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.
Amyloid fibrils associated with Alzheimer's disease and a wide range of other neurodegenerative diseases have a cross beta -sheet structure, where main chain hydrogen bonding occurs between beta ...-strands in the direction of the fibril axis. The surface of the beta -sheet has pronounced ridges and grooves when the individual beta -strands have a parallel orientation and the amino acids are in-register with one another. Here we show that in A beta amyloid fibrils, Met35 packs against Gly33 in the C-terminus of A beta 40 and against Gly37 in the C-terminus of A beta 42. These packing interactions suggest that the protofilament subunits are displaced relative to one another in the A beta 40 and A beta 42 fibril structures. We take advantage of this corrugated structure to design a new class of inhibitors that prevent fibril formation by placing alternating glycine and aromatic residues on one face of a beta -strand. We show that peptide inhibitors based on a GxFxGxF framework disrupt sheet-to-sheet packing and inhibit the formation of mature A beta fibrils as assayed by thioflavin T fluorescence, electron microscopy, and solid-state NMR spectroscopy. The alternating large and small amino acids in the GxFxGxF sequence are complementary to the corresponding amino acids in the IxGxMxG motif found in the C-terminal sequence of A beta 40 and Ap42. Importantly, the designed peptide inhibitors significantly reduce the toxicity induced by A beta 42 on cultured rat cortical neurons.
We have generated transgenic mice expressing the shortest human tau protein, the microtubule-associated protein that composes paired helical filaments in Alzheimer's disease. Transgenic tau ...transcripts and proteins were strongly expressed in neurons in the developing and adult brain. In contrast to the endogenous tau that progressively disappeared from neuronal cell bodies during development, the human transgenic tau remained abundant in cell bodies and dendrites of a subset of neurons in the adult. This somatodendritic transgenic tau was immunoreactive with antibodies to tau phosphorylated on Thr181 and Thr231 and with the conformation-dependent Alz50 antibody. A few astrocytes expressing the transgenic tau were strongly immunoreactive with antibodies to additional tau phosphorylation sites, ie, at Ser262/356 and Ser396/404. All of these phosphorylation sites have been identified in paired helical filaments-tau proteins. In electron microscopy, the transgenic tau was detected into microtubules in axons and in dendrites but not in cell bodies. Neurofibrillary tangles were not detected in transgenic animals examined up to the age of 19 months. These results indicate that transgenic manipulation of tau expression and intracellular targeting is sufficient per se to affect tau compartmentalization, phosphorylation, and conformation partly as it is observed at the pretangle stage in Alzheimer's disease.
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 Aβ is regulated by homodimerization, which involves both extracellular and juxtamembrane/transmembrane (JM/TM) regions.
Our aim is to understand the mechanisms controlling APP dimerization and the contribution of the ectodomain and JM/TM regions to this process.
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.
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.
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.
The cleavage of the transmembrane amyloid precursor protein (APP) by β-secretase leaves the C-terminal fragment of APP, C99, anchored in the plasma membrane. C99 is subsequently processed by ...γ-secretase, an unusual aspartyl protease activity largely dependent on presenilin (PS), generating the amyloid β-peptide (Aβ) that accumulates in the brain of patients with Alzheimer's disease. It has been suggested that PS proteins are the catalytic core of this proteolytic activity, but a number of other proteins mandatory for γ-secretase cleavage have also been discovered. The exact role of PS in the γ-secretase activity remains a matter of debate, because cells devoid of PS still produce some forms of Aβ. Here, we used insect cells expressing C99 to demonstrate that the expression of presenilin 1 (PS1), which binds C99, not only increases the production of Aβ by these cells but also increases the intracellular levels of C99 to the same extent. Using pulse-chase experiments, we established that this results from an increased half-life of C99 in cells expressing PS1. In Chinese hamster ovary cells producing C99 from full-length human APP, similar results were observed. Finally, we show that a functional inhibitor of γ-secretase does not alter the ability of PS1 to increase the intracellular levels of C99. This finding suggests that the binding of PS1 to C99 does not necessarily lead to its immediate cleavage by γ-secretase, which could be a spatio-temporally regulated or an induced event, and provides biochemical evidence for the existence of a substrate-docking site on PS1.
Alzheimer disease (AD), the most frequent cause of dementia, is characterized by an important neuronal loss. A typical histological hallmark of AD is the extracellular deposition of beta-amyloid ...peptide (A beta), which is produced by the cleavage of the amyloid precursor protein (APP). Most of the gene mutations that segregate with the inherited forms of AD result in increasing the ratio of A beta 42/A beta 40 production. A beta 42 also accumulates in neurons of AD patients. Altogether, these data strongly suggest that the neuronal production of A beta 42 is a critical event in AD, but the intraneuronal A beta 42 toxicity has never been demonstrated. Here, we report that the long term expression of human APP in rat cortical neurons induces apoptosis. Although APP processing leads to production of extracellular A beta 1-40 and soluble APP, these extracellular derivatives do not induce neuronal death. On the contrary, neurons undergo apoptosis as soon as they accumulate intracellular A beta 1-42 following the expression of full-length APP or a C-terminal deleted APP isoform. The inhibition of intraneuronal A beta 1-42 production by a functional gamma-secretase inhibitor increases neuronal survival. Therefore, the accumulation of intraneuronal A beta 1-42 is the key event in the neurodegenerative process that we observed.
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 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.
In contrast to the central nervous system (CNS), the peripheral nervous system (PNS) displays an important regenerative ability which is dependent, at least in part, on Schwann cell properties. The ...mechanisms which stimulate Schwann cells to adapt their behavior after a lesion to generate adequate conditions for PNS regeneration remain unknown. In this work, we report that adult rat dorsal root ganglion (DRG) neurons are able, after a lesion performed in vivo or when they are dissociated and cultured in vitro, to synthesize transforming growth factor beta (TGF beta), a pleiotropic growth factor implicated in wound healing processes and in carcinogenesis. This TGF beta is tentatively identified as the beta-1 isoform. Adult rat DRG neurons release a biologically active form of TGF beta which is able to elicit multiple Schwann cell responses including a stimulation to proliferate. Moreover, purified TGF beta-1 produces a Schwann cell morphology alteration and decreases the secretion of tissue-type plasminogen activator (tPA) and enhances the secretion of plasminogen activator inhibitor (PAI) by Schwann cells. This generates conditions which are thought to favor a successful neuritic regrowth. Furthermore, purified TGF beta-1 stimulates type IV collagen mRNA expression in Schwann cells. This subtype of collagen is associated with the process of myelinization. Finally, TGF beta-1 decreases nerve growth factor (NGF) mRNA expression by Schwann cells, an effect which could participate in the maintenance of a distoproximal NGF gradient during nerve regeneration. We propose that neuronal TGF beta plays an essential role as a neuronoglial signal that modulates the response of Schwann cells to injury and participates in the successful regeneration processes observed in the PNS.
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