Rachael L. Neve

Tissue distribution of human monoamine oxidase A and B mRNA

Abstract: Monoamine oxidase (MAO) A and B play important roles in the metabolism of biogenic amines. Northern analysis using 32P‐labeled subfragments of human liver MAO A and B cDNA clones detected a 5‐ and a 3‐kb transcript, respectively, in most human tissues examined. However, fetal heart and thymus express minute amounts of MAO A transcript, whereas fetal brain, muscle, thymus, spleen, meninges, and placenta express minute amounts of MAO B transcript. Small intestine and placenta express, in addition to the MAO A 5‐kb transcript, a 2‐kb transcript, which may arise from an alternative polyadenylation site. MAO A and B transcripts are expressed in similar regions of adult human brain. The highest concentrations of these transcripts were located in frontal cortex and locus coeruleus. This study demonstrates the tissue‐specific distribution of the MAO genes and will provide insight into the physiological functions of MAO A and B.

Contributions of Conserved Serine Residues to the Interactions of Ligands with Dopamine D2 Receptors

Abstract: Four dopamine D2 receptor mutants were constructed, in each of which an alanine residue was substituted for one of four conserved serine residues, i.e., Ser‐193, Ser‐194, Ser‐197, and Ser‐391. Wild‐type and mutant receptors were expressed transiently in COS‐7 cells and stably in C6 glioma cells for analysis of ligand‐receptor interactions. In radioligand binding assays, the affinity of D2 receptors for dopamine was decreased 50‐fold by substitution of alanine for Ser‐193, implicating this residue in the binding of dopamine. Each mutant had smaller decreases in affinity for one or more of the ligands tested, with no apparent relationship between the class of ligand and the pattern of mutation‐induced changes in affinity, except that the potency of agonists was decreased by substitution for Ser‐193. The potency of dopamine for inhibition of adenylyl cyclase was reduced substantially by substitution of alanine for Ser‐193 or Ser‐197. Mutation of Ser‐194 led to a complete loss of efficacy for dopamine and p‐tyramine, which would be consistent with an interaction between Ser‐194 and the p‐hydroxyl substituent of dopamine that is necessary for activation of the receptors to occur. Because mutation of the corresponding residues of β2‐adrenergic receptors has very different consequences, we conclude that although the position of these serine residues is highly conserved among catecholamine receptors, and the residues as a group are important in ligand binding and activation of receptors by agonists, the function of each of the residues considered separately varies among catecholamine receptors.

Molecular analysis of neuronal physiology by gene transfer into neurons with herpes simplex virus vectors

A genetic analysis of mammalian neuronal physiology might now be possible due to the development of defective herpes simplex virus vectors, which allow gene transfer directly into mature neurons, in culture or in the adult brain. Genetically altered proteins that play critical roles in neuronal physiology, including those responsible for the generation of action potentials, synthesis and release of neurotransmitters, and signal transduction enzymes, can now be stably expressed in neurons. The effect of such altered proteins on neuronal physiology can therefore be examined, using the tools of modern neuroscience. Genetic manipulation is biochemically specific and stable, and can be targeted both to a particular cell type and to a particular subregion of the cell to yield insights into the molecular basis for specific brain functions.

Expression of Plasma Membrane Calcium‐Pumping ATPase mRNAs in Developing Rat Brain and Adult Brain Subregions: Evidence for Stage‐Specific Expression

Abstract: The plasma membrane calcium‐pumping ATPases (Ca2+‐ATPases) maintain resting free cytosolic calcium concentrations in cells at the submicromolar level. These Ca2+‐ATPases are encoded by four genes that can be alternately spliced to produce nine different mRNAs, each of which has a unique tissue‐specific distribution. Examination of the expression of these mRNAs in rat brain during development revealed that transcripts from three of the four known genes are expressed by the end of gestation. However, the stage of transcription induction varies among the isoforms. The mRNA encoding plasma membrane Ca2+‐ATPase (PMCA) lb, the isoform thought to maintain a housekeeping function, was present from embryonic day 10. The other alternately spliced PMCA1 mRNAs, PMCAla and c, which are preferentially expressed in the brain, did not appear until embryonic day 14. PMCA2a mRNA and the alternatively spliced PMCA2b and c transcripts were coordinately induced on embryonic day 18. The PMCA3a transcript first appeared on embryonic day 18 but did not reach steady‐state levels until postnatal day 3, whereas production of PMCA3b mRNA first occurred on embryonic day 10 and reached steady‐state expression by embryonic day 18. Several PMCA mRNAs tested varied in expression in specific regions of the brain that were examined at three postnatal time points.

The Alzheimer amyloid precursor-related transcript lacking the β/A4 sequence is specifically increased in Alzheimer's disease brain

The deposition of cerebrovascular and plaque amyloid in the CNS is a primary feature of Alzheimers disease and aged Downs syndrome pathology. The localization of the Alzheimer amyloid protein precursor (APP) gene on chromosome 21, along with its overexpression in Downs syndrome brain compared with normal brain, suggests that alterations in APP gene expression may play a role in the development of the neuropathology common to the two diseases. In the present report, we demonstrate that a specific spliced form of mRNA that is transcribed from the APP gene and that lacks the beta/A4 sequence is elevated in the nucleus basalis, occipitotemporal cortex, and parahippocampal gyrus in Alzheimers disease brain relative to controls. These results are based on combined data from RNA slot blot analysis, in situ hybridization, and polymerase chain reaction quantification of specific mRNAs taken directly from tissue sections.

Antisense GAP-43 Inhibits the Evoked Release of Dopamine from PC12 Cells

Abstract: To investigate the role of the neuronal growth‐associated protein GAP‐43 (neuromodulin, B‐50, F1, P‐57) in neurotransmitter release, we transfected PC12 cells with a recombinant expression vector coding for antisense human GAP‐43 cRNA. Two stable transfectants, designated AS1 and AS2, were selected that had integrated the recombinant sequence and expressed antisense GAP‐43 RNA. Immunoblot analysis of proteins from AS1 and AS2 cells indicated that the level of GAP‐43 in these cell lines was reduced. In the presence of extracellular calcium, a depolarizing concentration of K+ (56 mM) evoked dopamine release from control cells, but not from AS1 and AS2 cells. Similarly, the calcium ionophore A23187 evoked dopamine release from control cells, but was ineffective in stimulating dopamine release from AS1 and AS2 cells. The antisense transfectants, as well as the control cells, contained appreciable quantities of dopamine and secretory granules with a normal appearance. Because the expression of antisense GAP‐43 RNA in PC12 cells leads to a decrease in GAP‐43 expression and to the loss of evoked dopamine release, these results provide evidence of a role for GAP‐43 in calcium‐dependent neurotransmitter release.

Determination of the nucleotide sequence and chromosomal localization of the ATP2B2 Gene Encoding Human Ca2+-Pumping ATPase Isoform PMCA2

The plasma membrane Ca(2+)-pumping ATPase (Ca(2+)-ATPase) is responsible for maintaining calcium homeostasis in eukaryotic cells. The Ca(2+)-ATPase is a family of pumps that are encoded by at least four genes. A cDNA for the human version of Ca(2+)-ATPase isoform PMCA2 was isolated and characterized. Comparison of the human and rat cDNA sequences showed that they were 95% homologous in the coding domain, and this homology was reflected in the deduced protein sequence where greater than 98% homology between the human and rat sequences was found. The amino acid differences that were found were almost all conservative. The PMCA2 cDNA was used to probe Southern blots of human-rodent somatic cell hybrid DNAs; the results indicated that the human PMCA2 gene was located on chromosome 3.

Molecular analysis of the function of the neuronal growth-associated protein GAP-43 by genetic intervention

GAP-43 is a presynaptic membrane phosphoprotein that has been implicated in both the development and the modulation of neural connections. The availability of cDNA clones for GAP-43 makes it possible to examine with greater precision its role in neuronal outgrowth and physiology. We used Northern blots andin situ hybridization with GAP-43 antisense RNA probes to show that GAP-43 is expressed selectively in associative regions of the adult brain. Immunocytochemical analyses showed alterations in the pattern of GAP-43 expression in the hippocampus during reactive synaptogenesis following lesions of the perforant pathway. Genetic intervention methodology was used to analyze the molecular nature of GAP-43 involvement in synaptic plasticity. GAP-43-transfected PC12 cells displayed an enhanced response to nerve growth factor, suggesting that GAP-43 may be directly involved in neurite extension and in the modulation of the neuronal response to extrinsic trophic factors. Studies of PC12 cell transfectants, in which the synthesis of GAP-43 was blocked by expression of GAP-43 antisense RNA, showed that evoked dopamine release was significantly attenuated in these cells. The use of gene transfer into neurons with the HSV-1 vector is presented as a method of analyzing the interaction of GAP-43 with signal transduction systems during neurotransmitter release.

Chapter 20 Trophic regulation of basal forebrain gene expression in aging and Alzheimer's disease

Publisher Summary An individual neurons vulnerability to the pathological consequences of Alzheimers disease (AD) may, in part, be determined by its ability to initiate appropriate “regenerative” or “trophic” programs of gene expression during periods of cellular stress. Stressors may include decreased trophic factor support, metabolic and oxidative stress, glucocorticoid or excitotoxin-mediated damage, loss of target or neighboring neurons, or pathological phenomena specific for the disease. This chapter focuses on the nerve growth factor (NGF)—the responsive pathway that originates in the cholinergic basal forebrain and terminates in the neocortex and hippocampal formation. NGF is the well-characterized member of a supergene family of growth factors that have been found in the CNS. It has been proposed that neurodegenerative changes, which occur in the cholinergic basal forebrain in AD, may be attributed to deficits in the NGF responsiveness of basal forebrain neurons. To determine the extent to which deficits in the NGF-responsiveness in the basal forebrain may contribute to neuronal atrophy and amyloid pathology in aging and AD, parallel studies in the rodent and human basal forebrain have been conducted in the chapter and several genes whose expression appears to be regulated by NGF have also been examined.