An old model with new insights: endogenous retroviruses drive the evolvement toward ASD susceptibility and hijack transcription machinery during development.

The BTBR T+Itpr3tf/J (BTBR/J) strain is one of the most valid models of idiopathic autism, serving as a potent forward genetics tool to dissect the complexity of autism. We found that a sister strain with an intact corpus callosum, BTBR TF/ArtRbrc (BTBR/R), showed more prominent autism core symptoms but moderate ultrasonic communication/normal hippocampus-dependent memory, which may mimic autism in the high functioning spectrum. Intriguingly, disturbed epigenetic silencing mechanism leads to hyperactive endogenous retrovirus (ERV), a mobile genetic element of ancient retroviral infection, which increases de novo copy number variation (CNV) formation in the two BTBR strains. This feature makes the BTBR strain a still evolving multiple-loci model toward higher ASD susceptibility. Furthermore, active ERV, analogous to virus infection, evades the integrated stress response (ISR) of host defense and hijacks the transcriptional machinery during embryonic development in the BTBR strains. These results suggest dual roles of ERV in the pathogenesis of ASD, driving host genome evolution at a long-term scale and managing cellular pathways in response to viral infection, which has immediate effects on embryonic development. The wild-type Draxin expression in BTBR/R also makes this substrain a more precise model to investigate the core etiology of autism without the interference of impaired forebrain bundles as in BTBR/J.

Abnormal behavior in a chromosome-engineered mouse model for human 15q11-13 duplication seen in autism.

Substantial evidence suggests that chromosomal abnormalities contribute to the risk of autism. The duplication of human chromosome 15q11-13 is known to be the most frequent cytogenetic abnormality in autism. We have modeled this genetic change in mice by using chromosome engineering to generate a 6.3 Mb duplication of the conserved linkage group on mouse chromosome 7. Mice with a paternal duplication display poor social interaction, behavioral inflexibility, abnormal ultrasonic vocalizations, and correlates of anxiety. An increased MBII52 snoRNA within the duplicated region, affecting the serotonin 2c receptor (5-HT2cR), correlates with altered intracellular Ca(2+) responses elicited by a 5-HT2cR agonist in neurons of mice with a paternal duplication. This chromosome-engineered mouse model for autism seems to replicate various aspects of human autistic phenotypes and validates the relevance of the human chromosome abnormality. This model will facilitate forward genetics of developmental brain disorders and serve as an invaluable tool for therapeutic development.

A common epigenetic mechanism across different cellular origins underlies systemic immune dysregulation in an idiopathic autism mouse model.

Immune dysregulation plays a key role in the pathogenesis of autism. Changes occurring at the systemic level, from brain inflammation to disturbed innate/adaptive immune in the periphery, are frequently observed in patients with autism; however, the intrinsic mechanisms behind them remain elusive. We hypothesize a common etiology may lie in progenitors of different types underlying widespread immune dysregulation. By single-cell RNA sequencing (sc-RNA seq), we trace the developmental origins of immune dysregulation in a mouse model of idiopathic autism. It is found that both in aorta-gonad-mesonephros (AGM) and yolk sac (YS) progenitors, the dysregulation of HDAC1-mediated epigenetic machinery alters definitive hematopoiesis during embryogenesis and downregulates the expression of the AP-1 complex for microglia development. Subsequently, these changes result in the dysregulation of the immune system, leading to gut dysbiosis and hyperactive microglia in the brain. We further confirm that dysregulated immune profiles are associated with specific microbiota composition, which may serve as a biomarker to identify autism of immune-dysregulated subtypes. Our findings elucidate a shared mechanism for the origin of immune dysregulation from the brain to the gut in autism and provide new insight to dissecting the heterogeneity of autism, as well as the therapeutic potential of targeting immune-dysregulated autism subtypes.

UBE3A regulates the transcription of IRF, an antiviral immunity.

UBE3A is a gene responsible for the pathogenesis of Angelman syndrome (AS), a neurodevelopmental disorder characterized by symptoms such as intellectual disability, delayed development and severe speech impairment. UBE3A encodes an E3 ubiquitin ligase, for which several targets have been identified, including synaptic molecules. Although proteolysis mainly occurs in the cytoplasm, UBE3A is localized to the cytoplasm and the nucleus. In fact, UBE3A is also known as a transcriptional regulator of the family of nuclear receptors. However, the function of UBE3A in the nucleus remains unclear. Therefore, we examined the involvement of UBE3A in transcription in the nuclei of neurons. Genome-wide transcriptome analysis revealed an enrichment of genes downstream of interferon regulatory factor (IRF) in a UBE3A-deficient AS mouse model. In vitro biochemical analyses further demonstrated that UBE3A interacted with IRF and, more importantly, that UBE3A enhanced IRF-dependent transcription. These results suggest a function for UBE3A as a transcriptional regulator of the immune system in the brain. These findings also provide informative molecular insights into the function of UBE3A in the brain and in AS pathogenesis.

Functional significance of rare neuroligin 1 variants found in autism.

Genetic mutations contribute to the etiology of autism spectrum disorder (ASD), a common, heterogeneous neurodevelopmental disorder characterized by impairments in social interaction, communication, and repetitive and restricted patterns of behavior. Since neuroligin3 (NLGN3), a cell adhesion molecule at the neuronal synapse, was first identified as a risk gene for ASD, several additional variants in NLGN3 and NLGN4 were found in ASD patients. Moreover, synaptopathies are now known to cause several neuropsychiatric disorders including ASD. In humans, NLGNs consist of five family members, and neuroligin1 (NLGN1) is a major component forming a complex on excitatory glutamatergic synapses. However, the significance of NLGN1 in neuropsychiatric disorders remains unknown. Here, we systematically examine five missense variants of NLGN1 that were detected in ASD patients, and show molecular and cellular alterations caused by these variants. We show that a novel NLGN1 Pro89Leu (P89L) missense variant found in two ASD siblings leads to changes in cellular localization, protein degradation, and to the impairment of spine formation. Furthermore, we generated the knock-in P89L mice, and we show that the P89L heterozygote mice display abnormal social behavior, a core feature of ASD. These results, for the first time, implicate rare variants in NLGN1 as functionally significant and support that the NLGN synaptic pathway is of importance in the etiology of neuropsychiatric disorders.

Correction: Functional significance of rare neuroligin 1 variants found in autism.

[This corrects the article DOI: 10.1371/journal.pgen.1006940.].

Behavioral and neuroanatomical analyses in a genetic mouse model of 2q13 duplication.

Duplications of human chromosome 2q13 have been reported in patients with neurodevelopmental disorder including autism spectrum disorder. Nephronophthisis-1 (NPHP1) was identified as a causative gene in the minimal deletion on chromosome 2q13 for familial juvenile type 1 nephronophthisis and Joubert syndrome, an autosomal recessive neurodevelopmental disorder characterized by a cerebellar and brain stem malformation, hypotonia, developmental delay, ataxia, and sometimes associated with cognitive impairment. NPHP1 encodes a ciliary protein, nephrocystin-1, which is expressed in the brain, yet its function in the brain remains largely unknown. In this study, we generated bacterial artificial chromosome-based transgenic mice, called 2q13 dup, that recapitulate human chromosome 2q13 duplication and contain one extra copy of the Nphp1 transgene. To analyze any behavioral alterations in 2q13 dup mice, we conducted a battery of behavioral tests. Although 2q13 dup mice show no significant differences in social behavior, they show deficits in spontaneous alternation behavior and fear memory. We also carried out magnetic resonance imaging to confirm whether copy number gain in this locus affects the neuroanatomy. There was a trend toward a decrease in the cerebellar paraflocculus of 2q13 dup mice. This is the first report of a genetic mouse model for human 2q13 duplication.

Model mice for 15q11-13 duplication syndrome exhibit late-onset obesity and altered lipid metabolism.

Copy number variations on human chromosome 15q11-q13 have been implicated in several neurodevelopmental disorders. A paternal loss or duplication of the Prader-Willi syndrome/Angelman syndrome (PWS/AS) region confers a risk of obesity, although the mechanism remains a mystery due to a lack of an animal model that accurately recreates the obesity phenotype. We performed detailed analyses of mice with duplication of PWS/AS locus (6 Mb) generated by chromosome engineering and found that animals with a paternal duplication of this region (patDp/+) show late-onset obesity, high sensitivity for high-fat diet, high levels of blood leptin and insulin without an increase in food intake. We show that prior to becoming obese, young patDp/+ mice already had enlarged white adipocytes. Transcriptome analysis of adipose tissue revealed an up-regulation of Secreted frizzled-related protein 5 (Sfrp5), known to promote adipogenesis. We additionally generated a new mouse model of paternal duplication focusing on a 3 Mb region (3 Mb patDp/+) within the PWS/AS locus. These mice recapitulate the obese phenotypes including expansion of visceral adipose tissue. Our results suggest paternally expressed genes in PWS/AS locus play a significant role for the obesity and identify new potential targets for future research and treatment of obesity.

Remodeling of the postsynaptic proteome in male mice and marmosets during synapse development.

Postsynaptic proteins play crucial roles in synaptic function and plasticity. During brain development, alterations in synaptic number, shape, and stability occur, known as synapse maturation. However, the postsynaptic protein composition changes during development are not fully understood. Here, we show the trajectory of the postsynaptic proteome in developing male mice and common marmosets. Proteomic analysis of mice at 2, 3, 6, and 12 weeks of age shows that proteins involved in synaptogenesis are differentially expressed during this period. Analysis of published transcriptome datasets shows that the changes in postsynaptic protein composition in the mouse brain after 2 weeks of age correlate with gene expression changes. Proteomic analysis of marmosets at 0, 2, 3, 6, and 24 months of age show that the changes in the marmoset brain can be categorized into two parts: the first 2 months and after that. The changes observed in the first 2 months are similar to those in the mouse brain between 2 and 12 weeks of age. The changes observed in marmoset after 2 months old include differential expression of synaptogenesis-related molecules, which hardly overlap with that in mice. Our results provide a comprehensive proteomic resource that underlies developmental synapse maturation in rodents and primates.

Increased self-triggered vocalizations in an epidermal growth factor-induced rat model for schizophrenia.

Rats elicit two types of ultrasonic vocalizations (USVs), positive (30-80 kHz; high pitch) and negative (10-30 kHz; low pitch) voices. As patients with schizophrenia often exhibit soliloquy-like symptoms, we explored whether an animal model for schizophrenia is similarly characterized by such self-triggered vocalizations. We prepared the animal model by administering an inflammatory cytokine, epidermal growth factor (EGF), to rat neonates, which later develop behavioral and electroencephalographic deficits relevant to schizophrenia. EGF model rats and controls at young (8-10 weeks old) and mature (12-14 weeks old) adult stages were subjected to acclimation, female pairing, and vocalization sessions. In acclimation sessions, low pitch USVs at the mature adult stage were more frequent in EGF model rats than in controls. In the vocalization session, the occurrences of low pitch self-triggered USVs were higher in EGF model rats in both age groups, although this group difference was eliminated by their risperidone treatment. Unlike conventional negative USVs of rats, however, the present low pitch self-triggered USVs had short durations of 10-30 ms. These results suggest the potential that self-triggered vocalization might serve as a translatable pathological trait of schizophrenia to animal models.

Comprehensive topographical map of the serotonergic fibers in the male mouse brain.

It is well established that serotonergic fibers distribute throughout the brain. Abnormal densities or patterns of serotonergic fibers have been implicated in neuropsychiatric disorders. Although many classical studies have examined the distribution pattern of serotonergic fibers, most of them were either limited to specific brain areas or had limitations in demonstrating the fine axonal morphology. In this study, we utilize male mice expressing green fluorescence protein under the serotonin transporter (SERT) promoter to map the topography of serotonergic fibers across the rostro-caudal extent of each brain area. We demonstrate previously unreported regional density and fine-grained anatomy of serotonergic fibers. Our findings include: (a) SERT fibers distribute abundantly in the thalamic nuclei close to the midline and dorsolateral areas, in most of the hypothalamic nuclei with few exceptions such as the median eminence and arcuate nuclei, and within the basal amygdaloid complex and lateral septal nuclei, (b) the source fibers of innervation of the hippocampus traverse through the septal nuclei before reaching its destination, (c) unique, filamentous type of straight terminal fibers within the nucleus accumbens, (d) laminar pattern of innervation in the hippocampus, olfactory bulb and cortex with heterogenicity in innervation density among the layers, (e) cortical labeling density gradually decreases rostro-caudally, (f) fibers traverse and distribute mostly within the gray matter, leaving the white fiber bundles uninnervated, and (g) most of the highly labeled nuclei and cortical areas have predominant anatomical connection to limbic structures. In conclusion, we provide novel, regionally specific insights on the distribution map of serotonergic fibers using transgenic mouse.

Cranioplastic Surgery and Acclimation Training for Awake Mouse fMRI.

MRI is a promising tool for translational research to link brain function and structure in animal models of disease to patients with neuropsychiatric disorders. However, given that mouse functional MRI (fMRI) typically relies on anesthetics to suppress head motion and physiological noise, it has been difficult to directly compare brain fMRI in anesthetized mice with that in conscious patients. Here, we developed a new system to acquire fMRI in awake mice, which includes a head positioner and dedicated radio frequency coil. The system was used to investigate functional brain networks in conscious mice, with the goal of enabling future studies to bridge fMRI of disease model animals with human fMRI. Cranioplastic surgery was performed to affix the head mount and the cupped-hand handling method was performed to minimize stress during MRI scanning. Here we describe the new mouse fMRI system, cranioplastic surgery and acclimation protocol. Graphic abstract: Awake fMRI system to investigate the neuronal activity in awaked mice.

Sensing the Sounds of Silence: A Pilot Study on the Detection of Model Mice of Autism Spectrum Disorder from Ultrasonic Vocalisations.

Studying the animal models of human neuropsychiatric disorders can facilitate the understanding of mechanisms of symptoms both physiologically and genetically. Previous studies have shown that ultrasonic vocalisations (USVs) of mice might be efficient markers to distinguish the wild type group and the model of autism spectrum disorder (mASD). Nevertheless, in-depth analysis of these 'silence' sounds by leveraging the power of advanced computer audition technologies (e. g., deep learning) is limited. To this end, we propose a pilot study on using a large-scale pre-trained audio neural network to extract high-level representations from the USVs of mice for the task on detection of mASD. Experiments have shown a best result reaching an unweighted average recall of 79.2 % for the binary classification task in a rigorous subject-independent scenario. To the best of our knowledge, this is the first time to analyse the sounds that cannot be heard by human beings for the detection of mASD mice. The novel findings can be significant to motivate future works with according means on studying animal models of human patients.

Genetic dissection identifies Necdin as a driver gene in a mouse model of paternal 15q duplications.

Maternally inherited duplication of chromosome 15q11-q13 (Dup15q) is a pathogenic copy number variation (CNV) associated with autism spectrum disorder (ASD). Recently, paternally derived duplication has also been shown to contribute to the development of ASD. The molecular mechanism underlying paternal Dup15q remains unclear. Here, we conduct genetic and overexpression-based screening and identify Necdin (Ndn) as a driver gene for paternal Dup15q resulting in the development of ASD-like phenotypes in mice. An excess amount of Ndn results in enhanced spine formation and density as well as hyperexcitability of cortical pyramidal neurons. We generate 15q dupΔNdn mice with a normalized copy number of Ndn by excising its one copy from Dup15q mice using a CRISPR-Cas9 system. 15q dupΔNdn mice do not show ASD-like phenotypes and show dendritic spine dynamics and cortical excitatory-inhibitory balance similar to wild type animals. Our study provides an insight into the role of Ndn in paternal 15q duplication and a mouse model of paternal Dup15q syndrome.

Development of serotonergic projections to the suprachiasmatic nucleus in the mouse brain.

Serotonin (5-HT) and its innervation have been implicated in various neural functions including circadian systems. Although classical studies have examined the 5-HT innervation pattern in the adult suprachiasmatic nucleus (SCN), the fine-grained morphological study of the development of pathway and terminal projections to the SCN remains scarce. Here, we utilize transgenic mice expressing GFP under the serotonin transporter (SERT) promoter to subserve our developmental mapping study. We demonstrate that the morphology of 5-HT pathway fibers decussating over the supraoptic commissure that projects to the SCN exhibits two distinct developmental patterns. The punctate fibers at the fetal stage gradually become smooth and filamentous, especially during postnatal one week and remain constant thereafter. The innervation field in the SCN develops properly only during postnatal two weeks. Its ventromedial area remains one of the highest 5-HT innervated areas in the adult brain, whereas the dorsolateral area is less innervated. Thus, we provide novel and specific insights on the developmental map of 5-HT system into the SCN using transgenic mouse.

Behavioral neuroscience of autism.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder. Several genetic causes of ASD have been identified and this has enabled researchers to construct mouse models. Mouse behavioral tests reveal impaired social interaction and communication, as well as increased repetitive behavior and behavioral inflexibility in these mice, which correspond to core behavioral deficits observed in individuals with ASD. However, the connection between these behavioral abnormalities and the underlying dysregulation in neuronal circuits and synaptic function is poorly understood. Moreover, different components of the ASD phenotype may be linked to dysfunction in different brain regions, making it even more challenging to chart the pathophysiological mechanisms involved in ASD. Here we summarize the research on mouse models of ASD and their contribution to understanding pathophysiological mechanisms. Specifically, we emphasize abnormal serotonin production and regulation, as well as the disruption in circadian rhythms and sleep that are observed in a subset of ASD, and propose that spatiotemporal disturbances in brainstem development may be a primary cause of ASD that propagates towards the cerebral cortex.

Altered microbiota composition reflects enhanced communication in 15q11-13 CNV mice.

Autism spectrum disorder (ASD) is a complex and heterogeneous neurodevelopmental disorder. In addition to the core symptoms of ASD, many patients with ASD also show comorbid gut dysbiosis, which may lead to various gastrointestinal (GI) problems. Intriguingly, there is evidence that gut microbiota communicate with the central nervous system to modulate behavioral output through the gut-brain axis. To investigate how the microbiota composition is changed in ASD and to identify which microbes are involved in autistic behaviors, we performed a 16S rRNA gene-based metagenomics analysis in an ASD mouse model. Here, we focused on a model with human 15q11-13 duplication (15q dup), the most frequent chromosomal aberration or copy number variation found in ASD. Species diversity of the microbiome was significantly decreased in 15q dup mice. A combination of antibiotics treatment and behavioral analysis showed that neomycin improved social communication in 15q dup mice. Furthermore, comparison of the microbiota composition of mice treated with different antibiotics enabled us to identify beneficial operational taxonomic units (OTUs) for ultrasonic vocalization.

Awake functional MRI detects neural circuit dysfunction in a mouse model of autism.

MRI has potential as a translational approach from rodents to humans. However, given that mouse functional MRI (fMRI) uses anesthetics for suppression of motion, it has been difficult to directly compare the result of fMRI in "unconsciousness" disease model mice with that in "consciousness" patients. We develop awake fMRI to investigate brain function in 15q dup mice, a copy number variation model of autism. Compared to wild-type mice, we find that 15q dup is associated with whole-brain functional hypoconnectivity and diminished fMRI responses to odors of stranger mice. Ex vivo diffusion MRI reveals widespread anomalies in white matter ultrastructure in 15q dup mice, suggesting a putative anatomical substrate for these functional hypoconnectivity. We show that d-cycloserine (DCS) treatment partially normalizes these anormalies in the frontal cortex of 15q dup mice and rescues some social behaviors. Our results demonstrate the utility of awake rodent fMRI and provide a rationale for further investigation of DCS therapy.

Gene expression profile data of the developing small intestine of Id2-deficient mice.

This article contains data related to the research article entitled "Id2 determines intestinal identity through repression of the foregut transcription factor, Irx5" [1]. Id2 deficient (Id2 -/-) mice developed gastric tumors and heterotopic squamous epithelium in the small intestine. These tumors and heterotopic tissues were derived from ectopic gastric cells and squamous cells formed in the small intestine respectively during development. In this study, microarray data of the developing small intestine of Id2 -/- mice was analyzed.

Behavioral analysis in mice deficient for GAREM2 (Grb2-associated regulator of Erk/MAPK subtype2) that is a subtype of highly expressing in the brain.

Grb2-associated regulator of Erk/MAPK (GAREM), is an adaptor protein related to the several cell growth factor receptor-signaling. The GAREM family has two subtypes, GAREM1 and GAREM2, both encoded in the human and mouse genome. Recent genome-wide research identified GAREM2 as a candidate of neurodegenerative diseases. Here, we use knockout (KO) mice to show the role of GAREM2, that is highly expressed in the brain. According to the comprehensive behavioral battery, they exhibited less anxiety both in elevated plus maze and open field tests, mildly increased social approaching behavior in the reciprocal social interaction test, and longer latency to immobility in the tail suspension test as compared to wild-type (WT). Additionally, the extension of neurites in the primary cultured neurons was suppressed in ones derived from GAREM2 KO mice. Furthermore, we also identified Intersectin, as a binding partner of GAREM2 in this study. Intersectin is also a multi-domain adaptor protein that regulates endocytosis and cell signaling, which can potentially alter the subcellular localization of GAREM2. The important molecules, such as the neurotrophin receptor and Erk family, that are involved in the signaling pathway of the neural cell growth in the mouse brain, have been reported to participate in emotional behavior. As GAREM plays a role in the cellular growth factor receptor signaling pathway, GAREM2 may have a common role related to the transduction of Erk signaling in the higher brain functions.