A ß-amyloid oligomer directly modulates P/Q-type calcium currents in Xenopus oocytes.

BACKGROUND AND PURPOSE: ß-amyloid (Aß) oligomers have been implicated in the early pathophysiology of Alzheimer's disease (AD). While the precise nature of the molecular target has not been fully revealed, a number of studies have indicated that Aß oligomers modulate neuron-specific ion channels. We recently provided evidence that Aß oligomers suppress isolated P/Q-type calcium currents in cultured nerve cells. Using a heterologous expression system, we aimed to prove a direct effect on the membrane channel mediating such current. EXPERIMENTAL APPROACH: The effects of a synthetically generated Aß oligomer, Aß globulomer, were investigated on P/Q-type currents recorded from Xenopus laevis oocytes expressing the full P/Q-type calcium channel or the pore-forming subunit only. We also examined the effects of Aß globulomer on recombinant NMDA receptor currents. Finally, we compared the modulation by Aß globulomer with that induced by a synthetic monomeric Aß. KEY RESULTS: Aß globulomer directly and dose-dependently modulated P/Q-type calcium channels. A leftward shift of the current-voltage curve indicated that the threshold for channel opening was reduced. The effect of Aß globulomer was also present when only the α1A subunit of the normally tripartite channel was expressed. In contrast, the monomeric Aß had no effect on P/Q current. Also globulomer Aß had no effect on glutamate-induced NMDA currents. CONCLUSIONS AND IMPLICATIONS: The α1A subunit of the P/Q-type calcium channel is directly modulated by oligomeric Aß. Threshold reduction as well as an increase in current at synaptic terminals may facilitate vesicle release and could trigger excitotoxic events in the brains of patients with AD.

Spectroscopic approaches to the conformation of tau protein in solution and in paired helical filaments.

The abnormal aggregation of the microtubule-associated protein tau into paired helical filaments is one the hallmarks of Alzheimer's disease. This aggregation is based in the partial formation of beta-structure. In contrast, the soluble protein shows a mostly random coil structure, as judged by circular dichroism, Fourier transform infrared, X-ray scattering and biochemical assays. Here, we review the basis of the natively unstructured character of tau, as well as recent studies of residual structure and long-range interactions between different domains of the protein. Analysis of the primary structure reveals a very low content of hydrophobic amino acids and a high content of charged residues, both of which tend to counteract a well-folded globular state of proteins. In the case of tau, the low overall hydrophobicity is sufficient to explain the lack of folding. This is in contrast to other proteins which also carry an excess charge at physiological pH. By tryptophan scanning mutagenesis and fluorimetry we found that most of the sequence is solvent exposed. Analysis of the hydrodynamic radii confirms a mostly random coil structure of various tau isoforms and tau domains. The proteins can be further expanded by denaturation with GdHCl which indicates some global folding. This was substantiated by a FRET-based approach where the distances between different domains of tau were determined. The combined data show that tau is mostly disordered and flexible but tends to assume a hairpin-like overall fold which may be important in the transition to a pathological aggregate.

Toward a unified scheme for the aggregation of tau into Alzheimer paired helical filaments.

Alzheimer's disease is characterized by aggregates of tau protein. Attempts to study the conditions for aggregation in vitro have led to different experimental systems, some of which appear mutually exclusive (e.g., oxidative vs reductive conditions, induction by polyanions vs fatty acids). We show here that different approaches and pathways can be viewed in a common framework, and that apparent differences can be explained by variations in the kinetics of subreactions. A unified view of PHF aggregation should help to analyze the causes of PHF aggregation and devise methods to prevent it.

Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local beta-structure.

The microtubule-associated protein tau is a natively unfolded protein in solution, yet it is able to polymerize into the ordered paired helical filaments (PHF) of Alzheimer's disease. In the splice isoforms lacking exon 10, this process is facilitated by the formation of beta-structure around the hexapeptide motif PHF6 ((306)VQIVYK(311)) encoded by exon 11. We have investigated the structural requirements for PHF polymerization in the context of adult tau isoforms containing four repeats (including exon 10). In addition to the PHF6 motif there exists a related PHF6* motif ((275)VQIINK(280)) in the repeat encoded by the alternatively spliced exon 10. We show that this PHF6* motif also promotes aggregation by the formation of beta-structure and that there is a cross-talk between the two hexapeptide motifs during PHF aggregation. We also show that two of the tau mutations found in hereditary frontotemporal dementias, DeltaK280 and P301L, have a much stronger tendency for PHF aggregation which correlates with their high propensity for beta-structure around the hexapeptide motifs.

Structure, microtubule interactions, and paired helical filament aggregation by tau mutants of frontotemporal dementias.

We have studied biochemical and structural parameters of several missense and deletion mutants of tau protein (G272V, N279K, DeltaK280, P301L, V337M, R406W) found in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). The mutant proteins were expressed on the basis of both full-length tau (htau40) and constructs derived from the repeat domain. They were analyzed with respect to the capacity to enhance microtubule assembly, binding of tau to microtubules, secondary structure content, and aggregation into Alzheimer-like paired helical or straight filaments. We find that the mutations cause a moderate decrease in microtubule interactions and stabilization, and they show no gross structural changes compared with the natively unfolded conformation of the wild-type protein, but the aggregation into PHFs is strongly enhanced, particularly for the mutants DeltaK280 and P301L. This gain of pathological aggregation would be consistent with the autosomal dominant nature of the disease.