calcimycin and Alzheimer-Disease

The microtubule associated tau protein becomes hyperphosphorylated in Alzheimer's disease (AD). While hyperphosphorylation promotes neurodegeneration, the cause and consequences of this abnormal modification are poorly understood. As impaired energy metabolism is an important hallmark of AD progression, we tested whether it could trigger phosphorylation of human tau protein in a transgenic Caenorhabditis elegans model of AD. We found that inhibition of a mitochondrial enzyme of energy metabolism, dihydrolipoamide dehydrogenase (DLD) results in elevated whole-body glucose levels as well as increased phosphorylation of tau. Hyperglycemia and tau phosphorylation were induced by either RNAi suppression of the dld-1 gene or by inhibition of the DLD enzyme by the inhibitor, 2-methoxyindole-2-carboxylic acid (MICA). Although the calcium ionophore A23187 could reduce tau phosphorylation induced by either chemical or genetic suppression of DLD, it was unable to reduce tau phosphorylation induced by hyperglycemia. While inhibition of the dld-1 gene or treatment with MICA partially reversed the inhibition of acetylcholine neurotransmission by tau, neither treatment affected tau inhibited mobility. Conclusively, any abnormalities in energy metabolism were found to significantly affect the AD disease pathology.

Topics: Alzheimer Disease; Animals; Caenorhabditis elegans; Calcimycin; Dihydrolipoamide Dehydrogenase; Disease Models, Animal; Glucose; Phosphorylation; tau Proteins

Ca(2+) dysregulation is an important factor implicated in Alzheimer's disease pathogenesis. The mechanisms mediating the reciprocal regulation of Ca(2+) homeostasis and amyloid precursor protein (APP) metabolism, function, and protein interactions are not well known. We have previously shown that APP interacts with Homer proteins, which inhibit APP processing toward amyloid-β. In this study, we investigated the effect of Ca(2+) homeostasis alterations on APP/Homer3 interaction. Influx of extracellular Ca(2+) upon treatment of HEK293 cells with the ionophore A23187 or addition of extracellular Ca(2+) in cells starved of calcium specifically reduced APP/Homer3 but not APP/X11a interaction. Endoplasmic reticulum Ca(2+) store depletion by thapsigargin followed by store-operated calcium entry also decreased the interaction. Interestingly, application of a phospholipase C stimulator, which causes inositol 1,4,5-trisphosphate-induced endoplasmic reticulum Ca(2+) release, caused dissociation of APP/Homer3 complex. In human neuroblastoma cells, membrane depolarization also disrupted the interaction. This is the first study showing that changes in Ca(2+) homeostasis affect APP protein interactions. Our results suggest that Ca(2+) and Homers play a significant role in the development of Alzheimer's disease pathology.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Calcimycin; Calcium; Calcium Ionophores; Carrier Proteins; Endoplasmic Reticulum; HEK293 Cells; Homer Scaffolding Proteins; Humans; Membrane Potentials; Neuroblastoma; Protein Binding; Thapsigargin; Tumor Cells, Cultured

The receptor for advanced glycation end products (RAGE) is a 55-kDa type I membrane glycoprotein of the immunoglobulin superfamily. Ligand-induced up-regulation of RAGE is involved in various pathophysiological processes, including late diabetic complications and Alzheimer disease. Application of recombinant soluble RAGE has been shown to block RAGE-mediated pathophysiological conditions. After expression of full-length RAGE in HEK cells we identified a 48-kDa soluble RAGE form (sRAGE) in the culture medium. This variant of RAGE is smaller than a 51-kDa soluble version derived from alternative splicing. The release of sRAGE can be induced by the phorbol ester PMA and the calcium ionophore calcimycin via calcium-dependent protein kinase C subtypes. Hydroxamic acid-based metalloproteinase inhibitors block the release of sRAGE, and by RNA interference experiments we identified ADAM10 and MMP9 to be involved in RAGE shedding. In protein biotinylation experiments we show that membrane-anchored full-length RAGE is the precursor of sRAGE and that sRAGE is efficiently released from the cell surface. We identified cleavage of RAGE to occur close to the cell membrane. Ectodomain shedding of RAGE simultaneously generates sRAGE and a membrane-anchored C-terminal RAGE fragment (RAGE-CTF). The amount of RAGE-CTF increases when RAGE-expressing cells are treated with a gamma-secretase inhibitor, suggesting that RAGE-CTF is normally further processed by gamma-secretase. Identification of these novel mechanisms involved in regulating the availability of cell surface-located RAGE and its soluble ectodomain may influence further research in RAGE-mediated processes in cell biology and pathophysiology.

Topics: ADAM Proteins; ADAM10 Protein; Alternative Splicing; Alzheimer Disease; Amyloid Precursor Protein Secretases; Calcimycin; Carcinogens; Cell Line; Cell Membrane; Diabetes Complications; Humans; Hydroxamic Acids; Ionophores; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Membrane Proteins; Protease Inhibitors; Protein Isoforms; Protein Structure, Tertiary; Receptor for Advanced Glycation End Products; Receptors, Immunologic; RNA, Small Interfering; Tetradecanoylphorbol Acetate

Overproduction of the amyloid beta (Abeta) peptide is a key factor in the pathogenesis of Alzheimer's disease (AD), but the mechanisms of its pathogenic effects have not been defined. Patients with AD have cerebrovascular alterations attributable to the deleterious effects of Abeta on cerebral blood vessels. We report here that NADPH oxidase, the major source of free radicals in blood vessels, is responsible for the cerebrovascular dysregulation induced by Abeta. Thus, the free-radical production and the associated alterations in vasoregulation induced by Abeta are abrogated by the NADPH oxidase peptide inhibitor gp91ds-tat and are not observed in mice lacking the catalytic subunit of NADPH oxidase (gp91phox). Furthermore, oxidative stress and cerebrovascular dysfunction do not occur in transgenic mice overexpressing the amyloid precursor protein but lacking gp91phox. The mechanisms by which NADPH oxidase-derived radicals mediate the cerebrovascular dysfunction involve reduced bioavailability of nitric oxide. Thus, a gp91phox-containing NADPH oxidase is the critical link between Abeta and cerebrovascular dysfunction, which may underlie the alteration in cerebral blood flow regulation observed in AD patients.

Topics: Acetylcholine; Adenosine; Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Bradykinin; Calcimycin; Cerebrovascular Circulation; Female; Glycoproteins; Humans; Hyperemia; Laser-Doppler Flowmetry; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Molecular Sequence Data; NADPH Oxidase 2; NADPH Oxidases; Nerve Tissue Proteins; Nitric Oxide Donors; Oxidative Stress; Peptide Fragments; Phenanthridines; Reactive Oxygen Species; S-Nitroso-N-Acetylpenicillamine; Vibrissae; X Chromosome

The E693G (Arctic) mutation of the amyloid precursor protein was recently found to lead to early-onset Alzheimer's disease in a Swedish family. In the present study, we report that the Arctic mutation decreases cell viability in human neuroblastoma cells. The cell viability, as measured by the MTT assay and propidium iodide staining, was further compromised following exposure to calcium ionophore A23187, microtubule-binding colchicine or oxidative stress inducer hydrogen peroxide. The manner of cell death was found to be apoptotic. During apoptosis, cells with the Arctic mutation also decreased their secretion of beta-secretase cleaved amyloid precursor protein. The enhanced sensitivity to toxic stress in cells with the Arctic mutation most likely contributes to the pathogenic pathway leading to Alzheimer's disease.

Topics: Alkaloids; Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Apoptosis; Blotting, Western; Calcimycin; Colchicine; Humans; Hydrogen Peroxide; Ionophores; Mutation; Neuroblastoma; Tumor Cells, Cultured

P35 or its truncated fragment p25 is required for cyclin dependent kinase (Cdk)5 activation. It has been reported that p25 is accumulated in the brain of Alzheimer's disease (AD) patients and that p25/Cdk5 induces high phosphorylation of tau and apoptosis in cultured neurons (Nature 402 (1999) 615). Our investigation of AD brain did not show specific accumulation of p25. Exposure to Ca ionophore (A23187) at 10(-6) M induced p25 accumulation in rat primary hippocampal neurons, causing neuronal death without showing hyperphosphorylation of tau. Transgenic mice expressing p25 showed the accumulation of p25 but neither hyperphosphorylation of tau nor neuronal death was shown in these mice. The feature of these mice was the progression of cell growth in pituitary gland. These results suggest that overexpression of p25 lead to the activation of cell cycle but not to the direct phosphorylation of tau.

Topics: Aged; Alzheimer Disease; Animals; Brain; Calcimycin; Cells, Cultured; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; Gene Expression Regulation; Hippocampus; Humans; Ionophores; Mice; Mice, Transgenic; Nerve Degeneration; Nerve Tissue Proteins; Neurofibrillary Tangles; Neurons; Phosphorylation; Pituitary Diseases; tau Proteins

Missense mutations in the human presenilin-1 (PS1) gene, which is found on chromosome 14, cause early-onset familial Alzheimer's disease (FAD). FAD-linked PS1 variants alter proteolytic processing of the amyloid precursor protein and cause an increase in vulnerability to apoptosis induced by various cell stresses. However, the mechanisms responsible for these phenomena are not clear. Here we report that mutations in PS1 affect the unfolded-protein response (UPR), which responds to the increased amount of unfolded proteins that accumulate in the endoplasmic reticulum (ER) under conditions that cause ER stress. PS1 mutations also lead to decreased expression of GRP78/Bip, a molecular chaperone, present in the ER, that can enable protein folding. Interestingly, GRP78 levels are reduced in the brains of Alzheimer's disease patients. The downregulation of UPR signalling by PS1 mutations is caused by disturbed function of IRE1, which is the proximal sensor of conditions in the ER lumen. Overexpression of GRP78 in neuroblastoma cells bearing PS1 mutants almost completely restores resistance to ER stress to the level of cells expressing wild-type PS1. These results show that mutations in PS1 may increase vulnerability to ER stress by altering the UPR signalling pathway.

Topics: Alzheimer Disease; Animals; Brain; Calcimycin; Carrier Proteins; Cell Death; Cell Line; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoribonucleases; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; Intracellular Membranes; Membrane Proteins; Mice; Mice, Transgenic; Molecular Chaperones; Mutation; Neuroblastoma; Neurons; Phosphorylation; Presenilin-1; Protein Binding; Protein Denaturation; Protein Folding; Protein Serine-Threonine Kinases; RNA, Messenger; Signal Transduction; Transfection; Tunicamycin

The beta-amyloid (A beta 1-40) peptide has previously been shown to enhance phenylephrine contraction of aortic rings in vitro. We have employed a novel observation, that A beta peptides enhance endothelin-1 (ET-1) contraction, to examine the relationship between vasoactivity and potential amyloidogenicity of A beta peptides, the role played by free radicals and calcium in the vasoactive mechanism, and the requirement of an intact endothelial layer for enhancement of vasoactivity. Rings of rat aortae were constricted with ET-1 before and after addition of amyloid peptide and/or other compounds, and a comparison was made between post- and pre-treatment contractions. In this system, vessel constriction is consistently dramatically enhanced by A beta 1-40, is enhanced less so by A beta 1-42, and is not enhanced by A beta 25-35. The endothelium is not required for A beta vasoactivity, and calcium channel blockers have a greater effect than antioxidants in blocking enhancement of vasoconstriction by A beta peptides. In contrast to A beta-induced cytotoxicity, A beta-induced vasoactivity is immediate, occurs in response to low doses of freshly solubilized peptide, and appears to be inversely related to the amyloidogenic potential of the A beta peptides. We conclude that the mechanism of A beta vasoactivity is distinct from that of A beta cytotoxicity. Although free radicals appear to modulate the vasoactive effects, the lack of requirement for endothelium suggests that loss of the free radical balance (between NO and O2-) may be a secondary influence on A beta enhancement of vasoconstriction. These effects of A beta on isolated vessels, and reported effects of A beta in cells of the vasculature, suggest that A beta-induced disruption of vascular tone may be a factor in the pathogenesis of cerebral amyloid angiopathy and Alzheimer's disease. Although the mechanism of enhanced vasoconstriction is unknown, it is reasonable to propose that in vivo contact of A beta peptides with small cerebral vessels may increase their tendency to constrict and oppose their tendency to relax. The subclinical ischemia resulting from this would be expected to up-regulate beta APP production in and around the vasculature with further increase in A beta formation and deposition. The disruptive and degenerative effects of such a cycle would lead to the complete destruction of cerebral vessels and consequently neuronal degeneration in the affected areas.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Aorta; Calcimycin; Calcium Channel Blockers; Calcium Channels; Calcium Channels, L-Type; Cerebral Amyloid Angiopathy; Drug Synergism; Endothelin-1; Endothelium, Vascular; Ionophores; Male; Peptide Fragments; Rats; Rats, Sprague-Dawley; Vasoconstriction; Vasoconstrictor Agents; Verapamil

Cerebral deposition of amyloid beta-protein (Abeta) as senile plaques is a pathological hallmark of Alzheimer's disease (AD). Abeta falls into two major subspecies defined by their C-termini, Abeta40 and Abeta42, ending in Val-40 and Ala-42, respectively. Although Abeta42 accounts for only approximately 10% of secreted Abeta, Abeta42 is the predominant species accumulated in senile plaques in AD brain and appears to be the initially deposited species. Its secretion level has recently been reported to be increased in the plasma or culture media of fibroblasts from patients affected by any of early-onset familial AD (FAD). Thus, inhibition of Abeta42 production would be one of the therapeutic targets for AD. However, there is little information about the cleavage mechanism via which Abeta40 and Abeta42 are generated and its relationship to intracellular protease activity. Here, we examined by well-characterized enzyme immunoassay the effects of calpain and proteasome inhibitors on the levels of Abeta40 and Abeta42 secretion by cultured cells. A calpastatin peptide homologous to the inhibitory domain of calpastatin, an endogenous calpain specific inhibitor, induced a specific increase in secreted Abeta42 relative to the total secreted Abeta level, a characteristic of the cultured cells transfected with FAD-linked mutated genes, while a proteasome specific inhibitor, lactacystin, showed no such effect. These findings suggest that the Abeta42 secretion ratio is modulated by the calpain-calpastatin system and may point to the possibility of exploring particular compounds that inhibit Abeta42 secretion through this pathway.

Topics: Acetylcysteine; Alzheimer Disease; Amyloid beta-Peptides; Calcimycin; Calcium-Binding Proteins; Calpain; Cell Line; Cysteine Proteinase Inhibitors; Dipeptides; Embryo, Mammalian; Humans; Kidney; Peptide Fragments; Transfection

The experiments in this paper identify multiple calcium compartments in cultured human fibroblasts and reveal abnormalities in one of these pools in cells from Alzheimer patients. In the presence of external calcium, bradykinin (BK) increased cytosolic free calcium ([Ca2+]i) about 3-fold and then [Ca2+]i rapidly declined. Omission of calcium from the media did not affect the BK-induced peak, which indicates that the peak reflects internal stores. Other compounds that also released calcium from internal stores included A23187 (a calcium ionophore), thapsigargin (Tg; an inhibitor of endoplasmic reticulum ATPase), and FCCP (an uncoupler of oxidative phosphorylation). The [Ca2+]i response to sequential addition of compounds in calcium-free media identified discrete internal calcium stores. BK depleted internal calcium pools such that subsequent stimulation with BK, FCCP or bombesin did not increase [Ca2+]i. However, A23187 or thapsigargin still elicited responses. A23187 depleted essentially all internal calcium pools. Either Tg or FCCP reduced the calcium stores that could be released by BK or A23187. Thus, cellular calcium compartments that respond to BK and A23187 partially overlap. The common pool includes Tg-and FCCP-sensitive compartments. Calcium stores were examined in cells from Alzheimer disease patients, because previous studies suggest that their calcium homeostasis is altered. A23187 addition to BK-treated cells produced a 95% greater response in cell lines from Alzheimer patients (n = 7) than in those from controls (n = 5). Thus, various calcium stores can be pharmacologically distinguished in fibroblasts and at least one of these compartments is abnormal in Alzheimer's disease.

Topics: Alzheimer Disease; Bradykinin; Calcimycin; Calcium; Calcium-Transporting ATPases; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Compartmentation; Cells, Cultured; Enzyme Inhibitors; Humans; Ionophores; Terpenes; Thapsigargin

Tetrahydroacridine (THA), or Cognex, is currently awaiting FDA approval for the treatment of Alzheimer's disease. In addition to reports indicating that THA improves the symptoms of patients with Alzheimer's disease, we have found that THA possesses potent antiplatelet activity. THA produced dose-dependent inhibition of human platelet aggregation induced by collagen, ADP, A23187, and phorbol ester. THA, when added to activated platelets, dispersed the platelet aggregates. We have also examined the effects of THA on intracellular Ca++ mobilization, ATP release, and production of cyclic AMP.

Topics: Adenosine Diphosphate; Aequorin; Alzheimer Disease; Blood Platelets; Calcimycin; Calcium; Collagen; Humans; Phorbol Esters; Platelet Aggregation; Platelet Aggregation Inhibitors; Tacrine