Association of phenotypic abnormalities of Down syndrome with an imbalance of genes on chromosome 21.

The phenotype of the brain in Down syndrome is different from that of a normal child both in its reduced size and altered gyral configuration. Underlying the mental retardation are neuronal abnormalities, including alterations of cortical lamination, reduced dendritic ramifications, and diminished synaptic formation. However, cholinergic enzymes such as choline acetyl transferase and acetyl cholinesterase have shown no abnormalities in young children with Down syndrome. The pace of dendritic maturation is altered in Down syndrome. In infancy, the normal dendritic tree continuously expands; in Down syndrome, at 4 months of age, the neurons show a relatively expanded tree, but during the first year, the dendrites stop growing and become atrophic relative to control neurons. To relate these phenotypic alterations to chromosome 21, we examined the gene products of several genes localized to chromosome 21. Identification of such genes and determination of their gene product allow the production of specific antibodies and the identification, through immunohistochemical techniques, of the expression of these proteins in both normal development and Down syndrome. Specifically, the localization and appearance during development of proteins such as S100 beta, beta A4-amyloid, superoxide dismutase, and OK-2 are providing links between genotype and phenotype. S100 beta protein is of particular interest because of its effect in vitro on neuritic outgrowth and its increased expression in the temporal lobe in Down syndrome. The brains of transgenic mice bearing multiple copies of the human S100 gene show some comparable changes to those in Down syndrome. These experimental approaches provide the means for better understanding the cellular and molecular basis for the mental retardation in Down syndrome.

Cell-specific expression of high levels of human S100 beta in transgenic mouse brain is dependent on gene dosage.

The beta-subunit of S100 protein (S100 beta) is highly conserved in the mammalian brain. The gene coding for human S100 beta has been mapped to chromosome 21. In order to study the consequences of overexpression of the S100 beta gene, transgenic mice were generated by microinjection of a 17.3 kilobase human genomic fragment containing the three exons and the transcription control elements of the human S100 beta gene. Mice from four transgenic lines carried approximately 10-100 transgene copies. Northern blotting demonstrated a tissue-specific and gene dose-dependent expression of human S100 beta mRNA in mouse brain. Increased expression of S100 beta mRNA was correlated with an increased production of S100 beta protein. Examination of brain sections by in situ hybridization and immunocytochemistry indicated that S100 beta was localized globally to astrocytes, as well as to discrete neurons in the mesencephalic and motor trigeminal, facial, and lemniscus nuclei in both normal and transgenic mice. In peripheral tissues, human S100 beta was expressed at 10-50-fold lower levels than in brain. The strict gene dosage dependence and cell specificity of transgene expression suggest the presence of a locus control region (LCR) in the human S100 beta gene. The mice tolerated 10-100-fold higher than normal levels of S100 beta gene expression in brain without any gross physical or behavioral abnormalities. The high-level expression and cell specificity of the S100 beta promoter/LCR suggest that it may provide a valuable tool to direct the expression of other transgenic products to specific cell types in the CNS.

Cloning and expression of the human S100 beta gene.

S100 protein is a low molecular weight, EF-hand, Ca2(+)-binding protein widely distributed and conserved in the central nervous system of vertebrates. The gene coding for the beta subunit of human S100 protein (S100 beta) has been recently mapped to chromosome 21. In order to study the expression of this gene in normal and abnormal brain development, we have isolated and characterized overlapping genomic clones spanning the region coding for human S100 beta and its flanking sequences. The intron-exon organization of the human S100 beta gene is similar to that of the genes coding for several other members of the S100 protein subfamily of EF-hand proteins. The human S100 beta gene is composed of 3 exons, the first of which specifies the 5'-untranslated region, while the second and third each encode a single EF-hand, Ca2(+)-binding domain. The promoter region contains several potential regulatory transcription elements including the cAMP-responsive elements CRE and AP-2. A novel sequence motif, the S100 protein element, situated in close proximity to the TATA box of the genes of several members of the S100 protein subfamily, has been identified. In addition, multiple repeats with similar nucleotide sequence and location to the recently reported beta globin direct repeat elements have been also found in the human S100 beta promoter. A full length (17.3 kilobases) copy of the human S100 beta gene was constructed and transfected into rat glioma C6 cells. Stable transfectants were shown to express correctly initiated transcripts of the human S100 beta gene, indicating that the cloned sequences contain functional regulatory transcription elements.

Effect of haloperidol and apomorphine treatment on dopamine receptors in pituitary and striatum.

Prompted by an interest in the similarity of brain and tuberoinfundibular systems, the authors studied butaclamol-specific neuroleptic and apomorphine binding in pituitary and striatum after chronic haloperidol and acute apomorphine treatment. Striatal binding increases but pituitary binding decreases in haloperidol-treated rats. Pituitary binding changes rapidly in response to apomorphine exposure and striatal binding does not. These findings suggest that factors influencing binding differ in these two tissues.

Hypothalamic catecholaminergic effects on prolactin release in vitro.

Hypothalamic catecholaminergic influences on prolactin release were investigated in vitro. Both dopamine and norepinephrine caused long lasting inhibition of prolactin release from either an isolated hemipituitary or a hemipituitary coincubated with a hypothalamus. Epinephrine also inhibited prolactin release. L-Dihydroxyphenylalanine (L-dopa) inhibited prolactin release from pituitaries in the presence of a hypothalamus but not in isolated pituitaries. DL-Threodihydroxyphenylserine (threodops), serotonin, 5-hydroxy-L-tryptophan (5-HTP), tyramine, octopamine, synephrine, thyrotropin-releasing hormone (TRH), luteinizing hormone releasing hormone (LH-RH), and somatostatin all failed to alter prolactin release. Results confirm that dopamine and norepinephrine directly inhibit prolactin release from pituitary and suggest that the hypothalamic mechanism inhibiting prolactin involves dopamine but not norepinephrine.

Dopamine receptors in the central nervous system.

Dopamine receptors in the central nervous system can be studied by measuring the specific binding of [3H]dopamine, [3H]haloperidol, d-[3H]LSD, [3H]dihydroergocryptine or [3H]apomorphine. The receptors are stereoselectively blocked by +)-butaclamol, a neuroleptic. All neuroleptics inhibit the specific binding of [3H]haloperidol in relation to their clinical potencies. The radioligand that desorbs most slowly from the receptor is [3H]apomorphine, thus making it a reliable ligand for dopamine receptors. Dopamine agonists that compete for [3H]apomorphine binding do so at concentrations that correlate with their potency in stimulating striatal adenylate cyclase. Structure-activity analysis, using [3H]apomorphine, confirms that the active dopamine-mimetic conformation is the beta rotamer of dopamine. Prolonged exposure in vitro of caudate homogenate to high concentrations of dopamine leads to increased binding of [3H]apomorphine or [3H]haloperidol, suggesting receptor "sensitization." Chronic haloperidol treatment of rats leads to an increased number of dopamine/neuroleptic receptors in the striatum, but a decrease in the pituitary.