FGF2 suppresses neuronogenesis of a cell line derived from rat olfactory epithelium.

Neurogenesis continues throughout adulthood in the mammalian olfactory epithelium (OE), and both neurons as well as nonneuronal cells are reconstituted following experimental injury. Underlying the capacity of the OE to replenish its mature elements is a population of progenitor basal cells. Although the precise lineage relationships among progenitor and mature cell types are incompletely understood, the population of globose basal cells (GBCs) contains immediate precursors to neurons as well as amplifying progenitors, and retroviral lineage analyses suggest that multipotential GBCs are activated following direct injury to the OE. To assess the controls on the process of epithelial regeneration, we have characterized a cell line derived from rat OE and studied the effects of serum and tissue extracts, fibroblast growth factor-2 (FGF2) and transforming growth factor-alpha (TGF alpha) on the cells. Using a panel of cell type-specific markers whose patterns of labeling in the OE are well defined, including recently developed markers for GBCs, we characterized the phenotype of the cell line under differing culture conditions. In complete medium, which contains serum and tissue extracts, the cell line displayed characteristics of GBCs that are prominent during regeneration. Serum and extract withdrawal induced the cells to differentiate into neurons. In contrast, FGF2 prevented neuronal differentiation and maintained a GBC phenotype. TGF alpha had a mitogenic or differentiative effect that was context dependent. Finally, we demonstrate here that FGF2 is contained in mature olfactory neurons and sustentacular cells in vivo, suggesting a physiologic role for this growth factor in OE cell regulation.

Differential regulation of APP secretion by apolipoprotein E3 and E4.

The apolipoprotein E isozyme, apolipoprotein E4, has been implicated as a risk factor for Alzheimer's disease. One reason for the increased risk may be that apolipoprotein E binds to the A beta peptide, but there may be other factors as well. We show that apolipoprotein E is a potent regulator of the secretion of amyloid precursor protein. In cultures of PC12 cells, nanomolar levels of apolipoprotein E3 induce a rapid decrease in the secretion of APP, being observable in 30 min. and stable over 24 hours. Apolipoprotein E4, in contrast, increases secretion of APP over a similar time course. Reciprocal changes occur in cellular amyloid precursor protein. Differential characteristics are also seen in apo E binding to the cells, where apo E4 binds over a slower time course than apo E3. These results suggest a novel mechanism by which apolipoprotein E may be influencing the metabolism of amyloid precursor protein.

Primary structure of the serotonin transporter in unipolar depression and bipolar disorder.

Genetic factors have been implicated in the etiology of affective disorders but due to the complex inheritance patterns of these disorders, identification of the responsible gene(s) has so far been unsuccessful. Decreased platelet serotonin (5-HT) transport and reduced binding of imipramine or paroxetine to brain and platelet 5-HT uptake sites/transporters in patients with depression and suicide victims define the 5-HT transporter (5-HTT) as a candidate gene. The primary structure of the 5-HTT was analyzed in 17 patients meeting DSM-III-R diagnostic criteria for major depressive or bipolar disorder and in 4 healthy controls using polymerase chain reaction (PCR-) amplification and sequencing of complementary deoxyribose nucleic acid (cDNA) synthesized from platelet 5-HTT messenger ribose nucleic acid (mRNA). Direct PCR sequencing of the protein coding region failed to reveal changes in the deduced amino acid sequence of the platelet/brain 5-HTT (40,000 base pairs sequence screened), although a conservative single-base substitution representing a silent polymorphism was found. The results provide preliminary evidence that alterations in the primary structure of 5-HTT are not generally involved in the pathogenesis of unipolar depression and manic-depressive illness.

Direct sequencing of the reserpine-sensitive vesicular monamine transporter complementary DNA in unipolar depression and manic depressive illness.

The reserpine model and the reduced monoamine hypothesis of the depressive symptom spectrum suggest that the reserpine-sensitive brain vesicular monoamine transporter (VMT) is a candidate for susceptibility to affective disorder. VMT nonselectively accumulates cytoplasmic biogenic monoamine neurotransmitters into the storage vesicles of presynaptic neurons and blood platelets. Complementary DNA (cDNA) synthesized from platelet VMT mRNA was analyzed in 17 patients meeting DSM-III-R diagnostic criteria for major depressive or bipolar disorder and in four healthy controls, using polymerase chain reaction (PCR) amplification and direct sequencing. PCR sequencing of the protein coding region failed to reveal changes in the deduced amino acid sequence of the platelet/brain VMT (approximately 36,000 base pairs sequence screened). The results indicate that alterations in the primary structure of the VMT are not generally involved in the pathogenesis of unipolar depression and manic depressive illness.

Organization of the human serotonin transporter gene.

The gene encoding the human serotonin transporter (5-HTT) has been isolated and characterized. The human 5-HTT gene is composed of 14 exons spanning approximately 31 kb. The sequence of all exons including adjacent intronic sequences and a tandem repeat DNA polymorphism (VNTR) has been determined and deposited in the EMBL/GenBank data base with the accession numbers X76753 to X76762. The characterization of 5-HTT gene will aid to advance molecular pharmacologic studies of 5-HT uptake regulation and facilitate investigations of its role in psychiatric disorders.

Primary structure of the human platelet serotonin uptake site: identity with the brain serotonin transporter.

A cDNA encoding the human platelet serotonin (5-HT) uptake site was isolated and sequenced using the PCR. The cDNA represents a approximately 3.1-kb mRNA transcript and contains an open reading frame encoding a hydrophobic polypeptide of 630 amino acids with 12 membrane-spanning segments, a calculated molecular mass of 70,320 Da, and an estimated isoelectrical point of 5.84. The human platelet 5-HT uptake site is identical with the human brain 5-HT transporter and approximately 92% homologous to the rat protein. Hydropathicity analysis indicates 12 membrane-spanning segments with two putative glycosylation sites within the second extracellular loop. The human platelet 5-HT uptake site contains two intraplasmatic consensus phosphorylation sites for cyclic AMP-dependent protein kinase recognition located in the cytoplasmatic N-terminal region and three potential protein kinase C phosphorylation sites. The identity of the human platelet 5-HT uptake site and the brain 5-HT transporter indicates that both proteins are encoded by the same single-copy gene, which has been assigned to the human chromosome 17. Our findings are likely to facilitate molecular pharmacologic and genetic investigations of the 5-HT transporter in psychiatric disorders.

Isolation of a cDNA encoding the human brain serotonin transporter.

A cDNA encoding a serotonin transporter (5-HTT) in the human dorsal raphe nucleus was isolated and sequenced using cross-species amplification of human 5-HTT partial cDNA by the polymerase chain reaction (PCR) and the RACE-PCR procedure, designed for rapid amplification of 3' and 5' cDNA ends. The cDNA contains an open reading frame encoding a hydrophobic polypeptide of 630 amino acids with a calculated molecular weight of approximately 70 kDa. The human 5-HTT is approximately 92% homologous to the rat protein but contains an additional consensus phosphorylation site for cAMP-dependent protein kinase recognition located in the cytoplasmic N-terminal region, while a potential protein kinase C phosphorylation site identified in the rat homolog is not conserved in the human 5-HTT. Hydropathicity analysis revealed twelve membrane spanning segments, a topology proposed for other cloned sodium-dependent transporters.

Extensive sequence divergence between the human and rat brain vesicular monoamine transporter: possible molecular basis for species differences in the susceptibility to MPP+.

A cDNA encoding the human brain vesicular monoamine transporter (VMT) was isolated and sequenced using PCR. The cDNA contains an open reading frame encoding a hydrophobic polypeptide of 514 amino acids with twelve membrane spanning segments, a calculated molecular weight of 55,709 Da, and an estimated isoelectrical point of 5.62. A structurally identical transporter is expressed in human platelets. Two intraplasmatic consensus phosphorylation sites of cAMP-dependent protein kinase recognition and two potential protein kinase C phosphorylation sites may be central to the regulation of the VMT. Although the human brain VMT is 90.7% homologous to the rat protein, an extensive sequence divergence occurs in the large luminal loop located between the first two transmembrane domains. This loop displays a remarkably reduced homology of 64.0% with several deletions and insertions, although four putative glycosylation sites are conserved. Since functional vesicular monoamine transport suppresses MPP+ toxicity and sequence divergence in the large luminal loop of the VMT expressed in rat brain and adrenal medulla may play a role in differential neurotoxic effects of MPP+, our findings indicate one possible molecular basis for the substantial species differences in the susceptibility to MPP+ demonstrated among humans, non-human primates, and rodents. They are also likely to facilitate molecular pharmacologic and genetic investigations of the human VMT in neurodegenerative disorders.

Regional brain expression of serotonin transporter mRNA and its regulation by reuptake inhibiting antidepressants.

Regional expression and antidepressant drug-induced regulation of mRNA encoding the serotonin (5-HT) transporter were studied in rat brain. While 5-HT transporter mRNA is abundantly expressed in the midbrain raphe complex, lower concentrations were also found in frontal cortex, hippocampus, and neostriatum using a combination of reverse transcriptase-polymerase chain reaction (RT-PCR), Southern hybridization, and sequence analysis. Long-term administration of antidepressants which inhibit 5-HT reuptake, but not monoamine oxidase inhibitors or 5-HT receptor agonists, decrease 5-HT transporter mRNA steady-state concentrations. Based on these observations, we conclude that (1) mRNA coding for the 5-HT transporter is present in several brain areas associated with ascending HT pathways, and (2) chronic treatment with reuptake inhibiting antidepressants may be associated with regulation of the 5-HT transporter at the level of gene expression which may contribute to the neuroadaptive mechanisms that likely underlie their therapeutic efficacy.

Fluoxetine modulates G protein alpha s, alpha q, and alpha 12 subunit mRNA expression in rat brain.

Signal-transducing G proteins are central to the coordination of receptor-effector communication. We have explored the effects of long-term fluoxetine administration of G alpha s, G alpha i1, G alpha i2, G alpha o, G alpha q and G alpha 12 mRNA expression in various rat brain regions using reverse transcriptase-polymerase chain reaction (RT-PCR)-mediated cross-species partial cDNA cloning. Northern blot analysis, and RNase protection assay techniques. Fluoxetine decreased G alpha s mRNA in midbrain, while mRNA expression of the novel G protein alpha subunits, G alpha q and G alpha 12, was increased in neostriatum and frontal cortex. We conclude that in addition to post-translational modification, regulation of G protein function by antidepressant drugs may occur at the level of gene expression.

3-(2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid decreases NMDA receptor mRNA.

Expression of the N-methyl-D-aspartate receptor gene during long-term administration of competitive and non-competitive NMDA antagonists was studied in rat brain using antisense cRNA transcribed from reverse transcriptase-polymerase chain reaction (RT-PCR)-generated rat NMDA receptor cDNA. Unlike non-competitive antagonists, 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) markedly decreased NMDA receptor mRNA steady-state concentrations in frontal cortex and hippocampus. Our results are consistent with a regulation of the NMDA receptor at the level of gene expression.

Differential effects of long-term lithium and carbamazepine administration on Gs alpha and Gi alpha protein in rat brain.

The G protein subunits, Gs alpha, Gi alpha, and Go alpha, have been quantitated in various rat brain regions using enzyme-linked immunosorbent assay (ELISA) techniques. Chronic (3-week) treatment with carbamazepine decreased Gs alpha in several brain regions reaching significance in the neostriatum, while chronic lithium treatment had no unequivocal effect. Lithium significantly increased Gi alpha in the hypothalamus and hippocampus, whereas carbamazepine decreased Gi alpha in the frontal cortex. Both treatments had no consistent effects on Go alpha. We conclude that long-term treatment with lithium and carbamazepine exerts differential effects on G protein alpha subunits, and that this modification of signal transduction represents a potential mechanism of antibipolar drug-induced neural plasticity.

Alz-50 antibody recognizes Alzheimer-related neuronal changes.

Alz-50 is a monoclonal antibody directed against an antigen present in Alzheimer's disease. Unlike conventional pathological stains that reveal only neurons with neurofibrillary tangles (NFTs), Alz-50 recognizes neurons that contain NFTs and additional neurons that do not. Our investigation of the topographical distribution of both NFT-containing and non-NFT-containing neurons recognized by Alz-50 reveals that they are both found primarily in certain cytoarchitectural areas and lamina that have been established as consistent sites for NFT pathology. Some of the neurons recognized by Alz-50 appear histopathologically normal. We suggest that Alz-50 recognizes an antigen in neurons that both precedes and accompanies NFT formation. Thus, it may mark a point early in cellular pathology before irreversible cytoskeletal and degenerative changes occur.

Recent advances in the neurochemistry of Alzheimer's disease.

This article reviews recent progress in research in the genesis of neurochemical changes in the Alzheimer brain, which has been primarily in two areas: the cellular proteins associated with the pathologic structures and the neurotransmitter changes. Research and clinical implications of recent studies and of works in progress are provided.

A neuronal antigen in the brains of Alzheimer patients.

A monoclonal antibody was prepared against pooled homogenates of brain tissue from patients with Alzheimer's disease. This antibody recognizes an antigen present in much higher concentration in certain brain regions of Alzheimer patients than in normal brain. The antigen appears to be a protein present in neurons involved in the formation of neuritic plaques and neurofibrillary tangles, and in some morphologically normal neurons in sections from Alzheimer brains. Partial purification and Western blot analysis revealed the antigen from Alzheimer brain to be a single protein with a molecular weight of 68,000. Application of the same purification procedure to normal brain tissue results in the detection of small amounts of a protein of lower molecular weight.

The binding of kappa- and sigma-opiates in rat brain.

Detailed displacements of [3H]dihydromorphine by ketocyclazocine and SKF 10,047, [3H]ethylketocyclazocine by SKF 10,047, and [3H]SKF 10,047 by ketocyclazocine are all multiphasic, suggesting multiple binding sites. After treating brain tissue in vitro with naloxazone, all displacements lose the initial inhibition of 3H-ligand binding by low concentrations of unlabeled drugs. Together with Scatchard analysis of saturation experiments, these studies suggest a common site which binds mu-, kappa, and sigma-opiates and enkephalins equally well and with highest affinity (KD less than 1 nM). The ability of unlabeled drugs to displace the low affinity binding of [3H]dihydromorphine (KD = 3 nM), [3H]ethylketocyclazocine (KD = 4 nM), [3H]SKF 10,047 (KD = 6 nM), and D-Ala2-D-Leu5-[3H]enkephalin (KD = 5 nM) remaining after treating tissue with naloxazone demonstrates unique pharmacological profiles for each. These results suggest the existence of distinct binding sites for kappa- and sigma-opiates which differ from those sites which selectively bind morphine (mu) and enkephalin (delta).

Specific chemical modification of the readily nitrated tyrosine of the RTEM beta-lactamase and of bacillus cereus beta-lactamase I. The role of the tyrosine in beta-lactamase catalysis.

The function of the hydroxyl group of the tyrosine residue readily nitrated by tetranitromethane (tyrosine-105) in the RTEM plasmid-derived beta-lactamase (penicillinase; penicillin amido beta-lactam-hydrolase, EC 3.5.1.6) from E. coli and in Bacillus cereus beta-lactamase I has been investigated by chemical modification methods. In the case of B. cereus beta-lactamase I the nitrated tyrosine can be acetylated by acetic anhydride without effect on beta-lactamase activity The nitrated tyrosine of the E. coli enzyme can also be acetylated but in this case beta-lactamase activity is lost in a manner which directly correlates with extent of acetylation. However, deacetylation of the nitrotyrosine does not restore activity. The dilemma created by the latter result has been resolved by development of a new method of tyrosine hydroxyl modification at low pH. The nitrated enzyme is reduced by dithionite and then treated with either carbonyldiimidazole or N-(2.2.2-trifluoroethoxycarbonyl)imidazole, both of which convert 3-aminotyrosine into benzoxazolinonylalanine. That the final modification has been achieved is demonstrated both by classical chemical methods and by employment of Fourier transform infrared spectroscopy to detect the characteristic benzoxazolinone carbonyl absorption. Further, it is shown that no significant loss of beta-lactamase activity is associated with this modification. Hence in neither the B. cereus or the E. coli enzyme does the readily nitrated tyrosine residue have a direct chemical function at the beta-lactamase active site.

Classification of multiple morphine and enkephalin binding sites in the central nervous system.

Detailed competitive displacement curves of 3H-labeled [D-Ala2,Met5]enkephalinamide, [D-Ala2,D-Leu5]enkephalin, and dihydromorphine by a series of opiates and enkephalins are biphasic, suggesting multiple sites. After treatment of tissue with naloxazone, the displacement of the three 3H-labeled ligands by all opiates and enkephalins tested becomes monophasic, losing the high-affinity displacement seen with low concentrations of both opiates and enkephalins. Coupled with Scatchard analysis of saturation experiments, these findings suggest a common site that binds both opiates and enkephalins equally well and with highest affinity (Kd values, less than 1 nM). Termed the mu 1 site, it corresponds to the previously described high-affinity site and appears to be the site responsible for analgesia under normal circumstances. The low-affinity binding of [3H]dihydromorphine (Kd, 3 nM) remaining after naloxazone treatment differs dramatically from low-affinity [D-Ala2,D-Leu5]-[3H]enkephalin binding (Kd, 5 nM). The mu 2 site, corresponding to the low-affinity [3H]dihydromorphine receptor sites, binds morphine (Ki, 10 nM) and dihydromorphine (Kd, 3 nM) far better than [D-Ala2,D-Leu5]enkephalin (Ki, 50 nM). Low-affinity [D-Ala2,D-Leu5]-[3H]enkephalin receptor sites bind [D-Ala2,D-Leu5]enkephalin (Ki, 5-8 nM) more potently than morphine (Ki, 71 nM) and correspond to the previously established delta receptor.