Insulin receptor substrate protein p53 localization in rats suggests mechanism for specific polyglutamine neurodegeneration.

Dentatorubral-pallidoluysian atrophy (DRPLA) is a neurodegenerative disease that results from the expansion of an unstable CAG repeat within the coding regions of the DRPLA gene. Recently it was shown that the DRPLA gene product, atrophin-1, interacts with the human insulin receptor tyrosine kinase substrate protein, IRSp53. We have isolated rat and mouse cDNA clones for IRSp53 and determined expression patterns in rat central nervous system. In situ hybridization analysis revealed enriched IRSp53 mRNA expression in rat forebrain structures, including the cerebral cortex (layers II/III, V and VI), striatum, hippocampus and olfactory bulb. IRSp53 hybridization signals were also detected in the cerebellum, subthalamic nucleus, pons, amygdala and hypothalamus. These findings support the idea that insulin and insulin growth factor-1 have a role in neurotransmission, one that is regionally specific. The expression of IRSp53 in regions similar to those that degenerate in DRPLA supports the notion that IRSp53 is a relevant atrophin-1 binding protein and may provide a mechanism for region-specific neurodegeneration.

TOGA: an automated parsing technology for analyzing expression of nearly all genes.

We have developed an automated, high-throughput, systematic cDNA display method called TOGA, an acronym for total gene expression analysis. TOGA utilizes 8-nt sequences, comprised of a 4-nt restriction endonuclease cleavage site and adjacent 4-nt parsing sequences, and their distances from the 3' ends of mRNA molecules to give each mRNA species in an organism a single identity. The parsing sequences are used as parts of primer-binding sites in 256 PCR-based assays performed robotically on tissue extracts to determine simultaneously the presence and relative concentration of nearly every mRNA in the extracts, regardless of whether the mRNA has been discovered previously. Visualization of the electrophoretically separated fluorescent assay products from different extracts displayed via a Netscape browser-based graphical user interface allows the status of each mRNA to be compared among samples and its identity to be matched with sequences of known mRNAs compiled in databases.

Rhes: A striatal-specific Ras homolog related to Dexras1.

We have characterized an apparently full-length cDNA corresponding to a rat mRNA, SE6C, previously identified by subtractive hybridization as being expressed predominantly in the striatal region of the brain. The SE6C mRNA encodes a 266 amino acid protein with significant similarity to members of the Ras-like GTP-binding protein family; thus, we have chosen the name Rhes, for Ras homolog enriched in striatum. The human homolog was found in a genomic sequence from human chromosome 22q13.1 and shares 95% identity with rat Rhes. Among the family of small G-proteins, Rhes shares 62% identity with Dexras1, a mouse dexamethasone-inducible Ras-like protein. Both Rhes and Dexras1 have substantially longer C-termini than other members of the Ras-like small G-protein family. Divergence between the C-terminal sequences of Rhes and Dexras1 suggests that, although their functions are probably similar, they have unique properties. Bacterially expressed Rhes binds GTP, suggesting that the protein indeed has GTPase functionality. Although Rhes was not induced by dexamethasone, its full expression is dependent upon thyroid hormone availability. Its accumulation is postnatal, consistent with the dependence upon thyroid hormone. It is noteworthy that most striatum-"specific" mRNAs characterized to date encode components of signal transduction cascades.

The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity.

We describe a hypothalamus-specific mRNA that encodes preprohypocretin, the putative precursor of a pair of peptides that share substantial amino acid identities with the gut hormone secretin. The hypocretin (Hcrt) protein products are restricted to neuronal cell bodies of the dorsal and lateral hypothalamic areas. The fibers of these neurons are widespread throughout the posterior hypothalamus and project to multiple targets in other areas, including brainstem and thalamus. Hcrt immunoreactivity is associated with large granular vesicles at synapses. One of the Hcrt peptides was excitatory when applied to cultured, synaptically coupled hypothalamic neurons, but not hippocampal neurons. These observations suggest that the hypocretins function within the CNS as neurotransmitters.

Cloning, mRNA expression, and chromosomal mapping of mouse and human preprocortistatin.

Cortistatin is a 14-residue putative neuropeptide with strong structural similarity to somatostatin and is expressed predominantly in cortical GABAergic interneurons of rats. Administration of cortistatin into the brain ventricles specifically enhances slow-wave sleep, presumably by antagonizing the effects of acetylcholine on cortical excitability. Here we report the identification of cDNAs corresponding to mouse and human preprocortistatin and the mRNA distribution and gene mapping of mouse cortistatin. Analysis of the nucleotide and predicted amino acid sequences from rat and mouse reveals that the 14 C-terminal residues of preprocortistatin, which make up the sequence that is most similar to somatostatin, are conserved between species. Lack of conservation of other dibasic amino acid residues whose cleavage by prohormone convertases would give rise to additional peptides suggests that cortistatin-14 is the only active peptide derived from the precursor. As in the rat, mouse preprocortistatin mRNA is present in GABAergic interneurons in the cerebral cortex and hippocampus. The preprocortistatin gene maps to mouse chromosome 4, in a region showing conserved synteny with human 1p36. The human putative cortistatin peptide has an arginine for lysine substitution, compared to the rat and mouse products, and is N-terminally extended by 3 amino acids.

1G5: a calmodulin-binding, vesicle-associated, protein kinase-like protein enriched in forebrain neurites.

We have characterized cDNA clones of 1G5, an mRNA highly enriched in the mammalian forebrain that encodes a 504-residue protein found in association with perikaryal membranes and neurites. The protein, which accumulates predominantly postnatally, is associated with vesicles in both axons and dendrites. The sequence of the 1G5 protein highly resembles those of protein kinases with serine/threonine specificity; however, although most residues universally conserved among protein kinases are present, a few signature residues are absent from the 1G5 protein. Furthermore, although recombinant 1G5 protein binds calmodulin in the presence of calcium, it lacks kinase activity with a sample substrate.

A novel adenylyl cyclase-activating serotonin receptor (5-HT7) implicated in the regulation of mammalian circadian rhythms.

We report the cloning and characterization of a novel serotonin receptor, designated as 5-HT7, which is coupled to the stimulation of adenylyl cyclase. 5-HT7 mRNA is expressed discretely throughout the CNS, predominantly in the thalamus and hypothalamus. 5-HT7 has a unique pharmacological profile that redefines agonist and antagonist classification of ligands previously thought to be "selective." The circadian phase of spontaneous neuronal activity of the rat suprachiasmatic nucleus of the hypothalamus advances in response to serotonin ligands with a pharmacological profile consistent exclusively with that of 5-HT7. These findings suggest a physiological role in the regulation of circadian rhythms for one subtype of serotonin receptor, 5-HT7, and provide a pharmacological test to evaluate its role in other neuronal systems.

Two members of a distinct subfamily of 5-hydroxytryptamine receptors differentially expressed in rat brain.

We report two serotonin (5-hydroxytryptamine, 5-HT) receptors, MR22 and REC17, that belong to the G-protein-associated receptor superfamily. MR22 and REC17 are 371 and 357 amino acids long, respectively, as deduced from nucleotide sequence and share 68% mutual amino acid identity and 30-35% identity with known catecholamine and 5-HT receptors. Saturable binding of 125I-labeled (+)-lysergic acid diethylamide to transiently expressed MR22 in COS-M6 cells was inhibited by ergotamine > methiothepin > 5-carboxamidotryptamine > 5-HT. For REC17, the rank of potency was ergotamine > 5-carboxamidotryptamine > methiothepin > methysergide > 5-HT. Both were insensitive to 5-HT1A, 5-HT1D or 5-HT2 serotonergic ligands [8-hydroxy-2-(di-n-propylamino)tetralin, sumatriptan, and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane]. The mRNAs encoding MR22 were detected in the CA1 region of hippocampus, the medial habenula, and raphe nuclei. In contrast, mRNAs encoding REC17 were found throughout the rat central nervous system. We propose that REC17 and MR22, designated as 5-HT5 alpha and 5-HT5 beta, represent a distinct subfamily of 5-HT receptors.