Karel Holada

Expression of Prion Protein in Mouse Erythroid Progenitors and Differentiating Murine Erythroleukemia Cells

Prion diseases have been observed to deregulate the transcription of erythroid genes, and prion protein knockout mice have demonstrated a diminished response to experimental anemia. To investigate the role of the cellular prion protein (PrPC) in erythropoiesis, we studied the proteins expression on mouse erythroid precursors in vivo and utilized an in vitro model of the erythroid differentiation of murine erythroleukemia cells (MEL) to evaluate the effect of silencing PrPC through RNA interference. The expression of PrPC and selected differentiation markers was analyzed by quantitative multicolor flow cytometry, western blot analysis and quantitative RT-PCR. The silencing of PrPC expression in MEL cells was achieved by expression of shRNAmir from an integrated retroviral vector genome. The initial upregulation of PrPC expression in differentiating erythroid precursors was detected both in vivo and in vitro, suggesting PrPCs importance to the early stages of differentiation. The upregulation was highest on early erythroblasts (16200±3700 PrPC / cell) and was followed by the gradual decrease of PrPC level with the precursors maturation reaching 470±230 PrPC / cell on most mature CD71−Ter119+ small precursors. Interestingly, the downregulation of PrPC protein with maturation of MEL cells was not accompanied by the decrease of PrP mRNA. The stable expression of anti-Prnp shRNAmir in MEL cells led to the efficient (>80%) silencing of PrPC levels. Cell growth, viability, hemoglobin production and the transcription of selected differentiation markers were not affected by the downregulation of PrPC. In conclusion, the regulation of PrPC expression in differentiating MEL cells mimics the pattern detected on mouse erythroid precursors in vivo. Decrease of PrPC protein expression during MEL cell maturation is not regulated on transcriptional level. The efficient silencing of PrPC levels, despite not affecting MEL cell differentiation, enables created MEL lines to be used for studies of PrPC cellular function.

Deletion of protease-activated receptor 2 prolongs survival of scrapie-inoculated mice.

Proteinase-activated receptor 2 (PAR2) has recently been identified to be a possible modulator of neurodegeneration. To investigate whether PAR2 plays a role in prion infection, we inoculated PAR2-deficient (PAR2(-/-)) and wild-type (WT) mice intracerebrally with the Rocky Mountain Laboratory strain of scrapie. PAR2(-/-) mice demonstrated a delayed onset of clinical symptoms, including weight loss, and demonstrated moderate but highly significant prolongation of survival over WT controls. Concomitantly, no apparent differences in brain pathology, infectivity or features of brain prion protein between deceased WT and PAR2(-/-) mice were found. Our study suggests that PAR2 deletion modulates dynamics of the disease without gross perturbation of its pathogenesis.

Gerstmann–Sträussler–Scheinker syndrome with the P102L pathogenic mutation presenting as familial Creutzfeldt–Jakob disease: a case report and review of the literature

Gerstmann–Sträussler–Scheinker syndrome is a rare autosomal dominant disease caused by a mutation in the prion gene, usually manifesting as progressive ataxia with late cognitive decline. A 44-year-old woman with a positive family history developed early personality and behavior changes, followed by paresthesias and ataxia, later associated with memory problems, pyramidal signs, anosognosia and very late myoclonus, spasticity, and severe dysexecutive impairment. Magnetic resonance showed caudate, mesio-frontal, and insular hyper-intensities, electroencephalography revealed generalized triphasic periodic complexes. A pathogenic P102L mutation in the prion gene was detected. Our case differed from classical Gerstmann–Sträussler–Scheinker syndrome by rapid progression, severe dementia, abnormal electroencephalography and magnetic resonance findings, which were highly suggestive of familial Creutzfeldt–Jakob disease.

Photodynamic inactivation of prions by disulfonated hydroxyaluminium phthalocyanine.

Sulfonated phthalocyanines (Pcs) are cyclic tetrapyrroles that constitute a group of photosensitizers. In the presence of visible light and diatomic oxygen, Pcs produce singlet oxygen and other reactive oxygen species that have known degradation effects on lipids, proteins and/or nucleic acids. Pcs have been used successfully in the treatment of bacterial, yeast and fungal infections, but their use in the photodynamic inactivation of prions has never been reported. Here, we evaluated the photodynamic activity of the disodium salt of disulfonated hydroxyaluminium phthalocyanine (PcDS) against mouse-adapted scrapie RML prions in vitro. PcDS treatment of RML brain homogenate resulted in a time- and dose-dependent inactivation of prions. The photodynamic potential of Pcs offers a new way to inactivate prions using biodegradable compounds at room temperature and normal pressure, which could be useful for treating thermolabile materials and liquids.

Precision in the design of an experimental study deflects the significance of proteinase-activated receptor 2 expression in scrapie-inoculated mice

Proteinase-activated receptor 2 (PAR2) is suspected to modulate the pathogenesis of various neurodegenerative conditions. We previously described delayed onset of clinical symptoms and prolonged survival of PAR2-deficient mice after intracerebral inoculation with prions. Here we report the results from a refined blinded study that aimed to investigate the effects of PAR2 deletion on scrapie pathogenesis after peripheral infection. This study failed to confirm that PAR2 deficiency impacts on the length of the incubation period, with PAR2-/- and PAR2+/+ littermates developing scrapie at the same time. To clarify the discrepancy between the two observations, we repeated the intracerebral inoculation study while utilizing our refined protocol, which aimed to limit possible sources of experimental bias. The study again failed to confirm the significant effect of PAR2 expression on the course of prion infection. Our report emphasizes and discusses the importance of unbiased experimental design and the selection of proper genetic controls when using genetically altered animal models for prion pathogenesis studies.

Blood storage affects the detection of cellular prion protein on peripheral blood leukocytes and circulating dendritic cells in part by promoting platelet satellitism.

BACKGROUNDnFlow cytometry represents an attractive approach for developing currently unavailable screening tests for prion diseases. Several studies have reported significant differences in the binding of antibodies directed against cellular prion protein (PrP(C)) to blood cells of prion-infected subjects compared with healthy controls. However, flow cytometry data usually show large individual variations in detected PrP(C) levels in both infected and control groups, rendering the interpretation of individual patient data difficult.nnnOBJECTIVESnTo determine how pre-analytical variables, such as the choice of anticoagulant, whether or not the blood was stored, and the storage temperature, affect the detection of PrP(C) in blood cells.nnnMETHODSnBlood from healthy donors was collected in EDTA or citrate anticoagulant and processed either immediately or after storage overnight at room temperature or at 4°C. The expression of PrP(C) by T cells, B cells, NK cells, monocytes and circulating dendritic cells was evaluated using quantitative flow cytometry with the PrP(C) monoclonal antibodies AG4 and AH6.nnnRESULTSnThe anticoagulation of blood with citrate resulted in decreased levels of PrP(C) on monocytes but not the other cell types. The storage of blood prior to analysis led to a significant decrease in the levels of PrP(C) on the cells studied, although there were substantial differences between the cell populations. This decrease was more pronounced when using mAb AG4, which targets the N-terminal portion of the PrP(C) molecule, or following storage at room temperature. Moreover, we identified platelet satellitism on leukocytes, especially on monocytes and granulocytes, as an additional factor contributing to the heterogeneity of PrP(C) detection in stored blood.nnnCONCLUSIONSnOur study demonstrates that the storage of blood prior to analysis greatly affects the detection of PrP(C) by flow cytometry. To limit the inclusion of storage-generated artifacts, we recommend the processing of blood samples immediately after their collection.

Expression of the cellular prion protein affects posttransfusion recovery and survival of red blood cells in mice

Cellular prion protein (PrPC) is expressed on various cell types including red blood cells (RBCs). The PrPC plays a key role in the pathogenesis of prion diseases, but its physiologic function remains unclear. PrPC is expressed on CD34+ hematopoietic stem cells and its expression is regulated during blood cell differentiation including the erythroid line.