Recurrent Connections Might Be Important for Hierarchical Categorization.

Visual short-term memory is an important ability of primates and is thought to be stored in area TE. We previously reported that the initial transient responses of neurons in area TE represented information about a global category of faces, e.g., monkey faces vs. human faces vs. simple shapes, and the latter part of the responses represented information about fine categories, e.g., facial expression. The neuronal mechanisms of hierarchical categorization in area TE remain unknown. For this study, we constructed a combined model that consisted of a deep neural network (DNN) and a recurrent neural network and investigated whether this model can replicate the time course of hierarchical categorization. The visual images were stored in the recurrent connections of the model. When the visual images with noise were input to the model, the model outputted the time course of the hierarchical categorization. This result indicates that recurrent connections in the model are important not only for visual short-term memory but for hierarchical categorization, suggesting that recurrent connections in area TE are important for hierarchical categorization.

Low-arginine and low-protein diets induce hepatic lipid accumulation through different mechanisms in growing rats.

BACKGROUND: Dietary protein deficiency and amino acid imbalance cause hepatic fat accumulation. We previously demonstrated that only arginine deficiency or total amino acid deficiency in a diet caused significant hepatic triglyceride (TG) accumulation in young Wistar rats. In this study, we explored the mechanisms of fatty liver formation in these models. METHODS: We fed 6-week-old male Wistar rats a control diet (containing an amino acid mixture equivalent to 15% protein), a low-total-amino acid diet (equivalent to 5% protein; 5PAA), and a low-arginine diet (only the arginine content is as low as that of the 5PAA diet) for 2 weeks. RESULTS: Much greater hepatic TG accumulation was observed in the low-arginine group than in the low-total-amino acid group. The lipid consumption rate and fatty acid uptake in the liver did not significantly differ between the groups. In contrast, the low-total-amino acid diet potentiated insulin sensitivity and related signaling in the liver and enhanced de novo lipogenesis. The low-arginine diet also inhibited hepatic very-low-density lipoprotein secretion without affecting hepatic insulin signaling and lipogenesis. CONCLUSIONS: Although the arginine content of the low-arginine diet was as low as that of the low-total-amino acid diet, the two diets caused fatty liver via completely different mechanisms. Enhanced lipogenesis was the primary cause of a low-protein diet-induced fatty liver, whereas lower very-low-density lipoprotein secretion caused low-arginine diet-induced fatty liver.

A translation repressor, 4E-BP1, regulates the triglyceride level in rat liver during protein deprivation.

Protein deprivation has been shown to induce fatty liver in humans and animals, but the molecular mechanisms underlying such induction are largely unknown. Our previous studies have shown that a low-protein diet increases eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) protein and triglyceride (TG) levels in rat liver. 4E-BP1 is known to repress translation by binding to eIF4E. There is also evidence indicating that 4E-BP1 regulates lipid metabolism. Here, we examined the role of 4E-BP1 on TG accumulation in the livers of rats under protein deprivation. The low-protein diet rapidly increased the hepatic 4E-BP1 mRNA level within 1 day, followed by the induction of hepatic TG accumulation. The knockdown of hepatic 4E-BP1 attenuated the TG accumulation in rat liver induced by the low-protein diet. 4E-BP1 knockdown also increased the protein level of carnitine palmitoyltransferase 1A (CPT1A), a regulator of fatty acid oxidation, in the liver of rats fed a low-protein diet. These results indicate that a low-protein diet increases the amount of 4E-BP1, leading to TG accumulation in rat liver. We thus conclude that 4E-BP1 plays an important role in inducing hepatic steatosis under protein deprivation.

Low-protein diet enhances adiponectin secretion in rats.

Previous studies including ours have shown that a low-protein diet up-regulates insulin signaling in the liver and muscle and induces fatty liver in rats. Adiponectin is known as an insulin-sensitizing adipocytokine. We, therefore, examined the effect of a low-protein diet on the adiponectin levels in rats. The low-protein diet significantly increased serum adiponectin level. However, mRNA and protein levels of adiponectin in white adipose tissue (WAT) were not changed by the low-protein diet. Since it is known that oligomerization is important to control serum adiponectin level, we examined the population of adiponectin oligomeric forms in WAT and found that low-protein diet did not change it. Despite these events, the amount of its secretion was significantly increased in the adipocytes isolated from WAT of low-protein diet-fed rats. These results indicate that a low-protein diet enhances adiponectin secretion, which is not due to the increased intracellular amount and oligomerization of adiponectin.

Branched-chain amino acid supplementation restores reduced insulinotropic activity of a low-protein diet through the vagus nerve in rats.

BACKGROUND: Previously, we reported that a low-protein diet significantly reduced insulin secretion in response to feeding within 1 h in rats, suggesting that the insulinotropic effect of dietary protein plays an important role in maintaining normal insulin release. The current study aimed to elucidate whether deficiency of certain amino acids could diminish the insulinotropic activity and to investigate whether reduced insulin secretion in response to a low-protein diet is restored by supplementation with certain amino acids. METHODS: First, we fed male Wistar rats (5-6 rats per group) with diets deficient in every single amino acid or three branched-chain amino acids (BCAAs); within 1-2 h after the onset of feeding, we measured the plasma insulin levels by using an enzyme-linked immunosorbent assay (ELISA). As insulin secretion was reduced in BCAA-deficient groups, we fed low-protein diets supplemented with BCAAs to assess whether the reduced insulin secretion was restored. In addition, we treated the pancreatic beta cell line MIN6 with BCAAs to investigate the direct insulinotropic activity on beta cells. Lastly, we investigated the effect of the three BCAAs on sham-operated or vagotomized rats to assess involvement of the vagus nerve in restoration of the insulinotropic activity. RESULTS: Feeding a low-protein diet reduced essential amino acid concentrations in the plasma during an absorptive state, suggesting that reduced plasma amino acid levels can be an initial signal of protein deficiency. In normal rats, insulin secretion was reduced when leucine, valine, or three BCAAs were deficient. Insulin secretion was restored to normal levels by supplementation of the low-protein diet with three BCAAs, but not by supplementation with any single BCAA. In MIN6 cells, each BCAA alone stimulated insulin secretion but the three BCAAs did not show a synergistic stimulatory effect. The three BCAAs showed a synergistic stimulatory effect in sham-operated rats but failed to stimulate insulin secretion in vagotomized rats. CONCLUSIONS: Leucine and valine play a role in maintaining normal insulin release by directly stimulating beta cells, and supplementation with the three BCAAs is sufficient to compensate for the reduced insulinotropic activity of the low-protein diet, through the vagus nerve.

Triglyceride synthesis in hepatocytes isolated from rats fed a low-protein diet is enhanced independently of upregulation of insulin signaling.

It is known that protein malnutrition develops fatty liver in rats. However, the mechanisms by which protein malnutrition enhances lipid accumulation in the liver are not fully understood. Our previous studies have demonstrated that protein malnutrition upregulates insulin signaling with an increase in TG levels in rat livers. Here, we examined whether the upregulated insulin signaling contributes to an enhancement of TG accumulation under protein malnutrition. As it is difficult to analyze insulin-induced hepatic TG synthesis in vivo, the isolated hepatocytes derived from rats fed a low-protein diet were used. The hepatocytes were isolated from rats fed a 15% casein diet (15C) as a control diet or a 5% casein diet (5C) as a low-protein diet and then treated with insulin. As shown in vivo, insulin signaling was upregulated in isolated hepatocytes from 5C-fed rats (5C hepatocytes). However, the insulin-induced increase in the mRNA levels of lipogenic enzymes, including acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FAS), was similar in both groups. The amounts of TG synthesized from both glucose and palmitate, as well as ACC1 and FAS protein levels, were increased at the basal state in 5C hepatocytes, but were not further increased by insulin. These results indicate that TG synthesis via both de novo fatty acid synthesis and esterification is enhanced in 5C hepatocytes, which is independent of the upregulation of insulin signaling.

Tissue-specific effects of protein malnutrition on insulin signaling pathway and lipid accumulation in growing rats.

Our previous studies have revealed that protein malnutrition enhances insulin signaling in rat liver and muscle in response to a bolus insulin injection. However, it has not been established whether protein malnutrition up-regulates insulin signaling under physiological conditions, such as feeding. Here, we studied the effects of protein malnutrition on insulin signaling after feeding in rat liver, muscle and white adipose tissue (WAT). Six-week-old rats were fed a 15% casein diet (15C) or a calorie-matched 5% casein diet (5C) for 8 h/day during 14 days. On the 15th day, blood and tissues were collected at various time points after feeding. Feeding-induced insulin secretion was reduced in 5C-fed rats compared to 15C-fed rats. The 5C-feeding suppressed immediate activation of insulin receptor after feeding in the liver, muscle, and WAT. However, 5C-feeding constantly increased tyrosine phosphorylation of insulin receptor substrate (IRS)-2 and threonine phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) in the liver during the examined periods, corresponding to the changes of their amounts. In skeletal muscle, 5C-feeding did not appreciably alter insulin signaling. In WAT, 5C-feeding decreased tyrosine phosphorylation of IRS-1 compared to 15C-feeding. Furthermore, hepatic triglyceride content was increased and feeding-induced acetyl-CoA carboxylase 1 gene expression was enhanced in 5C-fed rats. The 5C-feeding decreased insulin-dependent glucose uptake in adipocytes. These results suggest that enhanced insulin signaling through increased IRS-2 and 4E-BP1 levels in the liver and repressed insulin signaling through decreased IRS-1 levels in WAT contribute to the preferential hepatic lipid accumulation under protein malnutrition.

Amelioration of improper differentiation of somatostatin-positive interneurons by triiodothyronine in a growth-retarded hypothyroid mouse strain.

Thyroid hormone (TH) plays an important role in brain development, and TH deficiency during pregnancy or early postnatal periods leads to neurological disorders such as cretinism. Hypothyroidism reduces the number of parvalbumin (PV)-positive interneurons in the neocortex and hippocampus. Here we used a mouse strain (growth-retarded; grt) that shows growth retardation and hypothyroidism to examine whether somatostatin (Sst)-positive interneurons that are generated from the same pool of neural progenitor cells as PV-positive cells are also altered by TH deficiency. The number of PV-positive interneurons was significantly decreased in the neocortex and hippocampus of grt mice as compared with normal control mice. In contrast to the decrease in the number of PV neurons, the number of Sst-positive interneurons in grt mice was increased in the stratum oriens of the hippocampus and the hilus of the dentate gyrus, although their number was unchanged in the neocortex. These changes were reversed by triiodothyronine administration from postnatal day (PD) 0 to 20. TH supplementation that was initiated after PD21 did not, however, affect the number of PV- or Sst-positive cells. These results suggest that during the first three postnatal weeks, TH may be critical for the generation of subpopulations of interneurons.

Interaction, mobility, and phosphorylation of human orthologues of WD repeat-containing components of the yeast SSU processome t-UTP sub-complex.

We previously proposed a dynamic scaffold model for inner nuclear structure formation. In this model, structures in inter-chromatin regions are maintained through dynamic interaction of protein complex modules, and WD repeat- and disordered region-rich proteins and others act as scaffolds for these protein complexes. In this study, three WD-repeat proteins, i.e., CIRH1A, UTP15, and WDR43, were found in the nuclear matrix fraction and speculated to be present in the human t-UTP sub-complex of SSU processomes. The results obtained as to their subnuclear localization, binding with each other, mobilities, and phosphorylation were: (i) the majority of these proteins fused with GFP are localized to the fibrillar center region in nucleoli. (ii) these 3 proteins bind directly with each other in vitro. (iii) the movement of these proteins is very slow in living cells and independent of rDNA transcription. (iv) His-CIRH1A is phosphorylated at Thr(131) by a mitotic Xenopus egg extract, and binding with GST-UTP15 and GST-WDR43 is suppressed. These findings and others suggest that these 3 WD proteins found in the matrix fraction bind directly with each other, bind tightly to fibrillar center regions, and comprise a part of the nucleolar structure. These results are also consistent with our dynamic scaffold model.

Orally available pyridinylpyrimidine derivatives as novel RANKL-induced osteoclastogenesis inhibitors.

An HTS campaign led to the identification of 4-pyrroldino-2-(pyridin-2-yl)pyrimidine compound 1 as an RANKL-induced osteoclastogenesis inhibitor. The compound 1 showed high clearance values in microsomes, however. Modification of the pyrrolidino group to a benzylamino group improved human microsomal stability with a slight loss of in vitro activity. Substitution at the ortho position of the benzyl group ameliorated in vitro activity, and further fluorination of the benzyl group improved microsomal stability in rodents. Representative members of this series, compounds 20 and 23, exhibited efficacy in RANKL-induced osteopenic mice when administered orally at 0.3 mg/kg.

Acetylation of Prrp K150 regulates the subcellular localization.

Posttranslational modifications of proteins have profound effects on many aspects of their function and have received much attention due to the importance of these processes in epigenetic regulation. In this study, we report that deleted azoospermia associated protein 1 (DAZAP1)/proline-rich RNA binding protein (Prrp), a multifunctional RNA binding protein which is essential for spermatogenesis and normal cell growth, is acetylated at Lysine 150 within its RNA binding domain. The acetylation is predominantly observed in nuclear Prrp, and the nonacetylated form is in cytoplasm. Considering that Prrp is a shuttling protein, we suggest that the acetylation cycle at Prrp K150 regulates nucleocytoplasmic transport in cells.

Relationship between growth retardation and impaired glucose tolerance in hypothyroidal growth-retarded (grt) mice.

Growth-retarded (grt) mice exhibit congenital hypothyroidism and a characteristic growth pause followed by delayed onset of pubertal growth. This pattern of growth has never been reported in any other animal model exhibiting hypothyroidism; therefore, the growth retardation observed in grt mice is unlikely to be explained completely by the low plasma thyroid hormone levels. As growth is closely related to nutrient metabolism, we investigated the relationship between the appearance of growth retardation and glucose utilization, which is the main component of nutrient metabolism, in the peripubertal stage of grt mice. The relative weights of the organs involved in nutrient digestion and absorption were abnormal in grt mice. The intraperitoneal glucose tolerance test (IGTT) showed impaired glucose tolerance in grt mice. Moreover, this symptom appeared in parallel with the progression of growth retardation in grt mice. The impaired blood glucose levels on the IGTT in grt mice were considered to be attributable to decreased plasma insulin levels rather than to impaired insulin sensitivity. The pattern of anti-insulin antibody staining on sections of pancreatic islets from grt mice was almost the same as that in the corresponding sections from normal mice. Insulin treatment accelerated the growth of peripubertal grt mice. These findings suggest that the appearance of growth retardation in grt mice might be partially attributable to a reduction in glucose metabolism and impairment of insulin secretion during the early period of growth.

Impaired insulin secretion from the pancreatic islets of hypothyroidal growth-retarded mice.

The growth-retarded (grt) mouse shows thyroid dysfunction-related hyporesponsiveness to TSH. Thyroid hormone is a critical regulator of metabolism in many cells; thus, derangement of thyroid function affects many organs and systems. Experiments were conducted focusing on the function of the pancreatic islets in grt mice. We showed occurrence of a fasting hyperglycemia and a decreased plasma insulin level response to a glucose load in grt mice, despite normal insulin molecules being stored in secretory granules of pancreatic islets. We also demonstrated a reduction of insulin secretion in response to glucose administration from islets of grt mice in vitro, while the insulin release in response to KCl stimulation was comparable to that in normal mice, indicating that the isolated islets from grt mice have normal ATP-sensitive K(+) channels and postchannel activity. The mRNA expression levels of glucose transporter 2 and glucokinase in the islets of grt mice were similar to those in normal mice. Triiodothyronine administration to grt mice improved insulin secretion very slightly. On the other hand, mRNA for tyrosylprotein sulfotransferase 2 (Tpst2) was found to be expressed in the pancreatic islets of grt mice. Considering that Tpst2 is the responsible gene of grt mice, mutation of which is associated with a poor function of TSH receptor, the findings raise a possibility of involvement of factors including Tpst2 in the insulin hyposecretion in grt mice.

Expression levels of Protocadherin-alpha transcripts are decreased by nonsense-mediated mRNA decay with frameshift mutations and by high DNA methylation in their promoter regions.

The mouse protocadherin (Pcdh) clusters, Pcdh-alpha, -beta, and -gamma, are located on chromosome 18. Many polymorphic variations are found in the Pcdh-alpha genes in wild-derived and laboratory mouse strains. In comparing the expression levels of Pcdh-alpha isoforms among several strains, we observed lower expression levels of Pcdh-alpha9 in BLG2 and BFM/2, and of Pcdh-alpha8 in C57BL/6 (B6) than in the other strains. For Pcdh-alpha8, high DNA methylation (72.7%) in the promoter region was found only in B6, whereas 36.4-44.3% methylation was seen in the other strains. On the other hand, the Pcdh-alpha9 DNA-methylation levels were similar (23.6-36.3%) among the strains regardless of the difference in expression levels. Interestingly, however, the Pcdh-alpha9 variable exon in both BLG2 and BFM/2 included a premature termination codon (PTC) generated by a nucleotide deletion or insertion. Treatment with emetine, a potent inhibitor of nonsense-mediated mRNA decay (NMD), increased the expression level of Pcdh-alpha9 from the BLG2-Pcdh-alpha locus. These data indicate that the transcription levels of mature Pcdh-alpha mRNAs are decreased by the DNA-methylation state of the Pcdh-alpha promoter regions and by the NMD pathway during RNA maturation. And we correct some previous data on Sugino, H., Toyama, T., Taguchi, Y., Esumi, S., Miyazaki, M., Yagi, T., (2004) Negative and positive effects of an IAP-LTR on nearby Pcdaalpha gene expression in the central nervous system and neuroblastoma cell lines, Gene 337 91-103.

PSPC1, NONO, and SFPQ are expressed in mouse Sertoli cells and may function as coregulators of androgen receptor-mediated transcription.

In Sertoli cells of testis, androgen receptor-regulated gene transcription plays an indispensable role in maintaining spermatogenesis. Androgen receptor activity is modulated by a number of coregulators which are associated with the androgen receptor. Non-POU-domain-containing, octamer binding protein (NONO), a member of the DBHS-containing proteins, complexes with androgen receptor and functions as a coactivator for the receptor. Paraspeckle protein 1 alpha isoform (PSPC1, previously known as PSP1) and Splicing factor, proline- and glutamine-rich (SFPQ, previously known as PSF), other members of the DBHS-containing proteins, are also found in androgen receptor complexes, suggesting that these DBHS-containing proteins may cooperatively regulate androgen receptor-mediated gene transcription. We demonstrated that PSPC1, NONO, and SFPQ are coexpressed in Sertoli cell line TTE3 and interact reciprocally. The effect of the DBHS-containing proteins on the transcriptional activity was assessed using the construct containing androgen-responsive elements followed by a luciferase gene. The results showed that all the DBHS-containing proteins activate androgen receptor-mediated transcription, and PSPC1 is the most effective coactivator among them. Furthermore, we confirmed the presence of PSPC1, NONO, and SFPQ proteins in Sertoli cells of adult mouse testis sections. These observations suggest that PSPC1, NONO, and SFPQ form complexes with each other in Sertoli cells and may regulate androgen receptor-mediated transcriptional activity.

Molecular evolution of cadherin-related neuronal receptor/protocadherin(alpha) (CNR/Pcdh(alpha)) gene cluster in Mus musculus subspecies.

The mouse cadherin-related neuronal receptor/protocadherin (CNR/Pcdh) gene clusters are located on chromosome 18. We sequenced single-nucleotide polymorphisms (SNPs) of the CNR/Pcdh(alpha)-coding region among 12 wild-derived and four laboratory strains; these included the four major subspecies groups of Mus musculus: domesticus, musculus, castaneus, and bactrianus. We detected 883 coding SNPs (cSNPs) in the CNR/Pcdh(alpha) variable exons and three in the constant exons. Among all the cSNPs, 586 synonymous (silent) and 297 nonsynonymous (amino acid exchanged) substitutions were found; therefore, the K(a)/K(s) ratio (nonsynonymous substitutions per synonymous substitution) was 0.51. The synonymous cSNPs were relatively concentrated in the first and fifth extracellular cadherin domain-encoding regions (ECs) of CNR/Pcdh(alpha). These regions have high nucleotide homology among the CNR/Pcdh(alpha) paralogs, suggesting that gene conversion events in synonymous and homologous regions of the CNR/Pcdh(alpha) cluster are related to the generation of cSNPs. A phylogenetic analysis revealed gene conversion events in the EC1 and EC5 regions. Assuming that the common sequences between rat and mouse are ancestral, the GC content of the third codon position has increased in the EC1 and EC5 regions, although biased substitutions from GC to AT were detected in all the codon positions. In addition, nonsynonymous substitutions were extremely high (11 of 13, K(a)/K(s) ratio 5.5) in the laboratory mouse strains. The artificial environment of laboratory mice may allow positive selection for nonsynonymous amino acid variations in CNR/Pcdh(alpha) during inbreeding. In this study, we analyzed the direction of cSNP generation, and concluded that subspecies-specific nucleotide substitutions and region-restricted gene conversion events may have contributed to the generation of genetic variations in the CNR/Pcdh genes within and between species.

Identification and characterization of coding single-nucleotide polymorphisms within human protocadherin-alpha and -beta gene clusters.

The human protocadherin (Pcdh) gene clusters are located on chromosome 5q31. Single-nucleotide polymorphisms (SNPs) were detected in the Pcdh-alpha and -beta variable exons, and in the Pcdh-alpha constant exon, in samples from 104 individuals. Among coding SNPs (cSNPs), nonsynonymous (amino acid exchange) SNPs were 2.2 times more common than synonymous (silent) changes in the Pcdh-alpha variable exons, but only 1.2 times more common in the Pcdh-beta variable exons. The nonsynonymous SNPs were high in the ectodomain (EC) 1 encoding region of Pcdh-alpha but not of Pcdh-beta. One 48-kb region of extensive linkage disequilibrium (LD) is reported that has two haplotypes extending from the alpha1 to alpha7 genes in the Pcdh-alpha cluster. Here we identified 15 amino acid exchanges in these two major haplotypes; therefore, the two haplotypes encode different sets of Pcdh-alpha proteins in the brain. The distribution of cSNPs was different for each EC region of Pcdh-alpha or -beta. The frequency of cSNPs was negatively correlated with the paralogous sequence diversity. These results suggested that gene conversion events in homologous regions of the Pcdh-alpha and Pcdh-beta clusters generated the cSNPs. Within the cSNPs, gene conversions were found in Pcdh-alpha4 in the major haplotype, and in Pcdh-beta9. These gene conversions were caused by the unequal crossing-over of homologous sequence regions. Thus, nonsynonymous variations in the Pcdh-alpha and -beta genes are possible contributors to the variations in human brain function.

The RNA ligands for mouse proline-rich RNA-binding protein (mouse Prrp) contain two consensus sequences in separate loop structure.

Mouse proline-rich RNA-binding protein (mPrrp) is a mouse ortholog of Xenopus Prrp, which binds to a vegetal localization element (VLE) in the 3'-untranslated region (3'-UTR) of Vg1 mRNA and is expected to be involved in the transport and/or localization of Vg1 mRNA to the vegetal cortex of oocytes. In mouse testis, mPrrp protein is abundantly expressed in the nuclei of pachytene spermatocytes and round spermatids, and shifts to the cytoplasm in elongating spermatids. To gain an insight into the function of mPrrp in male germ cells, we performed in vitro RNA selection (SELEX) to determine the RNA ligand sequence of mPrrp. This analysis revealed that many of the selected clones contained both of two conserved elements, AAAUAG and GU1-3AG. RNA-binding study on deletion mutants and secondary structure analyses of the selected RNA revealed that a two-loop structure containing the conserved elements is required for high-affinity binding to mPrrp. Furthermore, we found that the target mRNAs of Xenopus Prrp contain intact AAAUAG and GU1-3AG sequences in the 3'-UTR, suggesting that these binding sequences are shared by Prrps of Xenopus and mouse.

Monoallelic yet combinatorial expression of variable exons of the protocadherin-alpha gene cluster in single neurons.

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.

Negative and positive effects of an IAP-LTR on nearby Pcdaalpha gene expression in the central nervous system and neuroblastoma cell lines.

Intracisternal A-particles (IAPs) are defective retrovirions encoded by members of a large family of endogenous proviral elements in the murine genome. An intact IAP element was found in the protocadherin alpha (Pcdhalpha) gene cluster of five laboratory mouse strains. However, IAP insertion was not detected in three wild mouse strains we investigated. This IAP insertion caused the disruption of one variable exon of laboratory mouse and down-regulated expression of the Pcdhalpha v8 exon, which is located just downstream of the IAP in the brain following the methylation of 5' regulatory region of Pcdhalpha v8. In contrast, the Pcdhalpha v8 exon was highly expressed in mouse neuroblastoma cell lines. This suggested that the IAP insertion activates the expression of the nearby Pcdhalpha v8 exon in these cell lines. In fact, the Pcdhalpha v8 exon expression was driven by the IAP-long terminal repeat (LTR) following the de-methylation of 5' regulatory region of Pcdhalpha v8. To investigate the promoter activity of the IAP, we constructed an IAP-LTR-ECFP reporter gene and introduced it into neuroblastoma, melanoma, lymphoma, and plasmacytoma cell lines. Interestingly, ECFP-positive cells were observed only in the neuroblastoma cell lines. Moreover, there were no differences in the promoter activities of the IAP-LTR whether it was in the sense or complimentary orientation. Thus, this IAP-LTR has negative and positive regulation on near by gene expression in the brain and neuroblastoma cell lines.