Emily K. Osterweil

Correction of fragile X syndrome in mice

Fragile X syndrome (FXS) is the most common form of heritable mental retardation and the leading identified cause of autism. FXS is caused by transcriptional silencing of the FMR1 gene that encodes the fragile X mental retardation protein (FMRP), but the pathogenesis of the disease is unknown. According to one proposal, many psychiatric and neurological symptoms of FXS result from unchecked activation of mGluR5, a metabotropic glutamate receptor. To test this idea we generated Fmr1 mutant mice with a 50% reduction in mGluR5 expression and studied a range of phenotypes with relevance to the human disorder. Our results demonstrate that mGluR5 contributes significantly to the pathogenesis of the disease, a finding that has significant therapeutic implications for fragile X and related developmental disorders.

Mutations causing syndromic autism define an axis of synaptic pathophysiology

Tuberous sclerosis complex and fragile X syndrome are genetic diseases characterized by intellectual disability and autism. Because both syndromes are caused by mutations in genes that regulate protein synthesis in neurons, it has been hypothesized that excessive protein synthesis is one core pathophysiological mechanism of intellectual disability and autism. Using electrophysiological and biochemical assays of neuronal protein synthesis in the hippocampus of Tsc2+/− and Fmr1−/y mice, here we show that synaptic dysfunction caused by these mutations actually falls at opposite ends of a physiological spectrum. Synaptic, biochemical and cognitive defects in these mutants are corrected by treatments that modulate metabotropic glutamate receptor 5 in opposite directions, and deficits in the mutants disappear when the mice are bred to carry both mutations. Thus, normal synaptic plasticity and cognition occur within an optimal range of metabotropic glutamate-receptor-mediated protein synthesis, and deviations in either direction can lead to shared behavioural impairments.

Hypersensitivity to mGluR5 and ERK1/2 Leads to Excessive Protein Synthesis in the Hippocampus of a Mouse Model of Fragile X Syndrome

Fragile X syndrome (FXS) is caused by loss of the FMR1 gene product FMRP (fragile X mental retardation protein), a repressor of mRNA translation. According to the metabotropic glutamate receptor (mGluR) theory of FXS, excessive protein synthesis downstream of mGluR5 activation causes the synaptic pathophysiology that underlies multiple aspects of FXS. Here, we use an in vitro assay of protein synthesis in the hippocampus of male Fmr1 knock-out (KO) mice to explore the molecular mechanisms involved in this core biochemical phenotype under conditions where aberrant synaptic physiology has been observed. We find that elevated basal protein synthesis in Fmr1 KO mice is selectively reduced to wild-type levels by acute inhibition of mGluR5 or ERK1/2, but not by inhibition of mTOR (mammalian target of rapamycin). The mGluR5-ERK1/2 pathway is not constitutively overactive in the Fmr1 KO, however, suggesting that mRNA translation is hypersensitive to basal ERK1/2 activation in the absence of FMRP. We find that hypersensitivity to ERK1/2 pathway activation also contributes to audiogenic seizure susceptibility in the Fmr1 KO. These results suggest that the ERK1/2 pathway, and other neurotransmitter systems that stimulate protein synthesis via ERK1/2, represent additional therapeutic targets for FXS.

Lovastatin Corrects Excess Protein Synthesis and Prevents Epileptogenesis in a Mouse Model of Fragile X Syndrome

Many neuropsychiatric symptoms of fragile X syndrome (FXS) are believed to be a consequence of altered regulation of protein synthesis at synapses. We discovered that lovastatin, a drug that is widely prescribed for the treatment of high cholesterol, can correct excess hippocampal protein synthesis in the mouse model of FXS and can prevent one of the robust functional consequences of increased protein synthesis in FXS, epileptogenesis. These data suggest that lovastatin is potentially disease modifying and could be a viable prophylactic treatment for epileptogenesis in FXS.

Cognitive dysfunction and prefrontal synaptic abnormalities in a mouse model of fragile X syndrome

Among the hallmark phenotypes reported in individuals with fragile X syndrome (FXS) are deficits in attentional function, inhibitory control, and cognitive flexibility, a set of cognitive skills thought to be associated with the prefrontal cortex (PFC). However, despite substantial clinical research into these core deficits, the PFC has received surprisingly little attention in preclinical research, particularly in animal models of FXS. In this study, we sought to investigate the molecular, cellular, and behavioral consequences of the loss of the fragile X mental retardation protein in the PFC of Fmr1 KO mice, a mouse model of FXS. We identify a robust cognitive impairment in these mice that may be related to the deficits in cognitive flexibility observed in individuals with FXS. In addition, we report that levels of proteins involved in synaptic function, including the NMDA receptor subunits NR1, NR2A, and NR2B; the scaffolding proteins PSD-95 and SAPAP3; and the plasticity-related gene Arc, are decreased in the prefrontal cortex of Fmr1 KO mice and are partly correlated with behavioral performance. Finally, we report that expression of c-Fos, a marker of neuronal activity, is decreased in the PFC of Fmr1 KO mice. Together, these data suggest that Fmr1 KO mice may represent a valuable animal model for the PFC-associated molecular, cellular, and behavioral abnormalities in FXS and that this model may be useful for testing the efficacy of therapeutic strategies aimed at treating the cognitive impairments in FXS.

Fragile X: Translation in Action

Fragile X is a synapsopathy—a disorder of synaptic function and plasticity. Recent studies using mouse models of the disease suggest that the critical defect is altered regulation of synaptic protein synthesis. Various strategies to restore balanced synaptic protein synthesis have been remarkably successful in correcting widely varied mutant phenotypes in mice. Insights gained by the study of synaptic plasticity in animal models of fragile X have suggested novel therapeutic approaches, not only for human fragile X but also for autism and mental retardation of unknown etiology.

Convergence of Hippocampal Pathophysiology in Syngap+/- and Fmr1-/y Mice.

Previous studies have hypothesized that diverse genetic causes of intellectual disability (ID) and autism spectrum disorders (ASDs) converge on common cellular pathways. Testing this hypothesis requires detailed phenotypic analyses of animal models with genetic mutations that accurately reflect those seen in the human condition (i.e., have structural validity) and which produce phenotypes that mirror ID/ASDs (i.e., have face validity). We show that SynGAP haploinsufficiency, which causes ID with co-occurring ASD in humans, mimics and occludes the synaptic pathophysiology associated with deletion of the Fmr1 gene. Syngap+/− and Fmr1−/y mice show increases in basal protein synthesis and metabotropic glutamate receptor (mGluR)-dependent long-term depression that, unlike in their wild-type controls, is independent of new protein synthesis. Basal levels of phosphorylated ERK1/2 are also elevated in Syngap+/− hippocampal slices. Super-resolution microscopy reveals that Syngap+/− and Fmr1−/y mice show nanoscale alterations in dendritic spine morphology that predict an increase in biochemical compartmentalization. Finally, increased basal protein synthesis is rescued by negative regulators of the mGlu subtype 5 receptor and the Ras–ERK1/2 pathway, indicating that therapeutic interventions for fragile X syndrome may benefit patients with SYNGAP1 haploinsufficiency. SIGNIFICANCE STATEMENT As the genetics of intellectual disability (ID) and autism spectrum disorders (ASDs) are unraveled, a key issue is whether genetically divergent forms of these disorders converge on common biochemical/cellular pathways and hence may be amenable to common therapeutic interventions. This study compares the pathophysiology associated with the loss of fragile X mental retardation protein (FMRP) and haploinsufficiency of synaptic GTPase-activating protein (SynGAP), two prevalent monogenic forms of ID. We show that Syngap+/− mice phenocopy Fmr1−/y mice in the alterations in mGluR-dependent long-term depression, basal protein synthesis, and dendritic spine morphology. Deficits in basal protein synthesis can be rescued by pharmacological interventions that reduce the mGlu5 receptor–ERK1/2 signaling pathway, which also rescues the same deficit in Fmr1−/y mice. Our findings support the hypothesis that phenotypes associated with genetically diverse forms of ID/ASDs result from alterations in common cellular/biochemical pathways.

Perturbed proteostasis in autism spectrum disorders

Dynamic changes in synaptic strength rely on de novo protein synthesis and protein degradation by the ubiquitin proteasome system (UPS). Disruption of either of these cellular processes will result in significant impairments in synaptic plasticity and memory formation. Mutations in several genes encoding regulators of mRNA translation and members of the UPS have been associated with an increased risk for the development of autism spectrum disorders. It is possible that these mutations result in a similar imbalance in protein homeostasis (proteostasis) at the synapse. This review will summarize recent work investigating the role of the UPS in synaptic plasticity at glutamatergic synapses, and propose that dysfunctional proteostasis is a common consequence of several genetic mutations linked to autism spectrum disorders.

Extinction of an instrumental response: a cognitive behavioral assay in Fmr1 knockout mice

Fragile X (FX) is the most common genetic cause of intellectual disability and autism. Previous studies have shown that partial inhibition of metabotropic glutamate receptor signaling is sufficient to correct behavioral phenotypes in a mouse model of FX, including audiogenic seizures, open‐field hyperactivity and social behavior. These phenotypes model well the epilepsy (15%), hyperactivity (20%) and autism (30%) that are comorbid with FX in human patients. Identifying reliable and robust mouse phenotypes to model cognitive impairments is critical considering the 90% comorbidity of FX and intellectual disability. Recent work characterized a five‐choice visuospatial discrimination assay testing cognitive flexibility, in which FX model mice show impairments associated with decreases in synaptic proteins in prefrontal cortex (PFC). In this study, we sought to determine whether instrumental extinction, another process requiring PFC, is altered in FX model mice, and whether downregulation of metabotropic glutamate receptor signaling pathways is sufficient to correct both visuospatial discrimination and extinction phenotypes. We report that instrumental extinction is consistently exaggerated in FX model mice. However, neither the extinction phenotype nor the visuospatial discrimination phenotype is corrected by approaches targeting metabotropic glutamate receptor signaling. This work describes a novel behavioral extinction assay to model impaired cognition in mouse models of neurodevelopmental disorders, provides evidence that extinction is exaggerated in the FX mouse model and suggests possible limitations of metabotropic glutamate receptor‐based pharmacotherapy.

Negative Allosteric Modulation of mGluR5 Partially Corrects Pathophysiology in a Mouse Model of Rett Syndrome.

Rett syndrome (RTT) is caused by mutations in the gene encoding methyl-CpG binding protein 2 (MECP2), an epigenetic regulator of mRNA transcription. Here, we report a test of the hypothesis of shared pathophysiology of RTT and fragile X, another monogenic cause of autism and intellectual disability. In fragile X, the loss of the mRNA translational repressor FMRP leads to exaggerated protein synthesis downstream of metabotropic glutamate receptor 5 (mGluR5). We found that mGluR5- and protein-synthesis-dependent synaptic plasticity were similarly altered in area CA1 of Mecp2 KO mice. CA1 pyramidal cell-type-specific, genome-wide profiling of ribosome-bound mRNAs was performed in wild-type and Mecp2 KO hippocampal CA1 neurons to reveal the MeCP2-regulated “translatome.” We found significant overlap between ribosome-bound transcripts overexpressed in the Mecp2 KO and FMRP mRNA targets. These tended to encode long genes that were functionally related to either cytoskeleton organization or the development of neuronal connectivity. In the Fmr1 KO mouse, chronic treatment with mGluR5-negative allosteric modulators (NAMs) has been shown to ameliorate many mutant phenotypes by correcting excessive protein synthesis. In Mecp2 KO mice, we found that mGluR5 NAM treatment significantly reduced the level of overexpressed ribosome-associated transcripts, particularly those that were also FMRP targets. Some Rett phenotypes were also ameliorated by treatment, most notably hippocampal cell size and lifespan. Together, these results suggest a potential mechanistic link between MeCP2-mediated transcription regulation and mGluR5/FMRP-mediated protein translation regulation through coregulation of a subset of genes relevant to synaptic functions. SIGNIFICANCE STATEMENT Altered regulation of synaptic protein synthesis has been hypothesized to contribute to the pathophysiology that underlies multiple forms of intellectual disability and autism spectrum disorder. Here, we show in a mouse model of Rett syndrome (Mecp2 KO) that metabotropic glutamate receptor 5 (mGluR5)- and protein-synthesis-dependent synaptic plasticity are abnormal in the hippocampus. We found that a subset of ribosome-bound mRNAs was aberrantly upregulated in hippocampal CA1 neurons of Mecp2 KO mice, that these significantly overlapped with FMRP direct targets and/or SFARI human autism genes, and that chronic treatment of Mecp2 KO mice with an mGluR5-negative allosteric modulator tunes down upregulated ribosome-bound mRNAs and partially improves mutant mice phenotypes.