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Amidation and Structure Relaxation Abolish the Neurotoxicity of the Prion Peptide PrP106–126 in Vivo and in Vitro

2005; Elsevier BV; Volume: 280; Issue: 24 Linguagem: Inglês

10.1074/jbc.m500210200

1083-351X

Ann‐Louise Bergström, Henriette Cordes, Nicole Zsürger, Peter M. H. Heegaard, Henning Laursen, Joëlle Chabry,

Trace Elements in Health

One of the major pathological hallmarks of transmissible spongiform encephalopathies (TSEs) is the accumulation of a pathogenic (scrapie) isoform (PrPSc) of the cellular prion protein (PrPC) primarily in the central nervous system. The synthetic prion peptide PrP106–126 shares many characteristics with PrPSc in that it shows PrPC-dependent neurotoxicity both in vivo and in vitro. Moreover, PrP106–126 in vitro neurotoxicity has been closely associated with the ability to form fibrils. Here, we studied the in vivo neurotoxicity of molecular variants of PrP106–126 toward retinal neurons using electroretinographic recordings in mice after intraocular injections of the peptides. We found that amidation and structure relaxation of PrP106–126 significantly reduced the neurotoxicity in vivo. This was also found in vitro in primary neuronal cultures from mouse and rat brain. Thioflavin T binding studies showed that amidation and structure relaxation significantly reduced the ability of PrP106–126 to attain fibrillar structures in physiological salt solutions. This study hence supports the assumption that the neurotoxic potential of PrP106–126 is closely related to its ability to attain secondary structure. One of the major pathological hallmarks of transmissible spongiform encephalopathies (TSEs) is the accumulation of a pathogenic (scrapie) isoform (PrPSc) of the cellular prion protein (PrPC) primarily in the central nervous system. The synthetic prion peptide PrP106–126 shares many characteristics with PrPSc in that it shows PrPC-dependent neurotoxicity both in vivo and in vitro. Moreover, PrP106–126 in vitro neurotoxicity has been closely associated with the ability to form fibrils. Here, we studied the in vivo neurotoxicity of molecular variants of PrP106–126 toward retinal neurons using electroretinographic recordings in mice after intraocular injections of the peptides. We found that amidation and structure relaxation of PrP106–126 significantly reduced the neurotoxicity in vivo. This was also found in vitro in primary neuronal cultures from mouse and rat brain. Thioflavin T binding studies showed that amidation and structure relaxation significantly reduced the ability of PrP106–126 to attain fibrillar structures in physiological salt solutions. This study hence supports the assumption that the neurotoxic potential of PrP106–126 is closely related to its ability to attain secondary structure. Prion diseases or transmissible spongiform encephalopathies (TSEs) 1The abbreviations used are: TSE, transmissible spongiform encephalopathy; CGN, cerebellar granular neurons; DAB, 3,3′-diaminobenzidine tetrachloride; dpi, days post injection; ECN, embryonal cortical neuron; ERG, electroretinography; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PBS, phosphate-buffered saline; PrPC, cellular prion protein; PrPSc, scrapie prion protein; ThT, thioflavin T; TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling. 1The abbreviations used are: TSE, transmissible spongiform encephalopathy; CGN, cerebellar granular neurons; DAB, 3,3′-diaminobenzidine tetrachloride; dpi, days post injection; ECN, embryonal cortical neuron; ERG, electroretinography; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PBS, phosphate-buffered saline; PrPC, cellular prion protein; PrPSc, scrapie prion protein; ThT, thioflavin T; TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling. represent a group of fatal, neurodegenerative diseases including e.g. Creutzfeldt-Jakob disease in humans, scrapie in sheep and goats, chronic wasting disease in deer, and bovine spongiform encephalopathy in cattle. The etiology of the diseases can be sporadic, hereditary, or infective. According to the “protein-only” hypothesis (1Prusiner S.B. Scott M.R. DeArmond S.J. Cohen F.E. Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (819) Google Scholar), the basic infectious mechanism is thought to be a conformational change of the normal (cellular) prion protein (PrPC) into the pathogenic (scrapie) PrPSc catalyzed by PrPSc itself. Thus, presence of PrPC is a prerequisite for prion infection (2Brandner S. Raeber A. Sailer A. Blattler T. Fischer M. Weissmann C. Aguzzi A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13148-13151Crossref PubMed Scopus (220) Google Scholar).PrPC is a glycosylphosphatidylinositol-anchored glycoprotein constitutively expressed on the surface of primarily neuronal cells. It consists of two structurally different parts, namely a C-terminal, globular part mainly α-helical in nature and an unstructured, N-terminal part (3Zahn R. Liu A. Luhrs T. Riek R. von Schroetter C. Lopez G.F. Billeter M. Calzolai L. Wider G. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 145-150Crossref PubMed Scopus (939) Google Scholar, 4Lopez G.F. Zahn R. Riek R. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8334-8339Crossref PubMed Scopus (364) Google Scholar). Misfolding of PrPC into PrPSc occurs post-translationally and results in increased β-sheet content and a gain of protease resistance. The central region of PrPC linking the unstructured N-terminal part with the globular C-terminal domain is believed to play a pivotal role in these conformational changes (5Muramoto T. Scott M. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15457-15462Crossref PubMed Scopus (175) Google Scholar, 6Rogers M. Yehiely F. Scott M. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3182-3186Crossref PubMed Scopus (156) Google Scholar, 7Chabry J. Caughey B. Chesebro B. J. Biol. 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Up-regulation of pro-apoptotic markers has been found in postmortem brains from Creutzfeldt-Jakob disease patients (17Puig B. Ferrer I. Acta Neuropathol. 2001; 102: 207-215Crossref PubMed Scopus (32) Google Scholar) and has additionally been found to precede the accumulation of PrPSc in scrapie-infected mice (18Jamieson E. Jeffrey M. Ironside J.W. Fraser J.R. Neuroreport. 2001; 12: 3567-3572Crossref PubMed Scopus (21) Google Scholar). The exact nature of the neurotoxic entity in the TSEs is still debated. Cytotoxicity of purified PrPSc has been shown in PrPC-expressing neuroblastoma cells in vitro (15Hetz C. Russelakis-Carneiro M. Maundrell K. Castilla J. Soto C. EMBO J. 2003; 22: 5435-5445Crossref PubMed Scopus (352) Google Scholar); however whether large aggregates or smaller oligomers of PrPSc are toxic is unknown (19Muller W.E. Ushijima H. Schroder H.C. Forrest J.M. Schatton W.F. Rytik P.G. Heffner-Lauc M. Eur. J. Pharmacol. 1993; 246: 261-267Crossref PubMed Scopus (157) Google Scholar).A synthetic peptide named PrP106–126 (numbering corresponding to the human prion protein sequence) resides within the central region of PrP near the N-terminal of the protease-resistant part of PrPSc. PrP106–126 shares many properties with PrPSc, as it readily forms amyloid fibrils with a high β-sheet content (20Tagliavini F. Prelli F. Verga L. Giaccone G. Sarma R. Gorevic P. Ghetti B. Passerini F. Ghibaudi E. Forloni G. Salmona M. Bugiani O. Frangione B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9678-9682Crossref PubMed Scopus (234) Google Scholar), shows partial proteinase K resistance (20Tagliavini F. Prelli F. Verga L. Giaccone G. Sarma R. Gorevic P. Ghetti B. Passerini F. Ghibaudi E. Forloni G. Salmona M. Bugiani O. Frangione B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9678-9682Crossref PubMed Scopus (234) Google Scholar, 21Forloni G. Angeretti N. Chiesa R. Monzani E. Salmona M. Bugiani O. Tagliavini F. Nature. 1993; 362: 543-546Crossref PubMed Scopus (893) Google Scholar), and is neurotoxic both in vivo (22Ettaiche M. Pichot R. Vincent J.P. Chabry J. J. Biol. Chem. 2000; 275: 36487-36490Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 23Chabry J. Ratsimanohatra C. Sponne I. Elena P.P. Vincent J.P. Pillot T. J. Neurosci. 2003; 23: 462-469Crossref PubMed Google Scholar) and in vitro (23Chabry J. Ratsimanohatra C. Sponne I. Elena P.P. Vincent J.P. Pillot T. J. Neurosci. 2003; 23: 462-469Crossref PubMed Google Scholar, 24Agostinho P. Oliveira C.R. Eur. J. Neurosci. 2003; 17: 1189-1196Crossref PubMed Scopus (88) Google Scholar, 25Brown D.R. J. Neurochem. 1999; 73: 1105-1113Crossref PubMed Scopus (93) Google Scholar, 26Florio T. Thellung S. Amico C. Robello M. Salmona M. Bugiani O. Tagliavini F. Forloni G. Schettini G. J. Neurosci. Res. 1998; 54: 341-352Crossref PubMed Scopus (79) Google Scholar, 27Peyrin J.M. Lasmezas C.I. Haik S. Tagliavini F. Salmona M. Williams A. Richie D. Deslys J.P. Dormont D. Neuroreport. 1999; 10: 723-729Crossref PubMed Scopus (113) Google Scholar, 28Salmona M. Malesani P. De Gioia L. Gorla S. Bruschi M. Molinari A. Della V.F. Pedrotti B. Marrari M.A. Awan T. Bugiani O. Forloni G. Tagliavini F. Biochem. J. 1999; 342: 207-214Crossref PubMed Scopus (105) Google Scholar). PrP106–126 contains the palindrome sequence AGAAAAGA, which makes it highly amyloidogenic. In contrast to other synthetic prion protein fragments that induce neuronal death independently of PrPC expression (23Chabry J. Ratsimanohatra C. Sponne I. Elena P.P. Vincent J.P. Pillot T. J. Neurosci. 2003; 23: 462-469Crossref PubMed Google Scholar, 29Haik S. Peyrin J.M. Lins L. Rosseneu M.Y. Brasseur R. Langeveld J.P. Tagliavini F. Deslys J.P. Lasmezas C. Dormont D. Neurobiol. Dis. 2000; 7: 644-656Crossref PubMed Scopus (42) Google Scholar), the neurotoxicity of PrP106–126 depends on the expression of endogenous PrPC (23Chabry J. Ratsimanohatra C. Sponne I. Elena P.P. Vincent J.P. Pillot T. J. Neurosci. 2003; 23: 462-469Crossref PubMed Google Scholar), which makes PrP106–126 a relevant model for PrPSc neurotoxicity. The in vivo neurotoxicity has been proposed to be linked to its aggregate-forming behavior and ability to form secondary structure in an aqueous environment (22Ettaiche M. Pichot R. Vincent J.P. Chabry J. J. Biol. Chem. 2000; 275: 36487-36490Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). PrP106–126 causes neuronal death via induction of apoptosis (15Hetz C. Russelakis-Carneiro M. Maundrell K. Castilla J. Soto C. EMBO J. 2003; 22: 5435-5445Crossref PubMed Scopus (352) Google Scholar, 22Ettaiche M. Pichot R. Vincent J.P. Chabry J. J. Biol. Chem. 2000; 275: 36487-36490Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 23Chabry J. Ratsimanohatra C. Sponne I. Elena P.P. Vincent J.P. Pillot T. J. Neurosci. 2003; 23: 462-469Crossref PubMed Google Scholar), and events such as mitochondrial disruption (30O'Donovan C.N. Tobin D. Cotter T.G. J. Biol. Chem. 2001; 276: 43516-43523Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), oxidative stress (31Turnbull S. Tabner B.J. Brown D.R. Allsop D. Neurosci. Lett. 2003; 336: 159-162Crossref PubMed Scopus (62) Google Scholar), and activation of caspases (15Hetz C. Russelakis-Carneiro M. Maundrell K. Castilla J. Soto C. EMBO J. 2003; 22: 5435-5445Crossref PubMed Scopus (352) Google Scholar) have been found to be involved in this process. Also, another prion-derived peptide, PrP118–135, has been found to cause neuronal death via induction of apoptosis (23Chabry J. Ratsimanohatra C. Sponne I. Elena P.P. Vincent J.P. Pillot T. J. Neurosci. 2003; 23: 462-469Crossref PubMed Google Scholar). The toxicity of PrP118–135 is, however, independent of endogenous PrPC expression. The toxicity of PrP106–126 has been found to be enhanced by activation of microglia cells (27Peyrin J.M. Lasmezas C.I. Haik S. Tagliavini F. Salmona M. Williams A. Richie D. Deslys J.P. Dormont D. Neuroreport. 1999; 10: 723-729Crossref PubMed Scopus (113) Google Scholar) and astrocytes (25Brown D.R. J. Neurochem. 1999; 73: 1105-1113Crossref PubMed Scopus (93) Google Scholar).The neuroretina represents an easily accessible and fully integrated part of the central nervous system. Normally, there is a very low level of protease activity in the corpus vitreum of young, healthy animals (32Vaughan-Thomas A. Gilbert S.J. Duance V.C. Investig. Ophthalmol. Vis. Sci. 2000; 41: 3299-3304PubMed Google Scholar), and injections of peptides can be made directly into the posterior chamber of the eye without causing damage to the eye. The neuronal cells of the retina express PrPC (33Giese A. Groschup M.H. Hess B. Kretzschmar H.A. Brain Pathol. 1995; 5: 213-221Crossref PubMed Scopus (146) Google Scholar, 34Chishti M.A. Strome R. Carlson G.A. Westaway D. Neurosci. Lett. 1997; 234: 11-14Crossref PubMed Scopus (20) Google Scholar) and are sensitive to PrP106–126 neurotoxicity, whereas the retinal neurons of PrP–/– mice are resistant to this effect (23Chabry J. Ratsimanohatra C. Sponne I. Elena P.P. Vincent J.P. Pillot T. J. Neurosci. 2003; 23: 462-469Crossref PubMed Google Scholar). Damage to retinal cells can be quantified with electroretinographic recordings.We recently showed that C-terminal amidation and structure relaxation of PrP106–126 significantly reduced the peptide's ability to form amyloid structure in water (35Heegaard P.M. Pedersen H.G. Flink J. Boas U. FEBS Lett. 2004; 577: 127-133Crossref PubMed Scopus (61) Google Scholar). Amidation of the C terminus has also been indicated by others to induce random coil structure in the peptide and to decrease its propensity to form amyloid fibrils (28Salmona M. Malesani P. De Gioia L. Gorla S. Bruschi M. Molinari A. Della V.F. Pedrotti B. Marrari M.A. Awan T. Bugiani O. Forloni G. Tagliavini F. Biochem. J. 1999; 342: 207-214Crossref PubMed Scopus (105) Google Scholar).Here, we found that amidation and structure relaxation of the PrP106–126 significantly reduced the peptide's in vivo and in vitro neurotoxicity and reduced its ability to form amyloid fibrils in a physiological salt solution. This finding supports the view that the fibril-forming capability of PrP106–126 is pivotal for its PrPC-dependent neurotoxicity and increases the biological relevance of this peptide as a model for PrPSc-induced pathology.MATERIALS AND METHODSAnimals—Adult male C57 black wild-type mice aged 9–11 weeks, were purchased from Centre d'élevage Janvier (Le Genest St Isle, France) or from Harlan Scandinavia (Allerød, Denmark). PrP–/– mice named Zurich I (36Bueler H. Fischer M. Lang Y. Bluethmann H. Lipp H.P. DeArmond S.J. Prusiner S.B. Aguet M. Weissmann C. Nature. 1992; 356: 577-582Crossref PubMed Scopus (1432) Google Scholar) were bred in the animal facilities at the Institute de Pharmacologie Moléculaire et Cellulaire, CNRS. Wistar Hannover Galas rat pups were purchased from Taconic M&B, Ry, Denmark.Peptides—Peptides were synthesized by solid phase Fmoc (N-(9-fluorenyl)methoxycarbonyl)-based synthesis using chlorotrityl resins for peptide acids and resins with the modified Rink linker for peptide amides (Novabiochem). After synthesis, cleavage by trifluoroacetic acid was performed, and the liberated peptides were analyzed by reverse phase (C5) high performance liquid chromatography-mass spectrometry (electrospray; Shimadzu LC20) and, if necessary, purified by preparative reverse phase chromatography on Lichrosorp C18 (Merck). The following peptides were used in this work: PrP106–126 (KTNMKHMAGAAAAGAVVGGLG, human PrP106–126, expected Mr = 1912.3); PrP106–126-amide (KTNMKHMAGAAAAGAVVGGLG-amide, expected Mr = 1911.3); scrambled control (LVGAHAGKMGANTAKAGAMVG, expected Mr = 1912.3); and RG2-amide (KTNMKHMAGAAAAGAVVGGLGRGGRGG-amide, expected Mr = 2722).The RG2-amide sequence was designed based on the principles of peptide structure relaxation as shown by Due Larsen and Holm (37Due Larsen B. Holm A. J. Pept. Res. 1998; 52: 470-476Crossref PubMed Scopus (26) Google Scholar). The peptides were dissolved in sterile PBS to a concentration of 1 mm and stored in aliquots at –20 °C until use. For intravitreal injections and for use in the primary mouse embryonal cortical neurons, the peptides were aged for 3 days at room temperature with slight agitation. When non-aged peptides were used for injections, they were kept on ice until use. For cellular experiments with rat cerebellar granular neurons, the peptides were aged for 2 days 37 °C at 2 mm in PBS.Thioflavin T (ThT) Assay for Amyloid Fibril Formation—A thioflavin T assay was performed as described by LeVine (38LeVine III, H. Methods Enzymol. 1999; 309: 274-284Crossref PubMed Scopus (1186) Google Scholar). Peptides were dissolved to 1 mm in sterile PBS and allowed to incubate at room temperature with slight agitation in the presence of 20 μm thioflavin T (1 mm stock solution in water; Sigma T3516), reading the fluorescence each day at 485 nm using an excitation wavelength of 440 nm (with a Spectra Flour Plus microplate fluorometer from Tecan). Readings were normalized to the same gain setting to allow comparisons from sample to sample. When comparing different plates, values were further corrected for background (fluorescence of ThT in water).Intravitreal Injections—Anesthesia was performed with intraperitoneal injections of pentobarbital (50 mg/kg). Topical application of a local anesthetic (Novesine®) was performed in both eyes. Injections of a 1-μl solution were done unilaterally (in the left eye) with a 30-gauge needle introduced into the posterior chamber on the upper pole of the eye directed toward the center of the vitreous body. The injections were performed slowly (over at least 60 s) to allow diffusion of the peptide and to avoid any ocular hypertension and backflow. For electroretinography (ERG) experiments 5–10 animals were used for each treatment group, and for histological analyses 3–5 animals per group were used. For histological analysis of eyes at 15 days post injection (dpi), 3 mice with representative ERG values (close to the group mean) from each treatment group were selected. For histological analyses of retinas at 4 dpi, the mice were not subjected to ERG before they were killed.ERG Measurements and Statistical Evaluation of Effect—Full field electroretinograms were prepared under dim red light on overnight dark-adapted animals. Anesthesia was performed with a mixture of 2% isoflurane (Forene®, Abbott Laboratories) and oxygen. The pupils of anesthetized animals were dilated with Mydriaticum®. The animals were kept on a heating mat during anesthesia. A ring-shaped recording electrode was placed on the cornea of each eye, and a reference electrode was placed behind each ear. Zero electrodes were placed on the hind legs. Light stimulus (8 ms) was provided by a single flash (10 candelas/s·m2) in front of the animal. The ERGs were recorded using Win7000b (Metrovision, Pérenchies, France). The amplitude of the a-wave was measured from the baseline to the bottom of the a-wave; the b-wave amplitude was measured from the bottom of the a-wave to the peak of the b-wave (Fig. 1). The averaged responses represent the mean of two white flashes (8-ms duration) delivered 2 min apart. ERGs were recorded before and then 4, 7, and 14 days after intravitreal injections. Only animals with approximately similar pre-injection values for the left and right eye were selected for injections. To quantify the effect of the injections, the non-injected eye was used as an internal control, and the absolute values for the ERG a- and b-waves of the injected eye were normalized to the non-injected control eye for each animal. Relative/normalized values for the different peptide groups were compared with the relative/normalized values for the group injected with the scrambled control peptide by using an unpaired t test.Tissue Preparation—Mice used for ERG recordings were killed on day 15 after injection, and other groups were killed on day 4 after injection. All mice were killed by cervical dislocation. The eyes were enucleated and fixed in ice-cold 4% paraformaldehyde for a minimum of 24 h and then cryoprotected overnight in PBS containing 20% sucrose. The eyes were then embedded in TissueTek (Sakura) and frozen at –80 °C. Cryosections (10 μm) throughout the whole eye were cut on a cryostat. The sections were then dried for 1–2 h at 55 °C before they were stored at –80 °C until further use.TUNEL Test on Cryosections—A TUNEL test (Roche Applied Science in situ cell death detection kit, POD) was performed on frozen sections from animals 4 or 15 dpi according to the manufacturer's instructions with slight modifications. The sections were defrosted for 30 min at room temperature and then washed 2× 5 min in PBS. Blocking was performed in methanol with 3% H2O2 for 30 min, after which the slides were washed 2× 5 min in PBS. Permeabilization was performed in 4 °C double distilled water with 0.5% sodium citrate and 0.5% Triton X-100 followed by a short wash in PBS. A positive control was digested with DNase for 20 min at 37 °C. A TUNEL mixture was then added to all slides (except a negative control, which received only the label solution from the TUNEL kit). Incubation with the TUNEL mixture was performed in a humid atmosphere for 3 h at 37 °C. Washing was performed 3× 5 min in PBS at 37 °C, and a converter solution from the TUNEL kit was added to all slides. Incubation was performed for 30 min, and the slides were then washed 3× 5 min in PBS. DAB solution (one thawed DAB tablet, Sigma catalog number D5905, was dissolved in 15 ml of PBS; 12 μl of 30% H2O2 was added immediately before use) was added, and development was performed for 6 min. The reaction was stopped in distilled water. The slides were dehydrated through a graded series of ethanols and mounted in xylol (Pertex). The slides were analyzed in a light microscope and photographed with a Leica DC300 digital camera.Primary Mouse Embryonal Cortical Neurons (ECNs) and Toxicity Assay—Primary cortical neuronal cultures were prepared from wild type C57 black embryos. Briefly, the embryos were dissected at embryonal day 13 or 14, and the brains were extracted and rinsed from meninges in 37 °C warm Dulbecco's PBS with 1% glucose. The PBS solution was replaced by Neurobasal medium (Invitrogen) with the addition of l-glutamine, 10% inactivated fetal calf serum, B27 supplement, and penicillin/streptomycin. Gentle homogenization was performed with a Pasteur pipette, after which the cells were washed once. The cell concentration was adjusted to the appropriate concentration, and plating of 5 × 104 cells/well was performed on Nunc 96-well plates precoated overnight with poly-d-lysine. Incubation was performed at 37 °C with 5% CO2 in a humidified incubator. After 30 min of incubation, the medium was changed. After 24 h of incubation, the medium was replaced by serum-free Neurobasal medium with a B27 supplement, l-glutamine, and penicillin/streptomycin. Peptides were added after 3–5 days of maturation and incubated for 12, 24, and 48 h. Viability was measured with an MTS assay (CellTiter96, AQueous One solution cell proliferation assay; Promega). A492 nm was measured on a spectrophotometer.Primary Rat Cerebellar Granular Neurons (CGNs) and Toxicity Assay—CGNs were isolated from 7–8 day-old Wistar Hannover Galas rat pups (Taconic M&B) as described by Patel & Kingsbury (39Patel A.J. Kingsbury A.E. Methods Neurosci. 1999; 18: 493-502Google Scholar) with some modifications. Briefly, cerebellar granules were isolated from pups after decapitation. The tissue was suspended in Krebs buffer and digested with trypsin before single cell suspensions were made in Neurobasal A medium supplemented with 10% fetal calf serum, 35 mm KCl, GlutaMAX-I supplement, penicillin/streptomycin, and 1 mm sodium pyruvate (Invitrogen). The cell concentration was adjusted to the appropriate concentration, and plating of 8 × 104 cells/well was performed on 96-well plates precoated with poly-d-lysine (BD Biosciences) and incubated for 2 h at 37 °C and 5% CO2 in a humidified incubator. The medium was changed to serum-free Neurobasal A with B27 (without antioxidants) and the same supplements as above. On day 6 the medium was replaced by fresh Neurobasal A (without phenol red) with the addition of B27 (without antioxidants), the same supplements as above, and peptides to a final concentration of 100 μm. Viability was measured day 15 using the WST-1 assay as described in the manufacturer's manual (cell proliferation reagent WST-1; Roche Applied Science). The cells were analyzed in light microscope and photographed with a Leica DC300 digital camera after 9 days of treatment.Caspase Induction—CGNs were isolated as described above and incubated for 6 days before peptides were added in serum-free Neurobasal A medium (without antioxidants, supplemented with 15 mm KCl and B27) in 96-well black microtiter plates (Optilux, BD Biosciences) precoated with poly-d-lysine. After incubation for 3 days, the induction of caspases was measured in a cell lysate described in the manufacturer's manual (homogenous caspases assay, Roche Applied Science). This assay measures mainly caspase-2, -3, and -7.Immunocytochemistry on CGN—CGN cultures were grown at 37 °C with 5% CO2 on eight-chamber Lab-Tek Permanox chamber slides (Nalge Nunc) precoated with poly-d-lysine. The cells were fixed in 4% paraformaldehyde in PBS, pH 7.4 (freshly prepared overnight with stirring), for 1 h at room temperature and washed with washing buffer (1% Triton X-100 in PBS, pH 7.4) 3× 5 min with gentle agitation before incubating overnight at 4 °C with 1 μg/ml of the mouse monoclonal anti-PrP antibody SAF32 (Spi-Bio, Montigny le Bretonneaux, France) or mouse IgG2b (catalog number X0944, DAKO) diluted in PBS, pH 7.4, with 1.5% horse serum. After rinsing, the slides were incubated with biotinylated anti-mouse immunoglobulin antibody diluted 1:200 in PBS, pH 7.4, with 1.5% horse serum for 1 h at room temperature, rinsed, and incubated with ABC reagent for 30 min (antibodies and ABC-reagent Vectastain ABC kit from Vector Laboratories). After rinsing, slides were developed with a DAB solution (one thawed DAB tablet from Sigma was diluted in 15 ml PBS; 12 μl of H2O2 was added immediately before use) before rinsing in distilled water. Slides were mounted with Aquamount mounting media (BDH Laboratories, Poole, UK) under glass coverslips.RESULTSAmidation and Structure Relaxation of PrP106–126 Reduce Its Tendency to Form Amyloid Fibrils in PBS—We analyzed the fibrillation tendency (amyloidogenicity) of the peptides at 1 mm in PBS with the ThT assay where fluorescence was followed for 7 days (Fig. 2). Fibrillation of PrP106–126 in PBS peaked after 3 days of incubation, after which it started to decline. The PrP106–126-amide and the RG2-amide showed much lower fibrillation tendency compared with PrP106–126, although they both rose to levels significantly higher than the scrambled control peptide. The fibrillation of the RG2-amide proceeded slower than that of both PrP106–126 and PrP106–126-amide, peaking after 5 days of incubation as opposed to 3 days of incubation.Fig. 2Thioflavin T binding of peptide variants. Thioflavin T binding of the peptides dissolved in PBS (1 mm) was analyzed by fluorescence measurements daily for 7 days. Each data point represents the mean of duplicate or quadruplicate determinations.View Large Image Figure ViewerDownload (PPT)Fibrillar PrP106–126 Induces Long Term Changes in the Retina—To test the peptide variants for their in vivo neurotoxicity, we performed intravitreous injections of the 3-day-aged peptides and performed ERG recordings at 4, 7, and 14 dpi. All animals were injected in their left eye only (the right was left as non-injected or PBS-injected controls). No difference was observed between non-injected and PBS-injected eyes (data not shown). Both eyes were measured by ERG, and for each animal the absolute values of the ERG a- and b-waves, respectively, for the injected eye were normalized to the control eye values (the a-wave and b-wave amplitude values from the control eye was set to 100%). This was done to avoid time-to-time variation in the ERG