Boe-Hyun Kim

A neuronal cell line that does not express either prion or doppel proteins.

Prions have been extensively studied since they represent a new class of infectious agents, the pathogenic prion protein (PrPSc). However, a central question on the physiological function of the normal prion protein (PrPC) remains unresolved. A cell model which was previously established from Rikn mice (PrP−/−) remains problematic because of its ectopic expression of the doppel (Dpl) which may have a neurotoxic effect. Here we established neuronal cell lines from Zürich I (PrP−/−) which do not express Dpl protein and ICR mice (PrP+/+) by transfecting with plasmid encoding for the large T antigen of SV40. The transformed cells have shown neuronal characteristics and, thus, these cell lines may provide a useful model to explore the function of neuronal PrPC.

The involvement of cellular prion protein in the autophagy pathway in neuronal cells

Apoptosis and autophagy are main mechanisms of neuronal death involved in prion diseases. Serum deprivation can induce both pathways to cell death in various types of cells. To investigate whether PrP(C) is involved in autophagy pathway, we analyzed the level of microtubule-associated protein 1 light chain 3 (LC3), an autophagy marker, by monitoring the conversion from LC3-I into LC3-II in Zürich I Prnp(-/-) hippocampal neuronal cells. We found that the expression level of LC3-II was increased in Prnp(-/-) compared to wild-type cells under serum deprivation. In electron microscopy, increased accumulation of autophagosomes in Prnp(-/-) cells was correlated with the increase in levels of LC3-II. Interestingly, this up-regulated autophagic activity was retarded by the introduction of PrP(C) into Prnp(-/-) cells but not by the introduction of PrP(C) lacking octapeptide repeat region. Thus, the octapeptide repeat region of PrP(C) may play a pivotal role in the control of autophagy exhibited by PrP(C) in neuronal cells.

Association of endothelial nitric oxide synthase and mitochondrial dysfunction in the hippocampus of scrapie-infected mice

The elevation of nitric oxide (NO) within the central nervous system (CNS) is known to be associated with the pathogenesis of neurodegenerative diseases such as HIV‐associated dementia (HAD), brain ischemia, Parkinsons disease, and Alzheimers disease. NO is enzymatically formed by the enzyme nitric oxide synthase (NOS). There are two forms of NOS, the constitutive and the inducible form. The constitutive form is present in endothelial cells (eNOS) and neurons (nNOS). The inducible form (iNOS) is expressed in various cell types including astroglia and microglia of the CNS. Using an animal model, we investigated the involvement of eNOS in the pathology of prion disease. We showed dramatic upregulation of eNOS immunoreactivity in reactive astroglial cells in the hippocampus in the prion disease animal model, scrapie in mice. Expression of eNOS was upregulated in cytosolic and mitochondrial fractions of whole brain. In the hippocampal region, eNOS was widely overexpressed in various components of the cell. We found that eNOS dramatically accumulated in hippocampal mitochondria and was particularly prevalent in structurally dysfunctional mitochondria. In association with the accumulation of eNOS in mitochondria, we showed that mitochondrial superoxide dismutase (Mn‐SOD or SOD2), cytochrome c, and ATP activity were downregulated both in whole brain and in the hippocampal region. These results indicate that eNOS plays a role in the development of dysfunctional mitochondria and this, in turn, could induce some of the histopathological changes seen in prion diseases.

Spontaneous immortalization of oligodendroglial cells derived from an SV40 T antigen-positive human glioblastoma multiforme.

The polyoma group of viruses, including SV40, is known to be oncogenic in certain species. Here we report for the first time naturally occurring, immortalized tumor cells from a patient with glioblastoma multiforme (GBM); the cells were shown to be oligodendroglia; cells had developed remarkable chromosomal changes and were positive for SV40 T antigen. Therefore, we postulated that the main cause of immortalization of these cells was the expression of SV40 T antigen gene and protein. Since the cells are naturally generated, they will provide a useful model to study the function of oligodendroglial cells and the development of GBM.

Physiological properties of astroglial cell lines derived from mice with high (SAMP8) and low (SAMR1, ICR) levels of endogenous retrovirus

Previous studies have reported that various inbred SAM mouse strains differ markedly with regard to a variety of parameters, such as capacity for learning and memory, life spans and brain histopathology. A potential cause of differences seen in these strains may be based on the fact that some strains have a high concentration of infectious murine leukemia virus (MuLV) in the brain, whereas other strains have little or no virus. To elucidate the effect of a higher titer of endogenous retrovirus in astroglial cells of the brain, we established astroglial cell lines from SAMR1 and SAMP8 mice, which are, respectively, resistant and prone to deficit in learning and memory and shortened life span. MuLV-negative astroglial cell lines established from ICR mice served as controls. Comparison of these cell lines showed differences in: 1) levels of the capsid antigen CAgag in both cell lysates and culture media, 2) expression of genomic retroelements, 3) the number of virus particles, 4) titer of infectious virus, 5) morphology, 6) replication rate of cells in culture and final cell concentrations, 7) expression pattern of proinflammatory cytokine genes. The results show that the expression of MuLV is much higher in SAMP8 than SAMR1 astrocyte cultures and that there are physiological differences in astroglia from the 2 strains. These results raise the possibility that the distinct physiological differences between SAMP8 and SAMR1 are a function of activation of endogenous retrovirus.

Oxidative Stress and Mitochondrial Dysfunction in Neurodegeneration of Transmissible Spongiform Encephalopathies (TSEs)

Transmissible spongiform encephalopathies (TSEs) or prion diseases are neurodegenerative disorders that are invariably fatal in humans and animals. An important component of the infectious agent is a glyco-protein, termed PrPSc, which is derived from a normal cellular protein, termed PrPc. The pathogenic mechanisms of TSEs are not clear, but several factors such as oxidative stress and mitochondrial dysfunction have been reported to be involved. In the current review, we will present data that supports a role for oxidative stress and mitochondrial dysfunction in the induction of these diseases. We will discuss the pathways whereby oxidative stress and mitochondrial dysfunction could lead to neuronal damage and the clinical manifestations of TSE diseases.

Cellular Prion Protein Combined with Galectin-3 and -6 Affects the Infectivity Titer of an Endogenous Retrovirus Assayed in Hippocampal Neuronal Cells.

Prion diseases are infectious and fatal neurodegenerative diseases which require the cellular prion protein, PrPC, for development of diseases. The current study shows that the PrPC augments infectivity and plaque formation of a mouse endogenous retrovirus, MuLV. We have established four neuronal cell lines expressing mouse PrPC, PrP+/+; two express wild type PrPC (MoPrPwild) and the other two express mutant PrPC (MoPrPmut). Infection of neuronal cells from various PrP+/+ and PrP-/- (MoPrPKO) lines with MuLV yielded at least three times as many plaques in PrP+/+ than in PrP-/-. Furthermore, among the four PrP+/+ lines, one mutant line, P101L, had at least 2.5 times as many plaques as the other three PrP+/+ lines. Plaques in P101L were four times larger than those in other PrP+/+ lines. Colocalization of PrP and CAgag was seen in MuLV-infected PrP+/+ cells. In the PrP-MuLV interaction, the involvement of galectin-3 and -6 was observed by immunoprecipitation with antibody to PrPC. These results suggest that PrPC combined with galectin-3 and -6 can act as a receptor for MuLV. P101L, the disease form of mutant PrPC results suggest the genetic mutant form of PrPC may be more susceptible to viral infection.

P4-124: Comparison of gene expression profiles of prion protein-deficient and wild type neuronal cell lines of mice

in various organs outside the central nervous system (CNS) where its detection may be used as an early marker of prion infection. Recently Goldstein et al. (Lancet 2003:361, 1258-1265) have demonstrated the accumulation of -amyloid (A ) aggregates in the ocular lens in Alzheimer’s disease (AD) patients and in AD transgenic mice. These A aggregates appear to produce increased light scattering effect and bind lipophilic A fluorescent dyes (e.g. methoxy-X04); therefore they can be detected in vivo using quasielastic light scattering (QLS) or fluorescent ligand scanning (FLS) approaches, respectively. Objective(s): Because of the molecular mimicry between A and PrP we investigated the presence of the PrP in the ocular lens of prion infected mice. Methods: Lenses obtained from presymptomatic and clinically sick prion infected animals were examined by ex vivo stereomicroscopy, immunohistochemistry, Western-blot and tandem mass spectroscopy (MS/MS). Results: Ex vivo stereomicroscopy revealed the presence of dense supranuclear cataracts in the lenses derived from both presymptomatic and symptomatic animals. Proteinaceous material forming these cataracts immunoreacted with anti-prion monoclonal antibodies. Western-blot analysis of the lens homogenate revealed the presence of proteinase K resistant PrP forming the classical three-band pattern that corresponds to diglycosylated, monoglycosylated, and non-glycosylated PrP isoforms. In gel trypsin digestion followed by MS/MS demonstrated that PrP co-exists in the lens in complexes with B-crystallin. Conclusions: Demonstration of PrP accumulation in the ocular lens during the course of prion infection in mice provides the basis for investigating the usefulness of QLS and FLS as a non-invasive tests for detection of the prion infectivity status in presymptomatic subjects.