In Vivo Cytotoxicity of the Prion Protein Fragment 106–126

Abstract

Transmissible spongiform encephalopathies are fatal neurological diseases characterized by astroglyosis, neuronal loss, and by the accumulation of the abnormal isoform of the prion protein. The amyloid prion protein fragment 106–126 (P106–126) has been shown to be toxic in cultured hippocampal neurons (1Forloni G. Angeretti N. Chiesa R. Monzani E. Salmona M. Bugiani O. Tagliavini F. Nature. 1993; 362: 543-546Crossref PubMed Scopus (896) Google Scholar). Here, we show that P106–126 is also cytotoxic in vivo. Taking advantage of the fact that retina is an integral part of the central nervous system, the toxic effect of the peptide was investigated by direct intravitreous injection. Aged solutions of P106–126 induced apoptotic-mediated retinal cell death and irreversibly altered the electrical activity of the retina. Neither apoptosis nor electroretinogram damages were observed with freshly diluted P106–126, suggesting that the toxicity is linked to the aggregation state of the peptide. The retina provides a convenient in vivo system to look for potential inhibitors of cytotoxicity associated with spongiform encephalopathies. Transmissible spongiform encephalopathies are fatal neurological diseases characterized by astroglyosis, neuronal loss, and by the accumulation of the abnormal isoform of the prion protein. The amyloid prion protein fragment 106–126 (P106–126) has been shown to be toxic in cultured hippocampal neurons (1Forloni G. Angeretti N. Chiesa R. Monzani E. Salmona M. Bugiani O. Tagliavini F. Nature. 1993; 362: 543-546Crossref PubMed Scopus (896) Google Scholar). Here, we show that P106–126 is also cytotoxic in vivo. Taking advantage of the fact that retina is an integral part of the central nervous system, the toxic effect of the peptide was investigated by direct intravitreous injection. Aged solutions of P106–126 induced apoptotic-mediated retinal cell death and irreversibly altered the electrical activity of the retina. Neither apoptosis nor electroretinogram damages were observed with freshly diluted P106–126, suggesting that the toxicity is linked to the aggregation state of the peptide. The retina provides a convenient in vivo system to look for potential inhibitors of cytotoxicity associated with spongiform encephalopathies. transmissible spongiform encephalopathies terminal deoxynucleotidyl transferase dUTP-end labeling phosphate-buffered saline 3,3′-diaminobenzidine electroretinograms Transmissible spongiform encephalopathies (TSE)1 are neurological disorders having common pathological signs like vacuolization of the neurophils, astrocytosis, and loss of neurons (2Fraser H. Br. Med. Bull. 1993; 49: 792-809Crossref PubMed Scopus (57) Google Scholar). TSE are characterized by the accumulation of large aggregates of the prion protein, PrP-res, in both human and animal brains. The abnormal PrP-res isoform has a high content of β-sheet secondary structure, forms amyloid fibrils, and is partially resistant to proteolysis (3Caughey B.W. Dong A. Bhat K.S. Ernst D. Hayes S.F. Caughey W.S. Biochemistry. 1991; 30: 7672-7680Crossref PubMed Scopus (742) Google Scholar, 4Pan K.-M. Baldwin M. Nguyen J. Gasset M. Serban A. Groth D. Mehlhorn I. Huang Z. Fletterick R.J. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10962-10966Crossref PubMed Scopus (2076) Google Scholar, 5Safar J. Roller P.P. Gajdusek D.C. Gibbs Jr., C.J. J. Biol. Chem. 1993; 268: 20276-20284Abstract Full Text PDF PubMed Google Scholar). The precursor of the pathological isoform PrP-res is a normal host glycoprotein (termed PrP-sen) widely expressed in the central nervous system and in peripheral tissues. Unlike PrP-res, PrP-sen is a protease-sensitive protein, soluble in mild detergents, and has mainly α-helix structures (6Meyer R.K. McKinley M.P. Bowman K.A. Braunfeld M.B. Barry R.A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2310-2314Crossref PubMed Scopus (517) Google Scholar, 7Riek R. Hornemann S. Wider G. Glockshuber R. Wuthrich K. FEBS Lett. 1997; 413: 282-288Crossref PubMed Scopus (664) Google Scholar, 8Riek R. Hornemann S. Wider G. Billeter M. Glockshuber R. Wuthrich K. Nature. 1996; 382: 180-182Crossref PubMed Scopus (1128) Google Scholar). Interestingly, the development of spongiform pathologies requires the presence of both isoforms PrP-res and PrP-sen (9Brandner S. Isenmann S. Raeber A.J. Fischer M. Sailer A. Kobayashi Y. Marino S. Weissmann C. Aguzzi A. Nature. 1996; 379: 339-343Crossref PubMed Scopus (718) Google Scholar), the level of PrP-sen expression being an essential factor in the pathology (10Büeler H. Aguzzi A. Sailer A. Greiner R.A. Autenried P. Aguet M. Weissmann C. Cell. 1993; 73: 1339-1347Abstract Full Text PDF PubMed Scopus (1810) Google Scholar). In experimental transmissions with transgenic mice, it was found that increasing the number ofPrP gene copies led to a decreased incubation time of the disease (11Carlson G.A. Ebeling C. Yang S.L. Telling G. Torchia M. Groth D. Westaway D. DeArmond S.J. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5690-5694Crossref PubMed Scopus (135) Google Scholar, 12Tremblay P. Meiner Z. Galou M. Heinrich C. Petromilli C. Lisse T. Cayetano J. Torchia M. Mobley W. Bujard H. DeArmond S.J. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12580-12585Crossref PubMed Scopus (138) Google Scholar). The in vivo mechanisms leading to the synaptic loss and neuronal death are not elucidated, but the possible involvement of apoptotic processes has been postulated and is consistent with the lack of inflammation stigmata in TSE-affected brains. The neural tissue damages could be due exclusively to PrP-res deposits, and/or to disruption of PrP-sen physiological functions, and/or to cytotoxic effects induced by PrP fragments or PrP-res aggregates. PrP-res and a synthetic peptide fragment (P106–126) have been shown to be cytotoxicin vitro likely via the programmed cell death pathway (1Forloni G. Angeretti N. Chiesa R. Monzani E. Salmona M. Bugiani O. Tagliavini F. Nature. 1993; 362: 543-546Crossref PubMed Scopus (896) Google Scholar,13Müller W.E.G. Ushijima H. Schröder H.C. Forrest J.M.S. Schatton W.F.H. Rytik P.G. Heffner-Lauc M. Eur. J. Pharmacol. 1993; 246: 261-267Crossref PubMed Scopus (157) Google Scholar). Like PrP-res, the P106–126 peptide is partially resistant to proteinase K, presents a high β-sheet enriched structure, and forms amyloid fibrils in vitro (14Selvaginni C. De Gioia L. Cantù L. Ghibaudi E. Diomede L. Passerini F. Forloni G. Bugiani O. Tagliavini F. Salmona M. Biochem. Biophys. Res. Commun. 1993; 194: 1380-1386Crossref PubMed Scopus (195) Google Scholar). Hope et al. (15Hope J. Shearman M.S. Baxter H.C. Chong A. Kelly S.M. Price N.C. Neurodegeneration. 1996; 5: 1-11Crossref PubMed Scopus (65) Google Scholar) have shown that the expression of PrP-sen is necessary for P106–126 to exert its cytotoxic effects. Whether the apoptotic process could account for in vivoneurodegeneration taking place during the development of TSE remains unclear. The induction of apoptosis has been suggested in scrapie-infected mouse brain (16Giese A. Groschup M.H. Hess B. Kretzschmar H.A. Brain Pathol. 1995; 5: 213-221Crossref PubMed Scopus (146) Google Scholar), as well as in Creutzfeldt-Jakob disease (17Gray F. Chretien F. Adle-Biassette H. Dorandeu A. Ereau T. Delisle M.B. Kopp N. Ironside J.W. Vital C. J. Neuropathol. Exp. Neurol. 1999; 58: 321-328Crossref PubMed Scopus (147) Google Scholar) and bovine spongiform encephalopathy (18Theil D. Fatzer R. Meyer R. Schobesberger M. Zurbriggen A. Vandevelde M. J. Comp. Pathol. 1999; 121: 357-367Crossref PubMed Scopus (20) Google Scholar). In the present report, we examined whether the PrP fragment P106–126 can induce apoptotic-mediated cell death in vivo. To address this question, we directly administrated P106–126 into rat eyes taking advantage of the fact that the retina is an integral part of the central nervous system. Apoptotic process induced by P106–126 was demonstrated by means of the terminal deoxynucleotidyl transferase dUTP-end labeling (TUNEL) staining and of the DNA fragmentation technique. Neuronal death was confirmed by measurement of the electrical activity of the retina. The human sequence of the prion protein fragment (P106–126; KTNMKHMAGAAAAGAVVGGLG) was purchased from Bachem. The prion fragment used as a control peptide (P89–103; GQGGGTHNQWNKPSK) was synthesized by Eurogentec. Lyophilized peptides were dissolved in deionized water at a concentration of 5 mm, distributed into 20-μl aliquots, and stored at −20 °C. Peptides were diluted twice with 2× PBS minus Ca2+ and immediately used or aged by incubation at room temperature for 3 days. Adult male albino Wistar rats (35–55 days old) were anesthetized with an intraperitoneal injection of 60 mg/kg sodium pentobarbital followed by a topical application of 0.4% oxybuprocaine hydrochloride. The injections of 2 μl of peptides or vehicle (phosphate-buffered solution) were done unilaterally with a 30-gauge needle introduced into the posterior chamber on the upper pole of the eye directed toward the center of the vitreous. The injections were performed slowly to allow a better diffusion of the peptide and avoid any ocular hypertension. For negative controls, we both considered the uninjected contralateral eyes and eyes injected in the same conditions with 2 μl of vehicle (PBS). At least four animals were used in each group of experimental conditions. Rats were euthanatized with an overdose of sodium pentobarbital 3 days post-intravitreal injections. The eyes were enucleated, and a puncture was made at the limbus to permit the infusion of the fixative solution. The eyes were immediately fixed in ice-cold 4% paraformaldehyde in PBS for 4 h then cryoprotected overnight in PBS containing 20% sucrose and embedded in Tissue-Tek® (Sakura). Frozen sections (10 μm) were cut on a cryostat (Leica), and slides were heated at 50 °C for 60 min then stored at −20 °C until use. Thein situ cell death detection was performed following the manufacturer's recommendations (Roche Molecular Biochemicals) then revealed using a 3,3′-diaminobenzidine (DAB) substrate kit (Vector Laboratories) and processed for detailed examination by light microscopy. Morphological and histological observations of retinas were done by staining the sections with 1% cresyl violet. 2 μl of PBS alone or of 3-day-aged peptide P106–126 diluted extemporarily in PBS (corresponding to 0.05, 0.5, and 5 nmol) were injected as described above. For each condition, four rats were treated, and retina sections were processed simultaneously for the TUNEL-labeling assay. To evaluate the number of TUNEL-positive cells, photographs of retinal sections were collected at an original magnification of × 10. The intensity of gray of each layer (ganglion cell, G; inner nuclear, INL; outer nuclear, ONL) was quantified using the NIH image software system. The gray intensity was expressed per surface unit in arbitrary units. After enucleation, the retinas were dissected from the retinal epithelium and choroid. Proteins were then digested with 200 μg/ml of proteinase K in lysis buffer (10 mm Tris, pH 7.4, 5 mm EDTA, 1% SDS) for 2 h at 55 °C. Retinal DNA was extracted by phenol-chloroform solutions, and the aqueous phase was incubated with Dnase-free RNase A (100 μg/ml) and then precipitated in a solution of 0.3 msodium acetate in ethanol overnight at −20 °C. Precipitated DNA samples were resuspended in distilled water, and the DNA concentration was determined by measuring the absorbance at 260 nm. Samples of 10 μg of DNA were electrophoresed through 1.2% agarose gel containing 1 μg/ml of ethidium bromide. DNA bands were visualized by a UV light transilluminator and photographed. Dark-adapted ERGs were performed as described previously (19Ettaiche M. Fillacier K. Widmann C. Heurteaux C. Lazdunski M. Invest. Ophthalmol. Visual Sci. 1999; 40: 729-736PubMed Google Scholar). Full-field ERG responses were obtained overnight with dark-adapted rats prepared under dim red light before recording. The pupils of anesthetized rats were dilated with a drop of 0.5% tropicamide. A silver chloride ring-recording electrode was placed on the cornea, and the reference electrode, with a silver-silver chloride tip, was connected to the ear. Light stimulus (10 μs) was provided by a stroboscopic flash (Grass Instruments Inc.) placed 0.25 m in front of the rat. The ERGs were recorded using the visual electroretinogram test system (LKC Technologies, Inc.) and then stored and analyzed. Amplitude of the a-wave was measured from the base line to the trough of the a-wave; b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave. The averaged responses represent the mean of five white flashes delivered four min apart. Electroretinograms were recorded before treatment and then at different times of recovery (1, 2, 3, and 7 days). The amplitude values of a- and b-waves obtained with each animal were normalized to base-line amplitudes at each time point of the study and expressed as percentage of base-line value. Data were expressed as mean ± S.E. of values obtained with 4 rats per group. Results from the ERG recordings were not distributed normally and had unequal variance. Therefore, the Mann WitneyU test was used to compare the peptide and the vehicle-treated group of rats. Differences were considered statistically significant when P values were less than 0.02. The effect of freshly prepared or 3-day-aged P106–126 and soluble P89–103 PrP peptides on the retina cell death was examined after intravitreal injection. Fig. 1 shows representative photomicrographs of treated retina sections processed through the TUNEL labeling assay (Fig. 1, B–E) and the histology of PBS-treated retina stained with cresyl violet (Fig. 1 A). After intravitreal injections of aged P106–126 (5 nmol), TUNEL-positive cells were observed in the inner and outer nuclear layers, as well as in the ganglion cells layer (Fig. 1 D). When freshly prepared P106–126 was intravitreally injected, only occasional TUNEL-positive cells were observed (Fig. 1 C) suggesting that the cytotoxic effect of the peptide was linked to its physicochemical state. Intact retinas (data not shown) and retinas treated with PBS alone (Fig. 1 B) or soluble PrP peptide P89–103 (Fig. 1 E) showed only few TUNEL-positive in labeling were observed the and the upper of the retina injections were To whether the cytotoxic effect was rat eyes were treated for h with PBS alone or containing 0.05, 0.5, or 5 of aged retina sections were processed through the TUNEL-labeling assay and revealed with the previously that the intensity of gray by the substrate was directly to the number of TUNEL-positive to a large surface layer and an number of retinal is a of the gray intensity obtained for the inner and outer nuclear and the ganglion cells The gray intensity of the with the of injected of injected P106–126 was not toxic for the retina cells, induced cells in ganglion cells and in inner and outer nuclear When 5 of P106–126 were injected, the TUNEL-labeling showed that the from the were To better and apoptotic cell the fragmentation of the retinal DNA was eyes were injected with vehicle alone or with 5 of aged peptide P106–126 and at different times after The retinas showed a of DNA with bands to of to 200 base (Fig. The DNA was but h after injection and was after and DNA was observed in the PBS-treated retinas (Fig. 3, control or in retinas treated with freshly prepared peptide P106–126 (data not The electrical activity of or retinas was by the Fig. 4 shows the effect of intravitreal injections on the electroretinograms and the of the a- and b-wave amplitudes days after When with vehicle-treated the aged group significant in both a- and b-wave direct of and damages to retinal In the the a-wave amplitude decreased at 1 ± and to slowly from 2 to Unlike the the b-wave amplitude decreased at 1 ± and then at 2 ± and decreased from 3 ± to 7 ± When electroretinogram recordings were done after injection of control peptide P89–103 or freshly prepared P106–126, significant on the a- and b-wave amplitudes could be observed (data not For the have shown that PrP peptide P106–126 could into the of of TSE pathology (1Forloni G. Angeretti N. Chiesa R. Monzani E. Salmona M. Bugiani O. Tagliavini F. Nature. 1993; 362: 543-546Crossref PubMed Scopus (896) Google Scholar, J. Shearman M.S. Baxter H.C. Chong A. Kelly S.M. Price N.C. Neurodegeneration. 1996; 5: 1-11Crossref PubMed Scopus (65) Google Scholar, J. Kretzschmar H.A. 1994; 5: PubMed Scopus Google Scholar, B. Kretzschmar H.A. Nature. 1996; PubMed Scopus Google Scholar). P106–126 not or induce in vitro of PrP-sen to PrP-res it a number of physicochemical with the PrP-res For P106–126 is partially proteinase and high β-sheet (14Selvaginni C. De Gioia L. Cantù L. Ghibaudi E. Diomede L. Passerini F. Forloni G. Bugiani O. Tagliavini F. Salmona M. Biochem. Biophys. Res. Commun. 1993; 194: 1380-1386Crossref PubMed Scopus (195) Google Scholar). fibrils in vitro by P106–126 are under light when stained by red F. F. L. G. R. P. B. Passerini F. Ghibaudi E. Forloni G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar). Like PrP-res, cell death in vitro W.E.G. Ushijima H. Schröder H.C. Forrest J.M.S. Schatton W.F.H. Rytik P.G. Heffner-Lauc M. Eur. J. Pharmacol. 1993; 246: 261-267Crossref PubMed Scopus (157) Google Scholar), P106–126 can induce apoptotic cell death (1Forloni G. Angeretti N. Chiesa R. Monzani E. Salmona M. Bugiani O. Tagliavini F. Nature. 1993; 362: 543-546Crossref PubMed Scopus (896) Google Scholar, T. S. C. M. Salmona M. Bugiani O. Tagliavini F. Forloni G. G. J. Res. 1998; PubMed Scopus Google Scholar). In P106–126 its toxic effects through direct and with cells J. Kretzschmar H.A. 1994; 5: PubMed Scopus Google Scholar, B. Groschup M.H. Kretzschmar H.A. Eur. J. Biol. 1998; PubMed Scopus Google Scholar). In with in vitro of P106–126 has been after direct into the can be for this the of the peptide by the this PrP-res is in the brain of a few days after then it to and the accumulation of the PrP-res R. D. J. 1996; PubMed Scopus Google Scholar). For the retina to be an in vivo retina is an integral part of the central nervous it is a system with a content of the PrP-sen isoform is expressed in cells of retina R. G.A. Westaway D. Lett. 1997; PubMed Scopus (20) Google Scholar). has been observed in N. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Prusiner S.B. Invest. Google Scholar, N.C. F. Exp. Neurol. PubMed Scopus Google Scholar). Therefore, the retina is a well to study the leading to neuronal death in the of the For the we show that aged P106–126 the death of retinal cells to an apoptotic process and provides a significant of the electrical activity of the retina. The observed effects of the peptide are by DNA fragmentation and electroretinogram and can be as as h after intravitreal injection. P106–126 damages on the physiological activity of recovery of the effects can be observed 7 days after peptide The mechanisms in the P106–126 toxicity are not The toxicity and physicochemical of amyloid peptides has been in vitro M. P. De Gioia L. S. M. A. F. B. T. Bugiani O. Forloni G. Tagliavini F. Biochem. J. 1999; PubMed Scopus Google Scholar, C. A. F. K. C.J. S.J. R. J. 1999; 73: PubMed Scopus Google Scholar). The β-sheet by P106–126 and its aggregation state are (1Forloni G. Angeretti N. Chiesa R. Monzani E. Salmona M. Bugiani O. Tagliavini F. Nature. 1993; 362: 543-546Crossref PubMed Scopus (896) Google Scholar, J. Shearman M.S. Baxter H.C. Chong A. Kelly S.M. Price N.C. Neurodegeneration. 1996; 5: 1-11Crossref PubMed Scopus (65) Google Scholar). are consistent with intravitreous injections of freshly diluted P106–126 to cell death and of electrical activity of the retina. al. A.J. A. S.M. A.J. Nature. 1998; Scopus Google Scholar) have shown that the amyloid peptides and in disease also induced cells apoptosis after intravitreous injections. cell death was observed only with aged freshly prepared solutions were of a control to the apoptotic induced by aged solutions of have been in the present (data not sequence and P106–126 both peptides the same to β-sheet structures and aggregates. that the of the aggregation of P106–126, than its is for the observed in vivo cell from mechanisms of P106–126 toxicity have been but are M. B. E. P. H. FEBS Lett. PubMed Scopus Google Scholar). The possible of the peptide into the cell could to the disruption of being for the cell death T. J. Biol. Chem. 1996; Full Text Full Text PDF Scopus Google Scholar, M. Y. N. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that at least part of the neuronal loss observed in TSE diseases is of the toxic effects of PrP-res and/or its In with disease in the of the amyloid peptides and is well the of the PrP remains unclear. fragment of PrP in and resistant to has been from of Creutzfeldt-Jakob disease P. P. L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). Caughey et al. B. Ernst D. J. 1991; PubMed Google Scholar) have also shown that a PrP-res isoform at is in scrapie-infected mouse fragments of PrP-sen have been in of normal and the has been to or P. P. L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). Interestingly, the in physiological conditions the of PrP-sen pathological conditions could the of PrP toxic fragments that are in physiological In study provides the in vivo of the involvement of an apoptotic process in the cytotoxic mechanisms of the prion protein fragment P106–126 suggesting that mechanisms also in The retina used in the present is a to the mechanisms leading to the neuronal loss that could be a of the retina for to cell and neuronal for with the of the and for animal for of this A to for and on the