1. Cover Sheet
ILHAC Final Report
Title: Molecular and Cellular Mechanisms Underlying Prion Protein (PrPsc)-induced Neurodegeneration
Principal Investigator:Anumantha Kanthasamy, Ph.D.
Eugene and Lloyd Professor
Department of Biomedical Sciences
College of Veterinary Medicine
Phone: 294-2516
Co-Investigators:Arthi Kanthasamy, Ph.D.
Assistant Professor
Department of Biomedical Sciences
College of Veterinary Medicine
Phone: 294-7238
Vellareddy Anantharam, Ph.D.
Affiliate Associate Professor
Department of Biomedical Sciences
College of Veterinary Medicine
Phone: 294-6179
Completion of the Project: 6/30/2006
2. Industry Summary
a. Problem that was studied: Prion diseases are fatal neurodegenerative conditions caused by transmissible protease-resistant prions. Considerable neuropathological and clinical similarities exist among the major prion diseases that include Creutzfeldt-Jacob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep, goats and other animals. Prion diseases cause severe neuronal damage mainly in the brain regions that control motor function, including the basal ganglia, cerebral cortex, thalamus, and cerebellum. Prion-induced neuronal damage is characterized pathologically bymassive neuronal degeneration associated with accumulation of the abnormal prion protein PrPsc (scrapie isoform) derived from the normal prion protein PrPc (cellular isoform) through unknown pathogenic mechanisms. However, it is important to mention that the physiological function of cellular non-pathogenic form of PrPcis still unclear. We recently determined that proteolytic activation of PKC not only mediates apoptosis but also, interestingly, amplifies the apoptotic cell death process through positive feedback activation of caspase-3 in neuronal cells (Kanthasamy et al., 2003, Anantharam et al., 2002). Since oxidative stress and apoptosis are well recognized in prion diseases, we proposed to determine the functional role of cellular isoform PrPc and scrapie isoform PrPsc in cell culture and animal models of Prion disease.
b. Objectives and results from the original proposal
Specific Aim I: To determine whether PrPsc treatment activates PKC by caspase-3-dependent proteolytic cleavage, and to investigate whether caspase–3-dependent proteolytic activation of PKC plays a role in PrPsc-induced neuronal apoptosis.
Results: We have identified that PrPc protects against oxidative stress and exacerbates ERstress-induced increases in caspase-3, -8, -9 and -12 activation, proteolytic activation of PKC and apoptotic cell death.
Specific Aim II: To further investigate whether the proteolytic activation of PKC amplifies PrPsc-induced apoptotic signaling through positive feedback activation of the caspase cascade.
Results: Using pharmacological and genetic tools, we found thatproteolytic activation of PKC not only promotes apoptotic cell death but also amplifies both ER and oxidative stress-induced multiple caspase activation and DNA fragmentation via a positive feedback mechanism.
Specific Aim III: To determine whether PKC knockout (PKC KO; PKC -/-) mice are resistant to PrPsc-induced neurobehavioral and neurodegenerative changes.
Results: In this study we found that proteolytic cleavage of PKC was more pronounced in the striatum of naïve PKC (+/+) C57 black mice inoculated with RML scrapie strain (Rocky Mountain Lab strains) compared to uninoculated animals.
c. Impact of the research results for the industry.The outcome of this proposal resulted in the identification of PrPc protein acting as an antioxidant protein,protecting cells against oxidative stress- and metal-induced neuronal degeneration. Interestingly, we also found that PrPc protein exacerbates ER stress-induced neuronal degeneration. This is the first report demonstrating the dual role of PrPc in oxidative stress- and ERstress-induced cell death mechanisms. We also determined PKC,a proapoptotic kinase, plays a critical role in oxidative and ERstress- induced neuronal neurodegenerationin invitro models of prion diseases,and could serve as potential therapeutic target.Our results may provide a long-term linkfor understanding the role of cellular and scrapie forms of prion protein in the physiology and pathobiology of pathogenesis of prion diseases, and development of therapeutics against select targets. During the course of this study we successfully established collaborations with Dr. Jeurgen A. Richt at NADC, Ames, IA and with Dr. Suzette A. Priola, NIH, Rocky Mountain Labs, Montana. The outcome of this study resulted inmajor funding from the Department of Defense to investigate the role of divalent cation metals, including manganese, in prion disease pathogenesis.
3. Scientific Report
Materials and methods
1. Generation of the brain-derived PrP0/0 cell line CF10 and establishment of stable PrPc and PrPko cells.
2. Transient expression of pPKCK376R-GFP, pPKCD327A-GFP, and pEGFP-N1 fusion proteins using a nucleofector device.
3. Treatment paradigm - After 2-4 days in culture, PrPc and PrPko cells were exposed to 10-100 M H2O2, 3-30 M BFA and 0.6-6 M TUN over 24 h. In inhibitor studies, MnTBAP (ROS inhibitor, 2 M), rottlerin (PKC inhibitor, 2 M), cyclosporine A (mitochondrial permeability pore inhibitor, 1 M), Z-VAD-FMK (pan-caspase inhibitor, 100 M), Z-DEVD-FMK (caspase-3-specific inhibitor, 100 M), Z-LEHD-FMK (caspase-9-specific inhibitor, 100 M), Z-IETD-FMK (caspase-8-specific inhibitor, 100 M) were added at the same time as H2O2, BFA, and TUN. After treatment, cells were harvested at 1, 3, 6, 12, and 24 h and were assessed for caspase-3, caspase-8, caspase-9, caspase-12 and proteolytic activation of PKC and DNA fragmentation. DMSO (dimethyl sulfoxide, 0.1%) and ethanol (0.1%)) were used as vehicles in control experiments.
4. Immunocytochemistry - After establishing PrPc and PrPko cells, they were assessed for PrPc expression using monoclonal antibody directed against 3F4 epitope. Cells were plated on poly-l-lysine (0.1 mg/ml)-coated coverslips and incubated with monoclonal antibody directed against the 3F4 epitope, followed by incubation with Alexa-488 conjugated secondary antibody.Cells were mounted on slides, viewed, and images were captured using a Nikon C1 confocal microscope.
5. Cytotoxicity assays, flow cytometric measurement of ROS generation, caspase enzymatic assays, Western blots for detecting PKC, caspase-12 and -actin, immunoprecipitation kinase assays for measuring PKC enzymatic activity, DNA fragmentation assay for measuring apoptotic cell death and data analysis have been previously described (Anantharam et al., 2002, Kitazawa, 2003, Kaul 20034, Yang et al., 2004.
Results and discussion
Although prion protein is abundantly expressed in the CNS, its biological function has not yet been established. To determine the role of prion protein in oxidative stress and protein processing, we compared the effects of H2O2 and the endoplasmic reticulum (ER) stress-inducers brefeldin A and tunicamycin on apoptotic cell signaling in a neural cell line derived from PrP knockout mice engineered to stably express non-pathogenic mouse prion protein (PrPc) and empty vector-expressing prion knockout cells (PrPko). Treatment with H2O2 (10-100 µM), brefeldin A (3-30 µM), and tunicamycin (0.6-6 µM) resulted in dose- and time-dependent increases in cytotoxic cell death, cytochrome c release, caspase-3 and -9 activation, and DNA fragmentation over a 24 hr period in both PrPc and PrPko cells. However, ERstress-induced caspase-3 (Fig. 1A), caspase-8 (Fig.1B), and caspase-9 activation (Fig.1C) and DNA fragmentation (Fig. 1D) were significantly higher in PrPc than PrPko. Alternatively, H2O2 stress-induced caspase-3 (Fig. 1A), and caspase-9 (Fig.1C) activation, and DNA fragmentation (Fig.1D) cell death was significantly reduced in PrPc compared to PrPko, suggesting that prion protein may be neuroprotective against oxidative damage. Additionally, caspase-12 (Fig. 2A) and caspase-8 (Fig. 1B) were activated only in cells treated with brefeldin A and tunicamycin but not with H2O2. Interestingly, PKC, an oxidative stress-sensitive kinase, was proteolytically cleaved and activated by caspase-3 during both oxidative stress and ER stress (Fig. 2B-C), and cleavage was accompanied by increases in PKC kinase activity in immunoprecipitation kinase assays (Fig. 3A). Comparative analysis of PKC proteolytic activation in PrPc and PrPkocells revealed that prion expressing cells were protected from oxidative stress-induced PKC activation; however, intensified ER-stress induced PKC activation.
Co-treatment with rottlerin, a PKC-specific inhibitor,almost completely suppressed oxidative and ERstress-induced apoptotic cell death (Fig. 3B). Overexpression of the kinase inactive PKC dominant negative mutant (PKCK376R) or the caspase-3 cleavage site-resistant PKC mutant (PKCD327A) significantly attenuated both ER and oxidative stress-induced apoptotic cell death (Fig. 4). The next logical step was to determine whether proteolytic activation of PKC occurs in scrpaie-infected animals. For this study mice were inoculated with Rocky Mountain Lab scrapie strain (RML) and 60 days post-infection (60 dpi) animals were sacrificed and different brain regions were harvested, homogenized and subjected to western blot analysis for detection of proteolytic cleavage of PKC. Uninoculated animals were used as controls. As shown in Fig 5, proteolytic cleavage of PKCoccurred in RMLinoculated animals. No PKC cleavage was observed in uninoculated animals. PKC cleavage was most pronounced in the striatum, moderately pronounced in thalamus, and to a much lesser extent in the frontal cortex and medulla oblongata. Together, these results suggested that prion protein enhancedthe susceptibility of neural cells to impairment inprotein processing and trafficking, but decreasedthe vulnerability to oxidative insults, and that PKC is a key downstream mediator of cellular stress-induced neuronal apoptosis. Using a combination of pharmacological and genetic tools, we developed a schematic model of the role of PrPc in oxidative and ERstress-induced apoptotic cell death (Fig. 6). We have also conducted in vivo studies, in which C57 black mice were inoculated inoculated with RML scrapie strain compared to uninoculated animals. The results showed a significant proteolytic cleavage of PKC in certain brain areas of infected animals. Future studies will focus on further cell signaling events associated with neurodegenerative processes in prion diseases.
Research results from ILHAC funding 2003-2006 have been presented at the following international meetings/conferences.
1.V.Anantharam; I.Vorberg; C.Choi; A.Kanthasamy; J.A.Richt; S.A.Priola; A.G.Kanthasamy, (2004), Differential Role of Prion Protein in Oxidative Stress- and ER Stress-induced Apoptotic Signaling in Neural Cells. United States. Animal Prion Diseases and the Americas International Symposium, Oct 14-16, Ames, IA.
- V.Anantharam; I.Vorberg; C.Choi; A.Kanthasamy; J.A.Richt; S.A.Priola; A.G.Kanthasamy, (2004), Proteolytic activation of PKC contributes to Oxidative and ERStress-induced Apoptotic Cell Death in Prion-expressing Mouse Neural Cells. 34rdAnnual Meeting of Society of Neuroscience, October, 23-27, San Diego, CA.
- V.Anantharam; I.Vorberg; C.Choi; A.Kanthasamy; J.A.Richt; S.A.Priola; A.G.Kanthasamy, (2005), Differential Role of Prion Protein in Oxidative Stress- and ER Stress-induced Apoptotic Signaling in Neural Cells. United States. Molecular Mechanisms of Transmissible Spongiform Encephalopathies (Prion Diseases), Keystone Symposium, Jan 11-15, Salt Lake City, UT.
- Dr. Kanthasamy also presented the research progress to the ILHAC advisory panel members during their visit to CVM Fall 2004. The slide presentation was well received by the panel.
Publications
- C. J. Choi; V. Anantharam;N. J. Saetveit;R. S. Houk;A. Kanthasamy; A.G. Kanthasamy, (2007)Normal cellular prion protein protects against manganese-induced oxidative stress and apoptotic cell death. Toxicological Sciences. In press.
- C. J. Choi; A. Kanthasamy; V. Anantharam; A. G. Kanthasamy, (2006) Interaction of metals with prion protein: possible role of divalent cations in the pathogenesis of prion diseases. Neurotoxicology. 27:777-87.
Manuscripts submitted
- V. Anantharam, I. Vorberg, C.J. Choi, A. Kanthasamy, J. A. Richt, S. A. Priola and A. G. Kanthasamy, Differential Role of Prion Protein in Oxidative Stress- and ER Stress-induced Apoptotic Signaling in Neural Cells. Submitted to J. Biol.Chem.
Extramural Funding:
Some of the preliminary data generated through ILHAC seed funding has helped us to secure from Department of Defense (DoD) to study the role of metals in pathogenesis of prion diseases. We thank ILHAC for the support.