBDA or THCA following Aβ 1-42 treatment performed better than those treated with Aβ 1-42 alone ( Figure 3B). On day 5 of the probe test, Aβ 1-42 -treated mice remained in the target quadrant (p < 0.001) and platform area (p = 0.035) for a significantly shorter time compared with mice treated with PBS. For Aβ 1-42 -treated mice, CBDA or THCA treatment resulted in a longer time in the target quadrant (CBDA; p = 0.008, THCA; p = 0.030) and platform area (CBDA; p = 0.059, THCA; p = 0.114) ( Figure 3C,D). The number of times crossing the platform area was significantly reduced in Aβ 1-42 -treated mice compared with the PBS-treated mice (p = 0.025). CBDA (p = 0.026) or THCA (p = 0.044) treatment resulted in an increase in the number of crossings in Aβ 1-42 -treated mice ( Figure 3E). During the novel object phase, mice treated with Aβ 1-42 + CBDA (p < 0.001) or THCA (p < 0.001) spent more time exploring the novel object and exhibited significantly higher discrimination ratios compared with Aβ 1-42 -treated mice ( Figure 3F). In the object location test, mice treated with Aβ 1-42 + CBDA (p < 0.001) or THCA (p < 0.001) also spent more time examining the displaced object. They exhibited significantly higher discrimination ratios compared with Aβ 1-42 -treated mice ( Figure 3G).
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CBDA and THCA Treatment Decreases Aβ and p-Tau Levels in the Hippocampus of Aβ1-42-Treated Mice
In the acute AD-like mouse model injected with amyloid beta, amyloid beta aggregation, and tau pathology, which are representative pathological markers of AD, occur [34]. So, to determine the effect of CBDA and THCA on hippocampal Aβ aggregation and ptau in Aβ1-42-treated mice, hippocampal tissue was collected from five mice from each group 19 days after the initial Aβ1-42 infusion. We conducted a Western blot analysis to measure hippocampal Aβ and p-tau (AT8) expression levels. The levels of hippocampal

Discussion
The typical symptoms of Alzheimer's disease are cognitive and memory impairment [39]. Representative pathological markers of AD are considered increased Aβ and p-tau, which result in neuronal cell death [40]. The cause of Alzheimer's disease is unclear, but studies have indicated that calcium dyshomeostasis is a major contributor [41]. In neurons, calcium activates and deactivates various signaling pathways. When calcium homeostasis is dysregulated, various signaling events collapse, causing impairment of learning and memory [42]. Patients with AD have higher calcium concentrations compared with ordinary people. As a result of increased calcium concentrations, long-term potentiation cannot occur, and memory cannot form [6,43]. In addition, in AD, the expression level of BDNF and the activity of the BDNF/CREB signaling pathway are decreased [44]. BDNF is attractive as a potential evaluation marker for the efficacy of AD treatments [45,46]. Several experiments have shown that BDNF overexpression or its injection into AD animal models demonstrated therapeutic efficacy for AD [47,48]. The BDNF/CREB signaling pathway is regulated by Ca 2+ [49]. p-CREB is a transcription factor involved in BDNF expression and is activated by Ca 2+ influx. However, excessive Ca 2+ influx causes dephosphorylation of p-CREB and reduction of BDNF expression [38].
The FDA approved Memantine, an antagonist of the N-methyl-D-aspartate (NMDA) calcium receptor, as a drug for treating AD [50]. Memantine lowers intracellular calcium concentrations by inhibiting the NMDA calcium receptor [51]. A decrease in calcium concentration following Memantine treatment results in neuroprotection, learning, and cognitive function, exhibits inhibitory effects on Aβ and p-tau production and activates the BDNF/CREB signaling pathway [52,53].
Although hemp is classified as a narcotic with many restrictions, studies have demonstrated its efficacy in treating various diseases [54]. There are various species of hemp, but only the Cannabis sativa. L strain, which is categorized according to THC and CBD content, is used [55]. Moreover, its receptor, the cannabinoid receptor, performs various functions in the nervous system. Cannabinoid receptors are expressed in astrocytes, microglia, and neurons [56,57]. Furthermore, cannabinoid receptors are involved in the survival of nerve cells, synaptic plasticity, and the development of dendrites [58][59][60]. Cannabinoid receptors are expressed not only in the peripheral nervous system but also in the central nervous system [61]. Thus, it affects various neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease [62,63]. Therefore, various studies have been conducted on the function of cannabinoid receptors in the central nervous system. In particular, many studies have been conducted on neurodegenerative diseases using CBD and THC, representative agonists of cannabinoid receptors [64,65]. In addition, CBD and THC have potential therapeutic efficacy in clinical trials for Parkinson's disease [66,67]. Accordingly, the development of drugs targeting cannabinoid receptors is progressing [68].
Several studies have shown that the activation of cannabinoids has various advantages, such as neuroprotective effects [69]. More than 120 types of phytocannabinoids exist in hemp, but most studies have focused on CBD and THC [70]. However, CBDA and THCA, the acidic variants of CBD and THC, account for a large proportion of hemp leaves [22]. In a pharmacokinetics study, CBDA and THCA were present in the serum at higher concentrations compared with CBD and THC [31]. CBDA can directly affect the brain because of its ability to penetrate the BBB [32]. CBDA showed anti-convulsant, anti-hyperalgesia, anti-inflammation, anti-nausea, anti-anxiety, and anti-seizure effects in animal models [23,25,71]. In addition, THCA has BBB penetration ability; it exhibits anti-convulsant, anti-inflammation, and anti-nausea effects in animal models as well as neuroprotection by inhibiting various inflammatory cytokines in cell models [29,32,72,73]. Furthermore, CBDA and THCA inhibit calcium influx by acting as antagonists in T-type calcium channels [30]. These characteristics offer a novel approach to treating AD.
Increased intracellular calcium concentration by Aβ reduces BDNF levels [74]. In the present study, we hypothesized that CBDA and THCA normalize calcium concentration to increase BDNF levels and inhibit Aβ and p-tau production, thereby inhibiting neuronal apoptosis and improving cognitive function. We found that CBDA and THCA modulate Ca 2+ influx, exhibit neuroprotective effects, and reduce Aβ and p-tau production against Aβ 1-42 in primary neurons. In addition, CBDA and THCA decreased the production of Aβ and p-tau, promoted CREB phosphorylation, a transcription factor of BDNF, and consequently increased the expression of BDNF and its receptor, p-TrkB, in the hippocampus of Aβ 1-42 -treated mice. CBDA and THCA rescued object and spatial cognitive function and memory deficits in Aβ 1-42 -treated mice. Overall, these results suggest that CBDA and THCA ameliorate AD-like features by modulating Ca 2+ homeostasis, which is fundamental to neuronal viability and function.

Animals
Female ICR mice (8 weeks) were purchased from the Koatech company (Pyeongtaek, Republic of Korea). The mice were housed in the animal care facility (temperature 22 ± 2 • C; humidity 40-60%, and a 12 h light/dark cycle) at the Korea Institute of Science and Technology (KIST). The mice were provided food and water ad libitum.

Primary Neuronal Culture
As previously described, the cerebral cortical tissue was dissected from day 15 embryonic ICR mice [75]. Cells were isolated by digestion with 0.05% trypsin and re-suspended in minimal essential medium containing 10% heat-inactivated horse serum, 10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin. The isolated cortical neurons were allowed to adhere to 0.2 mg/mL poly-D-lysine-coated culture dishes for 45 min and cultured in neurobasal medium supplemented with B27 (Gibco, Waltham, MA, USA), 1 mM glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin. Cultures at 6 days were treated with Aβ 1-42 and/or CBDA or THCA for 24 h, and the neurons were harvested for Western blot analysis to measure APP/Aβ, tau, and p-tau levels.

Cell Viability
Primary neurons (5 × 10 5 cells/well) were seeded into 96-well plates for 6 days. Aβ 1-42 and/or CBDA or THCA was added to the cells for 24 h. MTT solution (Invitrogen, Carlsbad, CA, USA) was added to the medium and incubated for 2 h. Cytotoxicity was measured using a microplate spectrophotometer (Bio-Tek Power Wave XS, Winooski, VT, USA) at 490 nm.

Fluorescence Ca 2+ Imaging
Primary neurons, 2 × 10 6 cells per well, were cultured in 6-well for 6 days to evaluate intracellular Ca 2+ . Cultures at 6 days were treated with Aβ 1-42 and/or CBDA or THCA for 24 h, washed with PBS, and loaded with 10 µM Ca 2+ indicator Fluo-4 AM (Invitrogen, Carlsbad, CA, USA) for 1 h. The stained sections were cleaned with PBS, a coverslip with Prolong Gold Antifide Reagent containing DAPI nuclear stain was added (Invitrogen, Carlsbad, CA, USA), and the samples were examined using a microscope (Carl Zeiss, Oberkochen, Germany). Regions of interest that were 422,500 µm 2 were randomly selected from each well and measured 3-4 areas per well. The Image J analysis program measured the entire fluo-4 AM fluorescence signal intensity (National Institute of Health, Bethesda, MD, USA).

Intrahippocampal Stereotaxic Injection of Aβ 1-42
Eight-week-old mice were acclimatized to laboratory conditions for one week and

Morris Water Maze Test
A modification of the water maze procedure described by Morris was used to examine cognitive function [76]. A circular tank (diameter 90 cm, height 50 cm; 22 ± 2 • C water temperature) was used for the test. The tank consisted of four quadrants filled with water. An escape platform (6 cm diameter and 29 cm height) was submerged 1 cm below the water surface at the center of one of the four quadrants. Each mouse was trained for 4 days to learn and memorize visual cues placed outside the tank, which indicated platform location. The swimming paths used by each mouse were recorded with a camera connected to a video recorder and path tracking software XT (EthoVision; Noldus Information Technology, Wageningen, The Netherlands). Four trials were performed each day during the 4-day training period. During each trial, each mouse was allowed 60 s to find the hidden platform and another 30 s to stay on the platform. If the mouse was unable to find the platform within 60 s, it was guided to it and allowed to remain for 30 s. The mean time (mean escape latency) that each mouse took to find the platform was recorded. The probe test was conducted after 4 days in the same manner without the platform. Each mouse was allowed 60 s to move freely and was recorded. The video was analyzed using tracking software (EthoVision; Noldus Information Technology, Wageningen) to count the time spent in the target quadrant area and platform areas and the number of crossovers.

The Novel Object Recognition Test
We used a modified novel object recognition test, which incorporates the natural tendency of a mouse to explore novel stimuli [77]. During a habituation session performed 2 days before testing, the mice were allowed to explore (for 10 min) a test environment consisting of an empty opaque, custom-made Plexiglas box (35 cm × 45 cm × 25 cm). The sample object phase was introduced 24 h later. Two identical white circular cylinders (the sample objects) were placed on the facing edge in the test environment, and the mice were given access to the objects for 10 min. After 24 h (the novel object phase), one of the sample objects in the test environment was replaced with a similar-sized novel object (a colored miniature animal), and the mice were given 5 min of contact with this new arrangement. The time that the animal's nose was <1 cm from an object was considered the time it navigated the object. The amount of time that the mouse stood on the object was excluded. The discrimination ratio was the time used to navigate the novel object over the time used to navigate both objects.

Object Location Test
The object location test was conducted in the same manner as described for the novel object recognition test. On the last day of the object location test, one of the two sample objects was moved to a different location.

Statistical Analysis
SPSS 19.0 for Windows (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. The results were presented as mean ± standard error of the mean (SEM) values. Mean escape latency results for the Morris water maze test were analyzed using two-way repeated measures ANOVA, followed by Bonferroni's test. The other data were analyzed using one-way ANOVA followed by Fisher's LSD.
Author Contributions: J.K. was responsible for the study design, performed the experiments, and wrote the initial draft of the manuscript. P.C. and T.K. provided materials and instruments. Y.-T.P. contributed to the interpretation of results. J.H. and J.-C.K. were responsible for the concept and design of the study and supervised the work. All authors have read and agreed to the published version of the manuscript.