sq-23377 and Alzheimer-Disease

CaMKIIα is a central mediator of bidirectional synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). To study how CaMKIIα movement during plasticity is affected by soluble amyloid-β peptide oligomers (Aβ), we used FingR intrabodies to simultaneously image endogenous CaMKIIα and markers for excitatory versus inhibitory synapses in live neurons. Aβ blocks LTP-stimulus-induced CaMKIIα accumulation at excitatory synapses. This block requires CaMKII activity, is dose and time dependent, and also occurs at synapses without detectable Aβ; it is specific to LTP, as CaMKIIα accumulation at inhibitory synapses during LTD is not reduced. As CaMKII movement to excitatory synapses is required for normal LTP, its impairment can mechanistically explain Aβ-induced impairment of LTP. CaMKII movement during LTP requires binding to the NMDA receptor, and Aβ induces internalization of NMDA receptors. However, surprisingly, this internalization does not cause the block in CaMKIIα movement and is observed for extrasynaptic, but not synaptic, NMDA receptors.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Disks Large Homolog 4 Protein; Female; Glutamic Acid; Hippocampus; Ionomycin; Long-Term Potentiation; Long-Term Synaptic Depression; Male; Membrane Proteins; N-Methylaspartate; Neuronal Plasticity; Protein Transport; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Synapses

Many neurodegenerative diseases present the loss of synapses as a common pathological feature. Here we have employed an in vitro model for synaptic loss to investigate the molecular mechanism of a therapeutic treatment, valproic acid (VPA). We show that amyloid-β (Aβ), isolated from patient tissue and thought to be the causative agent of Alzheimer's disease, caused the loss of synaptic proteins including synaptophysin, synapsin-1 and cysteine-string protein from cultured mouse neurons. Aβ-induced synapse damage was reduced by pre-treatment with physiologically relevant concentrations of VPA (10 μM) and a structural variant propylisopropylacetic acid (PIA). These drugs also reduced synaptic damage induced by other neurodegenerative-associated proteins α-synuclein, linked to Lewy body dementia and Parkinson's disease, and the prion-derived peptide PrP82-146. Consistent with these effects, synaptic vesicle recycling was also inhibited by these proteins and protected by VPA and PIA. We show a mechanism for this damage through aberrant activation of cytoplasmic phospholipase A2 (cPLA2) that is reduced by both drugs. Furthermore, Aβ-dependent cPLA2 activation correlates with its accumulation in lipid rafts, and is likely to be caused by elevated cholesterol (stabilising rafts) and decreased cholesterol ester levels, and this mechanism is reduced by VPA and PIA. Such observations suggest that VPA and PIA may provide protection against synaptic damage that occurs during Alzheimer's and Parkinson's and prion diseases.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Cells, Cultured; Cholesterol; Dose-Response Relationship, Drug; Embryo, Mammalian; Enzyme Inhibitors; HSP40 Heat-Shock Proteins; Humans; Ionomycin; Membrane Microdomains; Membrane Proteins; Mice; Peptide Fragments; Phospholipases A2; Prions; Signal Transduction; Synapses; Synaptophysin; Valproic Acid; Vesicle-Associated Membrane Protein 1

Down syndrome (DS), the most common genetic disorder, is caused by trisomy 21. DS is accompanied by heart defects, hearing and vision problems, obesity, leukemia, and other conditions, including Alzheimer's disease (AD). In comparison, most cancers are rare in people with DS. Overexpression of dual specificity tyrosine-phosphorylation-regulated kinase 1A and a regulator of calcineurin 1 located on chromosome 21 leads to excessive suppression of the calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway, resulting in reduced expression of a critical angiogenic factor. However, it is unclear whether the calcineurin-NFAT signaling pathway is involved in AD pathology in DS patients. Here, we investigated the association between the calcineurin-NFAT signaling pathway and AD using neuronal cells. Short-term pharmacological stimulation decreased gene expression of tau and neprilysin, and long-term inhibition of the signaling pathway decreased that of amyloid precursor protein. Moreover, a calcineurin inhibitor, cyclosporine A, also decreased neprilysin activity, leading to increases in amyloid-β peptide levels. Taken together, our results suggest that a dysregulation in calcineurin-NFAT signaling may contribute to the early onset of AD in people with DS.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Calcineurin; Calcineurin Inhibitors; Calcium Ionophores; Cell Line, Tumor; Cyclosporine; DNA-Binding Proteins; Down Syndrome; Humans; Intracellular Signaling Peptides and Proteins; Ionomycin; Luciferases; Muscle Proteins; Neprilysin; NFATC Transcription Factors; RNA, Messenger; Signal Transduction; tau Proteins; Tetradecanoylphorbol Acetate

Familial Alzheimer disease (FAD) is linked to mutations in the presenilin (PS) homologs. FAD mutant PS expression has several cellular consequences, including exaggerated intracellular Ca(2+) ([Ca(2+)](i)) signaling due to enhanced agonist sensitivity and increased magnitude of [Ca(2+)](i) signals. The mechanisms underlying these phenomena remain controversial. It has been proposed that PSs are constitutively active, passive endoplasmic reticulum (ER) Ca(2+) leak channels and that FAD PS mutations disrupt this function resulting in ER store overfilling that increases the driving force for release upon ER Ca(2+) release channel opening. To investigate this hypothesis, we employed multiple Ca(2+) imaging protocols and indicators to directly measure ER Ca(2+) dynamics in several cell systems. However, we did not observe consistent evidence that PSs act as ER Ca(2+) leak channels. Nevertheless, we confirmed observations made using indirect measurements employed in previous reports that proposed this hypothesis. Specifically, cells lacking PS or expressing a FAD-linked PS mutation displayed increased area under the ionomycin-induced [Ca(2+)](i) versus time curve (AI) compared with cells expressing WT PS. However, an ER-targeted Ca(2+) indicator revealed that this did not reflect overloaded ER stores. Monensin pretreatment selectively attenuated the AI in cells lacking PS or expressing a FAD PS allele. These findings contradict the hypothesis that PSs form ER Ca(2+) leak channels and highlight the need to use ER-targeted Ca(2+) indicators when studying ER Ca(2+) dynamics.

Topics: Alleles; Alzheimer Disease; Animals; Calcium; Calcium Channels; Calcium Signaling; Cell Line; Endoplasmic Reticulum; Fibroblasts; Gene Knockdown Techniques; Inositol 1,4,5-Trisphosphate Receptors; Ionomycin; Kinetics; Mice; Molecular Imaging; Mutation; Neurons; Presenilin-1; Presenilin-2; Ryanodine Receptor Calcium Release Channel; Up-Regulation

To investigate whether the observed association of intracellular neuronal calcium sensor (NCS) proteins with amyloid plaques and neurofibrillar tangles in Alzheimer brains is linked to a possible neuroprotective or neurotoxic activity of the protein, we performed cytotoxicity tests in PC12 cells transfected with the calcium sensor protein VILIP-1 (visinin-like protein) and the calcium buffer protein calbindin-D28K. Whereas VILIP-1 expression enhanced the neurotoxic effect of ionomycin already at low ionophore concentrations, calbindin-D28K protected against ionomycin-induced cytotoxicity only at high ionomycin and therefore calcium concentrations. However, in double-transfected cells calbindin-D28K rescued VILIP-1-mediated cytotoxicity at low ionomycin concentrations. Since VILIP-1 was found to be associated with fibrillar tangles in Alzheimer brains, we tested whether VILIP-1 has an influence on tau hyperphosphorylation. VILIP-1 expression enhanced hyperphosphorylation of tau protein compared to nontransfected or calbindin-D28K-transfected cells. These results raise the possibility that the observed reduction in VILIP-1-expressing cells may indicate a selective vulnerability of these neurons and that the calcium sensor protein is involved in the pathophysiology of Alzheimer's disease. The calcium sensor protein may influence tau phosphorylation and have a role in calcium-mediated neurotoxicity opposed to the previously discovered protective effect of calcium buffer proteins.

Topics: Alzheimer Disease; Animals; Brain; Brain Chemistry; Calbindin 1; Calbindins; Calcium; Calcium Signaling; Calcium-Binding Proteins; Humans; Ionomycin; Ionophores; Nerve Tissue Proteins; Neurocalcin; Neurofibrillary Tangles; PC12 Cells; Phosphorylation; Plaque, Amyloid; Protein Processing, Post-Translational; Rabbits; Rats; Receptors, Calcium-Sensing; Recombinant Fusion Proteins; S100 Calcium Binding Protein G; tau Proteins; Transfection

The majority of familial Alzheimer's disease (AD) cases are linked to mutations on presenilin 1 and 2 genes (PS1 and PS2). The normal function of the proteins and the mechanisms underlying early-onset AD are currently unknown. To address this, we screened an expression library for proteins that bind differentially to the wild-type PS1 and mutant in the large cytoplasmic loop (PS1L). Thus we isolated the C-terminal tail of the 170 kDa cytoplasmic linker protein (CLIP-170) and Reed-Sternberg cells of Hodgkin's disease-expressed intermediate filament-associated protein (Restin), cytoplasmic proteins linking vesicles to the cytoskeleton. PS1L binding to CLIP-170/restin requires Ca(2+). Treating cells with thapsigargin or ionomycin increased the mutated PS1 in CLIP-170 immunoprecipitates. Further, PS1 and CLIP-170 co-localize in transfected cells and neuronal cultures.

Topics: Alzheimer Disease; Conserved Sequence; Cytoskeleton; Humans; Intermediate Filament Proteins; Ionomycin; Membrane Proteins; Microtubule-Associated Proteins; Mutation; Neoplasm Proteins; Peptide Library; Precipitin Tests; Presenilin-1; Reed-Sternberg Cells; Thapsigargin

Cyclin-dependent kinase 5 (cdk5) and its neuron-specific activator p35 are required for neurite outgrowth and cortical lamination. Proteolytic cleavage of p35 produces p25, which accumulates in the brains of patients with Alzheimer's disease. Conversion of p35 to p25 causes prolonged activation and mislocalization of cdk5. Consequently, the p25/cdk5 kinase hyperphosphorylates tau, disrupts the cytoskeleton and promotes the death (apoptosis) of primary neurons. Here we describe the mechanism of conversion of p35 to p25. In cultured primary cortical neurons, excitotoxins, hypoxic stress and calcium influx induce the production of p25. In fresh brain lysates, addition of calcium can stimulate cleavage of p35 to p25. Specific inhibitors of calpain, a calcium-dependent cysteine protease, effectively inhibit the calcium-induced cleavage of p35. In vitro, calpain directly cleaves p35 to release a fragment with relative molecular mass 25,000. The sequence of the calpain cleavage product corresponds precisely to that of p25. Application of the amyloid beta-peptide A beta(1-42) induces the conversion of p35 to p25 in primary cortical neurons. Furthermore, inhibition of cdk5 or calpain activity reduces cell death in A beta-treated cortical neurons. These observations indicate that cleavage of p35 to p25 by calpain may be involved in the pathogenesis of Alzheimer's disease.

Topics: Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Peptides; Animals; Calcium; Calpain; Cells, Cultured; Egtazic Acid; Glutamic Acid; Hydrogen Peroxide; Ionomycin; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Nerve Tissue Proteins; Neurons; Peptide Fragments; Rats; Rats, Long-Evans; Rats, Sprague-Dawley; Recombinant Proteins

Abnormalities involving intracellular calcium homeostasis have been detected in Alzheimer's disease brain and fibroblasts as well as presenilin-1 mutation-bearing cells. In the present study we investigated inositol(1,4,5)trisphosphate-mediated calcium transients as well as calcium responses via mechanisms not related to surface receptors in Alzheimer's disease polymorphonuclear (PMN) granulocytes, using the tripeptide formyl-methionyl-leucyl-phenyl alanine (fMLP) and calcium ionophore ionomycin, respectively. fMLP elicited a biphasic response with an initial, fast increase in intracellular free calcium concentrations followed by a second, lower phase with no significant differences in either maximal response or time course between Alzheimer's disease granulocytes and controls. Similarly, the calcium signal elicited after ionomycin exposure was unchanged in Alzheimer's disease PMN. In conclusion, these results indicate that calcium mobilization from intracellular stores and via cross-membrane mechanisms is intact in Alzheimer's disease granulocytes.

Topics: Aged; Alzheimer Disease; Calcium; Female; Humans; Intracellular Membranes; Ionomycin; Ionophores; Male; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Osmolar Concentration; Reference Values; Time Factors

Reports of abnormalities of potassium-channel function in various cultured cells of Alzheimer's disease patients led us to attempt to characterise the pharmacological characteristics of the abnormal channel.. We studied platelets from 14 patients with Alzheimer-type dementia and 14 non-demented controls matched for age and sex. The effects of specific inhibitors of K+ channels on the efflux of rubidium-86 ions, a radioactive analogue of K+, from the platelets were measured.. Normal platelets contain three types of K+ channel, sensitive to the inhibitory actions of apamin (small-conductance calcium-dependent potassium channels), charybdotoxin (of less specificity, but probably intermediate-conductance calcium-dependent K+ channels), and alpha-dendrotoxin (voltage-sensitive K+ channels). However, 8Rb+ efflux from the platelets of patients with Alzheimer-type dementia was not inhibited by either apamin or charybdotoxin. By contrast, inhibition by alpha-dendrotoxin did occur.. Our results suggest that calcium-dependent K+ channels in platelets are selectively impaired in Alzheimer's disease. A similar abnormality in neurons could contribute to the pathophysiology of the disorder.

Topics: Aged; Alzheimer Disease; Analysis of Variance; Apamin; Case-Control Studies; Cells, Cultured; Charybdotoxin; Drug Interactions; Elapid Venoms; Female; Hemostatics; Humans; Ionomycin; Ionophores; Male; Middle Aged; Potassium Channel Blockers; Potassium Channels; Rubidium; Thrombin