The hypocretins (hcrts), also known as orexins, are two recently identified excitatory neuropeptides that in rat are produced by ϳ1200 neurons whose cell bodies are located in the lateral hypothalamus. The hypocretins/orexins have been implicated in the regulation of rapid eye movement (REM) sleep and the pathophysiology of narcolepsy. In the present study, we investigated whether the locus coeruleus (LC), a structure receiving dense hcrtergic innervation, which is quiescent during REM sleep, might be a target for hcrt to regulate REM sleep. Local administration of hcrt1 but not hcrt2 in the LC suppressed REM sleep in a dose-dependent manner and increased wakefulness at the expense of deep, slow-wave sleep. These effects were blocked with an antibody that neutralizes hcrt binding to hcrt receptor 1. In situ hybridization and immunocytochemistry showed the presence of hcrt receptor 1 but not the presence of hcrt receptor 2 in the LC. Iontophoretic application of hcrt1 enhanced the firing rate of LC neurons in vivo, and local injection of hcrt1 into the LC induced the expression of c-fos in the LC area. We propose that hcrt receptor 1 in the LC is a key target for REM sleep regulation and might be involved in the pathophysiological mechanisms of narcolepsy. Key words: norepinephrine; orexin; orexin receptors; c-fos; arousal; microinjection; immunocytochemistryThe hypocretins (hcrt1 and hcrt2), also called orexins, are two neuropeptides derived from the same precursor, which are expressed in a small set of neurons in the perifornical area of the hypothalamus Sakurai et al., 1998). The hypocretins are neuroexcitatory and bind to two different G-protein-coupled receptors, hcrt receptors 1 and 2 (hcrtr1 and hcrtr2, also known as OX1 and OX2 receptors) with different affinities (Sakurai et al., 1998). Recently, evidence has emerged that confirms a role for the hypocretins in arousal states. Lin et al. (1999) mapped the canine narcolepsy mutation (canarc-1) to hcrtr2. Knock-out experiments in mice demonstrated that the absence of hypocretin causes alterations in sleep architecture, particularly on the amount of rapid eye movement (REM) sleep during the dark period (Chemelli et al., 1999). In addition, hcrt-deficient mice display electroencephalographic patterns and behaviors that resemble those of narcoleptic attacks. Intracerebroventricular infusion of nanomolar amounts of hypocretin has recently been shown to increase arousal, reduce REM sleep, and affect neuroendocrine balance (Hagan et al., 1999). Nishino and colleagues (2000) found that seven of nine patients with narcolepsy had undetectable hcrt1 in CSF. These independent studies indicate that the hypocretins have a major role in the regulation of sleep, but the role of different brain structures and the contribution of each of the hcrt receptors remain unknown.The projections of hcrt-containing neurons extend widely throughout the brain Date et al., 1999). Four main hcrtergic afferent regions can be recognized from anatomical studies : an intrahypothalamic field...

The cellular influx and efflux of thyroid hormones are facilitated by transmembrane protein transporters. Of these transporters, monocarboxylate transporter 8 (MCT8) is the only one specific for the transport of thyroid hormones and some of their derivatives. Mutations in SLC16A2, the gene that encodes MCT8, lead to an X-linked syndrome with severe neurological impairment and altered concentrations of thyroid hormones. Histopathological analysis of brain tissue from patients who have impaired MCT8 function indicates that brain lesions start prenatally, and are most probably the result of cerebral hypothyroidism. A Slc16a2 knockout mouse model has revealed that Mct8 is an important mediator of thyroid hormone transport, especially T3, through the blood-brain barrier. However, unlike humans with an MCT8 deficiency, these mice do not have neurological impairment. One explanation for this discrepancy could be differences in expression of the T4 transporter OATP1C1 in the blood-brain barrier; OATP1C1 is more abundant in rodents than in primates and permits the passage of T4 in the absence of T3 transport, thus preventing full cerebral hypothyroidism. In this Review, we discuss the relevance of thyroid hormone transporters in health and disease, with a particular focus on the pathophysiology of MCT8 mutations.

The purpose of this review is to provide an up-to-date report on the molecular and physiologic processes involved in the role of thyroid hormone as an epigenetic factor in brain maturation. We summarize the available data on the control of brain gene expression by thyroid hormone, the correlation between gene expression and physiologic effects, and the likely mechanisms of action of thyroid hormone on brain gene expression. In addition we propose a role for unliganded thyroid hormone receptors in the pathogenesis of hypothyroidism. Finally, we review recent data indicating that thyroid hormone receptors have an impact on behavior.

Thyroid hormone (T3) controls critical aspects of cerebellar development, such as migration of postmitotic granule cells and terminal differentiation of Purkinje cells. T3 acts through nuclear receptors (TR) of two types, TR␣1 and TR␤, that either repress or activate gene expression. We have analyzed the cerebellar structure of developing mice lacking the TR␣1 isoform, which normally accounts for about 80% of T3 receptors in the cerebellum. Contrary to what was expected, granule cell migration and Purkinje cell differentiation were normal in the mutant mice. Even more striking was the fact that when neonatal hypothyroidism was induced, no alterations in cerebellar structure were observed in the mutant mice, whereas the wild-type mice showed delayed granule cell migration and arrested Purkinje cell growth. The results support the idea that repression by the TR␣1 aporeceptor, and not the lack of thyroid hormone, is responsible for the hypothyroid phenotype. This conclusion was supported by experiments with the TR␤-selective compound GC-1. Treatment of hypothyroid animals with T3, which binds to TR␣1 and TR␤, prevents any defect in cerebellar structure. In contrast, treatment with GC-1, which binds to TR␤ but not TR␣1, partially corrects Purkinje cell differentiation but has no effect on granule cell migration. Our data indicate that thyroid hormone has a permissive effect on cerebellar granule cell migration through derepression by the TR␣1 isoform.T he physiological actions of thyroid hormone (3,5,3Ј-triiodol-thyronine, T3), including the control of central nervous system (CNS) development, are mediated through interaction with nuclear receptors, which are ligand-modulated transcription factors (1). There are several T3 receptor proteins (TR), which are encoded by two distinct genes, TR␣ and TR␤. The TR␣ gene encodes three proteins, TR␣1, TRv␣2, and TRv␣3 that differ in their carboxyl terminus. From these, only TR␣1 is a bona fide receptor because it binds T3 and activates or represses target genes, whereas the two variants, TRv␣2 and TRv␣3, do not bind T3 and may antagonize T3 action (2-4). In addition to these protein isoforms, there are also truncated protein products of the ␣ gene known as ⌬␣1 and ⌬␣2, which may have a role in intestinal development (5). Several amino terminal protein variants are produced from the TR␤ gene: the two classical receptors, TR␤1 and TR␤2, and two newly identified, T3-binding proteins, TR␤3 and the truncated protein ⌬TR␤3 (6).Although the role of thyroid hormone in health and disease is well known, the physiological roles and specific functions of the T3 receptor isoforms still remain largely unidentified. Mutant mice have been generated that lack the expression of single or multiple products of the TR genes (7-9), and some discrete specific functions could be assigned to individual receptor isoforms. As a consequence of these studies, it is known that TR␤ is involved in the regulation of thyroid stimulating hormone (TSH) secretion, liver metabolism, and hearing (10, 11), whereas ...

Mutations of the gene expressing plasma membrane transporter for thyroid hormones MCT8 (SLC16A2) in humans lead to altered thyroid hormone levels and a severe neurodevelopmental disorder. Genetically engineered defect of the Mct8 gene in mice leads to similar thyroid hormone abnormalities but no obvious impairment of brain development or function. In this work we studied the relative role of the blood-brain barrier and the neuronal plasma cell membrane in the restricted access of T(3) to the target neurons. To this end we compared the effects of low doses of T(4) and T(3) on cerebellar structure and gene expression in wild-type (Wt) and Mct8 null male mice [Mct8-/y, knockout (KO)] made hypothyroid during the neonatal period. We found that compared with Wt animals, T(4) was considerably more potent than T(3) in the Mct8KO mice, indicating a restricted access of T(3), but not T(4), to neurons after systemic administration in vivo. In contrast, T(3) action in cultured cerebellar neurons was similar in Wt cells as in Mct8KO cells. The results suggest that the main restriction for T(3) entry into the neural target cells of the mouse deficient in Mct8 is at the blood-brain barrier.

Thyroid hormones influence brain development through the control of gene expression. The concentration of the active hormone T(3) in the brain depends on T(3) transport through the blood-brain barrier, mediated in part by the monocarboxylate transporter 8 (Mct8/MCT8) and the activity of type 2 deiodinase (D2) generating T(3) from T(4). The relative roles of each of these pathways in the regulation of brain gene expression is not known. To shed light on this question, we analyzed thyroid hormone-dependent gene expression in the cerebral cortex of mice with inactivated Mct8 (Slc16a2) and Dio2 genes, alone or in combination. We used 34 target genes identified to be controlled by thyroid hormone in microarray comparisons of cerebral cortex from wild-type control and hypothyroid mice on postnatal d 21. Inactivation of the Mct8 gene (Mct8KO) was without effect on the expression of 31 of these genes. Normal gene expression in the absence of the transporter was mostly due to D2 activity because the combined disruption of Mct8 and Dio2 led to similar effects as hypothyroidism on the expression of 24 genes. Dio2 disruption alone did not affect the expression of positively regulated genes, but, as in hypothyroidism, it increased that of negatively regulated genes. We conclude that gene expression in the Mct8KO cerebral cortex is compensated in part by D2-dependent mechanisms. Intriguingly, positive or negative regulation of genes by thyroid hormone is sensitive to the source of T(3) because Dio2 inactivation selectively affects the expression of negatively regulated genes.

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SFKs are frequently deregulated in cancer where they control cellular proliferation, migration, survival and metastasis. Here we study the role of SFKs catalytic activity in triple-negative/basal-like and metastatic human breast cancer MDA-MB-231 cells employing three well-established inhibitors: Dasatinib, PP2 and SU6656. These compounds inhibited migration and invasion. Concomitantly, they reduced Fak, paxillin, p130CAS, caveolin-1 phosphorylation and altered cytoskeletal structures. They also inhibited cell proliferation, but in different manners. Dasatinib and PP2 increased p27Kip1 expression and reduced c-Myc levels, restraining G1-S transition. In contrast, SU6656 did not modify p27Kip1 expression, slightly altered c-Myc levels and generated polyploid multinucleated cells, indicating inhibition of cytokinesis. These later effects were also observed in SYF fibroblasts, suggesting a SFKs-independent action. ZM447439, an Aurora B kinase inhibitor, produced similar cell cycle and morphological alterations in MDA-MB-231 cells, indicating that SU6656 blocked Aurora B kinase. This was confirmed by inhibition of histone H3 phosphorylation, the canonical Aurora B kinase substrate. Furthermore, hierarchical clustering analysis of gene expression profiles showed that SU6656 defined a set of genes that differed from Dasatinib and PP2. Additionally, Gene Set Enrichment Analyses revealed that SU6656 significantly reduces the Src pathway. Together, these results show the importance of SFKs catalytic activity for MDA-MB-231 proliferation, migration and invasiveness. They also illustrate that SU6656 acts as dual SFKs and Aurora B kinase inhibitor, suggesting its possible use as a therapeutic agent in breast cancer.