I. INTRODUCTION

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The hypothalamo-neurohypophysial system (HNS), this unique collection of diverse peptidergic neurons of the hypothalamus with its major axonal endings in the neurohypophysis, has guided physiological research to novel concepts for over 100 years. This guiding role is evident from our present understanding of neurosecretion, including biosynthesis of neuropeptides and electrophysiology, and of neuroendocrinology, including integration and communication between brain and periphery. The essence of these concepts has been discovered through investigation of the HNS, while proof of principle has been obtained from many other peptidergic systems. In physiological research today, the HNS contributes particularly to our understanding of the way peptidergic systems function at the molecular and cellular level. It tells us about the molecular make-up of a defined neuronal system, the developmental and regulatory functions of specific genes, and the integration of physiological signals to responses at the level of gene regulation. With the total overview of all our genes near, it is to be expected that the HNS will remain to serve as a guiding system to define functions of individual genes it expresses and will allow us to understand further the role and dynamics of gene expression programs during development and physiological functioning of a neuronal system. Here we review the current state of affairs about the HNS at the level of the HNS gene expression and function and their contribution to the physiological functioning of the HNS.

In the 19th century, endocrinology had become a novel discipline among physiologists. The pituitary gland appeared a rich source of preparations that could elicit a variety of physiological responses when administered to experimental animals. The traditional endocrinologists encountered a conceptual problem when studying extracts with vasopressor (578), antidiuretic (211, 835), oxytocic (166) (from  = rapid; o = birth) and milk-ejection activities (589). These were all located in the neural lobe of the pituitary gland, which consisted of neural elements rather than being glandular like the anterior lobe. These biological activities were, in the words of Sir Henry Dale (167) "... as complete a proof of a normal endocrine function for the neurohypophysis as any which has been given, or indeed, has since been given, for any other organ." Although this notion was accepted, it remained largely unexplained for several decades. Similarly, the vasopressor and antidiuretic activities could be separated from oxytocic and milk-ejecting activities (375), but the nature of the substances, although then already suspected to be peptides, remained elusive for several decades. In the meantime these preparations were essential instruments in the discovery of novel physiological concepts and disease in humans. In particular, in the field of kidney physiology and water homeostasis, antidiuretic preparations appeared to be crucial tools (826, 827; reviewed in Refs. 167, 225, 308, 692). During this period, all elementary physiological properties of the antidiuretic activity and responses of the posterior pituitary gland were discovered that we employ today in current research on the HNS.

In the hunt for "releasing factors" in the 1960s, it was noted that extirpation of the posterior pituitary gland not only affected water homeostasis, but also caused behavioral alterations that could be corrected by replacement of vasopressin (183). De Wied's vision that vasopressin and derivatives without endocrine activity could act directly on the brain led to the "neuropeptide concept." This concept encompasses the physiological functions of peptide neural communication and modulation of brain activity (184).

Although neurohypophysial extracts in which antidiuretic and oxytocic activities had been separated (375) were available commercially, e.g., Pitressin or Pitruitin (Parke-Davis) in the 1930s, it was not until the early 1950s that the chemical structure of the peptides responsible for the physiological activities could be identified (192, 193). The discovery and structural elucidation of the nonapeptides vasopressin (VP) and oxytocin (OT) was highly acknowledged; Du Vigneaud received the Nobel Prize for his achievements in 1955. With knowledge of the chemical structure, the route to chemical synthesis and design of analogs was now open and was highly exploited in the years to follow up to today (67, 485).

At the same time, other biochemical studies had identified longer peptides that often carried the biological activities in neurohypophysial extracts (797). This work led ultimately to another milestone, i.e., the elucidation of the biosynthetic pathway of VP and the prohormone concept. The complex termed "Van Dyke protein" was identified to contain VP carried by an associated protein, now known as neurophysin (NP) (2). Soon after, the common origin for VP and NP was proposed from studies on the biosynthesis of VP (654). Furthermore, the dynamic and hormone-NP interactions became elucidated. This culminated in understanding how peptide and NP binding promotes condensation of biosynthetic material and in the physical structure of the complex (643). Now we know that all biologically active peptides are biosynthesized from a precursor protein. Most if not all enzymes that process prohormones into the biologically active parts are also known.

The anatomical structure of the neurohypophysis, being typical for a neural element and very different from the glandular appearance of other endocrine organs, remained exceptional and unexplained to endocrinologists until Ernst Scharrer postulated the concept of production and release of hormones by neurons, which he termed "neurosecretion" (676). This concept was received with great skepticism, and although examples from a variety of vertebrate and invertebrate species accumulated (677), it was not fully appreciated until Bargmann was able to apply Gomori's pancreas staining to the hypothalamus to reveal neurons that had the appearance of the typical hormone-producing cells, which were so well known in pancreas and anterior pituitary gland (56). Moreover, fiber tracts from these neurosecretory cells appeared to innervate the neurohypophysis. Bargman and Scharrer formulated the anatomical structure that we now know as the HNS and proposed this structure to be responsible for the biosynthesis of the hormonal entities of the neurohypophysis (57). Early biochemical studies showing hypothalamic synthesis and transport of neurosecretory material supported this notion (720).

The Gomori staining employs chrome-alum-hematoxylin (273) or aldehyde fuchsine (274), which involves oxidized disulfide bonds in proteins. These are highly represented in VP, OT, and their NPs. Refinement of staining for oxidized disulfide bonds improved sensitivity and specificity, for instance, by using pseudo-isocyanine as in the work of Sterba (740), but the application of immunohistochemical (IHC) techniques provided the final answers on the anatomy of the HNS.

	II. THE HYPOTHALAMO-NEUROHYPOPHYSIAL SYSTEM

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A.  Organization and Connections

1.  Magnocellular nuclei of the hypothalamus

Classically, the hypothalamo-neurohypophysial neurosecretory system is defined as consisting of large neurons (20-40 µm cell body diameter) of the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei that have axons terminating on the blood capillaries of the posterior pituitary neural lobe (677). The use of immunocytochemistry (621, 728) and injection of retrograde tracers into the neural lobe, the MCNs (379, 700) have since defined accessory magnocellular cell groups in the preoptic area, the lateral hypothalamus, regions near the anterior commissure and the third ventricle, the perifornical nucleus, and the nucleus circularis. The SON is wholly magnocellular, while the PVN is divided into a lateral magnocellular subdivision and a more medial parvocellular subdivision. Parvocellular OT and VP neurons have smaller cell bodies (10-15 µm) and project to the median eminence (796), brain stem and spinal cord (753), as well as limbic and olfactory areas (108, 314, 729). Parvocellular neurons have different functions to MCNs, and it is not known whether their major genes are regulated by the same mechanisms. The phenotype of null mutants for several transcription factors (see sect. IVA) indicated that parvocellular neurons expressing corticotrophin- releasing hormone (CRH), thyrotropin-releasing hormone (TRH), and somatostatin may have a developmental origin common to MCNs. This review focuses on the magnocellular HNS. Here it is essential to realize that the magnocellular HNS is by no means a single system. It consists of different MCNs that can produce different products and have different anatomical positions.

The architecture of the HNS has been described in greatest detail for the rat and is exemplary summarized here. OT and VP MCNs are found intermingled in the magnocellular nuclei, although there is some topographical segregation (621, 728). Within the SON, OT MCNs are mainly rostral and dorsal, whereas VP MCNs are found mainly caudal and ventral. The unmyelinated, varicose axons of the MCNs leave the SON dorsomedially and then turn caudally to pass through the dorsal portions of the internal layer of the median eminence and pituitary stalk before reaching the neural lobe (16). As they leave the SON, the axons give rise to collaterals, many of which terminate close to the nucleus where they may contact lateral cholinergic neurons (304, 427, 496). In the magnocellular subdivision of the PVN, OT MCNs are predominantly rostral, whereas VP MCNs are more caudal and lateral. Their axons leave the PVN laterally, then course ventrally and medially over the SON, and pass through the ventral portion of the median eminence and pituitary stalk (16). Collaterals of PVN MCNs terminate within the nucleus or in the nearby perifornical region (304, 495). There is electrophysiological evidence to suggest that some magnocellular axons that terminate in the neural lobe may also give off collaterals to the median eminence, medial amygdaloid nucleus, and lateral septum (603). VP (551) and OT terminals (758) have been demonstrated contacting MCNs containing the same peptide, although the origin of these terminals, and their incidence, remains unclear.

MCNs of the SON have oval cell bodies with one to three thick dendrites that extend toward the ventral surface of the brain (44, 195), although some do project dorsally (575). At the surface of the brain, the dendrites turn in a rostrocaudal direction and can extend for over 200 µm within a ventral lamina of glial cell processes (195). The MCNs of the PVN are essentially the same in morphology, although their dendrites extend medially through the parvocellular subdivision of the nucleus toward the ependyma of the third ventricle (45, 788). In contrast to the SON, which is fairly homogeneous in that there are no other neuronal cell bodies in the nucleus, the magnocellular PVN contains a number of interneurons, at least some of which are GABAergic (51).

The soma and dendrites of the MCNs are sometimes directly adjacent to those of others, although normally interposed between them are thin astrocytic glial processes. During times of high hormone demand, alterations occur in the architecture of the magnocellular nuclei, bringing cell bodies and dendrites into direct contact and increasing synaptic inputs to the neurons (306, 760). MCNs make somatic and dendritic contacts via gap junctions, thus producing cytoplasmic coupling. The extent of this coupling varies with the structural plasticity within the magnocellular nuclei (306).

The varicose appearance of magnocellular axons is due to the presence of large swellings, which, in the neural lobe of the pituitary, bud to form palisades of terminals along the pericapillary basal lamina (541, 777). This, together with the fact that axons and swellings are themselves capable of releasing hormone (543), has left the definition of terminals in the neural lobe dependent only on their relative size and the presence of translucent microvesicles in addition to the hormone-containing electron-dense granules. Immunohistochemical examination at the light microscope level suggests that VP fibers enter the central region of the neural lobe of the pituitary gland, also termed neurohypophysis or posterior pituitary gland, while OT fibers enter the periphery of the gland (802). However, the fibers become dispersed and terminals of the two cell types can be found intermingled throughout the neural lobe (458). The neuronal elements of the pituitary are, as in the hypothalamic nuclei, interposed by the processes of resident astroglia, the pituicytes (869). The interrelationships of the magnocellular nerve endings, pituicytes, and capillaries also change dramatically during times of increased hormone secretion (306, 760).

It is now generally accepted that the HNS forms part of an osmoresponsive circuit with three structures within the lamina terminalis, the front wall of the third ventricle that defines the anterior extent of the hypothalamus (89, 330, 429, 501). In addition to direct projections from the subfornical organ (SFO) and the organum vasculosum of the lamina terminalis (OVLT), the HNS also receives indirect input from these via the median preoptic nucleus (MnPO; Fig. 1) (501, 525, 622, 661, 671, 690, 775, 863). Tract tracing has revealed that single neurons of the lamina terminalis can project to more than one of the magnocellular nuclei, providing the connections for concerted HNS responses (852). Finally, electrophysiological studies have also provided evidence for connections back to the lamina terminalis from the HNS, perhaps by collateral excitation of interneurons, thus completing the circuit (330).

View larger version (20K): [in this window] [in a new window]   Fig. 1. Major afferent inputs to the hypothalamo-neurohypophysial system (HNS). Schematic diagram showing the major afferent inputs to the oxytocin (OT) and vasopressin (VP) magnocellular neurons of the HNS. DBB, diagonal band of Broca; LC, locus coeruleus; MnPO, median preoptic nucleus; NTS, nucleus of the tractus solitarius; OB, main and accessory olfactory bulbs; OVLT, organum vasculosum of the lamina terminalis; PNZ, perinuclear zones adjacent to the supraoptic and tuberomammillary nuclei; VLM, ventrolateral medulla.

Although MCNs are themselves osmosensitive (88, 494), they require input from the lamina terminalis to respond fully to osmotic challenges (428, 330). Neurons in the lamina terminalis are also osmosensitive (89), and because the SFO and OVLT lie outside the blood-brain barrier, they can integrate this information with endocrine signals borne by circulating hormones, such as angiotensin II, relaxin, and atrial natriuretic peptide (516, 585). While circulating angiotensin II and relaxin excite both OT and VP MCNs, atrial natriuretic peptide inhibits VP neurons (221, 736, 847). In addition to an angiotensinergic path from the SFO (360), the OVLT and MnPO provide direct glutaminergic and GABAergic projections to the HNS (565, 888) (Fig. 1). Thus, because inputs from the lamina terminalis can be either excitatory or inhibitory, the view is that the osmoresponsive circuit increases the overall sensitivity of the HNS to osmotic stimuli. Furthermore, a role for nitric oxide (NO) acting throughout the forebrain osmoresponsive circuit to modulate neurohormone release has been suggested (456, 463).

A fall in arterial blood pressure produces a secretion of VP due to an inhibition of baroreceptors in the aortic arch and activation of chemoreceptors in the carotid body (691). Afferents from these receptors terminate in the dorsal medulla oblongata of the brain stem, including the nucleus of the tractus solitarius (NTS; Fig. 1). The resting inhibitory effect of baroreceptor activity on VP neurons is mediated indirectly via the A6 noradrenergic cell group of the locus coeruleus, the diagonal band of Broca in the forebrain and neurons lying just outside the magnocellular nuclei (76, 361, 362). Release from this inhibition after hemorrhage or direct electrical stimulation of the NTS causes the activation of VP neurons via the stimulatory A1 noradrenergic cell group of the ventrolateral medulla (175, 611). The A1 projects directly to VP MCNs to stimulate hormone release via ₁-adrenoreceptors (174).

The NTS appears to act also as an important relay for pathways ascending to OT MCNs. Whereas electrical stimulation of the NTS leads to indirect activation of VP neurons via the A1 cell group of the ventrolateral medulla, there is a direct excitatory input to OT neurons that is mediated, at least in part, by the A2 noradrenergic cell group lying within the NTS (175, 611) (Fig. 1). One stimulus that has been used to investigate this pathway is systemic administration of the peptide cholecystokinin (CCK) (171). Physiologically, this octapeptide activates CCK-A receptors on endings of the vagus nerve following distension of the gut after a meal leading to the release, at least in the rat, of OT but not VP into the bloodstream from the HNS (462, 618). With the use of c-fos and tyrosine hydroxylase immunocytochemistry combined with tract tracing, it has been possible to show that this information is relayed in A2 neurons of the NTS that project directly to the magnocellular nuclei of the hypothalamus (109, 429, 581, 628).

Activation of catecholaminergic neurons in the NTS has been associated with a number of other stimuli that cause the release of OT, including administration of nausea-producing agents (580), stress, interleukin-1 (206, 417), and nicotine (499). Furthermore, A2 neurons of the NTS are active during parturition (454) and suckling (725) and are, thus, possibly part of the pathway from the uterus and mammary glands to the HNS (584). These observations suggest that catecholaminergic neurons of the NTS may integrate information from a number of sources. However, it is not apparent whether this involves subsets of NTS neurons. Neuropeptide Y- and somatostatin-containing neurons are activated following CCK (817), while inhibin neurons appear to mediate at least part of the suckling stimulus (670).

There are a number of other inputs to the HNS, although the relevance of these is largely unknown (Fig. 1). They include scattered inputs from other hypothalamic nuclei, the preoptic area, septum, and limbic structures (431, 575, 662, 671, 757). The SON receives direct and indirect afferent information from both the suprachiasmatic nucleus (SCN) and the retina (159, 160). These inputs, together with the influence of the pineal gland on hormone release (867), provide substrates for the diurnal regulation of HNS function. There are well-documented direct projections from histamine neurons of the mammillary nuclei to the HNS (205, 853). These involve inputs to VP MCNs via histamine H₁ receptors that are electrically excitatory and inputs to OT MCNs via H₂ receptors that are electrically inhibitory (889). Interestingly, histamine administered intracerebroventrically induces c-fos in both OT and VP MCNs and increases the expression of both neurohormone genes (387, 388), but this may not be a direct effect. Roles for the histaminergic input have been implied during pregnancy and parturition (464), lactation (674), dehydration (386, 389), and novelty stress (881). Similarly, direct afferents from the main and accessory olfactory bulbs to the SON have been described (727, 726), the electrical stimulation of which can, like stimulation of the histaminergic input, lead to increased dye coupling between MCNs (306).

B.  Cell Biology

1.  Overview: the HNS as cell biological model

Over the past 20 years, neuropeptides have become increasingly prominent as intercellular messengers in the peripheral and central nervous system, acting as neurohormones, neuromodulators, neurotrophic factors, and/or neurotransmitters (327, 394, 742, 911). In contrast to "conventional" neurotransmitters (e.g., acetylcholine, excitatory and inhibitory amino acids, monoamines, etc.), which are synthesized in nerve terminals and can be packaged locally in recycled small vesicular membranes, the biosynthesis and secretion of neuropeptides such as OT and VP in the HNS requires continual de novo transcription and translation of peptide precursor proteins. The MCNs of the HNS have become prototypical of peptidergic neurons (Fig. 2). These precursor proteins must then be sorted via the Golgi apparatus to the "regulated secretory pathway," packaged into large dense core vesicles (LDCVs), in which they are posttranslationally processed to the biologically active peptides and axonally transported to nerve terminals before peptide secretion can occur. The secretion process itself may differ in detail between the small clear-cored synaptic vesicle- and LDCV-based secretion systems (825). Different isoforms of secretion-associated vesicular and membrane proteins often appear to be used in each (60, 253, 493), and secretion from LDCVs is rarely, if ever, evoked in response to single action potentials, and typically requires trains (bursts) of impulses to evoke peptide secretion (256, 467). Because conventional neurotransmitter and neuropeptide secretion very often coexist in the same neuron, it is clear that the mechanisms for both processes also must coexist in neurons throughout the nervous system (406).

View larger version (35K): [in this window] [in a new window]   Fig. 2. Peptidergic neuron. Cellular and molecular properties of a peptidergic neuron (neurosecretory cell) are shown. The structure of the neurosecretory cell is depicted schematically with notations of the various cell biological processes that occur in each topographic domain. Gene expression, protein biosynthesis, and packaging of the protein into large dense core vesicles (LDCVs) in the cell body, where the nucleus, rough endoplasmic reticulum (RER), and Golgi apparatus are located. Enzymatic processing of the precursor proteins into the biologically active peptides occurs primarily in the LDCVS (see inset), often during the process of anterograde axonal transport of the LDCVS to the nerve terminals on microtubule tracks in the axon. Upon reaching the nerve terminal, the LDCVS are usually stored in preparation for secretion. Conduction of a nerve impulse (action potential) down the axon and its arrival in the nerve terminal causes an influx of calcium ion through calcium channels. The increased calcium ion concentration causes a cascade of molecular events (see inset and text) that leads to neurosecretion (exocytosis). Recovery of the excess LDCV membrane after exocytosis is performed by endocytosis, but this membrane is not recycled locally, and instead is retrogradely transported to the cell body for reuse or degradation in lysosomes. TGN, trans-Golgi network; SSV, small secretory vesicles; PC1 or PC2, prohormone convertase 1 or 2, respectively; CP-H, carboxypeptidase H; PAM, peptiylglycine -amidating monooxygenase. [Adapted from Gainer and Chin (253).]

The MCNs of the HNS, which synthesize and secrete the nonapeptides OT and VP, represent a specialized class of peptidergic neurons called neurosecretory cells (APUD neuroendocrine cells or endocrine neurons), first described over 70 years ago in the fish central nervous system by Ernst Scharrer (675). The HNS neurons have served as important model systems for the study of peptide neurosecretion mechanisms in vivo, in large part due to their relatively compact nuclear organization in the CNS and physically distinct terminal field. The HNS neurons project via a well-defined axonal tract to the neural lobe where each axon is estimated to branch into hundreds of nerve terminals (302). These axonal branches and terminals have been estimated to represent ~50% of the total tissue mass of the neural lobe. The relatively easy access to the HNS cellular components, the cell bodies in the PVN and SON and axons in the median eminence by both stereotaxic and micropunch assay methods, and the nerve terminals by their presence outside of the blood-brain barrier in the posterior pituitary, have made these MCNs favorite subjects for many biochemical, morphological, and physiological studies (42, 43, 103, 256, 302, 303). Perhaps the most important virtue of the HNS as a cell-biological model is its requirement for very high rates of peptide biosynthesis and secretion. Because the OT and VP peptides are secreted directly into the bloodstream to act on distant target organs, the mammary gland, and kidney, respectively, substantial quantities of these peptides are secreted to compensate for their dilution in the general circulation. Consequently, transcriptional rates and mRNA levels for these peptides are very high in the HNS neurons, and peptide secretion from LDCVs in the neural lobe is exceptionally robust, thereby allowing for the analyses of all the cell biological aspects of neurosecretory mechanisms in an experimentally favorable context.

The gene organization of VP and OT genes is peculiar. They are located within the same chromosomal locus on a chromosome at a very short distance from each other (3-11 kb) in a head-to-head orientation (351, 534, 627, 745) (Fig. 3). Although the exon organization is simple, the genomic control regions are complex and likely dependent on large parts of the locus (374). Notably, in the VP-OT locus of the rat, long interspersed repeated DNA elements (LINEs) are present and transcribed. Expression predominates in the brain (681). A role of these LINEs in the expression of the VP and OT genes is not known. The VP gene consists of three exons and comprises ~2 kb. The structural organization of the VP gene is very similar to the OT gene. The first exon of the VP and OT gene, exon A, codes for a signal peptide, the hormone (VP or OT, respectively), a three-amino acid spacer (-Gly-Lys-Arg-), and the first nine NH₂-terminal amino acids of NP (NP-I or NP-II, respectively). Exon B codes for the highly conserved mid-portion of NP (amino acids 10-76), and exon C of the VP gene codes for the remaining COOH-terminal amino acids of NP, a monobasic cleavage site (-Arg-) and the COOH-terminal glycopeptide (GP) of 39 amino acids (Fig. 4). In contrast to the VP gene, exon C of the OT gene only codes for the COOH-terminal amino acids of OT-associated NP-I.

View larger version (23K): [in this window] [in a new window]   Fig. 3. Regulatory domains in the vasopressin (VP)-oxytocin (OT) locus. Structure of the rat and bovine VP and OT transgenes that have been studied in mice and rats is shown. The transgene structures are compared with the structure of the rat and mouse VP-OT locus. Rat transgenes are shown above the locus map, bovine transgenes are shown below. The horizontal arrows indicate the direction of transcription. Stippled boxes represent exons. Expression patterns for either VP (left) or OT (right) are described. HYP, hypothalamus; TEST, testis.

View larger version (13K): [in this window] [in a new window]   Fig. 4. Schematic representation of the vasopressin prohormone and the established diabetes insipidus mutations. The three moieties of the prohormone (VP, NP, and GP) as well as the sequences of the cleavage sites (GKR and R) and the position of the glucon (I) are indicated. SP indicates the signal peptide. The disulfide organization and known secondary elements are included (respectively, above and below the bar representing the prohormone) (146, 643):  = -strand; coiled springlike pattern = -helix; __ = loop. FNDI mutations identified in humans (126, 247, 278, 310, 616, 619, 620, 630, 778) are positioned in the prohormone. According to conventions in nomenclature of the familial nephrogenic diabetes insipidus mutations (630), amino acids of each moiety of the VP prohormone were numbered separately. Thus the abbreviation G1R underneath the NP moiety indicates the substitution of the G at position 14 of NP by an R. The symbol  indicates the deletion of the indicated residue(s). The asterisk marks the only recessive mutation (P7L); all other mutations are dominant.

VP and OT are closely related to each other in primary structure and differ in only two amino acids at position 3 and 8. It is generally believed that the mammalian VP/OT family developed from a common ancestral molecule by gene duplication about 450 million years ago (781). The strong conservation of the NP domain of the different VP-related prohormones further strengthens this assumption. The NP domain is remarkably well conserved and exists in all vertebrate and invertebrate species. Especially the exon B-encoded NP portion (NP_10-76) shows a considerable homology between different species. Two segments in the NP sequence, which fall within the region encoded by exon B, show 58% homology to each other and 60% at the nucleotide level. This indicates that the primordial NP gene itself arose from a partial gene duplication that extended an initially smaller structure (128). The secondary structure of NP also indicates a two-domain organization of the molecule in terms of the disulfide pairing, that clearly divides the molecule in a NH₂-terminal and a COOH-terminal domain (122). NP contains 14 cysteine residues, which are conserved with almost equal interdistances. The NH₂- and COOH-terminal domains, with each three cysteine, are held together by the extremely well-conserved amino acids C⁴⁴QEENYLPSPC⁵⁴ and a disulfide bridge between Cys¹⁰ and Cys⁵⁴.

All neuropeptides that are secreted from nerve terminals are first synthesized as protein precursors that are sorted to the regulated secretory pathway, and then processed by enzymatic mechanisms into biologically active peptides before secretion. The first step in the biosynthesis of a peptide is the expression of the gene that encodes the protein precursor. In the cases of the classical neurosecretory peptides, OT and VP, these nine-amino acid-long peptides are first made as part of two separate prohormones of 106 and 145 amino acids, respectively. Their mRNAs are translated on ribosomes attached to the rough endoplasmic reticulum (RER) in the cell body of the neurons, and their resultant precursor proteins undergo cotranslation processing steps in the RER which includes removal of the signal sequence amino acids from their NH₂ terminus of the precursor, the formation of disulfide bonds, and the initial stages of glycosylation of the precursor (139, 251).

Although the mechanisms of protein and pro-peptide sorting through the RER, Golgi, and secretory vesicle systems in cells are fundamental issues in cell biology, relatively little work has been done using the HNS. Sorting mechanisms have generally been divided into "receptor-mediated" (118, 445) versus "aggregation-mediated" (339, 769) models. These two views of how secretory proteins are sorted via the trans-Golgi network (TGN) into the regulated secretory pathway (usually via LDCVs) are controversial, and a full elucidation of them is beyond the scope of this review. In brief, the receptor-mediated view conceives of a "sorting receptor" in the TGN membrane that binds a "sorting signal" in the secretory protein to package the latter into the LDCV compartment. The aggregation-mediated model focuses on the self-aggregation of the secretory proteins in the TGN as the principle mechanism.

The experimental data underlying both in the above models usually focuses on specific disulfide-bonded loops, typically in the NH₂ termini of the secretory proteins and prohormones, which are essential for proper sorting to the regulated secretory pathway (339, 401, 444, 769). The OT and VP prohormones are extremely rich in disulfide-bonded loops (98), i.e., eight disulfide bridges exist in the ~10-kDa precursor proteins (see sect. IIIA). Both the biological functions of the peptides and their structural interactions with the NPs are completely dependent on the integrity of these disulfide bonds (98). The three-dimensional structures of NP-peptide complexes have been elucidated by crystallography (643). This structure emphasizes the function of the disulfide bridges for tightly folding of the NP protein and allowing a "peptide-binding" pocket to accommodate VP or OT by multiple intermolecular contacts.

For many years, the role of the NPs in the OT and VP prohormones has been something of a mystery, and its presumed role as a "carrier protein" (256) has been questioned, since no other prohormone is known to contain such a molecule. Nevertheless, both theoretical considerations (98) and experimental evidence (176) give increasing evidence to the view that the NPs serve as "chaperone-like" molecules serving intracellular transport of the MCNs. Experimental evidence for a role of NP-hormone interaction in sorting to the regulated secretory pathway includes the missorting of a mutant VP prohormone encoding a VP peptide, unable to bind to NP (116). When expressed in neuronal cells, this prohormone is partly retained in the endoplasmic reticulum (ER) and mostly secreted unprocessed via the constitutive secretory pathway. These data favor a mechanism in which the VP and NP domains of the prohormone need to interact for efficient passage of the prohormone through the ER, and for sorting to LDCVs. The intracellular fate of natural VP prohormone mutants causing pathology of the HNS (see sect. IID3) supports such a model.

The prohormones are selected from the Golgi and packaged in LDCVs. After sorting, subsequent posttranslational processing steps involve a variety of proteolytic (endo- and exopeptidase) as well as nonproteolytic modifications (e.g., amidation, sulfating, etc.). These steps occur within membrane-bound compartments in the cell, starting within the TGN and then primarily in the LDCV (Fig. 2). The first processing step is to proteolytically cleave the intact protein precursor into its correct peptide fragments. There are at least eight species of prohormone (precursor) convertases (PCs) found in mammalian cells, which are in the subtilisin-like (Kex-2) endoprotease family, that can process precursors of secreted proteins (253, 256, 644, 687-689, 739). Because the PCs usually cleave at paired-basic amino acid motifs, an exopeptidase (carboxypeptidase H/E) that trims the remaining COOH-terminal basic amino acid residue is required in most cells (234, 276). Another critical step in fashioning many biologically active peptides is the conversion of their COOH-terminal glycines into amides by an enzyme system referred to as peptidylglycine -amidating monooxygenase (PAM, see Ref. 199).

All of the above enzymatic mechanisms have been shown to operate in the MCNs of the HNS, and the presence of PCs has been demonstrated (253, 256). The PCs that have been reported to be present in the HNS are PC1 (73), PC2, PC5, and PACE4 (73, 172, 173, 189), and carboxypeptidase H/E (74, 234, 235, 470, 473) and PAM (91, 283, 433, 500, 673) have similarly been located in the HNS. In only a few studies has there been an effort to specifically localize the PC subtypes specifically to either the OT or the VP MCNs. In situ hybridization (ISH) studies have indicated that PC1/3 and PC5 are predominantly present in the VP and OT MCNs, respectively (73, 189). Studies on the regulation of these enzymes' gene expressions and activities have also been modest in number, although evidence for the osmotic regulation of carboxypeptidase H/E (84, 480) and glucocorticoid regulation of PAM (283) in the HNS has been reported.

Studies on the VP prohormone in cell lines differently equipped with PCs indicated that the VP prohormone can be cleaved first by furin at the level of the Golgi apparatus. This cleavage releases the COOH-terminal glycopeptide. The resulting VP-NP intermediate is cleaved by PC1 and/or PC2 (M. Nijenhuis and J. P. Burbach, unpublished data). One of the most intriguing differential regulations of the processing mechanisms occurs during development. There is a precocious expression of biologically active VP in the fetus, whereas in the OT MCNs there appears to be a cleavage of the OT prohormone to the OT COOH-terminally extended peptide intermediates, but processing of the extended OT forms by carboxypeptidase E/H does not occur until postnatal periods (21, 544). There are also reports of the secretion of the extended forms of OT in primates when they are stimulated by estrogen (23, 24). Because the COOH-terminally extended forms of OT and VP do not exhibit any known biological activities, the functional significance of this regulation of carboxypeptidase H/E activity in the HNS remains unclear.

LDCVs serve as the key intracellular sites for OT and VP processing, for axonal and dendritic transport of the peptides to storage and secretory sites, for long-term storage, and ultimately the secretion of the biologically active peptides (Fig. 2). As such, the LDCVs play multifunctional roles in the process of neurosecretion in the HNS. LDCVs are ~160-200 nm in diameter with a high peptide and protein content that cause them to exhibit electron-dense cores (139, 542) when viewed by electron microscopy. It is now clear that all the precursor processing enzymes activities can be found in the LDCVs of the HNS (251) and that these enzymes operate optimally at the mildly acidic condition (pH 5-6) that is found in these organelles (651, 652, 859). This acidic pH of the intravesicular environment is also essential for the binding of the OT and VP nonapeptides by the NPs, which is optimal at pH 5.5 (98, 141, 766), and which contributes to the stability of the intravesicular contents during LDCV storage in the cell. This acidic environment is maintained by a proton-translocating ATPase in the LDCV membrane (651, 652, 678). The LDCV membrane also contains cytochrome b₅₆₁ molecules that presumably contribute electrons to the enzymatic reaction catalyzed by PAM (194, 859).

After their formation in the TGN (Fig. 2), the LDCVs undergo anterograde transport (i.e., away from the cell body) on microtubular tracks (139), probably using members of the kinesin gene family as molecular motors. The kinesin gene superfamily has many members (94, 319), and it is not yet clear which of the specific molecules in this family are associated with LDCV axonal transport. The KIF2 and KIF3 kinesin proteins are reputed to transport vesicles in the 90- to 160-nm-diameter range and thus may be candidates for this function (253). Very few studies have been performed related to the axonal transport of LDCVs, although the association of LDCV transport with microtubules tracks has been indicated by its inhibition by colchicine (254, 255, 592). Direct measurements of LDCV transport rates using pulse-chase analyses have placed it in the "fast transport" category with rates approximating 140 mm/day (139, 223, 592).

In the MCNs of the HNS, most of the LDCVs are stored in the nerve terminals in the neurohypophysis where they are mobilized for secretion by electrical activity (139, 542). However, some LDCVs can also be stored in cell bodies and dendrites, and calcium-dependent secretion can also occur in the dendrites (465, 606). An example of this dual mode of secretion occurs in the OT MCNs in the hypothalamus of lactating animals. As part of the suckling reflex, these neurons secrete large boluses of OT from nerve terminals in the pituitary into the bloodstream to act on their distant targets, the mammary glands. They also secrete OT from their dendrites into the hypothalamus itself for a paracrine function, i.e., to modulate the synchrony of the OT neuron population's electrical response to suckling to generate bursts of OT release (465, 561).

The molecular mechanisms that are responsible for the secretion of peptides from LDCVs in neurosecretory terminals appear to be very similar to those that underlie secretion from small synaptic vesicles at synapses (493, 640). In both cases, the secretory event is preceded by an influx of calcium ions through a voltage-gated calcium channel located near a secretory vesicle (see Fig. 2, bottom inset) In neurotransmitter secretion, this apposition of the secretory vesicle to the calcium channel is critical, since the secretory event requires a relatively high concentration of calcium ions (10-100 µM) that occurs only immediately adjacent to open calcium channels in the plasma membrane. Secretory vesicles in such locations are said to be in "readily releasable" pools, and at active zones of synapses, they are referred to as being "docked" (640). While the classification of "docked" versus "cytoplasmic" reserve small vesicles has clear morphological correlates at synapses, this is not as clear for LDCVs in neuroendocrine cells. Nevertheless, ~1-5% of LDCVs in the posterior pituitary appear to be functionally docked, in that their contents are known to be readily releasable during excitation (139, 542). An intensive research effort is presently underway to identify the molecules that are involved in the cascade of events that cause "docked" secretory vesicles to fuse with plasma membranes and thereby release their intravesicular contents into the extracellular space. The latter event, termed "exocytosis," is the fusion event in the final step in secretion. After exocytosis, the nerve terminal membrane is increased in surface area, and this additional membrane is subsequently returned to the cell through a budding process known as "endocytosis." In recent years, extensive cloning studies have uncovered a very large number of protein families that are associated with either the secretory vesicles membranes or the active zones of secretion in the plasma membrane (see sect. IIIE for current views about molecular events underlying neurosecretion).

MCNs are classical neuroendocrine cells, specialized in the synthesis and secretion of vast quantities of the hormones VP and OT. These peptides are transported through the general circulation and exert their effects through interaction with receptors located at distal peripheral sites. The well-known functions of VP and OT in the regulation of salt and water homeostasis and reproduction have been studied for over a century (see sect. I). The physiological function of OT and VP can now be correlated with the presence and properties of the receptors for these neurohypophysial hormones, which are briefly summarized here. For detailed reviews, see References 55 and 684.

The OT receptor (OTR) and the VP receptors (V₁R, V₂R, and V₃R) form a subfamily within the much larger superfamily of G protein-coupled receptors (55). The receptors can be distinguished from each other by differential binding affinities for structural analogous of OT and VP and on the basis of their activation of different signaling pathways.

A) OTR. The multiple hormonal and neurotransmitter functions of OT are mediated by the specific OTR, which activates phospholipase C and increases in cytosolic calcium (487). OTRs are expressed in the uterus and mammary gland (381, 648). Expression has also been described in the brain (893), with patterns differing in different species, which may be related to different patterns of sexual behavior (346, 347). There is only one OTR gene. Therefore, the same receptor protein in brain and peripheral organs is expressed. However, posttranslational modification and interactions with downstream signal transduction components may modify OTR signaling (600, 658, 732). Notably, OT MCNs express the OTR (7) (see sect. IIID). The OTR can be considered as a "nonselective" receptor for neurohypophysial nonappetides, since it binds both OT and VP with almost similar affinities.

B) V₁R. The V₁R (formerly known as V_1a) is specific for VP. It activates the G_q/11 family of G proteins, the -subunit of which regulates the activity of the -isoform of phospholipase C. Receptor activation has been shown to stimulate phospholipases C, D, and A₂ and, through phosphatidylinositol hydrolysis, to an increase in intracellular calcium and an increase in cell acidification through stimulation of Na⁺/H⁺ exchange (101). V₁R is expressed in the liver, blood vessel smooth muscle cells, and most other peripheral tissues that express VP receptors (447). The V₁R is probably the most common receptor for VP in the brain (588).

C) V₂R. The V₂R is preferentially coupled to the -subunit of G_s, through which it stimulates activity of adenylate cyclase and cAMP production (583). V₂R is expressed only in the kidney of adult rats (447), but the mRNA encoding the V₂R is also expressed in the brain of newborns, although this declines to undetectable 2 wk after birth (318).

D) V₃R. The V₃R (formerly known as V_1b) stimulates phospholipases C and induces an increase in intracellular calcium. V₃ is expressed in the majority of anterior pituitary corticotroph cells, in multiple brain regions, and in a number of peripheral tissues, including kidney, thymus, heart, lung, spleen, uterus, and breast (446). It is of interest to note that MCNs also express the V₃ (314, 338; see sect. IIID).

There is a vast literature on the physiological mechanisms affected by VP and OT. For the context of this review, a brief summary of the main peripheral physiological functions established for VP and OT from the HNS is given here. For reviews on functions of VP and OT on the brain, mostly mediated by neurons other than MCNs, the reader is referred to Reference 782.

A) SALT AND WATER BALANCE. Mammals respond to changes in the osmolality of their extracellular fluid by altering their behavior and physiology. The behavioral response entails regulation of the salt and water intake through changes in sodium appetite and thirst. The physiological response entails modulation of renal excretion of water and sodium, which are achieved through changes in the plasma concentrations of the antidiuretic and natriuretic hormones VP and OT (617, 686). This, in turn, is achieved through an increase in the secretion of these hormones from MCN nerve terminals (103).

VP is classically known as the antidiuretic hormone. Renal V₂ receptors mediate VP-induced tubular reabsorption of water via induction of intracellular cAMP production in collecting duct cells (583), thereby conserving stores of bodily fluid during times of restricted intake. Whereas no mutations of the OTR, V₁, or V₃ receptors have been found in populations of any species, over 60 different genetic mutations of the V₂ receptor have been described that represent the cause for congenital X-linked nephrogenic diabetes insipidus (NDI) (572, 684). The functionally characterized mutants show a loss of function due to defects in their synthesis, processing, intracellular transport, VP binding, or interaction with the G protein/adenylate cyclase system.

Although OT is best known for its role in reproduction (see sect. IIC2D), it also stimulates natriuresis at physiological plasma levels (818, 866, 892), an effect mediated by estrogen-regulated OT receptors in macula densa and proximal tubule cells (99, 586, 587).

B) VASOCONSTRICTION. In animals subjected to hemorrhage, plasma VP concentrations increase to levels sufficient to cause vasoconstriction, thus attenuating the hypotensive response (686). In contrast, whereas hypotension also causes marked increases in plasma VP concentration in human subjects, VP does not seem to contribute to the maintenance of blood pressure (321).

C) CORTICOTROPH REGULATION. VP stimulates adrenocorticotropic hormone release from the anterior pituitary by acting on the V₃R, VP, derived from PVN parvocellular neurons, is released into the portal blood in the median eminence, and acts as a potent secretagogue of adrenocorticotropic hormone (32). Evidence suggests that VP released by MCNs may also be involved in the control of ACTH secretion, particularly after acute hyperosmotic and hypotensive stimuli (32, 613). The physiological contribution of VP to modulation of activity of the hypothalamo-pituitary-adrenal axis is particular dominant in situations of chronic stress.

D) REPRODUCTION. OT is involved in parturition (649) and lactation (899). The function of OT in lactation is dual. OT directly contributes to milk production through its stimulating activity on prolactin release (657). At term, OT is released from the pituitary in large pulses. This corresponds to a dramatic OTR upregulation in the uterine myometrium (648), which becomes extremely sensitive to OT. Thus is OT thought to induce contraction of uterine smooth muscle and expulsion of the young. During lactation, OT stimulates the contraction of the myoepithelial cells that surround the alveoli of the mammary gland, resulting in milk ejection.

However, the role of OT in parturition has recently been scrutinized and challenged. Knockout mice devoid of the OT gene (564, 898) are viable and fertile. OT-deficient females give birth normally and appear to demonstrate normal maternal behavior; however, all offspring die shortly after birth due to the dam's inability to nurse. Thus it appears that OT plays an essential role in milk ejection but not parturition, at least in the mouse. These observations are consistent with the normal delivery seen in humans and experimental animals with complete posterior pituitary dysfunction (27). The finding that the OT knockout mouse (564, 898) gives birth normally has reignited the long-term debate on the role of OT in parturition (649). The fact remains that OT is well placed in mammalian physiology to play a key role in all aspects of reproductive function. The peptide produced in the brain, ovary, uterus, or other peripheral tissues (31, 124, 229, 244, 424, 425, 513, 623) has been implicated in sexual and maternal behaviors, ovarian cyclicity, parturition, and lactation. It should be realized that the appearance of a phenotype of the OTR null mutatioin may require interaction with specific environmental and endogenous cues. Furthermore, the OT gene in the HNS is regulated during reproductive cycles, strongly implying functions for the neurohypophysial hormone.

However, there is ample evidence for a role for OT in parturition (649), and disruption of OT physiology can result in serious dysfunction. For example, OT antagonists delay the initiation of parturition and prolong labor (33). Most transgenic mice expressing a bovine OT gene (bOT3.5; see sect. VA1A) fail to deliver normally (323). High levels of transgene RNA were found in the ovary at the end of gestation and at the beginning of lactation in bOT3.5 mice, correlated with a parturition defect that results in considerable maternal mortality. Thus, whereas OT has a central role in parturition, it is clear that the organism can give birth in its absence and, thus, mechanisms must take the place of OT. The OT knockout mice will be an invaluable tool in the understanding of these mechanisms (649).

D.  Pathology of the HNS

1.  The homozygous diabetes insipidus (di/di) Brattleboro rat

The Brattleboro (di/di) rat was identified as a mutant of the Long Evans strain, suffering from a hereditary form of diabetes insipidus (784). This disease is transmitted as an autosomal recessive trait. This pathology was shown to be due to a lack of VP production by the HNS. Characteristically, water resorption is greatly impaired in the distal kidney tubules (785), leading to a massive loss of body fluid. The rats can excrete almost 70% of their body weight in hypotonic urine per day and compensate the resulting dehydration by drinking an equivalent amount of water. Urine output, urine osmolality, and water consumption are therefore commonly used to check for homozygosity. The disease state of the animal is reflected by a continuous osmotic stress that affects the MCNs of the HNS.

The genetic defect of the Brattleboro rat is the deletion of a single deoxyguanosine within exon B of the VP gene (680). The resulting frameshift alters the amino acid sequence of the NP moiety from residue 64 onward and the entire glycopeptide (GP) region (Fig. 4 and sect. IIIB). Due to the new amino acid sequence, 5 of the 14 cysteine residues in NP are missing, as well as additional PC cleavage sites and a recognition signal for glycosylation. In addition, the stop codon within the mRNA is missing, causing translation to continue through the Terrel normally noncoding part of the mRNA into the 3'-poly(A) tail creating a poly-lysine tail at the COOH terminus of the precursor. The poly(Lys) tail may continue as much as 70 Lys residues as estimated from the length of the poly(A) tail (353).

Although the mutant VP gene is correctly transcribed and spliced, mRNA is not efficiently translated (679). Therefore, it has been suggested that the expression of the mutant precursor is inhibited by a block at the translational level. In the di/di rat, the mutant di precursor can be detected in MCNs by IHC (286, 399). Immunoelectron microscopic studies have shown that the mutant precursor is restricted to the ER and small lysosomal-like bodies and does not reach the secretory granules (399).

Several other observations confirm that di/di rats are chronically osmotic stimulated animals. ISH of heteronuclear VP RNA revealed a significant increase in primary VP transcripts in the SON of di/di rats compared with the wild-type and heterozygous rat (754). No differences in levels of nuclear RNA were found in the PVN of these animals. These data imply an increased transcriptional activity in MCNs of the di/di rat.

Very early data by Sokol and Valtin (730) showed an extreme hypertrophy of perikarya, nuclei and nucleoli of MCNs in the SON of the di/di rat. The appearance of MCNs in the PVN of di/di rats is less different from wild-type cells. However, similar to the MCN neurons of the SON of di/di rats, the nucleolar size is significantly larger in di than in normal rats. The enlarged volume of the nucleoli can be normalized by VP substitution, which indeed suggests an activation of transcription in MCNs of the di/di rat due to osmotic stress (785). In addition, VP substitution of di/di rats has been shown to reduce the level of VP mRNA (895). A continuous osmotic stimulation of the di/di rat is further substantiated by a marked increase in mutant VP mRNA levels in di/di rats compared with the levels of mutant VP mRNA in heterozygous rats. In the heterozygous +/di rat, the mutant VP allele is expressed at levels that are only 5-7% of the expression of the wild-type allele. Instead of the expected twofold increase in mutant VP mRNA in di/di rats, a sevenfold increase has been observed (703).

Upon this continuous osmotic stress, VP gene expression in di/di rats can be further activated by chronic intermittent salt-loading. This is reflected by an increase in mRNA levels for both VP and dynorphin (703). An increase in VP mRNA levels in di/di rats could, however, not be observed after dehydration (483). These data indicate that the transcriptional regulation of the VP gene of the di/di rat is intact.

This, however, seems to contradict the fact that the continuous osmotic stress that affects the di/di rat does not lead to an increased level of VP mRNA compared with wild-type and heterozygous +/di rats. For instance, a decrease in the amount of VP mRNA in the di/di rats has been reported using Northern blot analysis (353, 702, 811) and ISH (285, 504, 895). Others, using similar methods, did not observe any differences in VP mRNA levels between these animals (243, 680, 779).

To explain the relatively low levels of mutant VP mRNA in both the di/di and +/di rat, it has been suggested that the expression of the mutant VP gene is affected at the posttranscriptional level (703). This may relate to the poor translatability of the mutant VP mRNAs that could be caused by the extended translation into the poly(A) tail (353, 679). Moreover, mutant VP mRNAs of di/di rats exhibit an up to 150 nucleotide longer poly(A) tail than normal VP mRNAs (215, 353).

The distribution of the VP neurons in the hypothalamus of the di/di rat is identical to that in wild-type rats, as determined by ISH (215, 504, 779) and in ICH (621, 750). However, the expression of the mutant VP precursor in the di/di rat considerably alters the morphology of the VP MCNs. The larger perikaryal cross sections, up to 90% greater than the VP neurons of the +/di rat, and the increased size of the nucleoli are typical of hypertrophy (540, 786, 895). Biochemically this hypertrophy is reflected by a high metabolic activity as demonstrated by an increased level of cytochrome oxidase (402) as well as glucose utilization (756) and increased staining of a Golgi and lysosomal marker (749). In addition, both the ER and Golgi apparatus of di/di cells are fragmented into small complexes, and increased numbers of lysosome-like bodies are present (350, 540). Whereas VP neurons of the wild-type rat contain large neurosecretory granules of 160-nm diameter, only small granules of ~100 nm are present in di/di cells (540, 859), and these contain the coexisting peptide dynorphin (856).

From 1985 onward, a number of startling papers have described the expression of apparently normal VP gene products in solitary MCNs of the di/di rat. Now, these observations can be clarified by a novel form of RNA mutation, discovered through analysis of the HNS. GP immunoreactivity has been observed in a small number of cells in the SON of di/di rats, suggesting a reversion of mutant VP neurons to the wild-type phenotype (520, 626, 800). Some years later, an extensive study on these reverted VP neurons demonstrated the colocalization of all VP gene products (i.e., VP, NP, and GP) in the same solitary cells together with the mutant di precursor (804). These solitary VP neurons of the di/di rat did not only display a heterozygous phenotype, but they also appeared to transport VP and related gene products into the neural lobe. Furthermore, this study showed an age-related increase in the number of these solitary reverted VP neurons. The number of solitary +/di cells in di/di rats increased linearly with age from only a few cell profiles in young rats to over 120 cell profiles in 2-yr-old adults of either sex. It has been shown by molecular cloning, ISH, and IHC that the apparent +/di phenotype is caused by further mutation of the di VP transcript by a dinucleotide deletion. The deletion involves the loss of a GA pair in one of two GAGAG motifs in the VP transcript (208). The deletion causes the 1 frame of the Brattleboro transcript to restore to the normal frame, but a stretch of 13 or 23 amino acids in the 1 frame remains. Thus this precursor is not identical to wild type. It can, however, be sorted partly to the regulated secretory pathway and results in the biosynthesis of normal VP (209).

Notably, the GA deletions do not exist at the genome level and are considered to be introduced in transcripts by a process termed "molecular misreading" (799). Based on the work on the Brattleboro rat, antisera were raised against predicted +1 frames of several proteins and tested for expression. The results show that molecular misreading is abundant in several neuropathologies, in particular Alzheimer's disease (801).

Despite the expression of apparently normal VP gene products, the solitary +/di cells of the di/di rat differ in appearance from the VP neurons of heterozygous (+/di) and wild-type rats. Similar to di/di cells, the ER appears disordered and fragmented into smaller complexes in the reverted +/di cells of the di/di rat, which may indicate a still hypertrophic state (288, 350). The VP immunoreactivity in these solitary cells was located, together with the mutant precursor, solely in the cisternae of the ER and not in any other compartment. Although small (80-100 nm) LDCVs indistinguishable from those in di/di cells, are present, none of them was reported to contain detectable levels of VP, NP, or GP immunoreactivity as determined by electron microscopy (607). This contradicts the presence of immunoreactivity for VP and related gene products in fibers in the neural lobe, which suggests they are axonally transported and thus packaged in granules (804).

The ultrastructural appearance of VP cells of the heterozygous +/di rat is similar to those of wild-type strains, which includes the presence of normal sized (160 nm) LDCVs (540). Thus the VP neurons of the di/di rat that have reverted to the +/di phenotype are different from the +/di VP cells in the heterozygous Brattleboro rat. With regard to the +/di rat, this may be explained by a differential expression of the wild-type and mutant allele, which results in a contribution of mutant VP mRNA of only 5-7% for the PVN and SON, respectively (703). Consequently, a very low amount of mutant precursor is present in VP neurons of the +/di rat.

Several peptides coexist within VP MCNs, which are differentially expressed in di/di and wild-type rats (see sect. IIIC). As in the wild-type rat, dynorphin, galanin, and NPY are synthesized in VP neurons of the di/di rat, but their turnover is enhanced (371, 641, 701). For example, dynorphin levels in di/di rats are reduced to 25% of wild-type estimates. Also, a reduction of galanin immunoreactivity in the neural lobe of di/di rats has been observed (641). In contrast, angiotensin II (ANG II) and the neuroendocrine polypeptide 7B2 immunoreactive cells are almost totally absent from di/di hypothalami (440, 488), except for a few solitary neurons (245, 325, 380). Remarkably, solitary neurons immunoreactive for ANG II or 7B2 were shown to colocalize, with the reverted neurons expressing VP and related gene products (245, 805), although no explanation for these observations is available. However, Van Leeuwen (798) has proposed the involvement of a compartmentalization of the ER, whereby ANG II, 7B2, and VP are synthesized and translocated within the same ER domains. In di/di cells, their expression would be obstructed by the mutant VP precursor, which then is alleviated by the revertance of phenotype. The peptides that are unaffected in expression (e.g., dynorphin, galanin, and NPY) would utilize other compartments of the ER.

In addition to the solitary neurons that express apparently normal VP gene products, a second subset of "reverted" cells has been described in di/di hypothalami. These solitary cells contain immunoreactivity for VP, OT-related NP, and the mutant di precursor, all located as large aggregates in the cisternae of the RER (607), but no OT, VP-related NP, and GP immunoreactivity could be detected. The number of this type of phenotypically changed neurons is about one-tenth of the +/di reverted VP cells described above. Thus this cell type is very rare among MCNs.

On the basis of the immunocytochemical data, it has been suggested that hybrid VP/OT-NP precursors exist in this second subset of solitary VP neurons of the di/di rat that may be derived by a conversion between the VP and OT genes. This is substantiated by the molecular identification of recombinant VP/OT transcripts in di/di as well as wild-type hypothalami (532). These are hybrids between VP and OT transcripts and encode the NH₂ terminus of the VP precursor fused to the COOH-terminal part of the OT precursor. Thus somatic gene conversion of the VP and OT may occur in both di/di and wild-type rats. Whether this underlies a similar age-related mechanism as the potential genetic alterations in the +/di VP cells of the di/di rat remains to be determined.

In summary, three types of VP MCNs have been recognized in di/di rats: 1) the di/di VP neurons that express the mutant di precursor and include ~4,500 MCNs of the di/di hypothalamus; 2) the solitary +/di VP neurons that express apparently normal VP gene products together with the mutant di precursor due to "molecular misreading." The number of these cells increases from a few in newborn animals to ~2% of the VP MCNs in 2-yr-old di/di rats. 3) The solitary VP/OT hybrid neurons that express VP/OT hybrid transcripts that encode the NH₂-terminal part of the VP precursor fused to the COOH-terminal part of the OT precursor, due to hybrid mRNA, comprise ~0.3% of the VP MCNs in aged di/di rats.

Familial neurohypophysial diabetes insipidus (FNDI) is the best-known inherited endocrine disease caused by prohormone defects (242, 635). In this autosomal dominant disease, mutations in the VP prohormone cause defects in the synthesis of VP and hence result in a large increase in urine production (polyuria) and fluid intake (polydipsia). In human FNDI, mutations have been observed in either the signal peptide, the VP moiety, or the NP moiety of the VP preprohormone (126, 247, 278, 310, 616, 619, 620, 630, 778). Thirty-two different mutations have been identified in more than 40 pedigrees (Fig. 4). The disease displays two unexpected features for a deficiency caused by a defective prohormone. First, the disease is dominant, demonstrating that one mutant prohormone allele suffices to cause the defect. Second, the onset of disease symptoms is delayed to several months or years of age. These peculiarities suggest that the mutant human VP prohormone somehow interferes with either synthesis, transport, and processing of the wild-type prohormone or with the viability of VP-producing cells.

Studies in which mutant VP precursors were expressed in cell lines with peptidergic properties (348, 562) showed that mutant proteins were largely retained in the ER and that accumulation of precursor caused morphological and likely functional derangement of the ER. Overexpression of mutant genes resulted in "accretions." It has been indicated that these accretions and the consequent disturbance of the ER are deleterious to the cell and will decrease functionality and/or viability of the MCNs, which express high amounts of VP prohormone in vivo. This hypothesis would explain both the dominant inheritance of human FNDI and the delayed onset. This mechanism bears similarities to observations on other neurodegenerative diseases in which protein aggregates are present as a consequence of altered protein structure, e.g., Alzheimer's disease and prion disease. It does not explain reports of diabetes insipidus symptoms in newborns. In addition to degeneration, it has also been shown that mutant and normal precursors can interact and negatively influence VP synthesis (349). Thus the mutation of a single allele in human FNDI may lead to immediate impairment of hormone synthesis due to protein-protein interactions in the ER, Golgi, and to gradual neurodegeneration as a result of permanent ER obstruction. This mechanism is fundamentally different from diabetes insipidus in the Brattleboro rat, which requires two affected alleles.

	III. GENE EXPRESSION

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The essential physiological function of the HNS is to convert centrally processed information on the physiological state of the organism into hormonal action on peripheral organs. Key for this function is the reception of chemical signals, transduction and appropriate reaction at the level of the gene and the release processes. Therefore, the expression of genes addressed in the section concerns the peptide "output" genes, the intracellular machinery of their secretion, and the receptors that transduce signals toward cellular activity of MCNs.