cytochalasin-d and Alzheimer-Disease

Alzheimer disease is characterized by neuronal loss and brain plaques of extracellular amyloid β (Aβ), but the means by which Aβ may induce neuronal loss is not entirely clear. Although high concentrations of Aβ (μM) can induce direct toxicity to neurons, we find that low concentration (nM) induce neuronal loss through a microglia-mediated mechanism. In mixed neuronal-glial cultures from rat cerebellum, 250 nM Aβ1-42 (added as monomers, oligomers or fibers) induced about 30% loss of neurons between 2 and 3 days. This neuronal loss occurred without any increase in neuronal apoptosis or necrosis, and no neuronal loss occurred with Aβ42-1. Aβ greatly increased the phagocytic capacity of microglia and induced phosphatidylserine exposure (an "eat-me" signal) on neuronal processes. Blocking exposed phosphatidylserine by adding annexin V or an antibody to phosphatidylserine or inhibiting microglial phagocytosis by adding either cytochalasin D (to block actin polymerization) or cyclo(RGDfV) (to block vitronectin receptors) significantly prevented neuronal loss. Loss of neuronal synapses occurred in parallel with loss of cell bodies and was also prevented by blocking phagocytosis. Inhibition of phagocytosis prevented neuronal loss with no increase in neuronal death, even after 7 days, suggesting that microglial phagocytosis was the primary cause of neuronal death induced by nanomolar Aβ.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Annexin A5; Cells, Cultured; Coculture Techniques; Cytochalasin D; Dose-Response Relationship, Drug; Humans; Integrin alphaVbeta3; Microglia; Neurons; Nucleic Acid Synthesis Inhibitors; Peptide Fragments; Peptides, Cyclic; Phagocytosis; Phosphatidylserines; Rats; Time Factors

Amyloid diseases are characterized by the formation of insoluble amyloid fibrils from previously soluble polypeptides. In Alzheimer's disease (AD), amyloid fibrils, formed from beta-amyloid peptides, are deposited as extracellular amyloid plaques only inside the brain. As previously shown, Alzheimer's-like plaque formation in human monocyte culture recapitulates the features of in vivo amyloid plaque formation. Here we show that this cell model can be used to screen compounds that potentially influence amyloid formation in a throughput manner. We found that cellular amyloid fibril formation can be enhanced by dextran sulfate as well as heparin and can be impaired by stabilization of a micell-like beta-amyloid conformer by using myoinositol or by inhibition of phagocytosis with cytochalasin D. Altogether, our data demonstrate the utility of this cell model for investigating pathways and molecular interactions critical to amyloidogenesis.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Cells, Cultured; Chondroitin; Cytochalasin D; Dextran Sulfate; Genistein; Heparin; Humans; Inositol; Macrophages; Microscopy, Electron; Monocytes; Nocodazole; Plaque, Amyloid; Probenecid

We studied a novel function of the presenilins (PS1 and PS2) in governing capacitative calcium entry (CCE), a refilling mechanism for depleted intracellular calcium stores. Abrogation of functional PS1, by either knocking out PS1 or expressing inactive PS1, markedly potentiated CCE, suggesting a role for PS1 in the modulation of CCE. In contrast, familial Alzheimer's disease (FAD)-linked mutant PS1 or PS2 significantly attenuated CCE and store depletion-activated currents. While inhibition of CCE selectively increased the amyloidogenic amyloid beta peptide (Abeta42), increased accumulation of the peptide had no effect on CCE. Thus, reduced CCE is most likely an early cellular event leading to increased Abeta42 generation associated with FAD mutant presenilins. Our data indicate that the CCE pathway is a novel therapeutic target for Alzheimer's disease.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Channels, L-Type; Calcium Channels, N-Type; Cells, Cultured; Cytochalasin D; Humans; Imidazoles; Ion Transport; Membrane Proteins; Mice; Mice, Transgenic; Mutagenesis, Site-Directed; Neurons; Patch-Clamp Techniques; Peptide Fragments; Presenilin-1; Presenilin-2; Transfection

During axotomy studies, we discovered that the beta A4-amyloid precursor protein (APP) participates in immune responses of the central nervous system. Since microglia constitute the main immune effector cell population of this response, we used the murine microglial cell line BV-2 to analyze immune response-related APP expression. We show that interaction of microglia with the extracellular environment, particularly components of the extracellular matrix, affects APP secretion as well as intracellular APP biogenesis and catabolism. Fibronectin enhanced APP secretion and decreased the level of cellular mature transmembrane APP, whereas laminin and collagen caused a decrease in secretion and an accumulation of cellular mature APP and APP fragments. Our results demonstrate that APP plays a fundamental role in the regulation of microglial mobility, i.e. migration, initial target recognition, and binding. The decrease in APP secretion and the concomitant increase in cellular mature APP were accompanied by an accumulation of C-terminal APP fragments. Enrichment of APP and APP fragments is assumedly based on inhibition of catabolic processes that is caused by a disorganization of the actin microfilament network. These observations provide evidence that microglia, which are closely associated with certain amyloid deposits in the brain of Alzheimer patients, can play a key role in initial events of amyloidogenesis by initiating accumulation of APP and also of amyloidogenic APP fragments in response to physiological changes upon brain injury.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Blotting, Western; Brain; Cell Line; Chlorocebus aethiops; Colchicine; Collagen; Cytochalasin B; Cytochalasin D; Cytoskeleton; Extracellular Matrix; Fibronectins; Gene Expression; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Kidney; Laminin; Microglia; Nocodazole; Peptide Fragments; Polylysine; Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic; Transfection