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.

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.

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.

Lactational exposure to perfluorooctane sulfonate remains a potential risk in brain function of middle-aged male mice.

Perfluorooctane sulfonate (PFOS) exerts adverse effects on neuronal development in young population. Limited evidences have shown that early-life PFOS exposure holds a potential risk for developing age-related neurodegenerative diseases such as Alzheimer's disease later in life. The present study investigated the effects of lactational PFOS exposure on cognitive function using one-year-old mice. Dams were exposed to PFOS (1 mg/kg body weight) through lactation by gavage. Male offspring were used for the behavior test battery to assess cognitive function. Western blot analysis was conducted to measure the levels of proteins related to the pathogenesis of Alzheimer's disease. PFOS-exposed mice displayed a mild deficiency in social recognition. In the hippocampus, the expression of tau protein was significantly increased. These results underline a mild effect of developing PFOS exposure on cognitive function and neurodegeneration. The present study presents the long-lasting effects of PFOS in middle-aged period and warrants a potential aftermath.

A Versatile, Behavioral Method to Investigate Thyroid Hormone Effects on Cerebellar Function.

Thyroid hormone (TH) action is essential during the development of the central nervous system, including the cerebellum. In case of TH deficiency in early life such as congenital hypothyroidism, patients display neurological disorders such as cognitive retardation and motor deficits. There are various studies using mouse models with tissue- or cell-specific TH deficiency to investigate the role of TH in the cerebellum. Compared to generalized congenital hypothyroid mice, cerebellar cell-specific TH-deficient mice display milder and subtler ataxic features, making the assessment of motor function difficult when using conventional tests such as the rotarod test. Due to the need for an alternative tool to assess motor function in TH-related animal models, we developed a versatile behavioral method called the "ladder beam test," in which we can design the various ladder tests depending on the severity of ataxia in model mice. We utilized transgenic mice expressing a dominant-negative TH receptor specifically in the cerebellar Purkinje cell, a sole output neuron in the cerebellar cortex modulating motor performance. The newly-built ladder beam test successfully detected robust impairments in motor performance in the transgenic mice at a greater level compared to the rotarod test. Disruption of motor learning was also detected in the ladder beam test but not in the rotarod test. The protocol with this novel behavioral apparatus can be applied to other animal models that may show mild ataxic phenotype to examine subtle changes in cerebellar function.

The neurotoxic effect of lactational PFOS exposure on cerebellar functional development in male mice.

Recent studies showed a possible association between perfluorooctane sulfonate (PFOS) and developmental disabilities. We previously found the specific effects of PFOS exposure on learning and memory, however, its effect on the other developmental disabilities such as motor and social deficits remains unclear. We examined the effect of early lactational PFOS exposure on motor coordination, social activity, and anxiety in male mice. We orally administered a PFOS solution to dams from postnatal day 1-14. At 10 weeks old, we conducted a behavior test battery to evaluate motor performance, social activity, and anxiety, followed by electrophysiology and Western blot analysis. PFOS-exposed mice displayed impaired motor coordination. Whole-cell patch-clamp recordings from Purkinje cells revealed that the short-term and long-term plasticity at parallel fiber-Purkinje cell synapses are affected by PFOS exposure. Western blot analysis indicated that PFOS exposure increased syntaxin binding protein 1 (Munc18-1) and glutamate metabotropic receptor 1 (mGluR1) protein levels, which may be associated with the change in neurotransmitter release from parallel fibers and the level of long-term depression, respectively. The present study demonstrates that lactational PFOS exposure may have disrupted the pre- and postsynaptic plasticity at parallel fiber-Purkinje cell synapses, causing profound, long-lasting abnormal effects on the cerebellar function.

Neurotoxic effects of lactational exposure to perfluorooctane sulfonate on learning and memory in adult male mouse.

The present study aims to examine the effect of early lactational perfluorooctane sulfonate (PFOS) exposures on learning and memory in male mice and reveal the underlying mechanisms involved. PFOS solution was orally administered to dams from the postpartum days 1-14, so that pups would be exposed through breast milk. At 8-10 weeks of age, we performed object location test (OLT), object recognition test (ORT), and pairwise visual discrimination (VD) task. We also performed in vivo microdialysis, and mRNA and protein analysis of genes responsible for hippocampal development and function. In both OLT and ORT, the performance of mice in the PFOS-exposed group was significantly lower than those in the control group. In the VD task, the PFOS-exposed group learned significantly slower than the control group. Concentrations of glutamate and gamma-aminobutyric acid in the dorsal hippocampus were significantly higher in the PFOS-exposed group than in the control group. No notable differences were shown in our mRNA and protein studies. The present study showed that lactational PFOS exposure has a profound, long-lasting neurotoxic effect in the hippocampus and consequently leads to learning and memory deficits.