Dominique P.V. de Kleijn

Molecular biology of neurohormone precursors in the eyestalk of Crustacea

Abstract Our knowledge concerning the primary structures of crustacean neuropeptides has been broadened considerably during the last few years and has greatly contributed to the successful application of molecular biological techniques to crustacean neuroendocrine research. In this review, we compare and discuss the preprohormones of the Red Pigment Concentrating Hormone (RPCH), the Pigment-Dispersing Hormone (PDH) and the different members of the Crustacean Hyperglycemic Hormone, Molt-Inhibiting and Gonad-Inhibiting Hormone family (CHH/MIH/ GIH peptide family), recently elucidated by cloning and sequencing of the respective cDNAs. Expression studies, using in situ hybridization, Northern blots and RNase protection assays, have demonstrated that the mRNAs encoding some of the aforementioned preprohormones (for example, preproPDH and preproCHH) are not only expressed in the eyestalk but also in other parts of the central nervous system. The combination of molecular biological techniques with (bio)chemical and immunochemical methods provides elegant tools to study neuropeptides at the level of mRNA and peptide in individual animals during different physiological conditions. The fundamental knowledge obtained by such a combined approach will give detailed insight into how neuropeptides are involved in the adaptation of Crustacea to a broad spectrum of natural and aquacultural conditions.

Cloning and expression of two mRNAs encoding structurally different crustacean hyperglycemic hormone precursors in the lobster Homarus americanus

The crustacean hyperglycemic hormone (CHH) of the X-organ sinus gland complex is a multifunctional neurohormone primarily involved in the regulation of blood sugar levels. HPLC analysis of lobster sinus glands revealed two CHH-immunoreactive groups, each consisting of two isoforms with identical amino acid sequences and molecular weights. In order to obtain more information concerning the number and sequences of preproCHHs, and to study their expression, we isolated two full-length cDNAs encoding two different CHH preprohormones. Both preprohormone structures consist of a signal peptide, a CHH-precursor-related peptide and a highly-conserved CHH peptide. Expression studies revealed that the X-organ is not the only source of CHH mRNA because the ventral nerve system also expresses this mRNA. Based on these findings and earlier studies on the effect of eyestalk ablation, implantation of thoracic/abdominal ganglia as well as the multifunctionality of CHH, we postulate that CHH, present in the ventral nerve system is a good candidate for a supplementary role in the control of reproduction and molting.

Cloning and expression of mRNA encoding prepro-gonad-inhibiting hormone (GIH) in the lobster Homarus americanus

The gonad‐inhibiting hormone (GIH) is produced in the eyestalk X‐organ sinus gland complex of male and female lobsters, and plays a prominent role in the regulation of reproduction, e.g. inhibition of vitellogenesis in female animals. To study this neurohormone at the mRNA level, we cloned and sequenced a cDNA which encodes GIH in the lobster Homarus americanus. The structure of preproGIH consists of a signal peptide and the GIH peptide itself. A comparative analysis revealed that lobster GIH, together with crab molt‐inhibiting hormone, belongs to a separate group of the crustacean hyperglycemic hormone (CHH) peptide family which seems to be unique for crustaceans. Expression studies showed that GIH mRNA is expressed in the eyestalk, indicating that the neuroendocrine center in this optic structure is the only source of GIH. As this center modulates the other (neuro)endocrine organs in crustaceans, it is postulated that GIH regulates production and release of hormones involved in reproduction/molting processes.

Involvement of the hyperglycemic neurohormone family in the control of reproduction in decapod crustaceans

Summary In the last few years, (bio)chemical and molecular biological studies have shown that several members of the hyperglycemic hormone family are present in different molecular forms. In vivo and in vitro bioassays revealed that some of these isoforms also play a role in the control of reproduction in decapod crustaceans. This communication gives a review of the cytological aspects of the eyestalk X-organ sinus gland complex, responsible for the synthesis, storage and release of these neuropeptides, and the molecular and functional aspects of those members involved in the control of reproduction. Finally, the role of the hyperglycemic hormone family in the regulation of reproduction in the female lobster is described as an example of the (possible) interactions of the members of the hyperglycemic hormone family with other (neuro)endocrine factors in the reproductive process of crustaceans.

Localization of crustacean hyperglycemic hormone (CHH) and gonad-inhibiting hormone (GIH) in the eyestalk ofHomarus gammarus larvae by immunocytochemistry and in situ hybridization

This study deals with the localization of crustacean hyperglycemic hormone (CHH) and gonad-inhibiting hormone (GIH) in the eyestalk of larvae and postlarvae ofHomarus gammarus, by immunocytochemistry and in situ hybridization. The CHH and GIH neuropeptides are located in the perikarya of neuroendocrine cells belonging to the X-organ of the medulla terminalis, in their tract joining the sinus gland, and in the neurohemal organ itself, at larval stages I, II and III and at the first postlarval stage (stage IV). In all the investigated stages, the mRNA encoding the aforementioned neuropeptides could only be detected in the perikarya of these neuroendocrine cells. In stage I, approximately 19 CHH-immunopositive and 20 GIH-immunopositive cells are present, both with a mean diameter of 7±1 μm. GIH cells are preferably localized at the periphery of the X-organ surrounding the CHH cells that are centrally situated. Colocalization of CHH and GIH immunoreactions can be observed in some cells. The cell system producing CHH and GIH in the larval and postlarval eyestalk is thus functional and is morphologically comparable to the corresponding neuroendocrine center in the adult lobster.

Structure and localization of mRNA encoding a pigment dispersing hormone (PDH) in the eyestalk of the crayfish Orconectes limosus

The pigment‐dispersing hormone (PDH) is produced in the eyestalks of Crustacea where it induces light‐adapting movements of pigment in the compound eye and regulates the pigment dispersion in the chromatophores. To study this hormone at the mRNA level, we cloned and sequenced cDNA encoding PDH in the crayfish Orconectes limosus. The structure of the PDH preprohormone consists of a signal peptide, a PDH precursor‐related peptide (PPRP) and the highly conserved PDH peptide at the carboxy‐terminal end. In situ hybridization in combination with immunocytochemistry revealed four cell clusters expressing PDH in the optic ganglia of the eyestalk. Three clusters stained both with the PDH cRNA probe and the PDH antiserum, however, the perikarya in the lamina ganglionaris (LG) only stained with the PDH antiserum, suggesting the presence of a PDH‐like peptide in the LG.

Chapter 27 Dinucleotide deletions in neuronal transcripts: A novel type of mutation in non-familial Alzheimer's disease and Down syndrome patients

Publisher Summary Familial Alzheimers disease (FAD) represents about 40% of the total Alzheimers disease (AD) cases. Most of these FAD cases ( 35% of all AD patients) do not inherit AD as an autosomaldominant trait. Although these patients have at least one other relative in the first degree suffering from the disease, the genetic factor causing AD in these cases is not known. Families with an autosomal-dominant inheritance pattern of AD, account for only 5% of the total number of AD patients. In a subset of these families, missense mutations in the genes for β-amyloid precursor protein (β-APP), presenilin-1, and -2 underlie the AD pathogenesis. The non-familial or sporadic form of AD comprises approximately 60% of the total AD cases. Aging is probably an important factor in the AD etiology. The central nervous system (CNS), however, displays a high degree of plasticity, such that initial or minor damage to the CNS will not directly lead to neuropathology and can be compensated for. On the other side, the aging CNS is very vulnerable, because it is not capable to compensate for lost neurons that are especially prominent in a number of areas. Thus, during life-time irreversible cellular damage caused by somatic mutations, oxidative stress, or synapse loss accumulate in the post-mitotic neurons. Especially in AD most of these neurons do not die, but appear to become less active and show shrinkage. This chapter proposes that transcript mutations occurring in neuronal genes might be one of these unknown aging factors and might be a part of a general mechanism that could contribute to the neuropathogenesis in the majority of the AD cases, apart from the autosomal dominant forms. Recently this process was designated as molecular misreading.

A method for the analysis of newly synthesized tritiated mRNA.

Molecular hybridization with RNA probes has been performed on unfractionated cells solubilized in guanidine thiocyanate solutions for the quantitative analysis of specific RNA transcripts (1, 2). A method utilizing this principle has been developed for the detection and quantification of newly synthesized mRNA. To evaluate the method, we analyzed the biosynthesis of proopiomelanocortin (POMC) mRNA in the neurointermediate lobe (NIL) of the pituitary gland of Xenopus laevis. Specificity of hybridization was assessed by comparing the results of sense and anti-sense probes at the experimentally determined optimal hybridization temperature of 35°C. Only the anti-sense probe protected newly synthesized [H]-labeled POMC mRNA (Figure 1A). Treatment of NILs during the labeling period with 5,6-dichloro-l-/S-D-ribofuranosylbenzimidazole (DRB), an mRNA synthesis inhibitor (3), decreased POMC mRNA synthesis by more than 90% (Figure IB). To show that the method is quantitative, increasing amounts of a [P]-labeled Xenopus POMC cDNA fragment were assayed instead of labeled tissue. This resulted in a linear increase of hybridization signals (r = 0.9924, n = 3) (Figure 2).