Autistic-like social behaviour in Shank2-mutant mice improved by restoring NMDA receptor function

Autism spectrum disorder (ASD) is a group of conditions characterized by impaired social interaction and communication, and restricted and repetitive behaviours. ASD is a highly heritable disorder involving various genetic determinants. Shank2 (also known as ProSAP1) is a multi-domain scaffolding protein and signalling adaptor enriched at excitatory neuronal synapses, and mutations in the human SHANK2 gene have recently been associated with ASD and intellectual disability. Although ASD-associated genes are being increasingly identified and studied using various approaches, including mouse genetics, further efforts are required to delineate important causal mechanisms with the potential for therapeutic application. Here we show that Shank2-mutant (Shank2(-/-)) mice carrying a mutation identical to the ASD-associated microdeletion in the human SHANK2 gene exhibit ASD-like behaviours including reduced social interaction, reduced social communication by ultrasonic vocalizations, and repetitive jumping. These mice show a marked decrease in NMDA (N-methyl-D-aspartate) glutamate receptor (NMDAR) function. Direct stimulation of NMDARs with D-cycloserine, a partial agonist of NMDARs, normalizes NMDAR function and improves social interaction in Shank2(-/-) mice. Furthermore, treatment of Shank2(-/-) mice with a positive allosteric modulator of metabotropic glutamate receptor 5 (mGluR5), which enhances NMDAR function via mGluR5 activation, also normalizes NMDAR function and markedly enhances social interaction. These results suggest that reduced NMDAR function may contribute to the development of ASD-like phenotypes in Shank2(-/-) mice, and mGluR modulation of NMDARs offers a potential strategy to treat ASD.     

Autistic-like behaviours and hyperactivity in mice lacking ProSAP1/Shank2

Autism spectrum disorders comprise a range of neurodevelopmental disorders characterized by deficits in social interaction and communication, and by repetitive behaviour. Mutations in synaptic proteins such as neuroligins, neurexins, GKAPs/SAPAPs and ProSAPs/Shanks were identified in patients with autism spectrum disorder, but the causative mechanisms remain largely unknown. ProSAPs/Shanks build large homo- and heteromeric protein complexes at excitatory synapses and organize the complex protein machinery of the postsynaptic density in a laminar fashion. Here we demonstrate that genetic deletion of ProSAP1/Shank2 results in an early, brain-region-specific upregulation of ionotropic glutamate receptors at the synapse and increased levels of ProSAP2/Shank3. Moreover, ProSAP1/Shank2(-/-) mutants exhibit fewer dendritic spines and show reduced basal synaptic transmission, a reduced frequency of miniature excitatory postsynaptic currents and enhanced N-methyl-d-aspartate receptor-mediated excitatory currents at the physiological level. Mutants are extremely hyperactive and display profound autistic-like behavioural alterations including repetitive grooming as well as abnormalities in vocal and social behaviours. By comparing the data on ProSAP1/Shank2(-/-) mutants with ProSAP2/Shank3αβ(-/-) mice, we show that different abnormalities in synaptic glutamate receptor expression can cause alterations in social interactions and communication. Accordingly, we propose that appropriate therapies for autism spectrum disorders are to be carefully matched to the underlying synaptopathic phenotype.     

Autistic-like behaviour and cerebellar dysfunction in Purkinje cell Tsc1 mutant mice

Autism spectrum disorders (ASDs) are highly prevalent neurodevelopmental disorders, but the underlying pathogenesis remains poorly understood. Recent studies have implicated the cerebellum in these disorders, with post-mortem studies in ASD patients showing cerebellar Purkinje cell (PC) loss, and isolated cerebellar injury has been associated with a higher incidence of ASDs. However, the extent of cerebellar contribution to the pathogenesis of ASDs remains unclear. Tuberous sclerosis complex (TSC) is a genetic disorder with high rates of comorbid ASDs that result from mutation of either TSC1 or TSC2, whose protein products dimerize and negatively regulate mammalian target of rapamycin (mTOR) signalling. TSC is an intriguing model to investigate the cerebellar contribution to the underlying pathogenesis of ASDs, as recent studies in TSC patients demonstrate cerebellar pathology and correlate cerebellar pathology with increased ASD symptomatology. Functional imaging also shows that TSC patients with ASDs display hypermetabolism in deep cerebellar structures, compared to TSC patients without ASDs. However, the roles of Tsc1 and the sequelae of Tsc1 dysfunction in the cerebellum have not been investigated so far. Here we show that both heterozygous and homozygous loss of Tsc1 in mouse cerebellar PCs results in autistic-like behaviours, including abnormal social interaction, repetitive behaviour and vocalizations, in addition to decreased PC excitability. Treatment of mutant mice with the mTOR inhibitor, rapamycin, prevented the pathological and behavioural deficits. These findings demonstrate new roles for Tsc1 in PC function and define a molecular basis for a cerebellar contribution to cognitive disorders such as autism.     

Deficient pheromone responses in mice lacking a cluster of vomeronasal receptor genes

The mammalian vomeronasal organ (VNO), a part of the olfactory system, detects pheromones--chemical signals that modulate social and reproductive behaviours. But the molecular receptors in the VNO that detect these chemosensory stimuli remain undefined. Candidate pheromone receptors are encoded by two distinct and complex superfamilies of genes, V1r and V2r (refs 3 and 4), which code for receptors with seven transmembrane domains. These genes are selectively expressed in sensory neurons of the VNO. However, there is at present no functional evidence for a role of these genes in pheromone responses. Here, using chromosome engineering technology, we delete in the germ line of mice an approximately 600-kilobase genomic region that contains a cluster of 16 intact V1r genes. These genes comprise two of the 12 described V1r gene families, and represent approximately 12% of the V1r repertoire. The mutant mice display deficits in a subset of VNO-dependent behaviours: the expression of male sexual behaviour and maternal aggression is substantially altered. Electrophysiologically, the epithelium of the VNO of such mice does not respond detectably to specific pheromonal ligands. The behavioural impairment and chemosensory deficit support a role of V1r receptors as pheromone receptors.     

Mechanism for the learning deficits in a mouse model of neurofibromatosis type 1.

Neurofibromatosis type I (NF1) is one of the most common single-gene disorders that causes learning deficits in humans. Mice carrying a heterozygous null mutation of the Nfl gene (Nfl(+/-) show important features of the learning deficits associated with NF1 (ref. 2). Although neurofibromin has several known properties and functions, including Ras GTPase-activating protein activity, adenylyl cyclase modulation and microtubule binding, it is unclear which of these are essential for learning in mice and humans. Here we show that the learning deficits of Nf1(+/-) mice can be rescued by genetic and pharmacological manipulations that decrease Ras function. We also show that the Nf1(+/-) mice have increased GABA (gamma-amino butyric acid)-mediated inhibition and specific deficits in long-term potentiation, both of which can be reversed by decreasing Ras function. Our results indicate that the learning deficits associated with NF1 may be caused by excessive Ras activity, which leads to impairments in long-term potentiation caused by increased GABA-mediated inhibition. Our findings have implications for the development of treatments for learning deficits associated with NF1.     

Cortico-striatal synaptic defects and OCD-like behaviours in Sapap3-mutant mice

Obsessive-compulsive disorder (OCD) is an anxiety-spectrum disorder characterized by persistent intrusive thoughts (obsessions) and repetitive actions (compulsions). Dysfunction of cortico-striato-thalamo-cortical circuitry is implicated in OCD, although the underlying pathogenic mechanisms are unknown. SAP90/PSD95-associated protein 3 (SAPAP3; also known as DLGAP3) is a postsynaptic scaffolding protein at excitatory synapses that is highly expressed in the striatum. Here we show that mice with genetic deletion of Sapap3 exhibit increased anxiety and compulsive grooming behaviour leading to facial hair loss and skin lesions; both behaviours are alleviated by a selective serotonin reuptake inhibitor. Electrophysiological, structural and biochemical studies of Sapap3-mutant mice reveal defects in cortico-striatal synapses. Furthermore, lentiviral-mediated selective expression of Sapap3 in the striatum rescues the synaptic and behavioural defects of Sapap3-mutant mice. These findings demonstrate a critical role for SAPAP3 at cortico-striatal synapses and emphasize the importance of cortico-striatal circuitry in OCD-like behaviours.     

Increased affiliative response to vasopressin in mice expressing the V1a receptor from a monogamous vole.

Arginine vasopressin influences male reproductive and social behaviours in several vertebrate taxa through its actions at the V1a receptor in the brain. The neuroanatomical distribution of vasopressin V1a receptors varies greatly between species with different forms of social organization. Here we show that centrally administered arginine vasopressin increases affiliative behaviour in the highly social, monogamous prairie vole, but not in the relatively asocial, promiscuous montane vole. Molecular analyses indicate that gene duplication and/or changes in promoter structure of the prairie vole receptor gene may contribute to the species differences in vasopressin-receptor expression. We further show that mice that are transgenic for the prairie vole receptor gene have a neuroanatomical pattern of receptor binding that is similar to that of the prairie vole, and exhibit increased affiliative behaviour after injection with arginine vasopressin. These data indicate that the pattern of V1a-receptor gene expression in the brain may be functionally associated with species-typical social behaviours in male vertebrates.     

MeCP2转基因食蟹猴所表征的类自闭症行为及种系传递

 自闭症是近年来公众关注度很高的一种神经系统疾病，甲基化CpG结合蛋白2（MeCP2）因其能够在转录水平调节基因表达和操控微小RNA（miRNA）的效应而在自闭症中扮演着重要的角色。当MeCP2因突变而功能缺失时会导致瑞特综合症（Rett syndrome），而当MeCP2拷贝数过多则会导致一种名为MeCP2重复综合症的自闭症。虽然目前科学家已经构建成功了MeCP2的转基因小鼠，但在这种小鼠模型中无法很好地观察到类似人类自闭症的表型。本研究组通过慢病毒侵染的方法构建了能在神经系统中特异表达人源MeCP2的转基因食蟹猴模型，并通过深度测序检测出了转基因插入位点以及通过免疫印迹（westernblot）确证了外源基因的表达。该转基因食蟹猴模型在行动、社交及情绪方面表现出明显的类似自闭症行为，并呈现转基因的种系传递现象。这些结果表明通过基因编辑技术构建非人灵长类模型在脑疾病研究中的重要性。     

CD38 is critical for social behaviour by regulating oxytocin secretion

CD38, a transmembrane glycoprotein with ADP-ribosyl cyclase activity, catalyses the formation of Ca2+ signalling molecules, but its role in the neuroendocrine system is unknown. Here we show that adult CD38 knockout (CD38-/-) female and male mice show marked defects in maternal nurturing and social behaviour, respectively, with higher locomotor activity. Consistently, the plasma level of oxytocin (OT), but not vasopressin, was strongly decreased in CD38-/- mice. Replacement of OT by subcutaneous injection or lentiviral-vector-mediated delivery of human CD38 in the hypothalamus rescued social memory and maternal care in CD38-/- mice. Depolarization-induced OT secretion and Ca2+ elevation in oxytocinergic neurohypophysial axon terminals were disrupted in CD38-/- mice; this was mimicked by CD38 metabolite antagonists in CD38+/+ mice. These results reveal that CD38 has a key role in neuropeptide release, thereby critically regulating maternal and social behaviours, and may be an element in neurodevelopmental disorders.     

Laboratory animal welfare: cage enrichment and mouse behaviour

Mice housed in standard cages show impaired brain development, abnormal repetitive behaviours (stereotypies) and an anxious behavioural profile, all of which can be lessened by making the cage environment more stimulating. But concerns have been raised that enriched housing might disrupt standardization and so affect the precision and reproducibility of behavioural-test results (for example, see ref. 4). Here we show that environmental enrichment increases neither individual variability in behavioural tests nor the risk of obtaining conflicting data in replicate studies. Our findings indicate that the housing conditions of laboratory mice can be markedly improved without affecting the standardization of results.     

A functional circuit underlying male sexual behaviour in the female mouse brain

In mice, pheromone detection is mediated by the vomeronasal organ and the main olfactory epithelium. Male mice that are deficient for Trpc2, an ion channel specifically expressed in VNO neurons and essential for VNO sensory transduction, are impaired in sex discrimination and male-male aggression. We report here that Trpc2-/- female mice show a reduction in female-specific behaviour, including maternal aggression and lactating behaviour. Strikingly, mutant females display unique characteristics of male sexual and courtship behaviours such as mounting, pelvic thrust, solicitation, anogenital olfactory investigation, and emission of complex ultrasonic vocalizations towards male and female conspecific mice. The same behavioural phenotype is observed after VNO surgical removal in adult animals, and is not accompanied by disruption of the oestrous cycle and sex hormone levels. These findings suggest that VNO-mediated pheromone inputs act in wild-type females to repress male behaviour and activate female behaviours. Moreover, they imply that functional neuronal circuits underlying male-specific behaviours exist in the normal female mouse brain.     

Functional identification of an aggression locus in the mouse hypothalamus

Electrical stimulation of certain hypothalamic regions in cats and rodents can elicit attack behaviour, but the exact location of relevant cells within these regions, their requirement for naturally occurring aggression and their relationship to mating circuits have not been clear. Genetic methods for neural circuit manipulation in mice provide a potentially powerful approach to this problem, but brain-stimulation-evoked aggression has never been demonstrated in this species. Here we show that optogenetic, but not electrical, stimulation of neurons in the ventromedial hypothalamus, ventrolateral subdivision (VMHvl) causes male mice to attack both females and inanimate objects, as well as males. Pharmacogenetic silencing of VMHvl reversibly inhibits inter-male aggression. Immediate early gene analysis and single unit recordings from VMHvl during social interactions reveal overlapping but distinct neuronal subpopulations involved in fighting and mating. Neurons activated during attack are inhibited during mating, suggesting a potential neural substrate for competition between these opponent social behaviours.     

Sex-specific peptides from exocrine glands stimulate mouse vomeronasal sensory neurons

In mammals, social and reproductive behaviours are modulated by pheromones, which are chemical signals that convey information about sex and strain. The vomeronasal organ, located at the base of the nasal septum, is responsible for mediating pheromone information in mice. Two classes of putative pheromone receptor gene families, V1R and V2R, are expressed by vomeronasal sensory neurons in mutually segregated epithelial zones of the vomeronasal organ. Although numerous studies have suggested that pheromones originate from urine, direct recordings of behaving mice have shown that neuronal firing in the vomeronasal system is modulated by physical contact with the facial area. Here we identify a male-specific 7-kDa peptide secreted from the extraorbital lacrimal gland. This peptide, which we named exocrine gland-secreting peptide 1 (ESP1), is encoded by a gene from a previously unrecognized large family clustered in proximity to the class I major histocompatibility complex (MHC) region. ESP1 is secreted from the eyes and is transferred to the female vomeronasal organ, where it stimulates V2R-expressing vomeronasal sensory neurons and elicits an electrical response. Our results indicate that mice respond to sex-specific peptides released from exocrine glands through the vomeronasal system during direct contact.     

Post-translational disruption of dystroglycan-ligand interactions in congenital muscular dystrophies

Muscle eye brain disease (MEB) and Fukuyama congenital muscular dystrophy (FCMD) are congenital muscular dystrophies with associated, similar brain malformations. The FCMD gene, fukutin, shares some homology with fringe-like glycosyltransferases, and the MEB gene, POMGnT1, seems to be a new glycosyltransferase. Here we show, in both MEB and FCMD patients, that alpha-dystroglycan is expressed at the muscle membrane, but similar hypoglycosylation in the diseases directly abolishes binding activity of dystroglycan for the ligands laminin, neurexin and agrin. We show that this post-translational biochemical and functional disruption of alpha-dystroglycan is recapitulated in the muscle and central nervous system of mutant myodystrophy (myd) mice. We demonstrate that myd mice have abnormal neuronal migration in cerebral cortex, cerebellum and hippocampus, and show disruption of the basal lamina. In addition, myd mice reveal that dystroglycan targets proteins to functional sites in brain through its interactions with extracellular matrix proteins. These results suggest that at least three distinct mammalian genes function within a convergent post-translational processing pathway during the biosynthesis of dystroglycan, and that abnormal dystroglycan-ligand interactions underlie the pathogenic mechanism of muscular dystrophy with brain abnormalities.     

Developmental defects of the ear, cranial nerves and hindbrain resulting from targeted disruption of the mouse homeobox gene Hox-1.6.

Gene targeting in mouse embryo-derived stem cells has been used to generate mice with a disruption in the homeobox gene Hox-1.6. Mice heterozygous at the Hox-1.6 locus appear normal, whereas Hox-1.6-/Hox-1.6- mice die at or shortly after birth. These homozygotes exhibit profound defects in the formation of the external, middle and inner ears as well as in specific hindbrain nuclei, and in cranial nerves and ganglia. The affected tissues lie within a narrow region along the anteroposterior axis of the mouse but are of diverse embryonic origin. The set of defects associated with the disruption of Hox-1.6 is distinct from and nonoverlapping with that of the closely linked Hox-1.5 gene. But both mutations cause loss, rather than homeotic transformation, of tissues and structures.     

Rewarding effects of opiates are absent in mice lacking the receptor for substance P

Modulation of substance P activity offers a radical new approach to the management of depression, anxiety and stress. The substance P receptor is highly expressed in areas of the brain that are implicated in these behaviours, but also in other areas such as the nucleus accumbens which mediate the motivational properties of both natural rewards such as food and of drugs of abuse such as opiates. Here we show a loss of the rewarding properties of morphine in mice with a genetic disruption of the substance P receptor. The loss was specific to morphine, as both groups of mice responded when cocaine or food were used as rewards. The physical response to opiate withdrawal was also reduced in substance P receptor knockout mice. We conclude that substance P has an important and specific role in mediating the motivational aspects of opiates and may represent a new pharmacological route for the control of drug abuse.     

DISC1-dependent switch from progenitor proliferation to migration in the developing cortex

Regulatory mechanisms governing the sequence from progenitor cell proliferation to neuronal migration during corticogenesis are poorly understood. Here we report that phosphorylation of DISC1, a major susceptibility factor for several mental disorders, acts as a molecular switch from maintaining proliferation of mitotic progenitor cells to activating migration of postmitotic neurons in mice. Unphosphorylated DISC1 regulates canonical Wnt signalling via an interaction with GSK3β, whereas specific phosphorylation at serine 710 (S710) triggers the recruitment of Bardet-Biedl syndrome (BBS) proteins to the centrosome. In support of this model, loss of BBS1 leads to defects in migration, but not proliferation, whereas DISC1 knockdown leads to deficits in both. A phospho-dead mutant can only rescue proliferation, whereas a phospho-mimic mutant rescues exclusively migration defects. These data highlight a dual role for DISC1 in corticogenesis and indicate that phosphorylation of this protein at S710 activates a key developmental switch.     

Keeping time with the human genome

The cloning and characterization of 'clock gene' families has advanced our understanding of the molecular control of the mammalian circadian clock. We have analysed the human genome for additional relatives, and identified new candidate genes that may expand our knowledge of the molecular workings of the circadian clock. This knowledge could lead to the development of therapies for treating jet lag and sleep disorders, and add to our understanding of the genetic contribution of clock gene alterations to sleep and neuropsychiatric disorders. The human genome will also aid in the identification of output genes that ultimately control circadian behaviours.