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The Journal of Neuroscience, February 2, 2005, 25(5):1071-1080; doi:10.1523/JNEUROSCI.2381-04.2005
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Neurobiology of Disease
Nucleation-Dependent Polymerization Is an Essential Component of Amyloid-Mediated Neuronal Cell Death
Mark Wogulis,
Sarah Wright,
Damian Cunningham,
Tamie Chilcote,
Kyle Powell, and
Russell E. Rydel
Elan Pharmaceuticals, South San Francisco, California 94080
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Abstract
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Accumulating evidence suggests that amyloid protein aggregation is pathogenic in many diseases, including Alzheimer's disease. However, the mechanisms by which protein aggregation mediates cellular dysfunction and overt cell death are unknown. Recent reports have focused on the potential role of amyloid oligomers or protofibrils as a neurotoxic form of amyloid- (A) and related amyloid aggregates. Here we describe studies indicating that overt neuronal cell death mediated by A1-40 is critically dependent on ongoing A1-40 polymerization and is not mediated by a single stable species of neurotoxic aggregate. The extent and rate of neuronal cell death can be controlled by conditions that alter the rate of A polymerization. The results presented here indicate that protofibrils and oligomeric forms of A most likely generate neuronal cell death through a nucleation-dependent process rather than acting as direct neurotoxic ligands. These findings bring into question the use of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide formazan assay (MTT assay) as a reporter of A-mediated neuronal cell death and suggest that diffusible A protofibrils and oligomers more likely mediate subtle alterations of synaptic function and long-term potentiation rather than overt neuronal cell death. These results have been extended to A1-42, the non-A component of Alzheimer's disease amyloid plaques, and human amylin, suggesting that nucleation-dependent polymerization is a common mechanism of amyloid-mediated neuronal cell death. Our findings indicate that ongoing amyloid fibrillogenesis may be an essential mechanistic process underlying the pathogenesis associated with protein aggregation in amyloid disorders.
Key words: Alzheimer's disease; amyloid-; neurotoxicity; neuronal cell death; nucleation-dependent polymerization; amyloidogenic proteins
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Introduction
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Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by extracellular amyloid plaques, neurofibrillary tangles, neuronal dysfunction, and overt neuronal cell death. A major constituent of amyloid plaques is the 39-43 amino acid amyloid- (A) protein (Glenner and Wong, 1984 ), which is constitutively produced by normal cleavage of the membrane-bound amyloid- precursor protein (Seubert et al., 1992). A variety of evidence suggests that A accumulation is a pathogenic event in Alzheimer's disease. Amyloid--containing extracellular plaques are an early and invariant feature of the disease. All known familial AD mutations lead to increased production or cerebral deposition (Selkoe, 2001) or aggregate state (Nilsberth et al., 2001) of A. Furthermore, the levels of soluble A correlate closely with synaptic loss and markers of disease severity (Lue et al., 1999; McLean et al., 1999).
A variety of studies have demonstrated neurotoxicity associated with the treatment of neuronal cultures with aggregated A (Yankner, 1996) and other amyloidogenic proteins (Caughey and Lansbury, 2003), supporting a pathogenic role for amyloid accumulation in various disease states. Amyloid- neurotoxicity has been shown to correlate with the presence of fibrils or -sheet structures (Simmons et al., 1994; Howlett et al., 1995; Seilheimer et al., 1997). However, the mechanisms by which A aggregation mediates neuronal cell death are unknown.
If protein aggregation is a critical component of A-mediated pathology, then manipulations that affect the aggregation state of A should provide insight into these neurotoxic properties. A aggregation has been shown to proceed by a multistep, nucleation-dependent process (Jarrett and Lansbury, 1993). Formation of the nucleation seed is rate limiting, so that, in the absence of preformed seed fibrils, there is a significant lag period for the formation of A fibrils, followed by a rapid fibril elongation phase once seed fibrils have been generated. The lag time for fibril formation can be dramatically shortened by the addition of preformed fibril seeds to monomer solutions of A (Jarrett and Lansbury, 1993). The rate of monomer incorporation into polymer increases with both increasing concentration of seed and increasing concentration of monomer (Naiki and Nakakuki, 1996). Therefore, the rate of A fibril formation is controlled by both fibril seed concentration and monomer concentration.
The results presented here demonstrate that treatments with fibrillar A or soluble A alone are not toxic to primary human or rodent neurons. However, when fibrillar A is used to seed the polymerization of soluble A, neuronal cell death develops that is directly proportional to the extent of polymerization. It is the process of A polymerization, rather than any neurotoxic oligomeric species, that correlates with neuronal cell death. Using this two-component system (fibrillar A and soluble A), we demonstrate precise control of both the initiation and termination of A-mediated neuronal cell death. We reveal that lot-to-lot variability of A neurotoxicity is attributable to variable amounts of fibrillar A and soluble A found in biochemically identical lots of A1-40. These studies demonstrate that neuronal cell death is mediated by nucleation-dependent amyloid polymerization. These findings reveal a novel conceptual framework for studying the structure-activity relationships of amyloidogenic peptides, allow for the precise control of cellular response to A and related peptides, and have implications for the therapeutic treatment of Alzheimer's disease and other amyloid disorders.
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Materials and Methods
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Atomic force microscopy. Samples of A solutions (100-200 µl) were added to freshly cleaved mica and incubated for 5-10 min at room temperature. Samples were gently washed twice with 200 µl/wash of filtered, deionized water. All images were generated by atomic force microscopy (AFM) (Molecular Imaging, Tempe, AZ) operated in Magnetic AC Mode. A samples were imaged under filtered, deionized water using tips with a spring constant of  0.5 N/m, with a resonance frequency generally between 20 and 50 kHz in water (depending on the particular tip or cantilever being used). An 18 x 18 µm area was typically scanned at three to five lines per second using 512 x 512 data points per line sampling. Images were collected using both height and deflection mode.
Transmission electron microscopy. Samples (10 µl) of A solutions were adsorbed to 400 mesh Formvar carbon-coated copper grids (FCF400-Cu; Electron Microscopy Sciences, Fort Washington, PA). A samples were left on for 1 min and then negatively stained for 1 min with 1% ammonium molybdate. The stain was wicked off, and the grids were air dried. Grids were examined on a Philips (Aachen, Germany) 400 electron microscope located at the University of California, San Francisco Electron Microscopy Facility in the Department of Biochemistry and Biophysics.
Freshly solubilized A. A1-40 lyophilized powder (catalog #641-10, lots MF-0641, MF-1041, MF-1141, and ME-0541; California Peptide Research, Napa, CA) was dissolved to 1 mM (1 mg of dry powder per 175 µl of water based on quantitative amino acid analysis) using glass vials, incubated at room temperature for 3 min, aliquoted into glass vials, and snap frozen on ethanol and dry ice.
Fibrillar A or "seed" preparation. A1-40 lyophilized powder (catalog #641-10, lots MF-0641, MF-1041, MF-1141, and ME-0541; California Peptide Research) was dissolved to 1 mM (1 mg of dry powder per 175 µl of water) using glass vials, aged for 3 d at 37°C, aliquoted into glass vials, and snap frozen on ethanol and dry ice. At least 90% of the total peptide present in these preparations was in the aggregated form, based on the observation that >90% of peptide was removed by a 100,000 x g spin (data not shown). Based on the morphology of aggregated A, as determined using transmission electron microscopy (TEM) (see Fig. 1), aggregated A is referred to as fibrillar A in this paper. These fibrillar A preparations are also likely to contain A protofibrils, as described previously (Harper and Lansbury, 1997; Walsh et al., 1999). The relative proportions of soluble and fibrillar A in different peptide preparations and treatments were determined by measuring the protein concentration of A after centrifugation (using A1-40 as a standard) and by measuring the characteristic 120 nm red shift of the excitation spectrum of the benzothiazole dye thioflavin-T (ThT) that occurs after ThT binding to fibrillar A (LeVine, 1997).
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Figure 1. The presence of fibrillar A is essential, but not sufficient, for A-mediated neuronal cell death. A, Synthetic lots of A1-40 (MF-0641, MF-1141, and ME-0541) were dissolved to a nominal 1 mM in water. They were then diluted to 30 µM in basal media (MEM) and either imaged by AFM or added at 100 µl/well (n = 3) to freshly aspirated human cortical neurons. Neuronal survival was determined using alamarBlue after 3 d of treatment. Note that lot ME-0541 has a significant number of fibrils present but was not toxic to the cells. B, Stocks (1 mM) of A1-40 (lot MF-0641) were diluted to 30 µM in basal media and spun at room temperature for 20 min at 20,000 x g or for 1 h at 100,000 x g. The unspun sample was kept at room temperature for 1 h. Aliquots were imaged by AFM or added to primary human cortical neuron cultures. Neurons were incubated for 3 d at 37°C and then assayed for neuronal viability using alamarBlue. All treatments were in triplicate wells. Error bars indicate ± SD. supe, Supernatant. C, DMSO stock (7.5 mM) of A1-40 (soluble A) and 3d aged 1 mM water stock (fibrillar A), both lot MF-0641, were diluted to 30 µM in basal media (MEM) and either imaged by AFM or TEM or added at 100 µl/well to primary human cortical neurons. All AFM images shown are deflection mode. Neuronal survival was determined using alamarBlue after 2 d of continuous treatment with either soluble A or fibrillar A. In additional experiments, continuous treatment with fibrillar A for up to 7 d also did not affect neuronal survival (data not shown). Note that the 3 d aged stock was difficult to image by AFM. Some fields would not produce clean images, perhaps attributable to an excessive amount of aggregated material in those fields. Furthermore, large aggregates that were visible by inspection of the mica plate after adsorption were lost after washing of the AFM samples. The TEM image (in which excess liquid is wicked off) is likely to be more representative of the aggregate state of the samples compared with the AFM images (in which the sample is washed with water).
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Soluble A preparation. Disaggregated A1-40 (catalog #641-10, lots MF-0641, MF-1041, MF-1141, and ME-0541; California Peptide Research) was prepared by dissolving lyophilized powder to 7.5 mM in dimethylsulfoxide (DMSO) (23.3 µl of DMSO per 1 mg of dry powder) using glass vials and sonicating for 30 min in a bath sonicator. After dilution in basal media, DMSO-disaggregated A shows no fibrillar or other large aggregates when examined by AFM (see Fig. 1). Based on previous findings that DMSO dissolution of A produces monomeric solutions containing variable amounts of A dimers or other small oligomers (Garzon-Rodriguez et al., 1997; Harper et al., 1999; Tseng et al., 1999), DMSO-disaggregated A is referred to as soluble A in this paper. Stock solutions of soluble A were snap frozen in small aliquots using glass vials and stored frozen at -30°C.
I125-A preparation. I125-A1-40 [2000 Ci/mmol, with lactose, no BSA present; catalog #IM294-25UCi (special order from Amersham Biosciences, Arlington Heights, IL)] was dissolved in DMSO at 100 µCi/ml, transferred to polypropylene tubes, and sonicated in a water-bath sonicator for 30 min. Aliquots of A were snap frozen.
Unsheared and sheared A preparation. A1-40 lyophilized powder (catalog #641-10, lot MF-1141; California Peptide Research) was dissolved to 1 mM (1 mg of dry powder per 175 µl of water) and snap frozen on ethanol and dry ice. A frozen 1 mM stock was diluted to 30 µM in 10 mM 2-(N-cyclohexylamino)ethanesulfonic acid, pH 9.0, and 150 mM NaCl in a final volume of 1 ml. The 30 µM stock was incubated for 2 h in a 37°C circulating water bath to allow for fibril growth; this solution was "unsheared fibrils." Sheared fibrils were produced by passing the solution 20 times through a 27 gauge needle. Shearing increased ThT signal 2.5-fold. By both ThT signal and filtration, 50% of total peptide was in the fibrillar form (data not shown).
Tissue culture. Human cortical cultures were established using dissociated human cerebral tissue at 13-16 weeks gestation. Cortical tissue was provide by Advanced Bioscience Resources (Alameda, CA), and the protocol for obtaining fetal brain tissue complied with federal guidelines for fetal research and with the Uniformed Anatomical Gift Act. Cortical tissue was washed twice in Ca2+/Mg2+-free HBSS (H-9394; Sigma, St. Louis, MO) and then dissociated by repeated pipetting in 10 ml of cold HBSS with 1 ml of DNase (D-4513; Sigma), and the solution was passed through a 100 µm nylon cell strainer (21008-950; VWR Scientific, West Chester, PA). The cells obtained were then centrifuged for 5 min at 200 x g, resuspended in a trypsin/EDTA solution (0.05% trypsin and 0.53 mM EDTA in HBSS; T-3924; Sigma), and incubated for 20 min at 37°C. After centrifugation, cells were resuspended in neuronal medium [MEM supplemented with B27 (17504-044; Invitrogen, Gaithersburg, MD), 1% glucose, 1 mM sodium pyruvate, and 1 mM glutamine] and plated onto polyethyleneimine-coated 96-well plates at a density of 125,000 cells per well. Cultures were maintained for 3 weeks before use, and culture media was replaced twice per week. By immunostaining, the cultures are >90% neurons (positive for neurofilament or microtubule-associated protein-2). The contaminating cells are glial fibrillary acidic protein positive (astrocytes) and complement receptor 3 (MAC-1) positive (microglia) (data not shown). These cells are likely present at the time of plating but do not appear to divide while in the defined medium.
Neuronal viability measurements. Neuronal viability was determined using several techniques: (1) cellular metabolic activity [determined using alamarBlue sodium 3'-[L-phenylaminocarbonyl-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)]; (2) membrane integrity [determined (a) by measuring the release of cytosolic enzyme lactate dehydrogenase (LDH) into the culture supernatant and (b) by staining with a membrane-permeable dye (Syto-13; S-7575; Molecular Probes, Eugene, OR) and a membrane-impermeable dye (propidium iodide; 1-348-639; Boehringer Mannheim, Indianapolis, IN)]; and (3) neuronal morphology (determined using phase contrast microscopy). Neuronal viability as determined using alamarBlue (catalog #DAL1100; Biosource, Camarillo, CA), XTT (catalog #1465015; Roche, Indianapolis IN), and LDH release were performed as described previously (Estus et al., 1997). Syto-13 and propidium iodide were used according to the instructions of the manufacturer. The MTT assay was obtained from Sigma (catalog #TOX-1) and was used per the instructions of the manufacturer.
A1-42, human amylin, and the non-A component of Alzheimer's disease amyloid plaques preparation. Seed or aggregated preparations of A1-42, human amylin, and the non-A component of Alzheimer's disease amyloid plaques (NAC) were made by dissolving lyophilized powder at 250 µM in PBS (A1-42), 1 mM in H20 (human amylin), or 1.3 mM in H20 (NAC) and aging for 3 d at 37°C. (All three peptides were synthesized by California Peptide Research: A1-42, catalog #641-15, lot MF-0639; human amylin, catalog #641-50, lot NG-0213; and NAC, catalog #641-80, lot ME-1125.) All seed preparations were aliquoted and frozen. Because A1-42 is not readily soluble in PBS, the cloudy suspension that resulted from adding PBS to A1-42 was aged and then sonicated for 30 min before aliquoting and freezing. Soluble fractions were made by dissolving lyophilized powder in DMSO at 5 mM, sonicating for 30 min, and snap freezing aliquots. A1-42 and human amylin solutions were diluted to 20 µM in media and then filtered through 30 kDa cutoff filters before treatment (filters were rinsed in 70% EtOH in H2O, aspirated, and dried before use; centriprep #4306; Amicon, Beverly, MA).
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Results
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Structural requirements for A-mediated neuronal cell death
To understand the morphological features required for A-mediated neurotoxicity, we used AFM to identify the aggregate forms of A present in three different lots of A1-40 that varied significantly in their neurotoxic properties when freshly solubilized at 1 mM in water and diluted to 30 µM in tissue culture medium (Fig. 1A). Although all three freshly solubilized peptide lots contained fibrillar A of comparable morphology, only lots MF-0641 and MF-1141 were neurotoxic to primary human cortical neurons. Lot ME-0541, which clearly contained fibrillar A of comparable size and morphology as lots MF-0641 and MF-1141, was not neurotoxic when freshly solubilized and added to primary human cortical neurons. Lot ME-0541 had a ThT signal that was fivefold higher than that of either lot MF-0641 or lot MF-1141, indicating that lot ME-0541 contained a greater proportion of aggregated or fibrillar A (data not shown.) These studies demonstrate that, although fibrillar A was present in neurotoxic lots of A, it was not sufficient to generate neuronal cell death.
To determine whether the presence of fibrillar A was required for mediating neuronal cell death, neurotoxic lot MF-0641 was freshly solubilized at 1 mM in water, diluted to 30 µM in tissue culture medium, and then centrifuged at 20,000 x g for 20 min or 100,000 x g for 60 min before addition of the supernatant to neuronal cultures. Centrifugation at 20,000 x g did not remove the shorter A fibrils, nor did it diminish the neuronal cell death associated with lot MF-0641 (Fig. 1B). However, centrifugation at 100,000 x g removed all fibrillar A and associated neuronal cell death. Although centrifugation at 100,000 x g did remove fibrillar A as measured by AFM (Fig. 1B), it did not have a measurable affect on the protein concentration of lot MF-0641 before or after centrifugation (data not shown). These findings suggest that neurotoxic lot MF-0641 contained very small quantities of fibrillar A. In fact, the ThT signal of neurotoxic lot MF-0641 shown in Figure 1A was comparable with the ThT signal obtained with DMSO-disaggregated lot MF-0641 shown in Figure 1C. In contrast, the ThT signal of neurotoxic lot MF-0641 increased 20-fold after aging for 3 d at 37°C (Fig. 1C and data not shown). This study clearly demonstrates that small quantities of fibrillar A are essential, but not sufficient, for A-mediated neuronal cell death.
Similar results were obtained when neurotoxic lot MF-0641 was prepared as a DMSO stock solution before dilution into tissue culture medium. DMSO-solubilized lot MF-0641 did not contain fibrillar A as demonstrated by AFM and was not toxic to cortical neurons (Fig. 1C). Based on the lack of fibrillar A in DMSO-solubilized A1-40 and a similar appearance of DMSO-solubilized A to preparations after centrifugation at 100,000 x g for 60 min, we will operationally refer to these preparations as "soluble A." Previous publications have demonstrated that similar preparations of A1-40 contain monomer A as well as A dimers and other low n-mer aggregates (Garzon-Rodriguez et al., 1997; Harper et al., 1999; Tseng et al., 1999). In addition to the lack of neuronal cell death associated with the removal of fibrillar A from peptide solutions, we found that neurotoxic activity was also abolished if peptide stocks were allowed to aggregate for extended periods of time (overaging). A lot MF-0641 was toxic when freshly prepared at 1 mM in water and then diluted into tissue culture medium (Fig. 1A). However, its neurotoxic properties were abolished when 1 mM stocks were allowed to aggregate for an additional 3 d at 37°C (Fig. 1C). Thus, both centrifugation experiments and solubilization/aging experiments demonstrate the lack of neuronal cell death associated with soluble A alone or fibrillar A alone.
Nucleation-dependent A polymerization is an essential component of A-mediated neuronal cell death
Previous studies using an all-D-enantiomer of A suggested that the neurotoxic actions of A are not attributable to a stereoisomer-specific ligand-receptor interaction but are more likely attributable to amyloid-mediated cellular interactions that are critically dependent on fibril formation (Cribbs et al., 1997). The lack of neuronal cell death found after neuronal cultures were treated with either fibrillar A alone or soluble A alone further indicated that A-mediated neuronal cell death might be a dynamic process associated with protein aggregation rather than a more conventional ligand-receptor interaction. Because A fibril elongation is a nucleation-dependent polymerization process, we sought to determine whether nucleation-dependent polymerization of A fibrils along the neuronal plasma membrane would elicit A-mediated neurotoxicity. To direct A polymerization to the neuronal cell surface, fibrillar A1-40 was incubated in cultures of primary human cortical neurons for 1 h, and then the culture wells were washed to remove unbound fibrils. Whereas treatment with 1 µM fibrillar A for up to 7 d was not toxic to human cortical neurons, the addition of 20 µM soluble A to cultures previously treated with fibrillar A resulted in robust neuronal cell death after 2-3 d (Fig. 2). Interestingly, neuronal cultures pretreated for 1 h with fibrillar A and then maintained for 7 d in culture demonstrated robust neuronal cell death 2-3 d after the addition of soluble A, suggesting that fibrillar A forms stable interactions with the neuronal cell membrane (data not shown). The fibrillar A used in these studies were prepared from solutions containing highly aggregated, short, bundled fibrils, as determined by TEM (Fig. 1C). Similar results have been obtained using fibrillar A and soluble A prepared from a number of different lots of A1-40. In addition, we found that freshly solubilized lots of A1-40 that contain fibrils, as determined by AFM, can also be used as a source of fibrillar A (data not shown). Although we have not extensively characterized the biochemical properties of the fibrillar A used in these studies, its appearance by AFM and TEM (fibril lengths >1 µm and height/width of 7-10 nm) is similar to the structural properties reported previously for A fibrils (Walsh et al., 1997).
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Figure 2. The presence of both soluble A and fibrillar A are required for A-mediated neuronal cell death. Cultures of human cortical neurons were treated with media alone or media containing 1 µM fibrillar A. After 1 h, culture medium was aspirated and replaced with media alone or media containing 20 µM soluble A. In total, four treatment conditions were used in this study: pretreatment with media alone followed by media-alone treatment, which was used as the control condition ("C"); pretreatment with fibrillar A, followed by media-alone treatment ("F"); pretreatment with media alone, followed by soluble A treatment ("S"); and pretreatment with fibrillar A, followed by soluble A treatment ("F + S"). Primary neuronal cultures were incubated for 3 d and then assayed for neuronal viability by phase contrast microscopy (A), live cell (green) and dead cell (red) staining (B), and by measuring cellular redox activity using alamarBlue, the tetrazolium redox dye XTT that forms a soluble formazan product, and the tetrazolium redox dye MTT that forms an insoluble formazan product (C). C, Third panel, The integrity of the neuronal cell membrane was quantitatively determined by measuring the release of the cytoplasmic enzyme LDH into the culture medium. Total LDH in cells was determined by lysing cells with 1% Triton X-100. This value was used to calculate percentage of total LDH released. The white and black bars represent data obtained using independent culture preparations. The modest effect of fibrillar A on LDH release obtained in one experiment (white bars), although significant, was not repeated in additional studies using fibrillar A. Error bars indicate ± SD. *p < 0.001 versus control cultures. Statistical analysis was based on the means ± SD (n = 6) using ANOVA, followed by Fisher's PLSD test using StatView software (version 5.0; SAS Institute, Cary, NC).
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The neuronal cell death associated with nucleation-dependent A polymerization was confirmed using methods that assess both the metabolic integrity of the neuronal cultures and the presence of an intact cell surface membrane (Fig. 2). Treatment with both fibrillar A and soluble A was required to demonstrate significant effects on neuronal viability as assessed by cell morphology (Fig. 2A), disruption of plasma membrane as determined by live/dead cell staining (Fig. 2B) and the release of the cytoplasmic enzyme LDH (Fig. 2C), and loss of mitochondrial potential as measured by two different redox dyes, XTT and alamarBlue (Fig. 2C). In addition to measuring overt neuronal cell death after treatment with fibrillar A and/or soluble A, biochemical responses associated with A-mediated neuronal cell death were also evaluated using this two-component experimental paradigm. In particular, activation and phosphorylation of Jun kinase and c-Jun, critical components of the cellular signaling pathway mediating A neurotoxicity (Bozyczko-Coyne et al., 2001; Morishima et al., 2001; Troy et al., 2001), were also found to require treatment with both fibrillar A and soluble A (data not shown), indicating that activation of this cell death signaling pathway was tightly coupled to nucleation-dependent A polymerization. Therefore, a variety of independent measurements of neuronal viability clearly demonstrated that ongoing A polymerization appeared to be an essential component of A-mediated neuronal cell death.
The reduction of the cellular MTT to MTT formazan is not a reliable early indicator of A-mediated neuronal cell death
Tetrazolium salts, such as MTT, are routinely used as colorimetric agents for measuring cell viability in cytotoxicity assays (Hansen et al., 1989). Cellular metabolic activity, or more precisely cellular redox activity, results in the reduction of soluble MTT to the insoluble MTT formazan. The MTT assay has been extensively used in studies addressing the neurotoxic properties of A. However, it is likely that use of the MTT assay in A neuronal cell death studies has confounded the interpretation of experiments using various aggregate forms of A. It has been reported by several laboratories that treatment with A can lead to a decrease in MTT formazan production in the absence of overt cell death (Shearman et al., 1994; Hertel et al., 1996; Liu et al., 1997; Soriano et al., 2003). A number of different explanations have been proposed for this observation, including A effects on membrane properties, MTT formazan exocytosis, and unknown intracellular targets whose interaction with A is mediated by endosomal/lysosomal acidification (Hertel et al., 1996; Liu et al., 1997; Kane et al., 1999). Although the mechanisms by which A can reduce insoluble MTT formazan formation in the absence of overt cell death are in dispute, the MTT formazan assay has been reported to be a specific early indicator of A-mediated cell death (Shearman et al., 1994).
We and others have found that amyloidogenic proteins such as A uniquely alter cellular MTT formazan crystal formation such that needle-shaped MTT formazan crystals are formed that puncture the cell plasma membrane (Shearman et al., 1995; Hertel et al., 1996) (data not shown). As reported previously (Shearman et al., 1995; Hertel et al., 1996), this effect of A on MTT formazan crystal formation is not shared with other structurally related tetrazolium redox dyes. Overall, these findings suggest that the MTT assay might not be a reliable indicator of overt A-mediated neuronal cell death.
To directly test the reliability of the insoluble MTT formazan assay as an early indicator of A-mediated neuronal cell death, we treated sister neuronal cultures with fibrillar A alone, soluble A alone, or both fibrillar A and soluble A and compared the neuronal viability determined using the MTT assay with that obtained by measurement of plasma membrane integrity using LDH release and cellular redox activity using two different soluble redox dyes, XTT and alamarBlue. As shown by the black bars in Figure 2C, treatment of primary human cortical neurons with either fibrillar A alone or soluble A alone had no effect on LDH release or cellular redox activity when assayed using soluble redox dyes. In contrast, treatment with either fibrillar A alone or soluble A alone had a significant affect on neuronal viability only when cellular redox activity was determined using the insoluble MTT formazan assay. We repeated these studies a number of times using primary mouse, rat, and human cortical neurons and have consistently found the MTT formazan assay to indicate A neurotoxicity under conditions in which there is no detectable A effect on neuronal viability as determined by phase contrast microscopy, live/dead cell staining, LDH release, and soluble redox dyes (data not shown). Because A-mediated neuronal cell death, as determined using a variety of methods other than the insoluble MTT formazan assay, requires treatment with both soluble A and fibrillar A, the reduction of insoluble MTT formazan found after treatment with either soluble A alone or fibrillar A alone cannot be interpreted as an early indicator of neuronal cell death.
Our observation that treatment of human cortical neurons with soluble A can lead to a reduction in MTT formazan formation in the absence of overt neuronal cell death is contrary to previous studies that did not show an effect of soluble A on MTT formazan formation using cultured rat primary cortical neurons (Walsh et al., 1999) or rat primary hippocampal neurons (Ward et al., 2000). However, these previous studies only evaluated the effects of soluble A on MTT formazan formation after relatively short treatment periods (0.5-2 h), whereas the studies reported here used treatment periods of 2-3 d. The results presented here demonstrate that, with longer incubation time, soluble A can lead to a reduction in the MTT assay in the absence of overt neuronal cell death.
Based on these and previous findings (Patel et al., 1996; Liu et al., 1997; Soriano et al., 2003), the studies reported here strongly indicate that the MTT formazan assay is an unreliable indicator of A-mediated neuronal cell death. Furthermore, these findings suggest that previous results on the neurotoxic properties of A fibrils, protofibrils, or other oligomeric species should be interpreted with caution if neuronal cell death was inferred using the MTT formazan assay.
The extent and rate of neuronal cell death can be precisely controlled by conditions that alter the rate of nucleation-dependent A polymerization
To further characterize the role of nucleation-dependent polymerization in A-mediated neurotoxicity, we evaluated the effect of fibrillar A pretreatment on the interaction of soluble A with primary human cortical neurons. Using trace amounts of soluble 125I-A1-40, we found that pretreatment of cultured cortical neurons with fibrillar A dramatically enhanced the incorporation of soluble A onto the neuronal monolayer (Fig. 3A). Under the conditions used (1 µM fibrillar A and 20 µM soluble A), the incorporation of soluble A was linear over 2 d of treatment. Overt neuronal cell death was only associated with the incorporation of soluble A along the neuronal cell surface (Fig. 3B), further suggesting that ongoing fibril polymerization might be critical for mediating A neurotoxicity.
If nucleation-dependent amyloid polymerization is a critical component of A-mediated neurotoxicity, then conditions that enhance or diminish the rate of A polymerization should have similar effects on the rate and extent of A-mediated neuronal cell death. Because the rate of nucleation-dependent polymerization is proportional to both seed concentration and monomer concentration, we evaluated the effects of fibrillar A concentration and soluble A concentration on A-mediated neuronal cell death. Human cortical neurons were pretreated with various concentrations of fibrillar A for 1 h, the culture medium was aspirated, and the neurons were then maintained for 3 d in the presence of 20 µM soluble A. As expected, increasing concentrations of fibrillar A from 0.1 nM to 1 µM dramatically enhanced the extent of A-mediated neuronal cell death (Fig. 4A). Similar results were obtained when fibrillar A concentration was kept constant and soluble A concentration was varied from 5 to 20 µM. As shown in Figure 4B, increasing the concentration of soluble A enhanced both the rate and extent of A-mediated neuronal cell death after 3, 7, 10, and 14 d of treatment. These studies clearly demonstrate a close relationship between the rate of A polymerization and both the rate and extent of A-mediated neuronal cell death.
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Figure 4. The extent of A-mediated neuronal cell death is dependent on both fibrillar A concentration and on soluble A concentration. A, Human cortical neurons were pretreated for 1 h with the indicated concentrations of fibrillar A. Nonadhering fibrillar A was removed by aspiration, and 20 µM soluble A was added to the cultures. Neuronal cultures were incubated for 3 d and then assayed for viability using alamarBlue. Three independent neuronal culture preparations were assayed on different days using frozen aliquots of the same fibrillar A preparation. We presume that the variability seen at the highest concentration of fibrillar A is attributable to variations in the amount of fibrillar A left behind after aspiration of the fibrillar A pretreatment. Sufficiently high concentrations of fibrillar A can inhibit toxicity, as seen in Figure 6, and this variability was eliminated when the fibrillar A pretreatment was washed with media alone before adding soluble A (data not shown). B, Human cortical neurons were pretreated for 1 h with 1 µM fibrillar A. Pretreatment was aspirated, and 5 µM (circles), 10 µM (diamonds), or 20 µM (squares) soluble A was added to the cultures. Neuronal viability was determined by alamarBlue after 3, 7, 10, and 14 d of treatment. All treatments were in triplicate wells. Error bars indicate ± SD.
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Studies on nucleation-dependent protein polymerization have demonstrated that the rate of polymerization is dependent on both the concentration of fibril seeds and the number of free fibril ends (Harper and Lansbury, 1997). Specifically, conditions that enhance the number of free fibril ends will enhance the rate of protein polymerization, even if the concentrations of fibrils and monomers are kept constant. Based on this relationship, we would expect treatments that increase the number of free A fibril ends, without changing the concentrations of fibrillar or soluble A, would also enhance the rate of A-mediated neuronal cell death. To address this relationship experimentally, we sheared A fibrils by repeated passage through a 27 gauge needle. Using AFM (Fig. 5A,B) and TEM (data not shown), we found that the sheared fibrils were significantly shorter than unsheared A fibrils. Pretreatment with sheared A fibrils, followed by treatment with soluble A, resulted in significantly greater neuronal cell death after 3 d of treatment compared with pretreatment with unsheared A fibrils. For example, similar effects on neuronal survival were found after 1 h pretreatment with either 1.2 µM unsheared A fibrils or 0.3 µM sheared A fibrils, followed by 3 d of treatment with 20 µM soluble A (Fig. 5C). Neuronal cultures treated with unsheared or sheared A fibrils alone did not exhibit cell death (data not shown), suggesting that both unsheared and sheared fibrils require soluble A to mediate overt neuronal cell death. Thus, the neurotoxic potency of A increases as the number of fibril ends increases, as would be expected for a nucleation-dependent polymerization process.
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Figure 5. Increasing the number of fibril ends by shearing leads to increased potency of A fibril seeds. Deflection mode AFM images of unsheared (A) and sheared (B) A fibrils generated at pH 9.0, 150 mM NaCl. These conditions were found to produce long, nonlaterally associated fibrils as determined by AFM. Samples (30 µM) were incubated for 5 min at room temperature on mica either immediately before (for unsheared samples) or immediately after (for sheared samples) shearing. The shorter, sheared fibrils do not stick well to mica at pH 9.0. NaCl was spiked in to a final concentration of 250 mM in the sheared fibrils immediately before adding to mica to enhance the adsorption of sheared fibrils to the mica surface. Although this treatment did enhance fibril adsorption, it is important to note that fibril adsorption to mica surface is not quantitative, and thus the apparent reduction in fibril content after shearing is likely attributable to differences in adsorption rather than loss of fibrils caused by shearing. Thioflavin-T signal was increased 2.5-fold in sheared samples compared with the unsheared samples (data not shown). C, Human cortical neurons were treated with the indicated concentration of sheared and unsheared fibrils (at pH 9.0, 150 mM NaCl) for 1 h. Dilutions were performed with 10 µM soluble A present to ensure that the fibrils did not disaggregate by loss of monomer from fibril ends. Nonadhering A fibrils were removed by aspiration, and 20 µM soluble A was added to the cultures. Cultures were maintained for 3 d, and neuronal viability was determined by alamarBlue. Statistical analysis was based on the means ± SD (n = 6). *p = 0.005 versus cultures treated with unsheared A fibrils based on ANOVA, followed by Fisher's PLSD test using StatView software (version 5.0; SAS Institute). ANOVA indicated that there was a group effect of cultures treated with unsheared A fibrils versus cultures treated with sheared A fibrils (p = 0.002), as well as an interaction effect between dose and treatment (p < 0.0001).
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The neurotoxic dependency on both fibrillar A concentration and soluble A concentration was reproduced using four different lots of A1-40 peptide, demonstrating that these findings were not unique to individual lots of A peptide. However, the neurotoxic potency of fibrillar A did vary from lot to lot, suggesting that the number of free fibril ends was peptide lot and preparation dependent (data not shown). Nonetheless, the neurotoxic potency of individual preparations of fibrillar A was highly reproducible, and frozen aliquots of fibrillar A showed consistent neurotoxic activity across multiple experiments (Fig. 4A). These findings provide a highly reproducible method by which to control the rate of A-mediated neuronal cell death and associated signaling pathways and should allow a greater understanding of the molecular events associated with amyloid-mediated pathology. Previous studies using human cerebrovascular smooth muscle cells have demonstrated that cell surface A fibril formation plays an important role in causing pathologic responses in this cell type (Van Nostrand et al., 1998). Confocal microscopy studies using anti-A and anti-neural cell adhesion molecule antibodies also indicates that A polymerization is closely associated with the neuronal cell surface under conditions that promote overt neuronal cell death (data not shown). Together, these series of studies strongly suggest that the nucleation-dependent polymerization of A1-40, most likely along the neuronal cell surface, is an essential component of A-mediated neuronal cell death. However, our interpretation of these studies did not take into consideration the potential for stable soluble A oligomers to be generated by the treatment conditions used in these studies. Such stable and toxic A oligomers could be generated by the process of A polymerization and then diffuse onto the neuronal cell surface. We therefore conducted a series of studies in which fibrillar A and soluble A were mixed in solution for various lengths of time before treatment of primary neuronal cultures to determine whether there was a soluble component of A-mediated neuronal cell death or whether A-mediated neuronal cell death was more tightly associated with A polymerization and overt fibril formation. In particular, we reasoned that, if A neuronal cell death was dependent on fibril elongation along the neuronal cell surface, then solutions of fibrillar A and soluble A would be toxic to neurons only if the solution conditions maintained ongoing A polymerization. However, if stable neurotoxic oligomers of A were generated in solution by the process of A polymerization, then solutions of fibrillar A and soluble A would be toxic to neurons even if solution conditions did not maintain ongoing A polymerization. As shown in Figure 6A, solutions containing 25 µM soluble A and 62, 125, or 250 nM fibrillar A were preincubated up to 6 h at 37°C before addition to neuronal cultures. After 30 min of preincubation, all three fibril concentrations generated solutions with very similar neurotoxic potencies. The neuronal cell death found in these treatments was comparable with that obtained using freshly solubilized neurotoxic lots of A (Fig. 1). The relatively small amount of fibrillar A required to induce neuronal cell death is consistent with our observation that neurotoxic lots of A1-40 contain very small amounts of fibrillar A (<5% of total peptide concentration of neurotoxic lots MF-0641 and MF-1141 were in fibrillar form based on ThT and centrifugation experiments; data not shown). However, with longer preincubation times up to 6 h, solutions containing the higher concentration of fibrillar A showed dramatic loss of neurotoxic potency. One potential interpretation of these studies was that the solutions containing higher concentrations of fibrillar A had consumed all of the soluble A and could no longer maintain ongoing A polymerization when added to neuronal cultures. To directly address this possibility, we repeated these studies using 62 nM fibrillar A and either 20 or 40 µM soluble A. Whereas the solution containing 20 µM soluble A lost its neurotoxic activity when preincubated for 24 h, the solution containing 40 µM soluble A maintained its ability to cause neuronal cell death (Fig. 6B). Thus, increasing the soluble A concentration directly extended the duration of neurotoxic activity associated with a fixed concentration of fibrillar A. Conversely, as predicted for a nucleation-dependent polymerization process, the addition of fibrillar A should inhibit neuronal cell death when amounts sufficient to consume all available soluble A are added to neuronal cultures. Cortical cultures were pretreated for 1 h with 1 µM fibrillar A, washed, and then treated with 20 µM soluble A, plus either 0 or 5 µM fibrillar A. After 3 d of treatment, neuronal viability was determined using the alamarBlue assay. As shown in Figure 6C, adding an additional 5 µM fibrillar A inhibited neuronal cell death by 85% compared with the cultures pretreated with 1 µM fibrillar A and 20 µM soluble A alone. Thus, through a variety of treatment conditions that altered the rate and extent of A polymerization, we have been able to demonstrate a strong correlation of the rate and extent of A polymerization with the rate and extent of A-mediated neuronal cell death.
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Figure 6. A-mediated neuronal cell death requires ongoing nucleation-dependent polymerization. A, Fibrillar A (62 nM) (open circles), 125 nM fibrillar A (filled squares), or 250 nM fibrillar A (filled diamonds) was added to 25 µM soluble A and left to incubate at 37°C for the indicated lengths of time before adding to primary human cortical neurons. Cells were treated for 3 d, and then neuronal survival was determined by alamarBlue. Note that increasing concentrations of fibrillar A led to a loss of neuronal cell death, presumably because of depletion of soluble A. B, Fibrillar A (62 nM) was added to either 20 or 40 µM soluble A and left to age at 37°C for the indicated times before adding to primary human cortical neurons. Note that the loss of neuronal cell death found using 62 nM fibrillar A at 24 h of aging is prevented by using a higher soluble A concentration. C, Human cortical neurons were pretreated with 1 µM fibrillar A for 1 h. Nonadhering fibrillar A was removed by aspiration, and cultures were treated with 20 µM soluble A with or without 5 µM fibrillar A. Cultures were incubated for 3 d, and then neuronal survival was determined by alamarBlue. The addition of 5 µM fibrillar A significantly inhibited the neuronal cell death normally found using 20 µM soluble A, presumably caused by depletion of soluble A. All treatments were intriplicate wells. Error bars indicate ± SD.
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To further investigate the possibility that a stable, soluble species of A could be mediating neurotoxicity under these conditions, we evaluated the conditioned media from neuronal cultures treated with fibrillar A and soluble A. Cortical cultures were pretreated for 1 h with fibrillar A and washed extensively, and then soluble A was added to the cultures. After 1, 2, or 3 d of treatment, the conditioned media from these neuronal cultures was transferred to cortical cultures that had been pretreated with fibrillar A or media alone. Donor cultures were assayed for neuronal viability at the time of media transfer, whereas recipient cultures were assayed for neuronal viability 3 d after treatment with conditioned media. As shown in Figure 7A, donor cultures displayed progressive neuronal cell death over the 3 d of treatment with fibrillar A and soluble A. Interestingly, recipient neuronal cultures that were pretreated with media alone did not demonstrate neurotoxicity after treatment with conditioned media, even when the conditioned media came from donor neuronal cultures displaying >80% neuronal cell death (Fig. 7). However, recipient neuronal cultures that were pretreated with fibrillar A displayed dramatic neuronal cell death under all treatment conditions (Fig. 7B). The failure of donor culture conditioned media to generate A neurotoxicity in the absence of pretreatment with fibrillar A strongly indicates that neuronal cell death was not caused by the formation of stable, soluble neurotoxic A species. If stable, neurotoxic A oligomers mediated this neuronal cell death, then there would not be an absolute requirement for fibrillar A pretreatment of recipient cultures. These studies provide additional support that nucleation-dependent polymerization is an essential component of A-mediated neuronal cell death.
Previous studies have demonstrated that microglia and astrocytes can modulate A-mediated neuronal cell death (Combs et al., 1999; Malchiodi-Albedi et al., 2001; Paradisi et al., 2004). This suggests the possibility that the neuronal cell death observed here is caused by soluble toxins released from contaminating glial cells rather than from a direct action of A polymerization on neurons. Two lines of evidence suggest that the results reported here are not attributable to soluble toxins released by contaminating glial cells. First, the use of B27 as a serum supplement dramatically reduces the number of contaminating microglial cells (<1%) and contaminating astrocytes (<10%). Second, we were unable to demonstrate neurotoxic activity in the conditioned media obtained in these experiments, strongly suggesting that the neurotoxic effects seen here are not attributable to soluble toxic factors released by contaminating glial cells.
Nucleation-dependent polymerization as a common component of amyloid-mediated neurotoxicity
Because protein aggregation is associated with a variety of neurodegenerative disorders, it was of interest to determine whether nucleation-dependent polymerization was a common component of the neurotoxic properties associated with other amyloidogenic proteins. In particular, we evaluated whether A1-42, human amylin, and NAC also require both fibrils and soluble peptide to be present to generate neuronal cell death. As shown in Figure 8, the neuronal cell death associated with all three peptides required the presence of both fibrillar and soluble peptide. Although the concentrations of fibrillar and soluble peptide required for neuronal cell death, as well as the method of generating fibrils, varies for different amyloidogenic peptides, a common feature of amyloid-mediated neuronal cell death is the presence of two components, fibrillar and soluble peptide. Thus, nucleation-dependent polymerization appears to be a common mechanistic feature of amyloid-mediated neuronal cell death.
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Abstract
Introduction
