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【摘要】
In patients affected by Creutzfeldt-Jakob disease and in animals affected by transmissible spongiform encephalopathies, retinal functions are altered, and major spongiform changes are observed in the outer plexiform layer where photoreceptors have their synaptic terminals. In the present study, the prion protein PrPc was found to form aggregates in rod photoreceptor terminals from both rat and human retina, whereas no labeling was observed in cone photoreceptors. Discrete staining was also detected in the inner plexiform layer where the prion protein was located at human amacrine cell synapses. In mixed porcine retinal cell cultures, the PrP106-126 prion peptide triggered a 61% rod photoreceptor cell loss by apoptosis as indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling, whereas cone photoreceptors were not affected. Amacrine cells were also reduced by 47% in contrast to ganglion cells. Although this cell loss was associated with a 5.5-fold increase in microglial cells, the strict correlation between the PrPc prion protein expression and the peptide toxicity suggested that this toxicity did not rely on the release of a toxic compound by glial cells. These results provide new insights into the retinal pathophysiology of prion diseases and illustrate advantages of adult retinal cell cultures to investigate prion pathogenic mechanisms.
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Transmissible spongiform encephalopathies in humans and in animals are linked to the transconformation of the prion protein PrPc into a proteinase-resistant form PrPres.1 These neurological disorders exhibit common pathological symptoms like vacuolization of the neurophils, astrocytosis, and loss of neurons.2 In patients with Creutzfeldt-Jakob disease, alteration of the retinal function was attested by the decrease in the electroretinogram b-wave amplitude,3-5 which reflects the activity of bipolar cells postsynaptic to photoreceptors. These early electroretinogram changes were even proposed as a diagnostic measurement for the disease.6 In histology, major spongiform changes were observed in the outer plexiform layer (OPL) where photoreceptors have their synaptic terminals, and only moderate changes were observed in the inner plexiform layer (IPL) and ganglion cell layer.5 These localizations were consistent with the reported accumulation of the pathogenic form of the prion protein, PrPres, throughout the plexiform layers of the human retina.7
The in vivo retina has been used on many occasions to study the progression of the disease in animal models because of its organized structure, its in vivo access for intraocular injection, and the possibility to correlate the histology to the measure of neuronal function by the electroretinogram. In mice inoculated with scrapie extract, the retina showed a progressive degeneration with a loss of photoreceptors and ganglion cells associated with optic nerve changes.8-10 These changes were correlated to a localization of the prion protein PrPc in the synaptic layers, especially in mice overexpressing the protein.11 When using prion peptides, both the PrP106-126 and PrP118-136 peptides were shown in vivo to induce cell death in all nuclear layers of the retina.12,13
When studying the molecular mechanisms of toxicity, both the PrP106-126 peptide and scrapie-infected extracts were found to have no effect on neurons from prion protein knockout mice (PrP0/0),14 whereas the PrP118-135 fragment induced neuronal cell death.13 In the presence of PrP-positive astrocytes, the PrP106-126 peptide may, however, become toxic to PrP0/0 neurons by promoting glutamate release from astrocytes.15 The toxicity of the PrP106-126 peptide was in fact related to its ability to aggregate.16
To understand further the retinal physiopathology of the transmissible spongiform encephalopathies, we examined the PrPc subcellular distribution in rat and human retina. PrPc was localized to rod spherules and amacrine cells, whereas cone pedicles did not express PrPc. In addition, we assessed the toxicity of the PrP106-126 peptide in adult mixed retinal cell cultures. A direct correlation was demonstrated between the in vitro peptide toxicity and the in vivo distribution pattern of PrPc. Mixed retinal cell cultures therefore represent an interesting model in which to study the transmissible spongiform encephalopathies physiopathological mechanisms on fully differentiated adult neurons.
【关键词】 toxicity cultured photoreceptors correlates distribution mammalian
Materials and Methods
Retinal Cell Culture
Adult mixed retinal cell cultures were prepared from the pig retina as previously described.17 In brief, the pig retina was isolated in a CO2-independent medium from pig eyes received from a local slaughterhouse. Retinal fragments were rinsed and incubated for 20 to 30 minutes at 37??C with activated papain (0.2%) in a Ca2+-free Ringer??s solution containing ethylenediamine tetraacetic acid (0.1 mmol/L). After stopping the enzymatic reaction in Dulbecco??s modified Eagle??s medium-10% fetal calf serum ganglion cell layer with DNase (0.2 mg/ml), retinal fragments were mechanically dissociated with a fire-polished Pasteur pipette. Retinal cells were isolated by centrifugation at 800 rpm for 5 minutes. Cells were finally seeded on laminin- and poly-D-lysine-coated coverslips in Dulbecco??s modified Eagle??s medium-10% fetal calf serum at a density of 4 x 104 cells/cm2. After 6 days in vitro, the human PrP106-126 prion peptide or its scrambled form was added to the culture medium for 4 days. Both the correct human prion protein peptide . The 80 µmol/L concentration was selected as it has been described as the lowest toxic concentration in primary neuronal cultures.18 Peptides were dissolved in deionized water at a concentration of 24 mmol/L and stored at C20??C. Subsequently, they were diluted twice with phosphate-buffered saline (PBS) and added to the culture medium. Culture solutions were changed every 2 days, and cells were finally fixed with 4% paraformaldehyde in PBS before cell labeling.
Cell Labeling
Human postmortem retinal tissues were obtained from the human tissue bank in Strasbourg in accordance with French legislation on the use of human tissues for medical and scientific research. Human tissues were fixed in 4% paraformaldehyde in PBS (0.1 mol/L, pH 7.4) at 4??C for 15 minutes. For rat retinal tissue, adult Long-Evans rats were sacrificed by cervical dislocation. Eyes were enucleated, the anterior segments removed, and the posterior eyecups fixed in 4% paraformaldehyde in PBS for 5 minutes. Following fixation, retinal tissues were dissected from the eyecup, cryoprotected in graded sucrose solutions (10%, 20%, 30%), and embedded in OCT to produce vertical retinal sections (10 µm) on a cryostat.
Sections and cell cultures were washed in PBS, permeabilized in PBS containing 0.1% Triton X-100 for 5 minutes, then bathed in PBS containing 1% bovine serum albumin, 1% goat serum for 1 hour at 37??C, and incubated in the same solution with the primary antibody for 2 hours at room temperature. For double-labeling experiments, a combination of primary antibodies was applied simultaneously. The sections were rinsed in PBS three times and incubated with the secondary antibodies for 1 hour at 37??C in the dark. The secondary antibodies included rabbit anti-mouse IgG conjugated to Alexa TM 568 and anti-rabbit IgG conjugated to Alexa TM 488 (all diluted 1:400; Molecular Probes, Eugene OR). Microglial cells were identified by the isolectin B4 bound to fluorescein isothiocyanate (ILB4, 1:50; Sigma Chemical Co, St. Louis, MO), whereas cone photoreceptors were stained by the peanut lectin agglutinin (PNA, 1:40; Sigma). Apoptotic cells were labeled with terminal deoxynucleotidal transferase dUTP nick-end labeling (TUNEL kit; Roche Diagnostics, Basel, Switzerland). 4,6-Diamidino-2-phenylindole nuclear dye was applied in PBS for 2 minutes. Finally, cells were washed four times before observation. Control experiments were performed by either omitting the primary prion antibody or adding the primary antibody with its corresponding synthetic prion peptide.
The primary antibodies used in the present study were anti-calbindin D-28K polyclonal antibody (1:1000; Chemicon); anti-PKC- polyclonal antibody (1:2000; Santa Cruz); anti-arrestin polyclonal antibody (1:2000; a generous gift of Dr. Y. Gery, National Eye Institute, National Institutes of Health, Bethesda, MD); anti-rhodopsin monoclonal antibody (Rho4D2, 1:1000; a generous gift of Dr. Hicks, Centre National de la Recherche Scientific Unit? Mixte de Recherche 7518, Strasbourg, France); anti-syntaxin antibody (1:500, HPC1; Sigma); anti-Bassoon polyclonal antibody19 (1:1000, anti-VGLUT1 polyclonal antibody, 1:4000; a generous gift from Dr. S. El Mestikawy, INSERM U513, Cr?teil, France); and the anti-PrP antibodies Prion-917 (protein A-purified IgG1k, 1:600; a generous gift from Dr J. Grassi, Commissariat
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作者单位：From the Laboratoire de Physiopathologie Cellulaire et Mol?culaire de la R?tine,* INSERM U592, Paris, France; Universit? Pierre et Marie Curie-Paris 6, Unit? Mixte de Recherche S592, Paris, France; Clinique de la souris, Institut de G?n?tique et Biologie Mol?culaire et Cellulaire, Illkirch, France;