Mutations or deletions of the SHANK3 gene are causative for Phelan–McDermid syndrome (PMDS), a syndromic form of autism spectrum disorders (ASDs). We analyzed Shank3Δ11(−/−) mice and organoids from PMDS individuals to study effects on myelin. SHANK3 was found to be expressed in oligodendrocytes and Schwann cells, and MRI analysis of Shank3Δ11(−/−) mice revealed a reduced volume of the corpus callosum as seen in PMDS patients. Myelin proteins including myelin basic protein showed significant temporal and regional differences with lower levels in the CNS but increased amounts in the PNS of Shank3Δ11(−/−) animals. Node, as well as paranode, lengths were increased and ultrastructural analysis revealed region-specific alterations of the myelin sheaths. In PMDS hiPSC-derived cerebral organoids we observed an altered number and delayed maturation of myelinating cells. These findings provide evidence that, in addition to a synaptic deregulation, impairment of myelin might profoundly contribute to the clinical manifestation of SHANK3 deficiency.

Background Autism spectrum disorder (ASD) is mainly characterized by deficits in social interaction and communication and repetitive behaviors. Known causes of ASD are mutations of certain risk genes like the postsynaptic protein SHANK3 and environmental factors including prenatal infections. Methods To analyze the gene-environment interplay in ASD, we combined the Shank3Δ11−/− ASD mouse model with maternal immune activation (MIA) via an intraperitoneal injection of polyinosinic/polycytidylic acid (Poly I:C) on gestational day 12.5. The offspring of the injected dams was further analyzed for autistic-like behaviors and comorbidities followed by biochemical experiments with a focus on synaptic analysis. Results We show that the two-hit mice exhibit excessive grooming and deficits in social behavior more prominently than the Shank3Δ11−/− mice. Interestingly, these behavioral changes were accompanied by an unexpected upregulation of postsynaptic density (PSD) proteins at excitatory synapses in striatum, hippocampus and prefrontal cortex. Limitations We found several PSD proteins to be increased in the two-hit mice; however, we can only speculate about possible pathways behind the worsening of the autistic phenotype in those mice. Conclusions With this study, we demonstrate that there is an interplay between genetic susceptibility and environmental factors defining the severity of ASD symptoms. Moreover, we show that a general misbalance of PSD proteins at excitatory synapses is linked to ASD symptoms, making this two-hit model a promising tool for the investigation of the complex pathophysiology of neurodevelopmental disorders.

Whole animal perfusion is a well-established method that has been used for the past decades in multiple research fields. Particularly, it has been very important for the study of the brain. The rapid and uniform fixation of tissue is essential for the preservation of its integrity and the study of complex structures. For small tissue pieces submerging in formaldehyde solution oftentimes is sufficient to get a good fixation, larger tissues or organs with a more complicated structure present a greater difficulty. Here, we report the precise parameters to successfully perform trans-cardiac perfusion of neonatal mouse pups that allows a uniform fixation of the whole body for subsequent structural analysis and immunohistochemistry. In comparison to standard perfusion procedures of adult mice, changes in the pump velocity, the buffer volume and in the needle size lead to high quality fixation of neonatal mice pups. Further, we present a whole-body section staining, which results in a highly specific immunofluorescence signal suited for detailed analysis of multiple tissues or systems at the same time. Thus, our protocol provides a reproducible and reliable method for neonatal perfusion and staining that can rapidly be applied in any laboratory. It allows a high quality analysis of cellular structures and expression profiles at early developmental stages.

SHANK2 (ProSAP1) is a postsynaptic scaffolding protein of excitatory synapses in the central nervous system and implicated in the development of autism spectrum disorders (ASD). Patients with mutations in SHANK2 show autism-like behaviors, developmental delay, and intellectual disability. We generated human induced pluripotent stem cells (hiPSC) from a patient carrying a heterozygous deletion of SHANK2 and from the unaffected parents. In patient hiPSCs and derived neurons SHANK2 mRNA and protein expression was reduced. During neuronal maturation, a reduction in growth cone size and a transient increase in neuronal soma size were observed. Neuronal proliferation was increased, and apoptosis was decreased in young and mature neurons. Additionally, mature patient hiPSC-derived neurons showed dysregulated excitatory signaling and a decrease of a broad range of signaling molecules of the ERK-MAP kinase pathway. These findings could be confirmed in brain samples from Shank2(−/−) mice, which also showed decreased mGluR5 and phospho-ERK1/2 expression. Our study broadens the current knowledge of SHANK2-related ASD. We highlight the importance of excitatory-inhibitory balance and mGluR5 dysregulation with disturbed downstream ERK1/2 signaling in ASD, which provides possible future therapeutic strategies for SHANK2-related ASD.

Whole animal perfusion is a well-established method that has been used for the past decades in multiple research fields. Particularly, it has been very important for the study of the brain. The rapid and uniform fixation of tissue is essential for the preservation of its integrity and the study of complex structures. For small tissue pieces submerging in formaldehyde solution oftentimes is sufficient to get a good fixation, larger tissues or organs with a more complicated structure present a greater difficulty. Here, we report the precise parameters to successfully perform trans-cardiac perfusion of neonatal mouse pups that allows a uniform fixation of the whole body for subsequent structural analysis and immunohistochemistry. In comparison to standard perfusion procedures of adult mice, changes in the pump velocity, the buffer volume and in the needle size lead to high quality fixation of neonatal mice pups. Further, we present a whole-body section staining, which results in a highly specific immunofluorescence signal suited for detailed analysis of multiple tissues or systems at the same time. Thus, our protocol provides a reproducible and reliable method for neonatal perfusion and staining that can rapidly be applied in any laboratory. It allows a high quality analysis of cellular structures and expression profiles at early developmental stages.