The overall goal of this project is to understand how retroviruses adversely affect the central nervous system. We are utilizing the mouse as an animal model and are currently focusing our attention on a virus, FrCasE, which causes a non-inflammatory spongiform encephalomyelopathy. Transcriptional profiling, using high density microarray technology, revealed that Endoplasmic Reticulum (ER) Stress responses may be involved in the pathogenesis of this disease. In vitro studies indicated that the ER stress was induced by the instability and misfolding of the viral envelope protein within the ER, leading to a prototypic Unfolded Protein Response. The Unfolded Protein Response induced by this virus is associated with the upregulation of ER chaperones, the stable binding of the one of these chaperones proteins (BiP) to the viral envelope protein and the shunting of this viral protein to the ubiquitin-proteasome degradative pathway. In addition, we have observed in cells infected by FrCasE the intracellular accumulation of reactive oxygen species. Interestingly, another virus, F43, which is neuroinvasive but avirulent, failed to induce any of these responses. F43 differs from FrCasE only in its envelope protein. Previous work from us and others have demonstrated that the neurotoxicity of FrCasE and related neuropathogenic viruses is determined by the sequence of the viral envelope protein. Thus, the viral envelope protein specifies neurovirulence in vivo and also mediates the activation of cellular stress responses demonstrable in vivo and in vitro. It is for these reasons that we now suspect that the brain disease induced by FrCasE and related murine retroviruses represents a virus-induced protein folding disease.? ? We have continued the characterization of ER stress in the brain and have focused our attention on the nature of cell death as well as the identity of the degenerating cells. While FrCasE infects a wide variety of cells in the brain including microvascular endothelia, perivascular and parenchymal microglia, oligodendrocytes and some populations of neurons that proliferate postnatally, the only cells that are damaged are cells of the oligodendrocyte lineage. This suggests that cells of the oligodendrocyte lineage are hypersensitive to ER stress. The degenerating cells are marked by the expression of the basic HLH transcription factor Olig2. Olig2 is expressed by neuronal-glial progenitors in the embryonic brain and is involved in lineage commitment of oligodendrocyte precursors cells. Olig2 is also expressed by fully differentiated, myelin producing oligodendrocytes as well. In the last year we have examined in more detail the level of differentiation of the degenerating cells. While the vast majority of these cells express the HMG transcription factor, SOX10, which is also expressed throughout oligodendrocyte differentiation, approximately half of these cells also express PDGF1alpha receptors and the Nkx2.2 transcription factor, both of which mark oligodendrocyte precursor cells (OPC?s). We are currently interested in the identification of the PDGFR1alpha/Nkx2.2 negative Olig2 positive population. These findings suggest the possibility that the cytopathology induced by FrCasE may be manifested at a critical stage of oligodendrocyte terminal differentiation from OPC?s to mature myelin producing cells. ? ? The type of cell death manifested by these cells is unusual and uncharacteristic of that generally thought to be induced by ER stress. Unsustained ER stress in vitro is generally associated with apoptosis. Yet ultrastructural studies of the degenerating cells in the brains of FrCasE-infected mice reveal extreme cytoplasmic swelling, dissolution of organelles including ribosomes, endoplasmic reticulum and Golgi, but with surprisingly well preserved nuclei. The cytoplasm of these cells often contain extensive double membrane structures resembling disorganized autophagic membranes, though intact autophagic vacuoles are not evident. We are currently examining in more detail the role of autophagy in the cellular stress induced by FrCasE. Preliminary results indicate that two different neurovirulent viruses FrCasE and Moloney MLVts1 induce extensive vacuolation of NIH3T3 cells and that the vacuolar membranes are associated with LC3, the mammalian homolog of the yeast autophagic membrane protein Atg8. A link between ER stress and induction of autophagy in yeast has recently be reported. We are currently very interested in understanding the nature of autophagic response induced by these neuropathogenic viruses.? ? Our major collaboration this year was with the laboratory of Drs. Nico Dantuma, Karolinska Institute, and Sarah Tabrizi, University College London. These studies have shown that PrPsc oligomers inhibit the Ubiquitin-Proteasome system by specifically interfering with caspase-like and chymotrypsin-like protease activity (beta1 and 5 proteolytic active sites) of the 20S core. We have carried out all of the in vivo studies of UPS function in scrapie-inoculated mice using a transgenic mouse line developed by Dr. Dantuma. This mouse carries a transgene consisting of GFP linked to uncleavable ubiquitin. The half-life of GFP under normal circumstances is short leading to nearly undetectable steady-state levels of GFP protein. When the UPS system is compromised, GFP levels increase. Indeed, we found evidence for accumulation of GFP in mice exhibiting clinical signs of scrapie and this was not due to the upregulation of GFP mRNA. This study represents the first clear evidence that the pathogenesis of TSE (prion) diseases may be linked to inhibition by the abnormally folded protein PrPsc of specific catalytic subunits of the proteasome. ? ? These studies are important because ER stress and dysfunction of the protein-degradative machinery of the cell have been found to be associated with a variety of acquired and heritable degenerative diseases of humans, including Parkinsons disease, Huntington?s and Alzheimer?s disease. Understanding the nature of the cellular stress responses induced by the neurovirulent murine retroviruses as well as the TSE agents, and the connection between these responses and the cytopathology induced by these infectious agents, promises to uncover important biochemical pathways to which therapeutic intervention can be directed.
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