Developmental and differential expression of beta amyloid protein precursor mRNAs in mouse brain.

S1 nuclease analysis was used to determine the levels and patterns of three beta amyloid protein precursor (BPP) mRNAs in mouse developmental brain and in primary neuronal and glial cultures. BPP695 mRNA lacking the Kunitz proteinase inhibitor (KPI) domain was detected exclusively in neuronal cultures and increased considerably in late embryonic and early postnatal periods. On the other hand, BPP751 and 770 mRNAs with KPI domain were detected predominantly in astrocyte- and microglia-enriched cultures and increased slightly only in embryonic stages. These results suggest that the product of each BPP mRNA may play a different role in the brain.     

GnRH-associated peptide (GAP) is cosecreted with GnRH into the hypophyseal portal blood of ovariectomized sheep

The human amyloid beta protein is a major component of brain amyloid found in patients with Alzheimer's disease. As an initial step to understand the biological function of its precursor protein, we have isolated cDNA for the mouse homolog of the human beta protein precursor. Comparison of the predicted amino acid sequence with that of human revealed a quite high degree of homology (96.8%), and the calculated evolutionary rate of the mRNA at amino acid substitution site was relatively low (0.1 x 10(-9)/site/year). The mRNA was abundant in brain and kidney, and also detected in other tissues at low level. These results indicated that this protein is highly conserved through mammalian evolution and may be involved in a basic biological process(es).     

MPTP delta, a putative murine homolog of HPTP delta, is expressed in specialized regions of the brain and in the B-cell lineage.

Protein tyrosine phosphatases (PTPs), together with protein tyrosine kinases (PTKs), are involved in the regulation of cell activation, growth, and differentiation. To further elucidate the fine tuning of cell growth and differentiation through tyrosine phosphorylation, we tried to isolate mouse receptor-type PTP (RPTP) cDNA clones by screening mouse brain cDNA libraries with mouse CD45 PTP domain probes under reduced-stringency conditions. Characterization of isolated cDNA clones for RPTP showed that the cytoplasmic region contains two tandem repeats of PTP domain of about 230 amino acids with intrinsic phosphatase activity. The extracellular region was composed of immunoglobulin (Ig)-like domains and fibronectin type III (FN-III)-like domains. The gene was highly homologous to human PTP delta (HPTP delta) and thus was named MPTP delta (murine counterpart of HPTP delta). The MPTP delta gene appeared to generate at least three species of mRNA, which differ in the composition of the extracellular domain: type A, one Ig-like and four FN-III-like domains; type B, one Ig-like and eight FN-III-like domains; and type C, three Ig-like and eight FN-III-like domains. Interestingly, the 5' untranslated region and the leader peptide of types A and B were completely different from those of type C. Northern (RNA) blot analysis demonstrated that brain, kidney, and heart cells express three mRNA species of about 7 kb. Antibody directed against part of the extracellular domain of type A MPTP delta recognized a 210-kDa protein in brain and kidney lysates. In situ hybridization of brain samples revealed that MPTP delta mRNA is present in the hippocampus, thalamic reticular nucleus, and piriform cortex, where some Src family PTKs have been also demonstrated to exist. Although MPTP delta mRNA was not detected in lymphoid tissues, all of the pre-B-cell lines tested and one of three B-cell lines tested expressed MPTP delta mRNA, whereas antibody-producing B-cell hybridomas and T-cell and macrophage lines did not. Finally, the MPTP delta locus was tightly linked to the brown (b) locus on mouse chromosome 4.     

Characterization of the adaptor protein ARH expression in the brain and ARH molecular interactions

Previously, pA134 was identified as one of the mRNAs present in the squid giant axon. Comparative sequence analyses revealed that the pA134 gene product manifested significant similarity to the mammalian lipoprotein receptor adaptor protein also known as ARH (autosomal recessive hypercholesterolemia). ARH mRNA and protein displayed very similar pattern of expression throughout the mouse brain. Significant levels of expression were observed in cells with a predominantly neuronal profile in the cerebellum, brainstem, olfactory bulb, hippocampus, and cortex. A yeast two hybrid screen for ARH protein interactions in mouse brain identified the following binders: amyloid precursor-like protein 1, low density lipoprotein receptor-related protein (LRP) 1, LRP8, and GABA receptor-associated protein-like 1. The interactions of ARH with LRP1 and GABA receptor-associated protein-like 1 were subsequently verified by co-immunoprecipitation of the protein complexes from transfected human embryonic kidney cells. The presence of ARH mRNA in axon of primary sympathetic neurons was established by RT-PCR analyses and confirmed by in situ hybridization. Taken together, our data suggest that ARH is a multifunctional protein whose spectrum of function in the brain goes beyond the traditionally known metabolism of lipoproteins, and that ARH may be locally synthesized in the axon.     

Characterization of amyloid beta peptides from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein

Alzheimer's disease (AD) is marked by the presence of neurofibrillary tangles and amyloid plaques in the brain of patients. To study plaque formation, we report on further quantitative and qualitative analysis of human and mouse amyloid beta peptides (Abeta) from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein (APP). Using enzyme-linked immunosorbant assays (ELISAs) specific for either human or rodent Abeta, we found that the peptides from both species aggregated to form plaques. The ratios of deposited Abeta1-42/1-40 were in the order of 2-3 for human and 8-9 for mouse peptides, indicating preferential deposition of Abeta42. We also determined the identity and relative levels of other Abeta variants present in protein extracts from soluble and insoluble brain fractions. This was done by combined immunoprecipitation and mass spectrometry (IP/MS). The most prominent peptides truncated either at the carboxyl- or the amino-terminus were Abeta1-38 and Abeta11-42, respectively, and the latter was strongly enriched in the extracts of deposited peptides. Taken together, our data indicate that plaques of APP-London transgenic mice consist of aggregates of multiple human and mouse Abeta variants, and the human variants that we identified were previously detected in brain extracts of AD patients.     

mPPP1R16B is a novel mouse protein phosphatase 1 targeting subunit whose mRNA is located in cell bodies and dendrites of neurons in four distinct regions of the brain

We cloned a cDNA encoding a novel mouse protein whose human homolog has been annotated in GenBank as a regulatory subunit of protein phosphatase 1, PPP1R16B. Both the primary protein sequence and the domain structure are highly conserved between PPP1R16B and proteins of unknown function from other species, such as Caenorhabditis elegans and Drosphila melanogaster. Besides a protein phosphatase 1 interaction motif, mouse PPP1R16B (mPPP1R16B) and the related proteins contain ankyrin repeats that may constitute binding sites for other proteins and C-terminal prenylation signals that are likely to target the proteins to the plasma membrane. In the adult mouse, Ppp1r16b mRNA is expressed in most tissues examined, with highest expression levels in kidney and brain. In the brain, Ppp1r16b message is particularly enriched in the olfactory bulb, striatum, dentate gyrus, and cerebellum. During postnatal cerebellar development, Ppp1r16b mRNA expression levels increase gradually and are maximal around postnatal day 30. In situ hybridization revealed that Ppp1r16b message is found in both the cell bodies and the dendrites in Purkinje cells of the cerebellum and granule neurons of the dentate gyrus.     

Molecular cloning and expression of the human and mouse homologues of the Drosophila dachshund gene

Recent genetic analysis of the Drosophila dachshund (dac) gene has established that dac encodes a novel nuclear protein that is involved in both eye and leg development. In the Drosophila eye, dac expression appears to be controlled by the product of the eyeless/Pax6 gene. In order to analyze the Pax6 pathway in vertebrates we have isolated and characterized the cDNA and genomic clones corresponding to the human and mouse homologues of Drosophila dac. A full-length human cDNA encoding dachshund (DACH) encodes the 706 amino acids protein with predicted molecular weight of 73 kDa. A 109 amino acid domain located at the N-terminus of the DACH showed significant sequence and secondary structure homologies to the ski/sno oncogene products. Northern blot analysis found human DACH predominantly in adult kidney, heart, and placenta, with less expression detected in the brain, lung, skeletal muscle and pancreas. A panel of human cell lines was studied and most notably a large proportion of neuroblastomas expressed DACH mRNA. Mouse Dach encodes a protein of 751 amino acids with predicted molecular weight of 78 kDa that is 95% identical to the human DACH. RNase protection analysis showed the highest Dach mRNA expression in the adult mouse kidney and lung, whereas lower expression was detected in the brain and testis. RT/PCR analysis readily detected Dach mRNA in the adult mouse cornea and retina. Dach mRNA expression in the mouse E11.5 embryo was observed primarily in the fore and hind limbs, as well as in the somites. Received: 9 February 1999 / Accepted: 19 April 1999     

The mouse homolog of the human amyloid beta protein (AD-AP) gene is located on the distal end of mouse chromosome 16: further extension of the homology between human chromosome 21 and mouse chromosome 16

The human amyloid beta protein is the major constituent of the brain amyloid plaques found in Alzheimer disease. The gene that encodes this protein is located on chromosome 21, and individuals with Down syndrome (trisomy 21) also exhibit an early onset form of Alzheimer disease. We have used the cloned human amyloid beta protein gene and a panel of somatic cell hybrids to map the location of the mouse homolog of this gene. We report here that the mouse gene is located on chromosome 16 within the region 16C3----ter, in common with three other genes which map within the Down syndrome region of human chromosome 21.     

Neuronal localization of amyloid beta protein precursor mRNA in normal human brain and in Alzheimer''s disease.

Clones for the amyloid beta protein precursor gene were isolated from a cDNA library prepared from the frontal cortex of a patient who had died with a histologically confirmed diagnosis of Alzheimer's disease; they were used to investigate the tissue and cellular distribution of amyloid beta protein precursor mRNA in brain tissues from control patients and from Alzheimer's disease patients. Amyloid beta protein precursor mRNA was expressed in similar amounts in all control human brain regions examined, but a reduction of the mRNA level was observed in the frontal cortex from patients with Alzheimer's disease. By in situ hybridization amyloid beta protein precursor mRNA was present in granule and pyramidal cell bodies in the hippocampal formation and in pyramidal cell bodies in the cerebral cortex. No specific labelling of glial cells or endothelial cells was found. The same qualitative distribution was observed in tissues from control patients and from patients with Alzheimer's disease. Senile plaque amyloid thus probably derives from neurones. The tissue distribution of amyloid beta protein precursor mRNA and its cellular localization demonstrate that its expression is not confined to the brain regions and cells that exhibit the selective neuronal death characteristic of Alzheimer's disease.     

Chronic overexpression of the calcineurin inhibitory gene DSCR1 (Adapt78) is associated with Alzheimer's disease

The DSCR1 (Adapt78) gene was independently discovered as a resident of the "Down syndrome candidate region"and as an "adaptive response"shock or stress gene that is transiently induced during oxidative stress. Recently the DSCR1 (Adapt78) gene product was discovered to be an inhibitor of the serine/threonine phosphatase, calcineurin, and its signaling pathways. We hypothesized that DSCR1 (Adapt78) might also be involved in the development of Alzheimer's disease. To address this question we first studied DSCR1 (Adapt78) in multiple human tissues and found significant expression in brain, spinal cord, kidney, liver, mammary gland, skeletal muscle, and heart. Within the brain DSCR1 (Adapt78) is predominantly expressed in neurons within the cerebral cortex, hippocampus, substantia nigra, thalamus, and medulla oblongata. When we compared DSCR1 (Adapt78) mRNA expression in post-mortem brain samples from Alzheimer's disease patients and individuals who had died with no Alzheimer's diagnosis, we found that DSCR1 (Adapt78) mRNA levels were about twice as high in age-matched Alzheimer's patients as in controls. DSCR1 (Adapt78) mRNA levels were actually three times higher in patients with extensive neurofibrillary tangles (a hallmark of Alzheimer's disease) than in controls. In comparison, post-mortem brain samples from Down syndrome patients (who suffer Alzheimer's symptoms) also exhibited DSCR1 (Adapt78) mRNA levels two to three times higher than controls. Using a cell culture model we discovered that the amyloid beta(1-42) peptide, which is a major component of senile plaques in Alzheimer's, can directly induce increased expression of DSCR1 (Adapt78). Our findings associate DSCR1 (Adapt78) with such major hallmarks of Alzheimer's disease as amyloid protein, senile plaques, and neurofibrillary tangles.     

Neural differentiation increases expression of Alzheimer amyloid protein precursor gene in murine embryonal carcinoma cells

Neural differentiation of the embryonal carcinoma P19 cell line markedly increased the abundance of mRNA encoding Alzheimer amyloid beta/A4-protein precursor (APP). In P19 cells treated with retinoic acid, the abundance of mRNA encoding APP695, which lacks the protease inhibitor domain, reached a maximum on days 2-4 and decreased thereafter, whereas the abundances of mRNAs encoding APP751 and APP770, both possessing the protease inhibitor domain, slowly increased to reach higher levels than APP695 mRNA at later stages of neural differentiation. The induction of APP695 mRNA was consistent with the appearance of neurons in the P19 cultures. A high abundance of APP695 mRNA was also detected in mouse brain at a stage of the period of neuroblast formation. Thus, neural differentiation of P19 cells may present a suitable model for studying the regulation of APP gene expression during early differentiation of brain cells in vivo.     

Mouse and human GTPBP2, newly identified members of the GP-1 family of GTPase

We earlier identified the GTPBP1 gene which encodes a putative GTPase structurally related to peptidyl elongation factors. This finding was the result of a search for genes, the expression of which is induced by interferon-gamma in a macrophage cell line, THP-1. In the current study, we probed the expressed sequence tag database with the deduced amino acid sequence of GTPBP1 to search for partial cDNA clones homologous to GTPBP1. We used one of the partial cDNA clones to screen a mouse brain cDNA library and identified a novel gene, mouse GTPBP2, encoding a protein consisting of 582 amino acids and carrying GTP-binding motifs. The deduced amino acid sequence of mouse GTPBP2 revealed 44.2% similarity to mouse GTPBP1. We also cloned a human homologue of this gene from a cDNA library of the human T cell line, Jurkat. GTPBP2 protein was found highly conserved between human and mouse (over 99% identical), thereby suggesting a fundamental role of this molecule across species. On Northern blot analysis of various mouse tissues, GTPBP2 mRNA was detected in brain, thymus, kidney and skeletal muscle, but was scarce in liver. Level of expression of GTPBP2 mRNA was enhanced by interferon-gamma in THP-1 cells, HeLa cells, and thioglycollate-elicited mouse peritoneal macrophages. In addition, we determined the chromosomal localization of GTPBP1 and GTPBP2 genes in human and mouse. The GTPBP1 gene was mapped to mouse chromosome 15, region E3, and human chromosome 22q12-13.1, while the GTPBP2 gene is located in mouse chromosome 17, region C-D, and human chromosome 6p21-12.