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Clustered protocadherin is a family of cell-surface recognition molecules implicated in neuronal connectivity that has a diverse isoform repertoire and homophilic binding specificity. Mice have 58 isoforms, encoded by

We generated cortical interneurons (cINs) from induced pluripotent stem cells derived from 14 healthy controls and 14 subjects with schizophrenia. Both healthy control cINs and schizophrenia cINs were authentic, fired spontaneously, received functional excitatory inputs from host neurons, and induced GABA-mediated inhibition in host neurons in vivo. However, schizophrenia cINs had dysregulated expression of protocadherin genes, which lie within documented schizophrenia loci. Mice lacking protocadherin-α showed defective arborization and synaptic density of prefrontal cortex cINs and behavioral abnormalities. Schizophrenia cINs similarly showed defects in synaptic density and arborization that were reversed by inhibitors of protein kinase C, a downstream kinase in the protocadherin pathway. These findings reveal an intrinsic abnormality in schizophrenia cINs in the absence of any circuit-driven pathology. They also demonstrate the utility of homogenous and functional populations of a relevant neuronal subtype for probing pathogenesis mechanisms during development.

Genome stability is essential for brain development and function, asde novomutations during neuronal development cause psychiatric disorders. However, the contribution of DNA repair to genome stability in neurons remains elusive. Here, we demonstrate that the base excision repair protein DNA polymerase β (Polβ) is involved in hippocampal pyramidal neuron differentiation via a TET-mediated active DNA demethylation during early postnatal stages usingNex-Cre/Polβfl/flmice of either sex, in which forebrain postmitotic excitatory neurons lack Polβ expression. Polβ deficiency induced extensive DNA double-strand breaks (DSBs) in hippocampal pyramidal neurons, but not dentate gyrus granule cells, and to a lesser extent in neocortical neurons, during a period in which decreased levels of 5-methylcytosine and 5-hydroxymethylcytosine were observed in genomic DNA. Inhibition of the hydroxylation of 5-methylcytosine by expression of microRNAs miR-29a/b-1 diminished DSB formation. Conversely, its induction by TET1 catalytic domain overexpression increased DSBs in neocortical neurons. Furthermore, the damaged hippocampal neurons exhibited aberrant neuronal gene expression profiles and dendrite formation, but not apoptosis. Comprehensive behavioral analyses revealed impaired spatial reference memory and contextual fear memory in adulthood. Thus, Polβ maintains genome stability in the active DNA demethylation that occurs during early postnatal neuronal development, thereby contributing to differentiation and subsequent learning and memory.SIGNIFICANCE STATEMENTIncreasing evidence suggests thatde novomutations during neuronal development cause psychiatric disorders. However, strikingly little is known about how DNA repair is involved in neuronal differentiation. We found that Polβ, a component of base excision repair, is required for differentiation of hippocampal pyramidal neurons in mice. Polβ deficiency transiently led to increased DNA double-strand breaks, but not apoptosis, in early postnatal hippocampal pyramidal neurons. This aberrant double-strand break formation was attributed to active DNA demethylation as an epigenetic regulation. Furthermore, the damaged neurons exhibited aberrant gene expression profiles and dendrite formation, resulting in impaired learning and memory in adulthood. Thus, these findings provide new insight into the contribution of DNA repair to the neuronal genome in early brain development.

Additional file 3: Figure S3. Pcdhβ deficiency does not affect neural activity at the cellular level (related to Figs. 1 and 2, and additional file 1). (a–b) Representative images of raster plots of all recorded cells from Wt (a) and Δβ (b) mice. (c) Cumulative curve of proportion of cells against number of active events during entire recording session (pre 10 min + sq 10 min). Proportions of cells according to the number of Ca2+ events during pre (d) and sq (e) sessions. Statistical values from Bonferroni’s multiple-comparison test are provided in Additional file 6. (f) The ratios of the number of Ca2+ events during the sq session to that in the pre session. Statistical values from Bonferroni’s multiple-comparison test are provided in Additional file 6 (n = 5 Wt mice, 4 Δβ mice). Data are means ± SEMs. No significant differences were observed (Kolmogorov–Smirnov test for panel c, Bonferroni’s multiple-comparison tests for panels d–f).

Individual neurons are basic functional units in the complex system of the brain. One aspect of neuronal individuality is generated by stochastic and combinatorial expression of diverse clustered protocadherins (Pcdhs), encoded by the Pcdha, Pcdhb, and Pcdhg gene clusters, that are critical for several aspects of neural circuit formation. Each clustered Pcdh gene has its own promoter containing conserved sequences and is transcribed by a promoter choice mechanism involving interaction between the promoter and enhancers. A CTCF/Cohesin complex induces this interaction by configuration of DNA-looping in the chromatin structure. At the same time, the semi-stochastic expression of clustered Pcdh genes is regulated in individual neurons by DNA methylation: the methyltransferase Dnmt3b regulates methylation state of individual clustered Pcdh genes during early embryonic stages prior to the establishment of neural stem cells. Several other factors, including Smchd1, also contribute to the regulation of clustered Pcdh gene expression. In addition, psychiatric diseases and early life experiences of individuals can influence expression of clustered Pcdh genes in the brain, through epigenetic alterations. Clustered Pcdh gene expression is thus a significant and highly regulated step in establishing neuronal individuality and generating functional neural circuits in the brain.

Genome stability is essential for brain development and function. However, the contribution of DNA repair to genome stability in neurons remains elusive. Here, we demonstrate that the base excision repair protein Polβ is involved in hippocampal neuronal differentiation via a TET-mediated active DNA demethylation during early postnatal stages. Polβ deficiency induced extensive DNA double-strand breaks (DSBs) in hippocampal neurons, and a lesser extent in cortical neurons, during a period in which decreased levels of 5-methylcytosine were observed in genomic DNA. Inhibition of the hydroxylation of 5-methylcytosine by microRNAs miR29a/b-1 expression diminished DSB formation. Conversely, its induction by TET1 overexpression increased DSBs. The damaged hippocampal neurons exhibited aberrant neuronal gene expression profiles and dendrite formation. Behavioral analyses revealed impaired spatial learning and memory in adulthood. Thus, Polβ maintains genome stability in the active DNA demethylation that occurs during postnatal neuronal development, thereby contributing to differentiation and subsequent behavior.

Alterations in the homeostasis of either cortical progenitor pool, namely the apically located radial glial (RG) cells or the basal intermediate progenitors (IPCs) can severely impair cortical neuron production. Such changes are reflected by microcephaly and are often associated with cognitive defects. Genes encoding epigenetic regulators are a frequent cause of intellectual disability and many have been shown to regulate progenitor cell growth, including our inactivation of the Smarca1 gene encoding Snf2l, which is one of two ISWI mammalian orthologs. Loss of the Snf2l protein resulted in dysregulation of Foxg1 and IPC proliferation leading to macrocephaly. Here we show that inactivation of the closely related Smarca5 gene encoding the Snf2h chromatin remodeler is necessary for embryonic IPC expansion and subsequent specification of callosal projection neurons. Telencephalon-specific Smarca5 cKO embryos have impaired cell cycle kinetics and increased cell death, resulting in fewer Tbr2+ and FoxG1+ IPCs by mid-neurogenesis. These deficits give rise to adult mice with a dramatic reduction in Satb2C upper layer neurons, and partial agenesis of the corpus callosum. Mice survive into adulthood but molecularly display reduced expression of the clustered protocadherin genes that may further contribute to altered dendritic arborization and a hyperactive behavioral phenotype. Our studies provide novel insight into the developmental function of Snf2h-dependent chromatin remodeling processes during brain development.

DNA repair is crucial for genome stability in the developing cortex, as somatic de novo mutations cause neurological disorders. However, how DNA repair contributes to neuronal development is largely unknown. To address this issue, we studied the spatiotemporal roles of DNA polymerase β (Polβ), a key enzyme in DNA base excision repair pathway, in the developing cortex using distinct forebrain-specific conditional knock-out mice, Emx1-Cre/Polβ(fl/fl) and Nex-Cre/Polβ(fl/fl) mice. Polβ expression was absent in both neural progenitors and postmitotic neurons in Emx1-Cre/Polβ(fl/fl) mice, whereas only postmitotic neurons lacked Polβ expression in Nex-Cre/Polβ(fl/fl) mice. We found that DNA double-strand breaks (DSBs) were frequently detected during replication in cortical progenitors of Emx1-Cre/Polβ(fl/fl) mice. Increased DSBs remained in postmitotic cells, which resulted in p53-mediated neuronal apoptosis. This neuronal apoptosis caused thinning of the cortical plate, although laminar structure was normal. In addition, accumulated DSBs also affected growth of corticofugal axons but not commissural axons. These phenotypes were not observed in Nex-Cre/Polβ(fl/fl) mice. Moreover, cultured Polβ-deficient neural progenitors exhibited higher sensitivity to the base-damaging agent methylmethanesulfonate, resulting in enhanced DSB formation. Similar damage was found by vitamin C treatment, which induces TET1-mediated DNA demethylation via 5-hydroxymethylcytosine. Together, genome stability mediated by Polβ-dependent base excision repair is crucial for the competence of neural progenitors, thereby contributing to neuronal differentiation in cortical development. SIGNIFICANCE STATEMENT DNA repair is crucial for development of the nervous system. However, how DNA polymerase β (Polβ)-dependent DNA base excision repair pathway contributes to the process is still unknown. We found that loss of Polβ in cortical progenitors rather than postmitotic neurons led to catastrophic DNA double-strand breaks (DSBs) during replication and p53-mediated neuronal apoptosis, which resulted in thinning of the cortical plate. The DSBs also affected corticofugal axon growth in surviving neurons. Moreover, induction of base damage and DNA demethylation intermediates in the genome increased DSBs in cultured Polβ-deficient neural progenitors. Thus, genome stability by Polβ-dependent base excision repair in neural progenitors is required for the viability and differentiation of daughter neurons in the developing nervous system.

We report locus-specific disintegration of megabase-scale chromosomal conformations in brain after neuronal ablation of Setdb1 (also known as Kmt1e; encodes a histone H3 lysine 9 methyltransferase), including a large topologically associated 1.2-Mb domain conserved in humans and mice that encompasses >70 genes at the clustered protocadherin locus (hereafter referred to as cPcdh). The cPcdh topologically associated domain (TADcPcdh) in neurons from mutant mice showed abnormal accumulation of the transcriptional regulator and three-dimensional (3D) genome organizer CTCF at cryptic binding sites, in conjunction with DNA cytosine hypomethylation, histone hyperacetylation and upregulated expression. Genes encoding stochastically expressed protocadherins were transcribed by increased numbers of cortical neurons, indicating relaxation of single-cell constraint. SETDB1-dependent loop formations bypassed 0.2-1 Mb of linear genome and radiated from the TADcPcdh fringes toward cis-regulatory sequences within the cPcdh locus, counterbalanced shorter-range facilitative promoter-enhancer contacts and carried loop-bound polymorphisms that were associated with genetic risk for schizophrenia. We show that the SETDB1 repressor complex, which involves multiple KRAB zinc finger proteins, shields neuronal genomes from excess CTCF binding and is critically required for structural maintenance of TADcPcdh.

SummaryIn the brain, enormous numbers of neurons have functional individuality and distinct circuit specificities. Clustered Protocadherins (Pcdhs), diversified cell-surface proteins, are stochastically expressed by alternative promoter choice and affect dendritic arborization in individual neurons. Here we found that the Pcdh promoters are differentially methylated by the de novo DNA methyltransferase Dnmt3b during early embryogenesis. To determine this methylation’s role in neurons, we produced chimeric mice from Dnmt3b-deficient induced pluripotent stem cells (iPSCs). Single-cell expression analysis revealed that individual Dnmt3b-deficient Purkinje cells expressed increased numbers of Pcdh isoforms; in vivo, they exhibited abnormal dendritic arborization. These results indicate that DNA methylation by Dnmt3b at early embryonic stages regulates the probability of expression for the stochastically expressed Pcdh isoforms. They also suggest a mechanism for a rare human recessive disease, the ICF (Immunodeficiency, Centromere instability, and Facial anomalies) syndrome, which is caused by Dnmt3b mutations.

The clustered protocadherin (Pcdh) genes are divided into the Pcdhα, Pcdhβ, and Pcdhγ clusters. Gene-disruption analyses in mice have revealed the in vivo functions of the Pcdhα and Pcdhγ clusters. However, all Pcdh protein isoforms form combinatorial cis-hetero dimers and enter trans-homophilic interactions. Here we addressed distinct and cooperative functions in the Pcdh clusters by generating six cluster-deletion mutants (Δα, Δβ, Δγ, Δαβ, Δβγ, and Δαβγ) and comparing their phenotypes: Δα, Δβ, and Δαβ mutants were viable and fertile; Δγ mutants lived less than 12 hours; and Δβγ and Δαβγ mutants died shortly after birth. The Pcdhα, Pcdhβ, and Pcdhγ clusters were individually and cooperatively important in olfactory-axon targeting and spinal-cord neuron survival. Neurodegeneration was most severe in Δαβγ mutants, indicating that Pcdhα and Pcdhβ function cooperatively for neuronal survival. The Pcdhα, Pcdhβ, and Pcdhγ clusters share roles in olfactory-axon targeting and neuronal survival, although to different degrees.

Figure S9. The genotyping of the iPS cells by PCR experiments. Genotyping by PCR on DNA extracted from wild-type (WT), cPcdh-KO, and Dnmt3b-KO iPS cells. PCR with primer sets for detection of (A) cPcdh and (B) Dnmt3b genotype. M: DNA molecular weight marker. (PDF 547 kb)

Figure S3. Comparison of morphological and electrophysiological properties of layer 4 neurons between P13–16 and P18–20 chimeric mice. (A) Traces of spiny stellate cells sampled from P13–16 wild-type chimeric mice (n = 9 cells, n = 8 barrels, n = 3 mice). Scale bar: 100 μm. (B–D) Comparison of the dendritic morphology between P13–16 (n = 9) and P18–20 (n = 16) chimeric mice. No significant differences in the total dendritic length (P = 0.26, t test, B) or the number of branches (P = 0.20, C) were observed, but there was a difference in the number of intersections near soma (*P = 0.046, D). A bar indicates the mean (B, C). Data presented as mean ± SEM in D. (E–G) Comparison of the GFP-negative neurons (N-cell) or GFP-positive neurons (P-cell) between P13–16 and P18–20 chimeric mice in the resting potential (P = 0.30 for GFP-negative neurons, P = 0.36 for GFP-positive neurons, t test, E), firing threshold (P = 0.44 for GFP-negative neurons, P = 0.20 for GFP-positive neurons, F), and input resistance (***P = 0.0007 for GFP-negative neurons, **P = 0.003 for GFP-positive neurons, G). Number of analyzed cells is shown above each box-and-whisker plot (median, 25th to 75th percentiles, minimum to maximum) (E–G). (PDF 636 kb)

Table S3. The combinations of mouse strains, staining methods, RNA probe label, and antibodies. (PDF 49 kb)

Figure S4. Synaptic connections in wild-type nonchimeric mice. (A) Representative average (n = 20) traces of presynaptic spikes (pre) and resultant excitatory postsynaptic currents (post) between layer 4 neuron pairs with a one-way (upper) or reciprocal (lower) connection in wild-type nonchimeric mice (C57BL/6 mice) at P18–20. (B) Percentage of neuron pairs with synaptic connections. There was no significant difference in the connectivity between the neuron pairs in wild-type nonchimeric mice and the nonclonal neuron pairs in wild-type chimeric mice at the same age (P > 0.17, χ2 test). Numbers of recorded pairs are indicated on the bars. (PDF 575 kb)

Figure S5. Synaptic connectivity is not affected by the mouse strain used for the blastocysts or iPS cell lines. (A) Percentage of synaptic connections between clonal neuron pairs (P-P pairs) in P18–20 wild-type chimeric mice produced with blastocysts prepared from BDF1 or C57BL/6 mice; iPS cells were produced using C57BL/6 mice. There was no significant difference in the connectivity between the two strains (P = 0.55, χ2 test). (B) Similar to A, but between nonclonal neuron pairs (P-N pairs). There was no significant difference in the connectivity between the two strains (P = 0.68, χ2 test). (C) Percentage of synaptic connections between GFP-positive neuron pairs (P-P pairs) in P18–20 chimeric mice produced from different wild-type iPS cell lines (iPS-1, iPS-2, and iPS-3). No significant difference was observed between any of the three iPS cell lines (P = 0.59, χ2 test). Numbers of recorded pairs are indicated on the bars. (PDF 540 kb)

Figure S7. Generation of cPcdh KO mice that have a Pcdh-abg del/del allele and a TG taf7 BAC transgene. (A) Schematic diagram of the a1MV targeting constructs. Filled triangles: loxP sites; open triangles: frt sites; M: Myc-tagged venus fluorescent protein gene; Neo: neomycin-resistance gene; DT-A: diphtheria toxin A fragment gene; α1: cPchd-a1 exon; α2: cPchd-a2 exon; α3: cPchd-a3 exon; B: BamHI; Sp: SpeI. (B, C) Southern blotting of homologous recombinant ES cells digested by BamHI, with Probe A (B), and digested by SpeI, with Probe B (C). (D) Genetic structures of loxP site insertions. The loxP sites were inserted 5′ of the Pcdh-α cluster and 3′ of the Pcdh-γ cluster in a1MV and gLacZ mutant mice, respectively. (E) Genetic structures of Pcdhαβγ del mice and TG taf7 BAC transgenic mice. Arrows indicate the primer positions used for genotyping. αCR: cPcdh-α constant region; γCR: cPcdh-γ constant region. (F, G) Genotyping by PCR of cPcdh-KO mutants with the Pcdh-αβγ del allele and the TG taf7 BAC transgene. (H) Expression of Pcdh-α, Pcdh-β, Pcdh-γ, and taf7 genes in the E18.5 brain of wild-type and cPcdh-KO mice, by RT-PCR analysis. All of the genotyping and gene expression experiments yielded similar results. (PDF 869 kb)

Figure S6. Morphological and electrophysiological properties of Dnmt3b-KO neurons. (A) Traces from iPS cell-derived Dnmt3b-KO spiny stellate cells in layer 4 of the barrel cortex in Dnmt3b-KO chimeric mice at P18–20. The neurons were stained by biocytin. Scale bar: 100 μm. (B) A representative trace of action potentials evoked by depolarizing current injection (300 pA) in current clamp mode in Dnmt3b-KO neurons. (C–E) Box-and-whisker plot showing the value (median, 25th to 75th percentiles, minimum to maximum) of the resting membrane potential (RMP, C), the threshold to induce action potentials (firing threshold, D), and the input resistance (E) in Dnmt3b KO neurons (purple) and wild-type GFP-positive neurons (green, the same as shown in Fig. 2) at P18–20. No significant differences in electrophysiological parameters between Dnmt3b-deficient cells and wild-type cells were observed; P = 0.72 (C), P = 0.71 (D), and P = 0.68 (E) (t test). The numbers of cells are indicated above the box-and-whisker plot. (PDF 728 kb)

Figure S8. Morphological and electrophysiological properties of cPcdh-KO neurons. (A) Traces from iPS-derived tdTomato-positive spiny stellate cells in layer 4 of the barrel cortex in cPcdh-KO chimeric mice at P18–20. Scale bar: 100 μm. (B) A representative trace of action potentials evoked by depolarizing current injection (300 pA) in current clamp mode in cPcdh-KO neurons. (C–E) Box-and-whisker plot showing the value (median, 25th to 75th percentiles, minimum to maximum) of the resting membrane potential (RMP, C), the threshold to induce action potentials (firing threshold, D), and the input resistance (E) in cPcdh-KO neurons (yellow) and wild-type GFP-positive neurons (green, the same as shown in Fig. 2) at P18–20. No significant differences in these electrophysiological parameters between cPcdh-deficient cells and wild-type cells were observed; P = 0.45 (C), P = 0.21 (D), and P = 0.21 (E) (t test). The number of cells is indicated above each plot. (F) Percentage of synaptic connections between GFP-positive neuron pairs (P-P pairs) in P13–16 chimeric mice produced from different cPcdh-KO iPS cell lines (iPS-1 and iPS-2). No significant difference was observed between any of the two iPS cell lines (P =0.55, χ2 test). Numbers of recorded pairs are indicated on the bars. (PDF 295 kb)

Figure S2. Synaptic connectivity obtained from each animal in four groups of neuron pairs. (A–C) Connection probability in wild-type (WT) P-P and P-N pairs, and Dnmt3b-KO and cPcdh-KO neuron pairs at P9–11 (A), P13–16 (B), and P18–20 (C). (D–F) Reciprocity in WT P-P and P-N pairs, and Dnmt3b-KO and cPcdh-KO neuron pairs at P9–11 (D), P13–16 (E), and P18–20 (F). Each symbol indicates an average value obtained from each animal. (PDF 416 kb)

The clustered protocadherins (Pcdhs), Pcdh-α, -β, and -γ, are transmembrane proteins constituting a subgroup of the cadherin superfamily. Each Pcdh cluster is arranged in tandem on the same chromosome. Each of the three Pcdh clusters shows stochastic and combinatorial expression in individual neurons, thus generating a hugely diverse set of possible cell surface molecules. Therefore, the clustered Pcdhs are candidates for determining neuronal molecular diversity. Here, we showed that the targeted deletion of DNase I hypersensitive (HS) site HS5-1, previously identified as a Pcdh-α regulatory element in vitro, affects especially the expression of specific Pcdh-α isoforms in vivo. We also identified a Pcdh-β cluster control region (CCR) containing six HS sites (HS16, 17, 17′, 18, 19, and 20) downstream of the Pcdh-γ cluster. This CCR comprehensively activates the expression of the Pcdh-β gene cluster in cis, and its deletion dramatically decreases their expression levels. Deleting the CCR nonuniformly down-regulates some Pcdh-γ isoforms and does not affect Pcdh-α expression. Thus, the CCR effect extends beyond the 320-kb region containing the Pcdh-γ cluster to activate the upstream Pcdh-β genes. Thus, we concluded that the CCR is a highly specific regulatory unit for Pcdh-β expression on the clustered Pcdh genomic locus. These findings suggest that each Pcdh cluster is controlled by distinct regulatory elements that activate their expression and that the stochastic gene regulation of the clustered Pcdhs is controlled by the complex chromatin architecture of the clustered Pcdh locus.

The protocadherin-alpha (Pcdh-alpha) gene encodes diverse transmembrane proteins that are differentially expressed in individual neurons in the vertebrate central nervous system. The Pcdh-alpha genomic structure contains variable first exons, each regulated by its own promoter. Here, we investigated the effect of DNA methylation on gene regulation in the Pcdh-alpha gene cluster. We studied two mouse cell lines, C1300 and M3, that expressed different combinations of Pcdh-alpha isoforms and found that 1) the transcription of specific Pcdh-alpha isoforms correlated significantly with the methylation state of the promoter and the 5' (but not the 3') region of the first exon and 2) mosaic or mixed methylation states of the promoters were associated with both active and inactive transcription. Demethylation of C1300 cells up-regulated all of the Pcdh-alpha isoforms, and, in a promoter assay, hypermethylation of the promoters repressed their transcriptional activity. Cell lines subcloned from the demethylated C1300 cells transcribed different combinations of Pcdh-alpha isoforms than the parental, nondemethylated cells, and the promoters showed differential mosaic or mixed methylation patterns. In vivo, the promoter and 5'-regions of the Pcdh-alphaC1 and alphaC2 exons, which are transcribed in all neurons, were extensively hypomethylated. In contrast, the promoters of the Pcdh-alpha1 to -alpha12 isoforms, which are transcribed differentially by individual Purkinje cells, exhibited mosaic methylation patterns. Overall, our results demonstrated that mosaic or mixed DNA methylation states in the promoter and 5'-region of the first exon may help regulate differential Pcdh-alpha transcription and that hypermethylation is sufficient to repress transcription.

Nuclei isolated from green fluorescent protein-marked neurons in the cerebral cortex of juvenile mice (14–21 d after birth) were injected into enucleated oocytes that were allowed to develop into blastocysts. Embryonic stem (ES) cell lines were established from the inner cell mass of 76 cloned blastocysts after injecting 2026 neuronal nuclei. Some ES cells were injected individually into enucleated oocytes (nuclear transfer). Other ES cells were transferred into the blastocoeles of tetraploid blastocysts (tetraploid complementation). Two-cell embryos after nuclear transfer were transferred to the oviducts of surrogate mothers. Four (1.5%) of 272 nuclear-transferred two-cell embryos developed to term, and two (0.7%) developed into fertile adults. Nineteen (1.9%) of 992 tetraploid blastocysts receiving ES cells reached term, and 10 (1.0%) developed into adults. These findings demonstrate that some of the nuclei of differentiated neurons in the cerebral cortex of juvenile mice maintain developmental pluripotency.

Diverse protocadherin-alpha genes (Pcdha, also called cadherin-related neuronal receptor or CNR) are expressed in the vertebrate brain. Their genomic organization involves multiple variable exons and a set of constant exons, similar to the immunoglobulin (Ig) and T-cell receptor (TCR) genes. This diversity can be used to distinguish neurons. Using polymorphisms that distinguish the C57BL/6 and MSM mouse strains, we analyzed the allelic expression of the Pcdha gene cluster in individual neurons. Single-cell analysis of Purkinje cells using multiple RT-PCR reactions showed the monoallelic and combinatorial expression of each variable exon in the Pcdha genes. This report is the first description to our knowledge of the allelic expression of a diversified receptor family in the central nervous system. The allelic and combinatorial expression of distinct variable exons of the Pcdha genes is a potential mechanism for specifying neuron identity in the brain.

Gene knockout experiments have revealed that Fyn-tyrosine kinase plays significant roles in neural network formation in the CNS. Using Fyn molecules, we have identified a novel CNR family from the CNS. CNR family proteins are located at the synaptic junction, and function as Reelin receptors during layer formation of the cerebral cortex. CNR family genes are clustered in the mouse and human genomes. The CNR family corresponds to the Pcdh α family, which is followed by the Pcdh β and Pcdh γ gene clusters. Each of the CNR / Pcdh α and Pcdh γ gene clusters is separated by variable and constant regions, similar to the arrangement of the BCR and TCR gene clusters. The constant regions are distinct between the CNR / Pcdh α and Pcdh γ genes in mice and humans. These respective gene clusters have undergone diversification during vertebrate evolution. In tereost fugu fish, the constant regions of the CNR/Pcdh α genes are duplicated, but in the clustered large exons, the Reelin-binding sequences are not conserved in their cadherin domains. Moreover, somatic mutations of CNR family transcripts accumulate during development of the cerebral cortex. Thus, characterization of CNR family genes will provide key insights into our understanding of the diversity and specificity of neural networks in the CNS during phylogeny and ontogeny.

We investigated whether anticoagulant therapy with heparin or a selective thrombin inhibitor, argatroban, may ameliorate the postischemic cerebral circulation and attenuate mortality after 10 min of forebrain ischemia. Postischemic subcutaneous injection of argatroban (5 mg/kg) significantly attenuated mortality (9.1%) compared with non-treatment (45.5%) during 14 days’ observation period. This effect coincided with: (1) increased cortical CBF after reperfusion; (2) attenuation of brain edema; and (3) less severe cell damages in the cerebral cortex. In contrast, nine of the 22 gerbils treated with heparin (830 IU/kg) were found dead on the next day due to massive bleeding in the surgical wound and 13 bleeding-avoided gerbils did not show significant amelioration in mortality (30.8%). These findings suggest that argatroban is an effective anticoagulant for prevention of cell damage after a relatively long forebrain ischemia.

遅発性神経細胞死にアポトーシス(programmed cell death)が関与しているという最近の報告は,「海馬CA1の神経細胞が短時間の前脳虚血に脆弱で選択的な細胞死を引き起こすことは,将来の長時間の虚血侵襲の際に脳全体と生命を守っていることに役立っている」という仮説が成り立つ可能性を示唆する.我々は,この仮説を検証する一つの実験方法として,砂ネズミを用い,予め5分間の前脳虚血を負荷せずに15分間前脳虚血を加えた群(A群)と,予め5分間の前脳虚血を負荷し海馬CA1の選択的な細胞死を生じさせておき,10日後に15分間前脳虚血を負荷した群(B群)において,2週間の観察で,B群では有意に生存率が高く,体重減少も軽減していることを前回報告した(脳卒中19: 145-152, 1997).しかし,2週間生き残った砂ネズミの組織学的検討では両群の違いはほとんどなかったため,今回は死亡する前の時点での脳浮腫測定および組織学的解析を新たに行った.大脳の水分含有量を15分間前脳虚血の2時間後,2日後,7日後について調べたところ,B群はA群より脳浮腫の増加が有意に抑制されていた(p

Neuronal diversity is a fundamental requirement for complex neuronal networks and brain function. The clustered protocadherin (Pcdh) family possesses several characteristic features that are important for the molecular basis of neuronal diversity. Clustered Pcdhs are expressed predominantly in the central nervous system, in neurites, growth cones, and synapses. They consist of about 60 isoforms, and their expression is stochastically and combinatorially regulated in individual neurons. The multiple clustered Pcdhs expressed in individual neurons form heteromultimeric protein complexes that exhibit homophilic adhesion properties. Theoretically, the clustered Pcdhs could generate more than 3×10(10) possible variations in each neuron and 12,720 types of cis-tetramers per neuron. The clustered Pcdhs are important for normal neuronal development. The clustered Pcdh genes have also attracted attention as a target for epigenetic regulation.

The molecular basis for providing the identity and diversity of single neurons is a key for realizing the brain system. Diverse protocadherin isoforms encoded by the Pcdh-alpha and Pcdh-gamma gene clusters are expressed in all of the vertebrates studied. For the Pcdh-alpha isoforms, differential expression patterns have been found in single Purkinje cells by unusual monoallelic and combinatorial types of gene regulation. Here we investigated total allelic gene regulation in the Pcdh-alpha and -gamma clusters, including the C-type variable exons (C1 to C5) and the Pcdh-gammaA and -gammaB variable exons in single Purkinje cells. Using split single-cell reverse transcription-PCR analysis, almost all of the Purkinje cells at postnatal day 21 biallelically expressed all the C-type isoforms, whereas the Pcdh-alpha isoforms showed both monoallelic and combinatorial expression. The Pcdh-gammaA and -gammaB isoforms also showed differential regulation in each cell with both monoallelic and combinatorial gene regulation. These data indicated that different types of allelic gene regulation (monoallelic versus biallelic) occurred in the Pcdh-alpha and -gamma clusters, although they were spliced into the same constant exons. It has been reported that each C-type Pcdh-alpha or -gamma transcript has a different expression pattern during brain development, suggesting that the different C-type variable exons may code temporal diversity, although the Pcdh-alpha, -gammaA, and -gammaB isoforms were differential and combinatorial gene regulation within a single cell. Thus, the multiple gene regulations in the Pcdh-alpha and -gamma clusters had a potential mechanism for increasing the diversity of individual neurons in the brain.

The clustered protocadherins comprise the largest subfamily of the cadherin superfamily and are predominantly expressed in the nervous system. The family of clustered protocadherins (clustered Pcdh family) is substructured into three distinct gene arrays in mammals: Pcdh-alpha, Pcdh-beta, and Pcdh-gamma. These are regulated by multiple promoters and cis-alternative splicing without DNA recombination. Pcdh-alpha proteins interact with beta1-integrin to promote cell adhesion. They also form oligomers with Pcdh-gamma proteins at the same membrane sites. During neuronal maturation, Pcdh-alpha expression is dramatically downregulated by myelination. The clustered Pcdh family has multiple variable exons that differ somewhat in number and sequence across vertebrate species. At the single-cell level, Pcdh-alpha mRNAs are regulated monoallelically, resulting in the combinatorial expression of distinct variable exons from each allele. These findings support the idea that diversified Pcdh molecules contribute to neural circuit development and provide individual cells with their specific identity.

Background The specificity of synaptic connections is fundamental for proper neural circuit function. Specific neuronal connections that underlie information processing in the sensory cortex are initially established without sensory experiences to a considerable extent, and then the connections are individually refined through sensory experiences. Excitatory neurons arising from the same single progenitor cell are preferentially connected in the postnatal cortex, suggesting that cell lineage contributes to the initial wiring of neurons. However, the postnatal developmental process of lineage-dependent connection specificity is not known, nor how clonal neurons, which are derived from the same neural stem cell, are stamped with the identity of their common neural stem cell and guided to form synaptic connections. Results We show that cortical excitatory neurons that arise from the same neural stem cell and reside within the same layer preferentially establish reciprocal synaptic connections in the mouse barrel cortex. We observed a transient increase in synaptic connections between clonal but not nonclonal neuron pairs during postnatal development, followed by selective stabilization of the reciprocal connections between clonal neuron pairs. Furthermore, we demonstrate that selective stabilization of the reciprocal connections between clonal neuron pairs is impaired by the deficiency of DNA methyltransferase 3b (Dnmt3b), which determines DNA-methylation patterns of genes in stem cells during early corticogenesis. Dnmt3b regulates the postnatal expression of clustered protocadherin (cPcdh) isoforms, a family of adhesion molecules. We found that cPcdh deficiency in clonal neuron pairs impairs the whole process of the formation and stabilization of connections to establish lineage-specific connection reciprocity. Conclusions Our results demonstrate that local, reciprocal neural connections are selectively formed and retained between clonal neurons in layer 4 of the barrel cortex during postnatal development, and that Dnmt3b and cPcdhs are required for the establishment of lineage-specific reciprocal connections. These findings indicate that lineage-specific connection reciprocity is predetermined by Dnmt3b during embryonic development, and that the cPcdhs contribute to postnatal cortical neuron identification to guide lineage-dependent synaptic connections in the neocortex. Electronic supplementary material The online version of this article (doi:10.1186/s12915-016-0326-6) contains supplementary material, which is available to authorized users.

中枢神経系と免疫系はともに、未知の環境に対して柔軟に対応できるという点で類似している。しかし、それを担う遺伝子の数には限りがある。これらを可能にする免疫系の分子メカニズムとしては、免疫グロブリンやT細胞受容体をコードする遺伝子の体細胞における再構成や免疫グロブリンにおける体細胞突然変異が知られている。また、抗原に対してより親和性の高い細胞が選択されることが知られている。一方、中枢神経系の機能には、複雑なネットワークつくる多様な神経細胞と可塑的な変化が重要な役割を担うと考えられるが、中枢神経系の多様性をもたらす分子メカニズムは明かとはいえない。 近年、チロシンリン酸化酵素であるFynと結合する分子として同定された多様化したCNRファミリーはシナプスに局在するカドヘリン様の接着分子ある。CNRファミリーは遺伝子構造の解析から可変領域と定常領域とからなり、免疫グロブリンやT細胞受容体の遺伝子構造と似ていることが明らかとなった。彼は、シナプスに発現するCNRファミリー遺伝子の発現メカニズムに免疫系類似の体細胞における変化があるか否かについて中枢神経系の発生にしたがい検討した。はじめにCNRファミリー遺伝子の発現メカニズムを解析する目的でC57BL/6（B6）とDBA/2（D2）の系統の異なるマウスの雑種1代目（F1）の大脳皮質を用いてCNRの転写産物の解析をおこなった。B6とD2とでは、CNR3の遺伝子の可変領域に1つ、定常領域に3つの異なる塩基配列がある。F1マウスの細胞は、B6由来の染色体とD2由来の染色体をそれぞれ1対ずつ持つことになるので通常ならばCNR3の転写産物は可変領域、定常領域ともにB6由来あるいはD2由来のCis型となる。しかし、生後60日目のF1マウスのCNR3転写産物をシークエンス解析した結果、約10％が可変領域がB6由来で定常領域がD2由来あるいはその逆のTrans型として発現していることを明かにした。また、Trans型の転写産物は、胎生15日目（E15）、生後1日目（P1）、生後60日目（P60）と発生が進むにつれて増加することがわかった。 同時に、彼はCNR3転写産物をシークエンス解析することで体細胞突然変異の有無についての検討をおこなった。P60の大脳皮質から抽出したCNR3転写産物には一塩基あたり2.6×10-3という高頻度の塩基置換が検出され、同じくP60の大脳皮質から抽出したE-カドヘリンの転写産物1.3×10-3との比較においても統計学的にも有意に高かった。P60において検出した高頻度の塩基置換が体細胞突然変異であるならば、脳の発生に伴い変異の頻度が変化することが予想された。E15、P1を解析したところ、1.7×10-3、2.4×10-3と発生に伴って増加することが明らかとなった。突然変異は、AからG、TからCへの塩基置換が多く、塩基置換の傾向はランダムではなかった。特に、CNR3の3’末端側の非翻訳領域にあるCU繰り返し配列はgerm lineのDNA配列では6回であるのに対し、P60のCNR3転写産物では約70％が7回のCU繰り返しに変わるという高頻度の変化を認めた。また、Cis型とTrans型の転写産物における突然変異率の比較ではTrans型の方がCis型にくらべて有意に突然変異率が高いことがわかった。これらの結果から、CNR遺伝子の転写産物に体細胞突然変異が起きていることが強く示され、突然変異の起こるメカニズムとTrans型のできるメカニズムに関連があることが示唆された。 興味深いことに、アミノ酸置換を伴う変異率は細胞外領域のEC1ドメインでのみ高頻度で起きているということである。EC1ドメインは、クラシカルなカドヘリンではホモフィリックな細胞接着に重要であることが示されており、CNRでは大脳皮質の層構造形成に重要なReelinと結合することから機能的にも重要な役割を果たしていると考えられている。よって、EC1ドメインのアミノ酸置換はCNRの機能に影響を与えるものと考えられる。一方、その他のドメインではアミノ酸置換を伴う変異率は発生が進むにつれて減少する。このことから、EC1ドメインでアミノ酸置換をおこしたもののうち、他のドメインではアミノ酸置換の少ないCNRが脳の発生に伴って選択されている可能性が示唆された。 本研究は、シナプスで発現するCNR遺伝子の転写産物に体細胞突然変異が起きてることをはじめて示し、何らかの選択機構が働いている可能性を示唆した。CNR遺伝子の転写産物の体細胞突然変異はCNRがシナプスに局在することから神経ネットワークの可塑的な変化を担う分子メカニズムとして興味深い。