The following article was published in the Newsletter of the Department of Medicine, Brown Medical School, October 2001, volume 3, issue 4

Neuroendocrinology Research in Dr. Eduardo A. Nillni’s Laboratory

Dr. Nillni’s laboratory is located in the Pierre M. Galletti research buildings at the Rhode Island Hospital Research complex. His laboratory personnel includes Dr. Charles Vaslet, an investigator in the Department of Pathology and Medicine, Brown University, two post-doctoral fellows Dr. Gregory Fox (Pediatric Endocrinology) and Dr. Elisabeth Volkstorf and Ronald Stuart, the Laboratory Manager. Also, Michelle Jurofcik, a graduate student from the Molecular Biology, Cell Biology & Biochemistry Graduate Program at Brown University, three undergraduate students, Alison Lee, Soraya Azari and Alissa Weinberg from Brown University, and Carla Levy, an international student. In addition, Dr. Nillni has an active collaboration with other scientists from the following institutions: the Division of Endocrinology at Beth Israel Hospital and Harvard Medical School; Maryland Psychiatric Research Center University of Maryland School of Medicine; Division of Endocrinology at the New England Medical Center Hospitals; Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal; Department of Psychiatry and Behavioral Sciences, Duke University Medical Center; Department of Psychiatry, Yale University; and Millennium Pharmaceurticals, Inc.

Dr. Nillni’s research is funded by the National Science Foundation, National Institute of Health, and recently has initiated a collaborative work with Millennium Pharmaceuticals, Inc. Since he joined the Brown faculty twelve years ago his research efforts were devoted to study the biosynthesis of neuropeptides, their regulation and biologic function.

In recent years considerable research has focused on the expression of neuropeptide genes and their tissue-specific regulation. However, it has become clear that the peptides derived from these genes play significant neuromodulatory roles in the control of other neuropeptides within the central nervous system (CNS) as well as endocrine cells outside the CNS. Even more astounding is the discovery that multiple neuropeptides with distinct physiological functions arise from the processing of single polypeptide precursors. Thus, to fully understand the biology of a neuropeptide, one must understand the post-translational processing of the preprohormone gene product, as well as the regulation of the gene’s transcription. How do cells produce different levels of one peptide with respect to another when both derive from the same protein sequence? This is achieved through differential processing and degradation by the action of specific processing enzymes acting in specific cellular and extracellular compartments. Post-translational processing of hormone precursor proteins is a critical mechanism by which cells increase their biological and functional diversity, such that two or more peptides with different biological functions originate from the same precursor.

Dr. Nillni’s laboratory has pioneered and consolidated in great part the current knowledge on the neurobiology of proThyrotropin Releasing Hormone (proTRH), a precursor protein to TRH (pyroGlu-His-ProNH₂). The biosynthesis of TRH and other proTRH-derived peptides follows the same processing mechanisms described for other secretory peptides, beginning with mRNA-directed ribosomal translation, followed by post-translational limited proteolysis of the larger precursor, proTRH. This process occurs while proTRH is transported from the transGolgi network to newly formed immature secretory granules. These granules then mature and are targeted to sites of secretion at the plasma membrane of the cell. Cleavage of the TRH precursor to generate biologically active TRH occurs at paired basic residues by the action of two members of the recently discovered family of prohormone convertases 1 and 2 (PC1 and PC2) followed by the action of carboxypeptidase E.

TRH, produced in the paraventricular nucleus of the hypothalamus (PVN), regulates the biosynthesis and secretion of thyroid-stimulating hormone (TSH) from the anterior pituitary. TSH in turn stimulates thyroid hormone biosynthesis and release. TRH is central in regulating the hypothalamic-pituitary-thyroid (HPT) axis. TRH influences the release of other hormones, including prolactin, growth hormone, vasopressin and insulin, and the classic neurotransmitters noradrenaline and adrenaline. Further, TRH is present in many brain loci outside of the hypothalamus, supporting a potential role as a neuromodulator or neurotransmitter outside of traditional HPT axis function. For example, TRH is implicated as a modulator of seizure activity and gastrointestinal function. TRH has been also found outside the central nervous system (CNS) in the gastrointestinal tract, pancreas, reproductive tissues including placenta, ovary, testis, seminal vesicles and prostate, retina, and blood elements. The widespread distribution of TRH within and outside the CNS supports a diverse range of roles for this molecule, roles likely to involve many functions outside of the traditional HPT axis.

The function of the thyroid gland is to produce the thyroid hormones, T3 and T4, which regulate gene transcription through binding to a family of nuclear receptors in cells throughout the body. In medical practice, the thyroid becomes a problem when it’s size or shape is abnormal, or when it produces too much or too little hormone. Thus, we typically think of the thyroid through the clinical states of goiter, or hyper or hypothyroidism. However, what is the physiology of the thyroid gland under conditions where the entire HPT axis is intact? This gland provides an unchanging basal level of hormone to keep the basic metabolic rate of all cells at a constant level.

Is this static view of thyroid physiology correct? Certainly not. A common situation in which thyroid hormone levels are subject to major physiologic modification is during the transition from the fed to the starved state. In the well studied rodent model, starvation produces a rapid suppression of the levels of T4 and T3, to an extent that suggests that a substantial effect upon thyroid dependent processes will result. What purpose might such suppression serve? Starvation is a severe threat to survival, and in rodents, the capacity to survive without nutrition is measured in days. Since thyroid hormone is best known for its ability to set the basal metabolic rate, a suppression of thyroid hormone and attendant reduction of metabolic rate would reduce the obligatory use of energy stores. Therefore, thyroid hormone is an important regulator of energy expenditure. Assuming that hypothyroidism did not impair the ability to obtain food, this adaptation would be expected to enhance survival. Since animals in the wild are thought to commonly experience periods of starvation, the thyroid response to starvation should be viewed as a major aspect of the regulatory biology of the thyroid gland.

How then is this nutritional adaptation regulated? As mentioned above the thyroid system is regulated downstream by TRH produced in the PVN and stimulating TSH produced in the pituitary. TSH acts on receptors on the thyroid to promote synthesis and release of the thyroid hormones T4 and T3. Starvation suppresses hypophysiotropic preproTRH gene expression in the region of hypothalamus (paraventricular nucleus) that is involved in control of the pituitary TSH-producing cells. This lowers TSH production, and appears also to reduce the bioactivity of TSH through altering its glycosylation. As a consequence of reduced bioactive TSH, T4 and T3 levels fall. Thus, starvation in rodents is a cause of “central hypothyroidism”.

But what is the signal to the brain that coordinates this adaptation? The signal to the brain that suppresses TRH expression in the PVN is the fall in levels of the hormone leptin. Leptin is a hormone produced principally in adipose tissue, whose central physiologic role is to provide information on energy stores and energy balance to brain centers that regulate appetite, energy expenditure and neuroendocrine function. To effectively deliver this information, leptin must reach its central targets, and engage receptors on specific hypothalamic neurons. The output of these neurons is then integrated with other signals, ultimately engaging final effector pathways. When leptin signaling is deficient, due either to mutation of the leptin hormone or leptin receptor genes, severe obesity results in both rodents and humans, underscoring the fundamental role of leptin in physiology. Some data suggest that the action of leptin on TRH neurons in the PVN of the hypothalamus occur through an indirect pathway involving the arcuate nucleus (AC) of the hypothalamus. This neuronal center releases neuropeptides such as neuropeptide Y (NPY), a-melanocyte stimulating hormone, (a-MSH) and AgRP that are leptin responsive and that project to the PVN where the hypophysiotropic neurons producing proTRH are located. However, a new line of work done recently in our laboratories strongly suggests that TRH neurons may also be regulated directly by leptin without intermediate pathways. Nillni’s laboratory provided the first direct evidence for leptin-mediated regulation of preproTRH mRNA expression or TRH prohormone processing. Although anatomical observations have implicated other neuropeptides NPY, a-MSH and AgRP peptides in regulation of the HPT axis, the stimulation of TRH neurons by a-MSH is also shown for the first time in his studies. Taken together his recent work suggests that leptin may regulate TRH neurons via both indirect pathways (a-MSH and NPY), and direct pathways via leptin receptors expressed on TRH neurons in the PVN, leading to regulation of both TRH expression and processing.

In addition, his laboratory has contributed to a better understanding of how other neuro-endocrine inputs such as norepinephrine and other hormones glucocorticoids affect proTRH gene regulation, its biosynthesis and processing. His laboratory also promoted the discovery of novel proTRH-derived peptides with potential biologic function(s). In physiological models, his laboratory has shown the effect that lactation has on hypothalamic proTRH processing, and discovered a proTRH-derived peptide (preproTRH_178-199) that is derived from proTRH processing and it is capable of inducing prolactin secretion in pituitary cells. Other studies involved the impact of cold stress on thermoregulation in the fat/fat mouse, which is obese, diabetic and infertile and, has a deficient level of hypophysiotropic TRH. In new studies his laboratory demonstrated that a distinct proTRH-derived peptide, preproTRH_83-106 increases in the periaquaductal gray matter during opiate withdrawal.

Understanding of the role of proTRH-derived peptides represents an exciting new frontier in proTRH research. During biosynthesis, these sequences within the precursor may function as structural or targeting elements that guide the folding and sorting of proTRH and its larger intermediates so that subsequent processing and secretion is properly regulated. The unique anatomical distribution of the proTRH end products, as well as regulation of their levels by neuroendocrine or pharmacological manipulations, argues that these peptides will have unique biological roles. Some of these roles will be within the HPT axis, while many others will be unrelated to traditional thyroid function. An extensive array of studies indicates that TRH can function far beyond the HPT axis, and should command significant future effort as a focus to develop new therapeutics. These therapeutics, in the form of TRH analogues or nonprotein peptidomimetics, and perhaps using novel delivery systems, will advance our ability to develop TRH and nonTRH proTRH-derived peptide agonists and antagonists that can target proTRH-derived peptides functioning in specific tissues or brain loci. It is hoped that these new drugs might provide novel treatment approaches for some of today’s most difficult health and societal issues, including drug abuse, depression, chronic pain disorders, and consequences of CNS injury.