Heike Heuer

Expression of thyrotropin-releasing hormone receptor 2 (TRH-R2) in the central nervous system of rats

The distribution of the recently discovered thyrotropin‐releasing hormone (TRH) receptor subtype TRH‐R2 was studied in rat brain, pituitary, and spinal cord by in situ hybridization histochemistry and compared with the distribution patterns of the other elements of TRH signaling, namely TRH, TRH‐R1, and the TRH‐degrading ectoenzyme (TRH‐DE). In contrast to the very restricted mRNA expression of TRH‐R1 in the central nervous system, TRH‐R2 mRNA was widely distributed with highest transcript levels throughout the thalamus, in the cerebral and cerebellar cortex, medial habenulae, medial geniculate nucleus, pontine nuclei, and throughout the reticular formation. In accordance with the well‐known endocrine function of TRH, TRH‐R1 is found predominantly expressed in hypothalamic regions. Expression of TRH‐R1 in various brainstem nuclei and spinal cord motoneurons seems to be associated with the described effects of TRH on the vegetative and autonomic system as well as on the somatomotor system. Furthermore, the fully complementary expression of both receptor subtypes, even in regions where transcripts for both receptors were found (e.g., medial septum, lateral hypothalamus superior colliculi, substantia nigra, etc.), indicates that in discrete neuroanatomical pathways the two receptors serve highly specific functions for the transmission of TRH signals. Together with TRH‐DE, the putative terminator of TRH actions that shows in various, but not all, brain areas, an overlapping mRNA distribution pattern with both receptors, the distribution of TRH‐R2 mRNA seems to provide the anatomical basis for the described effects of TRH on higher cognitive functions as well as its effect on arousal, locomotor activity, and pain perception. J. Comp. Neurol. 428:319–336, 2000.

Minireview: Pathophysiological Importance of Thyroid Hormone Transporters

Thyroid hormone metabolism and action are largely intracellular events that require transport of iodothyronines across the plasma membrane. It has been assumed for a long time that this occurs by passive diffusion, but it has become increasingly clear that cellular uptake and efflux of thyroid hormone is mediated by transporter proteins. Recently, several active and specific thyroid hormone transporters have been identified, including monocarboxylate transporter 8 (MCT8), MCT10, and organic anion transporting polypeptide 1C1 (OATP1C1). The latter is expressed predominantly in brain capillaries and transports preferentially T(4), whereas MCT8 and MCT10 are expressed in multiple tissues and are capable of transporting different iodothyronines. The pathophysiological importance of thyroid hormone transporters has been established by the demonstration of MCT8 mutations in patients with severe psychomotor retardation and elevated serum T(3) levels. MCT8 appears to play an important role in the transport of thyroid hormone in the brain, which is essential for the crucial action of the hormone during brain development. It is expected that more specific thyroid hormone transporters will be discovered in the near future, which will lead to a better understanding of the tissue-specific regulation of thyroid hormone bioavailability.

Transporters MCT8 and OATP1C1 maintain murine brain thyroid hormone homeostasis

Allan-Herndon-Dudley syndrome (AHDS), a severe form of psychomotor retardation with abnormal thyroid hormone (TH) parameters, is linked to mutations in the TH-specific monocarboxylate transporter MCT8. In mice, deletion of Mct8 (Mct8 KO) faithfully replicates AHDS-associated endocrine abnormalities; however, unlike patients, these animals do not exhibit neurological impairments. While transport of the active form of TH (T3) across the blood-brain barrier is strongly diminished in Mct8 KO animals, prohormone (T4) can still enter the brain, possibly due to the presence of T4-selective organic anion transporting polypeptide (OATP1C1). Here, we characterized mice deficient for both TH transporters, MCT8 and OATP1C1 (Mct8/Oatp1c1 DKO). Mct8/Oatp1c1 DKO mice exhibited alterations in peripheral TH homeostasis that were similar to those in Mct8 KO mice; however, uptake of both T3 and T4 into the brains of Mct8/Oatp1c1 DKO mice was strongly reduced. Evidence of TH deprivation in the CNS of Mct8/Oatp1c1 DKO mice included highly decreased brain TH content as well as altered deiodinase activities and TH target gene expression. Consistent with delayed cerebellar development and reduced myelination, Mct8/Oatp1c1 DKO mice displayed pronounced locomotor abnormalities. Intriguingly, differentiation of GABAergic interneurons in the cerebral cortex was highly compromised. Our findings underscore the importance of TH transporters for proper brain development and provide a basis to study the pathogenic mechanisms underlying AHDS.

The peptide transporter PepT2 is expressed in rat brain and mediates the accumulation of the fluorescent dipeptide derivative β-Ala-Lys-Nε-AMCA in astrocytes

We describe the synthesis of a fluorescent dipeptide derivative, β‐Ala‐Lys‐Nε‐AMCA, which could be used as an excellent reporter molecule for studying the oligopeptide transport system in brain cell cultures. Fluorescence microscopic and immunocytochemical studies revealed that the reporter peptide specifically accumulated in astrocytes (type I and II) and O‐2A progenitor cells but not in neurons or differentiated oligodendrocytes. In astroglia‐rich cell culture the dipeptide derivative is taken up in unmetabolized form by an energy dependent, saturable process with apparent kinetic constants of KM = 28 μM and Vmax = 6 nmol v̄mZ h−1 v̄mZ mg protein−1 at pH 7.2. Competition studies revealed that the accumulation of β‐Ala‐Lys‐Nε‐AMCA is strongly inhibited by dipeptides and pseudopeptides such as bestatin, arphamenine A and B. The biochemical data indicated that the properties of this high‐affinity oligopeptide carrier closely resemble those of the renal peptide transport system PepT2 and Northern blot analysis demonstrated that PepT2 mRNA is expressed in glial but not in neuronal cell cultures. In situ hybridization histochemistry also revealed a non‐neuronal localization of PepT2 transcripts and a diffuse, widespread distribution of PepT2 signals throughout the entire rat brain. The selective accumulation of the fluorescent reporter molecule by brain cells under viable conditions may provide a useful tool for studying peptide uptake systems and other aspects of astroglial physiology. GLIA 25:10–20, 1999.

Prolactin-Releasing Peptides Do Not Stimulate Prolactin Release in vivo

The prolactin (PRL)-releasing activity of the novel prolactin-releasing peptides (PrRPs) was studied in vivo using male and lactating female rats. Whereas thyrotropin-releasing hormone effectively stimulated PRL and thyrotropin release as expected, PrRP in both animal models neither stimulated PRL secretion nor affected the release of other pituitary hormones. At the anterior pituitary level, in situ hybridization (ISH) histochemistry and Northern blot analysis revealed significantly higher expression levels of PrRP receptor (UHR-1) transcripts in female compared to male rats but not between lactating and nonlactating animals. By ISH, expression of UHR-1 mRNA was also detected in the intermediate lobe but not in the posterior pituitary. UHR-1 transcripts were also readily detectable in various hypothalamic brain areas whereas expression of PrRP mRNA was restricted to the ventral part of the dorsomedial hypothalamic nucleus but was not detected in neuroendocrine hypothalamic nuclei (e.g. PVN, SON). We thus assume that in the central nervous system, PrRP may likely have functions as a neuromodulator. However, together with the detailed cytochemical studies of various investigators that failed to detect PrRP-immunopositive nerve endings in the median eminence, our results strongly suggest that the hypothalamic PrRPs cannot be classified as hypophysiotrophic factors.

Region‐specific expression of thyrotrophin‐releasing hormone‐degrading ectoenzyme in the rat central nervous system and pituitary gland

Thyrotrophin‐releasing hormone (TRH), a hypothalamic neuropeptide hormone and a putative neuromodulator/neurotransmitter in the central nervous system is inactivated by the TRH‐degrading ectoenzyme (TRH‐DE), a TRH‐specific metallopeptidase localized on the surface of neuronal brain cells in culture and on lactotrophic cells of the pituitary. After succeeding in cloning the cDNA of TRH‐DE we now report on the cellular distribution pattern of this enzyme in rat brain, spinal cord and pituitary gland using in situ hybridization histochemistry. In the pituitary, TRH‐DE mRNA was found both in the anterior and the neural lobe but not in the intermediate lobe. After treatment with triiodothyronine (T3) a dramatic increase in the mRNA levels of the TRH‐DE and a decrease in the intensity of the TRH receptor could be observed in the anterior lobe of the pituitary. In brain, TRH‐DE transcripts were predominantly found in neo‐ and allocortical regions with strongest signals in the olfactory bulb, the piriform cortex, the cerebral cortex, the granular layer of the cerebellar cortex and the pyramidal cells of the Ammons horn. In the diencephalon, the highest TRH‐DE mRNA levels were observed in the medial habenulae followed by several hypothalamic subregions. In the mesencephalon and brainstem, moderate signals were present in the superior colliculi, substantia nigra, dorsal raphe and in the periolivar region. In the spinal cord, TRH‐DE mRNA positive neurons were present in all layers. The very distinct distribution of TRH‐DE in the brain and the hormonal regulation of the adenohypophyseal enzyme support the concept that this peptidase serves very specialized functions.

Gene expression of connective tissue growth factor in adult mouse

The connective tissue growth factor (CTGF) is a well-known fibroblast mitogen and angiogenic factor that plays an important role in bone formation during embryogenesis. In the adult, CTGF is involved in wound healing as well as fibrotic and vascular disease. However, little is known about its physiological functions under non-pathological conditions in the adult organism. Here, we describe the cellular site of the CTGF mRNA expression in adult male and female mice as revealed by in situ hybridization histochemistry. Strong and persistent CTGF gene expression was particularly prominent in the mesenchyme of the cardiovascular system (aorta, auricular tissue, renal glomeruli), the mesenchyme surrounding the ovarian follicles or the testicular tubes in the gonadal tissue, and the subcapsular mesenchyme bordering densely innervated parts of whisker hair vibrissae. CTGF hybridization signals were not observed in the mesenchyme of many other organs including gut, muscle, liver or most parts of the lymphatic tissue. Strong expression was also present in the primary (early) ovarian follicles, the epithelium of the deep uterine glands and on myenteric ganglia neurons. These data suggest a selective and continuous mesenchymal function in the gonads and those tissues attracting very strong vascular supply or peripheral innervation. CTGF may also be involved in the cyclical proliferation of the uterine gland epithelium and in the early stages of follicular maturation, as well as in the neuropeptide regulation in the gut, cardiovascular and renal systems.

Thyrotropin Releasing Hormone (TRH), the TRH-Receptor and the TRH-Degrading Ectoenzyme; Three Elements of a Peptidergic Signalling System

The isolation and structural identification of thyrotropin releasing hormone (TRH) in 1969 (Boler et al. 1969; Burgus et al. 1969; see also Guillemin 1978; Schally 1978) ushered in the new area of modern neuroendocrinology because for the first time a hypothalamic hypophysiotrophic hormone had been deciphered at the molecular level. This discovery opened new avenues for studying the ever interesting question “How does the brain talk to the body?”. Since science can be defined as “knowledge put in order” the scientific milestones leading to this highlight of discovery should be acknowledged.

Analysis of the thyrotropin-releasing hormone-degrading ectoenzyme by site-directed mutagenesis of cysteine residues

Thyrotropin‐releasing hormone‐degrading ectoenzyme is a member of the M1 family of Zn‐dependent aminopeptidases and catalyzes the degradation of thyrotropin‐releasing hormone (TRH; Glp‐His‐Pro‐NH2). Cloning of the cDNA of this enzyme and biochemical studies revealed that the large extracellular domain of the enzyme with the catalytically active site contains nine cysteine residues that are highly conserved among species. To investigate the functional role of these cysteines in TRH‐DE we used a site‐directed mutagenesis approach and replaced individually each cysteine by a serine residue. The results revealed that the proteolytically truncated and enzymatically fully active enzyme consists of two identical subunits that are associated noncovalently by protein–protein interactions but not via interchain S‐S bridges. The eight cysteines contained within this region are all important for the structure of the individual subunit and the enzymatic activity, which is dramatically reduced in all mutant enzymes. This is even true for the four cysteines that are clustered within the C‐terminal domain remote from the Zn‐binding consensus sequence HEICH. In contrast, Cys68, which resides within the stalk region seven residues from the end of the hydrophobic membrane‐spanning domain, can be replaced by serine without a significant change in the enzymatic activity. Interestingly, this residue is involved in the formation of an interchain disulfide bridge. Covalent dimerization of the subunits, however, does not seem to be essential for efficient biosynthesis, enzymatic activity and trafficking to the cell surface.

TRH Action Is Impaired in Pituitaries of Male IGSF1-Deficient Mice

Loss-of-function mutations in the X-linked immunoglobulin superfamily, member 1 (IGSF1) gene cause central hypothyroidism. IGSF1 is a transmembrane glycoprotein of unknown function expressed in thyrotropin (TSH)-producing thyrotrope cells of the anterior pituitary gland. The protein is cotranslationally cleaved, with only its C-terminal domain (CTD) being trafficked to the plasma membrane. Most intragenic IGSF1 mutations in humans map to the CTD. In this study, we used CRISPR-Cas9 to introduce a loss-of-function mutation into the IGSF1-CTD in mice. The modified allele encodes a truncated protein that fails to traffic to the plasma membrane. Under standard laboratory conditions, Igsf1-deficient males exhibit normal serum TSH levels as well as normal numbers of TSH-expressing thyrotropes. However, pituitary expression of the TSH subunit genes and TSH protein content are reduced, as is expression of the receptor for thyrotropin-releasing hormone (TRH). When challenged with exogenous TRH, Igsf1-deficient males release TSH, but to a significantly lesser extent than do their wild-type littermates. The mice show similarly attenuated TSH secretion when rendered profoundly hypothyroid with a low iodine diet supplemented with propylthiouracil. Collectively, these results indicate that impairments in pituitary TRH receptor expression and/or downstream signaling underlie central hypothyroidism in IGSF1 deficiency syndrome.