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Biomedical subjects

R L Neve

Publications and source records attributed to R L Neve.

At least 19 recordsLinked to original sources
PubMed1992

Deposition of beta/A4 immunoreactivity and neuronal pathology in transgenic mice expressing the carboxyl-terminal fragment of the Alzheimer amyloid precursor in the brain.

The deposition of amyloid in senile plaques and along the walls of the cerebral vasculature is a characteristic feature of Alzheimer disease. The peptide comprising the carboxyl-terminal 100 amino acids of the beta-amyloid precursor protein (beta APP) has been shown to aggregate into amyloid-like fibrils in vitro and to be neurotoxic, suggesting that this fragment may play a role in the etiology of Alzheimer disease. To address this question, we expressed this carboxyl-terminal 100-amino acid peptide of beta APP in transgenic mice under the control of the brain dystrophin promoter. We used an antibody to the principal component of amyloid, beta/A4, to demonstrate cell-body and neuropil accumulation of beta/A4 immunoreactivity in the brains of 4- and 6-month-old transgenic mice. Only light cytoplasmic staining with this antibody was visible in control mice. In addition, immunocytochemical analysis of the brains with an antibody to the carboxyl terminus of beta APP revealed abnormal aggregation of this epitope of beta APP within vesicular structures in the cytoplasm and in abnormal-appearing neurites in the CA2/3 region of the hippocampus in transgenic mice, similar to its aggregation in the cells of Alzheimer disease brains. Thioflavin S histochemistry suggested accumulations of amyloid in the cerebrovasculature of transgenic mice with the highest expression of the beta APP-C100 transgene. These observations suggest that expression of abnormal carboxyl-terminal subfragments of beta APP in vivo may cause amyloidogenesis and specific neuropathology.

Amyloid beta-Protein Precursor↗
PubMed1992

Brain transplants of cells expressing the carboxyl-terminal fragment of the Alzheimer amyloid protein precursor cause specific neuropathology in vivo.

PC12 cells transfected with retroviral recombinants expressing the carboxyl-terminal 104 amino acids of the Alzheimer amyloid protein precursor (beta APP-C104) or PC12 cells transfected with the retroviral vector (DO) alone were transplanted into the brains of newborn mice. At 20 days after grafting, transplants could be detected in all of the mouse brains examined. At 4 months after transplantation, experimental animals exhibited significant cortical atrophy. Some also revealed immunoreactivity with Alz-50, an antibody that detects an Alzheimer disease-related protein, in the somatodendritic domain of neurons in the cortex surrounding the transplants. In addition, disorganization of the neuropil in the CA2/3 region of the hippocampus ipsilateral to the transplant was revealed by staining with an antibody to the carboxyl-terminal end of the amyloid protein precursor. A decrease in cell body immunoreactivity for this portion of the amyloid protein precursor was also detected with this antibody. Together, these results suggest that the carboxyl-terminal fragment of beta APP may cause specific neuropathology and neurodegeneration in vivo.

Aging↗
PubMed1992

Analysis of the tissue-specific distribution of mRNAs encoding the plasma membrane calcium-pumping ATPases and characterization of an alternately spliced form of PMCA4 at the cDNA and genomic levels.

The plasma membrane Ca(2+)-pumping ATPase (Ca(2+)-ATPase) mRNAs are encoded on four different genes designated PMCA1-PMCA4. The primary transcripts from some of these genes are known to be alternately spliced in the region encoding the regulatory domains of the enzymes. The known alternately spliced forms of these Ca(2+)-ATPase mRNAs and a new spliced variant of PMCA4 (PMCA4b), presented here, represent at least nine different mRNAs encoding the Ca(2+)-ATPases. In this report, the examination of the tissue-specific distribution of these alternately spliced mRNAs using polymerase chain reaction amplification of cDNA coupled with Southern blotting revealed that each spliced variant had a unique tissue distribution. PMCA1b and PMCA4a were present in all tissues examined. PMCA1a, PMCA1b, and PMCA4b were expressed in excitable tissues, whereas PMCA1d was expressed only in muscle tissues. PMCA2 was found in liver, adrenal gland, spinal cord, and brain. PMCA3a was present in spinal cord, and PMCA3b in thymus, adrenal gland, spinal cord, and brain. The mRNA for a new spliced variant of PMCA4 (PMCA4b) was detected in this study. Complementary DNAs for this isoform were isolated and characterized from human and bovine brain. This alternately spliced form of the PMCA4 mRNA contained an exon inserted at the splice junction immediately following the sequence encoding the calmodulin-binding domain. As has also been shown for PMCA1a, this insertion produced a shift in the reading frame at the 3'-end of the PMCA4 mRNA that yielded a sequence encoding a Ca(2+)-ATPase lacking a large portion of the C-terminal regulatory domain. When the human PMCA4 gene spanning this region of variable exon splicing was sequenced, it confirmed the intron-exon boundaries where alternate splicing occurs to produce PMCA4a and PMCA4b.

Amino Acid Sequence↗
PubMed1992

Determination of the nucleotide sequence and chromosomal localization of the ATP2B2 gene encoding human Ca(2+)-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.

Amino Acid Sequence↗
PubMed1992

Expression of plasma membrane calcium-pumping ATPase mRNAs in developing rat brain and adult brain subregions: evidence for stage-specific expression.

The plasma membrane calcium-pumping ATPases (Ca(2+)-ATPases) maintain resting free cytosolic calcium concentrations in cells at the submicromolar level. These Ca(2+)-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 Ca(2+)-ATPase (PMCA) 1b, the isoform though to maintain a housekeeping function, was present from embryonic day 10. The other alternatively spliced PMCA1 mRNAs, PMCA1a 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.

Adenosine Triphosphatases↗
PubMed1992

Contributions of conserved serine residues to the interactions of ligands with dopamine D2 receptors.

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 beta 2-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.

Adenylyl Cyclase Inhibitors↗
PubMed1992

The neurotoxic carboxy-terminal fragment of the Alzheimer amyloid precursor binds specifically to a neuronal cell surface molecule: pH dependence of the neurotoxicity and the binding.

One of the hallmarks of Alzheimer's disease neurodegeneration is the accumulation of deposits of amyloid in neuritic plaques and in the cerebral vasculature. Recent studies have implicated carboxy-terminal fragments of the Alzheimer amyloid precursor protein (beta APP) in the processes of amyloidogenesis and neurodegeneration. In particular, the carboxy-terminal 104 amino acids of beta APP (beta APP-C104) have been shown to cause amyloid-like fibrils when expressed in non-neuronal cells and to cause the degeneration of neuronal cells. These data suggest that it may play a role in the development of the progressive neuropathology of Alzheimer's disease. We hypothesized that beta APP-C104 may cause the degeneration of neurons by interacting with a cell surface receptor. In the present report, we show that beta APP-C104 synthesized in vitro binds specifically and with high affinity to the surface of NGF-treated PC12 cells. Both the cell surface binding and the neurotoxicity of beta APP-C104 are pH dependent and are not inhibited by tachykinins. Mutational analysis suggests that both the binding and the neurotoxicity are dependent at least in part on the presence of a tyrosine residue that is a potential site of phosphorylation at the carboxy terminus of the fragment.

Alzheimer Disease↗
PubMed1991

Increased abundance of alternatively spliced forms of D2 dopamine receptor mRNA after denervation.

The existence of two molecular forms of D2 dopamine receptors suggests that differences in the distribution or regulation of the two forms could be exploited for the pharmacological treatment of disease. Using probes selective for each alternatively spliced variant of D2 receptor mRNA, we determined that both variants were widely distributed in rat brain and pituitary but that the ratio of the forms varied among regions. mRNA for the 444-amino acid-long variant, D2(444), was the most abundant form in pituitary and neostriatum. Intermediate levels of both D2(444) mRNA and the short form, D2(415), were detected in midbrain, and low levels of D2(444) and D2(415) mRNAs were detected in all other regions examined, including hippocampus, cerebellum, and cortex. The D2(444)/D2(415) ratio was generally lower in the regions of low expression than in pituitary and neostriatum. Dopamine-depleting lesions increased the density of D2 receptors in the denervated neostriatum by 29% without altering the affinity of the receptors for [3H]spiperone. The proliferation of receptors appeared to be due to a lesion-induced increase of up to 120% in the abundance of both variants of mRNA in the neostriatum.

Animals↗
PubMed1991

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 and in 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.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

Pivotal role for aspartate-80 in the regulation of dopamine D2 receptor affinity for drugs and inhibition of adenylyl cyclase.

An aspartate residue corresponding to aspartate-80 of dopamine D2 receptors is strictly conserved among receptors that couple to guanine nucleotide-binding proteins. Mutation of this residue alters the function of several classes of neurotransmitter receptors. Dopamine D2 receptors couple to the guanine nucleotide-binding protein Gi to inhibit adenylyl cyclase (ATP-pyrophosphate-lyase, cyclizing; EC 4.6.1.1). Like other Gi-coupled receptors, the binding of agonists and some antagonists to D2 receptors is sensitive to pH and sodium. In the present report, we demonstrate that substitution of an alanine or glutamate residue for aspartate-80 severely impairs inhibition of adenylyl cyclase by D2 receptors and also abolishes or decreases the regulation of the affinity of D2 receptors for agonists and substituted benzamide antagonists by sodium and pH. Our data support the hypothesis that the conformation of D2 receptors is maintained by interactions of monovalent cations with aspartate-80. The regulation of D2 receptors by this interaction has important consequences for the affinity of D2 receptors for ligands and for signal transduction by D2 receptors.

Adenylyl Cyclase Inhibitors↗
PubMed1990

The primary structure and analysis of the squid kinesin heavy chain.

We report the cDNA sequence of the squid kinesin heavy chain and compared the predicted amino acid sequence with that of the Drosophila heavy chain as reported by Yang, J.T., Laymon, R.A., and Goldstein, L.S. B. (1989) Cell 56, 879-889). We compared the two kinesin sequences with regard to the predicted physicochemical parameters of hydrophobicity, charge, and propensities of the secondary conformations. A comparison of the sequences from the two species reveals the head, stalk, and tail domains because a reduced degree of conservation demarcates the stalk. The charge profile indicates that the head region is nearly neutral, the stalk region acidic, and the tail is basic. The Fourier transform analysis of the hydrophobic profile of the stalk shows predominant peaks at 1/3.5 and 1/2.3, which are indexed as the second and third orders of the period 7 residue. As in the Drosophila sequence, the rod domain is divided into an amino and a carboxyl subdomain by a predicted hinge region. We show that the disposition of hydrophobic residues is distinct in these two subdomains. In particular, the heptad repeat is more regular in the amino-terminal rod domain than in the carboxyl-terminal rod domain. The tail region is positively charged, a feature that is consistent with the known electrostatic interaction between the heavy chain and negatively charged surfaces such as glass coverslips and latex beads. Three monoclonal antibodies to the kinesin heavy chain have been mapped to a region within the carboxyl terminus of the stalk.

Adenosine Triphosphatases↗
PubMed1990

Homozygous deletion in Wilms tumours of a zinc-finger gene identified by chromosome jumping.

Cytogenetic analysis has identified chromosome 11p13 as the smallest overlap region for deletions found in individuals with WAGR syndrome, which includes Wilms tumour (a recessive childhood nephroblastoma), aniridia, genito-urinary abnormalities and mental retardation. The underlying loci have since been resolved into an aniridia (AN2) locus at a telomeric position, and a locus of closely spaced genes or a single pleiotropic gene involved in genito-urinary tract abnormalities and Wilms tumour at a more centromeric position. Pulsed-field gel analysis of the 11p13 region has revealed the presence of several putative CpG islands, structures which are frequently associated with the 5' ends of expressed sequences, mainly housekeeping genes and some tissue-specific genes. Starting from a CpG island, we have now isolated four neighbouring CpG islands, all within 650 kilobases (kb), by means of two consecutive bidirectional jumps in rare-cutting restriction-enzyme jumping libraries. In two instances, flanking sequences were conserved in other species and RNA transcripts were identified. A complementary DNA clone isolated for one of them derives from an RNA highly expressed in fetal kidney, and is predicted to encode a Krüppel-like zinc-finger protein that is probably a transcription factor. The entire cDNA region is included in two partially overlapping homozygous deletions found in Wilms tumour DNA samples. Cloning of the breakpoints in one tumour revealed a deletion size of 170 kb, one-third of which is covered by the cDNA. The expression pattern and sequence of this cDNA could point to an important role for its corresponding gene in the normal development of the renal system as well as in Wilms tumour.

Amino Acid Sequence↗
PubMed1990

The pattern of GAP-43 immunostaining changes in the rat hippocampal formation during reactive synaptogenesis.

The reactive synaptogenesis that takes place in the rat hippocampal formation after certain experimental manipulations affords an opportunity to investigate the molecular events that underlie structural remodeling in the adult CNS. Between 2 and 4 days after lesioning the perforant pathway, levels of the synaptic phosphoprotein, GAP-43 (B50, F1, pp46, neuromodulin), were found to increase markedly in the inner molecular layer (iml) of the dentate gyrus, coincident with the time at which commissural-associational (CA) fibers begin to sprout axon collaterals into dendritic portions denervated by the lesion. GAP-43 immunostaining in the iml began to decline by 8 days but continued to define an expanded CA projection for at least one month. In the outer molecular layer (oml), GAP-43 levels decreased after the loss of perforant pathway terminals and did not return for 2-3 weeks, the time at which sprouting of septal inputs into this layer can be visualized by cholinesterase histochemistry. These results demonstrate that GAP-43 levels change during reactive synaptogenesis, and point to differences among neural systems in their expression of this protein.

Animals↗
PubMed1990

Transfection of PC12 cells with the human GAP-43 gene: effects on neurite outgrowth and regeneration.

The neuronal growth associated protein GAP-43 is expressed at high levels during axonal growth and regeneration. In this report, we describe the transfection of the nerve growth factor (NGF)-responsive pheochromocytoma cell line PC12 with the human GAP-43 cDNA under the control of the Moloney murine leukemia virus long terminal repeat (MoMuLV LTR). Two PC12 subclones were isolated that constitutively expressed GAP-43 from the transfected cDNA and showed increased responsiveness to NGF. Of the two transfected PC12 subclones, the subclone expressing the most human GAP-43 RNA showed an accelerated initial neurite outgrowth response and a 10-fold increased sensitivity to NGF. Neurite regeneration was significantly enhanced in both transfected subclones and, in contrast to untreated PC12 cells, could occur transiently in the absence of added NGF. These results suggest that GAP-43 may potentiate the action of NGF on neurite initiation and regeneration.

Cell Differentiation↗
PubMed1990

The Alzheimer amyloid precursor-related transcript lacking the beta/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 Alzheimer's disease and aged Down's syndrome pathology. The localization of the Alzheimer amyloid protein precursor (APP) gene on chromosome 21, along with its overexpression in Down's 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 Alzheimer's 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.

Aged↗