CLINICAL NEUROSCIENCE AND NEUROPATHOLOGY NEUROREPORT 0959-4965 & Lippincott Williams & Wilkins Vol 11 No 17 27 November 2000 3931 PrP fragment 106-126 is toxic to cerebral endothelial cells expressing PrP C Ma Âria A. Deli, 1,5,CA Suehiro Sakaguchi, 4 Ryota Nakaoke, 1 Csongor S. A Â braha Âm, 1 Hideaki Takahata, 2 Juraj Kopac Ïek, 1 Kazuto Shigematsu, 3 Shigeru Katamine 4 and Masami Niwa 1 Departments of 1 Pharmacology 1, 2 Neurosurgery, 3 Pathology 2, School of Medicine; and 4 Department of Molecular Microbiology and Immunology, Graduate School of Medical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan; 5 Laboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Temesva Âri krt 62, H-6726 Szeged, Hungary CA,1 Corresponding Author and Address Received 22 September 2000; accepted 28 September 2000 A hydrophobic, ®brillogenic peptide fragment of human prion protein (PrP106-126) had in vitro toxicity to neurons expressing cellular prion protein (PrP C ). In this study, we proved that primary cultures of mouse cerebral endothelial cells (MCEC) express PrP C . Incubation of MCEC with PrP106±126 (25± 200 ìM) caused a dose-dependent toxicity assessed by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase release, bis-benzimide staining for nuclear morphology, and trypan blue exclusion test. Pentosan polysulphate (50-100 ìg/ml), a drug effective in scrapie prophy- laxis, dose-dependently attenuated the injury. MCEC cultures from mice homogenous for the disrupted PrP gene were resistant to the toxicity of PrP106-126. In conclusion, cerebral endothelium expressing PrP C may be directly damaged during spongiform encephalopathies. NeuroReport 11:3931±3936 & 2000 Lippincott Williams & Wilkins. Key words: Cellular prion protein; Cerebral endothelial cells; Pentosan polysulphate; PrP fragment 106-126; Toxicity INTRODUCTION In transmissible spongiform diseases, the most prominent histopathological features in the CNS are vacuolization and reactive gliosis with neuronal loss accompanied by accu- mulation of disease-speci®c proteinase K-resistant prion protein (PrP Sc ), an abnormal form of cellular prion protein (PrP C ) [1]. Forloni et al. [2] proved that a synthetic peptide containing residues 106±126 of the human PrP C and PrP Sc (PrP106-126) also possesses neurotoxic activity. Since then PrP106-126, a fragment adopting a â-sheet conformation and aggregating into amyloid ®brils resistant to digestion with protease, has been extensively used in in vitro studies [3±10]. Besides its neurotoxicity PrP106-126 induced hyper- trophy and proliferation of astrocytes, as well as activation of microglia [2±5,8,9]. PrP106-126 also had effects on cells originated outside the CNS: it activated circulating leuco- cytes [6], and proved to be toxic to muscle cells in vitro [7]. No study has been focused on the effects of PrP106-126 on cerebral endothelium, the morphological basis of the blood±brain barrier (BBB), and an active interface between circulation and CNS. Similarly, although mRNA of PrP C was demonstrated by in situ hybridization in vascular endothelial cells in normal hamster brain [11], and PrP Sc was detected in endothelial cells of scrapie-infected sheep brain [12], the presence and function of PrP C in cultured brain endothelial cells have not been examined yet. In the present experiments, we have investigated the expression of PrP C in cultured primary cerebral endothelial cells, and the effects of PrP106-126 on microvascular endothelial cells isolated from the brain of wild-type mice and a mouse strain de®cient in PrP C expression [13]. Effects of pentosan polysulphate (PPS), a prophylactic agent in scrapie [14], on PrP106-126 induced changes have also been tested. MATERIALS AND METHODS Animals: Wild type mice (C57BL6J 3 129/Sv strain) as well as mice de®cient in PrP C expression (Ngsk Prnp 0=0 ) homozygous for a disrupted Prnp gene were used [13]. Animals were cared for in accordance with Guidelines for Animal Experimentation of Nagasaki University. Cell culture: Primary cultures of mouse cerebral endothe- lial cells (MCEC) were prepared from 8-week old animals, similarly to the method described for rat cerebral endothe- lial cells [15]. Meninges were carefully removed from the forebrains and gray matter was minced, then digested with 1 mg/ml collagenase CLS2 (Worthington) in Dulbecco's modi®ed Eagle's medium (DME; Sigma) containing 50 ìg/ ml gentamycin and 2 mM glutamine in a shaker for 2 h at 378C. The cell pellet was separated by centrifugation in 20% bovine serum albumin (BSA)±DME (1000 3 g, 20 min). The microvessels obtained in the pellet were further digested with 1 mg/ml collagenase±dispase (Boehringer- Mannheim) in DME for 1.5 h at 378C. Microvessel endothe- lial cell clusters were separated on a 33% continuous