Long-term depression-inductive stimulation causes long-term potentiation in mouse Purkinje cells with a mutant thyroid hormone receptor.

Thyroid hormones (THs) regulate gene expression by binding to nuclear TH receptors (TRs) in the cell. THs are indispensable for brain development. However, we have little knowledge about how congenital hypothyroidism in neurons affects functions of the central nervous system in adulthood. Here, we report specific TH effects on functional development of the cerebellum by using transgenic mice overexpressing a dominant-negative TR (Mf-1) specifically in cerebellar Purkinje cells (PCs). Adult Mf-1 mice displayed impairments in motor coordination and motor learning. Surprisingly, long-term depression (LTD)-inductive stimulation caused long-term potentiation (LTP) at parallel fiber (PF)-PC synapses in adult Mf-1 mice, although there was no abnormality in morphology or basal properties of PF-PC synapses. The LTP phenotype was turned to LTD in Mf-1 mice when the inductive stimulation was applied in an extracellular high-Ca2+ condition. Confocal calcium imaging revealed that dendritic Ca2+ elevation evoked by LTD-inductive stimulation is significantly reduced in Mf-1 PCs but not by PC depolarization only. Single PC messenger RNA quantitative analysis showed reduced expression of SERCA2 and IP3 receptor type 1 in Mf-1 PCs, which are essential for mGluR1-mediated internal calcium release from endoplasmic reticulum in cerebellar PCs. These abnormal changes were not observed in adult-onset PC-specific TH deficiency mice created by adeno-associated virus vectors. Thus, we propose the importance of TH action during neural development in establishing proper cerebellar function in adulthood, independent of its morphology. The present study gives insight into the cellular and molecular mechanisms underlying congenital hypothyroidism-induced dysfunctions of central nervous system and cerebellum.

The effect of soy isoflavones in brain development: the emerging role of multiple signaling pathways and future perspectives.

Soybean is a source of protein, fibers, and phytochemical isoflavones which are considered to have numerous health benefits for children and adulthood. On the other hand, isoflavones are widely known as phytoestrogens that exert their action via the estrogen signaling pathway. With this regard, isoflavones are also considered as endocrine-disrupting chemicals. Endogenous estrogen plays a crucial role in brain development through binding to estrogen receptors (ERs) or G protein-coupled estrogen receptors 1 (GPER1) and regulates morphogenesis, migration, functional maturation, and intracellular metabolism of neurons and glial cells. Soy isoflavones can also bind to ERs, GPER1, and, furthermore, other receptors to modulate their action. Therefore, soy isoflavone consumption may affect brain development during the pre-and post-natal periods. This review summarizes the current knowledge on the mechanisms of isoflavone action, particularly in the early stages of brain development by introducing representative human, and animal models, and in vitro studies, and discusses their beneficial and adverse impact on neurobehavior. As a conclusion, the soy product consumption during the pre-and post-natal periods under proper range of dose showed beneficial effects in neurobehavior development, including improvement of anxiety, aggression, hyperactive behavior, and cognition, whereas their adverse effect by taking higher doses cannot be excluded. We also present novel research lines to further assess the effect of soy isoflavone administration during brain development.

Plasma prolactin axis shift from placental to pituitary origin in late prepartum mice.

The placenta secretes a prolactin (PRL)-like hormone PRL3B1 (placental lactogen II), a luteotropic hormone essential for maintaining pregnancy until labor in mice. A report from 1984 examined the secretion pattern of PRL3B1 in prepartum mice. In the current study, we found contradictory findings in the secretion pattern that invalidate the previous report. By measuring maternal plasma PRL3B1 and PRL every 4 hrs from gestational day 17 (G17), we newly discovered that maternal plasma PRL3B1 levels decrease rapidly in prepartum C57BL/6 mice. Interestingly, the onset of this decline coincided with the PRL surge at G18, demonstrating a plasma prolactin axis shift from placental to pituitary origin. We also found that maternal plasma progesterone regression precedes the onset of the PRL shift. The level of Prl3b1 mRNA was determined by RT-qPCR in the placenta and remained stable until parturition, implying that PRL3B1 peptide production or secretion was suppressed. We hypothesized that production of the PRL family, the 25 paralogous PRL proteins exclusively expressed in mice placenta, would decrease alongside PRL3B1 during this period. To investigate this hypothesis and to seek proteomic changes, we performed a shotgun proteome analysis of the placental tissue using data-independent acquisition mass spectrometry (DIA-MS). Up to 5,891 proteins were identified, including 17 PRL family members. Relative quantitative analysis between embryonic day 17 (E17) and E18 placentas showed no significant difference in the expression of PRL3B1 and most PRL family members except PRL7C1. These results suggest that PRL3B1 secretion from the placenta is suppressed at G18 (E18).

17ß-Estradiol (E2) Activates Matrix Mineralization through Genomic/Nongenomic Pathways in MC3T3-E1 Cells.

Estrogen plays an important role in osteoporosis prevention. We herein report the possible novel signaling pathway of 17ß-estradiol (E2) in the matrix mineralization of MC3T3-E1, an osteoblast-like cell line. In the culture media-containing stripped serum, in which small lipophilic molecules such as steroid hormones including E2 were depleted, matrix mineralization was significantly reduced. However, the E2 treatment induced this. The E2 effects were suppressed by ICI182,780, the estrogen receptor (ER)α, and the ERß antagonist, as well as their mRNA knockdown, whereas Raloxifene, an inhibitor of estrogen-induced transcription, and G15, a G-protein-coupled estrogen receptor (GPER) 1 inhibitor, had little or no effect. Furthermore, the E2-activated matrix mineralization was disrupted by PMA, a PKC activator, and SB202190, a p38 MAPK inhibitor, but not by wortmannin, a PI3K inhibitor. Matrix mineralization was also induced by the culture media from the E2-stimulated cell culture. This effect was hindered by PMA or heat treatment, but not by SB202190. These results indicate that E2 activates the p38 MAPK pathway via ERs independently from actions in the nucleus. Such activation may cause the secretion of certain signaling molecule(s), which inhibit the PKC pathway. Our study provides a novel pathway of E2 action that could be a therapeutic target to activate matrix mineralization under various diseases, including osteoporosis.

Calcium sensing and signaling are impaired in the lumbar spine of a rat model of congenital kyphosis.

PURPOSE: Kyphosis involves spines curving excessively backward beyond their physiological curvature. Although the normal structure of the spinal vertebrae is extremely important for maintaining posture and the normal function of the thoracic and abdominal organs, our knowledge concerning the pathogenesis of the disease is insufficient. We herein report that the downregulation of the calcium signaling pathway is involved in the pathogenesis of congenital kyphosis. METHODS: The third to fifth lumbar spine segments, the kyphotic region of Ishibashi (IS) rats, which are used as a model of congenital kyphoscoliosis, were collected. A DNA microarray, quantitative PCR, Western blotting, and immunohistochemistry were used to measure the expression of genes and proteins related to intracellular calcium signaling. RESULTS: We found that the expression of calcium-sensing receptor (CaSR) and transient receptor potential vanilloid 1 (Trpv1)-two receptors involved in the calcium signaling-was decreased in the lumbar spine of IS rats. We also observed that the number of CaSR-immunoreactive and Trpv1-immunoreactive cells in the lumbar spine of IS rats was lower than in wild-type rats. Furthermore, the expression of intracellular molecules downstream of these receptors, such as phosphorylated protein kinase C, c-Jun N-terminal kinase, and neural EGFL-like 1, was also reduced. In fact, the calcium content in the lumbar spine of IS rats was significantly lower than that in wild-type rats. CONCLUSION: These results indicate that adequate calcium signaling is extremely important for the regulation of normal bone formation and may also be a key factor for understanding the pathogenesis of congenital kyphosis.

Isoflavones Mediate Dendritogenesis Mainly through Estrogen Receptor α.

The nuclear estrogen receptor (ER) and G-protein-coupled ER (GPER1) play a crucial role during brain development and are involved in dendrite and spine growth as well as synapse formation. Soybean isoflavones, such as genistein, daidzein, and S-equol, a daidzein metabolite, exert their action through ER and GPER1. However, the mechanisms of action of isoflavones on brain development, particularly during dendritogenesis and neuritogenesis, have not yet been extensively studied. We evaluated the effects of isoflavones using mouse primary cerebellar culture, astrocyte-enriched culture, Neuro-2A clonal cells, and co-culture with neurons and astrocytes. Soybean isoflavone-augmented estradiol mediated dendrite arborization in Purkinje cells. Such augmentation was suppressed by co-exposure with ICI 182,780, an antagonist for ERs, or G15, a selective GPER1 antagonist. The knockdown of nuclear ERs or GPER1 also significantly reduced the arborization of dendrites. Particularly, the knockdown of ERα showed the greatest effect. To further examine the specific molecular mechanism, we used Neuro-2A clonal cells. Isoflavones also induced neurite outgrowth of Neuro-2A cells. The knockdown of ERα most strongly reduced isoflavone-induced neurite outgrowth compared with ERß or GPER1 knockdown. The knockdown of ERα also reduced the mRNA levels of ER-responsive genes (i.e., Bdnf, Camk2b, Rbfox3, Tubb3, Syn1, Dlg4, and Syp). Furthermore, isoflavones increased ERα levels, but not ERß or GPER1 levels, in Neuro-2A cells. The co-culture study of Neuro-2A cells and astrocytes also showed an increase in isoflavone-induced neurite growth, and co-exposure with ICI 182,780 or G15 significantly reduced the effects. In addition, isoflavones increased astrocyte proliferation via ER and GPER1. These results indicate that ERα plays an essential role in isoflavone-induced neuritogenesis. However, GPER1 signaling is also necessary for astrocyte proliferation and astrocyte-neuron communication, which may lead to isoflavone-induced neuritogenesis.

Effects of Perfluorooctane Sulfonate on Cerebellar Cells via Inhibition of Type 2 Iodothyronine Deiodinase Activity.

Perfluorooctane sulfonate (PFOS) has been used in a wide variety of industrial and commercial products. The adverse effects of PFOS on the developing brain are becoming of a great concern. However, the molecular mechanisms of PFOS on brain development have not yet been clarified. We investigated the effect of early-life exposure to PFOS on brain development and the mechanism involved. We investigated the change in thyroid hormone (TH)-induced dendrite arborization of Purkinje cells in the primary culture of newborn rat cerebellum. We further examined the mechanism of PFOS on TH signaling by reporter gene assay, quantitative RT-PCR, and type 2 iodothyronine deiodinase (D2) assay. As low as 10-7 M PFOS suppressed thyroxine (T4)-, but not triiodothyronine (T3)-induced dendrite arborization of Purkinje cells. Reporter gene assay showed that PFOS did not affect TRα1- and TRß1-mediated transcription in CV-1 cells. RT-PCR showed that PFOS suppressed D2 mRNA expression in the absence of T4 in primary cerebellar cells. D2 activity was also suppressed by PFOS in C6 glioma-derived cells. These results indicate that early-life exposure of PFOS disrupts TH-mediated cerebellar development possibly through the disruption of D2 activity and/or mRNA expression, which may cause cerebellar dysfunction.

Adult-onset hypothyroidism causes mechanical hypersensitivity due to peripheral nerve hyperexcitability based on voltage-gated potassium channel downregulation in male mice.

Thyroid hormones play an important role in the central and peripheral nervous system functions. Approximately 50% of adult-onset hypothyroid patients have sensory symptoms including pain, possibly caused by peripheral neuropathy. However, the mechanism causing the pain has not been clarified. We generated an adult-onset hypothyroid model animal by administering 50 ppm propylthiouracil (PTU) for 5 weeks to male mice. Female mice were not tested in this study. Mechanical hypersensitivity, determined by the von Frey hair test, was observed during the PTU exposure and recovered after the exposure termination. The sciatic nerve compound action potential was also analyzed. Under single-pulse stimulation, no significant change in the threshold and conduction velocity was observed in the PTU-administered group. On the other hand, under train-pulse stimulation, the latency delay in the Aδ-fiber component was less in the PTU-administered group in Week 4 of PTU exposure, indicating relative hyperexcitability. Fluticasone, which is the anti-inflammatory agent with an ability to activate the voltage-gated potassium channel subfamily A (Kv1), restored the decrease in the latency change ratio by PTU exposure under the train-pulse stimulation supporting our hypothesis that Kv1 may be involved in the conductivity change. Kv1.1 protein level decreased significantly in the sciatic nerve of the PTU-administered group. These results indicate that adult-onset hypothyroidism causes mechanical hypersensitivity owing to hyperexcitability of the peripheral nerve and that reduction of Kv1.1 level may be involved in such alteration.

Maternal prolactin levels during late pregnancy and nurturing behavior of offspring in mice.

Elucidating the mechanisms underlying nurturing and neglect behaviors is meaningful but challenging. Recently, we found that CIN85-deficient mice had reduced pituitary hormone prolactin secretion during late pregnancy, and their pups later showed an inhibited nurturing behavior. To examine whether this phenomenon could be reproduced in normal mice and not just CIN85-deficient mice, we investigated the nurturing behavior of offspring born to mothers whose blood prolactin levels had been reduced by bromocriptine administration during late pregnancy. First, to determine when bromocriptine treatment should be started, we investigated the detailed changes in blood prolactin levels in late pregnancy in mice, resulting in the identification of the prepartum prolactin surge. Furthermore, prolactin receptors in the fetal hypothalamus were expressed to the same extent as in the adult hypothalamus. Treatment with bromocriptine decreased the plasma concentrations of prolactin to the basal range throughout late pregnancy. However, against expectations, the proportion of the resultant pups exhibiting nurturing behaviors as adults was as high as that in the mice without bromocriptine treatment. In conclusion, the elimination of prolactin secretion during late pregnancy alone does not induce neglect-like behavior in offspring, suggesting that CIN85-deficient mice appear to involve another factor due to CIN85 deficiency besides prolactin deficiency.

Histone Deacetylase 3 Inhibitor Alleviates Cerebellar Defects in Perinatal Hypothyroid Mice by Stimulating Histone Acetylation and Transcription at Thyroid Hormone-Responsive Gene Loci.

Perinatal hypothyroidism impairs cerebellar organogenesis and results in motor coordination defects. The thyroid hormone receptor binds to corepressor complexes containing histone deacetylase (HDAC) 3 in the absence of ligands and acts as a transcriptional repressor. Although histone acetylation status is strongly correlated with transcriptional regulation, its role in cerebellar development remains largely unknown. We aimed to study whether the cerebellar developmental defects induced by perinatal hypothyroidism can be rescued by treatment with a specific HDAC3 inhibitor, RGFP966. Motor coordination was analyzed using three behavioral tests. The cerebella were subjected to RT-qPCR and chromatin immunoprecipitation assays for acetylated histone H3. The treatment with RGFP966 partially reversed the cerebellar morphological defects in perinatal hypothyroid mice. These findings were associated with the alleviation of motor coordination defects in these mice. In addition, the RGFP966 administration increased the mRNA levels of cerebellar thyroid hormone-responsive genes. These increases were accompanied by augmented histone acetylation status at these gene loci. These findings indicate that HDAC3 plays an important role in the cerebellar developmental defects induced by perinatal hypothyroidism. The HDAC3 inhibitor might serve as a novel therapeutic agent for hypothyroidism-induced cerebellar defects by acetylating histone tails and stimulating transcription at thyroid hormone-responsive gene loci.

GH-induced LH hyporesponsiveness as a potential mechanism for hypogonadism in male patients with acromegaly.

Male patients with acromegaly frequently have hypogonadism. However, whether excess GH affects gonadal function remains unclear. We retrospectively compared clinical features affecting total testosterone (TT) and free testosterone (FT) levels between 112 male patients with acromegaly and 100 male patients with non-functioning pituitary adenoma (NFPA) without hyperprolactinemia. Median maximum tumor diameter (14.4 vs. 26.5 mm) and suprasellar extension rate (33 vs. 100%) were lower in acromegaly, but LH, FSH, TT, and FT were not significantly different. In acromegaly, TT was less than 300 ng/dL in 57%, and FT was below the age-specific reference range in 77%. TT and FT were negatively correlated with GH, IGF-1, and the tumor size, and positively correlated with LH. In NFPA, they were positively correlated with IGF-1, LH, FSH, ACTH, cortisol, and free T4, reflecting hypopituitarism. Multiple regression analysis showed that TT and FT had the strongest correlation with GH in acromegaly, and with LH in NFPA. Surgical remission was achieved in 87.5% of 56 follow-up patients with acromegaly. TT and FT increased in 80.4 and 87.5%, respectively, with a significant increase in LH. In acromegaly, the degree of postoperative increase in TT(FT) correlated with the fold increase of TT(FT)/LH ratio, a potential parameter of LH responsiveness, but not with fold increase of LH, whereas in NFPA it correlated with both. These results suggest that excessive GH is the most relevant factor for hypogonadism in male acromegaly, and may cause impaired LH responsiveness as well as the suppression of LH secretion.

EID1 suppresses lipid accumulation by inhibiting the expression of GPDH in 3T3-L1 preadipocytes.

The imbalance between food intake and energy expenditure causes high accumulation of triglycerides in adipocytes. Obesity is related with the increased lipid accumulation in white adipose tissue, which is a major risk factor for the development of metabolic disorders, such as type 2 diabetes and cardiovascular disease. This study highlights the role of E1A-like inhibitor of differentiation 1 (EID1) in the modulation of adipogenesis through the downregulation of glycerol-3-phosphate dehydrogenase (GPDH), which is a key enzyme in the synthesis of triglycerides and is considered to be a marker of adipogenesis. By analyzing DNA microarray data, we found that when EID1 is overexpressed in preadipocytes (3T3-L1 cells) during adipocyte differentiation, EID1 inhibits lipid accumulation through the downregulation of GPDH. In contrast, EID1 is not involved in the regulation of intracellular glucose via the translocation of glucose transporter. A confocal image analysis showed that EID1 is located in the nucleus of preadipocytes in the form of speckles, which could be involved as a regulator of the transcriptional process. We further confirmed that EID1 is able to bind to the promoter sequence of GPDH in the nucleus. These findings provide a molecular explanation for the inhibitory effect of EID1 on lipid accumulation in adipocytes.

Dysfunction of the Cerebral Glucose Transporter SLC45A1 in Individuals with Intellectual Disability and Epilepsy.

Glucose transport across the blood brain barrier and into neural cells is critical for normal cerebral physiologic function. Dysfunction of the cerebral glucose transporter GLUT1 (encoded by SLC2A1) is known to result in epilepsy, intellectual disability (ID), and movement disorder. Using whole-exome sequencing, we identified rare homozygous missense variants (c.526C>T [p.Arg176Trp] and c.629C>T [p.Ala210Val]) in SLC45A1, encoding another cerebral glucose transporter, in two consanguineous multiplex families with moderate to severe ID, epilepsy, and variable neuropsychiatric features. The variants segregate with the phenotype in these families, affect well-conserved amino acids, and are predicted to be damaging by in silico programs. Intracellular glucose transport activity of the p.Arg176Trp and p.Ala210Val SLC45A1 variants, measured in transfected COS-7 cells, was approximately 50% (p = 0.013) and 33% (p = 0.008) lower, respectively, than that of intact SLC45A1. These results indicate that residues at positions 176 and 210 are critical for the glucose transport activity of SLC45A1. All together, our data strongly suggest that recessive mutations in SLC45A1 cause ID and epilepsy. SLC45A1 thus represents the second cerebral glucose transporter, in addition to GLUT1, to be involved in neurodevelopmental disability. Identification of additional individuals with mutations in SLC45A1 will allow better definition of the associated phenotypic spectrum and the exploration of potential targeted treatment options.

Secretory expression of thyroid hormone receptor using transgenic silkworms and its DNA binding activity.

Silkworms are economically important insects that have the ability to produce large amounts of silk. They have mass breeding methods and silk glands, which are specialized tissues that secrete silk fibroin and sericin. Thus, the production of recombinant proteins in a transgenic silkworm system is a promising approach. We developed a silkworm, Bombyx mori, as a host expression insect for recombinant proteins and successfully produced different proteins including antibodies, glycoproteins, and membrane receptors. The thyroid hormone receptor (TR) is a regulatory factor for many physiological phenomena. It is a lipophilic protein that has DNA-binding and ligand-binding domains. Based on our previous experiences, it was inferred that the recombinant TR easily formed aggregates and precipitates which is potentially due to an unstructured hinge domain. We applied the silkworm expression system to produce mice TRß1 that was fused with glutathione S-transferase. Using 160 larvae, the yield of the recombinant GST-TRß was approximately 4 mg, and the purified GST-TRß completely retained its physiological activity. Our results indicated that the recombinant TRß was secreted extracellularly using the silk fibroin signal peptide sequence. Moreover, we found that the expression system of silkworms was applicable to nuclear proteins.

Upregulated miR-224-5p suppresses osteoblast differentiation by increasing the expression of Pai-1 in the lumbar spine of a rat model of congenital kyphoscoliosis.

Congenital scoliosis is defined by the presence of structural anatomical malformations that arise from failures of vertebral formation or segmentation before and after birth. The understanding of genetic background and key genes for congenital scoliosis is still poor. We herein report that the excess expression of plasminogen activator inhibitor-1 (Pai-1) induced by the upregulation of miR-224-5p is involved in the pathogenesis of congenital kyphoscoliosis through impaired osteoblast differentiation. We first investigated the variety and progression of abnormalities of the lumbar spines in Ishibashi (IS) rats, a rat model of congenital kyphoscoliosis. The rats had already shown fusion and division of the primary ossification center at postnatal day 4. Over time, the rats showed various abnormalities of the lumbar spine, including the fusion of the annular epiphyseal nucleus. At postnatal day 42, spinal curvature was clearly observed due to the fusion of the vertebral bodies. Using a microRNA array, we found that the expression of miR-224-5p was increased in the lumbar spine of the rats at postnatal day 4. The expression of Pai-1, which is involved in osteoblast differentiation regulated by miR-224-5p, was also increased, while the levels of type I collagen, a marker of osteoblast differentiation, were decreased in the lumbar spine. These results indicate that the aberrant expression of miRNA-224-5p and its target genes is involved in the impaired osteoblast differentiation and may provide a partial molecular explanation for the pathogenesis of congenital scoliosis.

Chicken ovalbumin upstream promoter-transcription factor II protects against cisplatin-induced acute kidney injury.

The chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) plays essential roles in organogenesis of embryos. Recently COUP-TFII is also implicated in several diseases in adults. Here we focus on the role of COUP-TFII in cisplatin-induced acute kidney injury (AKI). COUP-TFII was the most abundantly expressed in the kidney among organs. Male tamoxifen-inducible COUP-TFII-knockout mice or control mice were intraperitoneally treated with 30 mg/kg body weight of cisplatin at 12 weeks old to induce AKI. The kidney samples were subject to morphological studies, terminal deoxynucleotidyl transferase-mediated deoxyuridine nick-end labeling (TUNEL) assay, immunohistochemistry and RT-qPCR. Serum levels of creatinine, blood urea nitrogen (BUN) and tumor necrosis factor alpha (TNF-α) were measured. Administration of cisplatin induced a more severe AKI in adult COUP-TFII-knockout mice. An increase in dead cells in both the proximal tubules and thick ascending limb of Henle's loop (TAL) was observed in the knockout mouse kidney. The expression levels of COUP-TFII decreased in the TAL by cisplatin administration. There was no difference in the expression levels of transporter mRNAs responsible for cellular cisplatin uptake between control and knockout mouse kidney. COUP-TFII-knockout mice and COUP-TFII-depleted cells exhibited an elevation in TNF-α levels, suggesting the involvement of the TNF-α pathway. Chromatin immunoprecipitation showed that COUP-TFII was enriched in the potential binding site, suggesting that COUP-TFII might directly suppress the TNF-α gene at transcriptional level. These results indicate the involvement of COUP-TFII in the pathophysiology of AKI and COUP-TFII may be a potential therapeutic target for AKI.

Maternal prolactin during late pregnancy is important in generating nurturing behavior in the offspring.

Although maternal nurturing behavior is extremely important for the preservation of a species, our knowledge of the biological underpinnings of these behaviors is insufficient. Here we show that the degree of a mother's nurturing behavior is regulated by factors present during her own fetal development. We found that Cin85-deficient (Cin85-/-) mother mice had reduced pituitary hormone prolactin (PRL) secretion as a result of excessive dopamine signaling in the brain. Their offspring matured normally and produced their own pups; however, nurturing behaviors such as pup retrieval and nursing were strongly inhibited. Surprisingly, when WT embryos were transplanted into the fallopian tubes of Cin85-/- mice, they also exhibited inhibited nurturing behavior as adults. Conversely, when Cin85-/- embryos were transplanted into the fallopian tubes of WT mice, the resultant pups exhibited normal nurturing behaviors as adults. When PRL was administered to Cin85-/- mice during late pregnancy, a higher proportion of the resultant pups exhibited nurturing behaviors as adults. This correlates with our findings that neural circuitry associated with nurturing behaviors was less active in pups born to Cin85-/- mothers, but PRL administration to mothers restored neural activity to normal levels. These results suggest that the prenatal period is extremely important in determining the expression of nurturing behaviors in the subsequent generation, and that maternal PRL is one of the critical factors for expression. In conclusion, perinatally secreted maternal PRL affects the expression of nurturing behaviors not only in a mother, but also in her pups when they have reached adulthood.

The retinol-retinoic acid metabolic pathway is impaired in the lumbar spine of a rat model of congenital kyphoscoliosis.

Although congenital scoliosis is defined as a genetic disease characterized by a congenital and abnormal curvature of the spinal vertebrae, our knowledge of the genetic underpinnings of the disease is insufficient. We herein show that the downregulation of the retinol-retinoic acid metabolism pathway is involved in the pathogenesis of congenital scoliosis. By analyzing DNA microarray data, we found that the expression levels of genes associated with the retinol metabolism pathway were decreased in the lumbar spine of Ishibashi rats (IS), a rat model of congenital kyphoscoliosis. The expression of Adh1 and Aldh1a2 (alcohol dehydrogenase), two enzymes that convert retinol to retinoic acid in this pathway, were decreased at both the gene and protein levels. Rarα, a receptor of retinoic acid and bone morphogenetic protein 2, which play a central role in bone formation and are located downstream of this pathway, were also downregulated. Interestingly, the serum retinol levels of IS rats were higher than those of wild-type control rats. These results indicate that the adequate conversion from retinol to retinoic acid is extremely important in the regulation of normal bone formation and it may also be a key factor for understanding the pathogenesis of congenital scoliosis.

Changes in biomarker levels and myofiber constitution in rat soleus muscle at different exercise intensities.

Although exercise affects the function and structure of skeletal muscle, our knowledge regarding the biomedical alterations induced by different intensities of exercise is incomplete. Here we report on the changes in biomarker levels and myofiber constitution in the rat soleus muscle induced by exercise intensity. Male adult rats at 7 weeks of age were divided into 3 groups by exercise intensity, which was set based on the accumulated lactate levels in the blood using a treadmill: stationary control (0 m/min), aerobic exercise (15 m/min), and anaerobic exercise (25 m/min). The rats underwent 30 min/day treadmill training at different exercise intensities for 14 days. Immediately after the last training session, the soleus muscle was dissected out in order to measure the muscle biomarker levels and evaluate the changes in the myofibers. The mRNA expression of citrate synthase, glucose-6-phosphate dehydrogenase, and Myo D increased with aerobic exercise, while the mRNA expression of myosin heavy-chain I and Myo D increased in anaerobic exercise. These results suggest that muscle biomarkers can be used as parameters for the muscle adaptation process in aerobic/anaerobic exercise. Interestingly, by 14 days after the anaerobic exercise, the number of type II (fast-twitch) myofibers had decreased by about 20%. Furthermore, many macrophages and regenerated fibers were observed in addition to the injured fibers 14 days after the anaerobic exercise. Constitutional changes in myofibers due to damage incurred during anaerobic exercise are necessary for at least about 2 weeks. These results indicate that the changes in the biomarker levels and myofiber constitution by exercise intensity are extremely important for understanding the metabolic adaptations of skeletal muscle during physical exercise.