Abstract
Variants of the SH3 and multiple ankyrin repeat domain 3 (SHANK3) gene, encoding excitatory postsynaptic core scaffolding proteins, are causally associated with numerous neurodevelopmental and neuropsychiatric disorders, including autism spectrum disorder (ASD), bipolar disorder, intellectual disability, and schizophrenia (SCZ). Although detailed synaptic changes of various Shank3 mutant mice have been well characterized, broader downstream molecular changes, including direct and indirect changes, remain largely unknown. To address this issue, we performed a transcriptome analysis of the medial prefrontal cortex (mPFC) of adult Shank3-overexpressing transgenic (TG) mice, using an RNA-sequencing approach. We also re-analyzed previously reported RNA-sequencing results of the striatum of adult Shank3 TG mice and of the prefrontal cortex of juvenile Shank3+/ΔC mice with a 50–70% reduction of Shank3 proteins. We found that several myelin-related genes were significantly downregulated specifically in the mPFC, but not in the striatum or hippocampus, of adult Shank3 TG mice by comparing the differentially expressed genes (DEGs) of the analyses side by side. Moreover, we also found nine common DEGs between the mPFC and striatum of Shank3 TG mice, among which we further characterized ASD- and SCZ-associated G protein-coupled receptor 85 (Gpr85), encoding an orphan Gpr interacting with PSD-95. Unlike the mPFC-specific decrease of myelin-related genes, we found that the mRNA levels of Gpr85 increased in multiple brain regions of adult Shank3 TG mice, whereas the mRNA levels of its family members, Gpr27 and Gpr173, decreased in the cortex and striatum. Intriguingly, in cultured neurons, the mRNA levels of Gpr27, Gpr85, and Gpr173 were modulated by the neuronal activity. Furthermore, exogenously expressed GPR85 was co-localized with PSD-95 and Shank3 in cultured neurons and negatively regulated the number of excitatory synapses, suggesting its potential role in homeostatic regulation of excitatory synapses in Shank3 TG neurons. Finally, we performed a gene set enrichment analysis of the RNA-sequencing results, which suggested that Shank3 could affect the directional expression pattern of numerous ribosome-related genes in a dosage-dependent manner. To sum up, these results reveal previously unidentified brain region-specific and broad molecular changes in Shank3-overexpressing mice, further elucidating the complexity of the molecular pathophysiology of SHANK3-associated brain disorders.
Highlights
SH3 and multiple ankyrin repeat domain 3 (SHANK3), known as proline-rich synapse-associated protein 2 (ProSAP2), is a gene that encodes excitatory synaptic core scaffolding proteins that organize the macromolecular protein complex of the postsynaptic density (PSD) (Naisbitt et al, 1999; Sheng and Kim, 2000)
To investigate molecular changes in the medial prefrontal cortex (mPFC) of Shank3 TG mice, we performed a transcriptome analysis (RNA sequencing [RNA-seq]) of mPFC tissue from adult (10- to 12-week-old) WT and Shank3 TG mice (Supplementary Tables S1, S2). We reasoned that this unbiased approach might highlight the major molecular changes or signaling pathways affected by mild Shank3 overexpression in the mPFC, as was done to reveal the altered mTORC1 signaling in the striatum of Shank3 TG mice (Lee et al, 2017d)
After applying adjusted P values (
Summary
SH3 and multiple ankyrin repeat domain 3 (SHANK3), known as proline-rich synapse-associated protein 2 (ProSAP2), is a gene that encodes excitatory synaptic core scaffolding proteins that organize the macromolecular protein complex of the postsynaptic density (PSD) (Naisbitt et al, 1999; Sheng and Kim, 2000). Shank3-dependent molecular, structural, and functional changes of excitatory synapses have been deeply characterized from in vitro and in vivo studies to elucidate some of the key underlying pathophysiological mechanisms (Han et al, 2013; Wang et al, 2016) and to provide potential therapeutic approaches for SHANK3-associated brain disorders, mainly ASDs (Bozdagi et al, 2013; Shcheglovitov et al, 2013; Duffney et al, 2015; Bidinosti et al, 2016; Vicidomini et al, 2016; Wang et al, 2016) It still remains largely unknown how different variants of a single gene, SHANK3, can lead to diverse phenotypic outcomes or clinical symptoms. Broader downstream molecular changes, including direct and indirect changes, of Shank that might be possible even in non-neuronal cell types remain scarcely investigated
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