NeurobiologyofAging, Vol. 13, pp. 587-590, 1992

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/3-Amyloid Neurotoxicity: A Discussion of In Vitro Findings C A R L W. C O T M A N , 1 C H R I S T I A N J. P I K E A N D A G A T A C O P A N I

Irvine Research Unit in Brain Aging, University of California, Irvine, CA 92717 R e c e i v e d 27 M a y 1992; A c c e p t e d 15 J u n e 1992 COTMAN, C. W., C. J. PIKE AND A. COPANI. 13-Amyloid neurotoxicity: A discussion of in vitrofindings'. NEUROBIOL AGING 13(5) 587-590, 1992.--Significant advances in Alzheimer's disease (AD) research require definitive, reproducible findings from all employed paradigms. Recently, the existing in vitro data addressing the possible contribution of ~-amyloid protein to AD neuropathology have been the subject of controversey. We summarize and interpret existing data and discuss relevant methodological issues. We suggest that in vitro data support the conclusion that/3-amyloid peptides decrease the viability of cultured neurons and that this effect can be enhanced by subsequent insults. Alzheimer's disease

/3-Amyloid

Neurotoxicity

Aggregation

THE selective neurodegeneration of AD has been hypothesized to result, at least in part, from the effects of f-amyloid protein, which is concentrated in an insoluble state within senile plaques. This position is grounded in histological observations of AD brain tissue, which typically reveal degenerative and regenerative neuritic processes surrounding and/or infiltrating plaques (4). In addition, within severely affected brain regions such as association cortices, both neuronal loss (7) and senile plaques (16) are often colocalized. Substantiating evidence for this position, and ultimately delineation of the relevant degenerative mechanisms and potential therapeutic interventions, will likely be derived initially from in vitro models, followed by application to in vivo paradigms. Thus, successful progress in AD research depends upon solid, reproducible in vitro findings. We will discuss possible sources of potential inconsistencies in the in vitro f-amyloid literature, including culture systems and the peptide choice, quality, and solubility. The reviewed findings and our own recent observations of the activity of/3amyloid peptides (lAPs) in vitro support the notion that f-amyloid can accelerate mechanisms that lead to cell death, rendering neurons less able to withstand insults.

Soluble/3APs Enhance Glutamate-Mediated Toxicity in Mature Cultues

CURRENT OBSERVATIONS

The hypothesis of a fAP-induced increase in neuronal vulnerability was first posited on the basis of our finding that soluble /31-42, although not toxic by itself to healthy, mature cortical cultures, potentiated the toxicity of the excitatory amino acid glutamate (11). Treatment of these cultures with/31-42 alone for as long as 4 days at the previously established effective concentration of 100 ug/ml ("-22 tsM) did not yield measurable toxicity. However, 131-42 pretreatment in combination with brief exposure to either glutamate, N-methyl-D-aspartate (NMDA),

Soluble lAPs Have Both Trophic-Like and Toxic" Effects on Developing Cultues In our initial investigations into the biological activities of lAPs, we utilized a culture system of low-density (3750 cells/ cm2), developing hippocampal neurons in defined medium that is designed for assessing trophic influences. Under these conditions, treatment with either full-length ( f l - 4 2 ) (18) or truncated (f 1-28) (19) water-solubilized l A P s resulted in increased

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survival for at least 2 days in vitro (DIV). In addition, the f l 42 peptide induced significant increases in neurite outgrowth parameters, including axonal length and dendritic branching; /31-28 did not measurably influence neurite growth. Using a somewhat higher density (I0,000 cells/cm2), serumtreated hippocampal culture system, Yankner and colleagues reported similar findings: soluble/31-28 and/31-40 enhanced neuronal survival between 0-2 DIV (21 ). A neurite enhancement effect was not reported; however, this inconsistency may reflect serum usage which can inhibit initial neurite outgrowth in hippocampal cultures (Pike, Copani, and Cotman, unpublished observations). Yankner et al. also reported that the initial trophic response to lAPs was transient and was replaced by a significantly toxic effect at four to five DIV. Since untreated control cultures exhibited > 70% cell loss by three DIV, one interpretation of these data is that soluble flAPs may not be directly neurotoxic but rather may accelerate the normal, time-dependent decline of cultured neurons. Thus, /3APs may increase neuronal vulnerability to a variety of insults by an as yet undetermined mechanism.

To whom reprint requests should be addressed.

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or kainate at otherwise minimally toxic concentrations, resulted in nearly complete neurodegeneration. These findings suggest that soluble flAPs may cause degeneration indirectly by rendering neurons more susceptible to excitotoxic injury. This hypothesis was supported and extended by our more recent finding in mature cortical cultures that/31-42 potentiates cell loss induced by transient glucose deprivation (3). Removal of glucose from culture medium causes an excitotoxic-mediated injury that, if prolonged, can lead to total neuronal loss. This in vitro paradigm has been employed as a means of investigating deficits in energy metabolism, which some have suggested may be an early event in AD pathology (6). In our experiments, normal maintenance medium (5.5 m M glucose) was temporarily replaced with glucose-free medium for a period of time (approximately 6 h) that consistently caused a slight morphological injury to untreated cultures. The insult was terminated by the addition of a concentrated glucose aliquot. In cultures exposed to/31-42 (50 ug/ml), this transient glucose deprivation was sufficient to induce not only marked somal swelling of most neurons, but also nearly complete neuronal degeneration within 24 h. The NMDA receptor antagonist MK801 attenuated the observed toxicity, indicating that/31-42 was somehow potentiating the effect of endogenous excitatory amino acids. The proposed indirect mode of soluble flAP-induced toxicity has been confirmed and further defined in a recently published report by Mattson and colleagues (13). Using differentiated cultures of human cortical neurons, they found that soluble flAPs were not directly neurotoxic but potentiated the toxicity of exogenously applied glutamate. In addition, they reported that flAPs destabilize intraneuronal levels of calcium. This disturbance in calcium homeostasis apparently reduces the ability of cells to effectively overcome subsequent calcium-related insults.

fl-Amyloid Forms Aggregates In Vivo and In Vitro One aspect of AD that these in vitro models do not directly address is the in vivo observation that fl-amyloid protein exists primarily in an insoluble, aggregated state within senile plaques rather than as a soluble, freely accessible substance. If fl-amyloid contributes to the progressive neurodegeneration in AD, the stable, preferred state of the protein may be particularly relevant to its proposed neurotoxicity, fl-Amyloid protein derived from both purification of AD brain tissue (12,17) and peptide synthesis (5,2) exhibits the unusual property of spontaneous self-assembly into large~ relatively insoluble aggregates. The aggregates from synthetic flAPs share many characteristics with AD-derived/3amyloid, including fl-sheet secondary structure (1,9), formation of 5-10 nm diameter fibrils (2), insolubility under reducing electrophoresis conditions (5,2), and positive staining with the senile plaque markers thioflavi n (2) and Congo red (5). Si nce aggregate formation occurs rapidly and appears relatively stable in aqueous medium, it seems probable that the aggregated form of fl-amyloid would predominate in vivo; this possibility is consistent with histological examination of AD brain tissue (20).

Aggregated flAPs Are Neurotoxic In Vitro Since flAPs tend to spontaneously aggregate, the possibility of quantitatively and/or qualitatively variable biological activities of flAPs dependent upon the state of aggregation requires examination. We directly tested this possibility by comparing the effects of soluble and aggregated fll-42 on the survival of cultured hippocampal neurons at one DIV (15). In order to effectively assess potentially toxic responses, we utilized a relatively high cell density (16,000 cells/cm 2) which increases short-term survival levels in comparison to lower density systems. We found

COTMAN, PIKE AND COPANI that soluble/31-42 did not significantly affect neuronal survival relative to the untreated condition; the absence of atrophic response was likely due to a ceiling effect resulting from minimal degeneration of controls. In agreement with our previous report (18), we did observe increased neuritic outgrowth in cultures treated with soluble /31-42. However, those cells treated with aggregated 131-42 exhibited severe degeneration at doses of 50 ~g/ml and 100 ~g/ml. Under the same conditions, the fll-28 peptide neither formed stable aggregates nor induced toxicity (14). We have recently examined an overlapping series of ten different flAPs and found that only aggregated flAPs were associated with toxicity in developing hippocampal cultures (Pike and Cotman, unpublished observations). Further, preventing or partially reversing the aggregation of flAPs attenuated their toxicity. CRITICAL METHODOLOGICALVARIABLES

Cultures Published in vitro findings have utilized several different culture paradigms as well as a variety of flAP fragments. In our laboratory, we have employed defined media conditions with both young, nearly pure neuronal cultures derived from embryonic rat hippocampus, and mature, mixed cortical cultures established from embryonic mice. We have observed many similarities in the reponses of these two culture systems to synthetic flAPs. These observations, in combination with findings reported by other laboratories, suggest that many culture models of central nervous system neurons may yield qualitatively similar responses to flAP treatment. However, culture variables including serumcontaining medium, age of cultures, and presence of glia may modify flAP-induced effects.

Peptide Synthesis A probable source of variability in studies of fl-amyloid's biological activity is the synthetic flAPs. The first obstacle is obtaining desired peptides with high purity. In order to accomplish this, we have used an Fmoc amino acid substitution technique with a confirmed coupling efficiency of > 99.5% for each amino acid addition (2). Difficult amino acid additions are double coupled to reach the efficiency criterion. Since automated synthesis cannot provide this level of accuracy, their use is not recommended. Such stringent requirements result in yields that are typically > 90% desired product. The peptides are purified by reverse-phase high performance liquid chromatography; their quality and purity are assured by mass spectroscopy and amino acid sequencing analyses. Purified peptides are aliquoted as HC1 salts in 0.5-1.0 mg samples and stored at - 2 0 ° C until solubilization with sterile, double-deionized water. Although our synthesis and solubilization procedures are performed in a consistent manner, slight variations in peptide solubility and activity are sometimes observed. Because of potential peptide variability, we generally repeat experiments with peptides from two or more separate syntheses.

Peptide Length A potentially serious confounding variable in studies of synthetic flAPs is the use of different peptides with the assumption that they have equivalent biological activities. Comparison of flAP physical properties show that flAPs varying by only three amino acid residues in length can exhibit significant differences in solubility and tendency to aggregate (1,2). In order to avoid this pitfall, we believe it is necessary to use a full-length, 40-43

/3-AMYLOID NEUROTOXICITY IN VITRO amino acid peptide to initially characterize activities before using shorter fragments such as/31-28 and/325-/335. Peptide Solubility

Because/3AP toxicity appears to be affected by aggregation which, in turn, is influenced by solvents, solution pH, 13AP concentration, time in solution (2), and perhaps other parameters as well, the solubilization and handling of/3APs may represent a critical source of variability in studies examining the biological activities of SAPs. We typically solubilize small aliquots (1 mg or less) of/3APs to a concentration of 250 #M with doubledeionized water. Since aggregate formation occurs over time, immediate use of newly solubilized ~AP solutions circumvents the aggregation process for most/3APs. Repeated freeze-thawing or incubation of/3AP solutions will yield aggregated samples. To overcome this obstacle, Yankner et al. have utilized a 35% acetonitrile/0.1% trifluoroacetic acid solvent system for SAP solubilization (21). We have observed that this system is an effective means for achieving initial solubility for most /3APs, however, aggregation can occur over time in such solutions. In addition, Mattson et al. have reported effective solubilization of /~APs in dimethylsulfoxide (DMSO) (13). Thus, reported findings have generally included solubilization techniques that should yield soluble peptide. However, without assessing the aggregation status of/3APs following addition to culture, definitive evidence of solubility is not possible. Since the typical effective concentrations of/3APs (-~ 20-25 uM) may be within the minimal concentration required for in vitro aggregate formation (Pike and Cotman, unpublished observations), soluble/3APs may, in fact, aggregate to some degree during experiments. ONGOING STUDIES Are Aggregated/gAPs Neurotoxic to Mature Cultures?

If soluble and aggregated/3APs affect cells according to the same mechanistic pathway, then aggregated/3APs should be directly neurotoxic not only to young cultures, but also to mature, differentiated cultures. In this case, the reported differences in the activities of soluble and aggregated /3AP may be merely quantitative, in order to address this issue, we are currently evaluating the effects of aggregated/31-42 on mature cortical cultures, a system we previously used to study soluble/3APs. At this time, preliminary data suggest that aggregated/31-42 is directly neurotoxic to healthy, mature cortical cultures at a concentration (100 ug/ml) at which soluble/31-42 is not toxic (Copani and Cotman, unpublished observations). In addition, aggregated/3142 effectively potentiates glutamate excitotoxicity in these cultures at concentrations as low as 25 ug/ml (Copani and Cotman, unpublished observations). Although premature, these initial data suggest that aggregation may yield a more potent neurotoxin than soluble/31-42, but may affect neurons by the same mechanism. Perhaps in contrast to these observations, Mattson has reported that/3APs solubilized in DMSO are effective at lower concentrations than /3APs solubilized in water (13), a solvent more likely to promote aggregation than DMSO. This possible inconsistency underscores the difficulty in achieving either completely soluble or aggregated peptides, which in solution are typically a heterogeneous population. How Do/3APs Interact with Neurons?

Some investigators have hypothesized that/3APs may induce biological changes via interaction with receptor(s) that also bind substance P (8,21). How aggregation of flAPs might affect this potential ligand binding has not been addressed. In our hippo-

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campal culture paradigm, we do not observe protection against /3AP toxicity by equimolar addition of substance P (one-way ANOVA, p = 0.78) (Pike and Cotman, unpublished observations). Alternatively, 13APs may be taken up into cells by endocytotic or related mechanisms. In this case, several aggregated proteins may be able to accumulate within cells and to induce pathologic changes. Such a possibility does not weaken the argument that/3-amyloid may contribute to AD neurodegeneration, since within the AD brain/3-amyloid is the specific protein exhibiting abnormal accumulation. Preliminary experiments investigating the potential toxicity of a synthetic fragment of pancreatic islet amyloid protein (amylin) show that the peptide forms aggregates but does not induce significant toxicity in developing hippocampal cultures at concentrations up to 100 uM (Pike and Cotman, unpublished observations). Although these data do not definitively address the topic ofamylin toxicity, they do suggest that, at the tested concentrations, the toxicity associated with aggregated t3-amyloid may represent a relatively selective effect as opposed to a general neuronal response to aggregated peptides. CONCLUSION In summary, recent in vitro data support the hypothesis that /3-amyloid protein contributes to AD neurodegeneration. One tentative conclusion is that/3-amyloid protein both increases the vulnerability of cultured neurons to injury by other insults and accelerates the normal pattern of in vitro degeneration. Thus, in the AD brain, neurons compromised by chronic exposure to /3-amyloid may be less able to overcome both acute insult and normally occurring challenges. On the basis of in vitro results, we propose a sequence of events responsible for the neurodegenerative effects of/3APs. In vitro aggregates of/3APs are attracted to cell membranes; this affinity is a prerequisite for /3AP uptake. Once bound to the membrane, the uptake process may occur according to an endocytotic pathway and/or a specific ligand-receptor interaction. Following internalization, /3APs may not be readily degraded and, thus, may accumulate over time. Similar observations have been recently reported in cultured flbroblasts (10) and may also apply to neurons. Cellular function(s) may be compromised by

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threshold for irreversible damage

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Cellular Accumulation of ~-Amyloid FIG. I. As neurons accumulatc/3-amyloid over timc, they have a decreased ability to successfully overcomc challenges. With substantial

accumulation, cells reach a threshold level at which the impairments induced by ~-amyloid are extensive enough to cause irreversible degeneration even in the absence of additional insults.

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C O T M A N , PIKE A N D COPANI

initial intracellular accumulation of 13-amyloid resulting in an enhanced cellular vulnerability to normal insults that otherwise would be overcome. Eventually, the continued accumulation and corresponding loss in viability would be sufficient to cause cell death (Fig. 1). Other fibrillar or aggregated peptides (e.g., amylin) that are less harmful to neurons than 13-amyloid are predicted either not to be internalized or successfully degraded following internalization. In vivo cellular exposure to/3-amyloid may result from both external sources and altered intracellular metabolism of APP. Following its proteolytic release from APP, t3-amyloid is initially soluble. U n d e r the influence of particular microenvironments

~-amyloid will aggregate, contributing to extracellular senile plaque formation and neuropil deposits and/or forming insoluble intracellular deposits./3-Amyloid aggregation in vivo likely follows a similar pathway as the in vitro process, which includes an equilibrium of many forms ranging from completely soluble peptides to large aggregated assemblies (2). Although such a process hampers identification of the active form(s), the primary effect of/3-amyloid on neurons in vitro is unambiguous. Accordingly, neuronal degeneration in vivo is predicted to be promoted by specific forms of t%amyloid, which could be accumulated from extracellular deposits and/or created by intracellular events.

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11. Koh, J.-Y.; Yang, L. L.; Cotman, C. W. i3-Amyloid protein increases the vulnerability of cultured cortical neurons to excitotoxic damage. Brain Res. 533:315-320; 1990. 12. Masters, C. L.; Simms, G.; Weinman, N. A.; Multhaup, G.; McDonald, B. L.; Beyreuther, K. Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc. Natl. Acad. Sci. USA 82:4245-4249; 1985. 13. Mattson+ M. P.; Cheng, B.; Davis, D.; Bryant, K.; Leieberburg, 1.; Rydel, R. ~-Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity. J. Neurosci. 12:376-389; 1992. 14. Pike, C. J.; Walencewicz, A. J.: Glabe, C. G.; Cotman, C. W. Aggregation-related toxicity of synthetic ~-amyloid protein in hippocampal cultures. Eur. J. Pharmacol. 207:367-368; 1991. 15. Pike, C. J.; Walencewicz, A. J.; Glabe, C. G.; Cotman, C. W. In vitro aging of g3-amyloid protein causes peptide aggregation and neurotoxicity. Brain Res. 563:311-314; 1991. 16. Rogers, J.; Morrison, J. H. Quantitative morphology and regional and laminar distributions of senile plaques in Alzheimer's disease. J. Neurosci. 5:2801-2808; 1985. 17. Selkoe, D. J.; Abraham, C. R.: Podlisny, M. B.; Duffy, L. D. Isolation of low-molecular-weight proteins from amyloid plaque fibers in Alzheimer's disease. J. Neurochem. 46:1820-1834; 1986. 18. Whitson, J. S.: Glabe, C. G.; Sfiitani, E.; Abcar, A.; Cotman, C. W. ¢3-Amyloid protein promotes neuritic branching in hippocampal cultures. Neurosci. Lett. 110:319-324: 1990. 19. Whitson, J. S.: Selkoe, D. J.; Cotman, C. W. Amyloid ~ protein enhances the survival of hippocampal neurons in vitro. Science 243: 1488-1490: 1989. 20. Wisniewski, H. M.: lqbal, K.; Bancher, C.; Miller, D.; Curie, J. Cytoskeletal protein pathology and the formation of beta-amyloid fibers in Alzheimer's disease. Neurobiol. Aging 10:409-412: 1989. 21. Yankner, B. A.: Duffy, L. K.; Kirschner, D. A. Neurotrophic and neurotoxic effects ofamyloid ~ protein: Reversal by tachykinin neuropeptides. Science 250:279-282: 1990.