Adnp-mutant mice with cognitive inflexibility, CaMKIIα hyperactivity, and synaptic plasticity deficits.

ADNP syndrome, involving the ADNP transcription factor of the SWI/SNF chromatin-remodeling complex, is characterized by developmental delay, intellectual disability, and autism spectrum disorders (ASD). Although Adnp-haploinsufficient (Adnp-HT) mice display various phenotypic deficits, whether these mice display abnormal synaptic functions remain poorly understood. Here, we report synaptic plasticity deficits associated with cognitive inflexibility and CaMKIIα hyperactivity in Adnp-HT mice. These mice show impaired and inflexible contextual learning and memory, additional to social deficits, long after the juvenile-stage decrease of ADNP protein levels to ~10% of the newborn level. The adult Adnp-HT hippocampus shows hyperphosphorylated CaMKIIα and its substrates, including SynGAP1, and excessive long-term potentiation that is normalized by CaMKIIα inhibition. Therefore, Adnp haploinsufficiency in mice leads to cognitive inflexibility involving CaMKIIα hyperphosphorylation and excessive LTP in adults long after its marked expressional decrease in juveniles.

Age, brain region, and gene dosage-differential transcriptomic changes in Shank3-mutant mice.

Shank3 is an abundant excitatory postsynaptic scaffolding protein implicated in various neurodevelopmental disorders, including autism spectrum disorder (ASD), Phelan-McDermid syndrome, intellectual disability, and schizophrenia. Shank3-mutant mice show various molecular, synaptic, and behavioral deficits, but little is known about how transcriptomic phenotypes vary across different ages, brain regions, and gene dosages. Here, we report transcriptomic patterns in the forebrains of juvenile and adult homozygous Shank3-mutant mice that lack exons 14-16 and also the prefrontal, hippocampal, and striatal transcriptomes in adult heterozygous and homozygous Shank3-mutant mice. The juvenile and adult mutant transcriptomes show patterns opposite from and similar to those observed in ASD (termed reverse-ASD and ASD-like patterns), respectively. The juvenile transcriptomic changes accompany synaptic upregulations and ribosomal and mitochondrial downregulations, whereas the adult transcriptome show opposite changes. The prefrontal, hippocampal, and striatal transcriptomes show differential changes in ASD-related gene expressions and biological functions associated with synapse, ribosome, mitochondria, and spliceosome. These patterns also differ across heterozygous and homozygous Shank3-mutant mice. These results suggest age, brain region, and gene dosage-differential transcriptomic changes in Shank3-mutant mice.

Brain region and gene dosage-differential transcriptomic changes in Shank2-mutant mice.

Shank2 is an abundant excitatory postsynaptic scaffolding protein that has been implicated in various neurodevelopmental and psychiatric disorders, including autism spectrum disorder (ASD), intellectual disability, attention-deficit/hyperactivity disorder, and schizophrenia. Shank2-mutant mice show ASD-like behavioral deficits and altered synaptic and neuronal functions, but little is known about how different brain regions and gene dosages affect the transcriptomic phenotypes of these mice. Here, we performed RNA-Seq-based transcriptomic analyses of the prefrontal cortex, hippocampus, and striatum in adult Shank2 heterozygous (HT)- and homozygous (HM)-mutant mice lacking exons 6-7. The prefrontal cortical, hippocampal, and striatal regions showed distinct transcriptomic patterns associated with synapse, ribosome, mitochondria, spliceosome, and extracellular matrix (ECM). The three brain regions were also distinct in the expression of ASD-related and ASD-risk genes. These differential patterns were stronger in the prefrontal cortex where the HT transcriptome displayed increased synaptic gene expression and reverse-ASD patterns whereas the HM transcriptome showed decreased synaptic gene expression and ASD-like patterns. These results suggest brain region- and gene dosage-differential transcriptomic changes in Shank2-mutant mice.

A Deficiency of the Psychiatric Risk Gene DLG2/PSD-93 Causes Excitatory Synaptic Deficits in the Dorsolateral Striatum.

Genetic variations resulting in the loss of function of the discs large homologs (DLG2)/postsynaptic density protein-93 (PSD-93) gene have been implicated in the increased risk for schizophrenia, intellectual disability, and autism spectrum disorders (ASDs). Previously, we have reported that mice lacking exon 14 of the Dlg2 gene (Dlg2 -/- mice) display autistic-like behaviors, including social deficits and increased repetitive behaviors, as well as suppressed spontaneous excitatory postsynaptic currents in the striatum. However, the neural substrate underpinning such aberrant synaptic network activity remains unclear. Here, we found that the corticostriatal synaptic transmission was significantly impaired in Dlg2 -/- mice, which did not seem attributed to defects in presynaptic releases of cortical neurons, but to the reduced number of functional synapses in the striatum, as manifested in the suppressed frequency of miniature excitatory postsynaptic currents in spiny projection neurons (SPNs). Using transmission electron microscopy, we found that both the density of postsynaptic densities and the fraction of perforated synapses were significantly decreased in the Dlg2 -/- dorsolateral striatum. The density of dendritic spines was significantly reduced in striatal SPNs, but notably, not in the cortical pyramidal neurons of Dlg2 -/- mice. Furthermore, a DLG2/PSD-93 deficiency resulted in the compensatory increases of DLG4/PSD-95 and decreases in the expression of TrkA in the striatum, but not particularly in the cortex. These results suggest that striatal dysfunction might play a role in the pathology of psychiatric disorders that are associated with a disruption of the Dlg2 gene.

Excitatory synapses and gap junctions cooperate to improve Pv neuronal burst firing and cortical social cognition in Shank2-mutant mice.

NMDA receptor (NMDAR) and GABA neuronal dysfunctions are observed in animal models of autism spectrum disorders, but how these dysfunctions impair social cognition and behavior remains unclear. We report here that NMDARs in cortical parvalbumin (Pv)-positive interneurons cooperate with gap junctions to promote high-frequency (>80 Hz) Pv neuronal burst firing and social cognition. Shank2-/- mice, displaying improved sociability upon NMDAR activation, show impaired cortical social representation and inhibitory neuronal burst firing. Cortical Shank2-/- Pv neurons show decreased NMDAR activity, which suppresses the cooperation between NMDARs and gap junctions (GJs) for normal burst firing. Shank2-/- Pv neurons show compensatory increases in GJ activity that are not sufficient for social rescue. However, optogenetic boosting of Pv neuronal bursts, requiring GJs, rescues cortical social cognition in Shank2-/- mice, similar to the NMDAR-dependent social rescue. Therefore, NMDARs and gap junctions cooperate to promote cortical Pv neuronal bursts and social cognition.

SLC6A20 transporter: a novel regulator of brain glycine homeostasis and NMDAR function.

Glycine transporters (GlyT1 and GlyT2) that regulate levels of brain glycine, an inhibitory neurotransmitter with co-agonist activity for NMDA receptors (NMDARs), have been considered to be important targets for the treatment of brain disorders with suppressed NMDAR function such as schizophrenia. However, it remains unclear whether other amino acid transporters expressed in the brain can also regulate brain glycine levels and NMDAR function. Here, we report that SLC6A20A, an amino acid transporter known to transport proline based on in vitro data but is understudied in the brain, regulates proline and glycine levels and NMDAR function in the mouse brain. SLC6A20A transcript and protein levels were abnormally increased in mice carrying a mutant PTEN protein lacking the C terminus through enhanced ß-catenin binding to the Slc6a20a gene. These mice displayed reduced extracellular levels of brain proline and glycine and decreased NMDAR currents. Elevating glycine levels back to normal ranges by antisense oligonucleotide-induced SLC6A20 knockdown, or the competitive GlyT1 antagonist sarcosine, normalized NMDAR currents and repetitive climbing behavior observed in these mice. Conversely, mice lacking SLC6A20A displayed increased extracellular glycine levels and NMDAR currents. Lastly, both mouse and human SLC6A20 proteins mediated proline and glycine transports, and SLC6A20 proteins could be detected in human neurons. These results suggest that SLC6A20 regulates proline and glycine homeostasis in the brain and that SLC6A20 inhibition has therapeutic potential for brain disorders involving NMDAR hypofunction.

A DLG2 deficiency in mice leads to reduced sociability and increased repetitive behavior accompanied by aberrant synaptic transmission in the dorsal striatum.

BACKGROUND: DLG2, also known as postsynaptic density protein-93 (PSD-93) or chapsyn-110, is an excitatory postsynaptic scaffolding protein that interacts with synaptic surface receptors and signaling molecules. A recent study has demonstrated that mutations in the DLG2 promoter region are significantly associated with autism spectrum disorder (ASD). Although DLG2 is well known as a schizophrenia-susceptibility gene, the mechanisms that link DLG2 gene disruption with ASD-like behaviors remain unclear. METHODS: Mice lacking exon 14 of the Dlg2 gene (Dlg2-/- mice) were used to investigate whether Dlg2 deletion leads to ASD-like behavioral abnormalities. To this end, we performed a battery of behavioral tests assessing locomotion, anxiety, sociability, and repetitive behaviors. In situ hybridization was performed to determine expression levels of Dlg2 mRNA in different mouse brain regions during embryonic and postnatal brain development. We also measured excitatory and inhibitory synaptic currents to determine the impacts of Dlg2 deletion on synaptic transmission in the dorsolateral striatum. RESULTS: Dlg2-/- mice showed hypoactivity in a novel environment. They also exhibited decreased social approach, but normal social novelty recognition, compared with wild-type animals. In addition, Dlg2-/- mice displayed strong self-grooming, both in home cages and novel environments. Dlg2 mRNA levels in the striatum were heightened until postnatal day 7 in mice, implying potential roles of DLG2 in the development of striatal connectivity. In addition, the frequency of excitatory, but not inhibitory, spontaneous postsynaptic currents in the Dlg2-/- dorsolateral striatum was significantly reduced. CONCLUSION: These results suggest that homozygous Dlg2 deletion in mice leads to ASD-like behavioral phenotypes, including social deficits and increased repetitive behaviors, as well as reductions in excitatory synaptic input onto dorsolateral spiny projection neurons, implying that the dorsal striatum is one of the brain regions vulnerable to the developmental dysregulation of DLG2.

The Neomycin Resistance Cassette in the Targeted Allele of Shank3B Knock-Out Mice Has Potential Off-Target Effects to Produce an Unusual Shank3 Isoform.

Variants of the SH3 and multiple ankyrin repeat domains 3 (SHANK3), which encodes postsynaptic scaffolds, are associated with brain disorders. The targeted alleles in a few Shank3 knock-out (KO) lines contain a neomycin resistance (Neo) cassette, which may perturb the normal expression of neighboring genes; however, this has not been investigated in detail. We previously reported an unexpected increase in the mRNA expression of Shank3 exons 1-12 in the brains of Shank3B KO mice generated by replacing Shank3 exons 13-16 with the Neo cassette. In this study, we confirmed that the increased Shank3 mRNA in Shank3B KO brains produced an unusual ∼60 kDa Shank3 isoform (Shank3-N), which did not properly localize to the synaptic compartment. Functionally, Shank3-N overexpression altered the dendritic spine morphology in cultured neurons. Importantly, Shank3-N expression in Shank3B KO mice was not a compensatory response to a reduction of full-length Shank3 because expression was still detected in the brain after normalizing the level of full-length Shank3. Moreover, in another Shank3 KO line (Shank3 gKO) with a similar Shank3 exonal deletion as that in Shank3B KO mice but without a Neo cassette, the mRNA expression levels of Shank3 exons 1-12 were lower than those of wild-type mice and Shank3-N was not detected in the brain. In addition, the expression levels of genes neighboring Shank3 on chromosome 15 were altered in the striatum of Shank3B KO but not Shank3 gKO mice. These results suggest that the Neo cassette has potential off-target effects in Shank3B KO mice.

Shank3 Exons 14-16 Deletion in Glutamatergic Neurons Leads to Social and Repetitive Behavioral Deficits Associated With Increased Cortical Layer 2/3 Neuronal Excitability.

Shank3, an abundant excitatory postsynaptic scaffolding protein, has been associated with multiple brain disorders, including autism spectrum disorders (ASD) and Phelan-McDermid syndrome (PMS). However, how cell type-specific Shank3 deletion affects disease-related neuronal and brain functions remains largely unclear. Here, we investigated the impacts of Shank3 deletion in glutamatergic neurons on synaptic and behavioral phenotypes in mice and compared results with those previously obtained from mice with global Shank3 mutation and GABAergic neuron-specific Shank3 mutation. Neuronal excitability was abnormally increased in layer 2/3 pyramidal neurons in the medial prefrontal cortex (mPFC) in mice with a glutamatergic Shank3 deletion, similar to results obtained in mice with a global Shank3 deletion. In addition, excitatory synaptic transmission was abnormally increased in layer 2/3 neurons in mice with a global, but not a glutamatergic, Shank3 deletion, suggesting that Shank3 in glutamatergic neurons are important for the increased neuronal excitability, but not for the increased excitatory synaptic transmission. Neither excitatory nor inhibitory synaptic transmission was altered in the dorsal striatum of Shank3-deficient glutamatergic neurons, a finding that contrasts with the decreased excitatory synaptic transmission in global and Shank3-deficient GABAergic neurons. Behaviorally, glutamatergic Shank3-deficient mice displayed abnormally increased direct social interaction and repetitive self-grooming, similar to global and GABAergic Shank3-deficient mice. These results suggest that glutamatergic and GABAergic Shank3 deletions lead to distinct synaptic and neuronal changes in cortical layer 2/3 and dorsal striatal neurons, but cause similar social and repetitive behavioral abnormalities likely through distinct mechanisms.

Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility.

Shank3, a postsynaptic scaffolding protein involved in regulating excitatory synapse assembly and function, has been implicated in several brain disorders, including autism spectrum disorders (ASD), Phelan-McDermid syndrome, schizophrenia, intellectual disability, and mania. Here we generated and characterized a Shank3 knock-in mouse line carrying the Q321R mutation (Shank3 Q321R mice) identified in a human individual with ASD that affects the ankyrin repeat region (ARR) domain of the Shank3 protein. Homozygous Shank3 Q321R/Q321R mice show a selective decrease in the level of Shank3a, an ARR-containing protein variant, but not other variants. CA1 pyramidal neurons in the Shank3 Q321R/Q321R hippocampus show decreased neuronal excitability but normal excitatory and inhibitory synaptic transmission. Behaviorally, Shank3 Q321R/Q321R mice show moderately enhanced self-grooming and anxiolytic-like behavior, but normal locomotion, social interaction, and object recognition and contextual fear memory. In addition, these mice show abnormal electroencephalogram (EEG) patterns and decreased susceptibility to induced seizures. These results indicate that the Q321R mutation alters Shank3 protein stability, neuronal excitability, repetitive and anxiety-like behavior, EEG patterns, and seizure susceptibility in mice.

Early Correction of N-Methyl-D-Aspartate Receptor Function Improves Autistic-like Social Behaviors in Adult Shank2-/- Mice.

BACKGROUND: Autism spectrum disorder involves neurodevelopmental dysregulations that lead to visible symptoms at early stages of life. Many autism spectrum disorder-related mechanisms suggested by animal studies are supported by demonstrated improvement in autistic-like phenotypes in adult animals following experimental reversal of dysregulated mechanisms. However, whether such mechanisms also act at earlier stages to cause autistic-like phenotypes is unclear. METHODS: We used Shank2-/- mice carrying a mutation identified in human autism spectrum disorder (exons 6 and 7 deletion) and combined electrophysiological and behavioral analyses to see whether early pathophysiology at pup stages is different from late pathophysiology at juvenile and adult stages and whether correcting early pathophysiology can normalize late pathophysiology and abnormal behaviors in juvenile and adult mice. RESULTS: Early correction of a dysregulated mechanism in young mice prevents manifestation of autistic-like social behaviors in adult mice. Shank2-/- mice, known to display N-methyl-D-aspartate receptor (NMDAR) hypofunction and autistic-like behaviors at postweaning stages after postnatal day 21 (P21), show the opposite synaptic phenotype-NMDAR hyperfunction-at an earlier preweaning stage (∼P14). Moreover, this NMDAR hyperfunction at P14 rapidly shifts to NMDAR hypofunction after weaning (∼P24). Chronic suppression of the early NMDAR hyperfunction by the NMDAR antagonist memantine (P7-P21) prevents NMDAR hypofunction and autistic-like social behaviors from manifesting at later stages (∼P28 and P56). CONCLUSIONS: Early NMDAR hyperfunction leads to late NMDAR hypofunction and autistic-like social behaviors in Shank2-/- mice, and early correction of NMDAR dysfunction has the long-lasting effect of preventing autistic-like social behaviors from developing at later stages.

GABA Neuronal Deletion of Shank3 Exons 14-16 in Mice Suppresses Striatal Excitatory Synaptic Input and Induces Social and Locomotor Abnormalities.

Shank3 is an excitatory postsynaptic scaffolding protein implicated in multiple brain disorders, including autism spectrum disorders (ASD) and Phelan-McDermid syndrome (PMS). Although previous neurobiological studies on Shank3 and Shank3-mutant mice have revealed diverse roles of Shank3 in the regulation of synaptic, neuronal and brain functions, whether Shank3 expression in specific cell types distinctly contributes to mouse phenotypes remains largely unclear. In the present study, we generated two Shank3-mutant mouse lines (exons 14-16) carrying global and GABA neuron-specific deletions and characterized their electrophysiological and behavioral phenotypes. These mouse lines show similar decreases in excitatory synaptic input onto dorsolateral striatal neurons. In addition, the abnormal social and locomotor behaviors observed in global Shank3-mutant mice are strongly mimicked by GABA neuron-specific Shank3-mutant mice, whereas the repetitive and anxiety-like behaviors are only partially mimicked. These results suggest that GABAergic Shank3 (exons 14-16) deletion has strong influences on striatal excitatory synaptic transmission and social and locomotor behaviors in mice.

Sexually dimorphic behavior, neuronal activity, and gene expression in Chd8-mutant mice.

Autism spectrum disorders (ASDs) are four times more common in males than in females, but the underlying mechanisms are poorly understood. We characterized sexually dimorphic changes in mice carrying a heterozygous mutation in Chd8 (Chd8+/N2373K) that was first identified in human CHD8 (Asn2373LysfsX2), a strong ASD-risk gene that encodes a chromatin remodeler. Notably, although male mutant mice displayed a range of abnormal behaviors during pup, juvenile, and adult stages, including enhanced mother-seeking ultrasonic vocalization, enhanced attachment to reunited mothers, and isolation-induced self-grooming, their female counterparts do not. This behavioral divergence was associated with sexually dimorphic changes in neuronal activity, synaptic transmission, and transcriptomic profiles. Specifically, female mice displayed suppressed baseline neuronal excitation, enhanced inhibitory synaptic transmission and neuronal firing, and increased expression of genes associated with extracellular vesicles and the extracellular matrix. Our results suggest that a human CHD8 mutation leads to sexually dimorphic changes ranging from transcription to behavior in mice.

NGL-2 Deletion Leads to Autistic-like Behaviors Responsive to NMDAR Modulation.

Netrin-G ligand 2 (NGL-2)/LRRC4, implicated in autism spectrum disorders and schizophrenia, is a leucine-rich repeat-containing postsynaptic adhesion molecule that interacts intracellularly with the excitatory postsynaptic scaffolding protein PSD-95 and trans-synaptically with the presynaptic adhesion molecule netrin-G2. Functionally, NGL-2 regulates excitatory synapse development and synaptic transmission. However, whether it regulates synaptic plasticity and disease-related specific behaviors is not known. Here, we report that mice lacking NGL-2 (Lrrc4-/- mice) show suppressed N-Methyl-D-aspartate receptor (NMDAR)-dependent synaptic plasticity in the hippocampus. NGL-2 associates with NMDARs through both PSD-95-dependent and -independent mechanisms. Moreover, Lrrc4-/- mice display mild social interaction deficits and repetitive behaviors that are rapidly improved by pharmacological NMDAR activation. These results suggest that NGL-2 promotes synaptic stabilization of NMDARs, regulates NMDAR-dependent synaptic plasticity, and prevents autistic-like behaviors from developing in mice, supporting the hypothesis that NMDAR dysfunction contributes to autism spectrum disorders.

IQ Motif and SEC7 Domain-containing Protein 3 (IQSEC3) Interacts with Gephyrin to Promote Inhibitory Synapse Formation.

Gephyrin is a central scaffold protein that mediates development, function, and plasticity of mammalian inhibitory synapses by interacting with various inhibitory synaptic proteins. Here, we show that IQSEC3, a guanine nucleotide exchange factor for ARF6, directly interacts with gephyrin, an interaction that is critical for the inhibitory synapse localization of IQSEC3. Overexpression of IQSEC3 increases inhibitory, but not excitatory, synapse density in a guanine nucleotide exchange factor activity-dependent manner. Conversely, knockdown of IQSEC3 decreases size of gephyrin cluster without altering gephyrin puncta density. Collectively, these data reveal that IQSEC3 acts together with gephyrin to regulate inhibitory synapse development.