Increasing evidence suggests that amyloid peptides associated with a variety of degenerative diseases induce neurotoxicity in their intermediate oligomeric state, rather than as monomers or fibrils. To test this hypothesis and investigate the possible involvement of Ca 2؉ signaling disruptions in amyloid-induced cytotoxicity, we made homogeneous preparations of diseaserelated amyloids (A␤, prion, islet amyloid polypeptide, polyglutamine, and lysozyme) in various aggregation states and tested their actions on fluo-3-loaded SH-SY5Y cells. Application of oligomeric forms of all amyloids tested (0.6 -6 g ml ؊1 ) rapidly (ϳ5 s) elevated intracellular Ca 2؉ , whereas equivalent amounts of monomers and fibrils did not. Ca 2؉ signals evoked by A␤42 oligomers persisted after depletion of intracellular Ca 2؉ stores, and small signals remained in Ca 2؉ -free medium, indicating contributions from both extracellular and intracellular Ca 2؉ sources. The increased membrane permeability to Ca 2؉ cannot be attributed to activation of endogenous Ca 2؉ channels, because responses were unaffected by the potent Ca 2؉ -channel blocker cobalt (20 m). Instead, observations that A␤42 and other oligomers caused rapid cellular leakage of anionic fluorescent dyes point to a generalized increase in membrane permeability. The resulting unregulated flux of ions and molecules may provide a common mechanism for oligomer-mediated toxicity in many amyloidogenic diseases, with dysregulation of Ca 2؉ ions playing a crucial role because of their strong trans-membrane concentration gradient and involvement in cell dysfunction and death.Alzheimer disease (AD) 1 is characterized by the appearance in the brain of plaques, containing extracellular deposits of amyloid ␤-peptide (A␤) that result from altered proteolytic processing of amyloid precursor protein, together with intracellular neurofibrillary tangles containing misfolded tau (1).Brain regions with plaques and tangles exhibit reduced numbers of synapses, and neurites associated with plaques and tangles are often damaged, suggesting a pivotal role for A␤ in the neuropathology of AD (2-5). Moreover, numerous other neurodegenerative disorders (including Huntington, Parkinson, and prion diseases) are also associated with the formation and accumulation of amyloid fibrils in specific brain areas (6, 7). These commonalities suggest a general mechanism of action for the more than 100 human amyloid-related diseases, whereby normally soluble peptides and proteins undergo aberrant folding (8).Aggregation of A␤ proceeds through several conformational states, including dimers, spherical oligomers composed of 10 -24 monomers, and strings of oligomers (protofibrils), before finally assuming an insoluble fibrillar conformation (9). The initial formulation of the "amyloid hypothesis" of AD specifically implicated fibrillar amyloid deposits (10). However, more recent evidence suggests that soluble oligomers may be the principal neurotoxic agent (11-15). Soluble A␤ oligomers are found in the cerebrospinal fluid of ...

Ca2+ release-activated Ca2+ (CRAC) channels underlie sustained Ca2+ signaling in lymphocytes and numerous other cells following Ca2+ liberation from the endoplasmic reticulum (ER). RNAi screening approaches identified two proteins, Stim1, 2 and Orai3-5, that together form the molecular basis for CRAC channel activity6, 7. Stim senses depletion of the ER Ca2+ store and physically relays this information by translocating from the ER to junctions adjacent to the plasma membrane (PM)1, 8, 9, and Orai embodies the pore of the PM calcium channel10-12. A close interaction between Stim and Orai, identified by co-immunoprecipitation12 and by Förster resonance energy transfer13, is involved in opening the Ca2+ channel formed by Orai subunits. Most ion channels are multimers of poreforming subunits surrounding a central channel, which are preassembled in the ER and transported in their final stoichiometry to the PM. Here we show by biochemical analysis after cross-linking in cell lysates and in intact cells, and by non-denaturing gel electrophoresis without cross-linking that Orai is predominantly a dimer in the PM under resting conditions. Moreover, single-molecule imaging of GFP-tagged Orai expressed in Xenopus oocytes revealed predominantly two-step photo-bleaching, consistent again with a dimeric basal state. In contrast, co-expression of GFP-tagged Orai with the C-terminus of Stim as a cytosolic protein to activate the Orai channel without inducing Ca2+ store depletion or clustering of Orai into punctae yielded predominantly four-step photobleaching, consistent with a tetrameric stoichiometry of the active Orai channel. Interaction with the C-terminus of Stim thus induces Orai dimers to dimerize, forming a tetramer that constitutes the Ca2+-selective pore. This represents a novel mechanism in which assembly and activation of the functional ion channel are mediated by the same triggering molecule.

No abstract

High-resolution imaging of calcium influx reveals that the Aβ peptides implicated in Alzheimer’s disease form highly toxic Ca2+-permeable pores.

Oligomeric forms of Aβ peptides associated with Alzheimer`s disease (AD) disrupt cellular Ca2+ regulation by liberating Ca2+ into the cytosol from both extracellular and intracellular sources. We elucidated the actions of intracellular Aβ42 by imaging Ca2+ responses to injections of Aβ oligomers into Xenopus oocytes. Two types of signal were observed: (i) local, 'channel-like' transients dependent on extracellular Ca2+ influx, which resembled signals from amlyoid pores formed by extracellular application of oligomers; (ii) local transients and global Ca2+ waves, resembling Ca2+ puffs and waves mediated by inositol trisphosphate (IP3). The latter responses were suppressed by antagonists of the IP3 receptor (caffeine and heparin), by pretreatment with the Gi/o-protein inhibitor pertussis toxin, and by pre-treatment with lithium to deplete membrane inositol lipids. We show that G-protein-mediated stimulation of IP3 production and consequent liberation of Ca2+ from the endoplasmic reticulum by intracellular Aβ oligomers is cytotoxic, potentially representing a novel pathological mechanism in AD which may be further exacerbated by AD-linked mutations in presenilins to promote opening of IP3 receptor/channels.