UBE3A and transsynaptic complex NRXN1-CBLN1-GluD1 in a hypothalamic VMHvl-arcuate feedback circuit regulates aggression.

The circuit origins of aggression in autism spectrum disorder remain undefined. Here we report Tac1-expressing glutamatergic neurons in ventrolateral division of ventromedial hypothalamus (VMHvl) drive intermale aggression. Aggression is increased due to increases of Ube3a gene dosage in the VMHvl neurons when modeling autism due to maternal 15q11-13 triplication. Targeted deletion of increased Ube3a copies in VMHvl reverses the elevated aggression adult mice. VMHvl neurons form excitatory synapses onto hypothalamic arcuate nucleus AgRP/NPY neurons through a NRXN1-CBLN1-GluD1 transsynaptic complex and UBE3A impairs this synapse by decreasing Cbln1 gene expression. Exciting AgRP/NPY arcuate neurons leads to feedback inhibition of VMHvl neurons and inhibits aggression. Asymptomatic increases of UBE3A synergize with a heterozygous deficiency of presynaptic Nrxn1 or postsynaptic Grid1 (both ASD genes) to increase aggression. Targeted deletions of Grid1 in arcuate AgRP neurons impairs the VMHvl to AgRP/NPY neuron excitatory synapses while increasing aggression. Chemogenetic/optogenetic activation of arcuate AgRP/NPY neurons inhibits VMHvl neurons and represses aggression. These data reveal that multiple autism genes converge to regulate the VMHvl-arcuate AgRP/NPY glutamatergic synapse. The hypothalamic circuitry implicated by these data suggest impaired excitation of AgRP/NPY feedback inhibitory neurons may explain the increased aggression behavior found in genetic forms of autism.

mGluR5 Negative Modulators for Fragile X: Treatment Resistance and Persistence.

Fragile X syndrome (FXS) is caused by silencing of the human FMR1 gene and is the leading monogenic cause of intellectual disability and autism. Abundant preclinical data indicated that negative allosteric modulators (NAMs) of metabotropic glutamate receptor 5 (mGluR5) might be efficacious in treating FXS in humans. Initial attempts to translate these findings in clinical trials have failed, but these failures provide the opportunity for new discoveries that will improve future trials. The emergence of acquired treatment resistance ("tolerance") after chronic administration of mGluR5 NAMs is a potential factor in the lack of success. Here we confirm that FXS model mice display acquired treatment resistance after chronic treatment with the mGluR5 NAM CTEP in three assays commonly examined in the mouse model of FXS: (1) audiogenic seizure susceptibility, (2) sensory cortex hyperexcitability, and (3) hippocampal protein synthesis. Cross-tolerance experiments suggest that the mechanism of treatment resistance likely occurs at signaling nodes downstream of glycogen synthase kinase 3α (GSK3α), but upstream of protein synthesis. The rapid emergence of tolerance to CTEP begs the question of how previous studies showed an improvement in inhibitory avoidance (IA) cognitive performance after chronic treatment. We show here that this observation was likely explained by timely inhibition of mGluR5 during a critical period, as brief CTEP treatment in juvenile mice is sufficient to provide a persistent improvement of IA behavior measured many weeks later. These data will be important to consider when designing future fragile X clinical trials using compounds that target the mGluR5-to-protein synthesis signaling cascade.

Selective inhibition of glycogen synthase kinase 3α corrects pathophysiology in a mouse model of fragile X syndrome.

Fragile X syndrome is caused by FMR1 gene silencing and loss of the encoded fragile X mental retardation protein (FMRP), which binds to mRNA and regulates translation. Studies in the Fmr1-/y mouse model of fragile X syndrome indicate that aberrant cerebral protein synthesis downstream of metabotropic glutamate receptor 5 (mGluR5) signaling contributes to disease pathogenesis, but clinical trials using mGluR5 inhibitors were not successful. Animal studies suggested that treatment with lithium might be an alternative approach. Targets of lithium include paralogs of glycogen synthase kinase 3 (GSK3), and nonselective small-molecule inhibitors of these enzymes improved disease phenotypes in a fragile X syndrome mouse model. However, the potential therapeutic use of GSK3 inhibitors has been hampered by toxicity arising from inhibition of both α and ß paralogs. Recently, we developed GSK3 inhibitors with sufficient paralog selectivity to avoid a known toxic consequence of dual inhibition, that is, increased ß-catenin stabilization. We show here that inhibition of GSK3α, but not GSK3ß, corrected aberrant protein synthesis, audiogenic seizures, and sensory cortex hyperexcitability in Fmr1-/y mice. Although inhibiting either paralog prevented induction of NMDA receptor-dependent long-term depression (LTD) in the hippocampus, only inhibition of GSK3α impaired mGluR5-dependent and protein synthesis-dependent LTD. Inhibition of GSK3α additionally corrected deficits in learning and memory in Fmr1-/y mice; unlike mGluR5 inhibitors, there was no evidence of tachyphylaxis or enhanced psychotomimetic-induced hyperlocomotion. GSK3α selective inhibitors may have potential as a therapeutic approach for treating fragile X syndrome.

Hypersociability in the Angelman syndrome mouse model.

Deletions and reciprocal triplications of the human chromosomal 15q11-13 region cause two distinct neurodevelopmental disorders. Maternally-derived deletions or inactivating mutations of UBE3A, a 15q11-13 gene expressed exclusively from the maternal allele in neurons, cause Angelman syndrome, characterized by intellectual disability, motor deficits, seizures, and a characteristic increased social smiling, laughing, and eye contact. Conversely, maternally-derived triplications of 15q11-13 cause a behavioral disorder on the autism spectrum with clinical features that include decreased sociability that we recently reconstituted in mice with Ube3a alone. Based on the unique sociability features reported in Angelman syndrome and the repressed sociability observed when Ube3a gene dosage is increased, we hypothesized that mice with neuronal UBE3A loss that models Angelman syndrome would display evidence of hypersocial behavior. We report that mice with maternally-inherited Ube3a gene deletion (Ube3amKO) have a prolonged preference for, and interaction with, social stimuli in the three chamber social approach task. By contrast, interactions with a novel object are reduced. Further, ultrasonic vocalizations and physical contacts are increased in male and female Ube3amKO mice paired with an unfamiliar genotype-matched female. Single housing wild type mice increased these same social behavior parameters to levels observed in Ube3amKO mice where this effect was partially occluded. These results indicate sociability is repressed by social experience and the endogenous levels of UBE3A protein and suggest some social behavioral features observed in Angelman syndrome may reflect an increased social motivation.

Autism gene Ube3a and seizures impair sociability by repressing VTA Cbln1.

Maternally inherited 15q11-13 chromosomal triplications cause a frequent and highly penetrant type of autism linked to increased gene dosages of UBE3A, which encodes a ubiquitin ligase with transcriptional co-regulatory functions. Here, using in vivo mouse genetics, we show that increasing UBE3A in the nucleus downregulates the glutamatergic synapse organizer Cbln1, which is needed for sociability in mice. Epileptic seizures also repress Cbln1 and are found to expose sociability impairments in mice with asymptomatic increases in UBE3A. This Ube3a-seizure synergy maps to glutamate neurons of the midbrain ventral tegmental area (VTA), where Cbln1 deletions impair sociability and weaken glutamatergic transmission. We provide preclinical evidence that viral-vector-based chemogenetic activation of, or restoration of Cbln1 in, VTA glutamatergic neurons reverses the sociability deficits induced by Ube3a and/or seizures. Our results suggest that gene and seizure interactions in VTA glutamatergic neurons impair sociability by downregulating Cbln1, a key node in the expanding protein interaction network of autism genes.

An image-based RNAi screen identifies SH3BP1 as a key effector of Semaphorin 3E-PlexinD1 signaling.

Extracellular signals have to be precisely interpreted intracellularly and translated into diverse cellular behaviors often mediated by cytoskeletal changes. Semaphorins are one of the largest families of guidance cues and play a critical role in many systems. However, how different cell types translate extracellular semaphorin binding into intracellular signaling remains unclear. Here we developed and performed a novel image-based genome-wide functional RNAi screen for downstream signaling molecules that convert the interaction between Semaphorin 3E (Sema3E) and PlexinD1 into cellular behaviors. One of the genes identified in this screen is a RhoGAP protein, SH3-domain binding protein 1 (SH3BP1). We demonstrate that SH3BP1 mediates Sema3E-induced cell collapse through interaction with PlexinD1 and regulation of Ras-related C3 botulinum toxin substrate 1 (Rac1) activity. The identification and characterization of SH3BP1 as a novel downstream effector of Sema3E-PlexinD1 provides an explanation for how extracellular signals are translated into cytoskeletal changes and unique cell behavior, but also lays the foundation for characterizing other genes identified from our screen to obtain a more complete picture of plexin signaling.

Increased gene dosage of Ube3a results in autism traits and decreased glutamate synaptic transmission in mice.

People with autism spectrum disorder are characterized by impaired social interaction, reduced communication, and increased repetitive behaviors. The disorder has a substantial genetic component, and recent studies have revealed frequent genome copy number variations (CNVs) in some individuals. A common CNV that occurs in 1 to 3% of those with autism--maternal 15q11-13 duplication (dup15) and triplication (isodicentric extranumerary chromosome, idic15)--affects several genes that have been suggested to underlie autism behavioral traits. To test this, we tripled the dosage of one of these genes, the ubiquitin protein ligase Ube3a, which is expressed solely from the maternal allele in mature neurons, and reconstituted the three core autism traits in mice: defective social interaction, impaired communication, and increased repetitive stereotypic behavior. The penetrance of these autism traits depended on Ube3a gene copy number. In animals with increased Ube3a gene dosage, glutamatergic, but not GABAergic, synaptic transmission was suppressed as a result of reduced presynaptic release probability, synaptic glutamate concentration, and postsynaptic action potential coupling. These results suggest that Ube3a gene dosage may contribute to the autism traits of individuals with maternal 15q11-13 duplication and support the idea that increased E3A ubiquitin ligase gene dosage results in reduced excitatory synaptic transmission.