Characterization of the gene and messages for vasoactive intestinal polypeptide (VIP) in rat and mouse.

We have studied the structure and expression of the gene for vasoactive intestinal polypeptide (VIP) in rodents. We used a human cDNA to identify and clone a fragment of the rat VIP gene. This genomic fragment contained two separate exons, one encoding VIP itself and the other encoding a closely related neuropeptide, peptide histidine-isoleucine (PHI-27). Probes containing either exon, or both, hybridized to two messages: a prominent 1700-base (b) mRNA and a rare 1000-b species. These messages are expressed together in a tissue-specific manner, with highest levels in polyadenylated RNA from cerebral cortex and from small intestine, paralleling the reported levels of the neuropeptides themselves in these tissues. Using the rat genomic fragment as a probe, we isolated the mouse VIP gene in its entirety. The mouse gene is similar in organization to its human counterpart, with a total of 7 exons spanning 8 kilobases (kb). The 7th and largest exon, which is transcribed into the bulk of the 3' untranslated region of the messages, bears two potential polyadenylation sites 700 basepairs (bp) apart. S-1 nuclease protection with a fragment of this exon indicated that the two identifiable VIP messages differ in the extent of their 3' untranslated regions. Conversely, we found no evidence for differential splicing to produce messages encoding only one of the neuropeptides. Instead, specific oligonucleotide-directed digestion with RNase H demonstrated that all of the detectable mRNA from this gene contains both VIP and PHI coding sequences.     

The complete structure of the rat VIP gene

Vasoactive intestinal polypeptide (VIP) is a regulatory neuropeptide/neurotransmitter of 28 amino acids involved in a wide variety of physiological functions. Using synthetic oligodeoxynucleotide probes related to the rat VIP-cDNA, we have isolated and characterized the gene encoding the rat pre-pro VIP/PHI-27 and compared it to the human VIP gene. The rat VIP gene spanned 7400 base pairs, and contained 7 exons interrupted by 6 introns. 100% identity was found between the gene exons and the cDNA sequence. Differences in sizes of introns 2, 4 and 5 (shorter in the rat gene) are the reason for the shorter rat gene compared with the human gene of 8837 base pairs. Comparison of the genes in the two species showed a high homology in the exon sequences, 80-90% in exons 2, 4, 5, 6 and 30-50% in exons 1 and 7. In addition, the exon-intron junctions shared high identity between the genes. The rat untranslated exon 1 had little homology (30%) with human exon 1 and was 13 base pairs shorter. Interestingly, the 160 base pairs at the 5'-flanking region upstream of the cap-site share more than 75% identity between the two genes, including the exact position of TATA-boxes in positions -28, -145, -155, a cAMP-responsive element in position -80 and a CAAT sequence in position -127. The conservation of the 5'-flanking region of the VIP gene in parallel with the conservation of its coding exons emphasize the importance of these sequences during evolution.     

Structural and functional analysis of the promoter region of the nerve growth factor gene

Molecular clones containing the NGF gene promoter regions and exons I were isolated from mouse and rat genomic libraries with synthetic oligonucleotide hybridization probes that corresponded to the 5' end of mouse submandibular gland NGF mRNA. The nucleotide sequences of the 5' flanking regions and exons I were determined and compared. There was 95% similarity in exons I and the adjacent promoter regions between mouse and rat sequences. Further upstream, the similarity decreased to 76%. Both mouse and rat promoter regions were only 33% similar to the presumptive human NGF gene promoter region. Upstream from the capsite of submandibular gland NGF mRNA as determined by an S1-nuclease protection assay, two promoter-like TATA-boxes were found at positions -28 and -49, resp., and two CAAT-like boxes at -379 and -546, resp. Both promoter regions contained a cluster of conserved CpG dinucleotide sequences in a GC-rich island whereas less conserved upstream regions contained only one CpG sequence. The promoter regions were fused to the human growth hormone gene reporter function. Transient expression in L929 cells yielded appropriate fusion mRNAs and secretion of hGH, demonstrating that the cloned promoters are functional.     

Mobilization of the cell adhesion glycoprotein F3/contactin to axonal surfaces is activity dependent

F3/contactin is a cell adhesion/recognition molecule of the immunoglobulin superfamily implicated in axonal growth. We examined its subcellular distribution and mobilization to the cell surface in oxytocin- (OT-) secreting neurons, which express it throughout life and the axons of which undergo activity-dependent remodelling. This was performed in hypothalamic organotypic slice cultures containing OT neurons with properties of adult neurosecretory cells. Immunocytochemistry and immunoblot analysis confirmed that OT neurons express high levels of F3/contactin in vitro. Light and confocal microscopy of cultures that underwent double immunofluorescence after fixation showed F3/contactin immunoreactivity throughout the cytoplasm of OT somata, dendrites and axons, and also in non-OT axons and in putative synaptic boutons which contacted OT neurons. By contrast, after treatment of live cultures with anti-F3/contactin antibodies followed by double immunofluorescence for the glycoprotein and OT, F3/contactin immunoreactivity was visible only on the surface of axons, whether or not OT-immunoreactivity was present. Because of its glycosylphosphatidyl-inositol (GPI) linkage, F3/contactin can occur in a membrane-bound or soluble form. As seen from immunocytochemistry of live cells and immunoblot analysis, treatment of cultures with a GPI-specific phospholipase C (GPI-PLC) resulted in loss of F3/contactin immunoreactivity from all cell surfaces. F3/contactin immunoreactivity reappeared on axonal surfaces within 5 h after enzyme washout. Such re-expression was accelerated by neuronal activity facilitation (by K+ depolarization or gamma-aminobutyric acid (GABA)-A receptor blockade with bicuculline) and inhibited by neuronal activity repression [by blockade of Ca2+ channels with Mn2+, Na+ channels with tetrodotoxin (TTX) or excitatory inputs with glutamate antagonists]. Our observations establish therefore that F3/contactin surface expression in hypothalamic neurons is polarized to the axons where it occurs mainly in a GPI-linked form. We also provide direct evidence that externalization of F3/contactin depends on Ca2+ entry and neuronal electrical activity. Taken together with our earlier finding that the glycoprotein is localized in neurosecretory granules, we demonstrate that F3/contactin is mobilized to the axonal surface via the activity-dependent regulated pathway, thus arriving at the correct place and time to intervene in activity-dependent remodelling of axons.     

Metal specificity of an iron-responsive element in Alzheimer's APP mRNA 5'untranslated region, tolerance of SH-SY5Y and H4 neural cells to desferrioxamine, clioquinol, VK-28, and a piperazine chelator

Iron closely regulates the expression of the Alzheimer's Amyloid Precursor Protein (APP) gene at the level of message translation by a pathway similar to iron control of the translation of the ferritin L- and H mRNAs by Iron-responsive Elements in their 5' untranslated regions (5'UTRs). Using transfection based assays in SH-SY5Y neuroblastoma cells we tested the relative efficiency by which iron, copper and zinc up-regulate IRE activity in the APP 5'UTR. Desferrioxamine (high affinity Fe3+ chelator), (ii) clioquinol (low affinity Fe/Cu/Zn chelator), (iii) piperazine-1 (oral Fe chelator), (iv) VK-28 (oral Fe chelator), were tested for their relative modulation of APP 5' UTR directed translation of a luciferase reporter gene. Iron chelation based therapeutic strategies for slowing the progression of Alzheimer's disease (and other neurological disorders that manifest iron imbalance) are discussed with regard to the relative neural toxic action of each chelator in SH-SY5Y cells and in H4 glioblastoma cells.     

Polymorphisms in the vicinity of the hypocretin/orexin are not associated with human narcolepsy.

Human narcolepsy/cataplexy is associated with reduced hypocretin (orexin) transmission. A common preprophypocretin (HCRT) polymorphism (-909C/T) was identified and tested in 502 subjects (105 trio families, 80 Caucasian narcolepsy cases, and 107 Caucasian control subjects). This polymorphism was not associated with the disease. The promoter and 5' untranslated (5'URT) regions of the HCRT gene (-320 to +21 from ATG) were also sequenced in 281 subjects. None of the subjects carried -22T, a rare 5'UTR polymorphism previously reported to be associated with narcolepsy. The HCRT locus is not a major narcolepsy susceptibility locus.