The ependymal route for insulin-like growth factor-1 gene therapy in the brain.

I.c.v. administration of the peptide insulin-like growth factor-1 (IGF-1) has been shown to be an effective neuroprotective strategy in the brain of different animal models, a major advantage being the achievement of high concentrations of IGF-1 in the brain without altering serum levels of the peptide. In order to exploit this therapeutic approach further, we used high performance recombinant adenoviral (RAd) vectors expressing their transgene under the control of the potent mouse cytomegalovirus immediate early (mCMV) promoter, to transduce brain ependymal cells with high efficiency and to achieve effective release of transgenic IGF-1 into the cerebrospinal fluid (CSF). We constructed RAd vectors expressing either a chimeric green fluorescent protein fused to HSV-1 thymidine kinase (TK/GFP)(fus), or the cDNA encoding rat IGF-1, both driven by the mCMV promoter. The vectors were injected into the lateral ventricles of young rats and chimeric GFP expression in brain sections was assessed by fluorescence microscopy. The ependymal cell marker vimentin was detected by immunofluorescence and nuclei were labeled with the DNA dye 4',6-diamidino-2-phenylindole. Blood and CSF samples were drawn at different times post-vector injection. In all cerebral ventricles, vimentin immunoreactive cells of the ependyma were predominantly transduced by RAd-(TK/GFP)(fus), showing nuclear and cytoplasmic expression of the transgene. For tanycytes (TK/GFP)(fus) expression was evident in their cytoplasmic processes as they penetrated deep into the hypothalamic parenchyma. I.c.v. injection of RAd-IGF-1 induced high levels of IGF-1 in the CSF but not in serum. We conclude that the ependymal route constitutes an effective approach for implementing experimental IGF-1 gene therapy in the brain.

Gene therapy in the neuroendocrine system.

The implementation of experimental gene therapy in animal models of neuroendocrine diseases is an area of growing interest. In the hypothalamus, restorative gene therapy has been successfully implemented in Brattleboro rats, an arginine vasopressin (AVP) mutant which suffers from diabetes insipidus, and in Koletsky (fa(k)/fa(k)) and in Zucker (fa/fa) rats which have leptin receptor mutations that render them obese, hyperphagic and hyperinsulinemic. In the above models, viral vectors expressing AVP, leptin receptor b and proopiomelanocortin, respectively, were stereotaxically injected in the relevant hypothalamic regions. In rats, aging brings about a progressive degeneration and loss of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons, which are involved in the tonic inhibitory control of prolactin secretion and lactotropic cell proliferation. Stereotaxic injection of an adenoviral vector expressing insulin-like growth factor I corrected their chronic hyperprolactinemia and restored TIDA neuron numbers. Spontaneous intermediate lobe pituitary tumors in a retinoblastoma (Rb) gene mutant mouse were corrected by injection of an adenoviral vector expressing the human Rb cDNA and experimental prolactinomas in rats were partially reduced by intrapituitary injection of an adenoviral vector expressing the HSV1-thymidine kinase suicide gene. These results suggest that further implementation of gene therapy strategies in neuroendocrine models may be highly rewarding.

In situ reverse transcription-polymerase chain reaction. Applications for light and electron microscopy.

Since its discovery in 1986 by Mullis, the polymerase chain reaction (PCR) has been extensively developed by morphologists in order to overcome the main limitation of in situ hybridization, the lack of sensitivity. In situ PCR combines the extreme sensitivity of PCR with the cell-localizing ability of in situ hybridization. The amplification of DNA (PCR) or a cDNA (RT-PCR) in cell or tissue sections has been developed at light and electron microscopic levels. A successful PCR experiment requires the careful optimization of several parameters depending on the tissue (or of cell types), and a compromise must be found between the fixation time, pretreatments and a good preservation of the morphology. Other crucial factors (primer design, concentration in MgCl2, annealing and elongation temperatures during the amplification steps) and their influence on the specificity and sensitivity of in situ PCR or RT-PCR are discussed. The necessity to run appropriate controls, especially to assess the lack of diffusion of the amplified products, is stressed. Current applications and future trends are also presented.

Receptor-mediated nuclear translocation of growth hormone.

We have previously shown that the growth hormone (GH) receptor-binding protein is associated with the nucleus. We show here both by electron microscopy and nuclear isolation that GH is subject to rapid nuclear translocation. The intracellular fate of intravenously injected 125I-bovine growth hormone (bGH) was examined in the rat hepatocyte by electron microscopic autoradiography. The hormone appeared rapidly at the plasma membrane, then sequentially in lysosomal and multivesicular bodies and/or the nuclear membrane before final translocation to the nuclear matrix. Maximal translocation to the nuclear matrix occurred within 30 min of injection. Nuclear translocation of 125I-hGH was also studied by isolation of nuclei from cells stably transfected with cDNAs encoding the GH receptor, GH-binding protein, and a membrane bound but cytoplasmic domain-deficient receptor. Specific internalization and nuclear translocation of hormone only occurred in cells transfected with the full-length receptor. The translocation was rapid and became saturated within 1 h after addition of hormone to the culture media. SDS-polyacrylamide gel electrophoresis of isolated nuclei showed that GH is transported to the nucleus as the intact molecule. Pretreatment of cells with lysosomotropic agents (chloroquine, ammonium chloride, and bacitracin) decreased hormone degradation and increased nuclear translocation of GH. The nuclear translocation of GH was achieved independent of the cytoskeletal system (microtubular, microfilament, and intermediate filament networks). Thus, GH is subject to rapid receptor-dependent nuclear translocation via the endosomal pathway.

[Ultrastructural in situ hybridization on ultrathin sections of frozen tissues]. / Hybridation in situ ultrastructurale sur couples ultrafines de tissus congelés.

In situ hybridization on ultrathin frozen sections can be performed with either complementary DNA or synthetic oligonucleotide probes, labeled with 35S or an antigen (biotin or digoxigenin) respectively revealed by ultrastructural autoradiographic and immunocytological techniques. Using this method, GH mRNA was found in somatotrophs, mainly in the cytoplasm (endoplasmic reticulum and cytoplasmic matrix). Ultrastructural deterioration is the main drawback of this method but is more than outweighed by high sensitivity.

Ultrastructural non-radioactive in situ hybridization of GH mRNA in rat pituitary gland: pre-embedding vs ultra-thin frozen sections vs post-embedding.

In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultra-thin frozen sections, or on ultra-thin sections of tissues embedded in hydrophilic resin such as Lowicryl. With the purpose of comparing the sensitivity, resolution, and ultrastructural preservation of these three methods, we examined the expression of the growth hormone (GH) gene in anterior pituitary cells by in situ hybridization at the ultrastructural level, using a synthetic oligonucleotide complementary to the codons of the mRNA from Gln 45 to Ser 54 labeled at the 3' end of biotin-21dUTP. All these methods gave similar results: mRNA was located on the lamellar endoplasmic reticulum of somatotrophs. The pre-embedding method gave the best ultrastructural preservation, with low resolution with the enzymatic detection system and an intermediate sensitivity. A probe concentration of 10 pmol/ml was sufficient to obtain a signal. With this method gold particles could not be used without pre-treatment. The frozen section method gave the best sensitivity (a signal was observed with 4 pmol/ml of probe) but the lowest ultrastructural preservation. On ultra-thin Lowicryl sections, resolution was as high as with the frozen-section method, ultrastructural conservation was intermediate, and sensitivity was low. These results indicate that the last method seems to be a good compromise between sensitivity and ultrastructural preservation.

ANF and exocrine pancreas: ultrastructural autoradiographic localization in acinar cells.

Atrial natriuretic factor (ANF) binding sites have been recently demonstrated to be present in exocrine pancreas by an in vitro autoradiographic approach. An autoradiographic study was carried out to identify the exocrine cells containing ANF binding sites and to monitor the fate of 125I-labeled ANF in acinar cells after removal of pancreas at specific time intervals (1-30 min) after intravenous administration. At the light microscopic level, silver grains were found over acinar and centroacinar cells. Concomitant injection of an excess of unlabeled ANF inhibited the binding of labeled peptide by approximately 60%. At the electron microscopic level, the time-course study in acinar cells has revealed that of the cell compartments examined, plasma membrane, Golgi apparatus, mitochondria, and zymogen granules, the nucleus had distinct labeling patterns. Plasma membrane was maximally labeled 1 and 2 min after injection with 125I-ANF. Golgi apparatus was significantly labeled from 2 to 30 min after injection, mitochondria from 1 to 30 min after injection, zymogen granules at 1 and 15 min, and the nucleus only at 30 min. The lysosomal compartment was not labeled during the 30-min observation period. These results suggest that after binding to the plasma membrane, ANF is rapidly internalized and distributed to the intracellular organelles as a function of time. Labeling of the zymogen granules suggests that they may bind ANF and that the atrial peptide may be secreted by acinar cells. The significance of association of radioactivity with mitochondria and nuclei remains to be elucidated but may represent intracellular sites of action of ANF complementary to those on plasma membranes.

Ontogeny of hypothalamic luteinizing hormone-releasing hormone (GnRH) and pituitary GnRH receptors in fetal and neonatal rats.

Although it is known that LH secretion starts at 17 days of gestation in the fetal rat and that this first LH release is most likely driven by hypothalamic GnRH, an earlier role for GnRH during fetal life has been postulated with the observation that presence of GnRH is important before day 13 of gestation for the differentiation of the pituitary anlage. In order to clarify the different roles of GnRH during fetal life, we have studied the first appearance of GnRH in the fetal brain, the expression of GnRH receptors in the fetal pituitary gland, and the presence of GnRH immunoreactivity within the fetal gonadotrophs. GnRH was present in the earliest brain tissue examined (12 days of gestation). From 12-17 days, GnRH content of fetal brain remained low and then increased markedly by the end of gestation. No immunoreactive GnRH-like material could be detected in rat placental tissue throughout gestation. Binding sites for GnRH were detected as early as 12 days of gestation in fetal pituitary glands. However, binding was very low until 16 days. At 17 days, Scatchard analysis indicated the presence of high affinity, low capacity binding sites [affinity constant (Ka) = 10(10) M-1]. Intracellular presence of GnRH as seen by immunocytochemistry using ultrathin sections prepared by cryoultramicrotomy was first visible at 14 days and started to increase at 16 days. LH was first detectable in the fetal pituitary by RIA at 17 days; FSH was first detectable at 21 days, and PRL at 1 day of postnatal life. Thereafter, neonatal pituitary contents of LH, FSH, and PRL increased linearly with-time, as did the number of pituitary GnRH receptors. At 10 days of postnatal life, pituitary contents of LH and FSH were significantly higher in females than in males. In summary, hypothalamic GnRH appears early in fetal life and potentially can induce differentiation of the pituitary anlage. Conversely, the presence at 15 days of gestation of specific binding sites for GnRH and of intracellular GnRH immunoreactivity in gonadotrophs indicates that the hypophysiotropic action of GnRH clearly precedes the start of LH biosynthesis.