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(American Journal of Pathology. 1999;154:1001-1007.)
© 1999 American Society for Investigative Pathology

Short Communication

A ßPP Peptide Carboxyl-Terminal to Aß Is Neurotoxic

Gabriella Marcon* , Giorgio Giaccone* , Barbara Canciani* , Laura Cajola* , Giacomina Rossi* , Luca De Gioia{dagger} , Mario Salmona{dagger} , Orso Bugiani* and Fabrizio Tagliavini*

From the Division of Neuropathology,* Istituto Nazionale Neurologico Carlo Besta, and the Istituto di Ricerche Farmacologiche Mario Negri,{dagger} Milano, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Extracellular Aß-amyloid and intraneuronal paired helical filaments (PHFs) composed of tau protein are the neuropathological hallmark of Alzheimer's disease. Aß is a 39- to 43-residue peptide derived by cleavage of a 695- to 770-amino-acid membrane-associate glycoprotein (termed ß-protein precursor, ßPP). Following the observation that an antiserum to an epitope located between residues 713 and 723 of ßPP770 (ie, the transmembrane region of the ßPP distal to Aß) labels PHFs and that a synthetic peptide homologous to residues 713 to 730 of ßPP770 (ßPP713–730) is highly fibrillogenic and interacts with tau in vitro, it has been hypothesized that ßPP fragments other than Aß may feature in the pathogenesis of Alzheimer's disease concurring with neuronal degeneration. To investigate this issue, we have analyzed the effects of the exposure of primary neuronal cultures to the synthetic peptide ßPP713–730. Cultures were prepared from rat hippocampus on embryonic day 17 and incubated with the peptide at 2.5 to 30 µmol/L concentration for 1 to 4 days. Cell viability was compared with that of control cultures exposed to a scrambled sequence of the peptide. A 4-day exposure to 20 µmol/L ßPP713–730 resulted in almost complete neuronal loss, whereas no changes were observed with the scrambled peptide. Degenerating neurons showed DNA fragmentation by agarose gel electrophoresis and apoptotic changes by light and electron microscopy. These findings support the view that ßPP sequences other than Aß may play a role in nerve cell degeneration in Alzheimer's disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Alzheimer's disease is a degenerative disorder of the brain that causes a progressive cognitive decline in mid to late adult life. The neuropathological hallmark of the disease is the co-occurrence of extracellular amyloid fibrils and intraneuronal neurofibrillary tangles (NFTs). The major constituent of amyloid fibrils is Aß, a 39- to 43-residue peptide1,2 derived by proteolytic cleavage of a 695- to 770-amino-acid membrane-associated glycoprotein (ß-protein precursor, ßPP) that has structural features of an integral transmembrane receptor.3-6 Aß is positioned partly in the extracellular domain and partly in the transmembrane domain, corresponding to residues 672–710/714 of ßPP770 (Figure 1) . NFTs are made up of paired helical filaments (PHFs) composed primarily of microtubule-associated protein tau in an abnormally phosphorylated form (see Ref. 7 for review).



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  		Figure 1. Schematic representation of ßPP770 with Aß (black box) and the synthetic peptide ßPP713–730 used in this study (dashed bar). Aß consists of 28 amino acids in the extracellular domain and 11 to 15 residues within the transmembrane domain. The synthetic peptide ßPP713–730 corresponds to the 11 last residues of the transmembrane domain and the 7 first residues of the intracytoplasmic domain.

 
The relationship between amyloid deposition, nerve cell degeneration, and NFT formation is undetermined.8,9 A direct link between Aß and neuronal degeneration has been suggested by the fact that fibrillar Aß is toxic to neurons in vitro.10 However, evidence indicates that Aß by itself is not sufficient to cause severe neuronal damage in vivo, as Aß deposition may occur also in the brain of aged nondemented individuals without significant neuronal loss or dysfunction.11-16

Recently it has been observed that the PHFs are immunoreactive with an antibody to a ßPP epitope distal to Aß and that a synthetic ßPP peptide containing this epitope (ßPP713–730) is highly fibrillogenic and binds to tau protein in vitro.17-19 These findings suggest that ßPP fragments other than Aß might have a role in the process of neuronal degeneration in Alzheimer's disease. To investigate this issue, we analyzed the effects of the peptide ßPP713–730 on rat hippocampal neurons in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Cultures

Primary cultures of hippocampal neurons were prepared from fetal rat brains on embryonic day 17. Hippocampal tissue was washed in phosphate-buffered saline (PBS; Dulbecco's without Ca2+/Mg2+, Biochrom KG-Seromed, Berlin, Germany); the cells were dissociated in Eagle's basal medium Eagle supplemented with 7% NaHCO3, 10% fetal calf serum, and 1% L-glutamine (BME-Hanks' salt, Gibco, Paisley, UK) and plated at a density of 3.5 x 105 cells per well in Primaria 15-mm wells (Falcon, Becton Dickinson, Meylan, France), precoated with poly-D-lysine (50 µg/ml; Sigma Chemical Co., St. Louis, MO). The cells were kept at 37°C in a humidified 5% CO2 atmosphere. Five days after plating, non-neuronal cell division was halted by exposure to 10-5 mol/L cytosine arabinoside (Sigma) to prevent overgrowth of glial cells. Additional cultures were prepared in tissue culture chamber slides (Nunc, Naperville, IL) for immunohistochemical investigation.

Peptide Synthesis and Purification

A peptide homologous to residues 713 to 730 of ßPP770 (ATVIVITLVMLKKKQYTS) and a scrambled sequence of this peptide (KVKTTKVTVAMIQYSILL) were synthesized by solid-phase chemistry on a 430A synthesizer (Applied Biosystems, Foster City, CA) and purified by reverse-phase high-pressure liquid chromatography (HPLC) as described previously.20 The identity and purity of the peptides were determined by analytical reverse-phase HPLC and amino acid sequencing (Applied Biosystems 477A microsequencer); purity was >95% for both peptides.

Treatment and Morphological Evaluation of Cultures

The synthetic peptides ßPP713–730 and scrambled ßPP713–730 were suspended in sterile distilled water at a concentration of 600 µmol/L. Under these conditions, ßPP713–730 aggregates into amyloid-like fibrils as previously described,17 whereas scrambled ßPP713–730 is soluble. Peptide suspensions were added directly to culture medium to a final concentration of 2.5, 5, 10, 20, and 30 µmol/L on day 4 and day 6 after plating. On day 8, cells were analyzed by light and electron microscopy, fluorescence microscopy after staining with the DNA-binding fluorochrome 4',6-diamidine-2-phenylindole-dihydrochloride (DAPI; Boehringer, Mannheim, Germany) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method for in situ labeling of fragmented DNA.21 For DAPI staining, the cultures were washed with PBS, incubated with aqueous solution of DAPI (1 µg/ml) at room temperature for 5 minutes and then at 37°C for 15 minutes, washed with 100% methanol, rehydrated, and mounted. TUNEL was performed on 4% paraformaldehyde-fixed cultures after microwave treatment (Miele M720 microwave oven, two cycles of 5 minutes at 150 W in 0.01 mol/L citrate buffer, pH 6),22 using the Boehringer kit for in situ detection detection of fragmented DNA (catalog item 1 684 817), according to the manufacturer's instructions. For electron microscopy, cultures were fixed with 2.5% glutaraldehyde in 100 mmol/L phosphate buffer, pH 7.4, at 4°C for 20 minutes, rinsed in Sörensen solution containing 10% saccharose, and post-fixed in 4% OsO4 at 4°C for 1 hour. After dehydration, the cultures were embedded in Epon (48 hours at 60°C). Ultrathin sections were stained with uranyl acetate and lead citrate and scanned with a ZEISS 109 electron microscope.

Viability of Cultures

Viability of cultured cells was assessed by crystal violet staining on day 8 after plating. Cultures were incubated with 0.5% crystal violet in water/methanol (4:1) for 4 minutes, washed with water, and dried. The mean optical density and the percent area covered by cultured cells with respect to the whole well area were determined using the image analyzer IBAS 2.0 (Kontron-Zeiss). These two parameters allowed us to calculate the weighted optical density (mean optical density x percent area), which has been demonstrated to be proportional to the number of living cells.23

DNA Analysis

To evaluate the DNA fragmentation, the hippocampal cells from 15-mm wells were washed with PBS, pH 7.4, and, after addition of cold lysis buffer (10 mmol/L Tris/HCl, 20 mmol/L EDTA, 0.5% Triton X-100, pH 8.0), collected, transferred to microtubes, and kept on ice for 15 minutes. After centrifugation at 13,000 x g for 20 minutes, the supernatant (containing low molecular weight DNA) was incubated with RNAse A (100 µg/ml at 37°C for 1 hour); the pellet (containing high molecular weight DNA) was incubated with proteinase K (100 µg/ml at 48°C overnight). DNA was extracted with phenol/chloroform solution and precipitated with NaCl (0.5 mol/L final concentration) and ethanol (3 vol) at -70°C overnight. It was then resuspended in 10 mmol/L Tris/HCl, 1 mmol/L EDTA, pH 7.4, and electrophoresed through 1.1% agarose gel. DNA bands were visualized by staining with ethidium bromide.

For a quantitative evaluation of DNA fragmentation, the cells were lysed with lysis buffer as described above and centrifuged at 13,000 x g for 20 minutes. Both the supernatant and the pellet were treated with trichloroacetic acid (12.5% final concentration) at 4°C overnight. After centrifugation at 13,000 x g for 5 minutes, the DNA in the pellet was quantified by the diphenylamine reaction24 with the spectrophotometrical analysis at 600 nm with an automated microplate reader (Titertek Multiskan, ICN, Costa Mesa, CA).

Immunocytochemistry

Cultures were washed with PBS, pH 7.2, fixed for 30 minutes in 4% paraformaldehyde in PBS, pH 7.2, washed with 50 mmol/L Tris buffer, pH 7.6, and permeabilized with 0.3% Triton X-100 in Tris buffer for 5 minutes. Cultures were immunostained with the monoclonal antibody TAU1 (1:100; Boehringer), which detects a nonphosphorylated epitope of tau including Ser199-Ser202, as well as with an anti-MAP2 monoclonal antibody (clone AP20, Boehringer) revealed by an avidin-biotin system with biotinylated anti-mouse serum raised in sheep (1:100; Amersham, Little Chalfont, UK) followed by streptavidin-horseradish peroxidase (1:100; Amersham), using 3–3'-diaminobenzidine as chromogen.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The normal hippocampal cultures initially consisted of undifferentiated round cells. After 2 to 3 days, the cells began to generate processes and to acquire neuronal morphology. Axonal processes elongated and organized in bundles that formed a loose network (Figure 2a) . The axonal processes were strongly immunoreactive with the monoclonal antibody TAU1 (Figure 2c) , whereas cell bodies and dendrites were labeled by anti-MAP2 antibody. These morphological and immunohistochemical features are typical of neuronal cultures of fetal rat hippocampus.25



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  		Figure 2. Primary cultures of hippocampal neurons exposed for 4 days to vehicle solution (a and c) or to the peptide ßPP713–730 at a concentration of 20 µmol/L (b, d, and e). a and b: Phase-contrast photomicrographs showing remarkable neuronal degeneration in culture treated with the ßPP peptide (b) as compared with control culture (a). c to e: TAU1 immunocytochemistry showing a dense network of strongly decorated axons in control culture (c) and almost complete loss of axonal processes in culture exposed to the ßPP peptide (d), the residual immunostaining being largely confined to a few cell bodies (e). Magnification, x210 (a), x420 (b), x155 (c and e), and x100 (d).

 
No difference from this pattern was observed in cell cultures treated with the scrambled ßPP713–730 peptide or the vehicle solution. Conversely, the neuronal cultures exposed to 20 µmol/L ßPP713–730 underwent severe degeneration. The first changes were detected 2 days after peptide treatment and consisted in loss of phase brightness of cell bodies followed by shrinkage. After a 4-day treatment, the few surviving cells displayed a collapse of the dendritic arbor and an almost complete loss of axonal processes (Figure 2, b and d) , the scanty residual Tau1 immunostaining being confined to some degenerating neuronal cell bodies (Figure 2e) . No other specific derangement of the cytoskeleton and in particular no abnormal cytoplasmic inclusions were observed in the ßPP713–730-treated neurons.

A semiquantitative evaluation of cell viability by crystal violet staining demonstrated that the neurotoxic effect of ßPP713–730 was dose dependent. The neuronal loss was 56%, 62%, and 75% at concentrations of 5 µmol/L, 10 µmol/L, and 20 µmol/L of ßPP713–730, respectively. By contrast, the viability of cultures treated with the scrambled peptide did not differ significantly from that of cultures exposed to vehicle solution (Figure 3) .



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  		Figure 3. Quantitative evaluation of the neurotoxic effect of ßPP713–730 as a function of peptide concentration. After a 4-day exposure of cultures to ßPP713–730 (5, 10, and 20 µmol/L) or to a scrambled sequence of ßPP713–730 (20 µmol/L), nerve cell viability was assessed by crystal violet staining followed by densitometry. Viable neurons are expressed as a percentage of the number of neurons in vehicle-treated cultures. The data are the mean + SE of 12 to 20 determinations.

 
To investigate whether a mechanism of programmed cell death was involved in the neurotoxicity induced by ßPP713–730, control cultures and cultures treated for 4 days with 20 µmol/L ßPP713–730 were stained with the DNA-binding fluorochrome DAPI or labeled with TUNEL. Microscopic examination showed that a large number of neurons exposed to ßPP713–730 exhibited pyknotic or fragmented nuclei that were marked by TUNEL (Figure 4, a and c) , whereas only rare apoptotic cells were present in cultures treated with scrambled ßPP713–730 or vehicle solution (Figure 4, b and d) . At the electron microscopic level, the altered neurons displayed changes corresponding to different stages of the apoptotic process. Some cells showed aggregation of chromatin into large compact granular masses lying on the nuclear membrane (Figure 4e) ; in others, the nucleus had condensed and broken up into fragments that formed intracytoplasmic apoptotic bodies.



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  		Figure 4. Apoptosis of hippocampal neurons treated with ßPP713–730. a to d: Photomicrographs of hippocampal cultures stained with the TUNEL method for in situ labeling of fragmented DNA (a and b) and with the DNA-binding fluorochrome DAPI (c and d) after a 4-day exposure to 20 µmol/L ßPP713–730 (a and c) or to vehicle solution (b and d). After ßPP713–730 treatment, several nuclei are strongly labeled by TUNEL (a) and show a typical pyknotic morphology (c). At variance, in control culture, nuclei labeled by TUNEL (b) or showing condensation and fragmentation after DAPI (d) are very rare. Magnification, x150 (a and b) and x420 (c and d). e: Electron micrograph of a neuron from hippocampal culture treated for 4 days with 10 µmol/L ßPP713–730, showing aggregation of chromatin into large compact granular masses lying on the nuclear membrane. Magnification, x14,000. f: Agarose gel electrophoresis of the supernatant (lanes 1 and 3) and the pellet (lanes 2 and 4) of DNA preparations from hippocampal neurons exposed for 4 days to 20 µmol/L ßPP713–730 (lanes 1 and 2) or to vehicle solution (lanes 3 and 4). Fragmented DNA, which separates in the supernatant as a ladder, is more abundant in cultures treated with ßPP713–730 (lane 1) than in control conditions (lane 3). Conversely, high molecular weight DNA, which separates in the pellet, is reduced in ßPP713–730-treated cultures (lane 2) as compared with controls (lane 4).

 
Agarose gel electrophoretic analysis of DNA extracted from cultured cells was carried out to evaluate the fragmented DNA (Figure 4f) that was then quantified by the diphenylamine method. In cultures treated with 20 µmol/L ßPP713–730 for 4 days, the fragmented DNA was 12.5 ± 3.4% (mean ± SD; n = 13) of total DNA, whereas in control cultures the fragmented DNA was 6.1 ± 3.1% (mean ± SD; n = 19) of total DNA. This difference was statistically significant (P < 0.001, Student's t-test).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The hypothesis of a neurotoxic effect of Aß originated from the observation that in Alzheimer brains Aß amyloid deposits are closely associated with abnormal neurites and was supported by the finding that Aß induces nerve cell death in vitro.10 A possible role of ßPP peptides other than Aß has not been extensively investigated to date, although immunohistochemical analyses indicated that ßPP and/or fragments thereof accumulate in dystrophic neurites of senile plaques.26,27 Our study shows that a synthetic peptide homologous to residues 713 to 730 of ßPP770, corresponding to the ßPP region located carboxyl-terminally to the Aß sequence, is neurotoxic and suggests that ßPP cleavage products other than Aß may feature in the pathogenesis of Alzheimer's disease.

Several lines of evidence support this view, including the early studies by Yankner and co-workers28 that showed that PC12 cells transfected with the 105 carboxyl-terminal amino acids of ßPP (ßPP-C105) degenerated when induced to differentiate into neuronal cells by nerve growth factor. Subsequently, this neurotoxic effect was entirely ascribed to Aß, based on the results obtained with relevant synthetic peptides.10 However, the neurotoxicity of ßPP-C105 to rat cortical neuronal cultures was stronger than that observed with any Aß fragment.29 Therefore, the possibility should be considered that Aß and ßPP sequences carboxyl-terminal to Aß act synergistically in determining the neurotoxic effect of ßPP-C105.

It is known that the processing of ßPP generates several carboxyl-terminal peptides including residues 713 to 730, although the biochemical characterization, cellular trafficking, and topology of such fragments have not been completely established. By the endosomal-lysosomal pathway, ßPP is cleaved by ß-secretase, yielding a 12-kd product containing Aß at its amino terminus. Additional enzymatic attack by {gamma}-secretase generates Aß and releases a complementary carboxyl-terminal fragment (usually indicated as p7) whose amino terminus corresponds to ßPP713–730.30,31 It is conceivable that p7 and possibly larger fragments containing the sequence 713 to 730 have physicochemical properties and biological activities analogous to those of the ßPP713–730 peptide. This scenario would parallel with the finding that peptides homologous to portions of Aß (eg, residues 25 to 35) retain the fibrillogenic and neurotoxic properties of the entire amyloid subunit.10 It is noteworthy in this regard that ßPP peptides corresponding to the 100 carboxyl-terminal residues accumulate consistently in neurons expressing any of the five known mutations of ßPP associated with familial Alzheimer's disease32 and that these fragments share the ability with Aß and ßPP713–730 to assemble into amyloid-like fibrils when expressed in COS cells.33

Our experiments also indicate that the dose-dependent neuronal death induced by the prolonged exposure of rat hippocampal neurons to ßPP713–730 peptide occurs by an apoptotic mechanism. This may be relevant to the process of neurodegeneration in Alzheimer's disease, considering that 1) cells that express ßPP mutants and accumulate carboxyl-terminal ßPP fragments undergo apoptosis34 and 2) extensive DNA fragmentation has been demonstrated in Alzheimer brains.35

Additional studies are needed to clarify how the ßPP713–730 peptide might affect neurons. On the other hand, despite extensive research in the past years, the mechanisms of neurotoxicity of Aß are not fully understood. Free radicals, increased sensitivity to excitotoxicity, disruption of Ca2+ homeostasis, and interference with ubiquitin-dependent protein degradation have been considered (see Ref. 9 for review). The analogy between the changes evoked in cultured neurons by Aß and ßPP713–730 peptides raises the possibility of similar mechanisms of action. Alternatively, as it has been suggested that the tau-binding properties of the ßPP region distal to Aß may reflect a physiological tau-mediated interaction between full-length ßPP and the cytoskeleton,18,19 the mechanism of neuronal damage induced by ßPP713–730 may involve a disruption of this binding.

In conclusion, our findings provide evidence for a neurotoxic activity of the synthetic peptide homologous to region 713 to 730 of ßPP770, which lies outside the extensively studied Aß region and may represent a novel molecular landmark for efforts to elucidate the relationship between the complex cascade of events triggered by ßPP metabolites and neurodegeneration in Alzheimer's disease.


   Footnotes
 
Address reprint requests to Dr. Giorgio Giaccone, Istituto Nazionale Neurologico Carlo Besta, via Celoria 11, 20133 Milano, Italy. E-mail: neuropatologia{at}istituto-besta.it

Supported by the Italian Ministry of Health, Department of Social Services, and by Telethon Italy (grant E360 to G. Giaccone and E482 to G. Marcon).

G. Marcon's present address: DPMSC, University of Udine.

Accepted for publication December 23, 1998.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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