Human genetic and genomic studies have supported a strong causal role of SHANK3 deficiency in autism spectrum disorder (ASD). However, the molecular mechanism underlying SHANK3 deficiency resulting in ASD is not fully understood. Recently, the zebrafish has become an attractive organism to model ASD because of its high efficiency of genetic manipulation and robust behavioral phenotypes. The orthologous gene to human SHANK3 is duplicated in the zebrafish genome and has two homologs, shank3a and shank3b. Previous studies have reported shank3 morphants in zebrafish using the morpholino method. Here, we report the generation and characterization of shank3b mutant zebrafish in larval and adult stages using the CRISPR/Cas9 genome editing technique. CRISPR/Cas9 was applied to generate a shank3b loss-of-function mutation (shank3b ^−/− ) in zebrafish. A series of morphological measurements, behavioral tests, and molecular analyses were performed to systematically characterize the behavioral and molecular changes in shank3b mutant zebrafish. shank3b^−/− zebrafish exhibited abnormal morphology in early development. They showed reduced locomotor activity both as larvae and adults, reduced social interaction and time spent near conspecifics, and significant repetitive swimming behaviors. Additionally, the levels of both postsynaptic homer1 and presynaptic synaptophysin were significantly reduced in the adult brain of shank3b-deficient zebrafish. We generated the first inheritable shank3b mutant zebrafish model using CRISPR/Cas9 gene editing approach. shank3b^−/− zebrafish displayed robust autism-like behaviors and altered levels of the synaptic proteins homer1 and synaptophysin. The versatility of zebrafish as a model for studying neurodevelopment and conducting drug screening will likely have a significant contribution to future studies of human SHANK3 function and ASD.

Searching for effective biomarkers is one of the most challenging tasks in the research field of Autism Spectrum Disorder (ASD). Magnetic resonance imaging (MRI) provides a non-invasive and powerful tool for investigating changes in the structure, function, maturation, connectivity, and metabolism of the brain of children with ASD. Here, we review the more recent MRI studies in young children with ASD, aiming to provide candidate biomarkers for the diagnosis of childhood ASD. The review covers structural imaging methods, diffusion tensor imaging, resting-state functional MRI, and magnetic resonance spectroscopy. Future advances in neuroimaging techniques, as well as cross-disciplinary studies and large-scale collaborations will be needed for an integrated approach linking neuroimaging, genetics, and phenotypic data to allow the discovery of new, effective biomarkers.

Repetitive Transcranial Magnetic Stimulation (rTMS)/ Deep-brain Magnetic Stimulation (DMS) is an effective therapy for various neuropsychiatric disorders including major depression disorder. The molecular and cellular mechanisms underlying the impacts of rTMS/DMS on the brain are not yet fully understood.

Recently, deleterious variants in the BR serine/threonine kinase 2 (BRSK2) gene have been reported in patients with autism spectrum disorder (ASD), suggesting that BRSK2 is a new high-confidence ASD risk gene, which presents an opportunity to understand the underlying neuropathological mechanisms of ASD. In this study, we performed clinical and neurobehavioral evaluations of a proband with a de novo non-sense variant in BRSK2 (p.R222X) with other reported BRSK2 mutant patients. To validate BRSK2 as an ASD risk gene, we generated a novel brsk2b-deficient zebrafish line through CRISPR/Cas9 and characterized its morphological and neurobehavioral features as well as performed molecular analysis of neurogenesis-related markers. The proband displayed typical ASD behaviors and language and motor delay, which were similar to other published BRSK2 mutant patients. Morphologically, brsk2b^–/– larvae exhibited a higher embryonic mortality and rate of pericardium edema, severe developmental delay, and depigmentation as well as growth retardation in the early developmental stage. Behaviorally, brsk2b^–/– zebrafish displayed significantly decreased activity in open field tests and enhanced anxiety levels in light/dark tests and thigmotaxis analysis. Specifically, brsk2b^–/– zebrafish showed a prominent reduction of social interaction with peers and disrupted social cohesion among homogeneous groups. Molecularly, the mRNA expression levels of homer1b (a postsynaptic density scaffolding protein), and mbpa, mpz, and plp1b (molecular markers of oligodendrocytes and myelination) were increased in the brain tissues of adult brsk2b^–/– zebrafish, while the expression level of isl1a, a marker of motor neurons, was decreased. Taken together, for the first time, we established a novel brsk2b-deficient zebrafish model that showed prominent ASD-like behaviors. In addition, the disturbed mRNA expression levels of neurogenesis-related markers implied that the processes of postsynaptic signaling as well as oligodendrocytes and myelination may be involved. This discovery may suggest a path for further research to identify the underlying neuropathological mechanisms between BRSK2 and ASD.

SHANK3 is a scaffolding protein that binds to various synaptic proteins at the postsynaptic density (PSD) of excitatory glutamatergic synapses. SHANK3 is not only strongly implicated in autism spectrum disorders (ASD) but also plays a critical role in human Phelan-McDermid syndrome (22q13.3 deletion syndrome). Accumulated experimental evidence demonstrates that the zebrafish model system is useful for studying the functions of ASD-related gene during early development. However, many basic features of shank3 transcript expression in zebrafish remain poorly understood. Here, we investigated temporal, spatial, and isoform-specific expression patterns of shank3 during zebrafish development on the basis of previous researches and the differential effects of each shank3 transcript expression after exposure to valproic acid (VPA), an ASD-associated drug. At first, we observed that both shank3a and shank3b were barely expressed at very early ages (before 24 h post-fertilization (hpf)), whereas their expression levels were increased and mainly enriched in the nervous system after 24 hpf. Secondly, all of the six shank3 transcripts gradually increased during the first 7 hpf and then decreased. Subsequently, they exhibited a second increasing peak between 1 month post-fertilization (mpf) and adulthood. Thirdly, VPA treatment affected the isoform-specific expression of zebrafish shank3. In particular, the mRNA expression levels of those isoforms that contain a SAM domain were significantly increased, whereas the mRNA expression level of those which contained an ANK domain but without a SAM domain was decreased. To conclude, our findings support the molecular diversity of shank3 in zebrafish and provide a molecular framework to understand the isoform-specific function of shank3 in zebrafish.

Mutations of the SHANK3 gene are found in some autism spectrum disorder (ASD) patients, and animal models harboring SHANK3 mutations exhibit a variety of ASD-like behaviors, presenting a unique opportunity to explore the underlying neuropathological mechanisms and potential pharmacological treatments. The histone deacetylase (HDAC) valproic acid (VPA) has demonstrated neuroprotective and neuroregenerative properties, suggesting possible therapeutic utility for ASD. Therefore, SHANK3-associated ASD-like symptoms present a convenient model to evaluate the potential benefits, therapeutic window, and optimal dose of VPA. We constructed a novel shank3-deficient (shank3ab^–/–) zebrafish model through CRISPR/Cas9 editing and conducted comprehensive morphological and neurobehavioral evaluations, including of core ASD-like behaviors, as well as molecular analyses of synaptic proteins expression levels. Furthermore, different VPA doses and treatment durations were examined for effects on ASD-like phenotypes. Compared to wild types (WTs), shank3ab^–/– zebrafish exhibited greater developmental mortality, more frequent abnormal tail bending, pervasive developmental delay, impaired social preference, repetitive swimming behaviors, and generally reduced locomotor activity. The expression levels of synaptic proteins were also dramatically reduced in shank3ab^–/– zebrafish. These ASD-like behaviors were attenuated by low-dose (5 μM) VPA administered from 4 to 8 days post-fertilization (dpf), and the effects persisted to adulthood. In addition, the observed underexpression of grm5, encoding glutamate metabotropic receptor 5, was significantly improved in VPA-treated shank3ab^–/– zebrafish. We report for the first time that low-dose VPA administered after neural tube closure has lasting beneficial effects on the social deficits and repetitive behavioral patterns in shank3-deficient ASD model zebrafish. These findings provide a promising strategy for ASD clinical drug development.