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Shank2 is an excitatory postsynaptic scaffolding protein strongly implicated in autism spectrum disorders (ASDs). Shank2-mutant mice with a homozygous deletion of exons 6 and 7 (Shank2-KO mice) show decreased NMDA receptor (NMDAR) function and autistic-like behaviors at juvenile [∼postnatal day (P21)] and adult (>P56) stages that are rescued by NMDAR activation. However, at ∼P14, these mice show the opposite change - increased NMDAR function; moreover, suppression of NMDAR activity with early, chronic memantine treatment during P7-21 prevents NMDAR hypofunction and autistic-like behaviors at later (∼P21 and >P56) stages. To better understand the mechanisms underlying this rescue, we performed RNA-Seq gene-set enrichment analysis of forebrain transcriptomes from wild-type (WT) and Shank2-KO juvenile (P25) mice treated early and chronically (P7-21) with vehicle or memantine. Vehicle-treated Shank2-KO mice showed upregulation of synapse-related genes and downregulation of ribosome- and mitochondria-related genes compared with vehicle-treated WT mice. They also showed a transcriptomic pattern largely opposite that observed in ASD (reverse-ASD pattern), based on ASD-related/risk genes and cell-type-specific genes. In memantine-treated Shank2-KO mice, chromatin-related genes were upregulated; mitochondria, extracellular matrix (ECM), and actin-related genes were downregulated; and the reverse-ASD pattern was weakened compared with that in vehicle-treated Shank2-KO mice. In WT mice, memantine treatment, which does not alter NMDAR function, upregulated synaptic genes and downregulated ECM genes; memantine-treated WT mice also exhibited a reverse-ASD pattern. Therefore, early chronic treatment of Shank2-KO mice with memantine alters expression of chromatin, mitochondria, ECM, actin, and ASD-related genes.

BACKGROUND: Autism spectrum disorder involves neurodevelopmental dysregulations that lead to visible symptoms at early stages of life. Many autism spectrum disorder-related mechanisms suggested by animal studies are supported by demonstrated improvement in autistic-like phenotypes in adult animals following experimental reversal of dysregulated mechanisms. However, whether such mechanisms also act at earlier stages to cause autistic-like phenotypes is unclear. METHODS: We used Shank2-/- mice carrying a mutation identified in human autism spectrum disorder (exons 6 and 7 deletion) and combined electrophysiological and behavioral analyses to see whether early pathophysiology at pup stages is different from late pathophysiology at juvenile and adult stages and whether correcting early pathophysiology can normalize late pathophysiology and abnormal behaviors in juvenile and adult mice. RESULTS: Early correction of a dysregulated mechanism in young mice prevents manifestation of autistic-like social behaviors in adult mice. Shank2-/- mice, known to display N-methyl-D-aspartate receptor (NMDAR) hypofunction and autistic-like behaviors at postweaning stages after postnatal day 21 (P21), show the opposite synaptic phenotype-NMDAR hyperfunction-at an earlier preweaning stage (∼P14). Moreover, this NMDAR hyperfunction at P14 rapidly shifts to NMDAR hypofunction after weaning (∼P24). Chronic suppression of the early NMDAR hyperfunction by the NMDAR antagonist memantine (P7-P21) prevents NMDAR hypofunction and autistic-like social behaviors from manifesting at later stages (∼P28 and P56). CONCLUSIONS: Early NMDAR hyperfunction leads to late NMDAR hypofunction and autistic-like social behaviors in Shank2-/- mice, and early correction of NMDAR dysfunction has the long-lasting effect of preventing autistic-like social behaviors from developing at later stages.

UNLABELLED: Shank2 is a multidomain scaffolding protein implicated in the structural and functional coordination of multiprotein complexes at excitatory postsynaptic sites as well as in psychiatric disorders, including autism spectrum disorders. While Shank2 is strongly expressed in the cerebellum, whether Shank2 regulates cerebellar excitatory synapses, or contributes to the behavioral abnormalities observed in Shank2-/- mice, remains unexplored. Here we show that Shank2-/- mice show reduced excitatory synapse density in cerebellar Purkinje cells in association with reduced levels of excitatory postsynaptic proteins, including GluD2 and PSD-93, and impaired motor coordination in the Erasmus test. Shank2 deletion restricted to Purkinje cells (Pcp2-Cre;Shank2fl/fl mice) leads to similar reductions in excitatory synapse density, synaptic protein levels, and motor coordination. Pcp2-Cre;Shank2fl/fl mice do not recapitulate autistic-like behaviors observed in Shank2-/- mice, such as social interaction deficits, altered ultrasonic vocalizations, repetitive behaviors, and hyperactivity. However, Pcp2-Cre;Shank2fl/fl mice display enhanced repetitive behavior in the hole-board test and anxiety-like behavior in the light-dark test, which are not observed in Shank2-/- mice. These results implicate Shank2 in the regulation of cerebellar excitatory synapse density, motor coordination, and specific repetitive and anxiety-like behaviors. SIGNIFICANCE STATEMENT: The postsynaptic side of excitatory synapses contains multiprotein complexes, termed the postsynaptic density, which contains receptors, scaffolding/adaptor proteins, and signaling molecules. Shank2 is an excitatory postsynaptic scaffolding protein implicated in the formation and functional coordination of the postsynaptic density and has been linked to autism spectrum disorders. Using Shank2-null mice and Shank2-conditional knock-out mice with a gene deletion restricted to cerebellar Purkinje cells, we explored functions of Shank2 in the cerebellum. We found that Shank2 regulates excitatory synapse density, motor coordination, and specific repetitive and anxiety-like behaviors, but is not associated with autistic-like social deficits or repetitive behaviors.