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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.

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.

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.

[EN] TrkB as the receptor for the neurotrophins BDNF and NT-4/5 mediates the survival of specific neuronal populations during development, and in adulthood participates in adaptative responses and pathological conditions. trkB gene encodes a full length isoform with a functional tyrosine kinase activity, and several truncated isoforms TrkB-T1, TrkB-T2 and TrkB-Shc which all lack kinase activity. In this study, we show that in cortical astrocytes, AMPc/PKA and ATP/CaMK signaling pathways mediate CREB activation leading to TrkB-T1 up-regulation. In cortical neurons, cAMP/PKA-dependent CREB activation mediates TrkB-Fl and TrkB-T1 up-regulation. This activation was mediated by the CRE sequences present in the TrkB P2 promoter. These findings provide the molecular basis for the increase of TrkB expression found in different adaptive/pathological responses in which CREB has been involved. Furthermore, we demonstrate that excitotoxic NMDA receptor overstimulation causes TrkB-T1 up-regulation, and TrkB-Fl down-regulation in cortical neuron cultures. This change in expression pattern of the trkB isoforms is directly linked to the excitotoxic neuronal cell death. Thus, neurospecific TrkB-Fl over-expression using a lentiviral system, reduced excitotoxicity in these cultures, meanwhile TrkB-T1 over-expression produced the opposite effect. Upregulation of truncated TrkB is also found in ischemia (an in vivo model of excitoxocity) which further supports that trkB isoform expression is regulated in pathological conditions associated with NMDA receptors over-stimulation., [ES] TrkB, receptor de las neurotrofinas BDNF y NT-4/5, media la supervivencia de poblaciones específicas de neuronas durante el desarrollo, y en estado adulto participa en respuestas adaptativas y situaciones patológicas. El ARNm de TrkB codifica para varias isoformas, una completa con un dominio tirosina kinasa (TrkB-Fl) y otras truncadas (TrkBT1, TrkB-T2 y TrkB-T-Shc) carentes de actividad catalítica. En el presente estudio demostramos que en astrocitos, la activación del factor de transcripción CREB de forma dependiente de las vías de señalización AMPc/PKA y ATP/CaMK, incrementa la expresión de TrkB-T1. En neuronas corticales la activación de CREB dependiente de AMPc/PKA aumenta la expresión de las isoformas TrkB-Fl y TrkB-T1. En ambos tipos celulares, esta activación es debida a dos secuencias CRE localizadas en el promotor P2 del gen trkB. Estos resultados aportan las bases moleculares que explican como se produce el incremento de la expresión de TrkB en respuesta a diferentes respuestas adaptativas en las que CREB está involucrado. Por otro lado, demostramos que la estimulación excitotóxica de los receptores de glutamato tipo NMDA cambia el patrón de expresión de las isoformas de TrkB en neuronas corticales, aumentado TrkB-T1 y disminuyendo TrkB-Fl, y que existe una relación directa entre la muerte neuronal y dicho cambio de expresión. Así, la sobre-expresión de TrkB-Fl reduce la muerte dependiente de NMDA, mientras que la sobreexpresión de TrkB-T1 la aumenta. De acuerdo con estos resultados, en isquemia (un modelo de excitotoxicidad in vivo) se produce un aumento de la expresión del receptor truncado, sugiriendo que el cambio de expresión de la isoformas de TrkB está involucrado en los procesos de muerte celular en condiciones de excitotoxicidad.

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.

Expression of the neurotrophin receptor trkB is regulated by thyroid hormone (T3) during development of the rat brain. trkB mRNA levels, coding for the full-length and the truncated isoforms, are increased in the cerebral cortex of neonatal experimental hypothyroid animals. Run-on transcription assays with nuclei from postnatal day 15, hypothyroid, and control cerebral cortices demonstrated that an increase in the transcription rate of the trkB gene accounts for the observed effect. Transient transfection experiments using a reporter plasmid containing a 7-kilobase pair DNA fragment upstream of the mouse trkB gene showed that unliganded thyroid hormone receptor (T3R) increases promoter activity, whereas addition of T3 reverses that activity below basal levels. Deletion analysis shows that the T3-dependent repression requires binding of the T3R to a specific region located downstream of the transcription start site. This region, at nucleotide position -465/-432, contains an array of thyroid hormone response half-sites that bind preferentially T3R as heterodimers with retinoid X receptor and whose deletion causes loss of the T3-dependent repression. These half-sites are able to confer negative regulation by T3 to a heterologous promoter, thus indicating the functionality of these sequences. These results demonstrate that, in the developing rat brain, T3 down-regulates the expression of the trkB gene through the active repression of a novel negative response element located downstream of its transcription initiation site.

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.

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.

Insulin-like growth factor I (IGF-I) strongly stimulates the generation of differentiated neurons in cultures of neuroepithelial cells of the embryonic chick neural retina in the presence of a laminin-1 tissue culture substrate. Treatment of cultured neuroepithelial cells with IGF-I rapidly up-regulated the mRNA coding for the alpha 6 integrin subunit whereas specific reduction of alpha 6 subunit levels by treatment with an alpha 6 integrin antisense oligonucleotide resulted in reduced neuronal differentiation in vitro. Although IGF-I immunoreactivity is seen throughout the neural retina, expression of IGF-I mRNA is confined to the pigment epithelium during the period of neurogenesis in vivo. Neutralization of the endogenous IGF-I with a blocking antibody down-regulated levels of alpha 6 integrin mRNA and reduced the production of differentiated retinal neurons in vivo. These data indicate a role for IGF-I in the generation of retinal neurons mediated by the interaction of laminin with its alpha 6 integrin subunit-containing receptor.

When chick embryos are treated with a monoclonal antibody specifically blocking the activity of neurotrophin-3 (NT-3), the development of the retina is profoundly affected. Fewer axons are found in the optic nerve, and the retina shows abnormalities in all layers. Early during retinogenesis, the proportion of dividing cells is higher in NT-3-deprived embryos compared with age-matched controls and that of differentiated neurons is smaller. The NT-3 receptor trkC is expressed early by a majority of retinal cells, and NT-3 is present in the retina at the earliest stage studied. Initially, it is located mainly in the pigmented epithelium, with a shift toward the neural retina as development proceeds. Thus, NT-3 seems to be an essential intrinsic signal acting early in development to promote the differentiation and survival of many retinal neurons.

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.

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.

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.

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.

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.

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.

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.

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.

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.