Deep brain stimulation of the Tbr1-deficient mouse model of autism spectrum disorder at the basolateral amygdala alters amygdalar connectivity, whole-brain synchronization, and social behaviors.

Autism spectrum disorders (ASDs) are considered neural dysconnectivity syndromes. To better understand ASD and uncover potential treatments, it is imperative to know and dissect the connectivity deficits under conditions of autism. Here, we apply a whole-brain immunostaining and quantification platform to demonstrate impaired structural and functional connectivity and aberrant whole-brain synchronization in a Tbr1+/- autism mouse model. We express a channelrhodopsin variant oChIEF fused with Citrine at the basolateral amygdala (BLA) to outline the axonal projections of BLA neurons. By activating the BLA under blue light theta-burst stimulation (TBS), we then evaluate the effect of BLA activation on C-FOS expression at a whole brain level to represent neural activity. We show that Tbr1 haploinsufficiency almost completely disrupts contralateral BLA axonal projections and results in mistargeting in both ipsilateral and contralateral hemispheres, thereby globally altering BLA functional connectivity. Based on correlated C-FOS expression among brain regions, we further show that Tbr1 deficiency severely disrupts whole-brain synchronization in the absence of salient stimulation. Tbr1+/- and wild-type (WT) mice exhibit opposing responses to TBS-induced amygdalar activation, reducing synchronization in WT mice but enhancing it in Tbr1+/- mice. Whole-brain modular organization and intermodule connectivity are also affected by Tbr1 deficiency and amygdalar activation. Following BLA activation by TBS, the synchronizations of the whole brain and the default mode network, a specific subnetwork highly relevant to ASD, are enhanced in Tbr1+/- mice, implying a potential ameliorating effect of amygdalar stimulation on brain function. Indeed, TBS-mediated BLA activation increases nose-to-nose social interactions of Tbr1+/- mice, strengthening evidence for the role of amygdalar connectivity in social behaviors. Our high-resolution analytical platform reveals the inter- and intrahemispheric connectopathies arising from ASD. Our study emphasizes the defective synchronization at a whole-brain scale caused by Tbr1 deficiency and implies a potential beneficial effect of deep brain stimulation at the amygdala for TBR1-linked autism.

Vcp overexpression and leucine supplementation extend lifespan and ameliorate neuromuscular junction phenotypes of a SOD1G93A-ALS mouse model.

Many genes with distinct molecular functions have been linked to genetically heterogeneous amyotrophic lateral sclerosis (ALS), including SuperOxide Dismutase 1 (SOD1) and Valosin-Containing Protein (VCP). SOD1 converts superoxide to oxygen and hydrogen peroxide. VCP acts as a chaperon to regulate protein degradation and synthesis and various other cellular responses. Although the functions of these two genes differ, in the current report we show that overexpression of wild-type VCP in mice enhances lifespan and maintains the size of neuromuscular junctions (NMJs) of both male and female SOD1G93A mice, a well-known ALS mouse model. Although VCP exerts multiple functions, its regulation of ER formation and consequent protein synthesis has been shown to play the most important role in controlling dendritic spine formation and social and memory behaviors. Given that SOD1 mutation results in protein accumulation and aggregation, it may direct VCP to the protein degradation pathway, thereby impairing protein synthesis. Since we previously showed that the protein synthesis defects caused by Vcp deficiency can be improved by leucine supplementation, to confirm the role of the VCP-protein synthesis pathway in SOD1-linked ALS, we applied leucine supplementation to SOD1G93A mice and, similar to Vcp overexpression, we found that it extends SOD1G93A mouse lifespan. In addition, the phenotypes of reduced muscle strength and fewer NMJs of SOD1G93A mice are also improved by leucine supplementation. These results support the existence of crosstalk between SOD1 and VCP and suggest a critical role for protein synthesis in ASL. Our study also implies a potential therapeutic treatment for ALS.

Quantification of the density and morphology of dendritic spines and synaptic protein distribution using Thy1-YFP transgenic mice.

Synaptopathy, which encompasses morphological deficits and any abnormal protein distribution of synapses, is a critical feature of many neurological diseases. We here provide a protocol using mice stably expressing a Thy1-YFP transgene to assess synaptic features in vivo. We describe steps for recording the entire morphology of projection neurons using confocal microscopy based on YFP signals. We detail assessment of the density and size of dendritic spines and the distributions of synaptic proteins using ImageJ and statistical analysis using Prism. For complete details on the use and execution of this protocol, please refer to Shih et al. (2020).1.

Sex bias in social deficits, neural circuits and nutrient demand in Cttnbp2 autism models.

Autism spectrum disorders caused by both genetic and environmental factors are strongly male-biased neuropsychiatric conditions. However, the mechanism underlying the sex bias of autism spectrum disorders remains elusive. Here, we use a mouse model in which the autism-linked gene Cttnbp2 is mutated to explore the potential mechanism underlying the autism sex bias. Autism-like features of Cttnbp2 mutant mice were assessed via behavioural assays. C-FOS staining identified sex-biased brain regions critical to social interaction, with their roles and connectivity then validated by chemogenetic manipulation. Proteomic and bioinformatic analyses established sex-biased molecular deficits at synapses, prompting our hypothesis that male-biased nutrient demand magnifies Cttnbp2 deficiency. Accordingly, intakes of branched-chain amino acids (BCAA) and zinc were experimentally altered to assess their effect on autism-like behaviours. Both deletion and autism-linked mutation of Cttnbp2 result in male-biased social deficits. Seven brain regions, including the infralimbic area of the medial prefrontal cortex (ILA), exhibit reduced neural activity in male mutant mice but not in females upon social stimulation. ILA activation by chemogenetic manipulation is sufficient to activate four of those brain regions susceptible to Cttnbp2 deficiency and consequently to ameliorate social deficits in male mice, implying an ILA-regulated neural circuit is critical to male-biased social deficits. Proteomics analysis reveals male-specific downregulated proteins (including SHANK2 and PSD-95, two synaptic zinc-binding proteins) and female-specific upregulated proteins (including RRAGC) linked to neuropsychiatric disorders, which are likely relevant to male-biased deficits and a female protective effect observed in Cttnbp2 mutant mice. Notably, RRAGC is an upstream regulator of mTOR that senses BCAA, suggesting that mTOR exerts a beneficial effect on females. Indeed, increased BCAA intake activates the mTOR pathway and rescues neuronal responses and social behaviours of male Cttnbp2 mutant mice. Moreover, mutant males exhibit greatly increased zinc demand to display normal social behaviours. Mice carrying an autism-linked Cttnbp2 mutation exhibit male-biased social deficits linked to specific brain regions, differential synaptic proteomes and higher demand for BCAA and zinc. We postulate that lower demand for zinc and BCAA are relevant to the female protective effect. Our study reveals a mechanism underlying sex-biased social defects and also suggests a potential therapeutic approach for autism spectrum disorders.

Macro Photography with Lightsheet Illumination Enables Whole Expanded Brain Imaging with Single-cell Resolution.

Macro photography allows direct visualization of the enlarged whole mouse brain by a combination of lightsheet illumination and expansion microscopy with single-cell resolution.  Taking advantage of the long working distance of a camera lens, we imaged a 3.7 cm thick, transparent, fluorescently-labeled expanded brain. In order to improve 3D sectioning capability, we used lightsheet excitation confined as the depth of field of the camera lens. Using 4x sample expansion and 5x optical magnification, macro photography enables imaging of expanded whole mouse brain with an effective resolution of 300 nm, which provides the subcellular structural information at the organ level.

Social behaviors and contextual memory of Vcp mutant mice are sensitive to nutrition and can be ameliorated by amino acid supplementation.

Both genetic variations and nutritional deficiency are associated with autism spectrum disorders and other neurological disorders. However, it is less clear whether or how nutritional deficiency and genetic variations influence each other under pathogenic conditions. "Valosin-containing protein" (VCP, also known as p97) is associated with multiple neurological disorders and regulates dendritic spine formation by controlling endoplasmic reticulum formation and protein synthesis efficiency. Increased protein synthesis ameliorates the dendritic spine defects of Vcp-deficient neurons. Therefore, we investigated if Vcp-deficient mice are sensitive to nutritional conditions. Here, we show that social interaction and contextual memory of Vcp-deficient mice are indeed influenced by different dietary protein levels. Moreover, leucine supplementation ameliorates the behavioral deficits and dendritic spine density of Vcp-deficient mice, strengthening evidence for the role of protein synthesis in VCP function. Our study illustrates that genetic variation and nutrient factors cross-talk to influence neuronal and behavioral phenotypes.

KLHL17/Actinfilin, a brain-specific gene associated with infantile spasms and autism, regulates dendritic spine enlargement.

BACKGROUND: Dendritic spines, the actin-rich protrusions emerging from dendrites, are the subcellular locations of excitatory synapses in the mammalian brain. Many actin-regulating molecules modulate dendritic spine morphology. Since dendritic spines are neuron-specific structures, it is reasonable to speculate that neuron-specific or -predominant factors are involved in dendritic spine formation. KLHL17 (Kelch-like 17, also known as Actinfilin), an actin-binding protein, is predominantly expressed in brain. Human genetic study has indicated an association of KLHL17/Actinfilin with infantile spasms, a rare form of childhood epilepsy also resulting in autism and mental retardation, indicating that KLHL17/Actinfilin plays a role in neuronal function. However, it remains elusive if and how KLHL17/Actinfilin regulates neuronal development and brain function. METHODS: Fluorescent immunostaining and electrophysiological recording were performed to evaluate dendritic spine formation and activity in cultured hippocampal neurons. Knockdown and knockout of KLHL17/Actinfilin and expression of truncated fragments of KLHL17/Actinfilin were conducted to investigate the function of KLHL17/Actinfilin in neurons. Mouse behavioral assays were used to evaluate the role of KLHL17/Actinfilin in brain function. RESULTS: We found that KLHL17/Actinfilin tends to form circular puncta in dendritic spines and are surrounded by or adjacent to F-actin. Klhl17 deficiency impairs F-actin enrichment at dendritic spines. Knockdown and knockout of KLHL17/Actinfilin specifically impair dendritic spine enlargement, but not the density or length of dendritic spines. Both N-terminal Broad-Complex, Tramtrack and Bric-a-brac (BTB) domain and C-terminal Kelch domains of KLHL17/Actinfilin are required for F-actin remodeling and enrichment at dendritic spines, as well as dendritic spine enlargement. A reduction of postsynaptic and presynsptic markers at dendritic spines and altered mEPSC profiles due to Klhl17 deficiency evidence impaired synaptic activity in Klhl17-deficient neurons. Our behavioral assays further indicate that Klhl17 deficiency results in hyperactivity and reduced social interaction, strengthening evidence for the physiological role of KLHL17/Actinfilin. CONCLUSION: Our findings provide evidence that KLHL17/Actinfilin modulates F-actin remodeling and contributes to regulation of neuronal morphogenesis, maturation and activity, which is likely relevant to behavioral impairment in Klhl17-deficient mice. Trial registration Non-applicable.

Vcp Overexpression and Leucine Supplementation Increase Protein Synthesis and Improve Fear Memory and Social Interaction of Nf1 Mutant Mice.

Neurofibromatosis type 1 (NF1) is a dominant genetic disorder manifesting, in part, as cognitive defects. Previous study indicated that neurofibromin (NF1 protein) interacts with valosin-containing protein (VCP)/P97 to control dendritic spine formation, but the mechanism is unknown. Here, using Nf1+/- mice and transgenic mice overexpressing wild-type Vcp/p97, we demonstrate that neurofibromin acts with VCP to control endoplasmic reticulum (ER) formation and consequent protein synthesis and regulates dendritic spine formation, thereby modulating contextual fear memory and social interaction. To validate the role of protein synthesis, we perform leucine supplementation in vitro and in vivo. Our results suggest that leucine can effectively enter the brain and increase protein synthesis and dendritic spine density of Nf1+/- neurons. Contextual memory and social behavior of Nf1+/- mice are also restored by leucine supplementation. Our study suggests that the "ER-protein synthesis" pathway downstream of neurofibromin and VCP is a critical regulator of dendritic spinogenesis and brain function.

CTTNBP2 Controls Synaptic Expression of Zinc-Related Autism-Associated Proteins and Regulates Synapse Formation and Autism-like Behaviors.

Synaptic dysregulation is a critical feature of autism spectrum disorders (ASDs). Among various autism-associated genes, cortactin binding protein 2 (CTTNBP2) is a cytoskeleton regulator predominantly expressed in neurons and highly enriched at dendritic spines. Here, using Cttnbp2 knockout and ASD-linked mutant mice, we demonstrate that Cttnbp2 deficiency reduces zinc levels in the brain, alters synaptic protein targeting, impairs dendritic spine formation and ultrastructure of postsynaptic density, and influences neuronal activation and autism-like behaviors. A link to autism, the NMDAR-SHANK pathway, and zinc-related regulation are three features shared by CTTNBP2-regulated synaptic proteins. Zinc supplementation rescues the synaptic expression of CTTNBP2-regulated proteins. Moreover, zinc supplementation and administration of D-cycloserine, an NMDAR coagonist, improve the social behaviors of Cttnbp2-deficient mice. We suggest that CTTNBP2 controls the synaptic expression of a set of zinc-regulated autism-associated genes and influences NMDAR function and signaling, providing an example of how genetic and environmental factor crosstalk controls social behaviors.

Anterior Commissure Regulates Neuronal Activity of Amygdalae and Influences Locomotor Activity, Social Interaction and Fear Memory in Mice.

The two hemispheres of the vertebrate brain are connected through several commissures. Although the anterior commissure (AC) is the most conserved white matter structure in the brains of different vertebrates, its complete physiological functionality remains elusive. Since the AC is involved in the connection between two amygdalae and because amygdalae are critical for emotional behaviors and social interaction, we assessed amygdalar activity and function to investigate the physiological role of the AC. We first performed ex vivo electrophysiological recording on mouse brains to demonstrate that the AC delivers a positive signal to facilitate synaptic responses and to recruit basolateral amygdalar neurons via glutamatergic synapses. Transection was then undertaken to investigate the role of the AC in vivo. Results from in vivo optogenetic stimulation suggest that AC transection impairs mutual activation between two basolateral amygdalae. Behavioral analyses were then used to assess if AC surgical lesioning results in hyperactivity, anxiety, social reduction or learning/memory impairment, which are behavioral features associated with neuropsychiatric disorders, such as autism spectrum disorders. We found that AC transection results in higher locomotor activity, aberrant social interaction and reduced associative memory, but not anxiety. Moreover, systemic administration of D-cycloserine, a coagonist of N-methyl-D-aspartate receptor, ameliorated auditory fear memory in AC-transected mice, reinforcing our evidence that the AC potentiates the activity of basolateral amygdalae. Our study suggests that the AC regulates basolateral amygdalar activity and influences neuropsychiatry-related behaviors in mice.

Ecm29-mediated proteasomal distribution modulates excitatory GABA responses in the developing brain.

Neuronal GABAergic responses switch from excitatory to inhibitory at an early postnatal period in rodents. The timing of this switch is controlled by intracellular Cl- concentrations, but factors determining local levels of cation-chloride cotransporters remain elusive. Here, we report that local abundance of the chloride importer NKCC1 and timely emergence of GABAergic inhibition are modulated by proteasome distribution, which is mediated through interactions of proteasomes with the adaptor Ecm29 and the axon initial segment (AIS) scaffold protein ankyrin G. Mechanistically, both the Ecm29 N-terminal domain and an intact AIS structure are required for transport and tethering of proteasomes in the AIS region. In mice, Ecm29 knockout (KO) in neurons increases the density of NKCC1 protein in the AIS region, a change that positively correlates with a delay in the GABAergic response switch. Phenotypically, Ecm29 KO mice showed increased firing frequency of action potentials at early postnatal ages and were hypersusceptible to chemically induced convulsive seizures. Finally, Ecm29 KO neurons exhibited accelerated AIS developmental positioning, reflecting a perturbed AIS morphological plastic response to hyperexcitability arising from proteasome inhibition, a phenotype rescued by ectopic Ecm29 expression or NKCC1 inhibition. Together, our findings support the idea that neuronal maturation requires regulation of proteasomal distribution controlled by Ecm29.

Interhemispheric Connectivity Potentiates the Basolateral Amygdalae and Regulates Social Interaction and Memory.

Impaired interhemispheric connectivity is commonly found in various psychiatric disorders, although how interhemispheric connectivity regulates brain function remains elusive. Here, we use the mouse amygdala, a brain region that is critical for social interaction and fear memory, as a model to demonstrate that contralateral connectivity intensifies the synaptic response of basolateral amygdalae (BLA) and regulates amygdala-dependent behaviors. Retrograde tracing and c-FOS expression indicate that contralateral afferents widely innervate BLA non-randomly and that some BLA neurons innervate both contralateral BLA and the ipsilateral central amygdala (CeA). Our optogenetic and electrophysiological studies further suggest that contralateral BLA input results in the synaptic facilitation of BLA neurons, thereby intensifying the responses to cortical and thalamic stimulations. Finally, pharmacological inhibition and chemogenetic disconnection demonstrate that BLA contralateral facilitation is required for social interaction and memory. Our study suggests that interhemispheric connectivity potentiates the synaptic dynamics of BLA neurons and is critical for the full activation and functionality of amygdalae.

Haploinsufficiency of autism causative gene Tbr1 impairs olfactory discrimination and neuronal activation of the olfactory system in mice.

Background: Autism spectrum disorders (ASD) exhibit two clusters of core symptoms, i.e., social and communication impairment, and repetitive behaviors and sensory abnormalities. Our previous study demonstrated that TBR1, a causative gene of ASD, controls axonal projection and neuronal activation of amygdala and regulates social interaction and vocal communication in a mouse model. Behavioral defects caused by Tbr1 haploinsufficiency can be ameliorated by increasing neural activity via D-cycloserine treatment, an N-methyl-D-aspartate receptor (NMDAR) coagonist. In this report, we investigate the role of TBR1 in regulating olfaction and test whether D-cycloserine can also improve olfactory defects in Tbr1 mutant mice. Methods: We used Tbr1+/- mice as a model to investigate the function of TBR1 in olfactory sensation and discrimination of non-social odors. We employed a behavioral assay to characterize the olfactory defects of Tbr1+/- mice. Magnetic resonance imaging (MRI) and histological analysis were applied to characterize anatomical features. Immunostaining was performed to further analyze differences in expression of TBR1 subfamily members (namely TBR1, TBR2, and TBX21), interneuron populations, and dendritic abnormalities in olfactory bulbs. Finally, C-FOS staining was used to monitor neuronal activation of the olfactory system upon odor stimulation. Results: Tbr1+/- mice exhibited smaller olfactory bulbs and anterior commissures, reduced interneuron populations, and an abnormal dendritic morphology of mitral cells in the olfactory bulbs. Tbr1 haploinsufficiency specifically impaired olfactory discrimination but not olfactory sensation. Neuronal activation upon odorant stimulation was reduced in the glomerular layer of Tbr1+/- olfactory bulbs. Furthermore, although the sizes of piriform and perirhinal cortices were not affected by Tbr1 deficiency, neuronal activation was reduced in these two cortical regions in response to odorant stimulation. These results suggest an impairment of neuronal activation in olfactory bulbs and defective connectivity from olfactory bulbs to the upper olfactory system in Tbr1+/- mice. Systemic administration of D-cycloserine, an NMDAR co-agonist, ameliorated olfactory discrimination in Tbr1+/- mice, suggesting that increased neuronal activity has a beneficial effect on Tbr1 deficiency. Conclusions: Tbr1 regulates neural circuits and activity in the olfactory system to control olfaction. Tbr1+/- mice can serve as a suitable model for revealing how an autism causative gene controls neuronal circuits, neural activity, and autism-related behaviors.

RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway.

Members of the ribonuclease A (RNase A) superfamily regulate various physiological processes. RNase A, the best-studied member of the RNase A superfamily, is widely expressed in different tissues, including brains. We unexpectedly found that RNase A can trigger proliferation of neuronal progenitor cells (NPC) both in vitro and in vivo. RNase A treatment induced cell proliferation in dissociated neuronal cultures and increased cell mass in neurosphere cultures. BrdU (5-Bromo-2'-Deoxyuridine) labeling confirmed the effect of RNase A on cell proliferation. Those dividing cells were Nestin- and SOX2-positive, suggesting that RNase A triggers NPC proliferation. The proliferation inhibitor Ara-C completely suppressed the effect of RNase A on NPC counts, further supporting that RNase A increases NPC number mainly by promoting proliferation. Moreover, we found that RNase A treatment increased ERK phosphorylation and blockade of the ERK pathway inhibited the effect of RNase A on NPC proliferation. Intracerebroventricular injection of RNase A into mouse brain increased the population of 5-ethynyl-2'-deoxyuridine (EdU) or BrdU-labeled cells in the subventricular zone. Those RNase A-induced NPCs were able to migrate into other brain areas, including hippocampus, amygdala, cortex, striatum, and thalamus. In conclusion, our study shows that RNase A promotes proliferation of NPCs via an ERK-dependent pathway and further diversifies the physiological functions of the RNase A family.

Mice lacking cyclin-dependent kinase-like 5 manifest autistic and ADHD-like behaviors.

Neurodevelopmental disorders frequently share common clinical features and appear high rate of comorbidity, such as those present in patients with attention-deficit hyperactivity disorder (ADHD) and autism spectrum disorders (ASD). While characterizing behavioral phenotypes in the mouse model of cyclin-dependent kinase-like 5 (CDKL5) disorder, a neurodevelopmental disorder caused by mutations in the X-linked gene encoding CDKL5, we found that these mice manifested behavioral phenotypes mimicking multiple key features of ASD, such as impaired social interaction and communication, as well as increased stereotypic digging behaviors. These mice also displayed hyper-locomotion, increased aggressiveness and impulsivity, plus deficits in motor and associative learning, resembling primary symptoms of ADHD. Through brain region-specific biochemical analysis, we uncovered that loss of CDKL5 disrupts dopamine synthesis and the expression of social communication-related key genes, such as forkhead-box P2 and mu-opioid receptor, in the corticostriatal circuit. Together, our findings support that CDKL5 plays a role in the comorbid features of autism and ADHD, and mice lacking CDKL5 may serve as an animal model to study the molecular and circuit mechanisms underlying autism-ADHD comorbidity.

Calcium/calmodulin-dependent serine protein kinase (CASK), a protein implicated in mental retardation and autism-spectrum disorders, interacts with T-Brain-1 (TBR1) to control extinction of associative memory in male mice.

BACKGROUND: Human genetic studies have indicated that mutations in calcium/calmodulin-dependent serine protein kinase (CASK) result in X-linked mental retardation and autism-spectrum disorders. We aimed to establish a mouse model to study how Cask regulates mental ability. METHODS: Because Cask encodes a multidomain scaffold protein, a possible strategy to dissect how CASK regulates mental ability and cognition is to disrupt specific protein-protein interactions of CASK in vivo and then investigate the impact of individual specific protein interactions. Previous in vitro analyses indicated that a rat CASK T724A mutation reduces the interaction between CASK and T-brain-1 (TBR1) in transfected COS cells. Because TBR1 is critical for glutamate receptor, ionotropic, N-methyl-D-aspartate receptor subunit 2B (Grin2b) expression and is a causative gene for autism and intellectual disability, we then generated CASK T740A (corresponding to rat CASK T724A) mutant mice using a gene-targeting approach. Immunoblotting, coimmunoprecipitation, histological methods and behavioural assays (including home cage, open field, auditory and contextual fear conditioning and conditioned taste aversion) were applied to investigate expression of CASK and its related proteins, the protein-protein interactions of CASK, and anatomic and behavioural features of CASK T740A mice. RESULTS: The CASK T740A mutation attenuated the interaction between CASK and TBR1 in the brain. However, CASK T740A mice were generally healthy, without obvious defects in brain morphology. The most dramatic defect among the mutant mice was in extinction of associative memory, though acquisition was normal. LIMITATIONS: The functions of other CASK protein interactions cannot be addressed using CASK T740A mice. CONCLUSION: Disruption of the CASK and TBR1 interaction impairs extinction, suggesting the involvement of CASK in cognitive flexibility.

AIM 2 inflammasomes regulate neuronal morphology and influence anxiety and memory in mice.

Inflammasomes are the protein assemblies that consist of inflammasome sensors, adaptor apoptosis-associated speck-like proteins containing a CARD (ASC) and inflammasome caspase. Inflammasomes sense multiple danger signals via various inflammasome sensors and consequently use caspase to trigger proteolytic processing and secretion of IL-1ß cytokines. Recent studies have suggested that neurons use their own innate immune system to detect danger signals and regulate neuronal morphology. Here, we investigate whether inflammasomes, the critical components of innate immunity, participate in regulation of neuronal morphology and function. Among various sensors, Absent in melanoma 2 (Aim2) expression in neurons is most prominent. Adding synthetic double-stranded DNA (dsDNA) to cultured neurons induces IL-1ß secretion in an AIM2-dependent manner and consequently downregulates dendritic growth but enhances axon extension. The results of Aim2 knockout and knockdown show that AIM2 acts cell-autonomously to regulate neuronal morphology. Behavioral analyses further reveal that Aim2-/- mice exhibit lower locomotor activity, increased anxious behaviors and reduced auditory fear memory. In conclusion, our study suggests that AIM2 inflammasomes regulate neuronal morphology and influence mouse behaviors.

Brain-specific transcriptional regulator T-brain-1 controls brain wiring and neuronal activity in autism spectrum disorders.

T-brain-1 (TBR1) is a brain-specific T-box transcription factor. In 1995, Tbr1 was first identified from a subtractive hybridization that compared mouse embryonic and adult telencephalons. Previous studies of Tbr1 (-∕-) mice have indicated critical roles for TBR1 in the development of the cerebral cortex, amygdala, and olfactory bulb. Neuronal migration and axonal projection are two important developmental features controlled by TBR1. Recently, recurrent de novo disruptive mutations in the TBR1 gene have been found in patients with autism spectrum disorders (ASDs). Human genetic studies have identified TBR1 as a high-confidence risk factor for ASDs. Because only one allele of the TBR1 gene is mutated in these patients, Tbr1 (+∕-) mice serve as a good genetic mouse model to explore the mechanism by which de novo TBR1 mutation leads to ASDs. Although neuronal migration and axonal projection defects of cerebral cortex are the most prominent phenotypes in Tbr1 (-∕-) mice, these features are not found in Tbr1 (+∕-) mice. Instead, inter- and intra-amygdalar axonal projections and NMDAR expression and activity in amygdala are particularly susceptible to Tbr1 haploinsufficiency. The studies indicated that both abnormal brain wiring (abnormal amygdalar connections) and excitation/inhibition imbalance (NMDAR hypoactivity), two prominent models for ASD etiology, are present in Tbr1 (+∕-) mice. Moreover, calcium/calmodulin-dependent serine protein kinase (CASK) was found to interact with TBR1. The CASK-TBR1 complex had been shown to directly bind the promoter of the Grin2b gene, which is also known as Nmdar2b, and upregulate Grin2b expression. This molecular function of TBR1 provides an explanation for NMDAR hypoactivity in Tbr1 (+∕-) mice. In addition to Grin2b, cell adhesion molecules-including Ntng1, Cdh8, and Cntn2-are also regulated by TBR1 to control axonal projections of amygdala. Taken together, the studies of Tbr1 provide an integrated picture of ASD etiology at the cellular and circuit levels.

Trans-synaptic zinc mobilization improves social interaction in two mouse models of autism through NMDAR activation.

Genetic aspects of autism spectrum disorders (ASDs) have recently been extensively explored, but environmental influences that affect ASDs have received considerably less attention. Zinc (Zn) is a nutritional factor implicated in ASDs, but evidence for a strong association and linking mechanism is largely lacking. Here we report that trans-synaptic Zn mobilization rapidly rescues social interaction in two independent mouse models of ASD. In mice lacking Shank2, an excitatory postsynaptic scaffolding protein, postsynaptic Zn elevation induced by clioquinol (a Zn chelator and ionophore) improves social interaction. Postsynaptic Zn is mainly derived from presynaptic pools and activates NMDA receptors (NMDARs) through postsynaptic activation of the tyrosine kinase Src. Clioquinol also improves social interaction in mice haploinsufficient for the transcription factor Tbr1, which accompanies NMDAR activation in the amygdala. These results suggest that trans-synaptic Zn mobilization induced by clioquinol rescues social deficits in mouse models of ASD through postsynaptic Src and NMDAR activation.