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, 9 (8), e104927
eCollection

Subchronic Treatment of Donepezil Rescues Impaired Social, Hyperactive, and Stereotypic Behavior in Valproic Acid-Induced Animal Model of Autism

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Subchronic Treatment of Donepezil Rescues Impaired Social, Hyperactive, and Stereotypic Behavior in Valproic Acid-Induced Animal Model of Autism

Ji-Woon Kim et al. PLoS One.

Abstract

Autism spectrum disorder (ASD) is a group of pervasive developmental disorders with core symptoms such as sociability deficit, language impairment, and repetitive/restricted behaviors. Although worldwide prevalence of ASD has been increased continuously, therapeutic agents to ameliorate the core symptoms especially social deficits, are very limited. In this study, we investigated therapeutic potential of donepezil for ASD using valproic acid-induced autistic animal model (VPA animal model). We found that prenatal exposure of valproic acid (VPA) induced dysregulation of cholinergic neuronal development, most notably the up-regulation of acetylcholinesterase (AChE) in the prefrontal cortex of affected rat and mouse offspring. Similarly, differentiating cortical neural progenitor cell in culture treated with VPA showed increased expression of AChE in vitro. Chromatin precipitation experiments revealed that acetylation of histone H3 bound to AChE promoter region was increased by VPA. In addition, other histone deacetyalse inhibitors (HDACIs) such as trichostatin A and sodium butyrate also increased the expression of AChE in differentiating neural progenitor cells suggesting the essential role of HDACIs in the regulation of AChE expression. For behavioral analysis, we injected PBS or donepezil (0.3 mg/kg) intraperitoneally to control and VPA mice once daily from postnatal day 14 all throughout the experiment. Subchronic treatment of donepezil improved sociability and prevented repetitive behavior and hyperactivity of VPA-treated mice offspring. Taken together, these results provide evidence that dysregulation of ACh system represented by the up-regulation of AChE may serve as an effective pharmacological therapeutic target against autistic behaviors in VPA animal model of ASD, which should be subjected for further investigation to verify the clinical relevance.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Drug treatment and behavioral study scheme with VPA mice.
VPA was s.c. injected at embryonic day 10 (E10) to pregnant mice. Donepezil was i.p. injected once daily from postnatal day 14 (P14) until the end of the study. Behavioral studies were performed from P21. Grooming and digging test and novel object recognition tests were performed at 4 weeks of age with different sets of animals.
Figure 2
Figure 2. Dysregulation of cholinergic enzyme in the prefrontal cortex region of VPA induced animal model of ASD.
Expression level of acetylcholinesterase and choline acetyltransferase was measured using Western blot (A), and acetylcholinesterase was also measured using immunohistochemistry (B) in the prefrontal cortex at post natal day 28. (A) Prenatally VPA-exposed SD rats (400 mg/kg) and ICR mice (300 mg/kg) showed increased acetylcholinesterase (AChE) but decreased choline acetyltransferase (ChAT); left panel rat (N = 6), right panel mouse (N = 4). Data are expressed as the mean ± S.E.M. *, **, p<0.05 and p<0.01 vs. control. (B) Immunohistochemical staining against acetylcholinesterase (green) in prefrontal cortex region from SD rat exposed to VPA at prenatal period. Nucleus was counter-stained with TO-PRO3 (blue).
Figure 3
Figure 3. Histone deacetylase inhibitors increased expression of AChE in cortical NPCs from SD rat.
Cortical neural progenitor cells from SD rat were treated with HDACIs: VPA (0.5 mM), TSA:Trichostatin A (0.1 µM), and SB:Sodium butyrate (0.1 mM). After 24 hours, mRNA and protein expressions were analyzed using RT-PCR (A) and Western blot (B). (A) Ache mRNA expression level was increased by both VPA and other HDACIs (N = 3). (B) Protein expression of AChE was also increased by VPA and other HDACIs treatment (N = 4). Data are expressed as the mean ± S.E.M. *, ***, p<0.05 and p<0.001 vs. control (Ache:Acetylcholinesterase mRNA, Gapdh:GAPDH mRNA, AChE:Acetylcholinesterase, AcH3:acetylated Histone H3, H3:Histone H3, VPA:valproic acid, TSA:Trichostatin A, SB:Sodium butyrate).
Figure 4
Figure 4. Acetylation of histone H3 bound to promoter region of Ache was increased by VPA treatment.
To confirm the effect of HDACIs on the upregulation of AChE, ChIP was performed using prefrontal cortex from VPA rat and VPA treated cortical NPCs from SD rat. (A) ChIP results using prefrontal cortex of rat prenatally exposed to VPA or PBS (con) (B) ChIP results from cortical neural progenitor cells of rat. Gapdh was used as a positive control for this analysis. (AcH:acetylated Histone H3, Ache:acetylcholinesterase).
Figure 5
Figure 5. Subchronic treatment of donepezil improved social behavior in VPA exposed mice.
In three chamber assay, sociability (A) and social preference (B) were measured. Upper panel showed stay duration (sec) in each compartment and lower panel showed social index and social preference index (N = 10). (C) Nest building test. The built nests during 16 hours period were scored by trained experimenters who were blind to the experimental conditions (1, poor; 5, good), N = 10. Data are expressed as the mean ± S.E.M. *, **, ***, p<0.05, p<0.01, p<0.001 vs. control in same compartment. #, ##, ###, p<0.05, p<0.01, p<0.001 vs. VPA exposed mice in same compartment.
Figure 6
Figure 6. Subchronic treatment of donepezil improved repetitive behaviors in VPA exposed mice.
(A) Marble burying test. Twenty marbles were equidistantly arranged (5×4) on the bedding surface. After 20 min, the number of buried marbles (>50% of its surface) was measured (N = 12). (B) Digging and grooming behavior tests were performed by simultaneously observing the home cage behavior of mice. Each mouse was placed individually in a mouse cage with 1 cm layered bedding. After 10 min habituation, accumulative time of digging and grooming behavior was measured for another 10 min (N = 12). Data are expressed as the mean ± S.E.M. *, ***, p<0.05, p<0.001: control mice vs. VPA mice. ###, p<0.001: VPA mice vs. donepezil treated VPA mice.
Figure 7
Figure 7. Subchronic treatment of donepezil improved hyperactive and abnormal anxiety behavior in VPA exposed mice.
(A) Open field test. Locomotor activity was measured for 20 min using CCD camera-assisted motion tracking apparatus and software (EthoVision 3.1, Noldus information Technology, the Netherlands). Left panel showed total distance moved and right panel showed their velocity (N = 12). (B) Elevated plus maze. Data expressed as a ratio of staying time in open arms divided by staying time in closed arms (N = 8 for Con and VPA, DPZ:8, N = 11 for V+D). Data are expressed as the mean ± S.E.M. *, ***, p<0.05, p<0.001: control mice vs. VPA mice. ##,###, p<0.01, p<0.001: VPA mice vs. donepezil treated VPA mice.
Figure 8
Figure 8. Subchronic treatment of donepezil improved impaired recognition in VPA exposed mice.
Novel object test was performed as described in methods. Data are expressed as a discrimination ratio (novel object interaction/total interaction with both objects, N = 12∼13, the mean ± S.E.M.) ***, p<0.001: control mice vs. VPA mice. ###, p<0.001: VPA mice vs. donepezil treated VPA mice.
Figure 9
Figure 9. Subchronic treatment of donepezil corrected increased AChE activity in the VPA exposed mice model.
AChE activity was measured in the prefrontal cortex at P35 (N = 6). Data are expressed as the mean ± S.E.M. **, p<0.01: control mice vs. VPA mice. ###, p<0.001: VPA mice vs. donepezil treated VPA mice.

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Grant support

This work was supported by a grant of the Korean Health Technology R&D Project, Ministry of health & welfare, Republic of Korea (No. A120029). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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