Increased expression of BIN1 mediates Alzheimer genetic risk by modulating tau pathology.

Genome-wide association studies (GWAS) have identified a region upstream the BIN1 gene as the most important genetic susceptibility locus in Alzheimer's disease (AD) after APOE. We report that BIN1 transcript levels were increased in AD brains and identified a novel 3 bp insertion allele ∼28 kb upstream of BIN1, which increased (i) transcriptional activity in vitro, (ii) BIN1 expression levels in human brain and (iii) AD risk in three independent case-control cohorts (Meta-analysed Odds ratio of 1.20 (1.14-1.26) (P=3.8 × 10(-11))). Interestingly, decreased expression of the Drosophila BIN1 ortholog Amph suppressed Tau-mediated neurotoxicity in three different assays. Accordingly, Tau and BIN1 colocalized and interacted in human neuroblastoma cells and in mouse brain. Finally, the 3 bp insertion was associated with Tau but not Amyloid loads in AD brains. We propose that BIN1 mediates AD risk by modulating Tau pathology.

Contribution of Kunitz protease inhibitor and transmembrane domains to amyloid precursor protein homodimerization.

BACKGROUND: The two major isoforms of the human amyloid precursor protein (APP) are APP695 and APP751. They differ by the insertion of a Kunitz-type protease inhibitor (KPI) sequence in the extracellular domain of APP751. APP-KPI isoforms are increased in Alzheimer's disease brains, and they could be associated with disease progression. Recent studies have shown that APP processing to Aß is regulated by homodimerization, which involves both extracellular and juxtamembrane/transmembrane (JM/TM) regions. OBJECTIVE: Our aim is to understand the mechanisms controlling APP dimerization and the contribution of the ectodomain and JM/TM regions to this process. METHODS: We used bimolecular fluorescence complementation approaches coupled to fluorescence-activated cell sorting analysis to measure the dimerization level of different APP isoforms and APP C-terminal fragments (C99) mutated in their JM/TM region. RESULTS: APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain of APP or C99 did not significantly affect fluorescence complementation. CONCLUSION: These findings indicate that the KPI domain plays a major role in APP dimerization. They set the basis for further investigation of the relation between dimerization, metabolism and function of APP.

A novel mRNA of the A4 amyloid precursor gene coding for a possibly secreted protein.

The gene, encoding the A4 peptide found in the amyloid core of senile plaques isolated from the cerebral cortex of patients with Alzheimer's disease, produces at least three precursors that resemble cell surface receptors. A clone isolated from a human brain complementary DNA library contained the structural sequence for an A4 amyloid peptide precursor with a serine protease inhibitor domain in which 208 amino acids at the carboxyl terminal are replaced by 20 amino acids derived from nucleotide sequences with homology to the Alu repeat family. This protein devoid of the transmembrane domain most likely represents a secreted form of the A4 amyloid peptide precursor.

[The precursor of amyloid peptide in Alzheimer disease: a protein with multiple functions]. / Le précurseur du peptide amyloïde de la maladie d'Alzheimer: une protéine aux multiples fonctions.

Cellular metabolism of the amyloid precursor protein (APP) has been widely studied, but the function of the protein remains elusive. APP knock out mice do not show any phenotype, due to in vivo compensation by APLP genes, encoding proteins similar to APP. In order to study the neuronal metabolism of APP, human APP has been expressed in rat cortical neurons in culture. Following differentiation in culture, rat cortical neurons are organized into networks of connected cells, which show neuronal activity in the form of spontaneous and synchronous calcium oscillations. Expression of human APP in these neuronal networks inhibits calcium oscillations, while downregulation of endogenous APP expression increases the frequency and decreases the amplitude of oscillations. Therefore, APP controls neuronal calcium homeostasis and excitability. In the same experimental model, APP is also able to control the neuronal synthesis of cholesterol. Finally, the APP carboxy terminal domain is involved in the epigenetic control of gene expression. Modulation of neuronal expression of APP allows to identify several important functions of the precursor of the amyloid peptide found in senile plaques of Alzheimer disease.

A GG nucleotide sequence of the 3' untranslated region of amyloid precursor protein mRNA plays a key role in the regulation of translation and the binding of proteins.

The alternative polyadenylation of the mRNA encoding the amyloid precursor protein (APP) involved in Alzheimer's disease generates two molecules, with the first of these containing 258 additional nucleotides in the 3' untranslated region (3'UTR). We have previously shown that these 258 nucleotides increase the translation of APP mRNA injected in Xenopus oocytes (5). Here, we demonstrate that this mechanism occurs in CHO cells as well. We also present evidence that the 3'UTR containing 8 nucleotides more than the short 3'UTR allows the recovery of an efficiency of translation similar to that of the long 3'UTR. Moreover, the two guanine residues located at the 3' ends of these 8 nucleotides play a key role in the translational control. Using gel retardation mobility shift assay, we show that proteins from Xenopus oocytes, CHO cells, and human brain specifically bind to the short 3'UTR but not to the long one. The two guanine residues involved in the translational control inhibit this specific binding by 65%. These results indicate that there is a correlation between the binding of proteins to the 3'UTR of APP mRNA and the efficiency of mRNA translation, and that a GG motif controls both binding of proteins and translation.

Iron mobilization from cultured rat bone marrow macrophages.

The reticuloendothelial system is responsible for removing old and damaged erythrocytes from the circulation, allowing iron to return to bone marrow for hemoglobin synthesis. Cultured bone marrow macrophages were loaded with 59Fe-labelled erythroblasts and iron mobilization was studied. After erythroblast digestion, iron taken up by macrophages was found in ferritin as well as in a low-molecular-weight fraction. The analysis of iron mobilization from macrophages shows: (1) the iron was mobilized as ferritin. (2) A higher mobilization was observed when apotransferrin was present in the culture medium. (3) In the presence of apotransferrin in the culture medium, part of the iron was found as transferrin iron. (4) Iron transfer from ferritin to apotransferrin was observed in a cell-free culture medium and this process was temperature independent. The results indicate that after phagocytosis of 59Fe-labelled erythroblasts by macrophages, iron is mobilized as ferritin. In the plasma, this iron can be transferred to apotransferrin.

Activation of protein kinase C increases the extracellular release of the transmembrane amyloid protein precursor of Alzheimer's disease.

The beta A4 peptide is the major constituent of the amyloid core of abundant senile plaques found in the cerebral cortex of patients with Alzheimer's disease. This amyloid peptide is synthesized as part of a large transmembrane amyloid protein precursor or APP. In addition to the highly expressed transmembrane APP isoforms, an mRNA encoding a secreted APP lacking the transmembrane domain has been identified. A cleavage of the transmembrane protein also yields an extracellular soluble APP fragment. The effect of phorbol esters on the release of the extracellular APP was studied in transfected Chinese hamster ovary cells which stably express either a transmembrane or a secreted APP isoform. The activation of protein kinase C by phorbol-12,13-dibutyrate increased the extracellular release of the transmembrane APP resulting from its proteolytic cleavage, while 4-beta-phorbol, which does not activate protein kinase C, did not significantly affect the recovery of the soluble APP. On the contrary, the recovery of APP secreted in the culture medium without proteolytic cleavage was not increased by protein kinase C-mediated phosphorylation.

Is aggregation of beta-amyloid peptides a mis-functioning of a current interaction process?

In a previous study, Hughes et al. [Proc. Natl. Acad. Sci. USA 93 (1996) 2065-2070] demonstrated that the amyloid peptide is able to interact with itself in a two-hybrid system and that interaction is specific. They further supported that the method could be used to define the sequences that might be important in nucleation-dependent aggregation. The sequence of the amyloid peptide can be split into four clusters, two hydrophilic (1-16 and 22-28) and two hydrophobic (17-21 and 29-42). We designed by molecular modeling and tested by the two-hybrid approach, series of mutations spread all over the sequence and changing the distribution of hydrophobicity and/or the spatial hindrance. In the two-hybrid assay, interaction of native Abeta is reproduced. Screening of mutations demonstrates that the C-domain (residues 29-40 (42)), the median domain (residues 17-22) and the N-domain (1-16) are all crucial for interaction. This demonstrates that almost all fragments of the amyloid peptide but a loop (residues 23-28) and the C-term amino acid are important for the native interaction. We support that the folded three-dimensional (3D) structure is the Abeta-Abeta interacting species, that the whole sequence is involved in that 3D fold which has a low secondary structure propensity and a high susceptibility to mutations and thus should have a low stability. The native fold of Abeta could be stabilized in Abeta-Abeta complexes which could in other circumstances facilitate the nucleation event of aggregation that leads to the formation of stable senile plaques.

[Alzheimer disease: cellular and molecular aspects]. / Maladie d'Alzheimer: aspects cellulaires et moléculaires.

A conclusive diagnosis of Alzheimer's disease (AD) can be made only by correlating clinical findings and neuropathological studies of post-mortem tissues. Two leading neuropathological changes correlate with the diagnosis of AD: first, the neurofibrillary tangles (NFTs) which accumulate in neuronal perikarya and are made of paired helical filaments (PHFs) containing the microtubule-associated protein tau; second, extracellular amyloid deposits in the form of diffuse or neuritic senile plaques which contain the amyloid peptide. In AD, NFTs can be easily visualized using antibodies recognizing the microtubule associated protein tau and are composed of bundles of PHFs. In the autopsy-derived AD brain, tau is hyperphosphorylated and more than 30 phosphorylation sites have been identified in PHF-tau proteins. The formation of NFTs is thought to be associated with a collapse of the microtubule network, disturbances of axoplasmic transports, synapse loss, neuritic atrophy, and neuronal death. Senile plaques are extracellular lesions which have been shown by electron micro-scopic studies to contain amyloid fibrils. Fibrils were isolated and a small 4.2 kDa poly-peptide was purified from this material. The amyloid peptide found in amyloid deposits of AD is designated Abeta. Since the Abeta peptide is small and unlikely to be a primary translational product, it was predicted to arise from a larger precursor. In 1987, this amyloid peptide precursor (APP) was characterised from the analysis of a full-length cDNA encoding a primary translational product of 695 residues. This protein is synthetized by neurons as a 100-kDa glycosylated transmembrane protein with a single membrane spanning domain. The use of cellular models has clearly identified two catabolic pathways for APP. A non amyloidogenic pathway, in which APP is cleaved by beta-secretase within the sequence of the amyloid peptide. This cleavage precludes the formation of the full-length Abeta found in the amyloid core of senile plaques. A second catabolic pathway of APP leads to the production of Abeta from its precursor. In this amyloidogenic pathway, APP is cleaved by beta-secretase at the N-terminus of Abeta. The C-terminal fragment of APP thus formed is in turn cleaved by beta-secretase to release the full-length amyloid peptide. In primary cultures of neurons over-expressing APP, the production of intraneuronal Abeta induces neuronal apoptosis. This neurotoxicity, which is not observed in epithelial cells, seems to be related to the formation of intraneuronal aggregates of Abeta 1-42. In AD, the specific inhibition of beta- or beta-secretase activities would decrease the production of Abeta from its precursor, in such a way that its relative concentration could be low enough to avoid the formation of aggregates. Molecules which can interact with Abeta in order to inhibit its aggregation are also being developed. Immunization against Abeta has also been tested in both animal models and clinical studies. Although these clinical studies had to be interrupted due to the development of T-lymphocyte meningoencephalitis in some patients, very preliminary results indicate that antibodies against Abeta slow cognitive decline in AD, and generate areas of neocortex devoid of senile plaques.

Yersinia enterocolitica can deliver Yop proteins into a wide range of cell types: development of a delivery system for heterologous proteins.

Y. enterocolitica translocates virulence proteins, called Yop effectors, into the cytosol of eukaryotic cells. Here we investigated whether Y. enterocolitica could translocate Yops into a range of eukaryotic cells including neurons and insect cells. Y. enterocolitica translocated the hybrid reporter protein YopE-Cya into each of the eukaryotic cell types tested. In addition, Y. enterocolitica was cytotoxic for each of the adherent cell types. Thus we detected no limit to the range of eukaryotic cells into which Y. enterocolitica can translocate Yops. The Yop effectors YopE, YopH and YopT were each cytotoxic for the adherent cell types tested, showing that not only is Y. enterocolitica not selective in its translocation of particular Yop effectors into each cell type, but also that the action of these Yop effectors is not cell type specific. Invasin and/or YadA, two powerful adhesins were required for translocation of Yop into non-phagocytic cells but not for translocation into macrophages. To use the Yersinia translocation system for broad applications, a Y. enterocolitica translocation strain and vector for the delivery of heterologous proteins into eukaryotic cells was constructed. This strain + vector combination lacks the translocated Yop effectors and allows delivery into eukaryotic cells of heterologous proteins fused to the minimal N-terminal secretion/translocation signal of YopE. Using this strategy translocation of a YopE-Diphtheria toxin subunit A hybrid protein into several cell types has been shown.

Quantification of mitochondrial DNA carrying the tRNA(8344Lys) point mutation in myoclonus epilepsy and ragged-red-fiber disease.

Myoclonus epilepsy and ragged-red-fiber syndrome (MERRF) is caused by a point mutation at nucleotide 8344 in the tRNA(Lys) gene of mitochondrial DNA. We analyzed leukocyte DNA from nine members of a large MERRF family using a new technique, solid-phase minisequencing. Quantitative analysis of the tRNA(8344Lys) mutation showed that the mutated mtDNA comprised from 9 to 72% of the total mtDNA in the leukocytes of these individuals. The minisequencing method is a promising tool for the diagnosis of MERRF. In addition to the identification of the tRNA(8344Lys) mutation, the relative amount of mutated mtDNA can be simultaneously determined in the same assay from one blood sample.

Specific increase of genetic expression of parvalbumin in fast skeletal muscles of mdx mice.

Parvalbumin mRNA was assayed by Northern blot analysis in muscles from normal and dystropic (mdx) mice. Its content was found to be specifically higher in mdx fast muscles than in control preparations. This suggests an increased expression of the protein in dystrophin-lacking fast fibres. A possible role in calcium homeostasis is discussed.

Inhibition of trypsin by the beta-amyloid protein precursor. A comparative study between transfected cells, human brain and cerebrospinal fluid.

Soluble beta-amyloid protein precursors (beta-APPs) were studied in human brain and cerebrospinal fluid (CSF) after partial purification by ion exchange chromatography. Proteins were analysed in immunoblotting experiments using a monoclonal antibody directed against the N-terminal segment of the beta-APP 770, and by reverse enzymography. In the human brain and CSF, a protein which comigrates with the beta-APP 770 expressed by transfected CHO cells was able to inhibit trypsin.

Baculovirus-infected cells do not produce the amyloid peptide of Alzheimer's disease from its precursor.

The amyloid peptide (Abeta) of Alzheimer's disease (AD) is produced by proteolytic cleavage of a larger precursor, the amyloid peptide precursor or APP. The discovery of pathogenic mutations in the APP gene provides strong evidence for the hypothesis that APP metabolism is involved in the etiology of AD. To study the metabolism of the protein, human APP has been expressed in several mammalian cell types. Insect cells, infected by a recombinant baculovirus carrying the human APP sequence, also provide an interesting expression system because these cells do not produce endogenous APP. Baculovirus-infected cells synthesize very high amounts of extracellular soluble APP, after cleavage of the transmembrane protein, as described for mammalian cells. However, we demonstrate here that insect cells do not produce Abeta from APP. These results suggest that while the enzymatic activity needed for the production of soluble APP is conserved between insect and mammalian cells, the enzymes required for the production of Abeta from APP are only expressed in mammalian cells.

Transferrin protein and iron uptake by cultured hepatocytes.

The binding and uptake of 59Fe-loaded 3H-labelled rat transferrin by cultured rat hepatocytes was investigated. At 4 degrees C, there is no evidence for a specific binding of transferrin which could be related to the association of neo-synthesized transferrin with plasma membrane receptors. At 37 degrees C, iron uptake is much more important than transferrin uptake; it proceeds linearly over the time of incubation, is largely proportional to the extracellular transferrin concentration, and is compatible with uptake by fluid phase endocytosis. The difference observed between iron and transferrin uptake implies the existence of a mechanism allowing the reutilization of transferrin after iron delivery.

Complete nucleotide sequence coding for rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase derived from a cDNA clone.

cDNA clones for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were isolated from rat liver expression libraries in lambda gt11 by antibody, oligonucleotide, and cDNA screening. One 1860 bp long clone contained a full-length nucleotide sequence coding for the 470 amino acids of each of the two identical subunits of the bifunctional enzyme. This clone also contained untranslated sequences, one 173 bp long upstream from the ATG start codon and one 271 bp long downstream from the TGA stop codon. The clone was terminated by a poly(A) tail of 29 nucleotides.

Neurofibrillary tangles and tau phosphorylation.

Neurofibrillary tangles (NFTs) are a characteristic neuropathological lesion of Alzheimer's disease (AD). They are composed of a highly-phosphorylated form of the microtubule-associated protein tau. We are investigating the relationship between NFTs and microtubule stability and how tau phosphorylation and function is affected in transgenic models and by co-expression with beta-amyloid precursor protein and presenilins. In most NFT-bearing neurons, we observed a strong reduction in acetylated alpha-tubulin immunoreactivity (a marker of stable microtubules) and a reduction of the in situ hybridization signal for tubulin mRNA. In transfected cells, mutated tau forms (corresponding to tau mutations identified in familial forms of frontotemporal dementias linked to chromosome 17) were less efficient in their ability to sustain microtubule growth. These observations are consistent with the hypothesis that destabilization of the microtubule network is an important mechanism of cell dysfunction in Alzheimer's disease. The glycogen synthase kinase-3 beta (GSK-3 beta) generates many phosphorylated sites on tau. We performed a neuroanatomical study of GSK-3 beta distribution showing that developmental evolution of GSK-3 beta compartmentalization in neurons paralleled that of phosphorylated tau. Studies on transfected cells and on cultured neurons showed that GSK-3 beta activity controls tau phosphorylation and tau functional interaction with microtubules. Tau phosphorylation was not affected in neurons overexpressing beta-amyloid precursor protein. Transgenic mice expressing a human tau isoform and double transgenic animals for tau and mutated presenilin 1 have been generated; a somatodendritic accumulation of phosphorylated transgenic tau proteins, as observed in the pretangle stage in AD, has been observed but NFTs were not found, suggesting that additional factors might be necessary to induce their formation.

The amyloid peptide and its precursor in Alzheimer's disease.

Alzheimer's disease, the most frequent cause of dementia, is characterized by the formation in the brain of neurofibrillary tangles and senile plaques. Neurofibrillary tangles are composed of bundles of paired helical filaments containing the microtubule-associated protein tau. In autopsy-derived brain samples from patients with Alzheimer's disease, tau is hyperphosphorylated and constitutes a promising disease marker. Senile plaques contain a small amyloid peptide derived from the amyloid precursor protein. Mutations of the amyloid precursor protein gene have been identified in rare cases of familial Alzheimer's disease, suggesting a causal role for amyloid peptide deposition in the disease. However, Alzheimer's disease has been demonstrated to be characterized by an important genetic heterogeneity. The identification of pathogenic DNA mutations, different from those of the amyloid precursor protein gene, will reveal whether the corresponding genes are involved in either an increased production of the amyloid peptide or a decrease of its removal, or in the fibrillogenic properties of the peptide, which seem to be related to its toxicity. Several mammalian cells are able to produce the amyloid peptide from its precursor. Understanding the cellular mechanisms that determine how cleavages occur in cells could help to identify new strategies for modulating amyloid peptide production. In attempts to produce animal models of Alzheimer's disease, investigators have used transgenic strategies. To date, these efforts have not been very successful. However, the expression in transgenic mice of both mutated amyloid peptide precursor and amyloid associated proteins should prove useful for examining the importance of putative etiological factors, and for testing novel therapies including anti-amyloidogenic strategies.

Distribution of the phosphorylated microtubule-associated protein tau in developing cortical neurons.

During brain development, the microtubule-associated protein tau presents a transient state of high phosphorylation. We have investigated the developmental distribution of the phosphorylated fetal-type tau in the developing rat cortex and in cultures of embryonic cortical neurons, using antibodies which react with tau in a phosphorylation-dependent manner. The phosphorylated fetal-type tau was present in the developing cortex at 20 days but not at 18 days of embryonic life and was not detected before four to five days in neuronal culture. The cyclin-dependent kinase p34cdc2 was expressed only in germinal layers in the embryonic brain and was not co-localized with phosphorylated tau. After 10 days of postnatal life, the phosphorylated tau progressively disappeared from cortical neurons, disappearing first from the deepest cortical layers where neurons are ontogenetically the oldest. Phosphorylated tau was found in axons and dendrites of cortical neurons at all developmental stages whereas unphosphorylated tau tended to disappear from dendrites during development. The timing of appearance of phosphorylated tau in the cortex, by comparison with the expression of other developmental markers, indicates that phosphorylated tau is present at a high level only during the period of intense neuritic outgrowth and that it disappears during the period of neurite stabilization and synaptogenesis, concomitantly to the expression of adult tau isoforms. In control cultures and in cultures treated with colchicine, the phosphorylated tau was not associated to cold-stable and to colchicine-resistant microtubules. These in vivo results suggest that the high expression of phosphorylated tau species is correlated with the presence of a dynamic microtubule network during a period of high plasticity in the developing brain.

The processing and biological function of the human amyloid precursor protein (APP): lessons from different cellular models.

One of the major neuropathological hallmarks of Alzheimer's disease is the presence of senile plaques in vulnerable regions of CNS. These plaques are formed of aggregated amyloid peptide. Amyloid peptide is released by the cleavage of its precursor (APP). The establishment of cell lines expressing human APP allowed to characterize both amyloidogenic and non-amyloidogneic pathways of APP catabolism and to identify some of the proteins involved in this processing (known as secretases). This led to a better comprehension of amyloid peptide production, which needs to be further characterized since gamma-secretase is as yet not identified; moreover, we still lack a clear overview of the interactions between APP and other proteins promoting Alzheimer's disease (tau, presinilinsellipsis). An important limitation of these cell lines for studying the mechanisms involved in Alzheimer's disease is supported by the observation that human APP expression does not modify transfected cells survival. The infection of primary neuronal cultures with full-length human APP indicates that APP expression induces neuronal apoptosis by itself; this neurotoxicity does not rely on extracellular production of APP derivatives (secreted APP, amyloid peptide). It is now essential to understand, in neuronal models, the production, localization and involvement of amyloid peptide in neurodegenerative processes.