Beta amyloid gene duplication in Alzheimer's disease and karyotypically normal Down syndrome.

With the recently cloned complementary DNA probe, lambda Am4 for the chromosome 21 gene encoding brain amyloid polypeptide (beta amyloid protein) of Alzheimer's disease, leukocyte DNA from three patients with sporadic Alzheimer's disease and two patients with karyotypically normal Down syndrome was found to contain three copies of this gene. Because a small region of chromosome 21 containing the ets-2 gene is duplicated in patients with Alzheimer's disease, as well as in karyotypically normal Down syndrome, duplication of a subsection of the critical segment of chromosome 21 that is duplicated in Down syndrome may be the genetic defect in Alzheimer's disease.

Combined assessment of DYRK1A, BDNF and homocysteine levels as diagnostic marker for Alzheimer's disease.

Early identification of Alzheimer's disease (AD) risk factors would aid development of interventions to delay the onset of dementia, but current biomarkers are invasive and/or costly to assess. Validated plasma biomarkers would circumvent these challenges. We previously identified the kinase DYRK1A in plasma. To validate DYRK1A as a biomarker for AD diagnosis, we assessed the levels of DYRK1A and the related markers brain-derived neurotrophic factor (BDNF) and homocysteine in two unrelated AD patient cohorts with age-matched controls. Receiver-operating characteristic curves and logistic regression analyses showed that combined assessment of DYRK1A, BDNF and homocysteine has a sensitivity of 0.952, a specificity of 0.889 and an accuracy of 0.933 in testing for AD. The blood levels of these markers provide a diagnosis assessment profile. Combined assessment of these three markers outperforms most of the previous markers and could become a useful substitute to the current panel of AD biomarkers. These results associate a decreased level of DYRK1A with AD and challenge the use of DYRK1A inhibitors in peripheral tissues as treatment. These measures will be useful for diagnosis purposes.

C21orf5, a human candidate gene for brain abnormalities and mental retardation in Down syndrome.

Mental retardation represents the more invalidating pathological aspect of trisomy 21 and has a hard impact on public health. The dosage imbalance of chromosome 21 genes could be the cause of neurological alterations and mental retardation seen in Down syndrome. We studied C21orf5 that we have demonstrated to be overexpressed in Down syndrome tissues, as a candidate gene for trisomy 21. A new optical technology (Rachidi et al., 2000) was used to compare signal intensity and cell density in presumptive embryonic brain compartments, at their boundaries and in higher specialized brain centres during fetal lifespan. We showed a developmentally regulated transcriptional activity of C21orf5 and a regional and cellular specific distribution of gene transcripts during human embryonic and fetal development. A wide but differential expression was detected in the nervous system during embryogenesis with a relatively lower level in the forebrain than in the midbrain and hindbrain and the highest transcription intensity in the future cerebellum. This developmentally regulated expression is maintained during post-embryogenesis and evolves selectively in fetal cerebral, hippocampal and cerebellar areas. Differential and cellular specificity were detected in hippocampus with higher C21orf5 mRNA level in the pyramidal cells compared to granular cells of the dentate gyrus. The expression pattern detected in cortical and cerebellar structures correlates well to the altered cortical lamination and to the lower size of the cerebellum observed in Down syndrome patients. In addition, the patterned differential expression detected in the medial temporal-lobe system, including hippocampal formation and perirhinal cortex, working as control centres of the memory circuits and involved in cognitive processes and memory storage, also corresponds to abnormal brain regions seen in Down syndrome patients. The C21orf5 selective expression in the key brain structures for learning and memory suggests that C21orf5 overexpression could participate in mental retardation pathogenesis in Down syndrome patients.

Expression of transfected human CuZn superoxide dismutase gene in mouse L cells and NS20Y neuroblastoma cells induces enhancement of glutathione peroxidase activity.

The human CuZn superoxide dismutase (superoxide dismutase 1) a key enzyme in the metabolism of oxygen free-radicals, is encoded by a gene located on chromosome 21 in the region 21 q 22.1 known to be involved in Down's syndrome. A gene dosage effect for this enzyme has been reported in trisomy 21. To assess the biological consequences of superoxide dismutase 1 overproduction within cells, the human superoxide dismutase 1 gene and a human superoxide dismutase 1 cDNA were introduced into mouse L cells and NS20Y neuroblastoma cells. Both cell types expressed elevated levels (up to 3-fold) of enzymatically active human superoxide dismutase 1. These human superoxide dismutase 1 overproducers, especially neuronal cell lines, showed an increased activity in the selenodependent glutathione peroxidase. These data are consistent with the possibility that gene dosage of superoxide dismutase 1 contributes to oxygen metabolism modifications previously described in Down's syndrome.

Developmentally regulated expression of mtprd, the murine ortholog of tprd, a gene from the Down syndrome chromosomal region 1.

The gene tprd, which contains three tetratricopeptide domains, has been recently localized in the Down syndrome (DS) chromosomal region 1. We have cloned a cDNA encoding part of the murine ortholog of tprd and used it to characterize the expression pattern of this gene during development and at the adult stage. At E8.5 the expression is uniform. In the later stages of embryogenesis, although expression remains ubiquitous, a pattern of tissues with particularly high expression develops: the strong expression is restricted to non proliferating zones of the nervous system such as the external layer of the cortex, the spinal cord, the cranial and root ganglia and the nerves. In the brain of adult mouse the strongest signals are observed in layers II-III and V-VI of the cortex, in the hippocampus and in the cerebellum, which correspond to the abnormal brain regions seen in DS patients.

Regional and cellular specificity of the expression of TPRD, the tetratricopeptide Down syndrome gene, during human embryonic development.

The TPRD gene (tetratricopeptide (TPR) containing Down syndrome gene) is one of the candidate genes in the Down syndrome chromosomal region-1. Duplication of this gene may be the cause of major phenotypic features of Down syndrome. Here we show that the TPRD expression is developmentally regulated during human embryogenesis. At the earliest stages of development (Carnegie 8-12) TPRD expression is ubiquitous. At later developmental stages (Carnegie stages 14, 16 and 18), it becomes restricted to the nervous system, as is the case for the mtprd gene during mouse development. We extended our analysis of TPRD expression during fetal development of the human nervous system (13, 22 and 24 weeks). A new oblique illumination technique was used to compare signal intensity and cell density. Some regions of the nervous system such as the external cortical layers of the brain, and the inner neuroblastic layer of the eye, strongly express the TPRD gene.

Molecular mapping of twenty-four features of Down syndrome on chromosome 21.

To determine which regions of chromosome 21 are involved in the pathogenesis of specific features of Down syndrome, we analysed, phenotypically and molecularly, 10 patients with partial trisomy 21. Six minimal regions for 24 features were defined by genotype-phenotype correlations. Nineteen of these features could be assigned to just 2 regions: short stature, joint hyperlaxity, hypotonia, major contribution to mental retardation and 9 anomalies of the face, hand and foot to the region D21S55, or Down syndrome chromosome region (DCR), located on q22.2 or very proximal q22.3, and spanning 0.4-3 Mb; 6 facial and dermatoglyphic anomalies to the region D21S55-MX1, including the DCR and spanning a maximum of 6 Mb on q22.2 and part of q22.3. Thus, the complex phenotype that constitutes Down syndrome may in large part simply result from the overdosage of only one or a few genes within the DCR and/or region D21S55-MX1.

Accurate evaluation of the sizes of DNA fragments (from 30 to 4700 kb) in pulse field gel electrophoresis.

Construction of long-range genomic maps by pulse field gel electrophoresis requires optimum resolution of large DNA fragments. Using the transverse-alternating field electrophoresis system, we describe a method to accurately evaluate the sizes of fragments generated by rare-cutter digestions within the 30-4700-kb range. A protocol generating large (greater than 1000 kb) molecules by partial digestion is also reported.

Chromosome 21 KIR channels in brain development.

Two KIR (K+ Inwardly Rectifying) channel genes have been identified on chromosome 21, in a region associated with important phenotypic features of trisomy 21, including mental retardation: KIR3.2 (GIRK2) and KIR4.2. We analysed the expression of these channel genes in developing human and mouse brains to determine the possible role of the corresponding channels in brain development and function. KIR3.2, which has been extensively studied in the mouse, was found to be expressed in the human cerebellum during development. The KIR4.2 channel is expressed later in development in both mice and humans. We compared the expression of these channels in terms of RNA and protein levels and discussed the potential synergy and consequences of the overexpression of these channels in Down's syndrome brain development.

Mapping of the Down syndrome phenotype on chromosome 21 at the molecular level.

Phenotypic and molecular analysis of individuals with partial trisomy 21 can be used to determine which regions of chromosome 21 are involved in the pathogenesis of specific features of Down's Syndrome. Using dosage analysis of 27 sequences we defined, at the molecular level, the extent of the chromosome 21 duplication in ten individuals with partial trisomy 21. Phenotype-genotype correlations led to the definition of minimal regions, the duplications of which are linked to the expression of 23 clinical features of Down's Syndrome. The D21S55 region or Down's Syndrome Chromosome Region 1 (DCR1) (1/20 of the long arm), on 21q22.2-21q22.3 proximal, is involved in four cardinal features of the disease: mental retardation, growth retardation, muscular hypotonia and joint hyperlaxity, and in eight of the 18 more common morphological anomalies of the face, hands and feet. Overlapping the DCR1, the D21S55-MX1 region or DCR2 (1/10 of the long arm), spanning 21q21.2 down to the 1/4th proximal part of 21q22.3, is involved in the features defined by the DCR1 plus congenital heart defect and five additional morphological anomalies. Thus, our results indicate that duplication of a relatively small region of chromosome 21 plays a critical role in the pathogenesis of the Down's phenotype.

DYRK1A promotes dopaminergic neuron survival in the developing brain and in a mouse model of Parkinson's disease.

In the brain, programmed cell death (PCD) serves to adjust the numbers of the different types of neurons during development, and its pathological reactivation in the adult leads to neurodegeneration. Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) is a pleiotropic kinase involved in neural proliferation and cell death, and its role during brain growth is evolutionarily conserved. Human DYRK1A lies in the Down syndrome critical region on chromosome 21, and heterozygous mutations in the gene cause microcephaly and neurological dysfunction. The mouse model for DYRK1A haploinsufficiency (the Dyrk1a(+/-) mouse) presents neuronal deficits in specific regions of the adult brain, including the substantia nigra (SN), although the mechanisms underlying these pathogenic effects remain unclear. Here we study the effect of DYRK1A copy number variation on dopaminergic cell homeostasis. We show that mesencephalic DA (mDA) neurons are generated in the embryo at normal rates in the Dyrk1a haploinsufficient model and in a model (the mBACtgDyrk1a mouse) that carries three copies of Dyrk1a. We also show that the number of mDA cells diminishes in postnatal Dyrk1a(+/-) mice and increases in mBACtgDyrk1a mice due to an abnormal activity of the mitochondrial caspase9 (Casp9)-dependent apoptotic pathway during the main wave of PCD that affects these neurons. In addition, we show that the cell death induced by 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), a toxin that activates Casp9-dependent apoptosis in mDA neurons, is attenuated in adult mBACtgDyrk1a mice, leading to an increased survival of SN DA neurons 21 days after MPTP intoxication. Finally, we present data indicating that Dyrk1a phosphorylation of Casp9 at the Thr125 residue is the mechanism by which this kinase hinders both physiological and pathological PCD in mDA neurons. These data provide new insight into the mechanisms that control cell death in brain DA neurons and they show that deregulation of developmental apoptosis may contribute to the phenotype of patients with imbalanced DYRK1A gene dosage.

Plasma DYRK1A as a novel risk factor for Alzheimer's disease.

To determine whether apparent involvement of DYRK1A in Alzheimer's disease (AD) pathology makes it a candidate plasma biomarker for diagnosis, we developed a method to quantify plasma DYRK1A by immunoblot in transgenic mouse models having different gene dosages of Dyrk1a, and, consequently, different relative protein expression. Then, we measured plasma DYRK1A levels in 26 patients with biologically confirmed AD and 25 controls (negative amyloid imaging available on 13). DYRK1A was detected in transgenic mouse brain and plasma samples, and relative levels of DYRK1A correlated with the gene copy number. In plasma from AD patients, DYRK1A levels were significantly lower compared with controls (P<0.0001). Results were similar when we compared AD patients with the subgroup of controls confirmed by negative amyloid imaging. In a subgroup of patients with early AD (CDR=0.5), lower DYRK1A expression was confirmed. In contrast, no difference was found in levels of DYRK1B, the closest relative of DYRK1A, between AD patients and controls. Further, AD patients exhibited a positive correlation between plasma DYRK1A levels and cerebrospinal fluid tau and phosphorylated-tau proteins, but no correlation with amyloid-ß42 levels and Pittsburgh compound B cortical binding. DYRK1A levels detected in lymphoblastoid cell lines from AD patients were also lower when compared with cells from age-matched controls. These findings suggest that reduced DYRK1A expression might be a novel plasma risk factor for AD.

Homocysteine threshold value based on cystathionine beta synthase and paraoxonase 1 activities in mice.

BACKGROUND: Hyperhomocysteinaemia is a metabolic disorder associated with the development of premature atherosclerosis. Among the determinants which predispose to premature thromboembolic and atherothrombotic events, serum activity of paraoxonase 1, mainly synthesized in the liver, has been shown to be a predictor of cardiovascular disease and to be negatively correlated with serum homocysteine levels in human. Even though treatments of hyperhomocysteinaemic patients ongoing cardiovascular complications are commonly used, it still remains unclear above which homocysteine level a preventive therapy should be started. MATERIALS AND METHODS: In order to establish a threshold of plasma homocysteine concentration we have analyzed the hepatic cystathionine beta synthase and paraoxonase 1 activities in a moderate to intermediate murine model of hyperhomocysteinaemia. Using wild type and heterozygous cystathionine beta synthase deficient mice fed a methionine enriched diet or a control diet, we first studied the link between cystathionine beta synthase and paraoxonase 1 activities and plasma homocysteine concentration. RESULTS: Among the animals used in this study, we observed a negative correlation between plasma homocysteine level and cystathionine beta synthase activity (rho=-0.52, P=0.0008) or paraoxonase 1 activity (rho=-0.49, P=0.002). Starting from these results, a homocysteine cut-off value of 15 microm has been found for both cystathionine beta synthase (P=0.0003) and paraoxonase 1 (P=0.0007) activities. CONCLUSIONS: Our results suggest that both cystathionine beta synthase and paraoxonase 1 activities are significantly decreased in mice with a plasma homocysteine value greater than 15 microm. In an attempt to set up preventive treatment for cardiovascular disease our results indicate that treatments should be started from 15 microm of plasma homocysteine.

Classification of human chromosome 21 gene-expression variations in Down syndrome: impact on disease phenotypes.

Down syndrome caused by chromosome 21 trisomy is the most common genetic cause of mental retardation in humans. Disruption of the phenotype is thought to be the result of gene-dosage imbalance. Variations in chromosome 21 gene expression in Down syndrome were analyzed in lymphoblastoid cells derived from patients and control individuals. Of the 359 genes and predictions displayed on a specifically designed high-content chromosome 21 microarray, one-third were expressed in lymphoblastoid cells. We performed a mixed-model analysis of variance to find genes that are differentially expressed in Down syndrome independent of sex and interindividual variations. In addition, we identified genes with variations between Down syndrome and control samples that were significantly different from the gene-dosage effect (1.5). Microarray data were validated by quantitative polymerase chain reaction. We found that 29% of the expressed chromosome 21 transcripts are overexpressed in Down syndrome and correspond to either genes or open reading frames. Among these, 22% are increased proportional to the gene-dosage effect, and 7% are amplified. The other 71% of expressed sequences are either compensated (56%, with a large proportion of predicted genes and antisense transcripts) or highly variable among individuals (15%). Thus, most of the chromosome 21 transcripts are compensated for the gene-dosage effect. Overexpressed genes are likely to be involved in the Down syndrome phenotype, in contrast to the compensated genes. Highly variable genes could account for phenotypic variations observed in patients. Finally, we show that alternative transcripts belonging to the same gene are similarly regulated in Down syndrome but sense and antisense transcripts are not.

Identification and characterisation of soluble epoxide hydrolase in mouse brain by a robust protein biochemical method.

The central nervous system is an important potential target for certain environmental prototoxins, but relatively little is known regarding brain-specific expression of biotransformation enzyme systems. On the other hand, developments in the field of molecular biology and advances in high-throughput screening methods continue to increase the number and amounts of available proteins. We used thus a robust and reliable technique, two-dimensional gel electrophoresis coupled to matrix assisted laser desorption/ionisation mass spectroscopy followed by tandem mass spectrometry and identified for the first time soluble epoxide hydrolase and added other biotransformation enzymes in the hippocampal region of mouse brain. Soluble epoxide hydrolase has an Mr of 61.5 kDa, pI of 5.9, twenty-six matching peptides and sequence coverage of 56% and was unambiguously identified by MS/MS. Since localised biotransformation events in regions of the central nervous system may account for pathologies and/or toxicities initiated by exposure to certain endogenous and/or environmental chemicals, identification of these enzymes would present an opportunity for developing novel therapeutic targets or would have critical toxicologic significance.

Nonrandom location of H1-H1 degree histones on chromatin of mouse liver and brain.

The electrophoretic behavior of nucleosome dimers reconstituted with H1 or H1 degrees and the features of the digestion of those reconstituted dimers with micrococcal nuclease were first investigated. Both criteria appear to support the notion that H1 degrees can replace H1 on the nucleosome on a one-to-one basis and that both proteins fulfill a similar structural role in chromatin. H1/H1 degrees ratios in different chromatin subfractions from mouse liver were then indirectly measured by means of an electrophoretic purification of H1 degrees- from H1-containing nucleosome monomers, followed by a titration of different specific nucleotide sequences in the corresponding DNAs. Satellite and globin containing chromatin subfractions were found to contain only about half the amount of H1 degrees which is normally encountered in bulk chromatin, indicating a nonrandom location of H1-H1 degrees variants on untranscribed sequences; in contrast, titrations with cDNA from poly(A+) RNA and albumin cDNA show an approximately 2-fold enrichment in H1 degrees for the corresponding chromatin when compared to the same bulk chromatin. In the brain, inactive albumin chromatin contains a relative amount of both H1 variants similar to that found in satellite or globin chromatin in liver. Amounts of H1 degrees can, therefore, be correlated with different states of chromatin: an inactive state with a lower amount of H1 degrees and a potentially active state with an enrichment in H1 degrees.

Mass spectrometrical analysis of phosphoprotein enriched in astrocytes of 15 kDa in mouse hippocampi.

Phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) is a small protein that was first identified as an abundant phosphoprotein in brain. PEA-15 was characterised so far at the immunochemical level and by a microsequencing attempt. In order to update characterisation of this important structure by advanced methodology unambiguously identifying proteins independent of antibody availability and specificity, we used a proteomic method for this purpose: Performing protein profiling in mouse hippocampi using two dimensional gel electrophoresis with subsequent mass spectrometrical (MS/MS) identification we detected this protein and demonstrate proteomic characterisation of PEA-15 (Q62048). This study enables further specific and unambiguous determination serving as an analytical tool.

Assessing implications between genotypic and phenotypic variables through lattice analysis.

A previous paper assessed a "Molecular Mapping of Twenty-Four Features of Down Syndrome on Chromosome 21" (Delabar et al., 1993), by analyzing the genotypes/phenotypes of patients suffering from partial trisomy. The mapping was defined through implications--each feature was mapped to the conjunction of cytogenetic bands that were shared by all patients having that feature. In the present paper, we extend that approach to determine how far those implications depart from defining equivalences. Finding equivalences is important. Local equivalences permit a genetic characterization of a feature. And if global equivalences held for all features, that set of bands would be sufficient to characterize the various phenotypes observed in individuals with partial trisomy 21. To extend the earlier approach, we examine the structure of equivalences as well as the structure of implications. We examine both conjunctions of bands and conjunctions of features. The use of Galois lattices permits simultaneous evaluation of both kinds of structures. Each Galois lattice is labeled with a basis (minimal generating set) of implications going from conjunctions of features into bands and those going from conjunctions of bands into features. Analysis reveals that about half of the conjunctions of bands that characterize the genetic structure embody equivalences. This allows us to improve the genetic description of features and to specify minimal sets of questions that need to be investigated to make the global genetic description more precise.