The intracellular C-terminal domain of mGluR6 contains ER retention motifs.

Metabotropic glutamate receptor 6 (mGluR6) predominantly localizes to the postsynaptic sites of retinal ON-bipolar cells, at which it recognizes glutamate released from photoreceptors. The C-terminal domain (CTD) of mGluR6 contains a cluster of basic amino acids resembling motifs for endoplasmic reticulum (ER) retention. We herein investigated whether these basic residues are involved in regulating the subcellular localization of mGluR6 in 293T cells expressing mGluR6 CTD mutants using immunocytochemistry, immunoprecipitation, and flow cytometry. We showed that full-length mGluR6 localized to the ER and cell surface, whereas mGluR6 mutants with 15- and 20-amino acid deletions from the C terminus localized to the ER, but were deficient at the cell surface. We also demonstrated that the cell surface deficiency of mGluR6 mutants was rescued by introducing an alanine substitution at basic residues within the CTD. The surface-deficient mGluR6 mutant still did not localize to the cell surface and was retained in the ER when co-expressed with surface-expressible constructs, including full-length mGluR6, even though surface-deficient and surface-expressible constructs formed heteromeric complexes. The co-expression of the surface-deficient mGluR6 mutant reduced the surface levels of surface-expressible constructs. These results indicate that basic residues in the mGluR6 CTD served as ER retention signals. We suggest that exposed ER retention motifs in the aberrant assembly containing truncated or misfolded mGluR6 prevent these protein complexes from being transported to the cell surface.

Involvement of the C-terminal domain in cell surface localization and G-protein coupling of mGluR6.

Metabotropic glutamate receptor 6, mGluR6, interacts with scaffold proteins and Gßγ subunits via its intracellular C-terminal domain (CTD). The mGluR6 pathway is critically involved in the retinal processing of visual signals. We herein investigated whether the CTD (residues 840-871) was necessary for mGluR6 cell surface localization and G-protein coupling using mGluR6-CTD mutants with immunocytochemistry, surface biotinylation assays, and electrophysiological approaches. We used 293T cells and primary hippocampal neurons as model systems. We examined C-terminally truncated mGluR6 and showed that the removal of up to residue 858 did not affect surface localization or glutamate-induced G-protein-mediated responses, whereas a 15-amino acid deletion (Δ857-871) impaired these functions. However, a 21-amino acid deletion (Δ851-871) restored surface localization and glutamate-dependent responses, which were again attenuated when the entire CTD was removed. The sequence alignment of group III mGluRs showed conserved amino acids resembling an ER retention motif in the CTD. These results suggest that the intracellular CTD is required for the cell surface transportation and receptor function of mGluR6, whereas it may contain regulatory elements for intracellular trafficking and signaling.

Brain ventriculomegaly in Down syndrome mice is caused by Pcp4 dose-dependent cilia dysfunction.

Down syndrome is a leading cause of congenital intellectual disability caused by an additional copy of the chromosome 21. Patients display physiological and morphological changes affecting the brain and its function. Previously we showed that Ts1Cje and Ts2Cje, Down syndrome mouse models carrying overlapping trisomic segments of different length, show similar ventriculomegaly and neurogenesis dysfunction leading to the hypothesis of a cause-consequence relationship between these phenotypes. However, we here discovered that Ts1Rhr Down syndrome model, carrying an even shorter trisomic segment, was sufficient to trigger ventricular enlargement and ependymal cilia beating deficiency without affecting neurogenesis. We further found that Pcp4 gene on the Ts1Rhr trisomic segment is expressed in ependymal cells, and its resumption to two copies rescued both ventricular enlargement and cilia dysfunction in Ts1Rhr mice. This work underlines a Pcp4-dependent ciliopathy in Down syndrome brain affecting cerebrospinal fluid flow.

Potentiation of excitatory synaptic transmission ameliorates aggression in mice with Stxbp1 haploinsufficiency.

Genetic studies point to a major role of de novo mutations in neurodevelopmental disorders of intellectual disability, autism spectrum disorders, and epileptic encephalopathy. The STXBP1 gene encodes the syntaxin-binding protein 1 (Munc18-1) that critically controls synaptic vesicle exocytosis and synaptic transmission. This gene harbors a high frequency of de novo mutations, which may play roles in these neurodevelopmental disorders. However, the system and behavioral-level pathophysiological changes caused by these genetic defects remain poorly understood. Constitutional (Stxbp1+/-), dorsal-telencephalic excitatory (Stxbp1fl/+/Emx), or global inhibitory neuron-specific (Stxbp1fl/+/Vgat) mice were subjected to a behavioral test battery examining locomotor activity, anxiety, fear learning, and social interactions including aggression. Furthermore, measurements of local field potentials in multiple regions of the brain were performed. Stxbp1+/- male mice exhibited enhanced aggressiveness and impaired fear learning associated with elevated gamma activity in several regions of the brain including the prefrontal cortex. Stxbp1fl/+/Emx mice showed fear-learning deficits, but neither Stxbp1fl/+/Emx nor Stxbp1fl/+/Vgat mice showed increased aggressiveness. Pharmacological potentiation of the excitatory transmission at active synapses via the systemic administration of ampakine CX516, which enhances the excitatory postsynaptic function, ameliorated the aggressive phenotype of Stxbp1+/- mice. These findings suggest that synaptic impairments of the dorsal telencephalic and subcortical excitatory neurons cause learning deficits and enhanced aggression in Stxbp1+/- mice, respectively. Additionally, normalizing the excitatory synaptic transmission is a potential therapeutic option for managing aggressiveness in patients with STXBP1 mutations.

Impairments in social novelty recognition and spatial memory in mice with conditional deletion of Scn1a in parvalbumin-expressing cells.

Loss of function mutations in the SCN1A gene, which encodes the voltage-gated sodium channel Nav1.1, have been described in the majority of Dravet syndrome patients presenting with epileptic seizures, hyperactivity, autistic traits, and cognitive decline. We previously reported predominant Nav1.1 expression in parvalbumin-expressing (PV+) inhibitory neurons in juvenile mouse brain and observed epileptic seizures in mice with selective deletion of Scn1a in PV+ cells mediated by PV-Cre transgene expression (Scn1afl/+/PV-Cre-TG). Here we investigate the behavior of Scn1afl/+/PV-Cre-TG mice using a comprehensive battery of behavioral tests. We observed that Scn1afl/+/PV-Cre-TG mice display hyperactive behavior, impaired social novelty recognition, and altered spatial memory. We also generated Scn1afl/+/SST-Cre-KI mice with a selective Scn1a deletion in somatostatin-expressing (SST+) inhibitory neurons using an SST-IRES-Cre knock-in driver line. We observed that Scn1afl/+/SST-Cre-KI mice display no spontaneous convulsive seizures and that Scn1afl/+/SST-Cre-KI mice have a lowered threshold temperature for hyperthermia-induced seizures, although their threshold values are much higher than those of Scn1afl/+/PV-Cre-TG mice. We finally show that Scn1afl/+/SST-Cre-KI mice exhibited no noticeable behavioral abnormalities. These observations suggest that impaired Nav1.1 function in PV+ interneurons is critically involved in the pathogenesis of hyperactivity, autistic traits, and cognitive decline, as well as epileptic seizures, in Dravet syndrome.

DYRK1A-haploinsufficiency in mice causes autistic-like features and febrile seizures.

Mutations and copy number variants affecting DYRK1A gene encoding the dual-specificity tyrosine phosphorylation-regulated kinase 1A are among the most frequent genetic causes of neurodevelopmental disorders including autism spectrum disorder (ASD) associated with microcephaly, febrile seizures and severe speech acquisition delay. Here we developed a mouse model harboring a frame-shift mutation in Dyrk1a resulting in a protein truncation and elimination of its kinase activity site. Dyrk1a+/- mice showed significant impairments in cognition and cognitive flexibility, communicative ultrasonic vocalizations, and social contacts. Susceptibility to hyperthermia-induced seizures was also significantly increased in these mice. The truncation leading to haploinsufficiency of DYRK1A in mice thus recapitulates the syndromic phenotypes observed in human patients and constitutes a useful model for further investigations of the mechanisms leading to ASD, speech delay and febrile seizures.

CUX2 deficiency causes facilitation of excitatory synaptic transmission onto hippocampus and increased seizure susceptibility to kainate.

CUX2 gene encodes a transcription factor that controls neuronal proliferation, dendrite branching and synapse formation, locating at the epilepsy-associated chromosomal region 12q24 that we previously identified by a genome-wide association study (GWAS) in Japanese population. A CUX2 recurrent de novo variant p.E590K has been described in patients with rare epileptic encephalopathies and the gene is a candidate for the locus, however the mutation may not be enough to generate the genome-wide significance in the GWAS and whether CUX2 variants appear in other types of epilepsies and physiopathological mechanisms are remained to be investigated. Here in this study, we conducted targeted sequencings of CUX2, a paralog CUX1 and its short isoform CASP harboring a unique C-terminus on 271 Japanese patients with a variety of epilepsies, and found that multiple CUX2 missense variants, other than the p.E590K, and some CASP variants including a deletion, predominantly appeared in patients with temporal lobe epilepsy (TLE). The CUX2 variants showed abnormal localization in human cell culture analysis. While wild-type CUX2 enhances dendritic arborization in fly neurons, the effect was compromised by some of the variants. Cux2- and Casp-specific knockout mice both showed high susceptibility to kainate, increased excitatory cell number in the entorhinal cortex, and significant enhancement in glutamatergic synaptic transmission to the hippocampus. CASP and CUX2 proteins physiologically bound to each other and co-expressed in excitatory neurons in brain regions including the entorhinal cortex. These results suggest that CUX2 and CASP variants contribute to the TLE pathology through a facilitation of excitatory synaptic transmission from entorhinal cortex to hippocampus.

Enlarged brain ventricles and impaired neurogenesis in the Ts1Cje and Ts2Cje mouse models of Down syndrome.

Down syndrome (DS) is the most common cause of mental retardation. Although structural and neurogenic abnormalities have been shown in the brains of DS patients, the molecular etiology is still unknown. To define it, we have performed structural and histological examinations of the brains of Ts1Cje and Ts2Cje, 2 mouse models for DS. These mice carry different length of trisomic segments of mouse chromosome 16 that are orthologous to human chromosome 21. At 3 months of age, ventricular enlargements were observed in both Ts1Cje and Ts2Cje brains at a similar degree. Both mice also showed decreases of the number of doublecortin-positive neuroblasts and thymidine-analog BrdU-labeled proliferating cells in the subventricular zone of the lateral ventricles (LVs) and in the hippocampal dentate gyrus at a similar degree, suggesting impaired adult neurogenesis. Additionally, at embryonic day 14.5, both strains of mice, when compared with diploid littermates, had smaller brains and decreased cortical neurogenesis that could possibly contribute to the ventricular enlargements observed in adulthood. Our findings suggest that the trisomic segment of the Ts1Cje mouse, which is shared with Ts2Cje, contains the genes that are responsible for these abnormal phenotypes and could be relevant to the mental retardation associated with DS.

DSCAM deficiency causes loss of pre-inspiratory neuron synchroneity and perinatal death.

Down syndrome cell adhesion molecule (DSCAM) is a neural adhesion molecule that plays diverse roles in neural development. We disrupted the Dscam locus in mice and found that the null mutants (Dscam(-/-)) died within 24 h after birth. Whole-body plethysmography showed irregular respiration and lower ventilatory response to hypercapnia in the null mutants. Furthermore, a medulla-spinal cord preparation of Dscam(-/-) mice showed that the C4 ventral root activity, which drives diaphragm contraction for inspiration, had an irregular rhythm with frequent apneas. Optical imaging of the preparation using voltage-sensitive dye revealed that the pre-inspiratory neurons located in the rostral ventrolateral medulla and belonging to the rhythm generator for respiration, lost their synchroneity in Dscam(-/-) mice. Dscam(+/-) mice, which survived to adulthood without any overt abnormalities, also showed irregular respiration but milder than Dscam(-/-) mice. These results suggest that DSCAM plays a critical role in central respiratory regulation in a dosage-dependent manner.

Impairment of spatial memory accuracy improved by Cbr1 copy number resumption and GABAB receptor-dependent enhancement of synaptic inhibition in Down syndrome model mice.

Down syndrome is a complex genetic disorder caused by the presence of three copies of the chromosome 21 in humans. The most common models, carrying extra-copies of overlapping fragments of mouse chromosome 16 that is syntenic to human chromosome 21, are Ts2Cje, Ts1Cje and Ts1Rhr mice. In electrophysiological analyses using hippocampal slices, we found that the later phase of the depolarization during tetanic stimulation, which was regulated by GABAB receptors, was significantly smaller in Ts1Cje and Ts2Cje mice than that in WT controls but not in Ts1Rhr mice. Furthermore, isolated GABAB receptor-mediated inhibitory synaptic responses were larger in Ts1Cje mice. To our knowledge, this is the first report that directly shows the enhancement of GABAB receptor-mediated synaptic currents in Ts1Cje mice. These results suggest that GABAB receptor-mediated synaptic inhibition was enhanced in Ts1Cje and Ts2Cje mice but not in Ts1Rhr mice. The Cbr1 gene, which is present in three copies in Ts1Cje and Ts2Cje but not in Ts1Rhr, encodes carbonyl reductase that may facilitate GABAB-receptor activity through a reduction of prostaglandin E2 (PGE2). Interestingly, we found that a reduction of PGE2 and an memory impairment in Ts1Cje mice were alleviated when only Cbr1 was set back to two copies (Ts1Cje;Cbr1+/+/-). However, the GABAB receptor-dependent enhancement of synaptic inhibition in Ts1Cje was unaltered in Ts1Cje;Cbr1+/+/- mice. These results indicate that Cbr1 is one of the genes responsible for DS cognitive impairments and the gene(s) other than Cbr1, which is included in Ts1Cje but not in Ts1Rhr, is responsible for the GABAB receptor-dependent over-inhibition.

Perturbation of the immune cells and prenatal neurogenesis by the triplication of the Erg gene in mouse models of Down syndrome.

Some mouse models of Down syndrome (DS), including Ts1Cje mice, exhibit impaired prenatal neurogenesis with yet unknown molecular mechanism. To gain insights into the impaired neurogenesis, a transcriptomic and flow cytometry analysis of E14.5 Ts1Cje embryo brain was performed. Our analysis revealed that the neutrophil and monocyte ratios in the CD45-positive hematopoietic cells were relatively increased, in agreement with the altered expression of inflammation/immune-related genes, in Ts1Cje embryonic brain, whereas the relative number of brain macrophages was decreased in comparison to wild-type mice. Similar upregulation of inflammation-associated mRNAs was observed in other DS mouse models, with variable trisomic region lengths. We used genetic manipulation to assess the contribution of Erg, a trisomic gene in these DS models, known to regulation hemato-immune cells. The perturbed proportions of immune cells in Ts1Cje mouse brain were restored in Ts1Cje-Erg+/+/Mld2 mice, which are disomic for functional Erg but otherwise trisomic on a Ts1Cje background. Moreover, the embryonic neurogenesis defects observed in Ts1Cje cortex were reduced in Ts1Cje-Erg+/+/Mld2 embryos. Our findings suggest that Erg gene triplication contributes to the dysregulation of the homeostatic proportion of the populations of immune cells in the embryonic brain and decreased prenatal cortical neurogenesis in the prenatal brain with DS.

A recurrent PJA1 variant in trigonocephaly and neurodevelopmental disorders.

OBJECTIVE: Neurodevelopmental disorders (NDDs) often associate with epilepsy or craniofacial malformations. Recent large-scale DNA analyses identified hundreds of candidate genes for NDDs, but a large portion of the cases still remain unexplained. We aimed to identify novel candidate genes for NDDs. METHODS: We performed exome sequencing of 95 patients with NDDs including 51 with trigonocephaly and subsequent targeted sequencing of additional 463 NDD patients, functional analyses of variant in vitro, and evaluations of autism spectrum disorder (ASD)-like phenotypes and seizure-related phenotypes in vivo. RESULTS: We identified de novo truncation variants in nine novel genes; CYP1A1, C14orf119, FLI1, CYB5R4, SEL1L2, RAB11FIP2, ZMYND8, ZNF143, and MSX2. MSX2 variants have been described in patients with cranial malformations, and our present patient with the MSX2 de novo truncation variant showed cranial meningocele and partial epilepsy. MSX2 protein is known to be ubiquitinated by an E3 ubiquitin ligase PJA1, and interestingly we found a PJA1 hemizygous p.Arg376Cys variant recurrently in seven Japanese NDD patients; five with trigonocephaly and one with partial epilepsy, and the variant was absent in 886 Japanese control individuals. Pja1 knock-in mice carrying p.Arg365Cys, which is equivalent to p.Arg376Cys in human, showed a significant decrease in PJA1 protein amount, suggesting a loss-of-function effect of the variant. Pja1 knockout mice displayed moderate deficits in isolation-induced ultrasonic vocalizations and increased seizure susceptibility to pentylenetetrazole. INTERPRETATION: These findings propose novel candidate genes including PJA1 and MSX2 for NDDs associated with craniofacial abnormalities and/or epilepsy.

Increased lipid peroxidation in Down's syndrome mouse models.

Elevated oxidative stress has been suggested to be associated with the features of Down's syndrome (DS). We previously reported increased oxidative stress in cultured cells from the embryonic brain of Ts1Cje, a mouse genetic DS model. However, since in vivo evidence for increased oxidative stress is lacking, we here examined lipid peroxidation, a typical marker of oxidative stress, in the brains of Ts1Cje and another DS mouse model Ts2Cje with an overlapping but larger trisomic segment. Accumulations of proteins modified with the lipid peroxidation-derived products, 13-hydroperoxy-9Z,11E-octadecadienoic acid and 4-hydroxy-2-nonenal were markedly increased in Ts1Cje and Ts2Cje brains. Analysis with oxidation-sensitive fluorescent probe also showed that reactive oxygen species themselves were increased in Ts1Cje brain. However, electron spin resonance analysis of microdialysate from the hippocampus of Ts1Cje showed that antioxidant activity remained unaffected, suggesting that the reactive oxygen species production was accelerated in Ts1Cje. Proteomics approaches with mass spectrometry identified the proteins modified with 13-hydroperoxy-9Z,11E-octadecadienoic acid and/or 4-hydroxy-2-nonenal to be involved in either ATP generation, the neuronal cytoskeleton or antioxidant activity. Structural or functional impairments of these proteins by such modifications may contribute to the DS features such as cognitive impairment that are present in the Ts1Cje mouse.

Impaired cortico-striatal excitatory transmission triggers epilepsy.

STXBP1 and SCN2A gene mutations are observed in patients with epilepsies, although the circuit basis remains elusive. Here, we show that mice with haplodeficiency for these genes exhibit absence seizures with spike-and-wave discharges (SWDs) initiated by reduced cortical excitatory transmission into the striatum. Mice deficient for Stxbp1 or Scn2a in cortico-striatal but not cortico-thalamic neurons reproduce SWDs. In Stxbp1 haplodeficient mice, there is a reduction in excitatory transmission from the neocortex to striatal fast-spiking interneurons (FSIs). FSI activity transiently decreases at SWD onset, and pharmacological potentiation of AMPA receptors in the striatum but not in the thalamus suppresses SWDs. Furthermore, in wild-type mice, pharmacological inhibition of cortico-striatal FSI excitatory transmission triggers absence and convulsive seizures in a dose-dependent manner. These findings suggest that impaired cortico-striatal excitatory transmission is a plausible mechanism that triggers epilepsy in Stxbp1 and Scn2a haplodeficient mice.

Ts1Cje Down syndrome model mice exhibit environmental stimuli-triggered locomotor hyperactivity and sociability concurrent with increased flux through central dopamine and serotonin metabolism.

Ts1Cje mice have a segmental trisomy of chromosome 16 that is orthologous to human chromosome 21 and display Down syndrome-like cognitive impairments. Despite the occurrence of affective and emotional impairments in patients with Down syndrome, these parameters are poorly documented in Down syndrome mouse models, including Ts1Cje mice. Here, we conducted comprehensive behavioral analyses, including anxiety-, sociability-, and depression-related tasks, and biochemical analyses of monoamines and their metabolites in Ts1Cje mice. Ts1Cje mice showed enhanced locomotor activity in novel environments and increased social contact with unfamiliar partners when compared with wild-type littermates, but a significantly lower activity in familiar environments. Ts1Cje mice also exhibited some signs of decreased depression like-behavior. Furthermore, Ts1Cje mice showed monoamine abnormalities, including increased extracellular dopamine and serotonin, and enhanced catabolism in the striatum and ventral forebrain. This study constitutes the first report of deviated monoamine metabolism that may help explain the basis for abnormal behaviors, including the environmental stimuli-triggered hyperactivity, increased sociability and decreased depression-like behavior in Ts1Cje mice.

Mitochondrial dysfunction and tau hyperphosphorylation in Ts1Cje, a mouse model for Down syndrome.

Trisomy 21 or Down syndrome (DS) is the most common genetic birth defect associated with mental retardation. The over-expression of genes on chromosome 21, including SOD1 (Cu/Zn superoxide dismutase) and APP (amyloid-beta precursor protein) is believed to underlie the increased oxidative stress and neurodegeneration commonly described in DS. However, a segmental trisomy 16 mouse model for DS, Ts1Cje, has a subset of triplicated human chromosome 21 gene orthologs that exclude APP and SOD1. Here, we report that Ts1Cje brain shows decreases of mitochondrial membrane potential and ATP production, increases of reactive oxygen species, hyperphosphorylation of tau without NFT formation, increase of GSK3beta and JNK/SAPK activities and unaltered AbetaPP metabolism. Our findings suggest that genes on the trisomic Ts1Cje segment other than APP and SOD1 can cause oxidative stress, mitochondrial dysfunction and hyperphosphorylation of tau, all of which may play critical roles in the pathogenesis of mental retardation in DS.