Amyloid beta protein (AbetaP) is the major constituent of senile plaques associated with Alzheimer's disease (AD). However, its mechanistic role in AD pathogenesis is poorly understood. Globular and nonfibrillar AbetaPs are continuously released during normal metabolism. Using techniques of atomic force microscopy, laser confocal microscopy, electrical recording, and biochemical assays, we have examined the molecular conformations of reconstituted globular AbetaPs as well as their real-time and acute effects on neuritic degeneration. Atomic force microscopy (AFM) of AbetaP1-42 shows globular structures that do not form fibers in physiological-buffered solution for up to 8 h of continuous imaging. AFM of AbetaP1-42 reconstituted in a planar lipid bilayer reveals multimeric channel-like structures. Consistent with these AFM resolved channel-like structures, biochemical analysis demonstrates that predominantly monomeric AbetaPs in solution form stable tetramers and hexamers after incorporation into lipid membranes. Electrophysiological recordings demonstrate the presence of multiple single channel currents of different sizes. At the cellular level, AbetaP1-42 allows calcium uptake and induces neuritic abnormality in a dose- and time-dependent fashion. At physiological nanomolar concentrations, rapid neuritic degeneration was observed within minutes; at micromolar concentrations, neuronal death was observed within 3-4 h. These effects are prevented by zinc (an AbetaP channel blocker) and by the removal of extracellular calcium, but are not prevented by antagonists of putative AbetaP cell surface receptors. Thus, AbetaP channels may provide a direct pathway for calcium-dependent AbetaP toxicity in AD.

A pathological hallmark in brain tissue from patients with Alzheimer's disease (AD) 1 is the accumulation of amyloid ␤ protein (A␤P), a 39 -43-amino acid-long polypeptide, as morphologically heterogeneous neuritic plaques and cerebrovascular deposits (1, 2). A␤P is derived primarily from a proteolytic cleavage of the ␤-amyloid precursor protein (A␤PP), a highly conserved and widely expressed integral membrane protein with a single membrane-spanning polypeptide. The amount and the nature of polypeptides vary considerably among various forms of ADs: A␤P 1-40 and A␤P 1-42 are differentially accumulated in sporadic Alzheimer's disease and non-demented brain samples (3) and a mutation in presenilins is linked with an increased ratio of A␤P 1-42 /A␤P 1-40 in familial Alzheimer's disease (4 -7). The early-onset familial AD has been correlated with an increased level of A␤P 1-42 . However, very little is known about the role of A␤P 1-42 in such pathology and about the mechanism(s) of its action.Accumulating evidence suggests an early and causative role of A␤Ps in the pathogenic cascade (8 -11). Postulated mechanisms of A␤P toxicity include, by its interaction with the tachykinin neuropeptide system, a surface membrane effect (12); by changing cellular ionic concentration via formation of plasma membrane channels (13-15); and by activating oxidative pathways and making cells more responsive to oxidative stress (for review see Refs. 16 and 17). Reactive oxygen species and the antioxidant defenses work probably by altering the lipid peroxidation and membrane composition. However, A␤P polypeptides associated with the reactive oxygen hypothesis have produced conflicting effects on cytoskeletal organization and cell lysis (18 -23).The commonly observed change in the cellular ion concentration involves increased calcium level (24 -26) either indirectly via modulating the existing Ca 2ϩ channel or directly via cation-selective channels formed by A␤Ps. Support for the cation-selective A␤P channels are accumulating. Arispe and his collaborators (13-15, 27) have reported cation-selective channels formed by A␤P 1-40 when reconstituted into lipid bilayers and in the membrane patches excised from hypothalamic gonadotropin-releasing hormone neurons. Kagan and his collaborators (28) have also recorded channel-like activity when A␤P [25][26][27][28][29][30][31][32][33][34][35] was reconstituted in lipid bilayers as well as for both A␤P 1-40 and A␤P 1-42 reconstituted in lipid bilayer, 2 though, with less reliability and reproducibility than the A␤P 25-35 current (28). Whether A␤P 1-42 toxicity is also mediated via A␤P 1-42 forming calcium-permeable ion channel is unclear.The molecular structure of A␤P oligomers, especially as an ion channel, is unknown. Durell et al. (29) have developed theoretical models for the structure of ion channel formed by the membrane-bound A␤P 1-40 . However, no direct structural data from EM, NMR, x-ray diffraction, or other microscopic techniques are available to support the presence of the A␤P channel.We h...

Amyloid beta peptides (AbetaP) deposit as plaques in vascular and parenchymal areas of Alzheimer's disease (AD) tissues and Down's syndrome patients. Although neuronal toxicity is a feature of late stages of AD, vascular pathology appears to be a feature of all stages of AD. Globular and nonfibrillar AbetaPs are continuously released during normal cellular metabolism, form calcium-permeable channels, and alter cellular calcium level. We used atomic force microscopy, laser confocal microscopy, and calcium imaging to examine the real-time and acute effects of fresh and globular AbetaP(1-42), AbetaP(1-40), and AbetaP(25-35) on cultured endothelial cells. AbetaPs induced morphological changes that were observed within minutes after AbetaP treatment and led to eventual cellular degeneration. Cellular morphological changes were most sensitive to AbetaP(1-42). AbetaP(1-42)-induced morphological changes were observed at nanomolar concentrations and were accompanied by an elevated cellular calcium level. Morphological changes were prevented by anti-AbetaP antibody, AbetaP-channel antagonist zinc, and the removal of extracellular calcium, but not by tachykinin neuropeptide, voltage-sensitive calcium channel blocker cadmium, or antioxidants DTT and Trolox. Thus, nanomolar fresh and globular AbetaP(1-42) induces rapid cellular degeneration by elevating intracellular calcium, most likely via calcium-permeable AbetaP channels and not by its interaction with membrane receptors or by activating oxidative pathways. Such rapid degeneration also suggests that the plaques, and especially fibrillar AbetaPs, may not have a direct causative role in AD pathogenic cascades.

Amyloid beta protein (A beta P) forms senile plaques in the cerebrocortical blood vessels and brain parenchyma of patients with Alzheimer's disease (AD). The nonfamilial or sporadic AD (SAD), the most prevalent form of AD, has been correlated with an increased level of 40-residue A beta P (A beta P1-40). However, very little is known about the role of A beta P1-40 in AD pathophysiology. We have examined the activity of A beta P1-40 reconstituted in phospholipid vesicles. A combined light fluorescence and atomic force microscope (AFM) was used to image the structure of reconstituted vesicles and 45Ca2+ uptake was used as an assay for calcium permeability across the vesicular membrane. Vesicles reconstituted with fresh and globular A beta P1-40 contain a significant amount of A0 beta P and exhibit strong immunofluorescence labeling with an antibody raised against the N-terminal domain of A beta P, suggesting the incorporation of A beta P1-40 peptide in the vesicular membrane. Vesicles reconstituted with A beta P1-40 exhibited a significant level of 45Ca2+ uptake. The vesicular calcium level saturated over time, showing an important ion channel characteristic. The 45Ca2+ uptake was inhibited by (i) a monoclonal antibody raised against the N-terminal region of A beta P and (ii) Zn2+. However, a reducing agent (DTT) did not inhibit the 45Ca2+ uptake, indicating that the oxidation of A beta P or its surrounding lipid molecules is not directly involved in A beta P-mediated Ca2+ uptake. These findings provide biochemical and structural evidence that fresh and globular A beta P1-40 forms calcium-permeable channels and thus may induce cellular toxicity by regulating the calcium homeostasis in nonfamilial or sporadic Alzheimer's disease.

The newly developed atomic force microscope (AFM) provides a unique window to the microworld of cells, subcellular structures, and biomolecules. The AFM can image the three-dimensional structure of biological specimens in a physiological environment. This enables real-time biochemical and physiological processes to be monitored at a resolution similar to that obtained for the electron microscope. The process of image acquisition is such that the AFM can also measure forces at the molecular level. In addition, the AFM can interact with the sample, thereby manipulating the molecules in a defined manner--nanomanipulation! The AFM has been used to image living cells and the underlying cytoskeleton, chromatin and plasmids, ion channels, and a variety of membranes. Dynamic processes such as crystal growth and the polymerization of fibrinogen and physicochemical properties such as elasticity and viscosity in living cells have been studied. Nanomanipulations, including dissection of DNA, plasma membranes, and cells, and transfer of synthetic structures have been achieved. This review describes the operating principles, accomplishments, and the future promise of the AFM.