Danielle Casanova

Successful Transmission of Three Mouse-Adapted Scrapie Strains to Murine Neuroblastoma Cell Lines Overexpressing Wild-Type Mouse Prion Protein

ABSTRACT Propagation of the agents responsible for transmissible spongiform encephalopathies (TSEs) in cultured cells has been achieved for only a few cell lines. To establish efficient and versatile models for transmission, we developed neuroblastoma cell lines overexpressing type A mouse prion protein, MoPrPC-A, and then tested the susceptibility of the cells to several different mouse-adapted scrapie strains. The transfected cell clones expressed up to sixfold-higher levels of PrPC than the untransfected cells. Even after 30 passages, we were able to detect an abnormal proteinase K-resistant form of prion protein, PrPSc, in the agent-inoculated PrP-overexpressing cells, while no PrPSc was detectable in the untransfected cells after 3 passages. Production of PrPSc in these cells was also higher and more stable than that seen in scrapie-infected neuroblastoma cells (ScN2a). The transfected cells were susceptible to PrPSc-A strains Chandler, 139A, and 22L but not to PrPSc-B strains 87V and 22A. We further demonstrate the successful transmission of PrPSc from infected cells to other uninfected cells. Our results corroborate the hypothesis that the successful transmission of agents ex vivo depends on both expression levels of host PrPC and the sequence of PrPSc. This new ex vivo transmission model will facilitate research into the mechanism of host-agent interactions, such as the species barrier and strain diversity, and provides a basis for the development of highly susceptible cell lines that could be used in diagnostic and therapeutic approaches to the TSEs.

Amphotericin B Inhibits the Generation of the Scrapie Isoform of the Prion Protein in Infected Cultures

ABSTRACT Transmissible spongiform encephalopathies form a group of fatal neurodegenerative disorders that have the unique property of being infectious, sporadic, or genetic in origin. Although some doubts about the nature of the responsible agent of these diseases remain, it is clear that a protein called PrPSc plays a central role. PrPSc is a conformational variant of PrPC, the normal host protein. Polyene antibiotics such as amphotericin B have been shown to delay the accumulation of PrPSc and to increase the incubation time of the disease after experimental transmission in laboratory animals. Unlike for Congo red and sulfated polyanions, no effect of amphotericin B has been observed in infected cultures. We show here for the first time that amphotericin B can inhibit PrPSc generation in scrapie-infected GT1-7 and N2a cells. Its activity seems to be related to a modification of the properties of detergent-resistant microdomains. These results provide new insights into the mechanism of action of amphotericin B and confirm the usefulness of infected cultures in the therapeutic research of transmissible spongiform encephalopathies.

Neural Stem Cell Model for Prion Propagation

The study of prion transmission and targeting is a major scientific issue with important consequences for public health. Only a few cell culture systems that are able to convert the cellular isoform of the prion protein into the pathologic scrapie isoform of the prion protein (PrPSc) have been described. We hypothesized that central nervous system neural stem cells (NSCs) could be the basis of a new cell culture model permissive to prion infection. Here, we report that monolayers of differentiated fetal NSCs and adult multipotent progenitor cells isolated from mice were able to propagate prions. We also demonstrated the large influence of neural cell fate on the production of PrPSc, allowing the molecular study of prion neuronal targeting in relation with strain differences. This new stem cell‐based model, which is applicable to different species and to transgenic mice, will allow thoughtful investigations of the molecular basis of prion diseases, and will open new avenues for diagnostic and therapeutic research.

The truncated 23-230 form of the prion protein localizes to the nuclei of inducible cell lines independently of its nuclear localization signals and is not cytotoxic

The mechanisms of prion-induced neurological dysfunction observed in prion diseases are poorly understood. Transgenic mice expressing a truncated form of the prion protein (23-230 PrP) acquire cerebellar degeneration (Ma and Lindquist, Science, 2002). To decipher the mechanisms of neurodegeneration induced by 23-230 PrP, we established inducible cell lines expressing this truncated form of PrP. We found that 23-230 PrP, expected to be cytosolic, accumulated mostly in the nucleus of the cells and was not cytotoxic. Nuclear localization of this mutant form of PrP is independent of its predicted nuclear localization signals. In contrast to what we previously described for PrPSc, nuclear accumulation of 23-230 PrP does not require a functional microtubule network. We observed that 23-230 PrP interacts with chromatin in vivo, as already described for recombinant PrP and for PrPSc. Our data demonstrate that the 23-230 PrP model does not reflect the situation of a cytosolic PrP but could represent a very useful tool to understand the consequences of the accumulation of the prion protein in the nucleus.

Effect of amphotericin B on wild-type and mutated prion proteins in cultured cells : Putative mechanism of action in Transmissible spongiform encephalopathies

Abstract: Transmissible spongiform encephalopathies form a group of fatal neurodegenerative disorders that have the unique property of being infectious, sporadic, or genetic in origin. Although some doubts remain on the nature of the responsible agent of these diseases, it is clear that a protein called PrPSc [the scrapie isoform of prion protein (PrP)] plays a central role. PrPSc represents a conformational variant of PrPC (the cellular isoform of PrP), the normal host protein. Polyene antibiotics, such as amphotericin B, have been shown to delay the accumulation of PrPSc and to increase the incubation time of the disease after experimental transmission in laboratory animals. Unlike agents such as Congo red, the inhibitory effect of amphotericin B on PrPSc generation has not been observed in infected cultures. Using transfected cells expressing wild‐type or mutated mouse PrPs, we show here that amphotericin B is able to interfere with the generation of abnormal PrP isoforms in culture. Its action seems related to a modification of PrP trafficking through the association of this glycosylphosphatidylinositol‐anchored protein with detergent‐resistant microdomains. These results represent a first step toward the comprehension of the mechanism of action of amphotericin B in transmissible spongiform encephalopathies.

Prion Replication Occurs in Endogenous Adult Neural Stem Cells and Alters Their Neuronal Fate: Involvement of Endogenous Neural Stem Cells in Prion Diseases

Prion diseases are irreversible progressive neurodegenerative diseases, leading to severe incapacity and death. They are characterized in the brain by prion amyloid deposits, vacuolisation, astrocytosis, neuronal degeneration, and by cognitive, behavioural and physical impairments. There is no treatment for these disorders and stem cell therapy therefore represents an interesting new approach. Gains could not only result from the cell transplantation, but also from the stimulation of endogenous neural stem cells (NSC) or by the combination of both approaches. However, the development of such strategies requires a detailed knowledge of the pathology, particularly concerning the status of the adult neurogenesis and endogenous NSC during the development of the disease. During the past decade, several studies have consistently shown that NSC reside in the adult mammalian central nervous system (CNS) and that adult neurogenesis occurs throughout the adulthood in the subventricular zone of the lateral ventricle or the Dentate Gyrus of the hippocampus. Adult NSC are believed to constitute a reservoir for neuronal replacement during normal cell turnover or after brain injury. However, the activation of this system does not fully compensate the neuronal loss that occurs during neurodegenerative diseases and could even contribute to the disease progression. We investigated here the status of these cells during the development of prion disorders. We were able to show that NSC accumulate and replicate prions. Importantly, this resulted in the alteration of their neuronal fate which then represents a new pathologic event that might underlie the rapid progression of the disease.

Proteomic consequences of expression and pathological conversion of the prion protein in inducible neuroblastoma N2a cells

Neurodegenerative diseases are often associated with misfolding and deposition of specific proteins in the nervous system. The prion protein, which is associated with transmissible spongiform encephalopathies (TSEs), is one of them. The normal function of the cellular form of the prion protein (PrPC) is mediated through specific signal transduction pathways and is linked to resistance to oxidative stress, neuronal outgrowth and cell survival. In TSEs, PrPC is converted into an abnormally folded isoform, called PrPSc, that may impair the normal function of the protein and/or generate toxic aggregates. To investigate these molecular events we performed a two-dimensional gel electrophoresis comparison of neuroblastoma N2a cells expressing different amounts of PrPC, and eventually infected with the 22L prion strain. Mass spectrometry and peptide mass fingerprint analysis identified a series of proteins with modified expression. They included the chaperones Grp78/BiP, protein disulfide-isomerase A6, Grp75 and Hsp60 which had an opposite expression upon PrPC expression and PrPSc production. The detection of these proteins was coherent with the idea that protein misfolding plays an important role in TSEs. Other proteins such as calreticulin, tubulin, vimentin or the laminin receptor had their expression modified in infected cells which was reminiscent of previous results. Altogether our data provide molecular information linking PrP expression and misfolding which could be the basis of further therapeutic and pathophysiological research in this field.

Genetic heterogeneity versus molecular analysis of prion susceptibility in neuroblasma N2a sublines

The neuroblastoma-derived cell line N2a is permissive to certain prion strains but resistant sublines unable to accumulate the pathological proteinase-K resistant form of the prion protein can be isolated. We compared for gene expression and phenotypes different N2a sublines that were susceptible or resistant to the 22L prion strain. Karyotypes and comparative genomic hybridization arrays revealed chromosomal imbalances but did not demonstrate a characteristic profile of genomic alterations linked to prion susceptibility. Likewise, we showed that this phenotype was not dependent on the binding of PrPres, the expression of the prion protein gene, or on its primary sequence. We completed this analysis by looking using real-time quantitative PCR at the expression of a set of genes encoding proteins linked to prion biology. None of the candidates could account by itself for the infection phenotype, nevertheless sublines had distinct transcriptional profiles. Taken together, our results do not support a role for specific genomic abnormalities and possible candidate proteins in N2a prion susceptibility. They also reveal genetic heterogeneity among the sublines and serve as a guidance for further investigation into the molecular mechanisms of prion infection.

Systemic Delivery of siRNA Down Regulates Brain Prion Protein and Ameliorates Neuropathology in Prion Disorder

One of the main challenges for neurodegenerative disorders that are principally incurable is the development of new therapeutic strategies, which raises important medical, scientific and societal issues. Creutzfeldt-Jakob diseases are rare neurodegenerative fatal disorders which today remain incurable. The objective of this study was to evaluate the efficacy of the down-regulation of the prion protein (PrP) expression using siRNA delivered by, a water-in-oil microemulsion, as a therapeutic candidate in a preclinical study. After 12 days rectal mucosa administration of Aonys/PrP-siRNA in mice, we observed a decrease of about 28% of the brain PrPC level. The effect of Aonys/PrP-siRNA was then evaluated on prion infected mice. Several mice presented a delay in the incubation and survival time compared to the control groups and a significant impact was observed on astrocyte reaction and neuronal survival in the PrP-siRNA treated groups. These results suggest that a new therapeutic scheme based an innovative delivery system of PrP-siRNA can be envisioned in prion disorders.

HEPES inhibits the conversion of prion protein in cell culture

HEPES is a well-known buffering reagent used in cell-culture medium. Interestingly, this compound is also responsible for significant modifications of biological parameters such as uptake of organic molecules, alteration of oxidative stress mechanisms or inhibition of ion channels. While using cell-culture medium supplemented with HEPES on prion-infected cells, it was noticed that there was a significant concentration-dependent inhibition of accumulation of the abnormal isoform of the prion protein (PrP(Sc)). This effect was present only in live cells and was thought to be related to modification of the PrP environment or biology. These results could modify the interpretation of cell-culture assays of prion therapeutic agents, as well as of previous cell biology results obtained in the field using HEPES buffers. This inhibitory effect of HEPES could also be exploited to prevent contamination or propagation of prions in cell culture.