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Prion Protein Does Not Confer Resistance to Hippocampus-Derived Zpl Cells against the Toxic Effects of Cu2+, Mn2+, Zn2+ and Co2+ Not Supporting a General Protective Role for PrP in Transition Metal Induced Toxicity.

Cingaram PK, Nyeste A, Dondapati DT, Fodor E, Welker E - PLoS ONE (2015)

Bottom Line: By employing a cell viability assay, we examined the effects of various concentrations of Cu2+, Zn2+, Mn2+, and Co2+ on Zpl (Prnp-/-) and ZW (Prnp+/+) hippocampus-derived mouse neuronal cells.However, when we introduced PrP or only the empty vector into Zpl cells, we could not discern any protective effect associated with the presence of PrP.Thus, our results on this mouse cell culture model do not seem to support a strong protective role for PrP against transition metal toxicity and also emphasize the necessity of extreme care when comparing cells derived from PrP knock-out and wild type mice.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary.

ABSTRACT
The interactions of transition metals with the prion protein (PrP) are well-documented and characterized, however, there is no consensus on their role in either the physiology of PrP or PrP-related neurodegenerative disorders. PrP has been reported to protect cells from the toxic stimuli of metals. By employing a cell viability assay, we examined the effects of various concentrations of Cu2+, Zn2+, Mn2+, and Co2+ on Zpl (Prnp-/-) and ZW (Prnp+/+) hippocampus-derived mouse neuronal cells. Prnp-/- Zpl cells were more sensitive to all four metals than PrP-expressing Zw cells. However, when we introduced PrP or only the empty vector into Zpl cells, we could not discern any protective effect associated with the presence of PrP. This observation was further corroborated when assessing the toxic effect of metals by propidium-iodide staining and fluorescence activated cell sorting analysis. Thus, our results on this mouse cell culture model do not seem to support a strong protective role for PrP against transition metal toxicity and also emphasize the necessity of extreme care when comparing cells derived from PrP knock-out and wild type mice.

No MeSH data available.


Related in: MedlinePlus

Expression levels of PrP in the ZW 13–2 and Zpl 2–1 cell lines.(A) Western blot of total cell lysates from wild type hippocampal neuronal cell line of ICR mice (ZW 13–2) and Prnp−/− hippocampal neuronal cell line of Zürich I mice (Zpl 2–1). Cell lysates were incubated either in the absence (lane 1 and lane 3) or in the presence of PNGase F (lane 2 and lane 4). Western blot analysis of PrP was carried out with monoclonal PrP antibody SAF 32. β-actin was used to confirm equal loading of proteins. (B) Immunocytochemistry verification of prion protein expression in ZW 13–2 and Zpl 2–1 cells. PrP is immunolabeled by using the monoclonal PrP antibody SAF 32 with an Alexa 568 conjugated secondary antibody (red), nucleus is stained with DAPI (cyanine blue) and merged images are shown in the last column. Pictures were recorded by using a 60X oil immersion objective and a zooming factor of 4.
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pone.0139219.g001: Expression levels of PrP in the ZW 13–2 and Zpl 2–1 cell lines.(A) Western blot of total cell lysates from wild type hippocampal neuronal cell line of ICR mice (ZW 13–2) and Prnp−/− hippocampal neuronal cell line of Zürich I mice (Zpl 2–1). Cell lysates were incubated either in the absence (lane 1 and lane 3) or in the presence of PNGase F (lane 2 and lane 4). Western blot analysis of PrP was carried out with monoclonal PrP antibody SAF 32. β-actin was used to confirm equal loading of proteins. (B) Immunocytochemistry verification of prion protein expression in ZW 13–2 and Zpl 2–1 cells. PrP is immunolabeled by using the monoclonal PrP antibody SAF 32 with an Alexa 568 conjugated secondary antibody (red), nucleus is stained with DAPI (cyanine blue) and merged images are shown in the last column. Pictures were recorded by using a 60X oil immersion objective and a zooming factor of 4.

Mentions: The role of the prion protein in a particular cellular process, including those that are concerned with metal-PrP interrelations in ex vivo and in vivo systems is generally studied by either genetically ablating [56,63–67] or siRNA silencing [68] the expression of the prion protein. In this respect, cells that are generated from Prnp−/− mice are especially valuable. While some Prnp−/− cells are generated from mice with the ectopic expression of Doppel in the CNS [63,67], Kim and coworkers have established a series of hippocampal neuronal cell lines from Zürich I Prnp−/− mice along with their respective controls from the ICR Prnp+/+ mice [63]. From this series, here we used the mouse hippocampal neuronal cell lines Zpl 2-1(Prnp−/−) and ZW 13–2 (Prnp+/+) that have previously been fully characterized [63]. The expression levels of the prion protein in the cell cultures maintained at our laboratory were verified before the experiments using immunoblotting and immunocytochemical analyses (Fig 1). ZW 13–2 cells have a high level of PrPC expression, which is confirmed by both untreated and PNGase treated samples (lanes 1 and 2, Fig 1A). Contrary, Zpl 2–1 cells are a knockout cell line with no expression of PrPC; accordingly, there was no detectable band for PrP on the immunoblot (lanes 3 and 4, Fig 1A). Expression and localization of the prion protein in the two cell lines was further tested by immunocytochemical analysis (Fig 1B). The bright red immunofluorescence-staining pattern in the ZW 13–2 cells revealed that the prion protein was distributed on the surface of the cells, whereas no immunoreactivity was detected in the Zpl 2–1 cells.


Prion Protein Does Not Confer Resistance to Hippocampus-Derived Zpl Cells against the Toxic Effects of Cu2+, Mn2+, Zn2+ and Co2+ Not Supporting a General Protective Role for PrP in Transition Metal Induced Toxicity.

Cingaram PK, Nyeste A, Dondapati DT, Fodor E, Welker E - PLoS ONE (2015)

Expression levels of PrP in the ZW 13–2 and Zpl 2–1 cell lines.(A) Western blot of total cell lysates from wild type hippocampal neuronal cell line of ICR mice (ZW 13–2) and Prnp−/− hippocampal neuronal cell line of Zürich I mice (Zpl 2–1). Cell lysates were incubated either in the absence (lane 1 and lane 3) or in the presence of PNGase F (lane 2 and lane 4). Western blot analysis of PrP was carried out with monoclonal PrP antibody SAF 32. β-actin was used to confirm equal loading of proteins. (B) Immunocytochemistry verification of prion protein expression in ZW 13–2 and Zpl 2–1 cells. PrP is immunolabeled by using the monoclonal PrP antibody SAF 32 with an Alexa 568 conjugated secondary antibody (red), nucleus is stained with DAPI (cyanine blue) and merged images are shown in the last column. Pictures were recorded by using a 60X oil immersion objective and a zooming factor of 4.
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Related In: Results  -  Collection

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pone.0139219.g001: Expression levels of PrP in the ZW 13–2 and Zpl 2–1 cell lines.(A) Western blot of total cell lysates from wild type hippocampal neuronal cell line of ICR mice (ZW 13–2) and Prnp−/− hippocampal neuronal cell line of Zürich I mice (Zpl 2–1). Cell lysates were incubated either in the absence (lane 1 and lane 3) or in the presence of PNGase F (lane 2 and lane 4). Western blot analysis of PrP was carried out with monoclonal PrP antibody SAF 32. β-actin was used to confirm equal loading of proteins. (B) Immunocytochemistry verification of prion protein expression in ZW 13–2 and Zpl 2–1 cells. PrP is immunolabeled by using the monoclonal PrP antibody SAF 32 with an Alexa 568 conjugated secondary antibody (red), nucleus is stained with DAPI (cyanine blue) and merged images are shown in the last column. Pictures were recorded by using a 60X oil immersion objective and a zooming factor of 4.
Mentions: The role of the prion protein in a particular cellular process, including those that are concerned with metal-PrP interrelations in ex vivo and in vivo systems is generally studied by either genetically ablating [56,63–67] or siRNA silencing [68] the expression of the prion protein. In this respect, cells that are generated from Prnp−/− mice are especially valuable. While some Prnp−/− cells are generated from mice with the ectopic expression of Doppel in the CNS [63,67], Kim and coworkers have established a series of hippocampal neuronal cell lines from Zürich I Prnp−/− mice along with their respective controls from the ICR Prnp+/+ mice [63]. From this series, here we used the mouse hippocampal neuronal cell lines Zpl 2-1(Prnp−/−) and ZW 13–2 (Prnp+/+) that have previously been fully characterized [63]. The expression levels of the prion protein in the cell cultures maintained at our laboratory were verified before the experiments using immunoblotting and immunocytochemical analyses (Fig 1). ZW 13–2 cells have a high level of PrPC expression, which is confirmed by both untreated and PNGase treated samples (lanes 1 and 2, Fig 1A). Contrary, Zpl 2–1 cells are a knockout cell line with no expression of PrPC; accordingly, there was no detectable band for PrP on the immunoblot (lanes 3 and 4, Fig 1A). Expression and localization of the prion protein in the two cell lines was further tested by immunocytochemical analysis (Fig 1B). The bright red immunofluorescence-staining pattern in the ZW 13–2 cells revealed that the prion protein was distributed on the surface of the cells, whereas no immunoreactivity was detected in the Zpl 2–1 cells.

Bottom Line: By employing a cell viability assay, we examined the effects of various concentrations of Cu2+, Zn2+, Mn2+, and Co2+ on Zpl (Prnp-/-) and ZW (Prnp+/+) hippocampus-derived mouse neuronal cells.However, when we introduced PrP or only the empty vector into Zpl cells, we could not discern any protective effect associated with the presence of PrP.Thus, our results on this mouse cell culture model do not seem to support a strong protective role for PrP against transition metal toxicity and also emphasize the necessity of extreme care when comparing cells derived from PrP knock-out and wild type mice.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary.

ABSTRACT
The interactions of transition metals with the prion protein (PrP) are well-documented and characterized, however, there is no consensus on their role in either the physiology of PrP or PrP-related neurodegenerative disorders. PrP has been reported to protect cells from the toxic stimuli of metals. By employing a cell viability assay, we examined the effects of various concentrations of Cu2+, Zn2+, Mn2+, and Co2+ on Zpl (Prnp-/-) and ZW (Prnp+/+) hippocampus-derived mouse neuronal cells. Prnp-/- Zpl cells were more sensitive to all four metals than PrP-expressing Zw cells. However, when we introduced PrP or only the empty vector into Zpl cells, we could not discern any protective effect associated with the presence of PrP. This observation was further corroborated when assessing the toxic effect of metals by propidium-iodide staining and fluorescence activated cell sorting analysis. Thus, our results on this mouse cell culture model do not seem to support a strong protective role for PrP against transition metal toxicity and also emphasize the necessity of extreme care when comparing cells derived from PrP knock-out and wild type mice.

No MeSH data available.


Related in: MedlinePlus