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Activation of cannabinoid receptor 2 protects rat hippocampal neuronsagainst A�-induced neuronal toxicity

Jingfu Zhao, Mengzhen Wang (Visualization) (Investigation), WeiLiu (Visualization) (Investigation), Zegang Ma (Supervision) (Writing- review and editing), Jie Wu

PII: S0304-3940(20)30477-8

DOI: https://doi.org/10.1016/j.neulet.2020.135207

Reference: NSL 135207

To appear in: Neuroscience Letters

Received Date: 24 April 2020

Revised Date: 29 May 2020

Accepted Date: 23 June 2020

Please cite this article as: Zhao J, Wang M, Liu W, Ma Z, Wu J, Activation of cannabinoidreceptor 2 protects rat hippocampal neurons against A�-induced neuronal toxicity,Neuroscience Letters (2020), doi: https://doi.org/10.1016/j.neulet.2020.135207

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© 2020 Published by Elsevier.

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Activation of cannabinoid receptor 2 protects rat hippocampal neurons against

Aβ-induced neuronal toxicity

Jingfu Zhao, Mengzhen Wang, Wei Liu, Zegang Ma, Jie Wu*

Department of Physiology, School of basic medicine, Institute of Brain Science and

Diseases, Qingdao University, Qingdao 266071, China

Correspondence:

*Jie Wu, MD, PhD

Professor

Institute of Brain Science and Diseases, Qingdao University

Qingdao, 266071, P.R. China

E-mail: jiewubni@gmail.com

Running head: Cannabinoid receptor 2 protects Aβ toxicity

Highlights

Chronic treatment with amyloid beta peptide 1-42 (Aβ1-42) oligomers for 7 days

induces hippocampal neuronal toxicity and upregulated cannabinoid receptor 2

(CB2R).

Activation of CB2Rs by agonist (JWH133) prevents Aβ1-42-induced

neurotoxicity.

Activation of CB2Rs enhances Akt signaling that is involved in CB2R’s neuronal

protective effects.

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Abstract

Alzheimer’s disease (AD) is a dementing, neurodegenerative disorder characterized by

increased accumulation of beta-amyloid peptides (Aβ), degeneration of hippocampal

neurons and the gradual development of learning and memory deficits. Therapeutically,

there are still no ideal medicines available and this represents an urgent need for the

development of new strategies to treat AD. Emerging lines of evidence suggest that

modulation of the cannabinoid system exhibits neuroprotective effects in various

neurological diseases, including AD. However, a consensus is yet to emerge as to the

impact of hippocampal cannabinoid receptor 2 (CB2R) in protection of hippocampal

neurons against Aβ induced neuronal toxicity. Here, we report that chronic treatment of

primary hippocampal neuronal cultures with 100 nM Aβ1-42 oligomers for 7 days results

in neurotoxicity, which includes increases in lactate dehydrogenase (LDH) levels,

suggesting an Aβ1-42 –induced neuron apoptosis. Further, chronic Aβ1-42 reduces the

ratio of phosphorylated Akt (pAkt)/Akt, in turn decreases neuronal Bcl-2/Bax ratio, and

leads to an increase of caspase-3, which likely underlines the signal pathway of chronic

Aβ1-42–induced neuron apoptosis. Interestingly, pre-treatments of CB2R agonist

(JWH133, 10 µM) with Aβ1-42 prevents Aβ1-42-induced the decrease of pAkt/Akt ratio,

the decrease of Bcl-2/Bax ratio, and the increase of caspase-3, and protects

hippocampal neurons against Aβ1-42-induced apoptosis. All neuroprotective effects of

JWH133 are abolished by a selective CB2R antagonist, AM630. Taken together, the

activation of hippocampal CB2Rs protects neurons against Aβ1-42 toxicity, and the

CB2R-mediated enhancement of the pAkt signaling is likely involved in the protection

of hippocampal neurons against Aβ1-42-induced neuronal toxicity.

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Abbreviations

Aβ , amyloid beta peptide; CB2R, cannabinoid receptor 2; LDH, lactate dehydrogenase;

AD, Alzheimer’s disease; Bcl-2, B-cell lymphoma

Keywords: CB2 receptor; Aβ1-42; hippocampal neurons; JWH133; Alzheimer’s disease

1. Introduction

Alzheimer’s disease (AD) is a common neurodegenerative disease that occurs

frequently in elderly patients and is characterized by loss of hippocampal neurons.

AD patients typically experience confusion, disorganized thinking, impaired judgment,

trouble expressing themselves and disorientation with regard to time, space, and

location [1]. The main pathological feature of AD is the appearance of senile plaques

(SP) in the brain and the formation of neurofibrillary tangles (NFT) in neurons [2].

Although the exact pathogenesis of AD remains unclear, genetic studies have indicated

that at least four mutations or genetic polymorphisms are associated with AD, including:

Amyloid precursor protein (APP) on chromosome 21, Presenilin-1 (PS-1) on

chromosome 14, Presenilin-2 (PS-2) on chromosome 1 and Apolipoprotein (APOE) on

chromosome 19 [3-6]. Moreover, mutations in these genes are related to the synthesis

and aggregation of pathologic Amyloid β-peptides (Aβ).

Usually, extracellular Aβ aggregates into soluble oligomers and gradually forms

fibrils. Both prefibrillar soluble oligomers and fibrillar Aβ show toxic effects on

neurons, causing neurofibrillary tangles, synaptic impairment, neuronal hyper-

excitation and eventually neuronal degeneration [7-10]. Accumulating lines of evidence

indicate that Aβ-induced neurotoxic effects are mediated through a reduction of

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PI3K/Akt signaling and the enhancement of PI3K/Akt signaling shows the

neuroprotective effects [11-15]. Although the downstream of PI3K/Akt is involved in

other signal pathways [16], the PI3K/Akt – Bcl/Bax – caspase-3 signaling plays an

important role in the mediation of cell apoptosis [17, 18] including the Aβ-induced

toxicity [19, 20]. Therefore, in this study, we focused on Aβ – PI3K/Akt – Bcl/Bax –

caspase-3 – cell apoptosis signal pathway and evaluate the roles of cannabinoid receptor

2 (CB2R) in modulation of this signal pathway and in protection of Aβ-induced

neuronal apoptosis.

Cannabinoid receptors belong to a G-protein-couple receptor family and includes

cannabinoid receptor 1 (CB1R) and CB2R. CB1R is prominently expressed in the central

nervous system (CNS) and CB2R is mainly expressed in the periphery where it is found

on cells of the immune system [21]. Recent studies have demonstrated that CB2R is

also expressed in the neurons of the CNS, albeit at a much lower level of expression

when compared to CB1R [22, 23]. However, CB2R exhibits a unique inducible feature,

which means its properties are alterable under different pathological conditions. For

example, the level of CB2R mRNA expression in the CNS is up regulated in the context

of some brain diseases such as stroke, epilepsy and drug addiction [24-28]. In addition,

activation of CB2Rs on brain microglia serve in a neuroprotective role following

intracerebral hemorrhage [29], and ischemic/reperfusion injury [30]. Furthermore, a

recent study shows that activation of CB2Rs on microglia in the hippocampal CA1

region exert neuroprotective effects in a model of vascular dementia [31] and can

control epileptic seizures. Together, this evidence clearly demonstrates that CB2Rs can

modulate immune function and neuroinflammatory responses in the CNS. However,

CB2Rs are not only expressed on glial cells, but expression has also been documented

in central neurons, including those of the hippocampus [32]. In hippocampal neurons,

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CB2Rs are expressed either on the cell body or on medium-sized dendrites [33] and

play a role in modulation hippocampal network function [32].

To assess the role of CB2R activation in protection following Aβ-induced

hippocampal neuronal apoptosis, we utilize rat hippocampal neuronal cultures to verify

the protective roles of CB2Rs and investigate possible signaling pathways involved.

2. Materials and Methods

2.1 Preparation of rat hippocampal primary neuron cultures

The protocol for preparation of neuronal cultures from rodents was approved by the

Institutional Animal Care and Use Committee of the Qingdao University. The day

before culture, poly D-lysine (0.02% solution) was added to culture dishes. Dishes were

swirled to make sure that the entire bottom was coated and then dishes were left in a

37˚C/5% CO2 incubator overnight. On the next day of culture, dishes were washed three

times with sterile water and left in the incubator after the final wash. 0-1-day-old,

postnatal SD rats were sacrificed and the CA1-CA3 region of the hippocampus was

dissected under a stereological microscope. Tissue was minced with scissors in ice-cold

Neurobasal medium (Invitrogen, Carlsbad, CA) and then digested with Papain (20

unit/mg, Worthington, Lakewood, NJ) at 30oC for 20 min in tubes shaken at 120 rpm

in a water bath shaker. After enzyme digestion, the reaction was halted by adding

inactivated fetal bovine serum to the medium. Then, the digested tissue was filtered and

transferred into 15 ml tubes. Following trituration, tissue was centrifuged at 1500 rpm

for 3 min to form pellets containing dissociated cells and the supernatant was removed

and replaced with Neurobasal medium, which was used to re-suspend the pellets and

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the process was repeated 3 times. After the final centrifugation, the supernatant was

replaced with Neurobasal medium supplemented with 0.5% (w/v) L-glutamine and 2%

B27 serum-free supplement. Cells were suspended and counted based on Trypan blue

exclusion and plated at a density of 1.0X106 cells per well in culture dishes. Cells were

kept within the 37˚C/5% CO2 incubator for future use.

2.2 Aβ preparation and treatment

Aβ1-42 peptides were purchased from the Sigma Aldrich. Based on the introduction of

the preparation of the oligomer form of Aβ1-42 peptide, it was dissolved in 1,1,1,3,3,3-

hexafluoro-2-propanol at a concentration of 1 mol/L in 2.217 mL aliquots, air dried in

the fume cupboard and stored at -20°C. The clear film obtained after HFIP volatilizing

was re-suspended in dimethyl-sulfoxide and was further diluted using the PBS (pH 7.4)

to a final concentration of 100 μM and incubated at 4 °C for 24h without shaking.

Following incubation, centrifuged at 13000 rpm, for 10 min in the cold, transferred

supernatant to a new tube, and the oligomeric Aβ1-42 was prepared.

Primary cultured neurons were maintained at 37°C 5% CO2 in an incubator for 7-

8 days before Aβ exposure. The Aβ-containing culture medium (100 nM) was

replenished daily for the next 7-8 days as previously described [34]. The same

procedure was followed with medium for the control group but Aβ exposure was

included. For pharmacological studies, the CB2R agonist (JWH133) or antagonist

(AM630) was applied for 40 minutes before Aβ exposure.

2.3 Neuronal viability Assay

The neuronal viability assay was performed using a lactate dehydrogenase (LDH)

reagent kit[34]. LDH is an oxidoreductase enzyme that catalyzes the interconversion of

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pyruvate and lactate. Cells release LDH into the culture supernatant after their

membrane is damaged. In a 96-well plate we added 5 μL of culture supernatant into

duplicate wells and the samples were brought to a final volume of 50 μL with LDH

Assay Buffer. Then, 50 μL of the Master Reaction Mix was added to each of the wells.

The plate was incubated at 37℃ and measurements (A450) were taken every 5 minutes

using a microplate reader (Rayto RT-2100C, Shenzhen, China). Calculate the change in

measurement using Tinitial to Tfinal from the samples.

2.4 Western blot

Primary cultured neurons were lysed with RIPA buffer and phenylmethylsulfonyl

fluoride (99:1) on ice for 30 minutes and the suspension was centrifuged at 4°C, 12000

rpm for 20 minutes. Then, the supernatant was collected, and protein concentrations

were detected. Proteins were separated using 12.5% SDS-polyacrylamide gel

electrophoresis and transferred to a methanol-activated PVDF membrane. The PVDF

membrane was blocked using TBST containing 5% skim milk powder for 2 h. The

membrane was then incubated with various monoclonal antibodies, such as β-actin

(1:10000, Affinity, AF7018), Bax (1:1000, Cell Signaling，5023S), Bcl-2 (1:1000,

Affinity, AF6139), phosphorylated Akt (p-Akt, 1:1000, Cell Signaling, 4060S) and Akt

(1:1000, Cell Signaling，4691S) at 4°C overnight. After being rinsed with TBST three

times, the membrane was incubated for 1 h with anti-rabbit HRP conjugated secondary

antibody (1:10000). The bands were detected with enhanced chemiluminescence (ECL)

reagent and images were collected using Tanon Image System.

2.5 Quantitative real-time PCR

qRT-PCR was performed using the TB Green QPCR Master Mix Kit (Takara Bio, Inc.,

Otsu, Japan) to measure CB2R mRNA and caspase-3 mRNA expression levels. Total

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RNA was extracted from hippocampal neuronal cultures with 1000 μL trizol. RNA was

evaluated spectrophotometrically for quantity and purity, and 0.5 μg of RNA was

reverse transcribed using a reverse transcription kit (Vazyme Biotech Co., Nanjing,

China). Then, the complementary DNA (cDNA) was obtained for qRT-PCR. Cycling

conditions contained an initial denaturation at 95 °C for 30 s followed by 40 cycles of

amplification at 95 °C for 5 seconds for denaturation, 60 °C for 30 seconds for

annealing. The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

was used to normalize target gene expression, and the 2-△△Ct method was used to

calculate the amount of the target gene.

The following primer sequences (5′-3′) were used for qPCR:

CB2R: forward 5’-TGG CAG CGT GAC TAT GAC-3’, reverse 5’- AAA GAG GAA

GGC GAT GAA-3’

Caspase-3: forward 5'-GTG GAA CTG ACG ATG ATA TGG C-3', reverse 5'-CGC

AAA GTG ACT GGA TGA ACC-3'

GAPDH: GAPDH: forward 5′-GGC ACA GTC AAG GCT GAG AAT G-3′, reverse

5′-ATG GTG GTG AAG ACG CCA GTA-3′.

2.6 Chemicals and reagents

Neurobasal-A(10888022) and B27(175 04044t) were purchased from Gibco(Grand

Island, New York, USA). Aβ1-42(SCP0038), 1,1,1,3,3,3-Hexafluoro-2-propanol

(52517), poly-D(p6407), Lactate Dehydrogenase Activity Assay Kit (MAK066) were

purchased from Sigma Chemical Co (St. Louis, MO, USA). CB2 receptor agonist JWH-

133 [3-(1,1-dimethylbutyl)-6aR,7,10,10aR-tetrahydro-6,6,9-trimethyl-6H-dibenzo

[b,d]pyran] and CB2 receptor antagonist AM-630 [6-Iodo-2-methyl-1-[2- （ 4-

morpholinyl ） ethyl]-1H-indol-3-yl]（4-methoxyphenyl）methanone] were purchased

from Sigma Chemical Co (St. Louis, MO, USA).

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2.7 Data analysis and statistics

Data are presented as mean ± SEM with number of samples (n). A probability level of

p<0.05 was considered to be statistically significant. Significant differences were

determined using the two-tailed Student’s t-test or one-way ANOVA as appropriate.

3. Results

3.1 Chronic Aβ1-42 upregulates CB2R mRNA expression in primary hippocampal

cultures

In initial experiments, we asked whether chronic treatment with Aβ1-42 (oligomer, 100

nM for 7 days) altered CB2R mRNA expression. The results collected from 4 repeated

experimental measurements, and demonstrated an increased level of CB2R mRNA

expression after chronic exposure of hippocampal cultures to Aβ1-42. As shown in Fig.

1, cell CB2R mRNA levels (CB2R/GAPDH gene expression) in control and Aβ1-42

treated groups were 1.01±0.05 and 2.00±0.10 (Unpaired Student T-test, t=9.360, df=6,

p<0.001, n=4), respectively. These results suggest that Aβ1-42 chronic treatment

enhances CB2R mRNA expression.

Figure 1 near here

3.2 The CB2R agonist JWH-133 protects hippocampal neurons against the Aβ1-42-

induced the enhancement of LDH and caspase-3 in hippocampal neuronal cultures

To examine the effects of the CB2R agonist, JWH133, on Aβ1-42-induced increases in

both LDH and caspase-3 levels, we measured LDH release and caspase-3 gene

expression in 4 experimental groups: control (untreated), Aβ1-42, Aβ1-42 + JWH133 and

Aβ1-42 + JWH133 + AM630 (CB2R antagonist). In these Aβ groups, hippocampal

neuronal cultures were treated with oligomeric Aβ1-42 100 nM for 7 days, and the culture

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medium (contained oligomeric Aβ1-42 100 nM) was changed every day. When JWH133

(or JWH133+AM630) was applied, it was pretreated to cells for 50 min, then, Aβ1-42

was added. Figure 2A shows that Aβ1-42 + JWH133 treatment prevented the Aβ1-42-

induced increase in LDH levels. As expected, in the Aβ1-42 + JWH133 + AM630

treatment group the effect of JWH133 was abolished due to the presence of the

antagonist, AM630 (One-Way ANOVA F3, 12=20.60, p<0.001, n=4). The same altered

pattern occurred in caspase-3 mRNA level changes in the above 4 experimental groups

(One-Way ANOVA F3, 12=19.38, p<0.001, n=4, Fig. 2B). These findings suggest a

pivotal role for CB2R activation in the protection of hippocampal neurons against the

Aβ-induced increase in both LDH and caspase-3 levels.

Figure 2 near here

3.3 Possible signaling pathways that underlie CB2R-mediated neuronal protection

Emerging evidence suggests that activation of PI3K-Akt signaling inhibits Aβ-induced

toxicity and formation of neurofibrillary tangles, leading to a protection of neurons

against apoptosis, and the activation of PI3K-Akt signaling has been considered as a

new approach to treat neurodegenerative diseases including AD [35]. To elucidate

whether PI3K-Akt signaling that may underlie the CB2R mediated neuronal protection

against Aβ1-42 toxicity, we examined Akt phosphorylation (pAkt) and Akt levels, and

compared pAkt/Akt ratio after chronic treatments with Aβ1-42, Aβ1-42 + JWH133, Aβ1-

42 + JWH133 + AM630, or control group, respectively. The results showed that the

difference of pAkt protein expression in 4 experimental groups is highly significant

(One-Way ANOVA F3, 12=11.29, p<0.001, n=4, Fig. 3B), and the difference of Akt

expression in 4 experimental groups is also highly significant (One-Way ANOVA F3,

12=6.87, p<0.01, n=4, Fig. 3D). Then, we compared pAkt/Akt ratio in 4 experimental

groups using One-Way ANOVA Tukey’s multiple comparison. Results showed that

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compared to control group, pAkt/Akt ratio in Aβ1-42 group was reduced, (p<0.05); in

Aβ1-42 + JWH133 group, pAkt/Akt ratio was up-regulated compared to the Aβ1-42 alone

group (p<0.01); and in Aβ1-42 + JWH133 + AM630 group, pAkt/Akt ratio was lower

than that in the Aβ1-42 + JWH133 group (p<0.01). These results suggest that JWH133

protects against Aβ1-42-induced apoptosis of hippocampal neurons, which is involved

in a CB2R-mediated enhancement of pAkt signaling.

Figure 3 near here

3.4 CB2R agonist, JWH-133 protected hippocampal neurons against the Aβ1-42-

induced reduction of Bcl-2/Bax ratio in hippocampal neuronal cultures

In addition, we evaluated the role of JWH133, and thus CB2R activation, in Aβ1-42 –

induced alterations of Bcl-2, Bax, and Bcl-2/Bax ratio. As shown Fig. 4 by Western-

blot measurements that the difference of Bcl protein expression in 4 experimental

groups is highly significant (One-Way ANOVA F3, 12=46.26, p<0.01, n=4, Fig. 4B),

and the Bax protein expression in 4 experimental groups is significant (One-Way

ANOVA F3, 12=12.52, p<0.05, n=4, Fig. 4D). Then, we compared Bcl/Bax ratio in 4

experimental groups. Results showed that the control, the Aβ1-42, the Aβ1-42 + JWH133,

or the Aβ1-42 + JWH133 + AM630 group had a ratio of Bcl/Bax of 1.79 ± 0.28, 0.51 ±

0.09, 2.18 ± 0.09, 0.51 ± 0.09, respectively (One-Way ANOVA F3, 12=22.12, p<0.001,

n=4, Fig. 4F). Further Tukey’s multiple comparison in Fig. 4F showed that compared

to the control group, Bcl/Bax ratio in Aβ1-42 group was reduced (p<0.01); in Aβ1-42 +

JWH133 group, Bcl/Bax ratio was increased compared to the Aβ1-42 alone group

(p<0.001); and in Aβ1-42 + JWH133 + AM630 group, Bcl/Bax ratio was lower than that

in the Aβ1-42 + JWH133 group (p<0.001). These results suggest that chronic treatment

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with Aβ1-42 induces a reduction of Bcl/Bax ratio in cultured hippocampal neurons, and

that JWH133, through activation of CB2Rs, reverses the effects of Aβ1-42.

Figure 4 near here

4. Discussion

The major and new finding put forward in this study is that the activation of

hippocampal CB2Rs protects these neurons against toxicity induced by Aβ1-42

application. We first demonstrated that chronic treatment of hippocampal primary

neuronal cultures with oligomeric Aβ1-42 enhanced levels of LDH in the culture medium

and caspase-3 mRNA expression, suggesting a caspase-3 signal mediated cell apoptosis.

We used this cellular model of Aβ1-42-induced toxicity to evaluate the protective effects

of CB2R agonist treatment on hippocampal neuronal toxicity. Our results showed that

chronic treatment with oligomeric Aβ1-42 increased CB2R mRNA expression levels,

suggesting that hippocampal CB2Rs participate in the process of Aβ1-42-induced toxicity.

Furthermore, we illustrated that JWH133, a selective CB2R agonist, prevented the Aβ1-

42-induced toxicity and that this effect can be abolished by application of AM630, a

selective CB2R antagonist, suggesting that the neuroprotective effect of JWH133 is

mediated through the neuronal CB2Rs of hippocampal cultures. Finally, we revealed

that the CB2R-mediated neuronal protection is likely regulated through activation of the

Akt signaling pathway.

AD is a neurodegenerative dementia characterized by increased accumulation of

beta-amyloid peptides (Aβ), gradual degeneration of neurons of the CNS and

progressive deficits in learning and memory. Additionally, Aβ accumulation and

aggregation in neuritic or senile plaques along with severe, selective cholinergic neuronal

deficits are characteristic hallmarks of AD [36]. Processes such as impairment of

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neurotrophic support and disorders of glucose metabolism have been implicated in

cholinergic neuronal loss and AD [37]. However, clear, neurotoxic effects of Aβ across

a range of in vivo or in vitro models suggests that Aβ plays a role in cholinergic neuronal

degeneration and the consequent deficits in learning and memory [36, 38], but the

mechanisms underlying this process are still unclear. Therefore, a better understanding

of such mechanisms is likely to help improve AD diagnosis and treatment. Recently,

we have established a cellular model of Aβ toxicity as a result of chronic exposure to

Aβ1-42 aggregates in rodent hippocampal primary cultures, in which, chronic Aβ1-42

aggregates-induced neuronal hyper-excitation and toxicity [39]. In this study, we used

this same experimental protocol to treat rat hippocampal cultures with Aβ1-42 (oligomers,

100 nM for 7 days), which we then evaluated for measures of neuronal degeneration

such as LDH level and caspase-3 mRNA levels. We found that chronic Aβ1-42 induced

an increase of both LDH release and caspase-3 levels. These results confirm that the

cell model of Aβ toxicity is repeatable and reliable and can be used to evaluate the impact

of CB2R activation in the protection of hippocampal neurons against Aβ induced toxicity.

Although the mechanisms of Aβ-induced neuronal toxicity are still unclear,

emerging evidence suggests that Aβ-induced reduction of PI3K/Akt signaling plays an

important role in mediation of cell apoptosis, and the activation of PI3K/Akt signaling

exhibits neuronal protection against the Aβ-induced toxicity and formation of

neurofibrillary tangles, leading to a protection of neurons against apoptosis, and the

activation of PI3K/Akt signaling has been considered as a new approach to treat

neurodegenerative diseases including AD [35]. The PI3K/Akt signaling pathway is

essential for cell survival as activated Akt affects numerous factors involved

in apoptosis, either by transcription regulation or direct phosphorylation[40]. Many

stimulants including neurotrophins are reported to activate this pathway in preclinical

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studies for neuronal protections [41]. Interestingly, it has been reported that activation

of CB2R induced a phosphorylation of Akt at the S473 and T308 residues [42],

suggesting a potential of CB2R-mediated neuroprotection is mediated through the

enhanced PI3K/Akt signal pathway.

CB2R is a G-protein-coupled receptor that was first cloned in 1993. CB2R has been

considered a “peripheral” cannabinoid receptor [43-45] but this concept has been

challenged by the identification of CB2Rs throughout the central nervous system [43,

44]. When compared with CB1Rs, brain CB2Rs possess some unique characteristics,

such as lower expression levels, highly inducible under some pathological conditions

(e.g., addiction, inflammation, anxiety, etc.), and characteristics patterns of distribution

in brain areas that extends into sub-neuronal compartments (e.g. neuronal

somatodendritic areas) [46]. Considering these features, CB2Rs appear to be an

important substrate for neuroprotection [47] and targeting CB2Rs is likely to offer a

novel therapeutic strategy for treating neuropsychiatric and neurological diseases

without typical CB1R-mediated side effects [48].

Emerging evidence demonstrates that CB2Rs exhibit neuroprotective roles during

the pathogenesis of AD. For example, it has been reported that an increase in CB2R

numbers expressed on microglia surrounding senile plaques [49, 50] and that this

increased expression is correlated with Aβ1-42 levels and plaque deposition, though not

with cognitive status [50]. These data suggest that enhanced CB2R expression and the

activation of these receptors stimulates amyloid removal by human macrophages [51].

Moreover, alternatively increased CB1R and CB2R expression observed during AD

pathogenesis is time dependent. For instance, the level of activity displayed by the

hippocampal and frontal cortex CB1R is greater in the early stages of AD but is reported

to decrease as the disease progresses [52]. This is in contrast to CB2Rs, which are

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expressed to a greater extent during the advanced stages of AD when

neuroinflammation is more evident and microglia and astrocytes are activated [53].

Collectively, there is a clear rationale to evaluate pharmacological effects of neuronal

protection using CB2R agents.

In this study, we examined the effects of a selective CB2R agonist, JWH133, on the

chronic Aβ1-42 treatment-induced neuronal toxicity in rat primary hippocampal cultures.

Our results showed that pre-treatment with JWH133 and Aβ1-42 significantly prevented

the Aβ1-42 –induced increases of both LDH levels and caspase-3 mRNA expression level,

suggesting a significant protection of the Aβ1-42 –induced neuronal apoptosis. These

effects of JWH133 were abolished by application of a selective CB2R antagonist

AM630, which suggests a neuroprotective effect for the activation of hippocampal

CB2Rs in the context of Aβ1-42 –induced neuronal toxicity. Since under our hippocampal

culture conditions, there are most the primary hippocampal neurons without glia cells,

our finding suggests that JWH133 activates neuronal, rather than glia cells such as

microglia or astrocytes, CB2Rs. Our finding is supported by the evidence that mouse

hippocampal neurons express CB2Rs, which plays a critical role in the modulations of

neuronal excitation, synaptic function the neuronal network synchronizations. Our

results extend these knowledges and suggest that CB2Rs on hippocampal neurons are

also involved in neuronal protection. Furthermore, we elucidated the possible signaling

pathways that mediated the CB2R-induced protective roles. We found that JWH133

prevented the reduced ratio of p-Akt/Akt induced by the Aβ1-42 chronic exposure,

suggesting that the CB2R-induced enhancement of p-Akt signaling likely underlies the

CB2R’s neuronal protection. Although the precise molecular mechanisms of the

CB2R— PI3K/Akt signaling pathway mediating neuronal protection are still unclear,

we postulate the interpretations of the CB2R-induced protective effects on the Aβ1-42 –

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induced neuronal toxicity based on our data and existed lines of evidence. After chronic

treatment of cultured hippocampal neurons with Aβ1-42 (100 nM, oligomers for 7 days),

hippocampal CB2Rs are doubly upregulated, and when JWH133 (10 µM, 50 min

pretreated before Aβ1-42) is treated with Aβ1-42, hippocampal CB2Rs are activated, and

the p85 regulatory subunit of PI3K moves to the vicinity of the cell membrane and

combines with the p110 subunit to convert PIP2 to PIP3, which then binds to the protein

kinase B (Akt) PH domain, and allows Akt to translocate to the cytoplasmic membrane

[54-56]. Then, the activated Akt plays a protective role by phosphorylating multiple

target proteins such as phosphorylated Bcl-2 family BAD to prevent binding with Bcl-

2 and Bcl-XL, and this enhanced signal signaling is opposite to Aβ’s effects, thereby

protects neurons against apoptosis [56, 57] (Fig. 5).

5. Conclusion

In hippocampal primary cultures, chronic treatment with Aβ1-42 induces neuronal

toxicity and an upregulation of CB2Rs mRNA. Pre-treatment with a selective CB2R

agonist, JWH133 enhances phosphorylated Akt signaling and prevents the Aβ1-42-

induced toxicity, and CB2R antagonist (AM630) abolishes JWH133’s protection. Taken

together, the activation of hippocampal CB2Rs protects neurons against Aβ1-42

toxicity, and the CB2R-mediated elevation of the Akt signaling is likely involved in the

protection of hippocampal neurons against Aβ1-42-induced neuronal toxicity.

Author contributions

J.W. bores the responsibility for the experimental design, analysis of the data, made the

figures and wrote the manuscript. J.Z., performed most experiments, participated in the

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study design and acquired the data. M.W., W.L. performed some experiments. Z.M.

participated in the study design and revised the article. All authors contributed

substantially to this work and approved the final manuscript.

Funding, declaration of interest, and acknowledgements

This study was supported by a research grant from the CNSF (81371437), China.

Disclosure: All authors have nothing to disclose. We thank Dr. Harrison Stratton for

his help to correct and edit the English writing of this manuscript.

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Figure 1 Chronic Aβ1-42 upregulated CB2R mRNA expression in primary hippocampal

cultures. Bar graph shows that compared to control cells (untreated), chronic Aβ1-42

treatment (oligomeric, 100 nM, for 7 days) upregulated CB2R mRNA expression in

primary hippocampal cultures. In this and following figures, the columns are presented

as the Mean±SE, and the *** indicates p<0.001.

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Figure 2 CB2R agonist, JWH-133 protected hippocampal neurons against the Aβ1-42-

induced increase of LDH and caspase-3 in hippocampal neuronal cultures. In primary

hippocampal cultures, the either culture medium LDH (A) or neuronal caspase-3

mRNA (B) were measured in 4 different experimental groups, including: control, Aβ1-

42, Aβ1-42 + JWH133, and Aβ1-42 + JWH133 + AM630. Compared to control, chronic

treatment with Aβ1-42 increased the levels of either LDH release (A) or caspase-3 mRNA

(B). However, with pre-treatment with CB2R agonist JWH133 for 50 min, the Aβ1-42-

induced increase of either LDH or caspase-3 has been prevented, and the JWH133’s

effects can be abolished by a CB2R antagonist, AM630.

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Figure 3 JWH133 protected hippocampal neurons against the Aβ1-42-induced changes

of protein expression of pAkt, Akt, and pAkt/Akt ratio in hippocampal neuronal

cultures. Raw data showed the changes of protein expression using Western-blot of

pAkt (A), Akt (C), and pAkt/Akt ratio (E) in control, Aβ1-42, Aβ1-42 + JWH133, and

Aβ1-42 + JWH133 + AM630 groups. Bar graph summarizes 4 groups of experiments

and showed that JWH133 prevented Aβ1-42 – induced protein alterations of pAkt (B),

Akt (D), and pAkt/Akt ratio (F), which could be abolished by the AM630.

Figure 4 JWH-133 protected hippocampal neurons against the Aβ1-42-induced changes

of protein expression of Bcl-2, Bax, and Bcl-2/Bax ratio in hippocampal neuronal

cultures. Raw data showed the changes of protein expression using Western-blot of Bcl

(A), Bax (C), and Ncl/Bax ratio (E) in control, Aβ1-42, Aβ1-42 + JWH133, and Aβ1-42 +

JWH133 + AM630 groups. Bar graph summarizes 4 groups of experiments and showed

that JWH133 prevented Aβ1-42 – induced protein alterations of Bcl (B), Bax (D), and

Bcl/Bax ratio (F), which could be abolished by the AM630.

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Figure 5 Carton picture shows a possible signal pathway of the Aβ-induced

neurotoxicity and the CB2R-mediated neuroprotection in hippocampal neurons. With

chronic Aβ1-42 treatments, Aβ1-42 reduces pAkt/Akt ratio, in turn decreases BcL/Bax

ratio on the mitochondria, leads to an increase of caspase-3, and results in neuron

apoptosis. On the other hand, chronic Aβ1-42 treatment with JWH133 pretreatment

increases pAkt/Akt ratio, in turn increases BcL/Bax ratio on the mitochondria, leads to

a decrease of caspase-3, and results in hippocampal neurons against the Aβ1-42 –induced

neuronal apoptosis.

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