FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Erratum (v16,p759)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by LIN, H.
Right arrow Articles by LAL, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by LIN, H.
Right arrow Articles by LAL, R.
(The FASEB Journal. 2001;15:2433-2444.)
© 2001 FASEB

Amyloid ß protein forms ion channels: implications for Alzheimer’s disease pathophysiology

HAI LIN1, RAJINDER BHATIA1 and RATNESHWAR LAL2

Neuroscience Research Institute, University of California, Santa Barbara, California 93016, USA

2Correspondence: Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, USA. E-mail: lal{at}lifesci.ucsb.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Amyloid ß protein (AßP) 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 AßPs 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 AßPs as well as their real-time and acute effects on neuritic degeneration. Atomic force microscopy (AFM) of AßP1–42 shows globular structures that do not form fibers in physiological-buffered solution for up to 8 h of continuous imaging. AFM of AßP1–42 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 AßPs 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, AßP1–42 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 AßP channel blocker) and by the removal of extracellular calcium, but are not prevented by antagonists of putative AßP cell surface receptors. Thus, AßP channels may provide a direct pathway for calcium-dependent AßP toxicity in AD.—Lin, H., Bhatia, R., Lal, R. Amyloid ß-protein forms ion channels: implications for Alzheimer’s disease pathophysiology.


Key Words: atomic force microscope • AßP • calcium uptake • AD


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Alzheimer’s disease (AD) is characterized pathologically by the presence of extracellular senile amyloid plaques and intracellular neurofibrillary tangles. The major component of AD senile plaques is a 39–43 residue peptide, the amyloid ß protein (AßP), which is the product of proteolytic processing of widely distributed ß-amyloid precursor protein (ßAPP) (1) . Clinical impairment in AD correlates with an early synaptic dysfunction, followed by more severe neuronal changes that include increased synaptic loss, widespread neuritic dystrophy, neurofibrillary tangles, and neuronal death (2 , 3) . Pathological and genetic analyses have shown a causal relationship for AßP and the AD pathogenesis, but the underlying mechanism is not well understood. Due to the presence of AßP fibrils in senile amyloid plaques and a correlation between the in vitro degenerative effect of AßP and the age and fibrillar morphology of AßP, formation of AßP fibrils has been proposed to be a prerequisite for AßP toxicity (4) .

On the other hand, globular and nonfibrillar AßPs are continuously released during normal cellular metabolism and are also present in AD tissues. Moreover, small nonfibrillar AßP alone is sufficient to cause cellular degeneration (5 , 6) and fibrillar AßPs may not induce toxicity (7) . One of the proposed mechanisms for AßP toxicity suggests that nonfibrillar AßPs form calcium-permeable ion channels in the cell plasma membrane. These channels would allow excessive calcium influx and disrupt the normal cellular calcium homeostasis. Consistent with this possibility, amyloid beta peptides of various lengths, including AßP25–35, AßP1–40, and AßP1–42, elicit cation-selective currents when reconstituted into lipid bilayers (8 9 10 11) as well as in the plasma membrane of neurons (12) . When reconstituted in liposome, AßP1–42 and AßP1–40 allow Ca2+ uptake in a dose-dependent manner (13 , 14) , and soluble AßPs induce ionophore-like calcium influx in neurons as well as non-neuronal cells (5 , 6 , 15 , 16) .

Based on the amino acid sequence of AßP and experimental evidence of multilevel AßP ion channel conductances, Durell et al. (17) have proposed a theoretical model for the channel structure formed by multimeric AßPs, with a subunit stoichiometry ranging from four to eight monomers. However, direct 3-dimensional structural data are not yet available to confirm such channel structures.

Moreover, neuronal responses mediated by these putative AßP channels are yet to be determined. More specifically, cellular changes mediated by physiological (nanomolar) concentrations of fresh and nonfibrillar AßPs are unknown. Such changes would include short-term and rapid neuritic degeneration in a dose-dependent manner, leading to eventual neuronal death during the period during when AßPs remain globular and do not form fibers.

We have imaged AßP1–42 incorporated into planar lipid bilayers with an atomic force microscope (AFM) (18) . These images show multimeric channel-like structures with four and six apparent subunits. Biochemical analysis also shows the formation of stable AßP1–42 tetramers and hexamers in lipid bilayers, and electrical recordings show heterogeneity of single channel currents. At the cellular level, AßP1–42 induced short-term neuritic degeneration and eventual neuronal death in a time- and concentration-dependent manner. AßP toxicity was prevented by zinc, previously shown to inhibit calcium uptake and conductance via AßP channels, but was not prevented by antagonists for the putative AßP plasma membrane receptors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
1,2-Dioleolyl-sn-glycero-3-phosphatidyl-choline (DOPC) was purchased from Avanti Polar Lipids (Birmingham, AL). Synthetic peptide AßP1–42 was obtained from Dr. Hirakura at UCLA and synthesized in Keck Laboratory at Yale University. AßP1–42 peptide was also purchased from Bachem (Torrance, CA). The monoclonal anti-AßP IgG (3D6) with an epitope at the extracellular amino terminus was a generous gift from Dr. Russell Rydel of Athena Neurosciences (South San Francisco, CA). Fura 2-AM was purchased from Molecular Probes (Eugene, OR). ZnCl2, Tris, Trolox, DTT, MK801 (dizocilpine maleate), and physalemin were purchased from Sigma-Aldrich Chemical Company (Milwaukee, WI).

Sample preparation for AFM imaging
To incorporate AßP1–42 into planner lipid bilayer, AßP1–42 was dissolved in chloroform and mixed with DOPC in chloroform at a 4:100 molar ratio. The mixture was dried with Argon gas and resuspended in 10 mM HEPES (pH 7.4) solution at 1 mg/ml. The lipid/AßP1–42 mixture was bath sonicated for 20 min to form liposomes. The liposomes were then deposited on freshly cleaved mica for 20 min, rinsed with HEPES solution, and heated at 65°C for 5 min to fuse liposomes into planar bilayers (19) . AFM imaging was carried out in Tapping mode in 10 mM HEPES (pH 7.4) solution.

Atomic force microscopy imaging
AFM images were obtained as described (13 , 14) using a Multimode AFM with an ExtenderTM electronics module (Digital Instruments, Santa Barbara, CA). Oxide sharpened silicon nitride tips with a nominal spring constant of ~0.32 N/m (Digital Instruments) were used for most of the experiments. Most of the imaging was carried out in the ‘Tapping mode‘ at frequencies near 9 kHz. The scanning speed ranged between 2 and 10 Hz. All imaging of AßP and lipid bilayers was conducted in 10 mM HEPES buffer (pH 7.4) at room temperature (22–24°C) as described (13) .

Polyacrylamide gel electrophoresis and immunoblotting
To extract AßP1–42 incorporated in the liposomes, the AßP-liposome mixture was frozen/thawed and pelleted by centrifugation and the pellet was washed to remove the unincorporated AßP1–42. This procedure was repeated three times to ensure that the unincorporated AßP was removed. The liposomes were resuspended in 10 mM HEPES buffer and solubilized with 1% SDS. Electrophoresis SDS sample buffer was added to the extracted AßP1–42-liposome sample as well as to AßP1–42 freshly dissolved in H2O and heated for 10 min at 90°C.

The peptides were separated by electrophoresis on 12% tris-trisine sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) under reducing conditions (20) , molecular weight markers (Sigma, St Louis, MO) were run parallel to the samples. Proteins were trans-blotted to a nitrocellulose membrane (Micron Separations, Westborough, MA) using a semidry blotter (Fisher Scientific, Tustin, CA). The blot was blocked with PBS containing 5% nonfat milk (30 min), followed by 1 h incubation at room temperature with PBS containing a monoclonal antibody (3D6, 2 µg/ml) and 5% nonfat milk. After washing with PBS, the blot was incubated for 1 h at room temperature with horseradish peroxidase-conjugated rabbit anti-mouse IgG (1:2000 dilution in PBS with 5% nonfat milk, Chemicon, Temecula, CA). The AßP bands on the blot were detected with a SuperSignal West chemiluminescence detection reagent (Pierce Chemical, Rockford, IL) on Fuji RX film (Fisher Scientific).

Ion channels current measurement
Phospholipid bilayer membranes were formed by a well-established method (21) . Solvent-containing membranes were formed by placing a bubble of lipid onto the end of a TeflonTM tube ~300 µ in diameter. The design of the chamber allowed rapid introduction of solution into immediate proximity with the membrane in a volume of only 50 ml. Agar salt bridges were used to connect the electrodes to the solutions and voltage clamp conditions were used in all experiments.

Ion channel currents were recorded with an Axopatch amplifier (Axon Instruments, Sunnyvale, CA) and stored on videotape for later playback and analysis. Membrane capacitance and resistance were monitored continuously in order to assure the formation and stability of reproducible membranes.

Cell culture
Neuro-2a (N2A) mouse neuroblastoma cells (American Type Culture collection, Rockville, MD) were cultured on sterile plastic Petri dishes by a standard method in Dulbecco’s modified Eagle’s medium (DMEM). The medium contained glucose, 10 mM HEPES, 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% heat-inactivated fetal bovine serum (Life Technologies, Rockville MD). For the fluorescent labeling experiments (cell viability, immunofluorescence labeling, and calcium imaging), cells were cultured on laminin-coated glass coverslips (Fisher Scientific). Cells were maintained at 37°C and 5% CO2.

AßP-induced neuronal degeneration
Monolayers of cultured N2A cells were imaged on days 2–3 after seeding. Calcein AM was used as a fluorescence tracer molecule to visualize neuritic reorganization and degeneration. Calcein AM is widely used as a probe for cell viability assay. To load the dye into the cells, N2A cells cultured on laminin-coated glass coverslips were incubated with calcein for 30 min at 37°C in HEPES-buffered DMEM. The glass coverslips were then rinsed and mounted in a chamber and placed on the stage of a Bio-Rad MRC 1024 laser confocal microscope. The objective used for the experiment was a 60x Nikon PlanApo oil immersion lens with a numerical aperture of 1.4. Images were collected at 10 min intervals.

For control experiments (i.e., without AßP addition), images were collected without any addition of AßP for 1 h at 10 min intervals. No adverse effect on neuronal processes or neurite morphology was observed over the time course of the experiment. To examine the effect of AßP1–42, AßP stock solutions were prepared by dissolving the peptides in deionized water. No DMSO or other solvents were used. The stock solutions were stored as aliquots at -20°C until used. AßPs were sonicated to disperse any fibrils and filtered before adding to the cells in culture. Image was captured before cells were treated with AßP; AßP was then added online and images were obtained at 10 min intervals for 1 h. The majority of AßP-induced cellular changes were observable within 15–45 min after the online addition of AßP. The repeatability of the effect was imaged in at least six to eight different Petri dishes for each perturbation.

Cell viability was assayed using a live/dead assay kit from Molecular Probes. Cells treated with AßP1–42 for 4 h and without AßP treatment were incubated with the dyes for 30–45 min at 37°C and 5% CO2 in opti-MEM. Immunofluorescence images were captured with a fluorescence microscope using a 20x objective lens.

Cell calcium imaging
The intracellular calcium level of N2A cells was imaged using a calcium-sensitive fluorescent dye Fura 2 (Molecular Probes). The N2A cells grown on laminin-coated glass coverslips were incubated with 5 µM fura 2-AM for 30 min. The glass coverslip was then rinsed, mounted in a chamber, and placed on the stage of a Zeiss Axiovert 100 microscope. The calcium imaging experiments were carried out using the Image Workbench Software and Image Lighting 2000 board from Axon Instruments (Foster City, CA). The excitation wavelengths were set at 350 ± 5 nm and 380 ± 5 nm and the emission wavelengths at 510 ± 10 nm, by using bandpass interference filters. Switching of excitation wavelengths was done with a 10–2 filter wheel from Sutter Instrument Co. (Novato, CA). The fluorescence images were collected with a MacroMax cooled CCD camera form Princeton Instruments (Princeton, NJ). The experiments were carried out in nominally HCO3-free HEPES-buffered DMEM at room temperature (22–24°C).

Immunofluorescence labeling
Cells grown on glass coverslips were incubated with 5 µM AßP for 3 h at 37°C, followed by a thorough wash with PBS. Cells with and without AßP treatment were then fixed with 4% paraformaldehyde for 10 min and washed with PBS and PBS containing 3% bovine serum albumin and 1% goat serum to minimize any nonspecific binding. Cells were then incubated with either 3D6 antibody against AßP (1 µg/ml) or normal mouse IgG (1 µg/ml) in PBS containing 3% BSA and 1% goat serum for 1 h. After washing, the sample was incubated for 1 h with a cy-3-conjugated goat anti-mouse antibody (1:200 dilution) under the same conditions as for the primary antibody. Immunofluorescence images were captured with a fluorescence microscope using a 20x objective lens.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Monomeric AßPs form stable multimeric complexes when reconstituted in lipid membrane
AFM images of freshly dissolved AßPs demonstrate that AßPs are globular proteins with an average diameter of 1–3 nm. These globular AßPs appear predominantly as monomers and dimers based on the molecular weight and size distribution of globular proteins in AFM images. This observation is consistent with results from previous AFM imaging of AßPs, which have shown that freshly dissolved AßPs is primarily monomeric and contains no large fibrillar formations (13 , 22 , 22a) .

Real-time AFM imaging of AßPs under buffered solution shows that AßPs retain the globular and nonfibrillar shape for an extended period and at nonphysiologically higher concentrations: even after 3 h of incubation at a relatively high concentration (2 mg/ml in PBS), no fibril formation is detected (Fig. 1 A). On the other hand, when allowed to oligomerize at room temperature for 48 h in PBS (pH 7.4), AßPs (2 mg/ml in PBS) assemble into linear fibrillar conformation. Figure 1B shows an AFM image of such oligomerized AßP fibrils after 48 h of incubation. However, a significant percentage of globular AßPs are still present. At lower concentrations (10–50 µg/ml in PBS), continuous AFM imaging shows that AßPs do not form fibrils after 8 h.



View larger version (54K):
[in this window]
[in a new window]
  		Figure 1. AFM height mode images of AßP1–42 (2 mg/ml) after incubation in PBS for A) 3 h and B) 48 h at room temperature. A) After 3 h of incubation, only globular forms of AbP1–42 were observed. B) After 48 h of incubation, fibrils of AßP1–42 were formed ranging between 20 and 500 nm in length. A large percentage of AßP1–42 was still in globular form.

We examined the possibility that freshly prepared soluble AßP monomers would undergo conformational changes and oligomerize when incorporated into lipid bilayers. AßP is highly lipophilic and spontaneously intercalates into the membrane bilayer and liposomes (13 , 14 , 23) . In the present study, AßP1–42 was reconstituted in liposomes, which were then fused to form planar lipid bilayers (13 , 14 , 19) . We were able to obtain both single and double lipid bilayers. Figure 2 A shows an AFM image of an imperfectly fused planer lipid bilayer that contained several lipid vesicles not completely fused into the planar membranes. To access the thickness of the planar lipid membranes, a portion of the membrane was force-dissected using very high imaging force (Fig. 2B ). The force dissection reveals two membrane leaflets. The total height of the planar lipid membranes measured from the thickness profile of the dissected area is ~12–14 nm (Fig. 2C ), which corresponds to thickness of two lipid bilayers.



View larger version (67K):
[in this window]
[in a new window]
  		Figure 2. A) AFM image (amplitude mode) of planar lipid membrane and some liposomes not completely fused into the membrane (arrows). B) A rectangular area of the planar lipid membrane was removed using a scanning tip with contact force, this area was imaged in tapping mode. C) Depth profile of planar lipid membrane cross-section along the dotted line in panel B. Two leaflets representing the overlapping lipid bilayers are visible (arrow).

After incorporation into lipid membranes, predominantly monomeric and dimeric globular AßPs (Fig. 1A ) form stable higher order oligomers. The evidence for such oligomeric structures is derived from complementary biochemical assay, recording of single channel currents, and direct AFM imaging of reconstituted bilayers. When freshly dissolved AßP1–42 is electrophoresed on a 12% reducing tris-trisine SDS-polyacrylamide gel, the peptides appear predominantly as a single band with an apparent size of ~4 kDa, the same as the monomeric AßP1–42 (Fig. 3 , right lane). A smaller portion of the peptides formed dimers; little protein with higher molecular weights was detected. However, when the membrane-incorporated AßPs were separated by SDS-PAGE under reducing conditions, two prominent bands with the molecular weights of tetrameric and hexameric AßPs appeared in addition to the bands representing monomeric AßP and dimeric AßPs (Fig. 3 , left lane). These results indicate that AßP1–42 formed stable, possibly covalently cross-linked, tetrameric and hexameric structures in the lipid bilayer membrane.



View larger version (82K):
[in this window]
[in a new window]
  		Figure 3. Electrophoresis of AßP1–42 on a 12% tris-trisine SDS-PAGE under reducing conditions. Right lane: AßP1–42 freshly dissolved in water; left lane: AßP1–42 after incorporation in liposomes. Positions of molecular weight markers are indicated on the left.

Electrophysiological studies from several laboratories have provided detailed characterizations of channel conductance when AßPs were reconstituted in lipid bilayers (9 , 10) . We had also previously reported that AßPs reconstituted liposomes allow calcium uptake (13 , 14) . Here, we provide electrophysiological data that show channel currents induced by the reconstitution of AßP1–42 in planer lipid bilayers. Consistent with earlier studies from other laboratories, our results also show the presence of a heterogeneous population of AßP channels. Figure 4 A shows single channel currents as a function of time across a planar lipid bilayer membrane exposed to 10 µM AßP1–42. At the start of the record, current is nearly zero, reflecting the low baseline conductivity of membranes, but shortly after the introduction of AßP, downward deflections of current representing the opening of individual ion channels are observed. The multiple different sizes of single channel currents correspond to various levels of channel conductance, which probably reflect the heterogeneity of aggregation of AßP1–42 subunits. A detailed biophysical characterization of the channel conductance, selectivity, and gating was beyond the scope of the present study. However, our results are consistent with previous electrophysiological recording data from other laboratories that suggest the presence of multiple number of subunits and stoichiometry of AßP channels (9 , 10 , 17) .



View larger version (65K):
[in this window]
[in a new window]
  		Figure 4. Ion channel activity and structure. AßP1–42 when incorporated in planer lipid bilayer membrane forms ion channels. A) Electrical current record as a function of time across a planar lipid bilayer membrane exposed to 10 µM AßP1–42. At the start of the recording, current is nearly zero, reflecting the low baseline permeability of membranes; shortly thereafter, downward deflections of current representing the opening of individual ion channels can be observed. Note the multiple different sizes of these channel currents, which probably reflect the heterogeneity of aggregation of AßP1–42. About halfway through, voltage polarity is reversed; after an initial decrease in current (of opposite sign), the AßP1–42-induced currents begin increasing rapidly. Solutions contained 100 mg KCl, 10 mM HEPES (pH 7.4). Lipid was Asolectin. B) Unprocessed AFM amplitude mode image of AßP1–42 reconstituted in planar bilayers. The images were obtained on the upper surface of the double bilayer membrane. At medium resolution (scan size, 700 nm, 512 pixels), individual donut-shaped structures protruding out of the membrane surface (background), presumably representing individual channels (arrows, B) are clearly visible. The image in B was obtained from left to right scans and thus the leading edge (left edge) is brighter. Note that some channels do not show clear pore opening. Images are color coded, brighter being greater in height.

AßP1–42 forms ion channels in reconstituted membrane
To obtain higher resolution AFM images of randomly distributed and noncrystalline arrays of AßP oligomers in reconstituted membranes, we fused liposomes into large planar bilayers before imaging rather than directly imaging on the smaller spherical liposomes. Both single and double bilayers were observed in our study (Fig. 2) . An example AFM image of the upper surface of the reconstituted bilayer membrane is shown in Fig. 4B . The oligomeric AßPs have channel-like structures: donut-shaped structures with an outer diameter of 8–12 nm, a centralized pore-like depression, and oligomeric walls protruding ~1 nm above the embedding lipid bilayer surface.

Closer examination of the individual AßP channel structures at higher resolution reveals at least two subunit arrangements: rectangular structure with four apparent subunits and hexagonal structures with six apparent subunits (Fig. 5 ). The extramembranous protrusions of each subunit are not uniform and differ by 0.1–0.3 nm. Whether this small variation in the topography is real or induced by the AFM imaging force is not clear. The resolution of AFM imaging in the present study is insufficient to resolve the structural details of each subunit. Subnanometer resolution AFM imaging has been achieved only on highly crystalline membrane protein samples.



View larger version (75K):
[in this window]
[in a new window]
  		Figure 5. High-resolution images of AßP1–42 channels. A, B) Surface plots. B) Off-line zoomed image. Subunit organizations are visible. Subunits’ protrusion vary by 0.1–0.3 nm as shown by the difference in the subunits’ brightness. C) Close-up (off-line zoomed) images of representative individual AßP1–42 channels in planar bilayer: three channel structures shown in the upper part of panel C contain four apparent subunits and the three channels shown in the lower panel contain six apparent subunits.

The present study provides the first structural evidence for the formation of AßP channel in a lipid bilayer membrane. AFM images of these channels show both open and occluded pore-like features (Fig. 5) . However, we are not certain whether the latter AßP structures truly represent closed AßP channels. Alternatively, they could be aggregated AßPs that did not assemble into proper channels.

As described above, the presence of both tetrameric and hexameric channel-like structures is also consistent with the biochemical analysis of oligomeric AßPs isolated from reconstituted liposomes (Fig. 3) as well as from electrophysiological recording of multiple single channel currents of different sizes (Fig. 4A ). The relative preponderance of tetrameric and hexameric arrangements in the AFM images was not quantified.

For AßP channels to be relevant to AD pathophysiology, they should induce cellular morphological changes at physiological (nanomolar) concentrations in a dose-dependent manner, these morphological changes should be blocked by AßP channel-specific inhibitors, and AßPs should form channel-like structures in the cell plasma membrane. The concentrations of peptides used for ion channel reconstitutions in artificial membrane and the concentrations of peptides necessary to show physiological effects at cellular level vary significantly. For reconstitution in artificial systems, a higher concentration (micromolar) of peptides is usually required although nanomolar concentration is usually considered physiological for cellular activity.

AßP1–42 induces rapid neuritic degeneration at physiological nanomolar concentration
We examined the effects of fresh and nonfibrillar AßP1–42 on neuritic arborization and neuronal death using a tracer fluorescent dye calcein AM. Neurons not treated with AßP remained stable and no significant neuritic reorganization or loss of neuronal processes was observed during 1 h of imaging (Fig. 6 A, B). After 15 min of incubation with AßP1–42, neurons began to undergo significant morphological changes including beading and granulation of neurites, loss of neuritic arborization, and swelling of neuronal bodies (Fig. 6 C--H). Since fibrillar AßPs had not been formed during such a short incubation period in our study (Fig. 1) , the AßP-induced changes in neuronal morphology are most likely induced by globular AßPs. Fifteen minutes of AßP incubation is sufficient to incorporate AßPs into the cell plasma membrane, for forming ion channels, and loading the cytoplasm with excess calcium to induce neuritic degeneration.



View larger version (89K):
[in this window]
[in a new window]
  		Figure 6. Effect of AßP1–42 on morphological changes in neuronal cells loaded with calcein AM. Cells before treatment with AßP1–42 are used as controls (time=0). AßP1–42 was then added online and images captured at 10 min intervals for 1 h. Only the images taken at time 0 (control) (A, C, E, G) and 45 min (B, D, F, H) are shown. A, B) Cells not treated with AßP1–42 did not show any appreciable morphological degeneration (for example, compare panels A and B) even after 45 min. Cells treated with 50 nM AßP1–42 (D) show significant neuritic degeneration compared with pre-AßP1–42 treatment (C) (compare regions denoted by arrows in panels C, D). Cells treated with 500 nM AßP1–42 (F) show significant changes in neuronal processes with neurite beading and loss of cell–cell contact compared with pre-AßP1–42 treatment (E) (compare regions denoted by arrows in panels E, F). Cells treated with 5 µM AßP1–42 (H) show significant changes in cellular morphology with loss of neuronal processes and fragmented neurites compared with pre-AßP1–42 treatment (G). The changes in cellular morphology can be observed within 15–25 min of adding AßP1–42. Scale bar: 10 µM.

AßP toxicity was dose and time dependent such that a lower concentration of AßP1–42 required a longer incubation to induce comparable structural changes. For example, 50 nM AßP1–42 induced neuritic degeneration within 45 min (Fig. 6C, D ) whereas no significant degeneration was observed for 20 nM AßP1–42 during this period (data not shown). To limit the possibility of forming fibrillar AßPs, we restricted the imaging to ~1 h; thus, we cannot exclude the possibility that with longer incubation at the same or lower concentrations, AßPs could induce neuritic degeneration. 200 nM AßP1–42 caused greater neuritic degeneration and loss of cell–cell contact (data not shown); increasingly significant neuritic losses were observed for 500 nM (Fig. 6E, F ) and 5 µM AßP1–42 (Fig. 6G, H ). The repeatability of the AßP-induced cell morphological changes observed within 1 h after the online addition of AßP was imaged in at least six to eight different cell preparations for each AßP concentration tested.

AßP1–42 induces neuronal death at micromolar concentration
We examined whether lower doses of AßPs with a shorter incubation would induce neurotoxicity and could correlate with the channel mechanism. Previous studies have shown that prolonged incubation with relatively high micromolar concentrations of AßPs induces neurotoxicity over an extended incubation period and is consistent with the prevailing fiber hypothesis. In our study, no fiber formation was observed over ~8 h for micomolar concentrations of AßPs. We thus confined our study for up to 4–5 h of incubation at concentrations ranging from 1 µM to 10 µM. Neuronal viability study using a live/dead assay kit comprising calcein AM and ethidium homodimer-1 shows death of a significant number of neurons within 4 h of AßP incubation (compare Fig. 7 A, B with Fig. 7C, D ). The percentage of dead neurons was AßP concentration dependent, such that more than 90% of neurons were dead upon incubation with 10 µM AßP1–42 and very few neurons were dead upon incubation with 1 µM AßP1–42 (data not shown).



View larger version (45K):
[in this window]
[in a new window]
  		Figure 7. Cell viability assay and Immunofluorescence imaging of cells treated with AßP1–42. AßP-induced cellular toxicity was blocked by Zn2+ and by the removal of extracellular calcium, but not by tachykinin or NMDA antagonist. Fluorescence images of neuronal cells treated with calcein (live cells: left panels A, C, E, G, I, K) and ethidium homodimer 1 (dead cells: right panels B, D, F, H, J, L) after treatment are shown. A, B) Control cells not treated with AßP1–42. C, D) Cells after treatment with 10 µM AßP1–42. E, F) Cells after treatment with AßP1–42 in the absence of extracellular calcium. G, H) Cells after treatment with AßP1–42 in the presence of 50 µM ZnCl2. Zn2+ protected cells from AßP-induced toxicity. I, J) Cells after treatment with AßP1–42 in the in the presence of 20 µM tachykinin (physalemin). K, L) Cells after treatment with AßP1–42 in the presence of 20 µM MK-801. No protection from AßP-induced cellular toxicity was observed for the cells treated with tachykinin or NMDA receptor antagonist. Immunofluorescence labeling: neurons were incubated with 5 µM AßP1–42 for 3 h and immunolabeled with either M) a monoclonal anti-AßP antibody (3D6) or N) normal mouse IgG as a negative control, followed by cy-3-conjugated secondary antibody. O) Neurons not treated with AßP1–42 exhibited none to little auto AßP immunofluorescence.

AßP toxicity is Ca2+ dependent and prevented by AßP channel blocker Zn2+
AßP toxicity is dependent on the presence of extracellular Ca2+: in the absence of external Ca2+, AßP1–42-treated neurons retained their morphology and viability (Fig. 7E, F ). This observation is consistent with the view that the altered cellular calcium homeostasis is a common denominator underlying AßP toxicity. AßP-induced Ca2+ uptake could occur indirectly via existing Ca2+-selective channels or directly via AßP-formed channels. Zn2+ was previously shown to inhibit ion conductance and Ca2+ uptake via AßP channels (9 10 11 12 13 14) . In our study, when Zn2+ was added along with or 5 min after AßP1–42 treatment, it protected neurons from AßP toxicity (Fig. 7G, H ).

At a concentration of more than 1 mM, zinc facilitates the precipitation of soluble AßPs from aqueous buffer (24 , 25) and thus could prevent AßPs from forming ion channels and hence prevent AßP toxicity. This is highly unlikely in our study, since the concentration of zinc we used was in the low micromolar range. More important, the blockage was observed even when zinc was added after sufficient time was allowed for AßPs to incorporate into the plasma membrane and form ion channels (see refs 13 , 14 ).

AßP toxicity could also be induced indirectly via its binding to plasma membrane receptors, including nAChR, NMDA, and tachykinin neuropeptide receptors (26 27 28) . Although it is difficult to rule out the possibility of receptor-mediated AßP toxicity, so far there has not been a replicated report that convincingly identified a bona fide AßP receptor. We did, however, examine the effects of antagonists/blockers of many putative AßP membrane receptors on AßP toxicity. In the present study, for example, physalemin, a tachykinin neuropeptide reported to prevent AßP toxicity (26) , had little effect on AßP1–42-induced morphological changes: in the presence of physalemin, AßP1–42 still caused cell death in ~90% of the population (Fig. 7I, J ). Similarly, MK-801, an antagonist of NMDA receptor, did not prevent AßP-induced toxicity in neuronal cells (Fig. 7K, L ). In the presence of MK-801, there was only a slight increase in the rate of cell survival (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Multiple AßP channel stoichiometries correlate with multilevel conductances
Previous electrophysiological studies suggest that ion channels formed by AßPs of various lengths (AßP25–35, AßP1–40, AßP1–42) share a common feature: they exhibit multilevel channel conductances. The multilevel conductances could result from multiple conformational changes in the AßP channel structure and/or from the difference in number of subunits that form a single channel (8 , 10 , 17) . A more recent study of AßP1–42 channel suggests that the multiple channel conductances reflect the difference in subunit number rather than changes in channel conformation (10) . The observation of single AßP1–42 channels with both four and six subunits (Fig. 5) and the formation of stable AßP tetramers and hexamers in lipid bilayers (Fig. 3) indicate that AßP channels can be composed of different numbers of subunits.

In the present study, predominantly monomeric AßP1–42 was directly solubilized into lipid. Thus, the aggregation and oligomerization of AßP1–42 should have occurred within the lipid bilayers. After incorporation into the lipid bilayer, there are more AßPs in stable tetrameric and hexameric forms than dimers (Fig. 3 , left lane). There are some AßP dimers, but few AßP tetramers and hexamers present before AßP membrane incorporation (Fig. 3 , right lane). This suggests that tetramers and hexamers are more stable in lipid bilayers. However, we cannot exclude the possibility of the presence of larger and less stable structures of AßP1–42 aggregates in lipid membrane, since a small number of larger aggregates of AßP1–42 (20–30 nm in diameter) were also observed with AFM imaging (data not shown). Although various lipids have been shown to possess differing propensity for AßP incorporations, once formed, channel activity and properties remain the same (B. Kagan, personal communication). Hirakura and Kagan have looked at the role of various lipids on channel formation by AßPs as well as other small peptides (10 , 11 , 21) .

Durell et al. (17) have proposed two different possible AßP channel structures with distinguishable alignments of the peptide backbone in the lipid bilayer. One of these structures could also convert to a third structural conformation. In the present study, we did not attempt to determine whether the AßP channels belong to either or both groups. By using AFM and antibodies against specific regions of AßP, the protein sequences of extramembrane domains and the pore region of the AßP channel can be identified and now provide further insight into the structure and functions of the AßP channels.

Progressive accumulation of AßPs accelerates toxicity
AßP is secreted by a variety of ßAPP-expressing neurons and non-neuronal cells, and is present up to nanomolar concentrations in the cerebrospinal fluid and blood circulation under physiological conditions (25 , 29) . In vivo and in vitro studies have shown that AßP can form small, stable oligomers but no fibrils at physiological concentrations (low nM) up to micromolar levels (22a , 30) .

Under normal conditions, soluble AßPs are bound to various AßP binding proteins and are quickly cleared from cerebrospinal fluid into the bloodstream (31) , most likely via receptor transport mechanisms across the blood–brain barrier (32 , 33) . The removal and degradation of AßPs from the brain could also be affected by certain AßP binding proteins (34 35 36) as well as by the production and activation of AßP-degrading proteases (37 38 39 40) .

In the brain of AD patients, the level of soluble AßP (especially AßP1–42) is significantly elevated (41 , 42) . This is probably due to an imbalance in the production and removal of AßP, which could lead to excessive accumulation of AßP in the cerebrospinal fluid, deposition of AßP, and formation of calcium-permeable AßP channels on cell plasma membranes. Continued accumulation of AßP channels over an extended period would allow considerably higher Ca2+ uptake, eventually disrupting calcium homeostasis and activating signal transduction pathways leading to cellular degeneration. The cellular toxicity data in our present study support such a scenario: AßP1–42 induces calcium uptake, rapid neuritic degeneration, and neuronal death at nanomolar and micromolar concentrations, respectively.

Consistent with the possibility that the neuritic degeneration observed in our study was caused by plasma membrane AßP channels, immunolabeling of AßP-treated cells with anti-AßP antibody indicates that a large amount of AßP was incorporated into and/or bound to cell plasma membrane (Fig. 7M-O ). Such intercalation has been reported previously by 1) direct structural analysis using light fluorescence immunolabeling or indirectly by freeze-fracture and/or negative staining EM or other high-resolution techniques or 2) electrophysiological recording of AßP-induced channel currents in the neurolemma.

Lane et al. (43) have reported ultrastructural evidence for the presence of intramembranous features in differentiated PC12 after AßP incubation. Hartley et al. (44) have shown immuno-EM labeling with anti-AßP antibody for all three preparations (globular AßP, protofilaments, and fibrils) on and around the cells. Torp et al. (45) have presented ultrastructural evidence for AßP association with neuronal membranes in aged dog brains: they used postembedding immunocytochemistry at the EM level to show AßP association with the plasma membrane. Pillot et al. (46) have shown by C14 radioactive labeling that most of the AßP (28 29 30 31 32 33 34 35 36 37 38 39 40 41) fusigenic portion was associated with an enriched plasma membrane fraction of rat cortical neurons. Our previous data on non-neuronal cells (5 , 6) and data from others labs using site-directed antibody against the outer portion of AßPs show strong immunolabeling.

No attempt was made in the present study to determine whether these membrane-associated AßPs form ion channel-like structures in native plasma membrane. Such a determination would provide even stronger direct evidence in support of the channel hypothesis of AD pathogenesis, but it is technically difficult to carry out such studies at this time. Several other studies have reported channel-like activity in AßP incubated neurons (12 , 15) . Moreover, Vargas et al. (47) have shown displacement currents associated with the direct insertion of AßPs into planar bilayer membrane. Kawahara et al. (12) have shown zinc-sensitive, cation-selective currents across membrane patches from hypothalamic neurons and recorded increased cellular calcium in hypothalamic neuronal cell line (48) .

AßP increases intracellular calcium
We have examined several postulated mechanisms of AßP toxicity, including 1) its interaction with plasma membrane receptors such as tachykinin receptors (26) , NMDA receptors (28) , or nAChR (27) and 2) changing cellular ionic concentration by modulating the activity of preexisting channels (49) or via its own ion channels (8 9 10 11 12 13 14) . Our data show that AßP toxicity is inhibited by the removal of external calcium, and thus calcium influx is required to induce cellular toxicity. When neurons were treated with 10 µM of AßP1–42 in the presence of normal extracellular calcium (1.8 mM), intracellular calcium increased in many of the observed cells (Fig. 8 ). However, the calcium response varied among different cells and many cells showed little change in the cellular calcium level. We have previously shown that AßPs induced calcium influx and intracellular calcium increases in both endothelial and fibroblast cells (5 , 6) .



View larger version (16K):
[in this window]
[in a new window]
  		Figure 8. Changes in intracellular calcium levels in response to 10 µM AßP1–42. After the addition of AßP1–42, intracellular calcium level increased in some but not all cells. Each trace in the figure represents the ratio of fluorescence emissions (510 nm) from an individual cell due to excitations at 350 and 380 nm. Each ratio corresponds to an intracellular calcium concentration. Detailed calibration was not carried out in the present study.

The AßP toxicity observed in our study is unlikely due to AßP’s interactions with its putative plasma membrane receptors, such as tachykinin receptors or NMDA receptors: neither physalemin or NMDA blocker MK-801 prevented the AßP toxicity. On the other hand, the AßP channel blocker Zn2+ prevented AßP toxicity, which suggests that it is induced by calcium uptake directly via AßP channels. Previous studies from other laboratories have also shown that several calcium channel blockers and antagonists of calcium mobilization such as {omega}-conotoxin, nifedipine, verapamil, APV, MK-801, cAMP, 8-bromo cAMP, cGMP did not inhibit neurotoxicity induced by AßP25–35 and AßP1–40 (50 , 51) . Thus, although it is difficult to exclude all possibilities of calcium uptake via existing channels, inhibition of cellular degeneration by zinc suggests that AßP toxicity is mediated by calcium influx via AßP channels.

In conclusion, we have shown that AßPs form ion channel-like structures in the lipid bilayer. We provide strong correlative evidence that AßP toxicity could be mediated by calcium influx via these AßP channels. Thus, AßP channels could provide a direct as well as complementary pathways for inducing cellular degeneration commonly associated with Alzheimer’s disease. Additional study is required to determine the structural conformation of AßPs in the cell plasma membrane. In addition to its direct relevance to AD, high-resolution imaging of AßP channel structures provides a new avenue for the study of the channel-like structures predicted for many cytotoxic peptides as well as other amyloidogenic proteins, including prion and {alpha}-synuclein (for review, see ref 52 ).


   ACKNOWLEDGMENTS
 
We thank Drs. Seung Rhee, Arjan Quist, and Nils Almqvist for insightful advice and suggestions and Maura Jess for help in preparing the figures. Electrophysiological recording of ion channel currents in reconstituted bilayers was performed in the laboratory of Dr. Bruce Kagan at UCLA. We thank Dr. Ashok Parbhu for providing preliminary data on AßP fibrillogenesis. We thank Dr. Russell Rydel from Athena Neurosciences for kindly providing us with the anti-AßP antibodies. We thank Drs. Paul Hansma and Stuart Feinstein for their suggestions to improve the quality and relevance of our results. This work was supported by the Alzheimer’s Disease Program, Department of Health, CA, Alzheimer’s Association of America, and the National Institutes of Health (GM-NIA).


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication May 15, 2001. Accepted for publication July 5, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Mattson, M. P. (1997) Cellular actions of beta-amyloid precursor protein and its soluble and fibrillogenic derivatives. Physiol. Rev. 77,1081-1132[Abstract/Free Full Text]
  2. Anderton, B. H., Callahan, L., Coleman, P., Davies, P., Flood, D., Jicha, G. A., Ohm, T., Weaver, C. (1998) Dendritic changes in Alzheimer’s disease and factors that may underlie these changes. Prog. Neurobiol. 55,595-609[Medline]
  3. Cummings, J. L., Vinters, H. V., Cole, G. M., Khachaturian, Z. S. (1998) Alzheimer’s disease: etiologies, pathophysiology, cognitive reserve, and treatment opportunities. Neurology 51,S2-S17[Medline]
  4. Jarrett, J. T., Lansbury, P. J. (1993) Seeding ‘one-dimensional crystallization’ of amyloid: a pathogenic mechanism in Alzheimer’s disease and scrapie?. Cell 73,1055-1058[Medline]
  5. Bhatia, R., Lin, H., Lal, R. (2000) Fresh and globular amyloid beta protein (1–42) induces rapid cellular degeneration: evidence for AbetaP channel-mediated cellular toxicity. FASEB J 14,1233-1243[Abstract/Free Full Text]
  6. Zhu, Y. J., Lin, H., Lal, R. (2000) Fresh and nonfibrillar amyloid beta protein(1–40) induces rapid cellular degeneration in aged human fibroblasts: evidence for AbetaP-channel-mediated cellular toxicity. FASEB J 14,1244-1254[Abstract/Free Full Text]
  7. Pallitto, M. M., Ghanta, J., Heinzelman, P., Kiessling, L. L., Murphy, R. M. (1999) Recognition sequence design for peptidyl modulators of beta-amyloid aggregation and toxicity. Biochemistry 38,3570-3578[Medline]
  8. Arispe, N., Pollard, H. B., Rojas, E. (1993) Giant multilevel cation channels formed by Alzheimer disease amyloid beta-protein [A beta P (1–40)] in bilayer membranes. Proc. Natl. Acad. Sci. USA 90,10573-10577[Abstract/Free Full Text]
  9. Arispe, N., Pollard, H. B., Rojas, E. (1996) Zn2+ interaction with Alzheimer amyloid beta protein calcium channels. Proc. Natl. Acad. Sci. USA 93,1710-1715[Abstract/Free Full Text]
  10. Hirakura, Y., Lin, M. C., Kagan, B. L. (1999) Alzheimer amyloid abeta1–42 channels: effects of solvent, pH, and Congo red. J. Neurosci. Res. 57,458-466[Medline]
  11. Mirzabekov, T. A., Lin, M. C., Kagan, B. L. (1996) Pore formation by the cytotoxic islet amyloid peptide amylin. J. Biol. Chem. 271,1988-1992[Abstract/Free Full Text]
  12. Kawahara, M., Arispe, N., Kuroda, Y., Rojas, E. (1997) Alzheimer’s disease amyloid beta-protein forms Zn2+-sensitive, cation-selective channels across excised membrane patches from hypothalamic neurons. Biophys. J. 73,67-75[Abstract/Free Full Text]
  13. Lin, H., Zhu, Y. J., Lal, R. (1999) Amyloid beta protein (1–40) forms calcium-permeable. Zn2+-sensitive channel in reconstituted lipid vesicles. Biochemistry 38,11189-11196[Medline]
  14. Rhee, S. K., Quist, A. P., Lal, R. (1998) Amyloid beta protein-(1–42) forms calcium-permeable. Zn2+-sensitive channel. J. Biol. Chem. 273,13379-13382[Abstract/Free Full Text]
  15. Sanderson, K. L., Butler, L., Ingram, V. M. (1997) Aggregates of a beta-amyloid peptide are required to induce calcium currents in neuron-like human teratocarcinoma cells: relation to Alzheimer’s disease. Brain Res 744,7-14[Medline]
  16. Simmons, M. A., Schneider, C. R. (1993) Amyloid beta peptides act directly on single neurons. Neurosci. Lett. 150,133-136[Medline]
  17. Durell, S. R., Guy, H. R., Arispe, N., Rojas, E., Pollard, H. B. (1994) Theoretical models of the ion channel structure of amyloid beta-protein. Biophys. J. 67,2137-2145[Abstract/Free Full Text]
  18. Lal, R., John, S. A. (1994) Biological applications of atomic force microscopy. Am. J. Physiol. 266,C1-C21[Abstract/Free Full Text]
  19. Mou, J., Czajkowsky, D. M., Shao, Z. (1996) Gramicidin A aggregation in supported gel state phosphatidylcholine bilayers. Biochemistry 35,3222-3226[Medline]
  20. Schägger, H., von Jagow, G. (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166,368-379[Medline]
  21. Mirzabekov, T. A., Silberstein, A. V., Kagan, B. L. (1998) The use of planar lipid bilayer membranes for rapid screening of membrane active compounds. Methods Enzymes 294,67-74
  22. Tseng, B. P., Esler, W. P., Clish, C. B., Stimson, E. R., Ghilardi, J. R., Vinters, H. V., Mantyh, P. W., Lee, J. P., Maggio, J. E. (1999) Deposition of monomeric, not oligomeric, Abeta mediates growth of Alzheimer’s disease amyloid plaques in human brain preparations. Biochemistry 38,10424-10431[Medline]
  23. Parbhu, A., Lin, H., Lal, R. (2001) Imaging real-time aggregation of amyloid beta protein (1-42) by atomic force microscopy. Peptides In press
  24. Mason, R. P., Jacob, R. F., Walter, M. F., Mason, P. E., Avdulov, N. A., Chochina, S. V., Igbavboa, U., Wood, W. G. (1999) Distribution and fluidizing action of soluble and aggregated amyloid beta-peptide in rat synaptic plasma membranes. J. Biol. Chem. 274,18801-18807[Abstract/Free Full Text]
  25. Bush, A. I., Pettingell, W. H., Multhaup, G., Paradis, M., Vonsattel, J. P., Gusella, J. F., Beyreuther, K., Masters, C. L., Tanzi, R. E. (1994) Rapid induction of Alzheimer A beta amyloid formation by zinc. Science 265,1464-1467[Abstract/Free Full Text]
  26. Garzon-Rodriguez, W., Sepulveda-Becerra, M., Milton, S., Glabe, C. G. (1997) Soluble amyloid Abeta-(1–40) exists as a stable dimer at low concentrations. J. Biol. Chem. 272,21037-21044[Abstract/Free Full Text]
  27. Yankner, B. A., Duffy, L. K., Kirschner, D. A. (1990) Neurotrophic and neurotoxic effects of amyloid beta protein: reversal by tachykinin neuropeptides. Science 250,279-282[Abstract/Free Full Text]
  28. Wang, H.-Y., Lee, D. H. S., D’Andreas, M. R., Peterson, P. A., Shank, R. P., Reitz, A. B. (2000) Beta-amyloid (1–42) binds to alpha7 nicotinic acetylcholine receptor with high affinity. Implications for Alzheimer’s disease pathology. J. Biol. Chem 275,5626-5632[Abstract/Free Full Text]
  29. Harkany, T., Mulder, J., Sasvari, M., Abraham, I., Konya, C., Zarandi, M., Penke, B., Luiten, P. G. M., Nyakas, C. (1999) N-Methyl-D-aspartate receptor antagonist MK-801 and radical scavengers protect cholinergic nucleus basalis neurons against beta-amyloid neurotoxicity. Neurobiol. Dis. 6,109-121[Medline]
  30. Haass, C., Schlossmacher, M. G., Hung, A. Y., Vigo-Pelfrey, C., Mellon, A., Ostaszewski, B. L., Lieberburg, I., Koo, E. H., Schenk, D., Teplow, D. B. (1992) Amyloid beta-peptide is produced by cultured cells during normal metabolism. Nature (London) 359,322-325[Medline]
  31. Huang, T. H., Yang, D. S., Plaskos, N. P., Go, S., Yip, C. M., Fraser, P. E., Chakrabartty, A. (2000) Structural studies of soluble oligomers of the Alzheimer beta-amyloid peptide. J. Mol. Biol. 297,73-87[Medline]
  32. Ghersi-Egea, J. F., Gorevic, P. D., Ghiso, J., Frangione, B., Patlak, C. S., Fenstermacher, J. D. (1996) Fate of cerebrospinal fluid-borne amyloid beta-peptide: rapid clearance into blood and appreciable accumulation by cerebral arteries. J. Neurochem. 67,880-883[Medline]
  33. Poduslo, J. F., Curran, G. L., Sanyal, B., Selkoe, D. J. (1999) Receptor-mediated transport of human amyloid beta-protein 1–40 and 1–42 at the blood–brain barrier. Neurobiol. Dis. 6,190-199[Medline]
  34. Zlokovic, B. V., Ghiso, J., Mackic, J. B., McComb, J. G., Weiss, M. H., Frangione, B. (1993) Blood–brain barrier transport of circulating Alzheimer’s amyloid beta. Biochem. Biophys. Res. Commun. 197,1034-1040[Medline]
  35. Biere, A. L., Ostaszewski, B., Stimson, E. R., Hyman, B. T., Maggio, J. E., Selkoe, D. J. (1996) Amyloid beta-peptide is transported on lipoproteins and albumin in human plasma. J. Biol. Chem. 271,32916-32922[Abstract/Free Full Text]
  36. Ghiso, J., Matsubara, E., Koudinov, A., Choi-Miura, N. H., Tomita, M., Wisniewski, T., Frangione, B. (1993) The cerebrospinal-fluid soluble form of Alzheimer’s amyloid beta is complexed to SP-40,40 (apolipoprotein J), an inhibitor of the complement membrane-attack complex. Biochem. J. 293,27-30
  37. Strittmatter, W. J., Weisgraber, K. H., Huang, D. Y., Dong, L. M., Salvesen, G. S., Pericak-Vance, M., Schmechel, D., Saunders, A. M., Goldgaber, D., Roses, A. D. (1993) Binding of human apolipoprotein E to synthetic amyloid beta peptide: isoform-specific effects and implications for late-onset Alzheimer disease. Proc. Natl. Acad. Sci. USA 90,8098-8102[Abstract/Free Full Text]
  38. Kurochkin, I. V., Goto, S. (1994) Alzheimer’s beta-amyloid peptide specifically interacts with and is degraded by insulin degrading enzyme. FEBS Lett 345,33-37[Medline]
  39. Naidu, A., Quon, D., Cordell, B. (1995) beta-Amyloid peptide produced in vitro is degraded by proteinases released by cultured cells. J. Biol. Chem. 270,1369-1374[Abstract/Free Full Text]
  40. Qiu, W. Q., Walsh, D. M., Ye, Z., Vekrellis, K., Zhang, J., Podlisny, M. B., Rosner, M. R., Safavi, A., Hersh, L. B., Selkoe, D. J. (1998) Insulin-degrading enzyme regulates extracellular levels of amyloid beta-protein by degradation. J. Biol. Chem. 273,32730-32738[Abstract/Free Full Text]
  41. Yamin, R., Malgeri, E. G., Sloane, J. A., McGraw, W. T., Abraham, C. R. (1999) Metalloendopeptidase EC 3.4.24.15 is necessary for Alzheimer’s amyloid-beta peptide degradation. J. Biol. Chem. 274,18777-18784[Abstract/Free Full Text]
  42. Jensen, M., Schreoder, J., Blomberg, M., Engvall, B., Pantel, J., Ida, N., Basun, H., Wahlund, L. O., Werle, E., Jauss, M., Beyreuther, K., Lannfelt, L., Hartmann, T. (1999) Cerebrospinal fluid A beta42 is increased early in sporadic Alzheimer’s disease and declines with disease progression. Ann. Neurol. 45,504-511[Medline]
  43. Kuo, Y. M., Emmerling, M. R., Vigo-Pelfrey, C., Kasunic, T. C., Kirkpatrick, J. B., Murdoch, G. H., Ball, M. J., Roher, A. E. (1996) Water-soluble Abeta (N-40, N-42) oligomers in normal and Alzheimer disease brains. J. Biol. Chem. 271,4077-4081[Abstract/Free Full Text]
  44. Lane, N. J., Balbo, A., Fukuyama, R., Rapoport, S. I., Galdzicki, Z. (1998) The ultrastructural effects of beta-amyloid peptide on cultured PC12 cells: changes in cytoplasmic and intramembranous features. J. Neurocytol. 27,707-718[Medline]
  45. Hartley, D. M., Walsh, D. M., Ye, C. P., Diehl, T., Vasquez, S., Vas4silev, P. M., Teplow, D. B., Selkoe, D. J. (1999) Protofibrillar intermediates of amyloid beta-protein induce acute electrophysiological changes and progressive neurotoxicity in cortical neurons. J. Neurosci. 19,8876-8884[Abstract/Free Full Text]
  46. Torp, R., Head, E., Cotman, C. W. (2000) Ultrastructural analyses of beta-amyloid in the aged dog brain: neuronal beta-amyloid is localized to the plasma membrane. Prog. Neuropsychopharmacol. Biol. Psychiatry 24,801-810[Medline]
  47. Pillot, T., Drouet, B., Queillé, S., Labeur, C., Vandekerchkhove, J., Rosseneu, M., Pinçon-Raymond, M., Chambaz, J. (1999) The nonfibrillar amyloid beta-peptide induces apoptotic neuronal cell death: involvement of its C-terminal fusogenic domain. J. Neurochem. 73,1626-1634[Medline]
  48. Vargas, J., Alarcón, J. M., Rojas, E. (2000) Displacement currents associated with the insertion of Alzheimer disease amyloid beta-peptide into planar bilayer membranes. Biophys. J. 79,934-944[Abstract/Free Full Text]
  49. Kawahara, M., Kuroda, Y. (2000) Molecular mechanism of neurodegeneration induced by Alzheimer’s beta-amyloid protein: channel formation and disruption of calcium homeostasis. Brain Res. Bull. 53,389-397[Medline]
  50. Etcheberrigaray, R., Ito, E., Kim, C. S., Alkon, D. L. (1994) Soluble beta-amyloid induction of Alzheimer’s phenotype for human fibroblast K+ channels. Science 264,276-279[Abstract/Free Full Text]
  51. Huang, H. M., Ou, H. C., Hsieh, S. J., Chiang, L. Y. (2000) Blockage of amyloid beta peptide-induced cytosolic free calcium by fullerenol-1, carboxylate C60 in PC12 cells. Life Sci 66,1525-1533[Medline]
  52. Whitson, J. S., Appel, S. H. (1995) Neurotoxicity of A beta amyloid protein in vitro is not altered by calcium channel blockade. Neurobiol. Aging 16,5-10[Medline]
  53. Kourie, J. I., Shorthouse, A. A. (2000) Properties of cytotoxic peptide-formed ion channels. Am. J. Physiol. 278,C1063-C1087[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Cell Biol.Home page
P. Van Damme, A. Van Hoecke, D. Lambrechts, P. Vanacker, E. Bogaert, J. van Swieten, P. Carmeliet, L. Van Den Bosch, and W. Robberecht
Progranulin functions as a neurotrophic factor to regulate neurite outgrowth and enhance neuronal survival
J. Cell Biol., April 3, 2008; 181(1): 37 - 41.
[Abstract] [Full Text] [PDF]

Home page
 Biophys. JHome page
J. Zheng, H. Jang, B. Ma