Amyloid fibrillization is multistep process involving soluble oligomeric intermediates, including spherical oligomers and protofibrils. Amyloid oligomers have a common, generic structure, and they are intrinsically toxic to cells, even when formed from non-disease related proteins, which implies they also share a common mechanism of pathogenesis and toxicity. Here we report that soluble oligomers from several types of amyloids specifically increase lipid bilayer conductance regardless of the sequence, while fibrils and soluble low molecular weight species have no effect. The increase in membrane conductance occurs without any evidence of discrete channel or pore formation or ion selectivity. The conductance is dependent on the concentration of oligomers and can be reversed by anti-oligomer antibody. These results indicate that soluble oligomers from many types of amyloidogenic proteins and peptides increase membrane conductance in a conformation-specific fashion and suggest that this may represent the common primary mechanism of pathogenesis in amyloid-related degenerative diseases.Soluble amyloid oligomers are a common intermediate in the pathway for amyloid fibril formation and have been implicated as the primary toxic species of amyloids related to neurodegenerative disease (1-6). More recent reports indicate that soluble amyloid oligomers are intrinsically toxic even when they are formed from proteins that are not normally related to degenerative disease (3), and the toxic activity of soluble oligomers may be related to a common generic structure that they share (6). Although the primary mechanism of amyloid toxicity is not clear, the fact that different amyloids reside in either the cytosolic or extracellular compartments and the observation that cytosolic amyloid aggregates are toxic when applied externally to cells (6, 7) points to the cell plasma membrane as a potential primary target of amyloid pathogenesis. Indeed, there are many reports of membrane perturbations caused by amyloids like A␤ (8), but it isn't clear whether these effects are specific to soluble oligomers nor whether they are common to other types of amyloids. Here we report that homogeneous populations of spherical amyloid oligomers and protofibrils increase the conductivity of membranes by a non-channel mechanism. This effect is observed for all soluble oligomers tested regardless of protein sequence and is not observed for amyloid fibrils or soluble low molecular weight species, suggesting that the increase in membrane conductivity may be a primary common mechanism of amyloid oligomer pathogenesis. MATERIALS AND METHODSPeptide Synthesis-Peptide synthesis: A␤ peptides, prion 106 -126, and IAPP 1 were synthesized by fluoren-9-ylmethoxy carbonyl chemistry using a continuous flow semiautomatic instrument as described previously (9). The purity was checked by analytical reverse phase-high performance liquid chromatography and by electrospray mass spectrometry. Polyglutamine KKQ40KK was a gift from Dr. Ronald Wetzel, and ␣-synuclein was a gif...

Human processed lipoaspirate (PLA) cells are multipotent stem cells, capable of differentiating into multiple mesenchymal lineages (bone, cartilage, fat, and muscle). To date, differentiation to nonmesodermal fates has not been reported. This study demonstrates that PLA cells can be induced to differentiate into early neural progenitors, which are of an ectodermal origin. Undifferentiated cultures of human PLA cells expressed markers characteristic of neural cells such as neuron-specific enolase (NSE), vimentin, and neuron-specific nuclear protein (NeuN). After 2 weeks of treatment of PLA cells with isobutylmethylxanthine, indomethacin, and insulin, about 20 to 25 percent of the cells differentiated into cells with typical neural morphologic characteristics, accompanied by increased expression of NSE, vimentin, and the nerve-growth factor receptor trk-A. However, induced PLA cells did not express the mature neuronal marker, MAP, or the mature astrocyte marker, GFAP. It was also found that neurally induced PLA cells displayed a delayed-rectifier type K+ current (an early developmental ion channel) concomitantly with morphologic changes and increased expression of neural-specific markers. The authors concluded that human PLA cells might have the potential to differentiate in vitro into cells that represent early progenitors of neurons and/or glia.

In Aplysia sensory and motor neurons in culture, the contributions of the major classes of calcium current can be selectively examined while transmitter release and its modulation are examined. A slowly inactivating, dihydropyridine-sensitive calcium current does not contribute either to normal synaptic transmission or to any of three different forms of plasticity: presynaptic inhibition, homosynaptic depression, and presynaptic facilitation. This current does contribute, however, to a fourth form of plasticity--modulation of transmitter release by tonic depolarization of the sensory neuron. By contrast, a second calcium current, which is rapidly inactivating and dihydropyridine-insensitive, contributes to release elicited by the transient depolarization of an action potential and to the other three forms of plasticity.

To determine whether the concentrations of calcium-binding proteins present in some neurons and sensory cells are sufficient to influence presynaptic calcium signaling, we studied the predominant calcium-binding protein in a class of sensory hair cells in the frog ear. Based on antibody affinity and molecular weight, we identified this protein as calretinin. We measured its cytoplasmic concentration to be approximately 1.2 mM, sufficient to bind approximately 6 mM Ca2+. Calcium signaling was altered when the diffusible cytoplasmic components were replaced by an intracellular solution lacking any fast calcium buffer, and was restored by the addition of 1.2 mM exogenous calretinin to the intracellular solution. We conclude that calretinin, when present at millimolar concentration, can serve as a diffusionally mobile calcium buffer/transporter capable of regulating calcium signaling over nanometer distances at presynaptic sites.

1. Spontaneous and evoked non-NMDA receptor-mediated EPSCs were recorded from cerebellar granule cells in slices at -24 and -34 'C. The EPSC decay was fitted with the sum of two exponential functions. 2. The time courses of non-NMDA receptor deactivation and desensitization were determined with fast concentration jumps of glutamate onto patches from cultured granule cells. Deactivation (decay time constant r = 0 6 ms at 24°C) was substantially faster than desensitization ('r = 4 ms). Both processes were fitted by single exponential functions.3. The decay of the fast component of the spontaneous EPSC (TEPSCfast = 09 ms at 23°C) was marginally slower than deactivation but too fast to be determined by desensitization. Our results suggest that the decay of this component is set by both the rate of decline of transmitter concentration and channel deactivation. The time course of excitatory postsynaptic currents (EPSCs) is determined by the time course of the concentration of transmitter in the synaptic cleft and the kinetic properties of the postsynaptic receptors. If the concentration of transmitter in the cleft declines slowly, relative to the EPSC, both the time course of transmitter decay and the rate of receptor desensitization would be expected to shape the EPSC (Trussell, Zhang & Raman, 1993;Barbour, Keller, Llano & Marty, 1994). In contrast, if the decay of transmitter is relatively fast, the EPSC decay would be determined predominantly by the rate of deactivation (defined here as the rate at which current declines after a jump to zero agonist concentration) (Magleby & Stevens, 1972;Katz & Miledi, 1973;Colquhoun, Jonas & Sakmann, 1992

KEY WORDS: NMDA receptors, synaptic currents 495 0066-4278/9510315-0495$05.00 Annu. Rev. Physiol. 1995.57:495-519. Downloaded from www.annualreviews.org Access provided by University of California -San Diego on 02/03/15. For personal use only. Quick links to online content Further ANNUAL REVIEWS Annu. Rev. Physiol. 1995.57:495-519. Downloaded from www.annualreviews.org Access provided by University of California -San Diego on 02/03/15. For personal use only. Annu. Rev. Physiol. 1995.57:495-519. Downloaded from www.annualreviews.org Access provided by University of California -San Diego on 02/03/15. For personal use only. Annu. Rev. Physiol. 1995.57:495-519. Downloaded from www.annualreviews.org Access provided by University of California -San Diego on 02/03/15. For personal use only.

The prevailing model of neurotransmitter release stipulates that Ca 2+ influx triggers the rapid fusion of vesicles that are docked at presynaptic active zones. Under this model, slower tonic release is supported by vesicles clustered nearby that have to translocate to the release sites before fusion. We have examined this hypothesis at the afferent synapse of saccular hair cells of the leopard frog, Rana pipiens. Detailed morphological measurements at this ribbon synapse show that on average 32 vesicles are docked at each active zone. We show that at this 'graded' synapse, depolarization produces an exocytotic 'burst' that is largely complete within 20 ms after fusion of 280 vesicles per active zone, almost an order of magnitude more than expected. Recovery from paired pulse depression occurs with a time constant of 29 ms, indicating that replenishment of this fast-fusing pool of vesicles is also fast. Our results suggest that non-docked vesicles are capable of fast fusion and that these vesicles constitute the vast majority of the fast-fusing pool. The view that the population of fast-fusing presynaptic vesicles is limited to docked vesicles therefore requires re-evaluation. We propose that compound fusion, i.e. the fusion of vesicles with each other before and/or after they fuse with the membrane can explain multivesicular release at this synapse.

Desformylflustrabromine (dFBr; 1) and desformylflustrabromine-B (dFBr-B; 2) have been previously isolated from natural sources, and the former has been demonstrated to be a novel and selective positive allosteric modulator of alpha4beta2 nicotinic acetylcholine (nACh) receptors. The present study describes the synthesis of water-soluble salts of 1 and 2, and confirms and further investigates the actions of 1 and 2 using two-electrode voltage clamp recordings.