CRISPR/Cas9-mediated disruption of SHANK3 in monkey leads to drug-treatable autism-like symptoms
(Full-text capture 2026-09-21; web artifacts lightly stripped; truncated.)
Zhuchi Tu
1Guangdong-Hongkong-Macau Institute of CNS Regeneration, Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Jinan University, Guangzhou, China
1,#, Hui Zhao
Hui Zhao
2State Key Laboratory of Molecular Developmental Biology, CAS Center for Excellence in Brain Science and Intelligence Technology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
3Key Laboratory of Regenerative Biology, South China Institute for Stem Cell, Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China
2,3,#, Bang Li
Bang Li
1Guangdong-Hongkong-Macau Institute of CNS Regeneration, Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Jinan University, Guangzhou, China
1,#, Sen Yan
Sen Yan
1Guangdong-Hongkong-Macau Institute of CNS Regeneration, Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Jinan University, Guangzhou, China
1, Lu Wang
Lu Wang
4Department of Nuclear Medicine and PET/CT-MRI Center, the First Affiliated Hospital of Jinan University & Institute of Molecular and Functional Imaging, Jinan University, Guangzhou China
4, Yongjin Tang
Yongjin Tang
4Department of Nuclear Medicine and PET/CT-MRI Center, the First Affiliated Hospital of Jinan University & Institute of Molecular and Functional Imaging, Jinan University, Guangzhou China
4, Zhujun Li
Zhujun Li
1Guangdong-Hongkong-Macau Institute of CNS Regeneration, Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Jinan University, Guangzhou, China
1, Dazhang Bai
Dazhang Bai
1Guangdong-Hongkong-Macau Institute of CNS Regeneration, Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Jinan University, Guangzhou, China
1, Caijuan Li
Caijuan Li
1Guangdong-Hongkong-Macau Institute of CNS Regeneration, Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Jinan University, Guangzhou, China
1, Yingqi Lin
Yingqi Lin
1Guangdong-Hongkong-Macau Institute of CNS Regeneration, Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Jinan University, Guangzhou, China
1, Yuefeng Li
Yuefeng Li
5Guangdong Landau Biotechnology Co. Ltd., Guangzhou, China
5, Jianrong Liu
Jianrong Liu
6Yuanxi Biotech Inc., Guangzhou China
6, Hao Xu
Hao Xu
4Department of Nuclear Medicine and PET/CT-MRI Center, the First Affiliated Hospital of Jinan University & Institute of Molecular and Functional Imaging, Jinan University, Guangzhou China
4, Xiangyu Guo
Xiangyu Guo
1Guangdong-Hongkong-Macau Institute of CNS Regeneration, Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Jinan University, Guangzhou, China
Yong-hui Jiang
7Department of Pediatrics and Department of Neurobiology, Duke University, Durham, NC, USA
7,✉, Yong Q Zhang
Yong Q Zhang
2State Key Laboratory of Molecular Developmental Biology, CAS Center for Excellence in Brain Science and Intelligence Technology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
2,✉, Xiao-Jiang Li
Xiao-Jiang Li
1Guangdong-Hongkong-Macau Institute of CNS Regeneration, Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Jinan University, Guangzhou, China
8Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA
1,8,✉
- Author information
- Article notes
- Copyright and License information
1Guangdong-Hongkong-Macau Institute of CNS Regeneration, Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Jinan University, Guangzhou, China
2State Key Laboratory of Molecular Developmental Biology, CAS Center for Excellence in Brain Science and Intelligence Technology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
3Key Laboratory of Regenerative Biology, South China Institute for Stem Cell, Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China
4Department of Nuclear Medicine and PET/CT-MRI Center, the First Affiliated Hospital of Jinan University & Institute of Molecular and Functional Imaging, Jinan University, Guangzhou China
5Guangdong Landau Biotechnology Co. Ltd., Guangzhou, China
6Yuanxi Biotech Inc., Guangzhou China
7Department of Pediatrics and Department of Neurobiology, Duke University, Durham, NC, USA
8Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA
✉
To whom correspondence should be addressed at: Xiao-Jiang Li, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Jinan University, Guangzhou 510632, China. Tel: +86 02085221351; Fax: +86 02085221351; Email: xli2@emory.edu; Yong Q. Zhang, State Key Laboratory of Molecular Developmental Biology, CAS Center for Excellence in Brain Science and Intelligence Technology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Tel: +86 1064807393; Fax: +86 01064806219; Email: yqzhang@genetics.ac.cn; Yong-hui Jiang, Department of Pediatrics and Department of Neurobiology, Duke University, Durham, NC 27710, USA. Tel: +919 6812789; Email: yong-hui.jiang@duke.edu
The first three authors contributed equally to this work.
Received 2018 Aug 31; Revised 2018 Oct 10; Accepted 2018 Oct 12; Issue date 2019 Feb 15.
© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model ( https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
PMCID: PMC6489410 PMID: 30329048
Abstract
Monogenic mutations in the SHANK3 gene, which encodes a postsynaptic scaffold protein, play a causative role in autism spectrum disorder (ASD). Although a number of mouse models with Shank3 mutations have been valuable for investigating the pathogenesis of ASD, species-dependent differences in behaviors and brain structures post considerable challenges to use small animals to model ASD and to translate experimental therapeutics to the clinic. We have used clustered regularly interspersed short palindromic repeat/CRISPR-associated nuclease 9 to generate a cynomolgus monkey model by disrupting SHANK3 at exons 6 and 12. Analysis of the live mutant monkey revealed the core behavioral abnormalities of ASD, including impaired social interaction and repetitive behaviors, and reduced brain network activities detected by positron-emission computed tomography (PET). Importantly, these abnormal behaviors and brain activities were alleviated by the antidepressant fluoxetine treatment. Our findings provide the first demonstration that the genetically modified non-human primate can be used for translational research of therapeutics for ASD.
Introduction
Mutations in SHANK3 gene , which encodes a scaffolding protein in the postsynaptic density of excitatory synapses, contribute to approximately 1–2% of autism spectrum disorder (ASD) cases ( 1–4). SHANK3 also plays a key role in the clinical presentations of Phelan–McDermid syndrome, a chromosome 22q13.3 deletion syndrome ( 5). Haploinsufficiency of SHANK3 is thought to be the mechanism responsible for the frequent presentation of ASD and other neurological comorbidity. Because of the well-established connection of SHANK3 to the synaptic development and function, SHANK3 causing ASD offers one of the best opportunities to model human ASD in model organisms.
Mutations in SHANK3 have been introduced and characterized in Drosophila, zebrafish, mouse and rat. Remarkably, a total of 14 lines of SHANK3 mutant mice have been reported ( 2, 4). Analyses of these Shank3 isoform or complete knock-out mutant mice have offered many novel insights regarding the role of SHANK3 in synapses and the pathophysiology caused by SHANK3 mutations. However, one of notable findings is that, in contrast to severe clinical presentation in humans with heterozygous SHANK3 mutations, there is no or only very mild phenotypes in heterozygous Shank3 mutant mice ( 2, 4, 6). This observation indicates that differences between human and rodent brains influence their response to the deficiency of SHANK3 protein. Indeed, our recent report has discovered that SHANK3 protein is the most abundant in the prefrontal cortex (PFC) in monkey but is expressed at the highest level in striatum (Str) in mice ( 4, 7, 8). Furthermore, it has been well recognized that replication or translation of the therapeutic effects on rodent models to human clinical trials is often difficult ( 9, 10). While there are many contributing factors for this difficulty, one of frequently debated subjects is whether an alternative model closer to human is needed for a preclinical study before launching an expensive clinical trial. Given those differences described, the importance of using non-human primates to model human diseases, especially psychiatric disorders, is clear.
We recently used clustered regularly interspersed short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) to disrupt exons 6 and 12 of SHANK3 gene in cynomolgus monkeys ( 8). Our recent studies found that the complete loss of SHANK3 in the PFC results in a significant neuronal loss which probably leads to the observed late embryonic lethality ( 8). However, one male monkey with a 2 bp deletion in exon 12 of SHANK3 ( SHANK3M3) has lived for 26 months now. Longitudinal investigation over the last 2 years revealed that the SHANK3M3 monkey developed the core features of behavioral phenotypes of ASD, including impaired social interaction and apparent stereotypical locomotion. Furthermore, after treatment with fluoxetine, a selective serotonin reuptake inhibitor commonly prescribed for major depression and obsessive–compulsive disorder (OCD) ( 11), the impaired social interaction and stereotypical behaviors were markedly improved. Thus, by generation of SHANK3 mutant monkey, we demonstrate that the non-human primate could faithfully recapitulate ASD-like behaviors, which holds a great potential for the development of effective therapeutics for ASD.
Results
Delayed growth and vocalization in SHANK3 mutant monkey
In our previous study, we used the Cas9/sgRNA method to target exons 6 and 12 of SHANK3 in cynomolgus monkeys ( Macaca fascicularis), which resulted in different mutations including a large deletion (11 456 bp) and small indels of −2, −5, −8, −22 and +1 bp in the SHANK3 gene ( 8). We observed a higher than expected embryonic and perinatal lethality associated with SHANK3 targeting, suggesting that SHANK3 is important for early development in primates. Nevertheless, we obtained a single male cynomolgus monkey ( SHANK3M3) that carries a 2 bp deletion in peripheral tissues ( 8) and has survived for 26 months up to now. Due to the mosaic nature of CRISPR/Cas9-mediated mutations and the lack of brain samples, the exact nature and extent of SHANK3 mutation in the brain of SHANK3M3 monkey remain to be determined. We examined the behaviors of SHANK3M3 monkey longitudinally after birth but more extensively after 12 months of age when abnormal behaviors started to emerge.
Although the fact of a single mutant monkey and the choice of control have posed a challenge for the experimental design, we believe that a longitudinal and repeated study of behavioral phenotypes could still offer valuable data as described in other behavioral studies using non-human primates ( 12, 13) and case reports of human patients. Three age- and gender-matched (male) monkeys were used as controls, and all the monkeys for examination were housed in the same facility under the same living conditions. Newborn monkeys were kept together with mothers until 12 months of age. The first noticeable difference was that SHANK3M3 monkey did not vocalize until 18 months, whereas three control monkeys did so immediately after birth ( Fig. 1A). The delayed vocalization in mutant monkey may be analogous to the delayed or no speech phenotype in young patients carrying SHANK3 mutations ( 14, 15).
Figure 1.
Delayed development and vocalization in SHANK3 mutant monkey. ( A) Schematic diagram summary of the development of behavioral phenotypes in SHANK3M3 monkey. ( B) Representative pictures of SHANK3M3 monkey and an age- and gender-matched control monkey at the age of 20 months. ( C) Body weight, ( D) body length and (E) head circumference of SHANK3M3 monkey compared to 15 wild-type control monkeys. Statistical analyses were performed using GraphPad Prism 6.0. Data are presented as mean ± SEM. ( F) Abnormal dental development of enamel dysplasia in SHANK3M3 monkey as compared to a control monkey.
We measured the growth parameters every 6 months after birth. The body weight and body length of SHANK3M3 monkey were significantly smaller and shorter, respectively, than those of control monkeys ( Fig. 1B–D). However, the head circumference of SHANK3M3 monkey is comparable to the controls ( Fig. 1E). No other behavioral difference between SHANK3M3 monkey and controls was noticed before they became one year old. We also noted enamel dysplasia, which had been reported in some autistic children with SHANK3 mutations ( 16), in SHANK3M3 monkey ( Fig. 1F).
Repetitive and anxiety-like behaviors in SHANK3 mutant monkey
Video recording of daily activity is considered a simple and effective method to assess the behaviors of monkeys ( 17). We assessed the activity of monkeys in the home cage and the adaptability in a new cage using video recording of two half-hour daily sessions for six consecutive days. The behaviors were scored by three trained experimenters unaware of animal identity. Compared with control monkeys, SHANK3M3 monkey exhibited significant stereotypic and anxiety-like behaviors in both home and new cages, which is evident by more frequent circling behavior ( Supplementary Material, Video S1). This typical behavior has been reported to associate with anxiety in non-human primates ( 18, 19). The stereotypical behavior was aggravated in the new cage ( Supplementary Material, Video S2). Quantification of the duration of inactivity, exploration and stereotypical behaviors verified that SHANK3M3 monkey displayed stereotypical behavior. Compared to individual control monkey, SHANK3M3 monkey displayed significantly reduced time on environment exploration in the home cage ( SHANK3M3 2.2 ± 0.50 min; Ctrl1 12.89 ± 1.47 min, ** P = 0.0012; Ctrl2 17.10 ± 2.66 min, ** P = 0.0038; Ctrl3 10.92 ± 2.38 min, * P = 0.0124) and in a novel cage ( SHANK3M3 7.71 ± 1.34 min; Ctrl1 28.05 ± 2.06 min, \\\* P = 0.0006; Ctrl2 21.38 ± 1.48 min, \\\* P = 0.0007; Ctrl3 18.59 ± 2.11, * P = 0.0169). For the locomotion activity, no significant differences were found between SHANK3M3 monkey and the control monkeys in both the home cage ( SHANK3M3 2.16 ± 0.62 min, Ctrl1 1.16 ± 0.62 min, P = 0.4469; Ctrl2 4.90 ± 1.67 min, P = 0.2273; Ctrl3 11.96 ± 1.57 min, ** P = 0.0016) and the novel cage ( SHANK3M3 14.56 ± 2.41 min, Ctrl 6.60 ± 2.80 min, P = 0.1479; Ctrl2 8.88 ± 3.03 min, P = 0.1425; Ctrl3 5.81 ± 2.71 min, P = 0.0623; Fig. 2A). However, SHANK3M3 monkey exhibited stereotypical behavior more frequently than control monkeys in both the home cage ( SHANK3M3 13.23 ± 3.16 min, Ctrl1 0.00 ± 0.00 min, ** P = 0.0085; Ctrl2 1.78 ± 0.87 min, * P = 0.0172; Ctrl3 7.58 ± 1.52 min, P = 0.1931) and the novel cage ( SHANK3M3 9.08 ± 1.50 min, Ctrl1 0.91 ± 0.49 min, ** P = 0.0065; Ctrl2 0.97 ± 0.83 min, ** P = 0.0019; Ctrl3 0.23 ± 0.08 min, ** P = 0.002; Fig. 2B).
Figure 2.
Impaired social and more repetitive behaviors in SHANK3 mutant monkey. ( A) Solo behaviors of SHANK3M3 and wild-type control monkeys in a familiar home cage and ( B) a novel cage. Compared to each control monkey, SHANK3M3 monkey displayed significantly lower environment exploration in home cage and significantly higher stereotypical behavior than control monkeys in both home and new cage. ( C) Representative pictures of SHANK3M3 monkey with crook-tail, a posture suggesting anxiety and fearfulness in juvenile monkeys. ( D) The frequency and ( E) duration of crook-tails in both home and novel cages. Three wild-type control monkeys were examined. The data are presented as mean ± SEM. NS, * P < 0.05, ** P < 0.01, Mann–Whitney U test. ( F) The frequency and ( G) duration of active social interaction, passive social interaction and stereotypical behaviors when a test monkey of SHANK3M3 or wild-type control was paired with a sociable monkey. Data are analyzed by one-way ANOVA and presented as mean ± SEM. NS, * P < 0.05; ** P < 0.01; \\\* P < 0.001; \\\** P < 0.0001. See detailed statistic results in the text.
Crook-tail is a behavior that also associates with anxiety or fearfulness in a juvenile monkey ( 20). SHANK3M3 monkey exhibited significantly longer time of crook-tail behavior than the control monkeys in the home cage ( SHANK3M3 34.28 ± 5.17 s, Ctrl1 1.67 ± 0.78 s, * P = 0.045; Ctrl2 1.30 ± 0.79 s, P = 0.1571; Ctrl3 1.12 ± 0.72 s, ** P = 0.0013) and the novel cage ( SHANK3M3 56.75 ± 21.08 s, Ctrl1 2.80 ± 1.26 s, * P = 0.0106; Ctrl2 3.37 ± 0.59 s, P = 0.1364; Ctrl3 1.85 ± 0.61 s, ** P = 0.0035; Fig. 2D). The mutant monkey also exhibited significantly high frequency of crook-tail in the home cage ( SHANK3M3 15.17 ± 2.89, Ctrl1 1 ± 0.37, * P = 0.0117; Ctrl2 0.83 ± 0.31, ** P = 0.0088; Ctrl3 0.5 ± 0.34, ** P = 0.0090) and the novel cage ( SHANK3M3 26.83 ± 11.54, Ctrl1 2.5 ± 0.76, ** P = 0.0074; Ctrl2 2.3 ± 0.42, ** P = 0.0070; Ctrl3 2.56 ± 0.73, ** P = 0.0078; Fig. 2E).
Impaired social interaction in SHANK3 mutant monkey
We next examined social interaction by performing a one-to-one interaction test using a protocol previously described ( 21), in which one monkey ( SHANK3M3 or its age- and gender-matched control monkey) was paired with a sociable wild-type monkey. A sociable monkey was selected based on its frequent interactions with others ( 22). SHANK3M3 monkey did not actively initiate or respond to social interaction, as shown by the significantly less frequent and shorter duration of interactions with its partner in both active and passive social interactions compared with control monkeys. SHANK3M3 monkey initiated social interaction with a significantly lower frequency ( SHANK3M3 1.17 ± 0.47, Ctrl1 11 ± 2.90 * P = 0.0176; Ctrl2 4.17 ± 0.87 * P = 0.0130; Ctrl3 24.67 ± 3.95, \\\* P = 0.0001; Fig. 2F) and shorter duration ( SHANK3M3 0.13 ± 0.10 min, Ctrl1 5.91 ± 1.36 min, * P = 0.0104; Ctrl2 6.65 ± 1.55 min, ** P = 0.0086; Ctrl3 13.43 ± 1.36 min, \\\* P = 0.0002; Fig. 2G). Consistently, SHANK3M3 mutant monkey received fewer interactions ( SHANK3M3 0.67 ± 0.33, Ctrl1 6.3 ± 1.65, ** P = 0.0048; Ctrl2 6.83 ± 1.35, * P = 0.0108; Ctrl3 9.33 ± 1.59, * P = 0.0283) with shorter duration ( SHANK3M3 0.03 ± 0.01 min, Ctrl1 11.35 ± 2.83 min, ** P = 0.0033; Ctrl2 13.41 ± 1.19 min, \\\* P < 0.0001; Ctrl3 10.83 ± 2.05 min, * P = 0.0105) than each control monkey. Instead, SHANK3M3 monkey displayed a greater frequency ( SHANK3M3 41.5 ± 6.3, Ctrl1 5.5 ± 5.5, ** P = 0.0016; Ctrl2 0 ± 0, \\\* P < 0.0001; Ctrl3 1.33 ± 1.15, ** P = 0.0012; Fig. 2F) and longer duration ( SHANK3M3 4.39 ± 0.83 min, Ctrl1 0.41 ± 0.41 min, ** P = 0.0039; Ctrl2 0.00 ± 0.00 min, ** P = 0.0033; Ctrl3 0.29 ± 0.24 min, ** P = 0.0010; Fig. 2G) of stereotypical behaviors, including circling in the cage than each control monkey. When grouped with other sociable control monkeys, SHANK3M3 monkey displayed social interaction deficits that prevented it from developing a close relationship with the control monkeys ( Supplementary Material, Video S3).
Reduced eye contact in SHANK3 mutant monkey
Eye contact is considered to be essential for communication and social interaction in both humans and other primate species ( 17, 23). Compared with control monkeys, SHANK3M3 monkey displayed significantly less gazing with shorter time ( SHANK3M3 18.99 ± 4.038 s, Ctrl1 69.81 ± 5.017 s, ** P = 0.0015; Ctrl2 68.38 ± 2.910 s, \\\* P = 0.0001; Ctrl3 88.60 ± 13.42 s, ** P = 0.0047; Fig. 3A) and lower frequency of eye contact with the person who was examining the monkey ( SHANK3M3 9.833 ± 1.537, Ctrl1 35.67 ± 3.148, ** P = 0.0045; Ctrl2 35.33 ± 5.364, \\\* P = 0.0004; Ctrl3 43.83 ± 11.44, \\\* P = 0.0001; Fig. 3B). Instead, the mutant monkey showed intense anxiety and fear by frequently circling in the cage ( Supplementary Material, Video S4).
Figure 3.
Impaired eye contact and decreased glucose metabolism in SHANK3 mutant monkey. ( A) Representative photos of eye contact of monkeys at 24 months old. ( B) SHANK3M3 monkey displayed significantly shorter eye contact duration with less frequency than control monkeys. ( C) PET-MRI imaging evaluation of [18F]FDG in control and SHANK3M3 monkey. Patterns of brain regions (the upper panel) and representative brain PET images overlaid on their individual MRI (control monkey in the middle panel and SHANK3M3 monkey in the bottom panel) are presented. Regional radioactivity was normalized to the injected radioactivity and body weight and expressed as the SUVr with Pon uptake as reference. ( D) Regional SUVr values in different brain regions in control ( n = 3) and SHANK3M3 monkeys. Data were obtained from three PET-MRI imaging tests, presented as mean ± SEM and are analyzed by two-way ANOVA. NS, * P < 0.05, ** P < 0.01, \\\* P < 0.001, \\\** P < 0.0001 (see detailed statistic results in the text).
Reduced brain activity in SHANK3 mutant brain
Positron emission computed tomography (PET) is a non-invasive in vivo molecular imaging technique that is widely used in clinical diagnosis and treatment evaluation ( 24–26). With the assistance of the most common PET tracer [18F] fluorodeoxyglucose ([18F]FDG), as well as the exquisite information on brain substructures provided by magnetic resonance imaging (MRI), PET can help us identify brain endophenotype associated with SHANK3 mutations. In order to investigate possible structural and functional changes of mutant brain, both MRI and PET/computerized tomography (CT) were conducted in mutant and control monkeys. Morphological T1-weighted 3-dimensional MRI demonstrated no obvious structural abnormality in SHANK3M3 mutant. However, the [18F]FDG-PET study revealed that the glucose metabolism in SHANK3M3 monkey brain tissues was significantly lower than that observed in normal controls. As shown in Figure 3C and D, using pons (Pon) as the reference region ( 27, 28), we observed that the regional brain standardized uptake value ratio (SUVr) values for SHANK3 were specifically reduced in a variety of brain regions when compared with the control [cerebellum (CB) SHANK3M3 1.36 ± 0.05, Ctrl 1.65 ± 0.21, P = 0.0756; Str SHANK3M3 1.65 ± 0.07, Ctrl 2.6 ± 0.1672, \\\* P < 0.0001; caudate (Cau) SHANK3M3 1.442 ± 0.08192, Ctrl 2.3 ± 0.19, \\\* P < 0.0001; putamen (Put) SHANK3M3 1.75 ± 0.07, Ctrl 2.69 ± 0.16, \\\* P < 0.0001; amygdala (Amyg) SHANK3M3 1.13 ± 0.05, Ctrl 1.6 ± 0.12, ** P = 0.0049; hippocampus (Hip) SHANK3M3 1.29 ± 0.05, Ctrl 1.93 ± 0.17, \\\ P = 0.0002; parahippocampal gyrus (Phg) SHANK3M3 1.12 ± 0.01, Ctrl 1.76 ± 0.05, \\\* P = 0.0002; temporal lobe (Ltemp) SHANK3M3 1.46 ± 0.10, Ctrl 2.12 ± 0.07, \\\* P = 0.0001; dorsal lateral prefrontal cortex (DLPFC) SHANK3M3 1.59 ± 0.12, Ctrl 2.14 ± 0.14, ** P = 0.0013; PFC SHANK3M3 1.44 ± 0.13, Ctrl 1.60 ± 0.10, P = 0.3170; anterior cingulate cortex (ACC) SHANK3M3 1.33 ± 0.05, Ctrl 2.23 ± 0.05, \\\** P < 0.0001]. These results showed significant decrease of glucose metabolism in the mutant monkey brain, which is consistent with the lower glucose metabolic activity in the brains of some ASD patients ( 29, 30).
Improved ASD core symptoms and brain metabolism in SHANK3 mutant monkey upon fluoxetine treatment
The robust autism-like behaviors observed in SHANK3M3 monkey prompted us to test whether this monkey model can be used for future drug development. Currently, there is no effective drug treatment for the core symptoms of ASD and SHANK3-related disorders. However, many clinicians have tried different anti-psychotic medications for managing the various behavioral issues associated with ASD. Fluoxetine, a commonly prescribed medication for major depression and OCD, has been used to treat repetitive behaviors in ASD patients ( 31, 32). We treated SHANK3M3 and control monkeys with fluoxetine at 2.5 mg/kg per day, a dose used in previous studies for non-human primate and ASD patients ( 32, 33). The behaviors of animals were then monitored by video recording daily. To better assess the therapeutic effects, we measured animal behavioral changes after fluoxetine treatment by comparing fold changes over the pre-treatment. We found that the treatment for 2 weeks markedly alleviated the repetitive behaviors and significantly increased active social interaction duration ( SHANK3M3 15.21 ± 1.38, Ctrl1 1.50 ± 0.48, \\\* P = 0.002; Ctrl2 2.07 ± 1.39, ** P = 0.0062; Ctrl3 0.59 ± 0.07, \\\* P = 0.0003) and frequency ( SHANK3M3 15.53 ± 1.46, Ctrl1 3.89 ± 1.41, ** P = 0.0028; Ctrl2 2.35 ± 0.29, ** P = 0.001; Ctrl3 0.92 ± 0.18, \\\* P = 0.0006) for SHANK3M3 monkey ( Fig. 4A; Supplementary Material, Video S5). Fluoxetine also significantly prolonged passive interaction duration (SHANK3M3 10.98 ± 1.12; Ctrl1 1.26 ± 0.26, \\\* P = 0.0006; Ctrl2 1.44 ± 0.25, ** P = 0.0011; Ctrl3 1.39 ± 0.49, ** P = 0.002) but moderately increased passive social interaction frequency ( SHANK3M3 4.58 ± 1.29; Ctrl1 1.60 ± 0.31, P = 0.32; Ctrl2 2.85 ± 1.05, P = 0.99; Ctrl3 2.57 ± 0.79, P = 0.98). In addition, fluoxetine treatment alleviated stereotypical behavior by reducing its duration ( SHANK3M3 0.14 ± 0.06; Ctrl1 0.93 ± 0.05, \\\* P = 0.004; Ctrl2 1.0 ± 0.0, \\\* P = 0.0001; Ctrl3 0.90 ± 0.09, ** P = 0.0017) and frequency ( SHANK3M3 0.05 ± 0.02; Ctrl1 0.78 ± 0.14, * P = 0.0112; Ctrl2 1.0 ± 0.0, \\\** P < 0.0001; Ctrl3 0.88 ± 0.12, ** P = 0.0026; Fig. 4C). Moreover, fluoxetine treatment increased eye contact duration ( SHANK3M3 3.41 ± 1.04; Ctrl1 0.65 ± 0.07, ** P = 0.004; Ctrl2 1.07 ± 0.11, * P = 0.0148; Ctrl3 0.60 ± 0.07, ** P = 0.0034) and frequency of SHANK3M3 monkey ( SHANK3M3 4.55 ± 0.79; Ctrl1 0.88 ± 0.16, * P = 0.0247; Ctrl2 1.21 ± 0.17, * P = 0.0201; Ctrl3 0.91 ± 0.11, * P = 0.0219; Fig. 4D; Supplementary Material, Video S6). Also, SHANK3M3 monkey became less frequently to stay or play alone by itself ( Supplementary Material, Video S5).
Figure 4.
Fluoxetine robustly improves autism symptoms and increased glucose metabolism to a normal level in SHANK3 mutant monkey. ( A, B) Fluoxetine treatment at 2.5 mg/kg/per day significantly increased active (A) and passive (B) social interaction duration and frequency of SHANK3M3 monkey, while control monkeys remained the same level of interaction with drug treatment. The results are presented as fold changes of post-treatment compared with pre-treatment. Fluoxetine also significantly prolonged passive interaction duration but moderately increased passive social interaction frequency. ( C) Fluoxetine alleviated stereotypical behavior by reducing its duration and frequency. ( D) Fluoxetine increased eye contact duration and frequency of SHANK3M3 monkey. ( E) Representative brain PET [18F]FDG images of pre- and post-fluoxetine treatment in a normal control or SHANK3M3 monkey. ( F) Regional brain SUVr value changes in control and SHANK3M3 monkey after fluoxetine treatment. Data are presented as mean ± SEM ( n = 3 for control for comparing to SHANK3M3 monkey) and analyzed by one-way ANOVA. * P < 0.05, ** P < 0.01, \\\* P < 0.001, \\\** P < 0.0001.
Furthermore, we also observed that brain glucose metabolism in SHANK3M3 monkey was increased to the level as the control monkeys, as indicated by PET imaging results ( Fig. 4E). Regional brain SUVr value changes in SHANK3M3 monkey after fluoxetine treatment was increased while those of controls remained unaltered (CB, SHANK3M3 1.21, Control 1.16 ± 0.08; Str, SHANK3M3 1.43, Control 1.647 ± 0.07; Cau, SHANK3M3 1.41, Control 0.92 ± 0.13; Put, SHANK3M3 1.45, Control 0.96 ± 0.10; Amyg, SHANK3M3 1.34, Control 0.91 ± 0.11; Hip, SHANK3M3 1.37, Control 0.94 ± 0.11; Phg, SHANK3M3 1.40, Control 0.97 ± 0.09; Ltemp, SHANK3M3 1.48, Control 0.93 ± 0.08; DLPFC, SHANK3M3 1.49, Control 0.97 ± 0.09; PFC, SHANK3M3 1.23, Control 0.94 ± 0.13; ACC, SHANK3M3 1.66, Control 0.97 ± 0.11). These results indicate that fluoxetine treatment corrected the network activities in various brain regions in SHANK3M3 monkey, consistent with the previous finding that the PET imaging functionality in ASD patients could be improved by fluoxetine ( 29). Taken together, these findings demonstrate that ASD-related abnormalities in SHANK3M3 monkey are reversible and would allow future studies to identify therapeutics that can be efficient to treat ASD.
Discussion
ASD represents one of the most important and common neurodevelopmental and neuropsychiatric diseases. Accordingly, there are considerable research efforts to tackle ASD from many different aspects. However, our understanding of the pathogenesis of ASD remains poor, and the effective treatment of ASD remains to be developed. These is an apparent question of whether many currently well-studied rodent models are sufficient to model the complex social behavior associated with ASD ( 34, 35). SHANK3 mutant monkeys generated by us ( 8) and other recently established MECP2 mutant monkeys using transgenic or TALEN-mediated methods ( 21, 36) have demonstrated the value of genetically modified non-human primate models for ASD research.
Using the CRISPR/Cas9-mediated genetic manipulation, we obtained a live mutant monkey that carries a 2 bp deletion in exon 12 of the SHANK3 gene. This mutation is predicted to result in a frameshift at Q419 (Q419fsX486) between ANK and SH3 domains for the majority of SHANK3 isoforms. It should be noted that a single base-pair insertion in an autism patient ( 37), which leads to a premature stop codon at the residue 503 (A447fsX503), is close to the 2 bp deletion of SHANK3M3 monkey. Because we were unable to analyze the brain tissues of this live SHANK3M3 monkey, we could not determine the SHANK3 mutation types and mosaicism in its brain. However, the robust core features of ASD and abnormal brain activity in SHANK3M3 monkey lend the support for SHANK3 haploinsufficiency, which is well established in ASD patients with SHANK3 mutations ( 3, 15).
Through longitudinal and repeated studies over 14 months by three experimenters and the comparison with three age- and gender-matched control monkeys, we found that SHANK3M3 monkey displayed abnormal behaviors that recapitulate core ASD features of impaired social interaction and repetitive behaviors. The delayed vocalization in SHANK3M3 monkey is noteworthy. Although the exact meaning of delayed vocalization is not clear, it is possible that monkeys communicate via vocalization, and the defect in vocalization may be analogous to the delayed or absent speech in many young patients with SHANK3 mutations ( 15, 38). The impaired social interaction of SHANK3M3 monkey is apparent and constant, which is in clear contrast to the subtle or no behavioral phenotypes of Shank3 heterozygous mutant mice ( 6). Similarly, the circling behavior of SHANK3M3 monkey in cage is apparent and reproducible. The robust behavioral phenotypes of SHANK3M3 monkey strongly indicate that the non-human primate offers a good alternative to model ASD.
Despite the similarity in the phenotypes of the SHANK3M3 monkey, there are some nontrivial caveats that need to be considered and discussed. First, our findings are based on the results from one mutant monkey. Due to the lower than expected rate of newborn SHANK3 mutant monkeys and the critical function of SHANK3 for early development of the monkey brain ( 8), it is difficult to obtain a large number of live founder animals by CRISPR/Cas9 gene editing. In addition, the genotype and phenotype correlation in humans has not been firmly established because of molecular heterogeneity of SHANK3 mutations ( 3, 15). Thus, it is expected to see a wide range of behavioral phenotypes in individual CRISPR/Cas9-targeted monkeys if their mutations are not identical. In the future, non-human primate cloning technique may facilitate the generation of monkeys with the same genetic modification ( 39). While it is reasonable to be cautious in drawing a conclusion from a single case study, like other typical case studies in clinic, our longitudinal observational studies presented here provided clear evidence for the abnormal behaviors in SHANK3M3 monkey.
From the studies of SHANK3 in both rodent and non-human primate models, we have observed clear and significant differences among them. In humans, there are no reports on any case with homozygous SHANK3 mutation, but patients with heterozygous SHANK3 mutation are typically severely affected. In rodents, a complete depletion of Shank3 in homozygotes does not affect the survival and early development. Heterozygotes of Shank3 mutant mice show very mild or no abnormal behavioral phenotypes ( 6). In contrast, the complete depletion of the SHANK3 gene in the certain monkey brain region results in significant neuronal loss and late embryonic lethality ( 8). This may suggest that complete loss of SHANK3 in human is likely early lethal, and the live SHANK3 mutant monkey is more likely to have a partial loss or haploinsufficiency of SHANK3.
Our findings also demonstrate that the ASD-like symptoms in SHANK3 mutant monkey can be effectively alleviated by the treatment with the antidepressant fluoxetine. Fluoxetine is a member of the class of selective serotonin reuptake inhibitors drug commonly prescribed as an antidepressant. Empirically, many clinicians prescribe fluoxetine to treat repetitive behaviors in ASD children, including SHANK3 mutation-causing ASD (Joan Jasien, personal communication). In a small scale of double-blind placebo-controlled fluoxetine trial, Holland et al. ( 32) reported a significant improvement for repetitive behavior in adult individuals with ASD. Although the mechanism underlying the efficacy of fluoxetine treatment in SHANK3M3 monkey remains to be investigated, the strong response to fluoxetine treatment provides the first example that the non-human primate model may be valuable for future drug development of ASD.
Materials and Methods
Animals
SHANK3M3 monkey and age- and gender-matched healthy control cynomolgus monkeys ( M. fascicularis) were housed in cages and examined for their behaviors at Guangdong Landau Biotechnology Co. Ltd., which is an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility. All animal-related protocols were approved in advance by the Institutional Animal Care and Use Committee of Guangdong Landau Biotechnology Co. Ltd and Jinan University. Commercial monkey diet (Ke-Ao, hfz-15kg, Beijing) was provided ad libitum twice per day. Health and behavior of the monkeys were monitored daily by the husbandry staff and veterinarians. All animals completed the whole study.
Mea
(Truncated: full text at source URL.)