NeurobiologyofAging. Vol. 13, pp. 605-607, 1992

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fl-Amyloid Peptide In Vitro Toxicity: Lot-to-Lot Variability P A T R I C K C. M A Y , *l B R U C E D. G I T T E R , * D I A N E C. W A T E R S , * L I N D A K. S I M M O N S , * G E R A L D W. B E C K E R , t J E N N I F E R S. S M A L L t A N D P A U L A M. R O B I S O N *

Departments of*CNS Research and 7Biotechnology Research, Lilly Research Laboratories, A Division of Eli Lilly and Co., Indianapolis, IN 46285 Received 30 April 1992; Accepted 15 J u n e 1992 MAY, P. C. , B. D. GITTER, D. C. WATERS, L. K. SIMMONS, G. W. BECKER, J. S. SMALL AND P. M. ROBISON. S-

Amyloid peptide in vitro toxicity: Lot-to-lot variability. NEUROBIOL AGING 13(5) 605-607, 1992.--SA4 peptide (SLAP)accumulates in amyloid plaques of Alzheimer's disease and may contribute to neuronal degeneration.Conflictingobservations have been reported regarding the direct in vitro and in vivo neurotoxicity of SAP. We have assessed in vitro SAP toxicity in high density primary rat hippocampal cultures and found marked lot-to-lot differences in the neurotoxic properties of SAP. One lot of SAP from a commercial supplier resulted in significantdirect neurotoxicity at 10 uM, while 2 other lots from the same supplier were essentiallynontoxic. Three additional lots of SAP from unrelated sources were also nontoxic at 10 #M. Initial biochemical characterization has not yet revealed any marked differencesamong the various lots of SAP. Low levels of endotoxin (ca., l EU/ ml) were detected in several SAP preparations but did not correlate with neurotoxicity. Our observation that lot-to-lot variability of SAP occurred even under identical in vitro culture conditions may account for part of the present controversy in this area. Alzheimer's disease

S-Amyloid

Rat hippocampal cultures

DEPOSITS of f-amyloid protein (lAP) accumulate in Alzheimer's disease (AD) brain, but their contribution to subsequent neuropathology is presently ill defined. A primary role for l A P in AD pathology was suggested by recent reports that l A P was directly toxic to neurons in vitro (8) and in vivo (3). However, conflicting observations regarding the direct neurotoxicity of l A P have been reported (2,4). We have focused on an in vitro model utilizing primary rat hippocampal cultures plated at high density to assess direct toxicity of flAP and discovered marked lot-tolot differences in its toxic properties.

Endotoxin

PolymyxinB

in 10% DMSO and either used immediately or aliquoted and frozen (-20°C).

Endotoxin Testing All l A P stock solutions were coded and assayed (at 10 #M) for endotoxin contamination by the Limulus Amoebocyte Lysate (LAL) assay (Quantitative Chromogenic LAL-1000 kit, Whittaker Bioproducts, Inc., Walkersville, MD). In addition, positive controls spiked with lipopolysaccharide (LPS, E. coli 055:B5; DIFCO) and negative controls (0.1% DMSO final concentration) were coded and assayed in parallel with fAP samples. One endotoxin unit (EU) equals 0.1 ng/ml LPS.

METHOD

flAP Sources Primary Rat Hippocampal Cell Culture

Unless otherwise indicated, 13AP were synthetic 40mers representing residues 1-40 of the 43mer native f-amyloid peptide isolated from AD plaque cores (6). Two lots of fAP (#ZI682 and #ZI960) were purchased from Bachem, Inc. (Torrance, CA), and one custom synthesized lot obtained from Multiple Peptide Systems (San Diego, CA). Two other lots of f A P (Bachem #ZI424 and Athena Lot # 164-OA-P232-1) were generously provided by Dr. Russell Rydel (Athena Neurosciences, S. San Francisco, CA). One lot of/3AP(1-43) peptide was synthesized at Eli Lilly and Co. in the lab of Dr. Richard DiMarchi and kindly provided by Dr. Sheila Little. fAP stocks of 1 m M were prepared

Primary hippocampal cultures were prepared from E 18 fetal Sprague-Dawley rat pups. Hippocampal cells were plated at high density (1.5 X 105/cm 2) in 24-well plates (Costar) coated with 0.5 mg/ml polyethyleneimine (Sigma, St. Louis). Hippocampal cultures were routinely maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 4 m M Gin, 5 g/l glucose, 1 m M pyruvate, 20 m M KC1, 50 units/ml penicillin and 50 #g/ ml streptomycin (Pen/Strep) plus 10% fetal bovine serum (GIBCO). All experimental treatments were conducted in chemically defined HEPES-buffered DMEM (5 tsg/ml bovine

Requests for reprints should be addressed to Patrick C. May, Ph.D., Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly and Co., Indianapolis, IN 46285.

605

606

MAY ET AL.

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to hippocampal cultures. Pretreatment with polymyxin B, at a concentration known to block endotoxin (LPS) effects on cultured human astrocytoma cells (7), had no effect upon #ZI682 HAP toxicity (Fig. 2). In addition, cotreatment with 10 #M Substance P also failed to significantly protect against #ZI682 HAP toxicity (Fig. 2).

!!!!!!

DISCUSSION

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:z: 30 c3

~;SSS~

._1

20 10

EU/ml

CTL

BAP ZI424

SAP ZI682

13AP ZI960

SAP Athena

0

1.1 ± .2

1.2 _+.1 .05 + .05 1.7 +_.6

BAP MPS 0

BAP(1-43) Lilly 0

FIG. 1. Direct toxicity of~AP in high density rat hippocampal cultures. ~AP from varioussourceswas added at 10 uM to rat hippocampalcultures (10 DIV) and viability assessed 4 days later. Results are expressed as units/ml of LDH (mean _+SD, n - 3) and are representative of two to six separate experiments. Total LDH determined in sister control-treated wells after osmotic lysis averaged 250 units/ml. Endotoxin levels (EU/ ml) determined at 10 pM peptide concentration are plotted below for each 13AP(mean + SD; n - two to four determinations).

insulin, 100 #g/ml human transferrin, 100 #g/ml BSA fraction V, 2 #M progesterone, 100 pM putrescine, 0.2/zM sodium selenite, 50 pM thyroxine, and 50 pM tri-iodo-thyronine; all purchased from Sigma) supplemented with Pen/Strep, 4 m M Gln, and 4.5 g/l glucose.

Lactate Dehydrogenase Assay Lactate dehydrogenase (LDH) was measured in 50 pl aliquots of conditioned defined-DMEM by a standard colorimetric endpoint assay for pyruvate (Sigma Kit 500). Quality control standards containing normal and elevated levels of serum LDH (Sigma Enzyme Control N and 2 N) were run with every assay. Results are expressed as units of LDH/ml where 1 unit is defined as the amount of LDH which reduces 4.8 × 10 -4 M pyruvate/ min at 25°C.

Our results indicate lot-to-lot variability in HAP preparations. Of three l A P lots obtained from Bachem, only #ZI682 HAP was neurotoxic to high density rat hippocampal cultures. Other lots of l A P from Bachem (nominally identical by standard criteria), as well as three unrelated sources of lAP, were nontoxic. Initial biochemical characterization (reverse phase HPLC chromatography, electrospray mass spectroscopy, and amino acid sequence analysis) has not yet revealed any obvious differences between these various HAP lots. Follow-up studies should evaluate aggregation properties of #ZI682 HAP as aggregation state-dependent neurotoxicity was recently reported for lAP(I-42) (5). Nonetheless, our observation that some but not all preparations of l A P are neurotoxic even under identical in vitro culture conditions may account for part of the present controversy in this area. Different cell culture conditions used in various labs (plating density, species, and brain region differences, etc.) also may contribute to the conflicting observations on direct HAP toxicity. Some prior studies have used low to very low density cultures (4,8) to optimize for morphological analysis of individual neurons. We have opted to use cultures plated at high density to improve neuronal survival. In addition, higher density cultures afford the opportunity to score cell death by a nonsubjective, easily quantifiable enzymatic assay. Despite the differences in our in vitro conditions, using one particular lot of flAP we have observed apparent direct HAP neurotoxicity similar but not identical to that reported by Yankner and colleagues (8) (see below). Z1682 HAP toxicity is unlikely to be acting indirectly through potentiation of glutamate excitotoxicity (2,4). Our experiments were conducted in serum-free media to limit exogenous glutamate addition and the low level

70

Statistical Analysis

60-

Main effects of treatment were assessed by analysis of variance and, if significant, group means were compared by post hoc analysis (StatView II, Abacus Concepts, Berkeley, CA).

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=

Y////////A

e.

RESULTS Multiple sources of flAP were evaluated for neurotoxicity. Only Bachem #ZI682 l A P produced significant neurotoxicity as judged visually and confirmed by elevated LDH levels in the culture media (p < 0.01, vehicle vs. #ZI682, Scheffe F-test); Bachem #ZI424 and #ZI960 HAP were essentially nontoxic (Fig. 1). Two additional HAP preparations, one each from Multiple Peptide Systems and Athena, as well as the Lilly lAP(I-43), were also nontoxic at 10 pM (Fig. 1). Many of the l A P preparations contained low levels of endotoxin ( 1-2 EU/ml), but endotoxin contamination did not appear to correlate with neurotoxicity (Fig. 1). To further dissociate endotoxin contamination from apparent HAP toxicity, polymyxin B (25 #g/ml) was preincubated (30 min at RT) with #ZI682 l A P in defined DMEM prior to addition

3:30 a "

*

~//////~

-

20 -

lO

CTL

SAP (ZI682)

RAP 1ZI682) + PMB

BAP 1ZI682) + Sub P

FIG. 2. Effect of polymyxin B and Substance P on #Z1682/3AP direct toxicity. Rat hippocampal cultures ( 10 DIV) were treated for 4 days with 10 uM #ZI682/3AP in the presence or absence of 25 pg/ml polymyxin B (PMB) or 10 pM Substance P. Wells treated with polymyxin B and Substance P alone were not different from control. Results are expressed as units/ml of LDH (mean + SD, n = 3) and are representative of two separate experiments.

V A R I A B L E /3AP IN V I T R O T O X I C I T Y

607

of glutamate formed from glutamine breakdown in the media should be rapidly taken up by numerous glia present in our cultures. Substance P afforded no protection against #Z1682/3AP neurotoxicity. Moreover, treatment of high density hippocampal cultures with the Substance P receptor antagonist, (+) CP-96,345, did not elicit cytotoxicity (data not shown). These observations suggest that the mechanism(s) of direct ¢/AP toxicity observed in high density and very low density rat hippocampal cultures may differ with respect to effects at tachykinin receptors (8). Finally, we caution investigators to evaluate possible endotoxin effects in their various in vitro models. In the present study, endotoxin contamination of several/~AP preparations did not

appear to account for the neurotoxicity of/3AP. However, endotoxin alone can elicit interleukin-1 release from nonneuronal cells (7) similar to that ascribed to flAP(l-42) (1). We suggest the use of polymyxin B at least in initial studies with any new lot of flAP to limit confounds caused by possible endotoxin contamination. ACKNOWLEDGEMENTS We thank Dr. Russell Rydel (Athena Neurosciences, S. San Francisco, CA) and Dr. Richard DiMarchi, Dr. Sheila Little, and Diane Stephenson (Eli Lily and Co., Indianapolis, IN) for providing/3APs used in this study. We also thank Dr. Jeffrey Howbert (Eli Lilly and Co.) for providing (_+) CP-96,345.

REFERENCES I. Arauju, D. M.; Cotman, C. W. B-Amyloid stimulates glial cells in vitro to produce growth factors that accumulate in senile plaques in Alzheimer's disease. Brain Res. 569:141 - 145; 1992. 2. Koh, J-Y.; Yang, L. L.; Cotman, C. W. ~-Amyloid protein increases the vulnerability of cultured cortical neurons to excitotoxic damage. Brain Res. 533:315-320; 1990. 3. Kowall, N. W.; Beal, M. F.; Busciglio, J. E.; Duffy, L. K.; Yankner, B. A. An in vivo model for the neurodegenerative effects of¢/amyloid and protection by substance P. Proc. Natl. Acad. Sci. USA 88:72477251; 1991. 4. Mattson, M. P.; Cheng, B.; Davis, D.; 13ryant, K.; Lieberburg, I.; Rydel, R. E. ¢~-Amyloidpeptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity. J. Neurosci. 12:376-389; 1992.

5. 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. 6. Selkoe, D. J. The molecular pathology of Alzheimer's disease. Neuron 6:487-498; 1991. 7. Velasco, S.; Tarlow, M.; Losen, K.; Shay, J. W.; McKracken, G. H., Jr.; Nisen, P. D. Temperature-dependent modulation oflipopolysaccharide-induced interleukin-l~ and tumor necrosis factor-alpha expression in cultured human astroglial cells by dexamethasone and indomethacin. J. Clin. Invest. 87:1674-1680; 1991. 8. Yankner, B. A.; Duffy, L K.; Kirschner, D. A. Neurotrophic and neurotoxic effects of amyloid fl protein: Reversal by tachykinin neuropeptides. Science 250:279-282; 1990.