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The N-methyl-D-aspartate receptor (NMDAR) is central to physiological and pathological functioning of neurons. Although promising results are beginning to be obtained in the treatment of dementias, clinical trials with NMDAR antagonists for stroke, trauma and neurodegenerative disorders, such as Hungtinton's disease, have failed before. In order to design effective therapies to prevent excitotoxic neuronal death, it is critical to characterize the consequences of excessive NMDAR activation on its expression and function. Previous data have reported partial downregulation of the NR1 and NR2B receptor subunits in response to excitotoxicity and cerebral ischemia. However, the effect of NMDAR overactivation on NR2A, a subunit fundamental to synaptic transmission and neuronal survival, is still elusive. In this study, we report the rapid and extensive proteolytic processing of NR2A, together with the scaffolding protein postsynaptic density-95 (PSD-95), induced by excitotoxic stimulation of cortical neurons in vitro and by transient focal cerebral ischemia. Processing of the C terminus of NR2A is irreversibly induced by brief agonist exposure of NR2B-containing receptors, and requires calcium influx and the activity of calpain, also responsible for PSD-95 cleavage. The outcome is a truncated NR2A subunit that is stable and capable to interact with NR1 at the surface of neurons, but lacking the structural domains required for association with scaffolding, downstream signaling and cytoskeletal proteins. Therefore, a rapid and significant uncoupling of synaptic NMDARs from downstream survival pathways is expected to occur during ischemia. This novel mechanism induced by excitotoxicity helps to explain the failure of most therapies based on NMDAR antagonists. © 2008 Nature Publishing Group All rights reserved., This work was supported by grant SAF2003-02327 (MD-G) from ‘Dirección General de Investigación Científica y Técnica’ (Ministerio de Ciencia y Tecnología). MD-G is a researcher of the Ramón y Cajal Program. SG is a recipient of a predoctoral fellowship from Comunidad de Madrid. We thank Dr T Yamamoto for the generous gift of pME18S-HA-NR2A and pME18S-HA-NR2B, and Dr J Luo for plasmid YFPNR2B.

Double and triple immunocytochemistry with stage-specific markers and specific antireceptor antibodies was used to study expression of nuclear thyroid hormone receptor (TR) isoforms in cultured mouse oligodendrocytes. To evaluate the coexpression of each TR isoform, antibodies were raised in rabbits and mice against specific regions of alpha1-TR and alpha2-TR common to both alpha isoforms and beta1-TR. Their specificities were assessed by Western blotting and by immunocytochemistry on rat hepatocytes. Oligodendrocyte subpopulations were found to coexpress the alpha- and beta1-TR epitopes at defined developmental stages. Both alpha- and beta1-TR isoforms are colocalized in oligodendrocytes during an early stage identified by the marker OL-1, before 2',3'-cyclic nucleotide 3'-phosphohydrolase is expressed. Expression of beta1-TR varies during maturation, and that of alpha-TR decreases during terminal maturation.

Mitochondrial respiratory function requires the expression of genes both from the mitochondrial and nuclear genomes. Nuclear respiratory factor 2 (NRF-2) is a transcription factor required for the expression of several nuclear-encoded mitochondrial proteins, including the specific mitochondrial transcription factor Tfam. This makes NRF-2 a likely candidate to coordinate expression of mitochondrial components. NRF-2 is a multisubunit complex of which the α subunit binds DNA and the β subunit enhances this binding, respectively. We have analysed in vivo the expression patterns of NRF-2 subunits both at the mRNA and protein level, in three rat tissues, liver, testis, and brain. In contrast with Tfam or the 'housekeeping' β-actin expressions in which a parallel gradient was observed, no correlation was found between NRF-2 mRNAs and proteins levels, thus suggesting post-transcriptional regulation. © 2000 Société française de biochimie et biologie moléculaire /Éditions scientifiques et médicales Elsevier SAS., This work was supported by grant PM97-0066 from the Dirección General de Enseñanza Superior e Investigación Científica (Ministerio de Educación y Cultura, Spain). H.E. was supported by a collaboration contract between CSIC and Biotools SA.

Mitochondrial function requires genes encoded in both mitochondrial and nuclear genomes. Tfam, the activator of mammalian mitochondrial transcription, is encoded in the nucleus and its expression has been shown in in vitro studies to be controlled by nuclear respiratory factors NRF-1 and NRF-2. In order to understand the physiological dependence of mitochondrial gene expression, we have analyzed in rat liver, testis and brain the expression level of mitochondrial genes in parallel with those of the three transcription factors. We found that: a) Tfam expression is down-regulated in rat testis, both at the protein and transcript level. The three-fold reduction in the abundance of Tfam protein in rat testis does not result in low steady-state levels of mitochondrial gene transcripts, suggesting that Tfam is in excess and does not limit transcription in vivo; and b) NRF-1 and NRF-2 (α, β and γ subunits) mRNAs were analyzed by Northern blotting; for each mRNA, several transcripts were observed as well as tissue-specific patterns of expression. The mRNA steady-state levels of NRF-1 and NRF-2 were higher in testis than in liver or brain. These data suggest that the low expression level of Tfam found in testis is not due to decreased NRF-1 and/or NRF-2 expression and further suggest the existence of tissue-specific post-transcriptional regulatory mechanisms for the expression of NRF-1/NRF-2 genes., This work was supported by grants, APC 97-0005 (C.G.V.) and PB7-0066 (A.R.P. and C.G.V.) from the Dirección General de Investigación Científica y Técnica (Ministerio de Educación y Cultura, Spain).

Thyroid hormone plays an important role in brain development and is essential to ensure a normal myelination. The effects of thyroid hormone are mediated by nuclear thyroid hormone receptors, which act as ligand-regulated transcription factors. There are several isoforms encoded by two genes, alpha and beta. Developmental studies have shown that alpha isoforms are widely expressed in the fetal brain, while beta isoforms expression is more restricted with a dramatic increase that begins at birth in the rat. Remarkably, receptor number reaches maximal levels by postnatal day 10, when serum thyroid hormone levels also peak and myelination is the most prominent event in the developing rat brain. Likewise, oligodendrocyte precursor cells express alpha isoforms and expression of the beta isoforms is confined to the differentiated oligodendrocytes, suggesting that these isoforms might mediate different thyroid hormone effects in the oligodendrocyte lineage. Thyroid hormone acts at multiple steps in the development of oligodendrocytes: (a) Early in development, it can function as an instructive signal for the generation of oligodendrocytes and enhance the proliferation of the committed preprecursor oligodendrocyte cells. (b) Thyroid hormone regulates the number of oligodendrocyte generated by directly promoting their differentiation. Since oligodendrocytes are produced in vitro after the same period in culture regardless of whether thyroid hormone was added to the cultures, it has been suggested that thyroid hormone is required for neither the timing nor the generation of oligodendrocytes, but is necessary to achieve adequate oligodendrocyte numbers. (c) Finally, thyroid hormone increases morphological and functional maturation of postmitoitic oligodendrocytes by stimulation of the expression of various myelin genes.

In the mammalian brain, thyroid hormones regulate myelination. Their actions are mediated by interactions with nuclear receptors that function as ligand-regulated transcription factors. Two genes, alpha and beta, encode different isoforms, of which only the beta and alpha1 isoforms are authentic nuclear triiodothyronine (T3)-receptors (NT3R). In agreement with the important role of T3 on myelination and oligodendrocyte generation, the presence of NT3Rs has been reported in oligodendrocytes and their precursors. We and others have shown that both progenitors and oligodendrocytes in vitro express the alpha1 and alpha2 isoforms, but the expression of the beta1 isoform is confined to differentiated oligodendrocytes, suggesting that they have different functions. To establish if this is the case during development in vivo, we have studied NT3R isoform expression in glial cells isolated by density gradient centrifugation from rat brains of various ages. We report the presence of the alpha1 NT3R and its variant alpha2, but not that of the beta1 isoform, in newborn rat glial progenitors. The pattern of expression of beta1, both at the level of mRNA and protein, parallels the increase in the number of oligodendrocytes. We found a significant change in the kinetic parameters of [125I]-T3 binding to NT3Rs in these cells during the first month of life, consisting of an increase in the binding capacity that peaks with myelination, and a significative decrease in Kd that coincides with the switch from the alpha to the beta1 isoform. Thus, the expression of NT3R isoforms in the rat oligodendrocyte lineage changes radically from the alpha to the beta1 isoform during the period when oligodendrocytes differentiate from progenitors.

We have been studying oligodendrocyte generationin vitroto obtain insights into how the timely generation of these cells might be regulated. Our studies suggest the existence of timing mechanisms quite different from those of existing models, wherein it is proposed that timely oligodendrocyte generation is associated with synchronous and symmetric differentiation controlled by cell-intrinsic biological clocks. Our results are most consistent with the hypothesis that the propensity of a clone of dividing oligodendrocyte type-2 astrocyte (O-2A) progenitors initially to generate at least one oligodendrocyte may be regulated by cell-intrinsic mechanisms, but that cell-extrinsic signals regulate the extent of further oligodendrocyte generation. In cultures of embryonic rat cortex grown in the presence of platelet-derived growth factor (PDGF), oligodendrocytes appeared in a timely manner in the absence of clonal differentiation. In contrast with previous suggestions, the presence or absence of thyroid hormone (T3) did not alter the probability of individual clones of O-2A progenitors generating at least one oligodendrocytein vitroat a time equal to the rat's day of birth. Instead, T3 increased the proportion of oligodendrocytes generated within clones. For postnatally derived progenitor cells, the initial appearance of oligodendrocytes also was followed by further asymmetric generation of these cells, with the ratio of progenitors to oligodendrocytes within clones being regulated by environmental signals. T3 and ciliary neurotrophic factor increased oligodendrocyte generation, while neurotrophin-3 (NT-3) suppressed oligodendrocyte generation. Also in contrast to previous reports, NT-3 was not required for the promotion of extensive division of O-2A progenitor cells by PDGF.

This work is licensed under the Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 Unported License.-- et al., A better understanding of the mechanisms underlying neuronal death in cerebral ischemia is required for the development of stroke therapies. Here we analyze the contribution of the tropomyosin-related kinase B (TrkB) neurotrophin receptor to excitotoxicity, a primary pathological mechanism in ischemia, which is induced by overstimulation of glutamate receptors of the N-methyl-D-aspartate type. We demonstrate a significant modification of TrkB expression that is strongly associated with neurodegeneration in models of ischemia and in vitro excitotoxicity. Two mechanisms cooperate for TrkB dysregulation: (1) calpain-processing of full-length TrkB (TrkB-FL), high-affinity receptor for brain-derived neurotrophic factor, which produces a truncated protein lacking the tyrosine-kinase domain and strikingly similar to the inactive TrkB-T1 isoform and (2) reverse regulation of the mRNA of these isoforms. Collectively, excitotoxicity results in a decrease of TrkB-FL, the production of truncated TrkB-FL and the upregulation of TrkB-T1. A similar neuro-specific increase of the TrkB-T1 isoform is also observed in stroke patients. A lentivirus designed for both neuro-specific TrkB-T1 interference and increased TrkB-FL expression allows recovery of the TrkB-FL/TrkB-T1 balance and protects neurons from excitotoxic death. These data implicate a combination of TrkB-FL downregulation and TrkB-T1 upregulation as significant causes of neuronal death in excitotoxicity, and reveal novel targets for the design of stroke therapies., This work was supported by Grants BFU2007-67695 and BFU2010-18380 from Ministerio de Ciencia e Innovación (MICINN), PIE 200420E601 and 200720I012 from CSIC to MD-G, and BFU2004-01498 to AR-P.

RC3 encodes a thyroid hormone-dependent, calmodulin-binding, protein kinase C substrate (neurogranin, p17) present in the dendritic spines of discrete neuronal populations in the forebrain. Its physiological role could be related to synaptic plasticity, memory, and other processes. In the present work we have isolated and sequenced 2.4 kbp of genomic DNA upstream from the origin of transcription and determined its nucleotide sequence. The major features of the RC3 promoter are the absence of TATA and CAAT boxes and the presence of an Initiator sequence surrounding the cap site. By sequence analysis we identified several cis-acting regulatory elements, among them response elements for retinoic acid and steroid (glucocorticoids/progesterone) hormone receptors. An oligonucleotide containing the retinoic acid responsive element bound to retinoic acid receptors specifically in vitro and conferred retinoic acid regulation to a heterologous promoter after transfection in COS-7 cells. Retinoic acid and dexamethasone, respectively, increased activity of the RC3 promoter in neuroblastoma cells when a deletion construct containing the retinoic acid and the glucocorticoid responsive elements was cotransfected with retinoic acid receptor or glucocorticoid receptor expression vectors. When added together all-trans retinoic acid and dexamethasone had additive effects. Despite the fact that RC3 expression in vivo is thyroid hormone-dependent, no evidence for the presence of a thyroid hormone responsive element was found within the 2.4 kbp flanking region analyzed and thyroid hormone did not increase reporter activity after cotransfection of suitable constructs with thyroid hormone receptor expression vectors. Our results suggest that the expression of RC3 in vivo could be subject to complex physiological signals, including retinoids and steroid hormones in addition to thyroid hormones., This work has been supported by grants from DGICYT PM92-0025, FIS 94/0273, Fundación Salud 2000, and NIH grant NS 22111.

We have previously described several cDNA clones whose expression is affected by thyroid hormone during rat brain development. We now report the identification of one of these, the E2 clone, as the brain-specific prostaglandin D2 (PGD2) synthetase gene. Sequence comparison shows a nearly complete identity between the 356 nucleotides of the E2 clone and nucleotides 403 to 759 of PGD2 synthetase cDNA. The pattern of E2 expression corresponds to that expected for brain specific PGD2 synthetase gene, i.e. the corresponding mRNA is not detected in any other tissue analyzed apart of the brain, and it was present at different levels in all brain regions. Hypothyroidism decreased E2 mRNA concentrations in cerebral cortex and cerebellum. Control of the level of expression of PGD2 synthetase gene may contribute the complex effects of thyroid hormone on brain development and function., This work was supported by grants from the DGICYT (PM92-0025 and SAF92/0396) and Fundación Ramón Areces.

RC3 is a brain-specific mRNA expressed in discrete neuronal groups of the forebrain that encodes a 78-amino acid protein, also called neurogranin, a calmodulin-binding, protein kinase-C substrate. Expression of RC3 mRNA was studied in normal and hypothyroid animals during the first month of life. Hypothyroid rats were produced by administration of methyl-mercapto-imidazol to the pregnant dams and subsequent surgical thyroidectomy on postnatal day 5 of the neonates. As studied by slot-blotting of total cerebrum poly(A)+ RNA, RC3 mRNA accumulates in normal brain from the fifth to seventh postnatal day, reaching maximal levels around days 10-12. RC3 mRNA accumulation in hypothyroid animals was blunted, and the maximal levels attained were about 30-50% of normal values. The effect of hypothyroidism on steady state mRNA levels was also observed by Northern blotting of RNA from cerebral cortex and striatum. As studied by immunoblotting using a polyclonal antibody, hypothyroidism also led to clear decreases in the amount of the RC3 protein in extracts from cerebral cortex, striatum, and hippocampus. A single administration of 10 μg T4 to hypothyroid rats on postnatal day 12 led to a steady increase in striatal RC3 mRNA from levels that were about 40% of normal to about 70% of normal at 16 h and 115% of normal at 48 h. In contrast to the effect on RC3, hypothyroidism did not affect developmental expression of the mRNA encoding GAP-43, another brain protein kinase-C substrate of axonal localization. RC3 is, thus, one of the few known neuronal genes whose expression is influenced by thyroid hormone in the brain. Thyroid hormone is required for an appropriate level of expression, not for the developmentally programmed timing of expression of the RC3 gene.

12 pages, 7 figures., Functional and protein interactions between the N-methyl-D-aspartate type of glutamate receptor (NMDAR) and neurotrophin or ephrin receptors play essential roles in neuronal survival and differentiation. A shared downstream effector for neurotrophin- and ephrin-receptor signaling is kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS). Because this molecule is obligatory for neurotrophin-induced differentiation, we investigated whether Kidins220/ARMS and NMDAR functions were related. Here, we identify an association between these proteins and discover that excitotoxicity, a specific form of neuronal death induced by NMDAR overstimulation, dramatically decreases Kidins220/ARMS levels in cortical neurons and in a model of cerebral ischemia. Kidins220/ARMS downregulation is triggered by overactivation of NMDARs containing NR2B subunits and subsequent Ca2+ influx, and involves a dual mechanism: rapid cleavage by the Ca2+-dependent protease calpain and calpain-independent silencing of Kidins220/Arms gene transcription. Additionally, Kidins220/ARMS knockdown decreases ERK activation and basal neuronal viability, and enhances neuronal death under excitotoxic conditions. Our results demonstrate Kidins220/ARMS participation in neuronal life and death pathways, and constitute the first report of its regulation under pathological conditions., This work was supported by grants BFU2007-67695 from `Ministerio de Ciencia e Innovación and `Fundación Mutua Madrileña' to M.D.-G.; and SAF2008-01951 from `Ministerio de Ciencia e Innovación', CAM S-SAL-0202-2006-01 from `Comunidad de Madrid' and CIBERNED from `Instituto de Salud Carlos III' to T.I. C.L.-M. and S.G. were recipients of a research contract and a predoctoral fellowship, respectively, funded by `Comunidad de Madrid'.

Understanding the molecular mechanisms that control cell proliferation requires the identification of the early signals important for initiating a mitogenic response. In this context, the activation of Ca2+-sensitive, phospholipid-dependent protein kinase (protein kinase C), which is stimulated by diacylglycerols and serves as a major phorbol ester receptor, may play an important part in signalling mitogenesis. This conclusion is based on two main lines of evidence. Firstly, activation of protein kinase C in intact quiescent fibroblasts is one of the earliest events elicited by a variety of growth-promoting agents including serum, platelet-derived growth factor (PDGF), vasopressin and bombesin, as judged by the increase in the phosphorylation of a cellular protein characterized by an Mr of 80 000 and a pI of 5. Secondly, the synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol, which directly competes with [3H]phorbol dibutyrate for binding to specific receptors in intact 3T3 cells and rapidly stimulates protein kinase C in these cells, is a potent mitogen for Swiss 3T3 cells, acting synergistically with other growth factors. We propose that activation of protein kinase C may be one of the early signals that mediate the mitogenic effects of a variety of growth factors and peptide hormones in quiescent fibroblastic cells.

The N-methyl-D-aspartate (NMDA) type of glutamate receptor (NMDAR) plays central roles in normal and pathological neuronal functioning. We have examined the regulation of the NR1 subunit of the NMDAR in response to excessive activation of this receptor in in vitro and in vivo models of excitotoxicity. NR1 protein expression in cultured cortical neurons was specifically reduced by stimulation with 100 μM NMDA or glutamate. NMDA decreased NR1 protein amounts by 71% after 8 h. Low NMDA concentrations (≤10 μM) had no effect. NR1 down-regulation was inhibited by the general NMDAR antagonist DL-AP5 and also by ifenprodil, which specifically antagonizes NMDARs containing NR2B subunits. Arrest of NMDAR signaling with DL-AP5 after brief exposure to NMDA did not prevent subsequent NR1 decrease. Down-regulation of NR1 did not involve calpain cleavage but resulted from a decrease in de novo synthesis consequence of reduced mRNA amounts. In contrast, NMDA did not alter the expression of NR2A mRNA or newly synthesized protein. In neurons transiently transfected with an NR1 promoter/luciferase reporter construct, promoter activity was reduced by 68% after 2 h of stimulation with NMDA, and its inhibition required extracellular calcium. A similar mechanism of auto-regulation of the receptor probably operates during cerebral ischemia, because NR1 mRNA and protein were strongly decreased at early stages of blood reperfusion in the infarcted brains of rats subjected to occlusion of the middle cerebral artery. Because NR1 is the obligatory subunit of NMDARs, this regulatory mechanism will be fundamental to NMDAR functioning. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc., This work was supported by Grants SAF2003-02327 (to M. D.-G.), SAF2001-1624 (to A. R.-P.) and PM99-0115 (to J. R.) from the “Dirección General de Investigación Científica y Técnica” (Ministerio de Ciencia y Tecnología).

An open issue in research on ageing is the extent to which responses to the environment during development can influence variability in life span in animals, and the health profile of the elderly in human populations. Both affluence and adversity in human societies have profound impacts on survivorship curves, and some of this effect may be traceable to effects in utero or in infancy. The Barker Hypothesis that links caloric restriction in very early life to disruptions of glucose-insulin metabolism in later life has attracted much attention, as well as some controversy, in medical circles. It is only rarely considered by evolutionary biologists working on phenotypic plasticity, or by biogerontologists studying model organisms such as C. elegans or Drosophila. One crucial mechanism by which animals can respond in an adaptive manner to adverse conditions, for example in nutrition or infection, during development is phenotypic plasticity. Here we begin with a discussion of adaptive plasticity in animals before asking what such phenomena may reveal of relevance to rates of ageing in animals, and in humans. We survey the evidence for effects on adult ageing of environmental conditions during development across mammalian and invertebrate model organisms, and ask whether evolutionary conserved mechanisms might be involved. We conclude that the Barker Hypothesis is poorly supported and argue that more work in human populations should be integrated with multi-disciplinary studies of ageing-related phenomena in experimental populations of different model species that are subjected to nutritional challenges or infections during pre-adult development.

Thyroid hormone (T3) regulates brain development and function and in particular ensures normal myelination. Animal models and in vitro systems have been employed to demonstrate the effects of T3, which acts via nuclear hormone receptors. T3 receptors (TRs) are transcription factors that activate or suppress target gene expression, such as myelin basic protein (MBP), in a hormone-dependent or -independent fashion. Two distinct genes, TRα and TRβ, encode several receptor isoforms with specific functions. This overview summarizes current knowledge on the cellular expression and the role of these isoforms and also examines the action of T3 on oligodendrocyte lineage cell types at defined developmental stages. Re-expression of TRs and also that of other transcription factors in oligodendrocytes may constitute some of the metabolic changes required for succesful remyelination in the adult central nervous system after demyelinating lesions., We thank ARSEP (Association pour la Recherche sur la Sclérose en Plaques), the Spanish Ministry of Science and Technology (Grant No. SAF2001-1624 to A.R-P.), and the “Accord INSERM/CSIC.”

trkB as receptor for neurotrophins brain-derived neurotrophic factor (BDNF)/neurotrophin (NT)-4/5 plays a crucial role during development, maintenance of the adult brain, and its adaptation to injury or pathological conditions. In spite of this, very little is known about the mechanisms that regulate its expression. Here, we show that forskolin (Fk) rapidly stimulates the expression of both the full-length and truncated trkB isoforms in primary cultures of cortical neurons. Gel shift assays and transient transfection experiments demonstrate that this activation occurs via a protein kinase A (PKA)/cyclic AMP-responsive element-binding protein (CREB)-dependent mechanism. Activated CREB binds to the second cyclic AMP (cAMP)-responsive element (CRE) of the two CRE sites located within the P2 promoter of the trkB gene, which is able to confer cAMP responsiveness to a heterologous promoter. Our results illustrate that the trkB gene is a target for CREB regulation and explain the increase of trkB expression produced in different adaptative responses of the nervous system where CREB is participating. © 2004 Elsevier Inc. All rights reserved., This work was supported by grant to A.R.-P., from “Dirección General de Investigación Cientı́fica y Técnica” (Ministerio de Ciencia y Tecnologı́a: SAF2001-1624). R.D. and G.E. were supported by predoctoral fellowships from “Ministerio de Ciencia y Tecnologı́a” and “Comunidad Autónoma de Madrid,” respectively.

Pleiotrophin (PTN) is a platelet-derived growth factor-inducible, 18-kDa heparin-binding cytokine that signals diverse phenotypes in normal and deregulated cellular growth and differentiation. To seek the mechanisms of PTN signaling, we studied the interactions of PTN with the receptor protein tyrosine phosphatase (RPTP) beta/zeta in U373-MG cells. Our results suggest that PTN is a natural ligand for RPTP beta/zeta. PTN signals through "ligand-dependent receptor inactivation" of RPTP beta/zeta and disrupts its normal roles in the regulation of steady-state tyrosine phosphorylation of downstream signaling molecules. We have found that PTN binds to and functionally inactivates the catalytic activity of RPTP beta/zeta. We also have found that an active site-containing domain of RPTP beta/zeta both binds beta-catenin and functionally reduces its levels of tyrosine phosphorylation when added to lysates of pervanidate-treated cells. In contrast, an (inactivating) active-site mutant of RPTP beta/zeta also binds beta-catenin but fails to reduce tyrosine phosphorylation of beta-catenin. Finally, in parallel to its ability to inactivate endogenous RPTP beta/zeta, PTN sharply increases tyrosine phosphorylation of beta-catenin in PTN-treated cells. The results suggest that in unstimulated cells, RPTP beta/zeta is intrinsically active and functions as an important regulator in the reciprocal control of the steady-state tyrosine phosphorylation levels of beta-catenin by tyrosine kinases and phosphatases. The results also suggest that RPTP beta/zeta is a functional receptor for PTN; PTN signals through ligand-dependent receptor inactivation of RPTP beta/zeta to increase levels of tyrosine phosphorylation of beta-catenin to initiate downstream signaling. PTN is the first natural ligand identified for any of the RPTP family; its identification provides a unique tool to pursue the novel signaling pathway activated by PTN and the relationship of PTN signaling with other pathways regulating beta-catenin.

We have analysed a 7-kb region upstream of the mouse trkB coding sequence. The region showed promoter activity in transient transfection experiments and conferred tissue-specific expression to a reporter gene. Deletion analysis of this region demonstrated the presence of two alternative promoters named P1 and P2 that have been mapped by RNase protection. P1 has been located to 1.8 kb and P2 to 0.5 kb upstream of the trkB translation start site. From the P1 promoter, alternative splicing generates various transcripts. Interestingly, P2 is located in an intron of the transcripts produced from the P1 promoter. This peculiar arrangement results in different mRNA species that encode the same protein(s) but differ in their 5'-untranslated regions. In addition, transcription of the trkB locus results in two different trkB isoforms (kinase and truncated receptors) originated by alternative splicing of the mRNA, that possess differential spatial and temporal expression patterns. Using RT-PCR, we demonstrated that there was no linkage between promoter usage and alternative splicing, since transcripts initiated from each promoter encoded both kinase and truncated receptor proteins. Copyright (C) 1999 Elsevier Science B.V., This work has been supported by grants from Dirección General de Enseñanza Superior (PB94-0090 and PM97-0066 to A.R.-P. and PM96-0073 to D.B.).

Detergent-insoluble glycosphingolipid-enriched membranes (DIGs) have been involved in the sorting and transport of specific proteins during oligodendrocyte maturation. The MAL (MAL, MVP17, VIP17) proteolipid, an integral membrane protein present in DIGs in mature oligodendrocytes, has been proposed as a component of the machinery for DIG-mediated transport in a restricted pattern of cell types including myelinating cells. We have previously shown that thyroid hormone regulates the expression of the myelin protein genes coordinately, and have suggested a major role for thyroid hormone in the control of oligodendrocytes generation. Here we show that the expression of the MAL gene is down-regulated by hypothyroidism and up- regulated by hyperthyroidism in myelinated regions of the brain. In contrast, adult-onset hypothyroidism has no effect on the steady-state levels of MAL mRNA. Taken together, our results show that MAL expression during oligodendrocyte maturation is modulated by thyroid hormone, suggesting that this hormone could play an important role in the myelin biogenesis during neonatal development., Funded by Fondo Investigaciones Sanitarias. Grant Number: FIS 94/0283; DGICYT. Grant Number: PB94–0090; Dirección General de Enseñanza Superior. Grant Number: PM96–0004 and Fundación Ramón Areces.

It is well known the need of systemic thyroid hormone for the normal central nervous system (CNS) development. One of the most striking features of the hypothyroid brain is a reduction in the total myelin content. This effect is correlated with a decrease in the amount of the myelin protein transcripts. Since the expression of the myelin protein genes is confined to the differentiated oligodendrocytes generated from their progenitors, the O-2A cells, we proposed that the major action of thyroid hormone on myelination is the promotion of the oligodendrocyte differentiation, leading to a correlation between the relative levels of myelin transcripts with the number of oligodendrocytes formed. In order to find out if this is the case, we have studied the cell composition in the optic nerve of eu- and hypothyroid rats. We have found that the oligodendrocyte number was reduced 50% in hypothyroid animals, but the number of progenitors and astrocytes were unchanged. Next, to obtain insights into how the timely generation of these cells is regulated by thyroid hormone (T3) we have used an “in vitro” model of oligodendrocyte differentiation from pure cultures of O2A progenitor cells. Our results show that T3 decreases the division of the O-2A progenitors and increases their differentiation to oligodendrocyte. In contrast with the existing model, the oligodendrocyte generation is associated with asymmetric differentiation controlled by cell-extrinsic signals, and their capability to generate at least one oligodendrocyte “in vitro” at a time equal to the rat’s day of birth is independent of T3 and seems to be a cell-intrinsic property.

To assess the role of thyroid hormone on myelin gene expression, we have studied the effect of hypothyroidism on the mRNA steady state levels for the major myelin protein genes: myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG) and 2′:3′-cyclic nucleotide 3′-phosphodiesterase (CNP) in different rat brain regions, during the first postnatal month. We found that hypothyroidism reduces the levels of every myelin protein transcript, with striking differences between the different brain regions. Thus, in the more caudal regions, the effect of hypothyroidism was extremely modest, being only evident at the earlier stages of myelination. In contrast, in the striatum and the cerebral cortex the important decrease in the myelin protein transcripts is maintained beyond the first postnatal month. Therefore, thyroid hormone modulates in a synchronous fashion the expression of the myelin genes and the length of its effect depends on the brain region. On the other hand, hyperthyroidism leads to an increase of the major myelin protein transcripts above control values. Finally, lack of thyroid hormone does not change the expression of the oligodendrocyte progenitor-specific gene, the platelet derived growth factor receptor α.

The levels of neurotrophins and their receptor mRNAs were measured in rat sciatic nerve, after 6 or 12 weeks of streptozotocin-induced diabetes. Expression of neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) was decreased by 50 and 29%, respectively, compared with age-matched controls after 12 weeks of diabetes. Expression of brain-derived neurotrophic factor (BDNF) was not detected. In addition, diabetes induced a reduction in the expression levels of the neurotrophin receptors; trk B mRNA decreased by 50% after 6 weeks of diabetes, but returned to control levels after 12 weeks; meanwhile the trk C and the p75 LNGFR transcripts were reduced by 20% of control at both times studied. trk A expression was below detection limits. Thus, these data suggest that a reduction in neurotrophin and neurotrophin receptors could contribute to the development and maintenance of diabetic neuropathy.

Thyroid hormone deficiency has dramatic effects on rat brain maturation. The expression of genes encoding neurotrophins and the trk family of neurotrophin receptors has been evaluated in several brain regions of normal and of neonatal or adult hypothyroid rats to analyze whether they are subject to thyroid hormone action. We found that hypothyroidism decreased trk mRNA levels in its major site of expression, the striatum, on postnatal days 5 (P5; 45%) and 15 (P15; 25%) and also in adults (35%). In contrast, no differences in trkB or trkC mRNAs levels were observed in any brain region at studied ages. According to previous reports, p75LNGFR mRNA was elevated in hypothyroid cerebellum as compared to age-matched controls on P5 and P15. We have also observed a distinct pattern for neurotrophin genes. The level of NGF mRNA was 20-50% lower in the cortex, hippocampus, and cerebellum of hypothyroid rats on neonatal hypothyroid rats on P15 and also after adult-onset hypothyroidism. Treatment of neonatally-induced hypothyroid rats with a single injection of triiodothyronine led to the recovery of hippocampal but not cortex NGF mRNA levels to that of control animals. On the contrary, no differences in the relatively high expression of the two mRNAs encoding BDNF were observed in any brain area. In contrast to a recent report, we did not find a reduction in brain NT-3 mRNA levels in hypothyroid animals. If any, the effect of thyroid deficiency in the hippocampus and cortex seems to be an early upregulation of NT-3 expression. In summary, our results support a region-specific developmental regulation by thyroid hormone of the expression of particular neurotrophic factors and their receptors in the rat brain. The observed alterations may contribute to the abnormal brain maturation caused by hypothyroidism., This work was supported by Grants from the Plan Nacional de I + D (SAF92/0396) and Fundación Ramón Areces.

The proteolipid protein (PLP) gene codes for the major central nervous system myelin protein. We have studied the effects of different agents on the expression of the PLP gene in C6 glioma cells. Retinoic acid (RA), but not dexamethasone, estradiol, insulin, growth hormone, or vitamin D3, had a drastic effect, increasing 10-20-fold the level of PLP mRNA. Concomitantly, RA also induced the appearance of the corresponding immunoreactive protein. The increase in PLP RNA level showed a slow kinetics and was blocked by cycloheximide, suggesting a posttranscriptional regulation by RA. Nuclear run-on assays confirmed that the rate of PLP gene transcription was unchanged by RA. In contrast, we found that retinoic acid augmented PLP mRNA stability, causing a substantial increase in its half-life. RA action was independent of cell density, serum, or PDGF but was partially inhibited by bFGF. On the other hand, thyroid hormone caused a moderate increase in PLP mRNA levels in C6 cells but only when the low numbers of thyroid receptors in these cells were increased by retrovirally mediated expression of an exogenous c-erbA/TR alpha-1 gene. Our results indicate that RA specifically up-regulates PLP expression in glioma C6 cells at a posttranscriptional level by increasing PLP RNA half-life.

7 pages, 7 figures., Congenital hypothyroidism strongly affects myelination. To assess the role of thyroid hormone on myelin gene expression, we have studied the effect of hypothyroidism on the steady state levels of myelin-associated glycoprotein (MAG) and its mRNA in rat brain during the first postnatal month. As studied by immunoblot analysis of several brain regions, MAG increased from days 10-15 onwards, reaching constant levels by days 20-25. Hypothyroid samples showed a delay in the accumulation of MAG that was more severe in rostral regions, such as cortex and hippocampus. The effect of hypothyroidism on the accumulation of the protein correlated with mRNA levels. MAG mRNA started to accumulate in the cerebrum of normal animals by postnatal day 7, reaching maximal levels by day 20. Hypothyroid rats showed a delay of several days in the onset of mRNA expression, increasing thereafter at the same rate as in normal animals, and eventually reaching similar values. When individual brain regions were analyzed, we found strong regional differences in the effect of hypothyroidism. The cerebral cortex was most affected, with messenger levels lower than in normal animals at all ages. In more caudal regions differences between control and hypothyroid rats were evident only at the earlier stages of myelination, with spontaneous recovery at later ages. By run on analysis, we found no differences in transcriptional activities of the MAG gene in normal, hypothyroid, or T4-treated rats. Therefore, the effects of hypothyroidism on MAG mRNA and protein levels were most likely caused by decreased mRNA stability. We propose that thyroid hormone contributes to enhanced myelin gene expression by affecting the stability of newly transcribed mRNA in the early phases of myelination., This work was supported by grants from Dirección General de Investigación Científica y Técnica (PM88-0006), from Antibióticos-Pharma S.A. (Madrid, Spain), and from the Community of Madrid (C254/90).

To define at the molecular biological level the effects of thyroid hormone on brain development we have examined cDNA clones of brain mRNAs and identified several whose expression is altered in hypothyroid animals during the neonatal period. Clones were identified with probes prepared by subtractive or differential hybridization, and those corresponding to mRNAs altered in hypothyroidism were further studied by Northern blot analysis. Using RNA prepared from whole brains, no effect of hypothyroidism was found on the expression of the astroglial gene coding for glial fibrillary acidic protein. Among genes of neuronal expression, no significant alterations were found in the steady state levels of mRNAs coding for neuron-specific enolase, microtubule-associated protein-2, Tau, or nerve growth factor. N-CAM mRNA increased slightly in hypothyroid brains. In contrast a 2- to 3-fold decrease was found in the mRNA coding for a novel neuronal gene, RC3. This is the first neuronal gene known to be significantly altered at the mRNA level by thyroid hormone deprivation. The abundance of the mRNAs for the major myelin proteins proteolipid protein, myelin basic protein, and myelin-associated glycoprotein, expressed by oligodendrocytes, were also decreased in hypothyroid brains. Developmental studies on RC3 and myelin-associated glycoprotein expression indicated that the corresponding mRNAs accumulate in the brain of normal rats during the first 15-20 days of neonatal life. A similar accumulation occurred in hypothyroid brains, but at much reduced levels. The results demonstrate that thyroid hormone controls the steady state levels of particular mRNAs during brain development.

Summary Treatment of intact 3T3 cells with biologically active phorbol esters causes a progressive decline in the total activity of Ca 2+ -sensitive, phospholipid-dependent protein kinase (protein kinase C) measured in cell-free, detergent-solubilized, extracts. The effect is dose-dependent and reversible; protein kinase C activity reappears when the phorbol esters are removed from the medium.

Addition of vasopressin to quiescent cultures of Swiss 3T3 cells caused a rapid increase in the phosphorylation of an acidic molecular weight 80,000 cellular protein (termed 80K). The effect was concentration- and time-dependent; enhancement in 80K phosphorylation could be detected as early as 30 sec after the addition of the hormone. Recently, a rapid increase in the phosphorylation of an 80K cellular protein following treatment with phorbol esters or diacylglycerol has been shown to reflect the activation of protein kinase C in intact Swiss 3T3 cells. Here we show that the 80K phosphoproteins generated in response to vasopressin and phorbol 12,13-dibutyrate (PBt2) were identical as judged by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) and peptide mapping following partial proteolysis with Staphylococcus aureus V8 protease. In addition, prolonged pretreatment of 3T3 cells with PBt2 which leads to the disappearance of protein kinase C activity blocked the ability of vasopressin to stimulate the phosphorylation of 80K. The effect of vasopressin on 80K phosphorylation and mitogenesis was selectively blocked by the vasopressin antagonist (Pmp1-O-Me-Tyr2-Arg8) vasopressin suggesting that these responses are mediated by its specific receptor in these cells. The removal of vasopressin leads to dephosphorylation (within minutes) of the 80K phosphoprotein. We conclude that vasopressin rapidly stimulates protein kinase C activity in intact 3T3 cells.

Los autores pertenencen al antiguo Departamento de Endocrinología Experimental, Instituto G. Marañón (CSIC)., Evidence is provided for a differential sensitivity to thyroid hormones of 2 nuclear polypeptides, shown to be under thyroid hormone control. In particular, the 81,000 M[r] polypeptide which is negatively regulated by thyroid hormones, appears to be extremely sensitive to T3. Very low doses of this hormone, having no effect on the 120,000 M[r] polypeptide or liver enzymes caused a clear decrease of the 81,000 M[r] polypeptide. The reason for the high sensitivity of the 81,000 M[r] polypeptide to T3, as compared to other responses, is unknown. The thyroid hormone control of protein concentrations in the cell is currently explained by effects at the level of mRNA formation. Both positive and negative control of hepatic gene expression by thyroid hormone has been shown. It is possible that the response of 81,000 M[r] to T3 represents a primary effect of the hormone, in contrast with the response of other proteins and enzymes which have been found to be modulated by both thyroid hormones and other factors.

Addition of serum to quiescent cultures of Swiss 3T3 cells and mouse embryo fibroblasts causes a rapid increase in the phosphorylation of an 80 000 mol. wt. cellular protein (termed 80 K). The effect is dose- and time-dependent; enhancement in 80 K phosphorylation can be detected as early as 10-15 s after adding serum. In contrast, platelet-derived growth factor elicits the response after a lag of 1.5 min suggesting that this growth factor does not mediate the response to serum. Recently a rapid increase in the phosphorylation of an 80 K cellular protein following treatment with phorbol esters or diacylglycerol has been shown to reflect the activation of protein kinase C in intact fibroblasts. The 80 K phosphoproteins generated in response to serum and to phorbol dibutyrate (PBt2) co-migrated in one- and two-dimensional PAGE and produced identical phosphopeptide fragments when subjected to partial digestion with Staphyloccocus aureus V8 protease. These observations suggest that the same 80 K protein is generated in response to serum and PBt2. We conclude that activation of protein kinase C in intact cells is one of the earliest effects elicited by serum in quiescent fibroblasts.

The mitogens phorbol 12,13-dibutyrate, bombesin and vasopressin stimulate the phosphorylation of an acidic Mr 80,000 cellular protein, a specific substrate of protein kinase C, in intact Swiss 3T3 cells. Phosphorylation of this substrate was rapidly reversed upon the removal of each of these agents. Dephosphorylation occurred with a similar half-life in each of the cases studied (2.2, 1.5 and 2 minutes for phorbol 12,13-dibutyrate, bombesin and vasopressin respectively) and agreed closely with the dissociation of the ligands from their specific high-affinity binding sites in Swiss 3T3 cells.

During incubation of rat liver nuclei at 22 C, a fraction of the T3 receptor was released to the medium. Ca2+ and Mg2+ decreased the amount of receptor released, thereby increasing specific T3 binding to the nuclei. However, the total specific binding (i.e. in the nuclei plus the incubation medium) was decreased at concentrations of divalent cations that induced maximal inhibition of receptor release. Neither Ca2+ nor Mg2+ influenced the binding of [125I]T3 to the receptor when a KCl extract of the nuclei was studied. Therefore, the decrease of total T3 binding when maximal amounts of receptor were chromatin bound was probably due to a decreased affinity of the chromatin-bound receptor. Furthermore, in the presence of 1 mM MgCl2 50% of the receptor was released to the incubation medium, and the affinity of the solubilized receptor was 1.7 times higher than the affinity of the receptor that remained associated to the chromatin; when CaCl2 was included in the incubation medium, 91% of the receptor was in the nuclei, and its affinity was lower than the affinity of the nuclear bound receptor in the presence of 1 mM MgCl2 and no Ca2+. These results suggest that divalent cations lower the affinity of the chromatin-bound receptor. Receptor dissociation from the nuclei was studied by diluting 15 times a nuclear suspension, at 0 C for 1 h. Receptor dissociation from the nucleus was enhanced by increasing dilution by salt, 25% of the receptor was released from the nuclei. Addition of either CaCl2 or MgCl2 up to a concentration of 10 mM receptor loss was 70% in the absence of divalent cations, and in either 10 mM CaCl2 or MgCl2 it was 90%. The main conclusion from this work is that divalent cations might influence the amount of chromatin-bound T3-receptor complex in two ways; 1) by lowering the affinity of the chromatin-bound receptor for T3, and 2) by increasing the dissociation of the receptor from its chromatin acceptor sites.

Antisera have been raised against human protein kinase C and also against a synthetic peptide based on the sequence of the bovine brain enzyme (LLNQEE-GEYYNVPIPE). These antibodies react with protein kinase C from a number of species (human, murine, rat, rabbit, bovine), indicating substantial conservation of epitopes. These antisera have been used to quantitate directly protein kinase C by immunoblot analysis. We show here that there is a strict correlation between the levels of immunoreactive polypeptide and extractable calcium- and phospholipid-dependent kinase activity for various cell lines. Treatment of murine, rat, and human cells with phorbol dibutyrate was found to deplete levels of immunoreactive protein kinase C severely. A detailed study of the time course of this depletion in Swiss 3T3 cells shows that it follows precisely the loss of extractable activity. On exposure to 400 nM phorbol 12,13-dibutyrate protein kinase C was essentially undetectable by 40 hours; the half-life of this down-regulation was 6.7 hours. This data thus demonstrate that the loss of immunoreactive protein kinase C and of extractable calcium- and phospholipid-dependent kinase activity precisely parallels the phorbol ester induced down-regulation of binding and responsiveness in Swiss 3T3 cells.

Los autores pertenencen al antiguo Departamento de Endocrinología Experimental, Instituto G. Marañón (CSIC)., Inhabitants of many severe endemic goiter areas have low serum T4 and high circulating TSH, despite normal levels of T3. This situation may be produced experimentally by chronically feeding rats a low iodine diet (LID). We fed rats a Remington-type LID and gave them 1% NaClO4 in their drinking water for 2 days. After this, the animals were divided into three groups. One group was fed LID, supplemented with 5 μg I/rat·day and was used as the control group. Another group was fed LID alone. The third group was fed LID and given 1% NaClO4 to drink. The latter treatment was used to induce severe hypothyroidism. Animals were killed 1, 2, 3, and 5 weeks after the onset of these treatment schedules. The following measurements were made on some or all groups of animals: body and thyroid weights; thyroidal I content; soluble labeled iodoprotein profile; thyroidal labeled iodoamino acid distribution pattern; plasma T4, T3) and TSH; pituitary GH content; and liver intramitochondrial α-glycerophosphate dehydrogenase and cytosolic malic enzyme activities. T4 and T3 concentrations were also measured in liver nuclei of the animals killed 5 weeks after the onset of treatment. As assessed from various indices of thyroid function, the LID rats became iodine deficient, although not as markedly as those given LID and ClO4-. The plasma T4 decreased to undetectable levels, and plasma TSH increased, whereas circulating T3 remained normal throughout in the LID rats. In rats given LID and ClO4-, plasma T4 decreased sooner than in rats given LID alone; plasma T3 levels also became undetectable, and TSH increased more markedly and sooner than in rats given LID alone. As measured at the end of 5 weeks of treatment, pituitary GH content, and liver α-glycerophosphate dehydrogenase and malic enzyme activities were lower in rats given LID than in the euthyroid LID- and I--treated controls. They were not, however, as markedly reduced as in the severely hypothyroid LID- and ClO4--treated rats. In spite of normal plasma T3 levels, the concentration of T3 in liver nuclei of the rats given LID alone was significantly lower than that of the LID- and I--treated controls. The results show that the thyrotrophs, somatotrophs, and livers of rats given LID alone are not like those of euthyroid rats despite normal circulating T3 levels. In iodine-deficient rats, there is a discrepancy between the measured indices of thyroid hormone action in the liver and the circulating T3 level, but not between biological activity and liver nuclear T3 concentration. It remains to be seen whether the same is true in the anterior pituitary.

Rats fed chronically a low iodine diet may have low serum T4 and high circulating TSH, despite normal serum T3. As the brain depends to a great extent on intracellular generation of T3 from T4 for its total and nuclear T3, we have carried out two experiments to determine whether the brain of iodine-deficient rats may become hypothyroid, despite normal serum T3 levels. In both experiments we confirmed previous data, showing that the pituitary and liver of iodine-deficient rats with very low plasma T4 levels are hypothyroid as compared to those of animals receiving the same diet supplemented with KI, though not as markedly as animals which had undetectable circulating levels of both T4 and T3 as a consequence of chronic ingestion of KClO4 -, or of surgical thyroidectomy. We have further found that the nuclear T3 content was decreased in the brain of iodine-deficient rats, as compared with the animals on the iodine-supplemented diet. The nuclear to plasma ratios of labeled T3 showed that the uptake of this hormone into liver and brain nuclei is not decreased in the iodine-deficient rats as compared with those on the iodine-supplemented diet. This finding indicates that the decreased liver and brain nuclear T3 contents of iodine-deficient rats are likely to be a consequence of the marked reduction of their T4 pool, leading to decreased amounts of intracellularly generated T3. The number of spines on shafts of pyramidal neurons from the visual cortex of iodine-deficient rats was lower than that of rats fed the same diet supplemented with KI. Their distributions along the shaft were also not the same. Such changes might well be an index of cerebral hypothyroidism, as they are similar to those found after thyroidectomy of adult rats. It is concluded from the present findings that normal circulating T3 levels may not be sufficient to maintain brain euthyroidism in rats fed a diet iodine deficient enough to result in very low circulating T4 levels.

The synthetic diacylglycerol 1-oleoyl-2-acetylglycerol competes directly with [3H]phorbol 12,13-dibutyrate for common binding sites in monolayer cultures of Swiss 3T3 cells and rapidly stimulates the phosphorylation of a Mr 80,000 cellular protein that has recently been shown to reflect the activation of protein kinase C in intact cells. Thus, this diacylglycerol provided a useful tool to determine whether exogenously added diacylglycerols can mimic the potent tumor promoter phorbol ester in eliciting DNA synthesis and cell division in quiescent cells. We found that OAG acts synergistically with insulin and other growth factors to stimulate reinitiation of cell proliferation, and several lines of evidence indicate that OAG shares with phorbol esters a common pathway of mitogenic action via stimulation of protein kinase C activity in intact 3T3 cells. The findings support the hypothesis that diacylglycerols represent endogenous analogs of phorbol esters and raise the possibility that diacylglycerols generated in the plasma membrane could act as a mitogenic signal for quiescent cells.

The mammalian brain is considered to be poorly responsive to thyroid hormone after the so called 'critical periods' of brain development, which occur in the rat before postnatal days 15-20. In a previous work (Munoz, A., A. Rodriguez-Pena, A. Perez-Castillo, B. Ferreiro, J. G. Sutcliffe, and J. Bernal. 1991. Mol. Endocrinol. 5:273-280) we have identified one neuronal gene, RC3, whose expression is influenced by early neonatal hypothyroidism and thyroid hormone treatment. In the present work we show that adult-onset hypothyroidism leads to a reversible decrease of RC3 mRNA. Rats thyroidectomized on postnatal day 40 and killed three months later showed a decreased RC3 mRNA concentration in the cerebral cortex and striatum. The same effect was observed in animals made hypothyroid on postnatal day 32 and killed on postnatal day 52. RC3 expression was normal when hypothyroid animals were treated with T4 five days before being killed. In contrast, the mRNA encoding myelin proteolipid protein showed no changes in either experimental situation. RC3 mRNA levels were not affected by food restriction demonstrating that the effect of hypothyroidism was not related to the lack of weight gain. The control of RC3 mRNA is so far the only molecular event known to be regulated by thyroid hormone once the critical periods of brain development are over and could represent a molecular correlate for the age- independent, reversible alterations induced by hypothyroidism in the adult brain., This work has been supported by grant PM88-0006 from Dirección General de Investigación Científica y Técnica.

Two nuclear proteins from rat liver were shown to be dependent on thyroid hormones. These proteins were present in the nucleosol or nucleoplasmic fraction, and were extracted from the nuclei with 0.15 m NaCl at pH8. After thyroidectomy, a 120 000-Mr polypeptide decreased in concentration to levels below 10% of normal control rats and another polypeptide of 81 000-Mr was increased. Treatment with T4 at physiological replacement doses for several days restored the levels of both proteins to normal. A single administration of 50 μg T3 induced a detectable increase of 120K after 14 h, with maximal effects at 48 h after administration. The minimum effective dose of T3 on 120K was 0.5 μg administered for three days. Preliminary observations suggest that the response of 81K to thyroid hormones is much more sensitive than that of 120K. The physiological role of these polypeptides is unknown, but they could be involved in the mode of T3 action at the nuclear level.