No abstract

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.

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.

BackgroundMutations in the STAMBP gene, which encodes a deubiquitinating isopeptidase called STAM-binding protein, are related to global developmental delay, microcephaly, and capillary malformation. Owing to the limited number of reported cases, the functional and phenotypic characteristics of STAMBP variants require further elucidation.Materials and methodsWhole exome sequencing was performed on a patient presenting with a neurodevelopmental disorder. Novel compound heterozygous mutations in STAMBP [c.843_844del (p.C282Wfs*11) and c.920G > A (p.G307E)] were identified and validated using Sanger sequencing. A 3D human cortical organoid model was used to investigate the function of STAMBP and the pathogenicity of the novel mutation (c.920G > A, p.G307E).ResultsThe patient was presented with global developmental delay, autism spectrum disorder, microcephaly, epilepsy, and dysmorphic facial features but without apparent capillary malformation on the skin and organs. Cortical organoids with STAMBP knockout (KO) showed significantly lower proliferation of neural stem cells (NSCs), leading to smaller organoids that are characteristic of microcephaly. Furthermore, STAMBP disruption did not affect apoptosis in early cortical organoids. After re-expressing wild-type STAMBP, STAMBPG307E, and STAMBPT313I (a known pathogenic mutation) within STAMBP KO organoids, only STAMBPWT rescued the impaired proliferation of STAMBP deficient organoids, but not STAMBPG307E and STAMBPT313I.ConclusionOur findings demonstrate that the clinical phenotype of STAMBP mutations is highly variable, and patients with different STAMBP mutations show differences in the severity of symptoms. The STAMBP missense mutation identified here is a novel pathogenic mutation that impairs the proliferation of NSCs in human brain development.