Abstract
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.
Highlights
To explore molecular and cellular mechanisms that underlie the rescue of phenotypes in Shank2-KO mice by early memantine treatment, we performed RNA-Seq analysis of transcriptomes in the forebrain region in WT and Shank2-KO mice at P25 treated early and chronically (P7–21) with vehicle or memantine using a dose (20 mg/kg, oral, twice-a-day) identical to that previously rescued synaptic and behavioral phenotypes in Shank2-KO mice (Chung et al, 2019; Figure 1A, Supplementary Table 1, and Supplementary Figure 1A)
KO-M/V transcripts were negatively enriched for mitochondria- and extracellular matrix (ECM)-related functions (Figure 4A), exhibiting EnrichmentMap patterns for mitochondria and ECM functions (Figure 4B). These results suggest that early memantine treatment of Shank2-KO mice upregulates chromatin- and splicing-related genes and downregulates mitochondria- and ECM-related genes, differences that are largely similar to the transcriptomic differences observed among Mem-K/W transcripts and are likely involved in memantine-dependent phenotypic rescue in Shank2-KO mice
Ribosome/mitochondriarelated genes showed strong positive enrichment, but negative enrichments among these transcripts were insignificant (Figures 5C,D, Supplementary Figure 7, and Supplementary Table 6). These results suggest that memantine treatment in WT mice compared with Shank2 gene deletion leads to both similar and distinct transcriptomic changes
Summary
Shank ( known as ProSAP1) is an abundant excitatory postsynaptic scaffolding protein [(Du et al, 1998; Boeckers et al, 1999; Lim et al, 1999; Naisbitt et al, 1999); reviewed in Boeckers et al (2002); Grabrucker et al (2011), Mossa et al (2017); Sala et al (2015), and Sheng and Kim (2000, 2011)] implicated in autism spectrum disorders (ASD), intellectual disability, developmental delay, and schizophrenia (Berkel et al, 2010, 2011; Pinto et al, 2010; Leblond et al, 2012, 2014; Rauch et al, 2012; Sanders et al, 2012; Chilian et al, 2013; Liu et al, 2013; Guilmatre et al, 2014; Costas, 2015; Peykov et al, 2015a,b; Homann et al, 2016; Mossa et al, 2017; Yuen et al, 2017; Bai et al, 2018; Lu et al, 2018; Satterstrom et al, 2020; Wang et al, 2020).Shank2-mutant mice with a homozygous deletion of exons 6 and 7, hereafter termed Shank2-KO mice, show decreased NMDA receptor (NMDAR) currents, through mechanisms affecting GluN2A- and GluN2B-containing NMDARs, additional details remain to be determined (Won et al, 2012). The same Shank2-KO mice show an opposite change in NMDAR function – NMDAR hyperactivity – at ∼P14 that is thought to cause NMDAR hypoactivity at ∼P21 and later stages, as supported by the fact that early, chronic (P7–21) treatment with memantine, a low-affinity uncompetitive antagonist of extrasynaptic NMDARs (Lipton, 2006) with beneficial effects on various brain disorders, including Alzheimer’s disease (Matsunaga et al, 2015; Folch et al, 2018), normalizes NMDAR function at juvenile (∼P21) and adult (>P56) stages (Chung et al, 2019; Verpelli et al, 2019) These results suggest that early NMDAR hyperactivity at P14 induces NMDAR hypoactivity at P21 that persists into adulthood, and that early correction of NMDAR hyperactivity may prevent the compensatory changes that lead to latestage NMDAR hypoactivity. It is unclear whether early memantine treatment in Shank2-KO mice restores wild-type (WT) mouse-like phenotypes by reversing molecular and cellular changes or induces some unique changes that account for the phenotypic reversal
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