Using Shank3 Model Mice to Probe the Neuroanatomic Basis of Autism

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2017

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Bey, Alexandra Lyndon

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Jiang, Yong-hui

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Abstract

Autism spectrum disorders (ASDs) are increasingly prevalent, and the costs associated with caring for affected patients across the lifespan are immense. However, the pathophysiology and brain regions involved in characteristic behavioral impairments remain poorly defined, which hinders progress towards targeted therapeutic development. Different brain regions have been suggested from human neuroimaging studies but the circuit mechanism is not known and cannot be easily defined in human studies. Genetic studies indicate that SHANK3, a gene encoding a scaffolding protein at the postsynaptic density, is a strong ASD causative gene. Studies of various isoform-specific knockout mice support these mice as valid models to dissect the pathophysiology of ASDs and implicate differential involvement of brain regions such as hippocampus and striatum. However, none of these mice recapitulate the most frequent SHANK-related mutation found in ASD patients: a deletion of the entire SHANK3 gene.

For this reason, we have created conventional complete knockout mice by deleting almost the entire coding region of exons 4 to 22, Shank3 Δe4-22, and performed a thorough characterization of their behavioral phenotypes. Their abnormalities in complex social and communication behaviors in addition to their profound display of repetitive and restrictive behaviors in combination with comorbid anxiety, locomotor, and learning phenotypes support them as a mouse model for SHANK3-causing autism with good construct and face validity. Additional studies by collaborators identified a striatal-centered model of circuit and synaptic dysfunction. Manipulation of metabotropic glutamate receptor 5 (mGluR5) activity attenuated the excessive grooming and instrumental learning differentially in Δe4-22-/- mice. These findings show that deficiency of the autism-associated Shank3 gene can impair mGluR5-Homer scaffolding, resulting in cortico-striatal circuit abnormalities which underlie deficits in learning and ASD-like behaviors. These data suggest causal links between genetic, molecular, and circuit mechanisms underlying the pathophysiology of ASDs.

However, because these and other existing Shank3 mutant mice are not region specific, causality between different brain regions and ASD-like behaviors cannot be firmly established. In order to define anatomic regions implicated in behavioral manifestations of ASD, conditional knockout mice lacking Shank3 proteins in different brain regions including forebrain excitatory neurons (NEX-Cre) and striatal inhibitory neurons (Dlx5/6-Cre), as well as distinct cell populations including direct (D1-Cre) and indirect (D2-Cre) medium spiny neurons, were generated and subjected to behavioral phenotyping. Different autism-relevant behaviors as well as comorbid behaviors were recapitulated by targeting Shank3 deletion to different brain regions or cell types. Electrophysiological and biochemical studies further identified synaptic defects resulting from region- or cell-autonomous loss of Shank3, with different biochemical pathways implicated when Shank3 deletion was targeted to the cortex and hippocampus versus the basal ganglia. This study demonstrates the impact of specific brain regions in modulating ASD-related behavior and identifies key molecular defects which are restricted to specific brain regions in SHANK3-deficient ASD, thus providing future therapeutic targets.

Lastly, as one of the major advantages of modeling ASDs in mice is their amenability for pre-clinical studies of interventions, we have tested two different cellular therapies hypothesized to modulate neuronal circuit function either through direct differentiation of stem cells into brain cells including neurons, glia, and microglia or through indirect effects on neuro-immune modulation. While neither perinatal nor young adulthood treatment with human umbilical cord blood derived stem cells affected significant improvements in the behaviors of Shank3 knockout mice, these experiments underscored the robust, reliable behavioral phenotypes of this animal model as well as supported the safety and tolerability of these treatments in a rodent pre-clinical model with a relevant genetic construct.

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Bey, Alexandra Lyndon (2017). Using Shank3 Model Mice to Probe the Neuroanatomic Basis of Autism. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/14354.

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