Increased expression of phospholipase D1 in the brains of scrapie-infected mice Jae-Kwang Jin,* Nam-Ho Kim,* Do Sik Min,à Jae-Il Kim,§ Jin-Kyu Choi,* Byung-Hoon Jeong,* Seung-Il Choi,* Eun-Kyoung Choi,* Richard I. Carp§ and Yong-Sun Kim* ,   *Ilsong Institute of Life Science and  Department of Microbiology, College of Medicine, Hallym University, Kyonggi-do, Korea àDepartment of Molecular Biology, College of Natural Science, Pusan National University, Busan, Korea §New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York, USA Abstract Mitochondrial dysfunction and free radical-induced oxidative damage are critical factors in the pathogenesis of neurode- generative diseases. Recently, phospholipid breakdown by phospholipase D (PLD) has been recognized as an important signalling pathway in the nervous system. Here, we examined the expression of PLD and alteration of membrane phosp- holipid in scrapie brain. We have found that protein expression and enzyme activity of PLD1 were increased in scrapie brains compared with controls; in particular, there was an increase in the mitochondrial fraction. PLD1 in mitochondrial membranes from scrapie brains, but not from control brains, was tyro- sine phosphorylated. Furthermore, the concentration of mitochondrial phospholipids such as phosphatidylcholine and phosphatidylethanolamine was increased and the content of phosphatidic acid, a product of PLD activity, was up-regulated in the mitochondrial membrane fractions. Immunohistochem- ically, PLD1 immunoreactivity was significantly increased in activated astrocytes in both cerebral cortex and hippocampus of scrapie brains. Taken together, these results suggest that PLD activation might induce alterations in mitochondrial lipids and, in turn, mediate mitochondrial dysfunction in the brains of scrapie-infected mice. Keywords: mitochondrial dysfunction, mitochondrial phosp- holipid, phospholipase D, scrapie. J. Neurochem. (2005) 92, 452–461. Mitochondria are one of the main targets of oxygen free radicals and dysfunction of this organelle is known to be a critical factor in the pathogenesis of prion diseases such as scrapie. Scrapie is the archetype of the group of diseases referred to as prion diseases or spongiform encephalopathies, and is characterized histologically by vacuolation and astrocytosis in the brains of sheep and goats (Carp and Callahan 1991). The etiological agent of the prion diseases is, at least in part, the scrapie prion protein (PrP Sc ) molecule which is formed from the normal cellular prion protein (PrP C ) by undetermined post-translational modification pro- cesses (Prusiner 1991). Previously, Choi et al. (1998) reported that mitochondrial dysfunction caused by oxidative stress gives rise to neurodegeneration in the brains of hamsters infected with scrapie agent. The involvement of oxidative stress in mitochondrial dysfunction has been suggested, but it is still unknown if or how oxidative stress induces neuropathology in prion diseases. It is possible that the breakdown of membrane phospholipids by oxidative stress induces mitochondrial dysfunction in scrapie. It has been shown that the loss of membrane phospholipids occurs by increased degradation and this enhanced degradation is induced by phospholipase activation after free radical attack Received July 16, 2004; revised manuscript received September 6, 2004; accepted September 7, 2004. Address correspondence and reprint requests to Yong-Sun Kim MD PhD, Ilsong Institute of Life Science and Department of Microbiology, College of Medicine, Hallym University, Ilsong Building, Kwanyang- dong, 1605–4 Dongan-gu, Anyang, Kyonggi-do 431–060, Korea. E-mail: yskim@hallym.ac.kr Abbreviations used: ARF, ADP-ribosylation factor; H 2 O 2 , hydrogen peroxide; LPA, lysophosphatidic acid; MDA, malondialdehyde; mGluRs, G-protein-coupled metabotropic glutamate receptors; PA, phosphatidic acid; PBS, phosphate-buffered saline; PC, phosphatidyl- choline; P-choline, phosphocholine; PE, phosphadidylethanolamine; PKC, protein kinase C; PLA 2 , phospholipase A 2 ; PLC, phospholi- pase C; PLD, phospholipase D; PrP C , normal cellular prion protein; PrP Sc , scrapie prion protein; PtdBut, phosphatidylbutanol; ROS, reactive oxygen species; RT, room temperature; TBA, thiobarbituric acid; TBARS, thiobarbituric-acid-reactive substances; TBS, Tris-buffered saline; TLC, thin layer chromatography. Journal of Neurochemistry , 2005, 92, 452–461 doi:10.1111/j.1471-4159.2004.02881.x 452 Ó 2005 International Society for Neurochemistry, J. Neurochem. (2005) 92, 452–461