QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

HOME  |   SEARCH  |   ARCHIVE  |   SUBSCRIBE  |   CONTACT  |   HELP

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (464) Citing Articles via Google Scholar Google Scholar Articles by Hartley, D. M. Articles by Selkoe, Dennis. J. Search for Related Content PubMed PubMed Citation Articles by Hartley, D. M. Articles by Selkoe, Dennis. J.

 Previous Article  |  Next Article 

The Journal of Neuroscience, October 15, 1999, 19(20):8876-8884

Protofibrillar Intermediates of Amyloid -Protein Induce Acute Electrophysiological Changes and Progressive Neurotoxicity in Cortical Neurons

Dean M. Hartley², Dominic M. Walsh², Chianping P. Ye^{1, 3}, Thekla Diehl², Sara Vasquez², Peter M. Vassilev^{1, 3}, David B. Teplow², and Dennis. J. Selkoe²

¹ Department of Neurology and Medicine, Harvard Medical School, and the ² Center for Neurologic Diseases and ³ Division of Endocrinology, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is thought to be caused in part by the age-related accumulation of amyloid -protein (A). The presence of neuritic plaques containing abundant A-derived amyloid fibrils in AD brain tissue supports the concept that fibril accumulation per se underlies neuronal dysfunction in AD. Recent observations have begun to challenge this assumption by suggesting that earlier A assemblies formed during the process of fibrillogenesis may also play a role in AD pathogenesis. Here, we present the novel finding that protofibrils (PF), metastable intermediates in amyloid fibril formation, can alter the electrical activity of neurons and cause neuronal loss. Both low molecular weight A (LMW A) and PF reproducibly induced toxicity in mixed brain cultures in a time- and concentration-dependent manner. No increase in fibril formation during the course of the experiments was observed by either Congo red binding or electron microscopy, suggesting that the neurotoxicity of LMW A and PF cannot be explained by conversion to fibrils. Importantly, protofibrils, but not LMW A, produced a rapid increase in EPSPs, action potentials, and membrane depolarizations. These data suggest that PF have inherent biological activity similar to that of mature fibrils. Our results raise the possibility that the preclinical and early clinical progression of AD is driven in part by the accumulation of specific A assembly intermediates formed during the process of fibrillogenesis.

Key words: Alzheimer; amyloid -protein; neurotoxicity; electrophysiology; fibrillogenesis; neurodegeneration

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Alzheimer's disease (AD) is a slowly progressive disorder at both the histopathological and clinical levels. Early symptoms of mild memory loss and minimal cognitive impairment lead gradually over 5-15 years to profound dementia and death. Biochemical and morphological studies suggest that clinical impairment in AD involves early synaptic dysfunction (Anderton et al., 1998; Cummings et al., 1998), followed by more severe neuronal changes that include increased synaptic loss, widespread neuritic dystrophy, neurofibrillary tangles, and frank neuronal death (Terry et al., 1991; Gómez-Isla et al., 1996; Sze et al., 1997; Anderton et al., 1998). The mechanism underlying the initiation of this progressive pathophysiology is thought to involve the age-related accumulation of amyloid -protein (A), which can form the abundant amyloid fibrils observed in neuritic plaques at autopsy (Esiri et al., 1997). The observation of these end-stage lesions in postmortem brain tissue has led to an assumption that accumulation of fibrils per se underlies the progression of AD. This impression has been supported in part by studies of neuronal cultures, in which progressive neurodegeneration can be induced by highly aggregated, fibrillar A but not by equivalent concentrations of A monomers (Mattson et al., 1993; Pike et al., 1993; Lorenzo and Yankner, 1994).

A is thought to start accumulating in vivo as low molecular weight species (LMW A) consisting principally of monomers that are constitutively secreted from brain cells. These may, under certain circumstances, progress to oligomers and ultimately to mature 7- to 10-nm-wide amyloid fibrils. A oligomers (dimers, trimers, tetramers, and possibly larger assemblies) have been identified in the conditioned media of certain cell lines that constitutively secrete A (Podlisny et al., 1995, 1998; Xia et al., 1997), and recently, in CSF (Pitschke et al., 1998). Fibril formation by synthetic A peptides is believed to proceed in vitro via a transition of LMW A to intermediate species that go on to form fibrils (Harper and Lansbury, 1997; Teplow, 1998). Recently, two laboratories identified such intermediates in the formation of synthetic A fibrils that are referred to as protofibrils (PF) (Harper et al., 1997a,b; Walsh et al., 1997).

In this paper, the electrophysiology and neurotoxicity of LMW A (monomers/dimers) and PF and their relationship to fibrillar A was investigated. We found that both of these earlier species reproducibly induce toxicity in cultured primary cortical neurons over a period of days. Furthermore, we show that submicromolar concentrations of PF can acutely increase the electrical activity of cortical neurons, whereas LMW A at the same concentrations elicits no electrophysiological response. Based on these and other data, we hypothesize that neuronal dysfunction is initiated by the formation of PF and that the PF can trigger neuronal loss directly and/or via their transition to higher MW species, including fibrils. Our model raises the possibility that the preclinical and early clinical progression of AD is driven, in part, by temporal changes in specific A assemblies formed during the process of fibrillogenesis. Elucidating the biological activity of PF should help to determine the role of early A intermediates in the mechanism of neuronal dysfunction in AD, with attendant therapeutic implications.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A preparation. Synthetic A peptides prepared as trifluoroacetic acid (TFA) salts are highly acidic, and care was taken to properly buffer the peptide to stabilize the effects of pH on morphology and neurotoxicity. A_1-40 (TFA salt) was purchased from Bachem (King of Prussia, PA; lot number ZN-571; 73.3% peptide). A at a concentration of 1 mM (4.3 mg/ml, based on total weight of peptide) was dissolved in 1 mM NaOH plus phenol red (0.1 mg/ml to monitor pH), pH ~3. To minimize isoelectric precipitation of A (pI 5.5), 10 mM NaOH (120-145 µl NaOH/mg of peptide) was added to achieve a rapid transition to a pH of ~7.0-7.5. The peptide was then further diluted to 500 µM in water and PBS (final concentration (in mM): 70 NaCl, 1.35 KCl, and 5 NaH₂PO4/Na₂HPO4). Fibrils were generated by incubating the peptide in a sealed tube at 37°C for 2-3 d. PF and LMW A were generated by incubating A peptide at room temperature (RT) for 2-3 d, centrifuging at 16,000 × g for 10 min, and then fractionated by size-exclusion chromatography (SEC) (see below), as previously described (Walsh et al., 1997).

Size-exclusion chromatography to isolate LMW A and PF. These species were isolated on a Superdex 75 SEC column using a Waters 650 Advanced Protein Purification System, as described (Walsh et al., 1997). However, for cell culture purposes, this procedure was modified by eluting the peaks with 70 mM NaCl and 5 mM Tris, pH 7.4 (TBS). Characterization by quasielastic light scattering and negative contrast electron microscopy (EM) indicated that LMW A and PF produced in this manner were indistinguishable from those previously reported (Walsh et al., 1997). As the LWM A and the PF peaks eluted from the column, each was collected in ~450 µl fractions, yielding four or five fractions per peak. Peptide content in individual fractions was quantified by amino acid analysis. The following tissue culture components were added to each fraction before applying them to cultures: MEM (1×); glucose (10 mM), penicillin streptomycin (500 U/ml and 500 µg/ml, respectively), HEPES (20 mM), and NaHCO₃ (26 mM) (all final concentrations).

Primary mixed brain cultures. Cultures were prepared according to Hartley et al. (1993), with slight modifications for rat tissue. Briefly, brain cells were isolated from the neocortex of embryonic day 15 (E15)-E17 rat embryos, plated at 200,000 cells/ml (or 1.3 × 10⁵ cells/cm²) in plating medium containing DMEM, 10% fetal bovine serum, Ham's F-12 (10%), HEPES (20 mM), glutamine (2 mM), and penicillin streptomycin (500 U/ml and 500 µg/ml, respectively) onto glial feeder layers in 48 well plates. Cultures were fed twice a week with this plating medium. After 9-10 d, cultures were inhibited with 10 µM ARA-C to stop glial growth and then changed into reduced-serum medium (plating medium with only 5% bovine calf serum). Upon initiation of all A toxicity experiments, the medium was completely removed and replaced with serum-free medium containing either no A, LMW A, PF, or fibrils (see previous section for final composition of toxicity medium).

The cultures, which contained neurons, astroglia, and microglia, were used after 3-4 weeks in vitro. Phase-contrast microscopy of typical cultures revealed phase-bright neurons resting on a darker, granular layer of astroglia. Neurons stained positively for the neuron-specific marker, microtubule-associated protein-2 (MAP-2), whereas the astroglia were positive for glial fibrillary acidic protein (GFAP). Microglia were identified by their uptake of rhodamine-conjugated low-density lipoprotein. These mixed brain cultures provided a useful model of cortical tissue, in that all major cell types were present, and neurons displayed appropriate electrophysiological activity (see Results).

Unless otherwise indicated, chemicals and tissue culture supplies were purchased from Sigma (St. Louis, MO) and Life Technologies (Gaithersburg, MD), respectively. Serum was purchased from Hyclone (Logan, UT).

Immunocytochemistry. Cultures were fixed with 4% paraformaldehyde in 0.15 M Na₂PO₄:KH₂PO₄, pH 7.4, for 20 min, washed, treated with 0.3% H₂O₂ in methanol for 30 min, and then blocked in blocking buffer (10% FBS, 0.3% Triton X-100, and 0.15 M Na₂PO₄:KH₂PO₄, pH 7.4). Primary antibodies were applied in this blocking buffer for 1 hr at RT using the following concentrations: anti-MAP2, 1:1000 (monoclonal, Sigma M4403); R1282, 1:750 (polyclonal) (Lemere et al., 1996). Primary antibody was visualized with anti-mouse or anti-rabbit biotinylated secondary antibodies bound to horseradish peroxidase or alkaline phosphatase, respectively, using an avidin-biotin kit (ABC Elite; Vector Laboratories, Burlingame, CA).

Cell death. Cell death was assessed visually by phase-contrast microscopy and quantitatively by measuring the release of the cytosolic enzyme, lactate dehydrogenase (LDH), into the medium, as previously described (Hartley et al., 1993).

Congo red binding. Congo red binding (CRB) was measured as described by Klunk et al. (1989), with slight modifications for reading in a microplate reader. Samples (25 µl) were mixed with 225 µl of 20 µM Congo red in 20 mM KH₂PO4 buffer, pH 7.4, and incubated at RT for 30 min. Absorbance at 480 and 540 nm were determined, and the amount of Congo red bound to fibrils was determined using the equation CRB (µmol/l) = A₅₄₀/25,295-A₄₈₀/46,306.

Search for fibrils by centrifugation and electron microscopy. Media of cultures treated with each of the three A assembly forms for 5 d were centrifuged for 20 min at 16,000 × g, and the pellets were resuspended in 10 µl of H₂0 and applied to EM grids. The grids were incubated with the samples for 20 min, rinsed with TBS, and blocked with 0.1% egg albumin for 30 min, all at RT. Polyclonal antibody R1282 to human A_1-40 was added at 1:400 to 1:800 dilution, incubated for 30 min at RT, and followed by washing. Goat anti-rabbit secondary antibodies conjugated to 10 nm gold particles were incubated for 30 min at RT, followed by rinsing. The proteins were then fixed to the grids with 2.5% glutaraldehyde for 5 min, and the grids were negatively stained with 2% uranyl acetate and then examined in a JEOL CX100 or a 1200LX electron microscope.

SDS-PAGE/Western blotting. Conditioned medium (15 µl) was mixed with 2× Tricine-SDS sample buffer (Novex, San Diego, CA), boiled for 5 min, and loaded onto 10-20% Tricine gels (Novex) and electrophoresed. Proteins were electroblotted for 2 hr at 400 mA (4°C) onto 0.2 µm polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). Membranes were blocked for 1 hr at RT with 5% milk in TBST, washed, and probed with A antibody R1282 (1:3000) in TBST for 1 hr (RT). After washing, a goat anti-rabbit secondary antibody conjugated to HRP was applied for 1 hr (RT). Immunopositive bands were visualized by chemiluminescence (ECL+Plus; Amersham, Arlington, IL), per manufacturer's instruction.

Electrophysiological studies. Patch-clamp electrophysiology in the cell-attached voltage-clamp (50 mV) mode was used for measuring EPSCs. Whole-cell and current-clamp mode was used for recording action potentials and membrane depolarizations. Mixed brain cultures (above) were perfused at RT in an extracellular bath solution containing (in mM): NaCl 140; KCl 4; MgCl₂ 1; CaCl₂ 1; HEPES 10; and Tris 5, pH 7.4. For cell-attached recordings, the pipette solution contained (in mM): NaCl 87; KCl 55; MgCl₂ 0.5; CaCl₂ 0.5; glucose 10; and HEPES 5, pH 7.4. The internal solution for whole-cell recordings contained (in mM): KCl 150; EGTA 10; MgCl₂ 1; HEPES 10; and Mg-ATP 3, pH 7.2 (using KOH). Patch pipettes (catalog #7052; Corning-Garner) were pulled and fire-polished to a tip diameter of <1 µm. High resistance (>10 G) seals were used, and currents were measured with an integrating patch-clamp amplifier (catalog #3900; Dagan). Freshly isolated LMW A and PF (see SEC section above) were used for electrophysiology experiments. To achieve the required sample volumes, the LMW A and PF peaks were not fractionated as described for neurotoxicity experiments (above); rather, the whole peak was collected as a single fraction. To these LMW A or PF peaks, stock electrophysiological buffer components were added to achieve the concentrations listed above (i.e., extracellular bath solution). Recordings were completed within 4-5 hr of peptide collection from the SEC. A preparations were held at 4°C until applied. Baseline recordings were monitored for 3-5 min before applying the A preparations, which were applied by adding an equal volume of the A preparation directly into the bath solution (no perfusion system was used).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Structural and biochemical characterization of LMW A and PF preparations

Two laboratories have previously isolated and characterized intermediate structures, termed protofibrils (PF), that are formed during the assembly of synthetic A into typical 4-8 nm amyloid fibrils (Harper et al., 1997a,b; Walsh et al., 1997). Using SEC, we separated and collected the PF as well as the LMW A_1-40 peptides from which the PF are derived (Fig. 1a). No structures could be detected in the LMW A fraction by EM. Quasielastic light-scattering spectroscopy has shown that LMW A has a hydrodynamic radius of 1.5-2.0 nm, too small to be resolved by EM (Walsh et al., 1997). EM of the PF showed individual, curvilinear structures of 4-11 nm diameter and <200 nm length, as previously described (Walsh et al., 1997) (Fig. 1b, PF). This appearance contrasted with that of mature fibrils, which were straighter and much longer (indeterminate length with a width of 6-10 nm) and often found to occur in dense mats of multiple fibrils (Fig. 1b, F).

View larger version (107K): [in this window] [in a new window]   Figure 1.   Flow diagram depicting generation of LMW A and PF and their structural differences. a, A was dissolved in NaOH:PBS, incubated for 2-3 d, centrifuged, and then injected onto a size-exclusion column. As the PF or LMW A peak emerged from the column, it was collected in fractions, to which tissue culture reagents were added and then applied to mixed brain cultures. Neuronal viability was monitored by phase-contrast microscopy and by measuring the release of LDH into the medium. b, Protofibrils (PF) were present as individual, dispersed, curvilinear structures of 4-11 nm diameter and <200 nm length. Mature fibrils (F) were 6-10 nm in diameter, much longer (indeterminate length; mean diameter, 8 nm), and often occurred in dense mats of multiple fibrils.

In addition to their EM characterization, LMW A, PF, and fibrils were analyzed by SDS-PAGE. Freshly prepared LMW A, PF, and fibrils were each diluted into the buffer used for neurotoxicity experiments (see Materials and Methods) and subjected to Western blotting with the A antibody R1282 (Lemere et al., 1996). Significant differences were observed among the preparations. The LMW A material showed only an ~4 kDa band (monomer) (Fig. 2a), whereas this band and two or three higher molecular weight bands (oligomers between 6-12 kDa) were detected in the PF material (Fig. 2b). The fibrillar material produced bands corresponding to monomer and oligomers and an additional band at the very top of the gel (Fig. 2b). Thus, a portion of fibrils does not enter the gel or forms a smear after entering the gel. Centrifuging the fibril preparation at 16,000 × g for 10 min yielded a pellet that contained solely the band at the top of the gel (Pike et al., 1993); no 4 kDa monomer or oligomers were observed (data not shown). It is important to note that the LMW A and PF are purified by SEC, whereas our standard A fibril preparations are mixed fractions similar to those used in numerous published A neurotoxicity studies, i.e., they were prepared by incubating ("aging") high concentrations of A (100-500 µM) at 37°C for several days. After denaturing in SDS, the latter preparations showed not only gel-excluded A-reactive material [i.e., SDS-insoluble fibrils (Pike et al., 1993)], but also A monomers and oligomers (Fig. 2c), demonstrating that fibril preparations contain a mixture of species. Our unfractionated fibril preparations thus contained LMW A, PF, and higher MW species and are similar, in this regard, to A preparations used by others for neurotoxicity experiments.

View larger version (48K): [in this window] [in a new window]   Figure 2.   Western blotting reveals electrophoretic differences between LMW A, PF, and fibril preparations. Freshly collected fractions of LMW A (a) and PF (b) from the size-exclusion column and preformed fibrils (c) were subjected to SDS-PAGE. Proteins were immunoblotted with an anti-A1-40 polyclonal antibody, R1282, and immunopositive bands were visualized by chemiluminescence. The numbers under each lane represent the concentrations of A in individual SEC fractions for LMW A and PF (and similar amounts of fibrils), as determined by amino acid analysis. Molecular weight of protein standards (×1000) are at right. This gel depicts one of three independent experiments that gave similar results.

Neuronal injury as a function of A assembly state

As the LMW A and PF peaks emerged from the SEC column, each peak was collected in four or five fractions of 450 µl. The A concentrations in these fractions varied from 5-53 µM, as determined by amino acid analysis, and these were usually within the concentration range of our fibril preparations, i.e., 7-42 µM. The PF and LMW SEC fractions were each individually added to wells of mixed brain cultures and incubated for up to 6 d. At ~24 hr intervals, aliquots of the conditioned media were collected and assayed for the release of intracellular LDH (see below). After termination of the experiment, the cultures were fixed and immunostained to assess A deposition and neuronal loss. Control cultures receiving no A showed little neuronal loss after 5 d, as judged by MAP2 immunostaining, (Fig. 3a, blue stain). In contrast, substantial neuronal loss was consistently observed after 5 d exposure to LMW A (Fig. 3b; 18 µM in experiment illustrated). The neuronal loss as judged by MAP2 immunostaining was concentration-dependent (data not shown; however, see LDH data below as a quantitative marker of cell injury). Similar results were obtained with PF; a majority of the neurons were lost in the presence of PF for 5 d (Fig. 3c; 21 µM in this experiment). The degree of cell death from either the LMW A or PF preparations approached that observed with fibrils (Fig. 3d; 28 µM in this experiment). With each of the three preparations, A deposition could be observed on and around the cells at day 5, as detected by the A-specific antibody, R1282 (Fig. 3b-d, red stain). However, no detectable difference in the light microscopic pattern of this A immunoreactivity could be observed among the three preparations. Cell death was primarily neuronal, as indicated by the clear loss of MAP2-stained neurons compared to the control (untreated) cultures, whereas the glial monolayer showed little or no cell loss by phase-contrast microscopy (Fig. 3). Neuronal loss in controls (untreated) cultures over the 5-6 d toxicity experiments was usually <10%, whereas the A-induced neuronal loss was usually >80%.

View larger version (144K): [in this window] [in a new window]   Figure 3.   Immunocytochemistry of neurons and of A in primary mixed cortical cultures exposed to various A preparations. Mixed cortical cultures were exposed to medium only (a), LMW A (b), PF (c), or fibrils (d) for 5 d, fixed and double-labeled for A deposition (R1282) (red staining) and neurons (MAP2) (blue staining). Significant neuron loss is observed with each of the A preparations (LMW A, 18 µM; PF, 21 µM; fibrils, 28 µM). Scale bar, 50 µm.

View larger version (63K): [in this window] [in a new window]   Figure 4.   Comparison of neurotoxicity induced by LMW A, PF, and fibrils. Various concentrations of LMW A, PF, and fibrils [µM = concentration of A applied (top numbers, x-axis)] were applied to mixed cortical cultures established 3-4 weeks earlier. Fraction number refers to the order the fractions were collected from either the LMW A or PF peak (bottom numbers, x-axis)(also see Fig. 1). After each day of treatment (z-axis), an aliquot of conditioned medium was analyzed for LDH release (y-axis). Values were normalized to blanks (0 µM A), which were given a value of 1. The graph represents one experiment, with each bar being the mean of duplicate assays; this graph is representative of four independent experiments in which four separate brain dissections were used (see Table 1 for comparison of the relative neurotoxicity of the preparations using LDH values from day 3).

We quantified LDH release induced by each of the three A preparations for all 6 d in vitro (Fig. 4). Consistent with the MAP2 immunostaining (above), concentrations of any of LMW A, PF, or fibrils at concentrations >7 µM all induced LDH release. The amount of LDH released was both time- and concentration-dependent. In general, day 3 (Fig. 4, red area) was the first day that a substantial rise in LDH could be detected, and on this day, LDH levels were significantly (p < 0.05) increased by the LMW A, PF, and fibril preparations versus controls (Table 1). The principal difference among the LMW A, PF, and fibril preparations was the earlier and more rapid induction of LDH release by the fibrils. In four separate experiments, the relative degree of LDH release by day 3 was similar for both the LMW A and PF cultures (each significantly higher than controls; p < 0.05), whereas that of fibrils was significantly (p < 0.05) higher than those of the LMW A and PF as well as the control cultures (Table 1).

View this table: [in this window] [in a new window]   Table 1.   LDH release from mature mixed brain cultures on day 3 of in vitro treatment with different A assemblies

Assaying for fibrillar A in the culture media by Congo red binding and EM

We performed several different kinds of experiments to determine whether the reproducible neurotoxicity induced by the LMW A and PF preparations (above) was caused by their gradual conversion to fibrils during the culture experiment or whether intermediate forms of A may themselves induce cell injury. One approach involved the use of CRB, which has been used to identify both synthetic and endogenous A fibrils (Klunk et al., 1989, 1999; Wood et al., 1996; Dickson, 1997). Conditioned media were collected at the end of the experiment (day 6) and assayed for CRB (Klunk et al., 1989). Fibril-containing cultures showed a concentration-dependent rise in CRB in their media: no binding was observed at 6 µM, whereas significant increases were detected at 13, 26, and 39 µM (Fig. 5a). In contrast, LMW A and PF at the highest concentrations used in these cultures (25 and 19 µM, respectively), produced no significant CRB. These results were observed consistently in three separate experiments examined for CRB: a clear dose response was observed for fibrillar A, but no significant CRB occurred in cultures treated with the SEC fractions containing LMW A or PF, despite their producing definite and significant neurotoxicity. Thus, the neuronal injury and loss produced by PF did not correlate with the development of significant CRB-positive fibrillar material during our experiments.

View larger version (125K): [in this window] [in a new window]   Figure 5.   Lack of detectable fibril formation in mixed brain cultures treated for 5-6 d with LMW A or PF. a, Mixed cortical cultures were exposed to LMW, PF, or preformed fibrils for 6 d, at which time the medium was removed and analyzed for fibril formation by Congo red binding. Each column is the mean of duplicate assays. Graph represents one of three identical experiments yielding similar results. b-d, Lack of fibril formation as determined by immuno-EM in 5 d conditioned media of LMW A (b), PF (c), or fibril (d) preparation. PF-treated media (c) predominantly contained large electron-dense mats of distinct protofibrils. In contrast, distinct fibrils (d) could readily be detected in the media of cultures treated with preformed fibrils. Results represent one of two experiments, in both of which few or no fibrils were detected in the media of LMW A and PF cultures.

Because the CRB assay may lack the sensitivity to detect small amounts of fibrillar A, we performed additional assays for the development of insoluble, fibrillar aggregates in the conditioned media using centrifugation and EM. The culture media of cultures conditioned for 5 d in the presence of LMW A, PF, or fibrils were centrifuged (16,000 × g, 20 min), and the pellets were examined by EM for fibril content. No fibrils were detected by EM in the cultures exposed to control (A-free) medium (data not shown) or LWM A (Fig. 5b). However, large mats of PF were readily observed in the pellets of the media of the PF-treated cultures, with occasional fibrils evident (Fig. 5c). Although evaluation by EM is only semiquantitative, there was a large difference in the ease with which fibrils were detected in the media pellets of cultures treated with fibrils (Fig. 5d) versus those treated with LMW A or PF. These data, in combination with the lack of Congo red binding (Fig. 5a), suggest that the consistent neurotoxicity obtained with the LMW A and PF is caused by intermediate forms of A, not to any substantial conversion to mature fibrils during our experiments.

Electrophysiological evidence of early neuronal alterations caused by protofibrils

In view of the above evidence that intermediate forms of A may have biological activity that can induce neuronal injury, we sought to confirm such activity at a time point so early and at concentrations so low that any progressive changes in the physical state of the A material were very unlikely to have occurred. To this end, we undertook electrophysiological recordings of our mixed brain cultures in the absence versus presence of either LMW A, PF, or fibrillar A. Three- to 4-week-old cultures containing both neurons and glia (indistinguishable from those used for the toxicity studies above) were examined in electrophysiological studies using the patch-clamp technique (see Materials and Methods). For these experiments, LMW A and PF were isolated by SEC just before their addition to the culture being recorded. The interval between the generation of LMW A and PF and their addition to the cultures was as brief as 30 min and always <5 hr. Once a stable baseline recording was established in the voltage-clamp mode, EPSCs were measured. In each case, the baseline was established for 4 min before the addition of an A preparation. The addition of LMW A at 3 µM produced either no increase or a very transient increase in EPSCs followed by a rapid return to baseline (Fig. 6a, LMW). Concentrations approaching 20 µM (i.e., neurotoxic) of LMW A still showed no increase in EPSCs (data not shown). In sharp contrast, addition of either PF or fibrils at ~3 µM concentrations invariably produced a rapid and sustained increase in electrical activity (Fig. 6a, PF, Fibrils). Each trace represents a recording from a single cell. Traces in Figure 6a were then graphed as the number of EPSCs per minute versus time (Fig. 6b). The normalized mean values (±SEM) of the EPSCs per minute for the first and the eighth minute of treatment with PF (6.4 ± 1.4 and 7.0 ± 1.3, respectively; n = 8) and fibrils (6.2 ± 1.8 and 7.4 ± 1.5, respectively; n = 5) were found to be approximately sixfold greater (p < 0.05) than that of LMW A (1.0 ± 0.34; n = 7) (Fig. 6c). PF and fibrils were both statistically different from medium only and LMW A, but no difference was found between the PF and fibril preparations. This neuronal activation was concentration-dependent for both the PF and fibrils, with EC₅₀ values of 760 and 560 nM, respectively (data not shown). Interestingly, at concentrations >2 µM, no further increase in activation was observed. The lowest A concentrations that caused an increase in EPSCs during an 8-10 min monitoring period were ~144 nM for PF and ~300 nM for fibrils.

View larger version (48K): [in this window] [in a new window]   Figure 6.   Sustained increases in EPSCs caused by PF and fibrils but not LMW A. Cell-attached patch-clamp mode was used to record inward EPSCs in voltage-clamp mode. a, Examples of current traces are shown in left panels. Baseline activities were recorded for 4 min (the ends of which periods are shown as dotted lines). Addition of LMW A (a, LMW, solid line) at a concentration of 3 µM caused only a brief, transient increase in EPSCs compared with the preceding control period. In contrast, addition of PF (3-5 µM) (a, PF) or fibrils (3-5 µM) (a, Fibrils, solid lines) caused a rapid, sustained increase in EPSCs. b, The number of EPSCs per minute plotted versus time for the experiment shown in a. Each plot in b graphs data from a representative single cell exposed to one of the three A preparations. Data from multiple experiments (c) were then compiled by normalizing the EPSCs per minute to the basal activity, which was given a value of one. The normalized mean EPSC values (5-8 experiments; mean ± SEM) for the basal, first, and eighth minute revealed a significant increase in EPSCs with PF and fibril application compared with the basal or LMW A activity (c). PF and fibrils were found to be statistically different from both medium alone and LMW A (p < 0.05); no significant difference between the PF and fibril preparations was observed.

To further evaluate the effects of PF on the electrical activity of neurons, we investigated the relative change of action potentials (APs) and membrane depolarizations (MDs) in the presence of LMW A, PF, and fibrils. Whole-cell recording in current-clamp mode was used to monitor the baseline activity for 3-5 min. Freshly prepared LMW A (3.9 µM), PF (2.2 µM), or preformed fibrils (0.75 µM) were then applied to the mixed brain cultures (Fig. 7). Application of either PF or fibrils increased the number of APs per minute by 2.59 ± 0.24-fold (n = 6) or 3.04 ± 1.03-fold (n = 8), respectively, versus the values of controls [i.e., no A (normalized to 1)] or LMW A (1.15 ± 0.10, n = 4) (Fig. 7). The values for PF and fibrils were found to be statistically different from control and LMW A (p < 0.05), but not from each other. Furthermore, increased frequencies and larger sizes of MDs were observed after application of PF or fibrils compared to controls or LMW A (Fig. 7, arrows; compare MDs of control or LMW A vs PF or fibrils). These data suggest that a consequence of the increase in EPSCs is an increase in the rates of APs and large membrane depolarizations, which should have a significant impact on the physiology of the cell because of the large ion fluxes that occur.

View larger version (26K): [in this window] [in a new window]   Figure 7.   Increased frequency of action potentials and membrane depolarizations caused by PF but not LMW A. Whole-cell recordings in current-clamp mode were used to measure APs (single sharp deflections) and MDs (arrows). Baseline activities were recorded for 3-5 min (dotted lines). Addition of LMW A (solid line) produced no significant increase in APs and MDs, whereas PF and fibrils (solid lines) increased the frequency of APs and the amplitude of MDs.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this study, we have compared the biological effects of different A assemblies on primary mixed brain cultures to determine whether nonfibrillar or immature, prefibrillar forms of A may be neurotoxic. This potentially important source of neuronal injury has been overlooked until very recently, perhaps because the neuropathological diagnosis of AD requires the presence of abundant fibrillar amyloid in the form of myriad neuritic plaques in postmortem brain tissue. Because mature amyloid plaques surrounded by dystrophic neurites, activated microglia, and reactive astrocytes are composed principally of A fibrils, it has been generally assumed that fibrillar A is the form most likely to be responsible for neuronal and glial injury in AD. The apparent importance of amyloid fibrils has been reinforced by cell culture studies that consistently show that the aggregation state of A, most notably the formation of amyloid fibrils, is associated with neuronal alteration and loss (Pike et al., 1991, 1993; Mattson et al., 1993; Lorenzo and Yankner, 1994; Estus et al., 1997; Seilheimer et al., 1997). Therefore, in vitro toxicity and human neuropathological studies have heretofore not provided clear evidence that intermediate species formed during the A fibrillogenesis process play a significant role in neuronal injury and death.

Based on the results of the experiments reported here, we hypothesize that nonfibrillar or immature fibrillar A species have specific biological activities that may underlie, at least in part, the slowly progressive neuronal/synaptic changes that occur in AD. In support of this hypothesis, we present the novel finding that a recently described protofibrillar intermediate formed during fibrillogenesis of synthetic A (Harper et al., 1997b; Walsh et al., 1997), consistently and significantly alters the electrical activity of neurons as measured by EPSCs, action potentials, and membrane depolarization at biologically relevant concentrations. Interestingly, electrophysiological effects of PF and mature fibrils were similar, because both increased membrane conductance and depolarizations. In contrast, LMW A, consisting solely of monomers and/or dimers, had no significant electrophysiological effect at the same concentration. These results suggest that PFs have biological activity that mimics in part the ability of mature amyloid fibrils to alter ion fluxes across membranes (Mark et al., 1992; Mattson et al., 1992; Arispe et al., 1993; Wu et al., 1995; Kawahara et al., 1997; Ye et al., 1997).

The work reported here is consistent with the recent observation that another soluble, diffusible form of synthetic A, referred to as A-derived diffusible ligands (ADDLs), can alter the electrical activity of neurons, observed as an attenuation of long-term potentiation (LTP) (Lambert et al., 1998). However, our rapid increase in electrical activity (e.g., EPSCs), would not necessarily be associated with a reduction in LTP. These differences in effects are currently being investigated, but it is of interest to note that the structural and biochemical features of ADDLs appear to be distinct from those of PF. First, the ADDLs are much smaller, globular structures (~5 nm diameter), compared to the curvilinear PF that have an average length of 25 nm and lengths as high as 200 nm. Second, SDS-PAGE of fresh preparations suggests that ADDLs contain two principal A species, 17 and 22 kDa, whereas our PF yield two or three oligomeric bands of ~6, 8, and 12 kDa. This physical disparity between PF and ADDLs supports the concept that different A assemblies have distinct neurobiological activities, which may be manifested differently using an electrophysiological readout.

In support of the hypothesis that intermediate species formed during fibrillogenesis may play an important role in the disease process are the findings that some transgenic mice overexpressing human APP have shown altered behavior and/or electrophysiological responses before or during the initiation of A plaque formation, before any substantial histological lesions or neuronal pathology were observed (Holcomb et al., 1998; Chapman et al., 1999; Hsia et al., 1999). The decline in synaptic activity in many of these animal models appears to take several months to develop (Hsiao et al., 1996; Holcomb et al., 1998; Chapman et al., 1999; Hsia et al., 1999), perhaps reflecting a gradual increase in A levels to a concentration that allows formation of intermediates that noticeably alter neuronal activity. In humans, alterations in neuronal physiology caused by PF or other A oligomeric intermediates could underlie the earliest mild amnesiac symptoms observed in elderly subjects with minimal cognitive impairment (Morris et al., 1996), as observed in the above mouse models (Holcomb et al., 1998; Chapman et al., 1999).

Recent data also suggest that intermediate species formed during A fibrillogenesis could not only be responsible for early synaptic dysfunction, but may contribute to frank cell death. For example, an A complex sedimenting more slowly than amyloid fibrils was shown to have a neurotoxic effect on cultured PC12 cells, as quantified by the MTT conversion assay (Oda et al., 1995). In addition, -1-antichymotrypsin is capable of blocking fibril formation without decreasing A-induced neurotoxicity (Aksenova et al., 1996). A soluble A species isolated from AD cerebral cortex that kills neurons in mixed brain cultures has been identified (Giulian et al., 1996; Roher et al., 1996). Furthermore, the ADDLs mentioned above can also induce neuronal death (Lambert et al., 1998).

These various data are consistent with our findings that neurons exposed to LMW A or PF show evidence of progressive injury leading to frank cell loss. This effect was similar to that observed with A fibrils, although fibrillar preparations generally produced a more rapid injury. Interestingly, we found that LMW A induced neuronal death at similar concentrations and time points. LMW A could either progress in vitro to PF that cause the neurotoxicity or it could induce neurotoxicity by a different mechanism than PF, as supported by the fact that LMW A did not acutely activate ion channels (this study) or cause the reduction of MTT (Walsh et al., 1999).

Because the induction of neurotoxicity in our cultures took 3-5 d in vitro, a major concern was that LMW A and PF were transitioning to fibrils, and these caused the neuronal injury. However, several observations suggest that neither of these A assembly forms principally converted to fibrils. Conditioned media from the LMW A and PF cultures (day 5-6) showed no rise in Congo red binding, whereas media exposed to fibrils from time 0 showed a dose-dependent recovery of Congo red-positive (fibrillar) A. EM analysis of day 5-6 media that were centrifuged to concentrate any fibrils revealed few or no detectable fibrils in the LMW A and PF cultures. These data clearly suggest that it is not the formation of fibrils in our LMW A and PF experiments that explains the reproducible and profound neurotoxicity that these preparations induce. Furthermore, we have found by another relatively rapid biological readout, MTT reduction, that PF can directly alter cortical neurons biochemically, presumably before significant formation of fibrils occurs (Walsh et al., 1999). Finally, and most importantly, the almost instantaneous and highly reproducible enhancement in electrical activity of neurons by freshly prepared PF strongly suggests that PF can initiate a cell death process or render neurons more vulnerable to further insult without having to age to mature fibrils.

An important goal of our future work will be to search for the existence of protofibril-like assemblies of A in vivo. In this regard, we have observed stable oligomers (dimers, trimers) of natural A in the conditioned media of some cells overexpressing APP (Podlisny et al., 1995, 1998). Their potential pathological relevance was shown by observing an increase in the amounts of such A_1-42 oligomers when AD-causing presenilin 1 or 2 mutations, which increase A_1-42 secretion, are coexpressed with APP (Xia et al., 1997). These findings suggest that natural A under physiological conditions (nanomolar concentrations) can form oligomeric species, which could then seed the aggregation process as cerebral A levels begin to rise with age in AD (Harper and Lansbury, 1997; Teplow, 1998). This hypothesis is consistent with fluorescence correlation spectroscopy study that identified soluble higher order, A aggregates in the CSF of AD patients (Pitschke et al., 1998). Furthermore, soluble oligomeric A, primarily A_1-42, has been shown to be elevated in AD cortex compared to age-matched control subjects (Kuo et al., 1996). Similarly, specific increases principally in A_1-42 rather than A_1-40 have been observed in other studies of AD brain tissue (Gravina et al., 1995) or transgenic mice expressing mutant human APP (Johnson-Wood et al., 1997). Even though the A aggregation state was not determined in the latter studies, the documented rise in A_1-42 would increase the probability of aggregation occurring.

In summary, we have shown that soluble, intermediate forms of A assemblies, particularly PF, can induce acute as well as delayed neurotoxic effects in the absence of significant conversion to fibrils. If oligomeric A species resembling PF are present in human brain tissue before or during neuritic plaque (i.e., amyloid fibril) formation, the PF-like assemblies could well have a greater neuropathological impact than amyloid fibrils. As fibrils clump and become sequestered in glial-rich neuritic plaques, their ability to interact with surrounding cells may decrease. Soluble assemblies could have a more detrimental effect than mature fibrils because of their diffusibility. Based on the results reported here, identifying the presence of PF in vivo and determining their concentrations and half-life will be the next critical step in assessing the importance of prefibrillar species in AD. From a therapeutic point of view, blocking formation of mature fibrils could be harmful if toxic intermediates resembling those described here are allowed to accumulate.

	FOOTNOTES

Received June 18, 1999; revised Aug. 2, 1999; accepted Aug. 9, 1999.

This work was supported by National Institutes of Health Grants AG12749 and AG06173 (D.J.S.), AG00891 (C.P.Y.), and AG14266 and NS38328 (D.B.T.). We thank Margaret Condron for expert technical assistance.

Correspondence should be addressed to Dean M. Hartley or Dennis J. Selkoe, Center for Neurologic Diseases, Harvard Institutes of Medicine, Room 740, 77 Avenue Louis Pasteur, Boston, MA 02115. 

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

• Aksenova MV, Aksenov MY, Butterfield DA, Carney JM (1996) -1-antichymotrypsin interaction with A (1-40) inhibits fibril formation but does not affect the peptide toxicity. Neurosci Lett 211:45-48[Web of Science][Medline].
• Anderton BH, Callahan L, Coleman P, Davies P, Flood D, Jicha GA, Ohm T, Weaver C (1998) Dendritic changes in Alzheimer's disease and factors that may underlie these changes. Prog Neurobiol 55:595-609[Web of Science][Medline].
• Arispe N, Pollard HB, Rojas E (1993) Giant multilevel cation channels formed by Alzheimer disease amyloid -protein [A P-(1-40)] in bilayer membranes. Proc Natl Acad Sci USA 90:10573-10577[Abstract/Free Full Text].
• Chapman PF, White GL, Jones MW, Cooper-Blacketer D, Marshall VJ, Irizarry M, Younkin L, Good MA, Bliss TV, Hyman BT, Younkin SG, Hsiao KK (1999) Impaired synaptic plasticity and learning in aged amyloid precursor protein transgenic mice. Nat Neurosci 2:271-276.[Web of Science][Medline]
• Cummings JL, Vinters HV, Cole GM, Khachaturian ZS (1998) Alzheimer's disease: etiologies, pathophysiology, cognitive reserve, and treatment opportunities. Neurology 51:S2-S17[Abstract/Free Full Text].
• Dickson DW (1997) The pathogenesis of senile plaques. J Neuropathol Exp Neurol 56:321-339[Web of Science][Medline].
• Esiri M, Hyman B, Beyreuther K, Masters C (1997) Aging and dementia. In: Greenfield's neuropathology, Vol 2 (Graham D, Lantos P, eds), pp 153-233. London: Arnold.
• Estus S, Tucker HM, van Rooyen C, Wright S, Brigham EF, Wogulis M, Rydel RE (1997) Aggregated amyloid- protein induces cortical neuronal apoptosis and concomitant apoptotic pattern of gene induction. J Neurosci 17:7736-7745[Abstract/Free Full Text].
• Giulian D, Haverkamp LJ, Yu JH, Karshin W, Tom D, Li J, Kirkpatrick J, Kuo LM, Roher AE (1996) Specific domains of -amyloid from Alzheimer plaque elicit neuron killing in human microglia. J Neurosci 16:6021-6037[Abstract/Free Full Text].
• Gómez-Isla T, Price JL, McKeel Jr DW, Morris JC, Growdon JH, Hyman BT (1996) Profound loss of layer II entorhinal cortex neurons occurs in very mild Alzheimer's disease. J Neurosci 16:4491-4500[Abstract/Free Full Text].
• Gravina SA, Ho L, Eckman CB, Long KE, Otvos Jr L, Younkin LH, Suzuki N, Younkin SG (1995) Amyloid  protein (A) in Alzheimer's disease brain. Biochemical and immunocytochemical analysis with antibodies specific for forms ending at A 40 or A 42(43). J Biol Chem 270:7013-7016[Abstract/Free Full Text].
• Harper JD, Lansbury Jr PT (1997) Models of amyloid seeding in Alzheimer's disease and scrapie: mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins. Annu Rev Biochem 66:385-407[Web of Science][Medline].
• Harper JD, Wong SS, Lieber CM, Lansbury Jr PT (1997a) Observation of metastable A amyloid protofibrils by atomic force microscopy. Chem Biol 4:119-125[Web of Science][Medline].
• Harper JD, Lieber CM, Lansbury Jr PT (1997b) Atomic force microscopic imaging of seeded fibril formation and fibril branching by the Alzheimer's disease amyloid- protein. Chem Biol 4:951-959[Web of Science][Medline].
• Hartley DM, Kurth MC, Bjerkness L, Weiss JH, Choi DW (1993) Glutamate receptor-induced ⁴⁵Ca²⁺ accumulation in cortical cell culture correlates with subsequent neuronal degeneration. J Neurosci 13:1993-2000[Abstract].
• Holcomb L, Gordon MN, McGowan E, Yu X, Benkovic S, Jantzen P, Wright K, Saad I, Mueller R, Morgan D, Sanders S, Zehr C, O'Campo K, Hardy J, Prada CM, Eckman C, Younkin S, Hsiao K, Duff K (1998) Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes. Nat Med 4:97-100[Web of Science][Medline].
• Hsia A, Masliah E, McConlogue L, Yu G, Tatsuno g, Hu K, Kholodenko D, Malenka R, Nicoll R, Mucke L (1999) Plaque-independent disruption of neural circuits in Alzheimer's disease mouse models. Proc Natl Acad Sci USA 96:3228-3233[Abstract/Free Full Text].
• Hsiao K, Chapman P, Nilsen S, Ekman C, Harigaya Y, Younkin S, Yang F, Cole G (1996) Correlative memory deficits, A elevation, and amyloid plaques in transgenic mice. Science 274:99-102[Abstract/Free Full Text].
• Johnson-Wood K, Lee M, Motter R, Hu K, Gordon G, Barbour R, Khan K, Gordon M, Tan H, Games D, Lieberburg I, Schenk D, Seubert P, McConlogue L (1997) Amyloid precursor protein processing and A42 deposition in a transgenic mouse model of Alzheimer disease. Proc Natl Acad Sci USA 94:1550-1555[Abstract/Free Full Text].
• Kawahara M, Arispe N, Kuroda Y, Rojas E (1997) Alzheimer's disease amyloid -protein forms Zn(2+)-sensitive, cation-selective channels across excised membrane patches from hypothalamic neurons. Biophys J 73:67-75[Web of Science][Medline].
• Klunk WE, Pettegrew JW, Abraham DJ (1989) Quantitative evaluation of congo red binding to amyloid-like proteins with a -pleated sheet conformation. J Histochem Cytochem 37:1273-1281[Abstract/Free Full Text].
• Klunk WE, Jacob RF, Mason RP (1999) Quantifying amyloid -peptide (A) aggregation using the Congo red-A (CR-a) spectrophotometric assay. Anal Biochem 266:66-76[Web of Science][Medline].
• Kuo Y-M, Emmerling MR, Vigo-Pelfrey C, Kasunic TC, Kirkpatrick JB, Murdoch GH, Ball MJ, Roher AE (1996) Water-soluble A (N-40, N-42) oligomers in normal and Alzheimer disease brains. J Biol Chem 271:4077-4081[Abstract/Free Full Text].
• Lambert MP, Barlow AK, Chromy BA, Edwards C, Freed R, Iosatos M, Morgan TE, Rozovsky I, Trommer B, Viola KL, Wals P, Zhang C, Finch CE, Krafft GA, Klein WL (1998) Diffusible, nonfibrillar ligands derived from A_1-42 are potent central nervous system neurotoxins. Proc Natl Acad Sci USA 95:6448-6453[Abstract/Free Full Text].
• Lemere CA, Blustzjan JK, Yamaguchi H, Wisniewski T, Saido TC, Selkoe DJ (1996) Sequence of deposition of heterogeneous amyloid -peptides and Apo E in Down syndrome: implications for initial events in amyloid plaque formation. Neurobiol Dis 3:16-32[Web of Science][Medline].
• Lorenzo A, Yankner B (1994) -amyloid neurotoxicity requires fibril formation and is inhibited by Congo red. Proc Natl Acad Sci USA 91:12243-12247[Abstract/Free Full Text].
• Mark RJ, Hensley K, Butterfield DA, Mattson MP (1992) Amyloid -peptide impairs ion-motive ATPase activities: evidence for a role in loss of neuronal Ca²⁺ homeostasis and cell death. J Neurosci 15:6239-6249[Abstract].
• Mattson MP, Cheng B, Davis D, Bryant K, Lieberburg I, Rydel RE (1992) -Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity. J Neurosci 12:379-389.
• Mattson MP, Tomaselli KJ, Rydel RE (1993) Calcium-destabilizing and neurodegenerative effects of aggregated -amyloid peptide are attenuated by basic FGF. Brain Res 621:35-49[Web of Science][Medline].
• Morris JC, Storandt M, McKeel Jr DW, Rubin EH, Price JL, Grant EA, Berg L (1996) Cerebral amyloid deposition and diffuse plaques in "normal" aging: evidence for presymptomatic and very mild Alzheimer's disease. Neurology 46:707-719[Free Full Text].
• Oda T, Wals P, Osterburg HH, Johnson SA, Pasinetti GM, Morgan TE, Rozovsky I, Stine WB, Snyder SW, Holzman TF, Krafft GA, Finch CE (1995) Clusterin (apoJ) alters the aggregation of amyloid -peptide (A 1-42) and forms slowly sedimenting A complexes that cause oxidative stress. Exp Neurol 136:22-31[Web of Science][Medline].
• Pike CJ, Walencewicz AJ, Glabe CG, Cotman CW (1991) In vitro aging of -amyloid protein causes peptide aggregation and neurotoxicity. Brain Res 563:311-314[Web of Science][Medline].
• Pike CJ, Burdick D, Walencewicz AJ, Glabe CG, Cotman CW (1993) Neurodegeneration induced by -amyloid peptides in vitro: the role of peptide assembly state. J Neurosci 13:1676-1687[Abstract].
• Pitschke M, Prior R, Haupt M, Riesner D (1998) Detection of single amyloid B-protein aggregates in the cerebrospinal fluid of Alzheimer's patients by fluorescence correlation spectroscopy. Nat Med 4:832-834[Web of Science][Medline].
• Podlisny MB, Ostaszewski BL, Squazzo SL, Koo EH, Rydel RE, Teplow DB, Selkoe DJ (1995) Aggregation of secreted amyloid -protein into SDS-stable oligomers in cell culture. J Biol Chem 270:9564-9570[Abstract/Free Full Text].
• Podlisny MB, Walsh DM, Amarante P, Ostaszewski BL, Stimson ER, Maggio JE, Teplow DB, Selkoe DJ (1998) Oligomerization of endogenous and synthetic amyloid -protein at nanomolar levels in cell culture and stabilization of monomer by Congo red. Biochemistry 37:3602-3611[Medline].
• Roher AE, Chaney MO, Kuo Y-M, Webster SD, Stine WB, Haverkamp LJ, Woods AS, Cotter RJ, Tuohy JM, Krafft GA, Bonnell BS, Emmerling MR (1996) Morphology and toxicity of A-(1-42) dimer derived from neuritic and vascular amyloid deposits of Alzheimer's disease. J Biol Chem 271:20631-20635[Abstract/Free Full Text].
• Seilheimer B, Bohrmann B, Bondolfi L, Müller F, Stüber D, Döbeli H (1997) The toxicity of the Alzheimer's -amyloid peptide correlates with a distinct fiber morphology. J Struct Biol 119:59-71[Web of Science][Medline].
• Sze CI, Troncoso JC, Kawas C, Mouton P, Price DL, Martin LJ (1997) Loss of the presynaptic vesicle protein synaptophysin in hippocampus correlates with cognitive decline in Alzheimer disease. J Neuropathol Exp Neurol 56:933-944[Web of Science][Medline].
• Teplow DB (1998) Structural and kinetic features of amyloid -protein fibrillogenesis. Amyloid 5:121-42[Web of Science][Medline].
• Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, Hill R, Hansen LA, Katzman R (1991) Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is the major correlate of cognitive impairment. Ann Neurol 30:572-580[Web of Science][Medline].
• Walsh DM, Hartley DM, Kusumoto Y, Fezoui Y, Condron MM, Lomakin A, Benedek GB, Selkoe DJ, Teplow DB (1999) Amyloid -protein fibrillogenesis: structure and biological activity of protofibrillar intermediates. J Biol Chem 274:25945-25952[Abstract/Free Full Text].
• Walsh DM, Lomakin A, Benedek GB, Maggio JE, Condron MM, Teplow DB (1997) Amyloid -protein fibrillogenesis: detection of a protofibrillar intermediate. J Biol Chem 272:22364-22374[Abstract/Free Full Text].
• Wood SJ, Maleeff B, Hart T, Wetzel R (1996) Physical, morphological and functional differences between pH 5.8 and 7.4 aggregates of the Alzheimer's amyloid peptide A. J Mol Biol 256:870-877[Web of Science][Medline].
• Wu J, Anwyl R, Rowan MJ (1995) -Amyloid selectively augments NMDA receptor-mediated synaptic transmission in rat hippocampus. NeuroReport 6:2409-2413[Web of Science][Medline].
• Xia W, Zhang J, Kholodenko D, Citron M, Podlisny MB, Teplow DB, Haas C, Seubert P, Koo EH, Selkoe DJ (1997) Enhanced production and oligomerization of the 42-residue amyloid -protein by Chinese hamster ovary cells stably expressing mutant presenilins. J Biol Chem 272:7977-7982[Abstract/Free Full Text].
• Ye C, Ho-Pao CL, Kanazirska M, Quinn S, Rogers K, Seidman CE, Seidman JG, Brown EM, Vassilev PM (1997) Amyloid- proteins activate Ca(2+)-permeable channels through calcium-sensing receptors. J Neurosci Res 47:547-554[Web of Science][Medline].

---

Copyright © 1999 Society for Neuroscience  0270-6474/99/19208876-09$05.00/0

This article has been cited by other articles:

				S. K. Maji, R. R. Ogorzalek Loo, M. Inayathullah, S. M. Spring, S. S. Vollers, M. M. Condron, G. Bitan, J. A. Loo, and D. B. Teplow Amino Acid Position-specific Contributions to Amyloid {beta}-Protein Oligomerization J. Biol. Chem., August 28, 2009; 284(35): 23580 - 23591. [Abstract] [Full Text] [PDF]

				M. W. van der Kamp and V. Daggett The consequences of pathogenic mutations to the human prion protein Protein Eng. Des. Sel., August 1, 2009; 22(8): 461 - 468. [Abstract] [Full Text] [PDF]

				C. Nerelius, A. Sandegren, H. Sargsyan, R. Raunak, H. Leijonmarck, U. Chatterjee, A. Fisahn, S. Imarisio, D. A. Lomas, D. C. Crowther, et al. {alpha}-Helix targeting reduces amyloid-{beta} peptide toxicity PNAS, June 9, 2009; 106(23): 9191 - 9196. [Abstract] [Full Text] [PDF]

				Z. Liu, R. K. Meray, T. N. Grammatopoulos, R. A. Fredenburg, M. R. Cookson, Y. Liu, T. Logan, and P. T. Lansbury Jr. Membrane-associated farnesylated UCH-L1 promotes {alpha}-synuclein neurotoxicity and is a therapeutic target for Parkinson's disease PNAS, March 24, 2009; 106(12): 4635 - 4640. [Abstract] [Full Text] [PDF]

				S. E. Perez, S. Lumayag, B. Kovacs, E. J. Mufson, and S. Xu {beta}-Amyloid Deposition and Functional Impairment in the Retina of the APPswe/PS1{Delta}E9 Transgenic Mouse Model of Alzheimer's Disease Invest. Ophthalmol. Vis. Sci., February 1, 2009; 50(2): 793 - 800. [Abstract] [Full Text] [PDF]

				L. W. Hung, G. D. Ciccotosto, E. Giannakis, D. J. Tew, K. Perez, C. L. Masters, R. Cappai, J. D. Wade, and K. J. Barnham Amyloid-{beta} Peptide (A{beta}) Neurotoxicity Is Modulated by the Rate of Peptide Aggregation: A{beta} Dimers and Trimers Correlate with Neurotoxicity J. Neurosci., November 12, 2008; 28(46): 11950 - 11958. [Abstract] [Full Text] [PDF]

				A. Jan, O. Gokce, R. Luthi-Carter, and H. A. Lashuel The Ratio of Monomeric to Aggregated Forms of A{beta}40 and A{beta}42 Is an Important Determinant of Amyloid-{beta} Aggregation, Fibrillogenesis, and Toxicity J. Biol. Chem., October 17, 2008; 283(42): 28176 - 28189. [Abstract] [Full Text] [PDF]

				J. B. Paulson, M. Ramsden, C. Forster, M. A. Sherman, E. McGowan, and K. H. Ashe Amyloid Plaque and Neurofibrillary Tangle Pathology in a Regulatable Mouse Model of Alzheimer's Disease Am. J. Pathol., September 1, 2008; 173(3): 762 - 772. [Abstract] [Full Text] [PDF]

				X. Wang, N. D. Hammer, and M. R. Chapman The Molecular Basis of Functional Bacterial Amyloid Polymerization and Nucleation J. Biol. Chem., August 1, 2008; 283(31): 21530 - 21539. [Abstract] [Full Text] [PDF]

				E. Herczenik and M. F. B. G. Gebbink Molecular and cellular aspects of protein misfolding and disease FASEB J, July 1, 2008; 22(7): 2115 - 2133. [Abstract] [Full Text] [PDF]

				D. M. Hartley, C. Zhao, A. C. Speier, G. A. Woodard, S. Li, Z. Li, and T. Walz Transglutaminase Induces Protofibril-like Amyloid {beta}-Protein Assemblies That Are Protease-resistant and Inhibit Long-term Potentiation J. Biol. Chem., June 13, 2008; 283(24): 16790 - 16800. [Abstract] [Full Text] [PDF]

				I. H. Cheng, K. Scearce-Levie, J. Legleiter, J. J. Palop, H. Gerstein, N. Bien-Ly, J. Puolivali, S. Lesne, K. H. Ashe, P. J. Muchowski, et al. Accelerating Amyloid-beta Fibrillization Reduces Oligomer Levels and Functional Deficits in Alzheimer Disease Mouse Models J. Biol. Chem., August 17, 2007; 282(33): 23818 - 23828. [Abstract] [Full Text] [PDF]

				Y. Hori, T. Hashimoto, Y. Wakutani, K. Urakami, K. Nakashima, M. M. Condron, S. Tsubuki, T. C. Saido, D. B. Teplow, and T. Iwatsubo The Tottori (D7N) and English (H6R) Familial Alzheimer Disease Mutations Accelerate Abeta Fibril Formation without Increasing Protofibril Formation J. Biol. Chem., February 16, 2007; 282(7): 4916 - 4923. [Abstract] [Full Text] [PDF]

				L. Fioriti, N. Angeretti, L. Colombo, A. De Luigi, A. Colombo, C. Manzoni, M. Morbin, F. Tagliavini, M. Salmona, R. Chiesa, et al. Neurotoxic and Gliotrophic Activity of a Synthetic Peptide Homologous to Gerstmann-Straussler-Scheinker Disease Amyloid Protein J. Neurosci., February 14, 2007; 27(7): 1576 - 1583. [Abstract] [Full Text] [PDF]

				N. Yamamoto, E. Matsubara, S. Maeda, H. Minagawa, A. Takashima, W. Maruyama, M. Michikawa, and K. Yanagisawa A Ganglioside-induced Toxic Soluble Abeta Assembly: ITS ENHANCED FORMATION FROM AbetaBEARING THE ARCTIC MUTATION J. Biol. Chem., January 26, 2007; 282(4): 2646 - 2655. [Abstract] [Full Text] [PDF]

				P. N. Lacor, M. C. Buniel, P. W. Furlow, A. Sanz Clemente, P. T. Velasco, M. Wood, K. L. Viola, and W. L. Klein A{beta} Oligomer-Induced Aberrations in Synapse Composition, Shape, and Density Provide a Molecular Basis for Loss of Connectivity in Alzheimer's Disease J. Neurosci., January 24, 2007; 27(4): 796 - 807. [Abstract] [Full Text] [PDF]

				A. Sanbe, J. Yamauchi, Y. Miyamoto, Y. Fujiwara, M. Murabe, and A. Tanoue Interruption of CryAB-Amyloid Oligomer Formation by HSP22 J. Biol. Chem., January 5, 2007; 282(1): 555 - 563. [Abstract] [Full Text] [PDF]

				T. Muller, C. G. Concannon, M. W. Ward, C. M. Walsh, A. L. Tirniceriu, F. Tribl, D. Kogel, J. H.M. Prehn, and R. Egensperger Modulation of Gene Expression and Cytoskeletal Dynamics by the Amyloid Precursor Protein Intracellular Domain (AICD) Mol. Biol. Cell, January 1, 2007; 18(1): 201 - 210. [Abstract] [Full Text] [PDF]

				M. A. Abad, M. Enguita, N. DeGregorio-Rocasolano, I. Ferrer, and R. Trullas Neuronal Pentraxin 1 Contributes to the Neuronal Damage Evoked by Amyloid-{beta} and Is Overexpressed in Dystrophic Neurites in Alzheimer's Brain J. Neurosci., December 6, 2006; 26(49): 12735 - 12747. [Abstract] [Full Text] [PDF]

				H.-W. Klafki, M. Staufenbiel, J. Kornhuber, and J. Wiltfang Therapeutic approaches to Alzheimer's disease Brain, November 1, 2006; 129(11): 2840 - 2855. [Abstract] [Full Text] [PDF]

				A. M. Isaacs, D. B. Senn, M. Yuan, J. P. Shine, and B. A. Yankner Acceleration of Amyloid beta-Peptide Aggregation by Physiological Concentrations of Calcium J. Biol. Chem., September 22, 2006; 281(38): 27916 - 27923. [Abstract] [Full Text] [PDF]

				E. Cohen, J. Bieschke, R. M. Perciavalle, J. W. Kelly, and A. Dillin Opposing Activities Protect Against Age-Onset Proteotoxicity Science, September 15, 2006; 313(5793): 1604 - 1610. [Abstract] [Full Text] [PDF]

				A. Deshpande, E. Mina, C. Glabe, and J. Busciglio Different conformations of amyloid beta induce neurotoxicity by distinct mechanisms in human cortical neurons. J. Neurosci., May 31, 2006; 26(22): 6011 - 6018. [Abstract] [Full Text] [PDF]

				V. Novitskaya, O. V. Bocharova, I. Bronstein, and I. V. Baskakov Amyloid Fibrils of Mammalian Prion Protein Are Highly Toxic to Cultured Cells and Primary Neurons J. Biol. Chem., May 12, 2006; 281(19): 13828 - 13836. [Abstract] [Full Text] [PDF]

				K. M. Rosen, V. Veereshwarayya, C. E-H. Moussa, Q. Fu, M. S. Goldberg, M. G. Schlossmacher, J. Shen, and H. W. Querfurth Parkin Protects against Mitochondrial Toxins and beta-Amyloid Accumulation in Skeletal Muscle Cells J. Biol. Chem., May 5, 2006; 281(18): 12809 - 12816. [Abstract] [Full Text] [PDF]

				E. B. Lee, L. Z. Leng, B. Zhang, L. Kwong, J. Q. Trojanowski, T. Abel, and V. M.-Y. Lee Targeting Amyloid-beta Peptide (Abeta) Oligomers by Passive Immunization with a Conformation-selective Monoclonal Antibody Improves Learning and Memory in Abeta Precursor Protein (APP) Transgenic Mice J. Biol. Chem., February 17, 2006; 281(7): 4292 - 4299. [Abstract] [Full Text] [PDF]

				O. Lazarov, L. D. Peterson, D. A. Peterson, and S. S. Sisodia Expression of a Familial Alzheimer's Disease-Linked Presenilin-1 Variant Enhances Perforant Pathway Lesion-Induced Neuronal Loss in the Entorhinal Cortex J. Neurosci., January 11, 2006; 26(2): 429 - 434. [Abstract] [Full Text] [PDF]

				M. T. Maloney, L. S. Minamide, A. W. Kinley, J. A. Boyle, and J. R. Bamburg {beta}-Secretase-Cleaved Amyloid Precursor Protein Accumulates at Actin Inclusions Induced in Neurons by Stress or Amyloid {beta}: A Feedforward Mechanism for Alzheimer's Disease J. Neurosci., December 7, 2005; 25(49): 11313 - 11321. [Abstract] [Full Text] [PDF]

				I-H. Liu, V. N. Uversky, L. A. Munishkina, A. L. Fink, W. Halfter, and G. J. Cole Agrin binds {alpha}-synuclein and modulates {alpha}-synuclein fibrillation Glycobiology, December 1, 2005; 15(12): 1320 - 1331. [Abstract] [Full Text] [PDF]

				T. Luhrs, C. Ritter, M. Adrian, D. Riek-Loher, B. Bohrmann, H. Dobeli, D. Schubert, and R. Riek 3D structure of Alzheimer's amyloid-{beta}(1-42) fibrils PNAS, November 29, 2005; 102(48): 17342 - 17347. [Abstract] [Full Text] [PDF]

				R. A. Velliquette, T. O'Connor, and R. Vassar Energy Inhibition Elevates {beta}-Secretase Levels and Activity and Is Potentially Amyloidogenic in APP Transgenic Mice: Possible Early Events in Alzheimer's Disease Pathogenesis J. Neurosci., November 23, 2005; 25(47): 10874 - 10883. [Abstract] [Full Text] [PDF]

				H. A. Lashuel Membrane Permeabilization: A Common Mechanism in Protein-Misfolding Diseases Sci. Aging Knowl. Environ., September 21, 2005; 2005(38): pe28 - pe28. [Abstract] [Full Text]

				M. H. Magdesian, A. A. Nery, A. H. B. Martins, M. A. Juliano, L. Juliano, H. Ulrich, and S. T. Ferreira Peptide Blockers of the Inhibition of Neuronal Nicotinic Acetylcholine Receptors by Amyloid {beta} J. Biol. Chem., September 2, 2005; 280(35): 31085 - 31090. [Abstract] [Full Text] [PDF]

				H. Mukai, T. Isagawa, E. Goyama, S. Tanaka, N. F. Bence, A. Tamura, Y. Ono, and R. R. Kopito Formation of morphologically similar globular aggregates from diverse aggregation-prone proteins in mammalian cells PNAS, August 2, 2005; 102(31): 10887 - 10892. [Abstract] [Full Text] [PDF]

				Y.-J. Kim, R. Nakatomi, T. Akagi, T. Hashikawa, and R. Takahashi Unsaturated Fatty Acids Induce Cytotoxic Aggregate Formation of Amyotrophic Lateral Sclerosis-linked Superoxide Dismutase 1 Mutants J. Biol. Chem., June 3, 2005; 280(22): 21515 - 21521. [Abstract] [Full Text] [PDF]

				J. D. Best, M. T. Jay, F. Otu, J. Ma, A. Nadin, S. Ellis, H. D. Lewis, C. Pattison, M. Reilly, T. Harrison, et al. Quantitative Measurement of Changes in Amyloid-{beta}(40) in the Rat Brain and Cerebrospinal Fluid following Treatment with the {gamma}-Secretase Inhibitor LY-411575 [N2-[(2S)-2-(3,5-Difluorophenyl)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide] J. Pharmacol. Exp. Ther., May 1, 2005; 313(2): 902 - 908. [Abstract] [Full Text] [PDF]

				D. M. Walsh, M. Townsend, M. B. Podlisny, G. M. Shankar, J. V. Fadeeva, O. E. Agnaf, D. M. Hartley, and D. J. Selkoe Certain Inhibitors of Synthetic Amyloid {beta}-Peptide (A{beta}) Fibrillogenesis Block Oligomerization of Natural A{beta} and Thereby Rescue Long-Term Potentiation J. Neurosci., March 9, 2005; 25(10): 2455 - 2462. [Abstract] [Full Text] [PDF]

				D. V. Laurents, P. M. Gorman, M. Guo, M. Rico, A. Chakrabartty, and M. Bruix Alzheimer's A{beta}40 Studied by NMR at Low pH Reveals That Sodium 4,4-Dimethyl-4-silapentane-1-sulfonate (DSS) Binds and Promotes {beta}-Ball Oligomerization J. Biol. Chem., February 4, 2005; 280(5): 3675 - 3685. [Abstract] [Full Text] [PDF]

				M. Wogulis, S. Wright, D. Cunningham, T. Chilcote, K. Powell, and R. E. Rydel Nucleation-Dependent Polymerization Is an Essential Component of Amyloid-Mediated Neuronal Cell Death J. Neurosci., February 2, 2005; 25(5): 1071 - 1080. [Abstract] [Full Text] [PDF]

				M. Yao, T.-V. V. Nguyen, and C. J. Pike {beta}-Amyloid-Induced Neuronal Apoptosis Involves c-Jun N-Terminal Kinase-Dependent Downregulation of Bcl-w J. Neurosci., February 2, 2005; 25(5): 1149 - 1158. [Abstract] [Full Text] [PDF]

				M. R. Nichols, M. A. Moss, D. K. Reed, S. Cratic-McDaniel, J. H. Hoh, and T. L. Rosenberry Amyloid-{beta} Protofibrils Differ from Amyloid-{beta} Aggregates Induced in Dilute Hexafluoroisopropanol in Stability and Morphology J. Biol. Chem., January 28, 2005; 280(4): 2471 - 2480. [Abstract] [Full Text] [PDF]

				P. J. Crouch, R. Blake, J. A. Duce, G. D. Ciccotosto, Q.-X. Li, K. J. Barnham, C. C. Curtain, R. A. Cherny, R. Cappai, T. Dyrks, et al. Copper-Dependent Inhibition of Human Cytochrome c Oxidase by a Dimeric Conformer of Amyloid-{beta}1-42 J. Neurosci., January 19, 2005; 25(3): 672 - 679. [Abstract] [Full Text] [PDF]

				B. Urbanc, L. Cruz, S. Yun, S. V. Buldyrev, G. Bitan, D. B. Teplow, and H. E. Stanley In silico study of amyloid {beta}-protein folding and oligomerization PNAS, December 14, 2004; 101(50): 17345 - 17350. [Abstract] [Full Text] [PDF]

				R. Kayed, Y. Sokolov, B. Edmonds, T. M. McIntire, S. C. Milton, J. E. Hall, and C. G. Glabe Permeabilization of Lipid Bilayers Is a Common Conformation-dependent Activity of Soluble Amyloid Oligomers in Protein Misfolding Diseases J. Biol. Chem., November 5, 2004; 279(45): 46363 - 46366. [Abstract] [Full Text] [PDF]

				I. Dolev and D. M. Michaelson A nontransgenic mouse model shows inducible amyloid-{beta} (A{beta}) peptide deposition and elucidates the role of apolipoprotein E in the amyloid cascade PNAS, September 21, 2004; 101(38): 13909 - 13914. [Abstract] [Full Text] [PDF]

				H. Koike, K. Misu, M. Sugiura, M. Iijima, K. Mori, M. Yamamoto, N. Hattori, E. Mukai, Y. Ando, S. Ikeda, et al. Pathology of early- vs late-onset TTR Met30 familial amyloid polyneuropathy Neurology, July 13, 2004; 63(1): 129 - 138. [Abstract] [Full Text] [PDF]

				G. Ying, P. Iribarren, Y. Zhou, W. Gong, N. Zhang, Z.-X. Yu, Y. Le, Y. Cui, and J. M. Wang Humanin, a Newly Identified Neuroprotective Factor, Uses the G Protein-Coupled Formylpeptide Receptor-Like-1 as a Functional Receptor J. Immunol., June 1, 2004; 172(11): 7078 - 7085. [Abstract] [Full Text] [PDF]

				E. A. Stern, B. J. Bacskai, G. A. Hickey, F. J. Attenello, J. A. Lombardo, and B. T. Hyman Cortical Synaptic Integration In Vivo Is Disrupted by Amyloid-{beta} Plaques J. Neurosci., May 12, 2004; 24(19): 4535 - 4540. [Abstract] [Full Text] [PDF]

				Q. Wang, D. M. Walsh, M. J. Rowan, D. J. Selkoe, and R. Anwyl Block of Long-Term Potentiation by Naturally Secreted and Synthetic Amyloid {beta}-Peptide in Hippocampal Slices Is Mediated via Activation of the Kinases c-Jun N-Terminal Kinase, Cyclin-Dependent Kinase 5, and p38 Mitogen-Activated Protein Kinase as well as Metabotropic Glutamate Receptor Type 5 J. Neurosci., March 31, 2004; 24(13): 3370 - 3378. [Abstract] [Full Text] [PDF]

				Z. Suo, M. Wu, B. A. Citron, G. T. Wong, and B. W. Festoff Abnormality of G-Protein-Coupled Receptor Kinases at Prodromal and Early Stages of Alzheimer's Disease: An Association with Early {beta}-Amyloid Accumulation J. Neurosci., March 31, 2004; 24(13): 3444 - 3452. [Abstract] [Full Text] [PDF]

				J. D. Green, C. Goldsbury, J. Kistler, G. J. S. Cooper, and U. Aebi Human Amylin Oligomer Growth and Fibril Elongation Define Two Distinct Phases in Amyloid Formation J. Biol. Chem., March 26, 2004; 279(13): 12206 - 12212. [Abstract] [Full Text] [PDF]

				I. Sirangelo, C. Malmo, C. Iannuzzi, A. Mezzogiorno, M. R. Bianco, M. Papa, and G. Irace Fibrillogenesis and Cytotoxic Activity of the Amyloid-forming Apomyoglobin Mutant W7FW14F J. Biol. Chem., March 26, 2004; 279(13): 13183 - 13189. [Abstract] [Full Text] [PDF]

				H. Lin, J. Zhai, R. Canete-Soler, and W. W. Schlaepfer 3' Untranslated Region in a Light Neurofilament (NF-L) mRNA Triggers Aggregation of NF-L and Mutant Superoxide Dismutase 1 Proteins in Neuronal Cells J. Neurosci., March 17, 2004; 24(11): 2716 - 2726. [Abstract] [Full Text] [PDF]

				M. L. DeMarco and V. Daggett From conversion to aggregation: Protofibril formation of the prion protein PNAS, February 24, 2004; 101(8): 2293 - 2298. [Abstract] [Full Text] [PDF]

				N. Iwata, H. Mizukami, K. Shirotani, Y. Takaki, S.-i. Muramatsu, B. Lu, N. P. Gerard, C. Gerard, K. Ozawa, and T. C. Saido Presynaptic Localization of Neprilysin Contributes to Efficient Clearance of Amyloid-{beta} Peptide in Mouse Brain J. Neurosci., January 28, 2004; 24(4): 991 - 998. [Abstract] [Full Text] [PDF]

				M. A. Moss, M. R. Nichols, D. K. Reed, J. H. Hoh, and T. L. Rosenberry The Peptide KLVFF-K6 Promotes {beta}-Amyloid(1-40) Protofibril Growth by Association but Does Not Alter Protofibril Effects on Cellular Reduction of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Mol. Pharmacol., November 1, 2003; 64(5): 1160 - 1168. [Abstract] [Full Text] [PDF]

				J. R. Kim, T. J. Gibson, and R. M. Murphy Targeted Control of Kinetics of {beta}-Amyloid Self-association by Surface Tension-modifying Peptides J. Biol. Chem., October 17, 2003; 278(42): 40730 - 40735. [Abstract] [Full Text] [PDF]

				A. Ferrari, F. Hoerndli, T. Baechi, R. M. Nitsch, and J. Gotz {beta}-Amyloid Induces Paired Helical Filament-like Tau Filaments in Tissue Culture J. Biol. Chem., October 10, 2003; 278(41): 40162 - 40168. [Abstract] [Full Text] [PDF]

				D. D. Mousseau, S. Chapelsky, G. De Crescenzo, M. D. Kirkitadze, J. Magoon, S. Inoue, D. B. Teplow, and M. D. O'Connor-McCourt A Direct Interaction between Transforming Growth Factor (TGF)-{beta}s and Amyloid-{beta} Protein Affects Fibrillogenesis in a TGF-{beta}Receptor-independent Manner J. Biol. Chem., October 3, 2003; 278(40): 38715 - 38722. [Abstract] [Full Text] [PDF]

				J. R. Cirrito, P. C. May, M. A. O'Dell, J. W. Taylor, M. Parsadanian, J. W. Cramer, J. E. Audia, J. S. Nissen, K. R. Bales, S. M. Paul, et al. In Vivo Assessment of Brain Interstitial Fluid with Microdialysis Reveals Plaque-Associated Changes in Amyloid-{beta} Metabolism and Half-Life J. Neurosci., October 1, 2003; 23(26): 8844 - 8853. [Abstract] [Full Text] [PDF]

				H. Hiruma, T. Katakura, S. Takahashi, T. Ichikawa, and T. Kawakami Glutamate and Amyloid {beta}-Protein Rapidly Inhibit Fast Axonal Transport in Cultured Rat Hippocampal Neurons by Different Mechanisms J. Neurosci., October 1, 2003; 23(26): 8967 - 8977. [Abstract] [Full Text] [PDF]

				G. Bitan, S. S. Vollers, and D. B. Teplow Elucidation of Primary Structure Elements Controlling Early Amyloid {beta}-Protein Oligomerization J. Biol. Chem., September 12, 2003; 278(37): 34882 - 34889. [Abstract] [Full Text] [PDF]

				M. Tanaka, Y. Machida, Y. Nishikawa, T. Akagi, T. Hashikawa, T. Fujisawa, and N. Nukina Expansion of Polyglutamine Induces the Formation of Quasi-aggregate in the Early Stage of Protein Fibrillization J. Biol. Chem., September 5, 2003; 278(36): 34717 - 34724. [Abstract] [Full Text] [PDF]

				Y. Gong, L. Chang, K. L. Viola, P. N. Lacor, M. P Lambert, C. E. Finch, G. A. Krafft, and W. L. Klein Alzheimer's disease-affected brain: Presence of oligomeric A{beta} ligands (ADDLs) suggests a molecular basis for reversible memory loss PNAS, September 2, 2003; 100(18): 10417 - 10422. [Abstract] [Full Text] [PDF]

				G. Merlini and V. Bellotti Molecular Mechanisms of Amyloidosis N. Engl. J. Med., August 7, 2003; 349(6): 583 - 596. [Full Text] [PDF]

				I. Qahwash, K. L. Weiland, Y. Lu, R. W. Sarver, R. F. Kletzien, and R. Yan Identification of a Mutant Amyloid Peptide That Predominantly Forms Neurotoxic Protofibrillar Aggregates J. Biol. Chem., June 13, 2003; 278(25): 23187 - 23195. [Abstract] [Full Text] [PDF]

				M. Hoshi, M. Sato, S. Matsumoto, A. Noguchi, K. Yasutake, N. Yoshida, and K. Sato Spherical aggregates of {beta}-amyloid (amylospheroid) show high neurotoxicity and activate tau protein kinase I/glycogen synthase kinase-3{beta} PNAS, May 27, 2003; 100(11): 6370 - 6375. [Abstract] [Full Text] [PDF]

				R. Kayed, E. Head, J. L. Thompson, T. M. McIntire, S. C. Milton, C. W. Cotman, and C. G. Glabe Common Structure of Soluble Amyloid Oligomers Implies Common Mechanism of Pathogenesis Science, April 18, 2003; 300(5618): 486 - 489. [Abstract] [Full Text] [PDF]

				W. B. Stine Jr., K. N. Dahlgren, G. A. Krafft, and M. J. LaDu In Vitro Characterization of Conditions for Amyloid-beta Peptide Oligomerization and Fibrillogenesis J. Biol. Chem., March 21, 2003; 278(13): 11612 - 11622. [Abstract] [Full Text] [PDF]

				G. Bitan, M. D. Kirkitadze, A. Lomakin, S. S. Vollers, G. B. Benedek, and D. B. Teplow Amyloid beta -protein (Abeta ) assembly: Abeta 40 and Abeta 42 oligomerize through distinct pathways PNAS, January 7, 2003; 100(1): 330 - 335. [Abstract] [Full Text] [PDF]

				M. Buttini, G.-Q. Yu, K. Shockley, Y. Huang, B. Jones, E. Masliah, M. Mallory, T. Yeo, F. M. Longo, and L. Mucke Modulation of Alzheimer-Like Synaptic and Cholinergic Deficits in Transgenic Mice by Human Apolipoprotein E Depends on Isoform , Aging, and Overexpression of Amyloid beta Peptides But Not on Plaque Formation J. Neurosci., December 15, 2002; 22(24): 10539 - 10548. [Abstract] [Full Text] [PDF]

				H. A. Lashuel, D. M. Hartley, D. Balakhaneh, A. Aggarwal, S. Teichberg, and D. J. E. Callaway New Class of Inhibitors of Amyloid-beta Fibril Formation. IMPLICATIONS FOR THE MECHANISM OF PATHOGENESIS IN ALZHEIMER'S DISEASE J. Biol. Chem., November 1, 2002; 277(45): 42881 - 42890. [Abstract] [Full Text] [PDF]

				D. J. Selkoe Alzheimer's Disease Is a Synaptic Failure Science, October 25, 2002; 298(5594): 789 - 791. [Abstract] [Full Text] [PDF]

				L. Hou, I. Kang, R. E. Marchant, and M. G. Zagorski Methionine 35 Oxidation Reduces Fibril Assembly of the Amyloid Abeta -(1-42) Peptide of Alzheimer's Disease J. Biol. Chem., October 18, 2002; 277(43): 40173 - 40176. [Abstract] [Full Text] [PDF]

				Y. Luo, J. V. Smith, V. Paramasivam, A. Burdick, K. J. Curry, J. P. Buford, I. Khan, W. J. Netzer, H. Xu, and P. Butko Inhibition of amyloid-beta aggregation and caspase-3 activation by the Ginkgo biloba extract EGb761 PNAS, September 17, 2002; 99(19): 12197 - 12202. [Abstract] [Full Text] [PDF]

				Y.-H. Suh and F. Checler Amyloid Precursor Protein, Presenilins, and alpha -Synuclein: Molecular Pathogenesis and Pharmacological Applications in Alzheimer's Disease Pharmacol. Rev., September 1, 2002; 54(3): 469 - 525. [Abstract] [Full Text] [PDF]

				K. N. Dahlgren, A. M. Manelli, W. B. Stine Jr., L. K. Baker, G. A. Krafft, and M. J. LaDu Oligomeric and Fibrillar Species of Amyloid-beta Peptides Differentially Affect Neuronal Viability J. Biol. Chem., August 23, 2002; 277(35): 32046 - 32053. [Abstract] [Full Text] [PDF]

				R. B. DeMattos, M. A. O'dell, M. Parsadanian, J. W. Taylor, J. A. K. Harmony, K. R. Bales, S. M. Paul, B. J. Aronow, and D. M. Holtzman Clusterin promotes amyloid plaque formation and is critical for neuritic toxicity in a mouse model of Alzheimer's disease PNAS, August 6, 2002; 99(16): 10843 - 10848. [Abstract] [Full Text] [PDF]

				K. T. Dineley, K. A. Bell, D. Bui, and J. D. Sweatt beta -Amyloid Peptide Activates alpha 7 Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes J. Biol. Chem., July 5, 2002; 277(28): 25056 - 25061. [Abstract] [Full Text] [PDF]

				K. Zou, J.-S. Gong, K. Yanagisawa, and M. Michikawa A Novel Function of Monomeric Amyloid beta -Protein Serving as an Antioxidant Molecule against Metal-Induced Oxidative Damage J. Neurosci., June 15, 2002; 22(12): 4833 - 4841. [Abstract] [Full Text] [PDF]

				K. T. Dineley, X. Xia, D. Bui, J. D. Sweatt, and H. Zheng Accelerated Plaque Accumulation, Associative Learning Deficits, and Up-regulation of alpha 7 Nicotinic Receptor Protein in Transgenic Mice Co-expressing Mutant Human Presenilin 1 and Amyloid Precursor Proteins J. Biol. Chem., June 14, 2002; 277(25): 22768 - 22780. [Abstract] [Full Text] [PDF]

				M. A. Westerman, D. Cooper-Blacketer, A. Mariash, L. Kotilinek, T. Kawarabayashi, L. H. Younkin, G. A. Carlson, S. G. Younkin, and K. H. Ashe The Relationship between Abeta and Memory in the Tg2576 Mouse Model of Alzheimer's Disease J. Neurosci., March 1, 2002; 22(5): 1858 - 1867. [Abstract] [Full Text] [PDF]

				M. Koistinaho, M. Ort, J. M. Cimadevilla, R. Vondrous, B. Cordell, J. Koistinaho, J. Bures, and L. S. Higgins Specific spatial learning deficits become severe with age in beta -amyloid precursor protein transgenic mice that harbor diffuse beta -amyloid deposits but do not form plaques PNAS, November 20, 2001; (2001) 261562998. [Abstract] [Full Text] [PDF]

				S. S.-S. Wang, D. L. Rymer, and T. A. Good Reduction in Cholesterol and Sialic Acid Content Protects Cells from the Toxic Effects of beta -Amyloid Peptides J. Biol. Chem., November 2, 2001; 276(45): 42027 - 42034. [Abstract] [Full Text] [PDF]

				K. H. Ashe Learning and Memory in Transgenic Mice Modeling Alzheimer's Disease Learn. Mem., November 1, 2001; 8(6): 301 - 308. [Abstract] [Full Text] [PDF]

				H. LIN, R. BHATIA, and R. LAL Amyloid {beta} protein forms ion channels: implications for Alzheimer's disease pathophysiology FASEB J, November 1, 2001; 15(13): 2433 - 2444. [Abstract] [Full Text] [PDF]

				H. YAZAWA, Z.-X. YU, TAKEDA, Y. LE, W. GONG, V. J. FERRANS, J. J. OPPENHEIM, C. C. H. LI, and J. M. WANG {beta} Amyloid peptide (A{beta}42) is internalized via the G-protein-coupled receptor FPRL1 and forms fibrillar aggregates in macrophages FASEB J, November 1, 2001; 15(13): 2454 - 2462. [Abstract] [Full Text] [PDF]

				W. T. Kimberly, J. B. Zheng, S. Y. Guenette, and D. J. Selkoe The Intracellular Domain of the beta -Amyloid Precursor Protein Is Stabilized by Fe65 and Translocates to the Nucleus in a Notch-like Manner J. Biol. Chem., October 19, 2001; 276(43): 40288 - 40292. [Abstract] [Full Text] [PDF]

				M. Michikawa, J.-S. Gong, Q.-W. Fan, N. Sawamura, and K. Yanagisawa A Novel Action of Alzheimer's Amyloid {beta}-Protein (A{beta}): Oligomeric A{beta} Promotes Lipid Release J. Neurosci., September 15, 2001; 21(18): 7226 - 7235. [Abstract] [Full Text] [PDF]

				K. T. Dineley, M. Westerman, D. Bui, K. Bell, K. H. Ashe, and J. D. Sweatt {beta}-Amyloid Activates the Mitogen-Activated Protein Kinase Cascade via Hippocampal {alpha}7 Nicotinic Acetylcholine Receptors: In Vitro and In Vivo Mechanisms Related to Alzheimer's Disease J. Neurosci., June 15, 2001; 21(12): 4125 - 4133. [Abstract] [Full Text] [PDF]

				D. J. Selkoe Alzheimer's Disease: Genes, Proteins, and Therapy Physiol Rev, April 1, 2001; 81(2): 741 - 766. [Abstract] [Full Text] [PDF]

				J.-H. Kim, R. Anwyl, Y.-H. Suh, M. B. A. Djamgoz, and M. J. Rowan Use-Dependent Effects of Amyloidogenic Fragments of {beta}-Amyloid Precursor Protein on Synaptic Plasticity in Rat Hippocampus In Vivo J. Neurosci., February 15, 2001; 21(4): 1327 - 1333. [Abstract] [Full Text] [PDF]

				R. Siman, A. G. Reaume, M. J. Savage, S. Trusko, Y.-G. Lin, R. W. Scott, and D. G. Flood Presenilin-1 P264L Knock-In Mutation: Differential Effects on Abeta Production, Amyloid Deposition, and Neuronal Vulnerability J. Neurosci., December 1, 2000; 20(23): 8717 - 8726. [Abstract] [Full Text] [PDF]

				S. Kumar-Singh, C. De Jonghe, M. Cruts, R. Kleinert, R. Wang, M. Mercken, B. De Strooper, H. Vanderstichele, A. Lofgren, I. Vanderhoeven, et al. Nonfibrillar diffuse amyloid deposition due to a {gamma}42-secretase site mutation points to an essential role for N-truncated A{beta}42 in Alzheimer's disease Hum. Mol. Genet., November 1, 2000; 9(18): 2589 - 2598. [Abstract] [Full Text] [PDF]

				L. Mucke, E. Masliah, G.-Q. Yu, M. Mallory, E. M. Rockenstein, G. Tatsuno, K. Hu, D. Kholodenko, K. Johnson-Wood, and L. McConlogue High-Level Neuronal Expression of Abeta 1-42 in Wild-Type Human Amyloid Protein Precursor Transgenic Mice: Synaptotoxicity without Plaque Formation J. Neurosci., June 1, 2000; 20(11): 4050 - 4058. [Abstract] [Full Text] [PDF]

				D. L. Rymer and T. A. Good The Role of G Protein Activation in the Toxicity of Amyloidogenic Abeta -(1-40), Abeta -(25-35), and Bovine Calcitonin J. Biol. Chem., January 19, 2001; 276(4): 2523 - 2530. [Abstract] [Full Text] [PDF]

				L. Tong, P. L. Thornton, R. Balazs, and C. W. Cotman beta -Amyloid-(1-42) Impairs Activity-dependent cAMP-response Element-binding Protein Signaling in Neurons at Concentrations in Which Cell Survival Is Not Compromised J. Biol. Chem., May 11, 2001; 276(20): 17301 - 17306. [Abstract] [Full Text] [PDF]

				M. Koistinaho, M. Ort, J. M. Cimadevilla, R. Vondrous, B. Cordell, J. Koistinaho, J. Bures, and L. S. Higgins Specific spatial learning deficits become severe with age in beta -amyloid precursor protein transgenic mice that harbor diffuse beta -amyloid deposits but do not form plaques PNAS, December 4, 2001; 98(25): 14675 - 14680. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (464) Citing Articles via Google Scholar Google Scholar Articles by Hartley, D. M. Articles by Selkoe, Dennis. J. Search for Related Content PubMed PubMed Citation Articles by Hartley, D. M. Articles by Selkoe, Dennis. J.

Home  |   Search  |   Archive  |   Subscribe  |   Contact  |   Help