Sex bias in social deficits, neural circuits and nutrient demand in Cttnbp2 autism models.

Autism spectrum disorders caused by both genetic and environmental factors are strongly male-biased neuropsychiatric conditions. However, the mechanism underlying the sex bias of autism spectrum disorders remains elusive. Here, we use a mouse model in which the autism-linked gene Cttnbp2 is mutated to explore the potential mechanism underlying the autism sex bias. Autism-like features of Cttnbp2 mutant mice were assessed via behavioural assays. C-FOS staining identified sex-biased brain regions critical to social interaction, with their roles and connectivity then validated by chemogenetic manipulation. Proteomic and bioinformatic analyses established sex-biased molecular deficits at synapses, prompting our hypothesis that male-biased nutrient demand magnifies Cttnbp2 deficiency. Accordingly, intakes of branched-chain amino acids (BCAA) and zinc were experimentally altered to assess their effect on autism-like behaviours. Both deletion and autism-linked mutation of Cttnbp2 result in male-biased social deficits. Seven brain regions, including the infralimbic area of the medial prefrontal cortex (ILA), exhibit reduced neural activity in male mutant mice but not in females upon social stimulation. ILA activation by chemogenetic manipulation is sufficient to activate four of those brain regions susceptible to Cttnbp2 deficiency and consequently to ameliorate social deficits in male mice, implying an ILA-regulated neural circuit is critical to male-biased social deficits. Proteomics analysis reveals male-specific downregulated proteins (including SHANK2 and PSD-95, two synaptic zinc-binding proteins) and female-specific upregulated proteins (including RRAGC) linked to neuropsychiatric disorders, which are likely relevant to male-biased deficits and a female protective effect observed in Cttnbp2 mutant mice. Notably, RRAGC is an upstream regulator of mTOR that senses BCAA, suggesting that mTOR exerts a beneficial effect on females. Indeed, increased BCAA intake activates the mTOR pathway and rescues neuronal responses and social behaviours of male Cttnbp2 mutant mice. Moreover, mutant males exhibit greatly increased zinc demand to display normal social behaviours. Mice carrying an autism-linked Cttnbp2 mutation exhibit male-biased social deficits linked to specific brain regions, differential synaptic proteomes and higher demand for BCAA and zinc. We postulate that lower demand for zinc and BCAA are relevant to the female protective effect. Our study reveals a mechanism underlying sex-biased social defects and also suggests a potential therapeutic approach for autism spectrum disorders.

Vcp Overexpression and Leucine Supplementation Increase Protein Synthesis and Improve Fear Memory and Social Interaction of Nf1 Mutant Mice.

Neurofibromatosis type 1 (NF1) is a dominant genetic disorder manifesting, in part, as cognitive defects. Previous study indicated that neurofibromin (NF1 protein) interacts with valosin-containing protein (VCP)/P97 to control dendritic spine formation, but the mechanism is unknown. Here, using Nf1+/- mice and transgenic mice overexpressing wild-type Vcp/p97, we demonstrate that neurofibromin acts with VCP to control endoplasmic reticulum (ER) formation and consequent protein synthesis and regulates dendritic spine formation, thereby modulating contextual fear memory and social interaction. To validate the role of protein synthesis, we perform leucine supplementation in vitro and in vivo. Our results suggest that leucine can effectively enter the brain and increase protein synthesis and dendritic spine density of Nf1+/- neurons. Contextual memory and social behavior of Nf1+/- mice are also restored by leucine supplementation. Our study suggests that the "ER-protein synthesis" pathway downstream of neurofibromin and VCP is a critical regulator of dendritic spinogenesis and brain function.

CTTNBP2 Controls Synaptic Expression of Zinc-Related Autism-Associated Proteins and Regulates Synapse Formation and Autism-like Behaviors.

Synaptic dysregulation is a critical feature of autism spectrum disorders (ASDs). Among various autism-associated genes, cortactin binding protein 2 (CTTNBP2) is a cytoskeleton regulator predominantly expressed in neurons and highly enriched at dendritic spines. Here, using Cttnbp2 knockout and ASD-linked mutant mice, we demonstrate that Cttnbp2 deficiency reduces zinc levels in the brain, alters synaptic protein targeting, impairs dendritic spine formation and ultrastructure of postsynaptic density, and influences neuronal activation and autism-like behaviors. A link to autism, the NMDAR-SHANK pathway, and zinc-related regulation are three features shared by CTTNBP2-regulated synaptic proteins. Zinc supplementation rescues the synaptic expression of CTTNBP2-regulated proteins. Moreover, zinc supplementation and administration of D-cycloserine, an NMDAR coagonist, improve the social behaviors of Cttnbp2-deficient mice. We suggest that CTTNBP2 controls the synaptic expression of a set of zinc-regulated autism-associated genes and influences NMDAR function and signaling, providing an example of how genetic and environmental factor crosstalk controls social behaviors.

Interhemispheric Connectivity Potentiates the Basolateral Amygdalae and Regulates Social Interaction and Memory.

Impaired interhemispheric connectivity is commonly found in various psychiatric disorders, although how interhemispheric connectivity regulates brain function remains elusive. Here, we use the mouse amygdala, a brain region that is critical for social interaction and fear memory, as a model to demonstrate that contralateral connectivity intensifies the synaptic response of basolateral amygdalae (BLA) and regulates amygdala-dependent behaviors. Retrograde tracing and c-FOS expression indicate that contralateral afferents widely innervate BLA non-randomly and that some BLA neurons innervate both contralateral BLA and the ipsilateral central amygdala (CeA). Our optogenetic and electrophysiological studies further suggest that contralateral BLA input results in the synaptic facilitation of BLA neurons, thereby intensifying the responses to cortical and thalamic stimulations. Finally, pharmacological inhibition and chemogenetic disconnection demonstrate that BLA contralateral facilitation is required for social interaction and memory. Our study suggests that interhemispheric connectivity potentiates the synaptic dynamics of BLA neurons and is critical for the full activation and functionality of amygdalae.