A polymorphism in the regulatory region of APOE associated with risk for Alzheimer's dementia.

The epsilon4 allele of the apolipoprotein E gene (APOE) has been associated with an increased risk of developing Alzheimer's disease (AD; refs 1,2). However, it is apparent that the APOEepsilon4 allele alone is neither necessary nor sufficient to cause the disease. We have recently found three new polymorphisms within the APOE transcriptional regulatory region (M.J.A. et al., manuscript submitted) and now establish an association between one of these polymorphisms (-491A/T) and dementia as observed in Alzheimer's disease, in two independent clinical populations. The results suggest that homozygosity of a common variant (-491A) is associated with increased risk for AD, and that this association is independent of APOEepsilon4 status. In vitro studies suggest that the -491A/T polymorphism may increase risk for AD by altering the level of ApoE protein expression.

APOE and Alzheimer disease: a major gene with semi-dominant inheritance.

Apolipoprotein E (APOE) dependent lifetime risks (LTRs) for Alzheimer Disease (AD) are currently not accurately known and odds ratios alone are insufficient to assess these risks. We calculated AD LTR in 7351 cases and 10 132 controls from Caucasian ancestry using Rochester (USA) incidence data. At the age of 85 the LTR of AD without reference to APOE genotype was 11% in males and 14% in females. At the same age, this risk ranged from 51% for APOE44 male carriers to 60% for APOE44 female carriers, and from 23% for APOE34 male carriers to 30% for APOE34 female carriers, consistent with semi-dominant inheritance of a moderately penetrant gene. Using PAQUID (France) incidence data, estimates were globally similar except that at age 85 the LTRs reached 68 and 35% for APOE 44 and APOE 34 female carriers, respectively. These risks are more similar to those of major genes in Mendelian diseases, such as BRCA1 in breast cancer, than those of low-risk common alleles identified by recent GWAS in complex diseases. In addition, stratification of our data by age groups clearly demonstrates that APOE4 is a risk factor not only for late-onset but for early-onset AD as well. Together, these results urge a reappraisal of the impact of APOE in Alzheimer disease.

Laboratory exposure to Coccidioides: lessons learnt in a non-endemic country.

Coccidioides is a primary pathogenic fungus, which infects humans through highly infectious arthroconidia, causing substantial morbidity including life-threatening disseminated infections. Due to the low infectious dose, laboratory personnel might become infected during diagnostic procedures. Accordingly, coccidioidomycosis is reported as the most frequent laboratory-acquired systemic mycosis worldwide. This risk is aggravated in non-endemic countries, where the diagnosis may not be suspected. We report on an inadvertent exposure of 44 persons to Coccidioides posadasii in a clinical microbiology laboratory in Chile, the measures of containment after rapid diagnosis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and the lessons learnt in a non-endemic setting.

Ketone body transport in renal brush border membrane vesicles.

Ketone body uptake by renal brush border vesicles has been investigated. Ketone bodies enter into the brush border vesicles by a carrier-mediated process. The uptake is dependent on an Na+ gradient ([Na+]outside>[Na+]inside) and is electroneutral. The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. A pH gradient (alkaline inside) also stimulates the ketone body uptake. Acetoacetate and 3-hydroxybutyrate share the same carrier as demonstrated by the accelerated exchange diffusion and mutual inhibitory effects.

Glycine transport in rat brain and liver mitochondria.

Transport of glycine by rat brain and liver mitochondria has been investigated by both [14C]glycine uptake and swelling experiments. Glycine enters mitochondria passively down its concentration gradient by a respiratory-independent carrier-mediated process. This view is supported by the following observations: (a) glycine inside the mitochondria reaches the incubation medium concentration; (b) mitochondria swell in the presence of isoomotic solutions of glycine in a concentration-dependent fashion; (c) the uptake of glycine is not influenced by respiratory inhibitors such as KCN or by uncouplers such as carbonylcyanide p-trifluoromethoxyphenylhydrazone; (d) initial rates of uptake approach saturation kinetics, the apparent Km of the rat brain mitochondria for glycine being 1.7 mM and that of the liver mitochondria being 5.7 mM; (e) the rate of swelling is inhibited by methylmalonate, propionate and, at pH 6.5, by mersalyl, and (f) uptake is inhibited by phosphoserine, methylmalonate and propionate, but not by alanine or proline.

Tyrosine transport by membrane vesicles isolated from rat brain.

Tyrosine uptake by membrane vesicles derived from rat brain has been investigated. The uptake is dependent on an Na+ gradient ([Na+]outside greater than [Na+]inside). The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. The process is stimulated by a membrane potential (negative inside) as demonstrated by the effect of the ionophores valinomycin and carbonyl cyanide m-chlorophenylhydrazone and anions with different permeabilities. Kinetic data show that tyrosine is accumulated by two systems with different affinities. Tyrosine uptake is inhibited by the presence of phenylalanine and tryptophan.

Location of an epitope shared by Alzheimer's amyloid peptide and brain creatine kinase using a newly developed monoclonal antibody.

Amyloid plaques, composed mainly by a peptide termed A4-amyloid, derived by proteolytic processing from the amyloid precursor protein (APP), are a hallmark in the brain of Alzheimer's disease patients. We have prepared a collection of monoclonal antibodies as tools to study APP expression and proteolysis in different systems. One of these, 5AH10, raised against residues 9-22 of A4-peptide, was selected for its ability to recognize only A4 subpeptides having the intact APP-secretase target sequence, as well as whole recombinant APP. By using synthetic subpeptides, we have located 5AH10 epitope between amino acids 15 and 22 of A4. In addition, 5AH10 showed a strong immunoreactivity to a 47 kDa protein present in rat brain extracts, that was identified as the B (brain specific) subunit of creatine kinase by immunochemical data and direct N-terminal sequencing. The cross-reaction observed is most probably due to a high degree of sequence identity between amino acids 15 to 22 of A4 peptide and amino acids 9 to 16 of rat B creatine kinase. 5AH10 did not recognize the muscle specific isoform (M subunit) of rat creatine kinase, nor the B subunit of human and rabbit creatine kinase, suggesting that glutamine at first position of the epitope is essential for antigen recognition by 5AH10.

Alzheimer's amyloid precursor protein is expressed on the surface of hematopoietic cells upon activation.

A4-amyloid is the major component of senile plaques and neurofibrillary tangles found in the brain of patients suffering Alzheimer's disease. This 39-42 amino acid peptide is derived from a larger precursor protein (APP). Since APP gene encodes for a putative membrane protein, the study of APP expression at the cell surface may provide useful data for understanding its physiological function. In this report, we present data on APP expression, that was detected by APP specific mAbs in cells of the hematopoietic system. APP was weakly expressed on the cell surface of resting human lymphocytes and monocytes, but it could be induced to the surface of those cells upon stimulation. The cell activators capable of inducing APP membrane expression comprehended mitogenic lectins, calcium ionophores, phosphatase inhibitors, and anti mu-chain or anti-CD3 antibodies in B and T cells, respectively. Interestingly, phorbol esters were able to induce APP membrane expression in monocytic, but not in lymphoid cells. In contrast to lymphocytes and monocytes, granulocytes never expressed cell surface or cytoplasmic APP, even after the activation. The induction of membrane APP in response to lymphocyte activation signals, including antibodies to the antigen receptor of B and T cells, raises the possibility that APP might play the role of a cell surface receptor in the immune system.

Proteolysis of Alzheimer's disease beta-amyloid precursor protein by factor Xa.

Amyloid beta-protein is a 4-kDa peptide which originates from proteolysis of a larger protein precursor (APP) and accumulates in senile plaques in brains of Alzheimer's disease (AD) patients. Since secreted APP inhibits factors IXa, Xa and XIa, and thrombin appears to play a role in APP secretion and proteolysis, a relationship between hemostasis system and APP metabolism seems to exist. In this work we investigate the susceptibility to proteolytic cleavage by factor Xa of a fusion construct containing full-length APP prepared in bacteria, and demonstrate that both APP695 and APP770 are substrates for this protease. Factor Xa was found to cleave APP after arginines 102, 268, 510, 573 and 601 (APP695 numeration); most of these sites appear to be common for different coagulation factors. In addition, APP incubation with factor Xa generates an array of six potentially amyloidogenic fragments. Comparative kinetic analysis of APP695 and APP770 cleavage by factor Xa suggests that Kunitz-type inhibitor-containing isoforms exert an inhibitory effect on the protease. However, this inhibition is far from complete even at a 5-fold molar excess of inhibitor. Our results raise the possibility that proteases from the coagulation cascade may contribute to APP proteolysis, and support the notion that these proteases play a role in AD pathogenesis.

Identification of amino acid residues of transcription factor AP-2 involved in DNA binding.

AP-2 is a cell-type specific, developmentally regulated transcription factor which has been described as a critical regulator of gene expression during vertebrate development and embryogenesis. Although the overall domains of this factor necessary for their activity have been identified, the exact identity of AP-2 amino acid residues responsible for its interaction with the DNA structure has not yet been described. Here, we describe the identification of a region of AP-2 which was protected by an oligonucleotide probe containing its binding site from trypsin digestion, monitored by peptide mapping by MALDI-TOF mass spectrometry. Furthermore, we analyzed the relative in vitro DNA-binding activity, the stimulatory potency on the AP-2-dependent APOE promoter, as well as the ability to inhibit the effect of the wild-type protein of each one of a set of single-site substitution AP-2 mutants spanning the identified region. Taken together, our data clearly demonstrate that the region between amino acid residues 252-260 of AP-2 is essential for its DNA-binding activity. Particularly, the individual substitution in any of the residues 253, 254, 255, 257 or 260 is sufficient for completely abolishing the interaction with DNA and the stimulation of APOE promoter activity. These results indicate a crucial role of this region in the formation of an active DNA-binding domain and strongly suggest that these residues provide direct contacts with the DNA structure at the AP-2 binding site.

Transcription factor AP-2 activity is modulated by protein kinase A-mediated phosphorylation.

We recently reported that APOE promoter activity is stimulated by cAMP, this effect being mediated by factor AP-2 [Garcia et al. (1996) J. Neurosci. 16, 7550-7556]. Here, we study whether cAMP-induced phosphorylation modulates the activity of AP-2. Recombinant AP-2 was phosphorylated in vitro by protein kinase A (PKA) at Ser239. Mutation of Ser239 to Ala abolished in vitro phosphorylation of AP-2 by PKA, but not the DNA binding activity of AP-2. Cotransfection studies showed that PKA stimulated the effect of AP-2 on the APOE promoter, but not that of the S239A mutant. Therefore, cAMP may modulate AP-2 activity by PKA-induced phosphorylation of this factor.

Allelic polymorphisms in the transcriptional regulatory region of apolipoprotein E gene.

In this work, we explored the existence of genetic variants within the apolipoprotein E gene transcriptional regulatory region, using a denaturing gradient gel electrophoresis screening of a region comprising nucleotides -1017 to +406. Upon a population study, three new polymorphic sites (-491, -427 and -219) and two mutations were found. Functional effects of the polymorphisms, assayed by transient transfection and electrophoretic mobility shift assays in a human hepatoma cell line, showed that polymorphisms at sites -491 and -219 of the APOE promoter produce variations in the transcriptional activity of the gene, most probably through differential binding of nuclear proteins.

Contribution of APOE promoter polymorphisms to Alzheimer's disease risk.

OBJECTIVE: To determine whether the effects of APOE promoter polymorphisms on AD are independent of the APOE-epsilon4 allele. BACKGROUND: Recently, the -491 A-->T and -219 G-->T polymorphisms located in the APOE promoter have been suggested to be risk factors for AD. However, the effects of these polymorphisms have not always been reproduced in case-control studies, possibly because of the strong linkage disequilibrium existing at this locus or the characteristics of the populations studied. METHODS: Data collection was performed from six independent samples (1,732 patients with AD and 1,926 control subjects) genotyped for APOE exon 4 and the two APOE promoter polymorphisms. The risks associated with the APOE polymorphisms for developing AD were estimated using logistic regression procedures and calculation of odds ratios with 95% CI adjusted by age, sex, and collection center. Independence of the APOE promoter polymorphisms was tested by stratification for APOE-epsilon4 and tertile design was used for age stratification. RESULTS: The independence of the -491 AA genotype was observed in the whole sample whereas the independence of the -219 TT genotype was observed only in the oldest population. CONCLUSION: The -491 and -219 APOE promoter polymorphisms incur risk for AD in addition to risk associated with the APOE-epsilon4 allele, with age accentuating the effect of the -219 TT genotype. Because these polymorphisms appear to influence apoE levels, these results suggest that APOE expression is an important determinant of AD pathogenesis.

Anti-brain spectrin immunoreactivity in Alzheimer's disease: degradation of spectrin in an animal model of cholinergic degeneration.

In a previous work, we described the existence of anti-brain spectrin auto antibodies in Alzheimer's disease (AD) patients (J. Neuroimmunol. 68 (1996) 39-44). In this report, we further support our previous observations, showing that sera from 9 out of 18 AD patients, but none of 14 control subjects, immunoreacted with spectrin synthesized by PC12 cells. In addition, degradation of brain spectrin was found to be greatly enhanced in the frontal cortex of rats subjected to an animal model of cholinergic degeneration. Our data suggest that spectrin degradation and generation of anti-spectrin auto antibodies may be related to the cholinergic degeneration encountered in AD.

Antibodies to human brain spectrin in Alzheimer's disease.

We investigated the existence of antibodies in sera of Alzheimer's disease patients which immunoreact with specific antigens from crude human brain extracts. We found that 49% of patients, per only 5% of control subjects, had increased levels of antibodies to a 240 kDa protein. On the basis of immunological criteria and internal amino acid sequencing, this antigen was identified as brain spectrin, a cytoskeletal protein which appears to be implicated in synaptic plasticity. Our data raises the possibility that anti-spectrin antibodies could be implicated in Alzheimer's disease pathogenesis.

Inhibition by valproic acid of pyruvate uptake by brain mitochondria.

The anticonvulsive drug, valproic acid, inhibits competitively the pyruvate carrier in rat brain and liver mitochondria. Due to this inhibition the oxygen consumption supported by pyruvate oxidation is also affected. In our experimental conditions, pyruvate oxidation is partially inhibited by VPA concentration as low as 0.05 mM. Valproic acid, however, is unable, even at 10 mM, to fully inhibit pyruvate oxidation. Concentrations of VPA higher than 1 mM have an uncoupling effect on mitochondrial respiration. The oxidation of other mitochondrial substrates such as isocitrate, 2-ketoglutarate, DL-3-hydroxybutyrate and succinate is uncoupled but not inhibited by VPA. The effects of VPA on mitochondrial metabolism may be related to the therapeutic and/or toxicologic properties of this drug.

Molecular epidemiology of human rotaviruses in Santiago, Chile.

BACKGROUND: Protective immunity against rotavirus infection is directed against antigenic epitopes on the outer capsid proteins VP7 and VP4. Our aim was to characterize the epidemiology of rotavirus antigenic types over time in Santiago, Chile. METHODS: We prospectively obtained 2097 stool samples for rotavirus testing, VP7 (G1 to G4) and VP4 (P4, P6, P8, P9) typing from children with diarrhea evaluated in emergency rooms of 5 base hospitals of Santiago. In addition 256 rotavirus-positive samples collected between 1985 and 1987 in the north health care area of Santiago were studied. RESULTS: Of 995 rotavirus-positive samples obtained 825 (82%) were typable for 1 or more VP7 types. G1 represented 81% of the G-typed samples during 1993 through 1995 and 77% during 1985 through 1987, predominating in all health care areas. G2 was next most common in all 5 areas, representing 6 to 23% of typed samples, with 1 area, the Southeast concentrating a significantly higher number of G2 infections. G2 declined from 35% of rotavirus-positive samples in 1993 to 0% in 1995 (P < 0.001), and from 25% to 2% in the north health care area from 1985 to 1987 (P < 0.001). G4 was uncommon and significantly more prevalent in 1985 through 1987 than in 1993 through 1995 (7% vs. 3%, P = 0.015). G3 was not detected. G1P8 (53%) and G2P4 (16%) combinations were by far the most commonly detected G-P associations. CONCLUSIONS: In Santiago, Chile, rotavirus antigenic type G1P8 has been highly prevalent and G2P4 has circulated in cycles. Differences in epidemiology of rotavirus antigenic types worldwide may prove to be relevant in efficacy of rotavirus vaccines.

Interaction of bilirubin with gangliosides.

Gangliosides seem to play an important role in the interaction of the neurotoxic pigment bilirubin with the synaptosomal plasma membrane (Vázquez et al. [1988] J. Biol. Chem. 263, 1255-1265). In this report, a further characterization of the bilirubin-ganglioside interaction is presented. The interaction is fast, and it is observed at any pH in the range 7.0-9.0. The characteristics of the interaction are different from those observed with other membrane lipids, including sphingomyelin. A model of binding to a single population of sites is able to adequately fit the experimental data. This model predicts a decrease in the tendency of bilirubin to interact with gangliosides and an increase in the binding capacity as the pH is decreased from 8.0 to 7.0. Our data would suggest a role for gangliosides in explaining the preferential accumulation of bilirubin in some areas of the brain and the toxic effect of this pigment in neuronal membrane-related functions.

Apolipoprotein E gene promoter polymorphisms in Alzheimer's disease.

Alzheimer's disease, the most frequent form of senile dementia, presents in the vast majority of cases as a multifactorial trait, where a series of genetic and environmental risk factors converge. The increasing body of data, both epidemiological and functional, is strengthening the evidence that apolipoprotein E (APOE, gene; apoE, protein) is a true susceptibility factor for the onset of the common form of Alzheimer's disease. The E4 isoform of apoE remains to date as the main genetic risk factor for the disease, although the mechanisms responsible for this association are not well understood. It is also clear that apoE4 is not necessary or sufficient to cause the disease, indicating that other risk and protecting factors exist. ApoE is upregulated in response to nervous system injury, suggesting that it could have a neuroprotective role; on the other hand, there is evidence indicating that apoE is neurotoxic when present at high levels. Thus, apoE levels seem to be relevant for the functionality of the protein. The APOE proximal promoter hosts numerous regulatory elements, raising the possibility that polymorphisms in this region could produce variation in apoE levels by altering APOE transcriptional activity, which could finally result in AD susceptibility. We will review here the current evidence on the relationship between APOE proximal promoter polymorphisms, APOE gene transcriptional activity and apoE protein levels, and risk for AD.

Developmental studies on the uptake of tyrosine by synaptosomes and plasma membrane vesicles derived from rat brain. Effect of thyroid hormones.

The uptake of L-tyrosine at various stages of development was examined in synaptosomes and in plasma membrane vesicles derived from rat brain. The total uptake has two components, Na+-dependent and Na+-independent, respectively. The Na+-dependent component of the transport system appears around the 5th postnatal day and increases with age. The affinity of the transport system for tyrosine does not vary substantially during development. The Vmax increases more than six-fold between day 15 and adulthood. Plasma membrane vesicles derived from T3-treated rats accumulate more tyrosine than those obtained from the control animals. The results support the view that thyroid hormones during development promote the establishment of the systems implicated in neurotransmission in the developing nervous system.