Thyrotropin releasing hormone (TRH) in the hippocampus of Alzheimer patients.

Thyrotropin-releasing hormone (TRH) is best known for its hypothalamic neuroendocrine role in regulating thyroid function. In extra-hypothalamic regions in vitro, we have shown TRH to have a protective effect against synaptic loss and neuronal apoptosis. A role for TRH in Alzheimer's disease (AD) has not been established previously. In this study, we examined the content of the TRH peptide in the hippocampus of elderly controls (n=5) and AD patients (n=7) by radioimmunoassay (RIA). The TRH concentration was decreased in the AD hippocampus compared to normal elderly controls (p < 0.01). In a separate series of experiments utilizing primary cell cultures made from rat hippocampus, TRH peptide concentration was depleted by the addition of TRH antiserum. TRH withdrawal was found to enhance the activity of glycogen synthetase kinase-3 (GSK-3beta), a critical enzyme necessary for the phosphorylation of tau, as well as the phosphorylation of the tau protein itself. This TRH depletion induced upregulation in phosphorylation that was observed to initiate axonal retraction in cultured neurons. These data suggest that TRH within the hippocampus can regulate the activity of various proteins by phosphorylation/dephosphorylation that may be involved in the pathogenesis of AD.

Effect of preproTRH antisense on thyrotropin-releasing hormone synthesis and viability of cultured rat diencephalic neurons.

To investigate a possible neurotropic role for thyrotropin-releasing hormone (TRH) in the central nervous system, we used recombinant antisense TRH adenovirus (TRHav) to "knock out" TRH in cultured 17-d fetal rat diencephalon. The morphology along with beta-galactosidase (beta-gal) enzyme histochemistry (X-gal staining) and TRH content (femtomoles/well) were used to measure the effect of antisense TRH virus. Control adenovirus mediated beta-gal transfection efficiency was nearly 85%, as shown by positive X-gal staining, and was without effect on cell morphology, TRH content, or the normal response to glucocorticoid (dexamethasone) exposure with enhanced TRH expression. A significant 90% decline in TRH content as well as changes in neuronal morphology (shrunken cell bodies and short dendrites) were observed after 14 but not 7 d following TRHav treatment. The addition of synthetic TRH peptide at 2.5 microM along with TRHav, but not dexamethasone, partly prevented the morphologic changes. No morphologic changes were seen in wild-type AtT20 cells, a pituitary cell line that does not produce TRH. To investigate whether neuronal death from loss of proTRH was owing to apoptosis, neuronal DNA change by means of fluorescent dye H-33342 staining, TUNEL staining, and DNA laddering analysis was examined. Eighty to 90% positive H-33342 and TUNEL staining as well as a 180- to 200-bp DNA fragment on DNA laddering analysis were found as compared to control. These results indicate that proTRH gene expression prevents neuronal apoptosis and may play a role in neuronal development and function.

MR imaging of pituitary adenomas after gamma knife stereotactic radiosurgery.

OBJECTIVE: The purpose of this study was to evaluate the response of pituitary adenomas to radiosurgery as manifested by changes in size and appearance on serial MR imaging. MATERIALS AND METHODS: Over a mean follow-up period of 36 months, changes in 44 pituitary adenomas were assessed on 147 enhanced MR imaging studies. Prior surgery had been performed in 36 tumors (82%). RESULTS: At the time of radiosurgery, mean tumor volume was 5.9 +/- 0.8 cm(3) (mean diameter, 2.2 cm). The mean reduction in volume at last follow-up was 41% (+/- 5%, p < 0.001), and a decrease in tumor volume of 25-100% was observed in 34 tumors (77%). Mean reduction in tumor volume at 6 months after radiosurgery was 9% (p = 0.095); at 1 year, 24% (p < 0.001); at 2 years, 34% (p < 0.001); at 3 years, 41% (p < 0.001); and at 4 years, 50% (p = 0.008). Six months after radiosurgery a slight and transient increase in size was observed in 21% of tumors. During follow-up, neither decreased contrast enhancement nor cyst development was associated with changes in tumor volume. CONCLUSION: Tumor control was observed for most pituitary adenomas after radiosurgery and occurred gradually over a period of several years. A small increase in tumor size might be observed in the first 6 months after radiosurgery. In most cases, reductions in tumor size were not accompanied by a change in contrast enhancement or cyst formation.

Porcine CTLA4-Ig lacks a MYPPPY motif, binds inefficiently to human B7 and specifically suppresses human CD4+ T cell responses costimulated by pig but not human B7.

The CTLA4 receptor (CD152) on activated T lymphocytes binds B7 molecules (CD80 and CD86) on APC and delivers a signal that inhibits T cell proliferation. Several regions involved in binding to B7 are known, but the relative importance of these is not clear. We have cloned porcine CTLA4 (pCTLA4). Although highly homologous to human CTLA4 (hCTLA4), the predicted protein sequence contains a leucine for methionine substitution at position 97 in the MYPPPY sequence. A fusion protein constructed from the extracellular regions of pCTLA4 and the constant regions of human IgG1 (pCTLA4-Ig) bound porcine CD86 with equivalent affinity to that of hCTLA4-Ig. However, pCTLA4-Ig bound poorly to human CD80 and CD86 expressed on transfectants and EBV-transformed human B cells. In functional assays with MHC class II-expressing porcine endothelial cells and human B cells, pCTLA4-Ig blocked human CD4+ T cell responses to pig but not human cells, whereas control hCTLA4-Ig inhibited responses to both. Comparison between mouse, human, and porcine CTLA4-Ig suggests that the selective binding of pCTLA4-Ig to porcine CD86 molecules is due to the L for M substitution at position 97. Our results indicate that pCTLA4-Ig may be a useful reagent to define the precise nature of the interaction between B7 and CTLA4. By failing to inhibit the delivery of costimulatory signals provided by human B7, it may also prove to be a relatively specific inhibitor of the direct human T cell response to immunogenic pig tissue.

Association of cocaine- and amphetamine-regulated transcript-immunoreactive elements with thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus and its role in the regulation of the hypothalamic-pituitary-thyroid axis during fasting.

Because cocaine- and amphetamine-regulated transcript (CART) coexists with alpha-melanocyte stimulating hormone (alpha-MSH) in the arcuate nucleus neurons and we have recently demonstrated that alpha-MSH innervates TRH-synthesizing neurons in the hypothalamic paraventricular nucleus (PVN), we raised the possibility that CART may also be contained in fibers that innervate hypophysiotropic thyrotropin-releasing hormone (TRH) neurons and modulate TRH gene expression. Triple-labeling fluorescent in situ hybridization and immunofluorescence were performed to reveal the morphological relationships between pro-TRH mRNA-containing neurons and CART- and alpha-MSH-immunoreactive (IR) axons. CART-IR axons densely innervated the majority of pro-TRH mRNA-containing neurons in all parvocellular subdivisions of the PVN and established asymmetric synaptic specializations with pro-TRH neurons. However, whereas all alpha-MSH-IR axons in the PVN contained CART-IR, only a portion of CART-IR axons in contact with pro-TRH neurons were immunoreactive for alpha-MSH. In the medial and periventricular parvocellular subdivisions of the PVN, CART was co-contained in approximately 80% of pro-TRH neuronal perikarya, whereas colocalization with pro-TRH was found in <10% of the anterior parvocellular subdivision neurons. In addition, >80% of TRH/CART neurons in the periventricular and medial parvocellular subdivisions accumulated Fluoro-Gold after systemic administration, suggesting that CART may serve as a marker for hypophysiotropic TRH neurons. CART prevented fasting-induced suppression of pro-TRH in the PVN when administered intracerebroventricularly and increased the content of TRH in hypothalamic cell cultures. These studies establish an anatomical association between CART and pro-TRH-producing neurons in the PVN and demonstrate that CART has a stimulatory effect on hypophysiotropic TRH neurons by increasing pro-TRH gene expression and the biosynthesis of TRH.

Evidence from studies with N-ethyl-maleimide and 12-O-tetradecanoylphorbol-13-acetate that AP-1 and CREB are involved in the glucocorticoid activation of TRH gene expression in hypothalamic cultures.

To determine whether c-fos/c-jun (AP-1) and CREB mediate glucocorticoid stimulated TRH gene regulation, we investigated the effect of N-ethyl-maleimide (NEM), an alkylating agent, and 12-O-tetradecanoylphorbol-13-acetate (TPA) on this process. NEM decreased while TPA increased TRH levels in rat hypothalamic culture, changes similar to their effects on CREB and Fos/Jun proteins in the AtT20 cell line. This suggests that glucocorticoid stimulation of TRH gene expression may be regulated by the AP-1 complex and CREB pathway.

Advantage of double labeled in situ hybridization for detecting the effects of glucocorticoids on the mRNAs of protooncogenes and neural peptides (TRH) in cultured hypothalamic neurons.

In order to study the effect of glucocorticoids on thyrotropin-releasing hormone (TRH) and protooncogenes, we describe a double labeled in situ hybridization method to explore this issue. The development of non-isotopic in situ hybridization histochemistry has proven to be an important tool for cellular and molecular studies in neurobiology [C.L.E. Moine, E. Normand, B. Bloch, Use of non-radioactive probes for mRNA detection by in situ hybridization: interests and applications in the central nervous system, Cell. Mol. Biol. 41 (1995) 917-923]. These methods involve the anatomic localization of labeled RNA or DNA molecules which hybridize with complementary target RNA or DNA sequences in the cell. With regard to gene expression, in situ hybridization allows the study of specific mRNA levels and the distribution between various cell types. It also allows the comparison of mRNA levels at various stages of development. Double labeled in situ hybridization is able to detect the colocalization of two different mRNAs simultaneously. Accordingly, this approach is utilized for specific studies involving the expression and distribution of TRH mRNA and the protooncogenes, c-fos/c-jun, in cultured rat hypothalamic neurons [L. G. Luo, I.M.D. Jackson, Glucocorticoids stimulate TRH and c-fos/c-jun gene co-expression in cultured hypothalamic neurons, Brain Research 791 (1998) 56-62]. Our protocol for double labeled in situ hybridization reflects a modification of a number of original protocols developed by others [H. Breitschopf, G. Suchanek, R.M. Gould, D.R. Colman, H. Lassmann, In situ hybridization with digoxigenin-labeled probes: sensitive and reliable detection method applied to myelinating rat brain, Acta Neurropathol. 84 (1992) 581-587; S. McQuaid, J. McMahon, G.M. Allan, A comparison of digoxigenin and biotin labeled DNA and RNA probes for in situ hybridization, Biotech. Histochem. 70 (1995) 147-154; E. Hrabovszky, M.E. Vrontakis, S.L. Petersen, Triple-labeling method combining immunocytochemistry and in situ hybridization histochemistry: demonstration of overlap between Fos-immunoreactive and galanin mRNA-expressing subpopulations of luteinizing hormone-releasing hormone neurons in female rats, J. Histochem. Cytochem. 43 (1995) 363-370]. This technique can be readily applied to various studies of cellular gene expression in the mammalian nervous system involving other neural peptides and transcription factors.

Role of gamma knife therapy in the management of pituitary tumors.

1. Gamma knife therapy is an effective method of delivering radiation to pituitary tumors that have failed surgery and may be used as primary treatment in circumstances in which the patient refuses or is unsuitable for a transsphenoidal procedure. 2. Stereotactic radiosurgery with the gamma knife unit is generally administered in a single session unlike fractionated radiotherapy, which is administered four to five times per week over a 6-week period. 3. Preliminary data suggest that resolution of pituitary hypersecretion is faster with gamma knife therapy than with conventional radiotherapy. 4. Because of the nature of the gamma knife therapy and the fact that the radiation dose conforms to the tumor shape, there is a steep fall-off of radiation to surrounding tissue. Accordingly, the radiation dose to extrapituitary brain is substantially less with gamma knife radiosurgery than with conventional radiotherapy. This suggests that the development of second brain tumors and neurocognitive complications, which are significant risks with conventional radiotherapy, is much less likely with gamma knife surgery. 5. Gamma knife radiosurgery can be used to ablate tumors invading the cavernous sinus. 6. Gamma knife radiosurgery is safe as long as the dose of radiation to the optic structures is kept under 10 Gy. 7. Long-term follow-up is required for pituitary tumors treated by gamma knife therapy so as to determine its efficacy as well as its effects on pituitary function and any resultant complications.

Role of gamma knife radiosurgery in acromegaly.

Stereotactic radiosurgery with the Gamma Knife allows the delivery of focused radiation in a single session from a Cobalt-60 source to a pituitary tumor with little radiation to surrounding normal brain tissue. At this time the major role for Gamma Knife radiosurgery in acromegaly is for the treatment of failed pituitary surgery although it may also by used as primary treatment for patients unwilling or unsuitable, for medical reasons, to undergo transsphenoidal surgery. The major risk from Gamma Knife radiosurgery appears to be radiation damage to the visual pathways, but this can be obviated by limiting the radiation dose to the optic chiasm under 10 Gy. In contrast, the neuronal and vascular structures running in the cavernous sinus are much less radiosensitive allowing an ablative dose to be administered to tumors showing lateral invasion and impinging on cranial nerves III, IV, V and VI. Gamma Knife radiosurgery appears to produce effects in GH secreting tumors faster than with fractionated radiotherapy without the potential long-term risk of developing a second extrapituitary brain tumor as well as the neuropsychiatric effects associated with conventional radiation administration.

Gamma knife radiosurgery for pituitary tumours.

Stereotactic radiosurgery with the gamma knife delivers focused radiation from a cobalt-60 source in a single session to a pituitary tumour with minimal radiation to the adjacent normal brain tissue. Currently, gamma knife radiosurgery is predominantly used to treat failed pituitary surgery, although it has a role as a primary treatment for patients unwilling or unsuitable, for medical reasons, to undergo trans-sphenoidal surgery. The major risk from gamma knife radiosurgery is radiation damage to the visual pathways, but this can be avoided by limiting the radiation dose to the optic chiasm to under 10 Gy. In contrast, the neuronal and vascular structures running in the cavernous sinus are much less radiosensitive, allowing an ablative dose to be administered to tumours showing lateral invasion and impinging on cranial nerves III, IV, V and VI. Gamma knife radiosurgery appears to produce remission in secretory tumours faster than fractionated radiotherapy. Furthermore, the potential long-term risk of developing a second extra-pituitary brain tumour, as well as the neuropsychiatric effects associated with conventional radiation administration, seems less likely to occur with this form of treatment.

Antidepressants inhibit the glucocorticoid stimulation of thyrotropin releasing hormone expression in cultured hypothalamic neurons.

BACKGROUND: The pituitary thyroid axis is frequently effected in human depression possibly due to alteration in hypothalamic thyrotropin releasing hormone (TRH) secretion. Since clinical recovery is associated with normalization of thyroid function, the direct effect of antidepressants on TRH expression in a well established fetal rat hypothalamic neuronal culture system was investigated. METHODS: Fetal rat hypothalamic neurons (day 17) in culture were treated with different concentrations of antidepressants with or without glucocorticoids for 7 days following which TRH content was measured by radioimmunoassay (RIA). RESULTS: The results showed that Imipramine (IMIP), a tricyclic antidepressant (TCA), decreased the TRH content in a dose-dependent manner (from 80.7 +/- 4.9, at 10(-9) mol/L, to 14.1 +/- 0.6, at 10(-5) mol/L, fmol/well; P < 0.05). Desipramine (DESI), another tricyclic antidepressant, also decreased the TRH content (from 63.6 +/- 2.5, at 10(-9) mol/L, to 12.6 +/- 0.4, at 10(-5) mol/L, fmol/well; P < 0.05). Sertraline (SERT) and Fluoxetine (FLUO), serotonin selective reuptake inhibitors (SSRI), also decreased TRH content in a dose dependent manner (from 83.9 +/- 7.9, at 10(-10) mol/L, to 7.6 +/- 0.4, at 10(-5) mol/L, and from 41.66 +/- 2.5, at 10(-8) mol/L, to 17.54 +/- 0.92, at 10(-6) mol/L, fmol/well, respectively; both P < 0.05). We then tested the effect of these antidepressants on the Dex stimulation of TRH content. IMIP, DESIP and FLUO at 10(-6) mol/L reduced the TRH response to glucocorticoid stimulation (36.4 +/- 4.0, 56.6 +/- 2.4, 23.75 +/- 4.0, respectively vs 107 +/- 7.5 fmol/well; P < 0.05). CONCLUSION: This raises the possibility that the enhanced thyroid function in depression, which we postulate, may result in part from glucocorticoid stimulation of TRH gene expression, can be reversed by antidepressants through a direct effect on the TRH neuron. However, other mechanisms may need to be invoked in addition since basal TRH content was also reduced.

The thyroid axis and depression.

Hypothyroidism may give rise to frank depression that usually responds to thyroxine therapy. Depressed subjects with subclinical hypothyroidism and/or autoimmune thyroiditis should probably also be treated similarly. Most patients with depression, although generally viewed as chemically euthyroid, have alterations in their thyroid function including slight elevation of the serum thyroxine (T4), blunted thyrotropin (TSH) response to thyrotropin-releasing hormone (TRH) stimulation, and loss of the nocturnal TSH rise. These changes are generally reversed following alleviation of the depression. The role of adjuvant triiodothyronine (T3) treatment in resistant depression has not been established, but the data suggest that it will be beneficial in about 25% of cases. However, controlled trials to establish this approach are needed. The underlying mechanism leading to the beneficial response from T3 is unknown, but may reflect brain hypothyroidism in the context of systemic euthyroidism. The hypothalamus in culture, which is analogous to a deafferentated hypothalamus in vivo, shows a paradoxic increase in TRH production after glucocorticoid stimulation. It is known that in human depression there is a functional disconnection of the hypothalamus with impairment of the inhibitory glucocorticoid feedback pathway from the hippocampus to the hypothalamus that results in the typical elevated cortisol levels and impaired dexamethasone suppression. It is postulated that a similar situation prevails with regards to the thyroid axis and that the increased T4 in depression, as well as the blunted TSH response to exogenous TRH, reflects glucocorticoid activation of the TRH neuron leading to increased TRH secretion with resultant down regulation of the TRH receptor on the thyrotrope. Normalization of thyroid function after treatment may result in part from an inhibitory response of the TRH neuron to antidepressant medication, although other effects may also be responsible.

Antisense oligomers of cfos and cjun block glucocorticoid stimulation of thyrotropin-releasing hormone (TRH) gene expression in cultured anterior pituitary cells.

The proto-oncogenes, cfos/cjun, are co-localized with thyrotropin-releasing hormone (TRH) in cultured anterior pituitary cells and increase following exposure to dexamethasone (Dex). To assess the role of cfos and cjun in the Dex stimulation of TRH gene expression, we used antisense oligonucleotides to block cfos and cjun expression in order to reduce formation of activating protein-1 (AP-1). The results showed that the antisense oligonucleotides together effectively reduced cfos/cjun gene expression and consequently the glucocorticoid stimulation of TRH peptide and mRNA. The findings indicate that cfos/cjun are involved in the glucocorticoid activation of TRH gene expression.

Thyrotropin-releasing hormone gene expression in the anterior pituitary. IV. Evidence for paracrine and autocrine regulation.

Disulfiram (Dis), an inhibitor of peptidyl-glycine alpha-amidating monooxygenase, the enzyme responsible for the production of alpha-amidated peptides from their immediate, glycine-extended precursors was used to investigate the paracrine effects of TRH on anterior pituitary (AP) hormone secretion. It reduces the production of TRH without directly affecting the classical pituitary hormones, none of which is amidated. Dis (8 microM) decreased the accumulation of TRH accompanied by an equimolar increase in TRH-Gly levels, indicating that pro-TRH biosynthesis persisted. TRH and TSH release into the medium was significantly lowered, whereas other pituitary hormones were unaffected. In contrast, dexamethasone (10 nM), which up-regulates TRH gene expression in this system, increased TRH (+89.5%) and TSH (+61.3%) secretion. The combination of dexamethasone and Dis further diminished the release of TRH (-73%) and TSH (-40.3%) observed with Dis alone, indicating that TRH synthesized within the AP regulates TSH secretion. Dis significantly elevated prepro-TRH (25-50) and pro-TRH messenger RNA levels, suggesting that reduced TRH formation leads to increased pro-TRH biosynthesis and that TRH regulates its own secretion. Thus, TRH synthesized by cultured AP cells not only stimulates TSH release through a paracrine effect, but has a negative feedback on its own biosynthesis by an autocrine mechanism.

Glucocorticoids stimulate TRH and c-fos/c-jun gene co-expression in cultured hypothalamic neurons.

To explore whether the protooncogenes, c-fos/c-jun, might be involved in regulating the effect of glucocorticoids on thyrotropin-releasing hormone in fetal rat diencephalic neurons, their localization and transcriptional activity were investigated using double-labeled in situ hybridization, Northern blot and nuclear run-on assays. The results showed that TRH mRNA was coexpressed with both c-jun and c-fos in the same neurons. Treatment with dexamethasone, a synthetic glucocorticoid, at 10(-8) M, enhanced transcriptional activity resulting in an increase in both cell number and intensity of all three mRNAs. The existence of c-fos/c-jun in thyrotropin-releasing hormone neurons and the increased transcriptional activity following dexamethasone treatment suggests that these protooncogenes could mediate the effect of glucocorticoids on thyrotropin-releasing hormone gene expression.

Identification of the thyrotropin-releasing hormone precursor, its processing products, and its coexpression with convertase 1 in primary cultures of hypothalamic neurons: anatomic distribution of PC1 and PC2.

The processing of pro-TRH, has been extensively studied in our laboratory using a corticotropic cell line, AtT20, transfected with the pro-TRH gene. We have also demonstrated that the convertases PC1 and PC2 process pro-TRH to cryptic peptides in vitro. However, although these processing pathways have been well characterized in vitro, little is known about the processing and subcellular distribution of pro-TRH and its derived peptides in hypothalamic neurons, an endogenous source of pro-TRH and PC enzymes. In this study we used multiple approaches to identify, both biochemically and anatomically, the presence and localization of pro-TRH (26 kDa) and its processing products. We also investigated the presence of PC1 and PC2 enzymes and the coexpression of pro-TRH and PC1 messenger RNAs. Identification of the TRH precursor was demonstrated by 1) Western blot analysis of cellular extracts, 2) immunoprecipitation of radiolabeled pro-TRH followed by analysis on acrylamide gel electrophoresis, 3) fluorescence immunocytochemistry, and 4) immunoelectron microscopy. The presence of the convertases PC1 and PC2 was determined by Western blot analysis of cellular extracts and fluorescence immunocytochemistry. The coexpression of pro-TRH with PC1 was shown by double in situ hybridization. Our findings support three main conclusions. First, this primary culture system of hypothalamic neurons is suitable for characterizing pro-TRH processing as well as identifying the anatomical location of its processing products. Second, prohormome processing takes place during axonal transport after removal of the signal peptide in the endoplasmic reticulum, and subsequent cleavages of the prohormone occur as intermediate peptides move down the axon toward the nerve terminal. This coupled transport-processing phenomenon may provide the necessary mechanism to ensure flexibility in differential processing of specific protein sequences that are determined by the secretory needs of cells. It appears that certain intermediate peptides differ in their subcompartmental distribution, suggesting the possibility of a differential processing and maturation of pro-TRH-derived peptides. Thirdly, the 87-kDa form of PC 1 may initiate the processing of pro-TRH at the Golgi complex level, which then continues to be processed by PC1 and PC2 in later stages of the secretory pathway.

Opiate withdrawal increases ProTRH gene expression in the ventrolateral column of the midbrain periaqueductal gray.

The midbrain periaqueductal gray matter (PAG) has a critical role in the modulation of behavioral and autonomic manifestations of the opiate withdrawal syndrome. We report a nearly 5-fold increase in proTRH gene expression in neurons of the ventrolateral column of the PAG following naltrexone precipitated morphine withdrawal. The accumulation of immunoreactive proTRH-derived peptides, but not the mature TRH tripeptide was concomitantly observed in these cells. These findings indicate that proTRH-derived peptides synthesized in neurons of the ventrolateral PAG may function as modifiers of opiate withdrawal responses.

Activation of thyrotropin-releasing hormone gene expression in cultured fetal diencephalic neurons by differentiating agents.

The present studies were undertaken to investigate the effect of 5-bromo-2'-deoxyuridine (BrdU; 50 microM) or forskolin/3-isobutyl-1-methylxanthine (F/I; 10/500 microM) on TRH gene expression in cultured fetal diencephalic cells. BrdU as well as drugs such as F/I that raise intracellular cAMP levels had been previously termed differentiating agents because they cause morphological and functional differentiation of IMR-32 neuroblastoma cells. We postulated that neurons of fetal diencephalons may remain relatively undifferentiated in vitro and that this might be the reason for low or undetectable TRH production. We hypothesized that treatment with differentiating agents might increase neuropeptide expression. Both BrdU and F/I dramatically (P < 0.01) raised intracellular TRH and pro-TRH messenger RNA concentrations in cultured diencephalic neurons. Although a short BrdU exposure during the first 4 days of culture was sufficient to irreversibly change TRH neurons and to cause maintenance of high TRH levels after withdrawal of the drug, F/I had to be present continuously throughout the observation period of 16 days to significantly elevate TRH expression. This suggests that BrdU and F/I act at different intracellular sites to activate TRH expression in cultured diencephalic neurons. The reduction of glial cells that occurs concurrent with the BrdU treatment was not observed after F/I exposure, and therefore, this effect does not appear to be a key factor for the induction of TRH expression. As the intracellular accumulation of somatostatin and arginine vasopressin, which were determined in parallel, was similarly enhanced after treatment with BrdU or F/I, our culture system might provide a valuable tool for the study of these and possibly other neuropeptides in vitro.

Does thyroid hormone have a role as adjunctive therapy in depression?

Depression is associated with abnormalities of the thyroid axis, but the role of thyroid hormone therapy is controversial. In patients presenting with depression, the thyroid status should be carefully evaluated since hypothyroidism can cause depression. Frank hypothyroidism should be treated in the usual fashion with L-thyroxine, which may reverse the depressive state. If subclinical hypothyroidism and/or autoimmune thyroiditis are present, T3 adjuvant administration (25 micrograms/day) should be seriously considered in patients resistant to tricyclic antidepressant (TCA) (and probably also) serotonin selective reuptake inhibitor (SSRI) medication. The possible efficacy of adjuvant T4 in reversing the depression of such subjects appears less than T3. In depressed patients with TCA or SSRI resistance and no evidence of hypothyroidism, the data available do not establish the therapeutic role of T3 in this situation. Multicenter controlled studies of T3 adjuvant therapy are required. The possible mechanisms through which T3 adjuvant therapy might be efficacious are discussed.

Pro-thyrotropin-releasing hormone processing by recombinant PC1.

Pro-thyrotropin-releasing hormone (proTRH) is the precursor to thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH2), the hypothalamic releasing factor that stimulates synthesis and release of thyrotropin from the pituitary gland. Five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven cryptic peptides are formed following posttranslational proteolytic cleavage of the 26-kDa rat proTRH precursor. The endopeptidase(s) responsible for the physiological conversion of proTRH to the TRH progenitor form is currently unknown. We examined the in vitro processing of [3H]leucine-labeled or unlabeled proTRH by partially purified recombinant PC1. Recombinant PC1 processed the 26-kDa TRH precursor by initially cleaving the prohormone after the basic amino acid at either position 153 or 159. Based on the use of our well-established antibodies, we propose that the initial cleavage gave rise to the formation of a 15-kDa N-terminal peptide (preproTRH25-152 or pre-proTRH25-158) and a 10-kDa C-terminal peptide (pre-proTRH154-255 or preproTRH160-255). Some initial cleavage occurred after amino acid 108 to generate a 16.5-kDa C-terminal peptide. The 15-kDa N-terminal intermediate was further processed to a 6-kDa peptide (prepro-TRH25-76 or preproTRH25-82) and a 3.8-kDa peptide (preproTRH83-108), whereas the 10-kDa C-terminal intermediate was processed to a 5.4-kDa peptide (prepro-TRH206-255). The optimal pH for these cleavages was 5.5. ZnCl2, EDTA, EGTA, and the omission of Ca2+ inhibited the formation of pYE27 (preproTRH25-50), one of the proTRH N-terminal products, by 48, 82, 72, and 45%, respectively. This study provides evidence, for the first time, that recombinant PC 1 enzyme can process proTRH to its predicted peptide intermediates.