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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 generated stable cell lines.(A) Total cell lysates of stably expressing cells made from Prnp−/− hippocampal neuronal cell line of Zürich I mice (Zpl 2–1) transfected with either the empty vector (Zpl 2-1-vector) or with mouse PrP gene (Zpl 2-1-PrP). Cell lysates were incubated in the absence (lane 1 and lane 3) or presence of PNGase F (lane 2 and lane 4). Western blot analysis was carried out with monoclonal PrP antibody SAF 32. β-actin was used to confirm equal loading of proteins. (B) Immunocytochemistry performed to verify prion protein expression in Zpl 2-1- PrP and Zpl 2-1-vector cells. PrP is immunolabeled by the monoclonal PrP antibody SAF 32 and an Alexa 568 conjugated secondary antibody (red), nucleus is stained with DAPI (cyanine blue) and merged image is shown in the last column. Pictures were recorded using a 60X oil immersion objective with no zooming. (C) Relative expressions of PrP from ZW 13–2 and Zpl 2-1-PrP (left panel). The expression of PrPC is quantified using densitometry analysis (right panel), the bars represent the average value obtained from three independent western blots and the error bars represent the standard deviations.
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pone.0139219.g003: Expression levels of PrP in the generated stable cell lines.(A) Total cell lysates of stably expressing cells made from Prnp−/− hippocampal neuronal cell line of Zürich I mice (Zpl 2–1) transfected with either the empty vector (Zpl 2-1-vector) or with mouse PrP gene (Zpl 2-1-PrP). Cell lysates were incubated in the absence (lane 1 and lane 3) or presence of PNGase F (lane 2 and lane 4). Western blot analysis was carried out with monoclonal PrP antibody SAF 32. β-actin was used to confirm equal loading of proteins. (B) Immunocytochemistry performed to verify prion protein expression in Zpl 2-1- PrP and Zpl 2-1-vector cells. PrP is immunolabeled by the monoclonal PrP antibody SAF 32 and an Alexa 568 conjugated secondary antibody (red), nucleus is stained with DAPI (cyanine blue) and merged image is shown in the last column. Pictures were recorded using a 60X oil immersion objective with no zooming. (C) Relative expressions of PrP from ZW 13–2 and Zpl 2-1-PrP (left panel). The expression of PrPC is quantified using densitometry analysis (right panel), the bars represent the average value obtained from three independent western blots and the error bars represent the standard deviations.

Mentions: The appropriate expression level and the proper processing of the prion protein were confirmed in the established cell populations using immunoblot and immunocytochemical analyses. Total cell lysates of Zpl 2-1-vector and Zpl 2-1-PrP hippocampal neuronal cells were collected and were either left untreated or were treated by PNGase F before immunoblotting with monoclonal PrP antibody SAF 32 (Fig 3A). The Zpl 2-1-vector cells, not expected to express PrPC, show no detectable bands for PrP (lane 3 and 4, Fig 3A), whereas, the Zpl 2-1-PrP cells, exhibit a well-detectable level of PrPC expression with proper N-glycosylation as judged by the bands in the PNGase treated and untreated samples (lanes 1 and 2, Fig 3A). Expression and correct localization of the prion protein was further confirmed by immunocytochemical analysis (Fig 3B). The bright red immunofluorescence-staining pattern in the Zpl 2-1-PrP cells revealed that the prion protein was distributed on the surface of the cells, whereas no immunoreactivity was detected in the Zpl 2-1-vector cells. The percentage of transformant cells was estimated based on the microscopy pictures using the counts of DAPI stained cells as the total cells, and the Alexa568 positive cells as counts of the transformant cells. We found that 90 (+/- 1.7) % of the Zpl 2-1-PrP cells express PrP, whereas, we found no positively stained cells of the vector-expressing cells. To compare the expression levels of PrPC in ZW 13–2 and Zpl 2-1-PrP cells, western blot analysis was performed (Fig 3C, left panel). The expression of PrPC was quantified using densitometry analysis of the bands with normalization to the band of β-actin as loading control (Fig 3C, right panel) and shows that PrPC expression is comparable in Zpl 2-1-PrP and ZW 13–2 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 generated stable cell lines.(A) Total cell lysates of stably expressing cells made from Prnp−/− hippocampal neuronal cell line of Zürich I mice (Zpl 2–1) transfected with either the empty vector (Zpl 2-1-vector) or with mouse PrP gene (Zpl 2-1-PrP). Cell lysates were incubated in the absence (lane 1 and lane 3) or presence of PNGase F (lane 2 and lane 4). Western blot analysis was carried out with monoclonal PrP antibody SAF 32. β-actin was used to confirm equal loading of proteins. (B) Immunocytochemistry performed to verify prion protein expression in Zpl 2-1- PrP and Zpl 2-1-vector cells. PrP is immunolabeled by the monoclonal PrP antibody SAF 32 and an Alexa 568 conjugated secondary antibody (red), nucleus is stained with DAPI (cyanine blue) and merged image is shown in the last column. Pictures were recorded using a 60X oil immersion objective with no zooming. (C) Relative expressions of PrP from ZW 13–2 and Zpl 2-1-PrP (left panel). The expression of PrPC is quantified using densitometry analysis (right panel), the bars represent the average value obtained from three independent western blots and the error bars represent the standard deviations.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591282&req=5

pone.0139219.g003: Expression levels of PrP in the generated stable cell lines.(A) Total cell lysates of stably expressing cells made from Prnp−/− hippocampal neuronal cell line of Zürich I mice (Zpl 2–1) transfected with either the empty vector (Zpl 2-1-vector) or with mouse PrP gene (Zpl 2-1-PrP). Cell lysates were incubated in the absence (lane 1 and lane 3) or presence of PNGase F (lane 2 and lane 4). Western blot analysis was carried out with monoclonal PrP antibody SAF 32. β-actin was used to confirm equal loading of proteins. (B) Immunocytochemistry performed to verify prion protein expression in Zpl 2-1- PrP and Zpl 2-1-vector cells. PrP is immunolabeled by the monoclonal PrP antibody SAF 32 and an Alexa 568 conjugated secondary antibody (red), nucleus is stained with DAPI (cyanine blue) and merged image is shown in the last column. Pictures were recorded using a 60X oil immersion objective with no zooming. (C) Relative expressions of PrP from ZW 13–2 and Zpl 2-1-PrP (left panel). The expression of PrPC is quantified using densitometry analysis (right panel), the bars represent the average value obtained from three independent western blots and the error bars represent the standard deviations.
Mentions: The appropriate expression level and the proper processing of the prion protein were confirmed in the established cell populations using immunoblot and immunocytochemical analyses. Total cell lysates of Zpl 2-1-vector and Zpl 2-1-PrP hippocampal neuronal cells were collected and were either left untreated or were treated by PNGase F before immunoblotting with monoclonal PrP antibody SAF 32 (Fig 3A). The Zpl 2-1-vector cells, not expected to express PrPC, show no detectable bands for PrP (lane 3 and 4, Fig 3A), whereas, the Zpl 2-1-PrP cells, exhibit a well-detectable level of PrPC expression with proper N-glycosylation as judged by the bands in the PNGase treated and untreated samples (lanes 1 and 2, Fig 3A). Expression and correct localization of the prion protein was further confirmed by immunocytochemical analysis (Fig 3B). The bright red immunofluorescence-staining pattern in the Zpl 2-1-PrP cells revealed that the prion protein was distributed on the surface of the cells, whereas no immunoreactivity was detected in the Zpl 2-1-vector cells. The percentage of transformant cells was estimated based on the microscopy pictures using the counts of DAPI stained cells as the total cells, and the Alexa568 positive cells as counts of the transformant cells. We found that 90 (+/- 1.7) % of the Zpl 2-1-PrP cells express PrP, whereas, we found no positively stained cells of the vector-expressing cells. To compare the expression levels of PrPC in ZW 13–2 and Zpl 2-1-PrP cells, western blot analysis was performed (Fig 3C, left panel). The expression of PrPC was quantified using densitometry analysis of the bands with normalization to the band of β-actin as loading control (Fig 3C, right panel) and shows that PrPC expression is comparable in Zpl 2-1-PrP and ZW 13–2 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