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Cell type-specific neuroprotective activity of untranslocated prion protein.

Restelli E, Fioriti L, Mantovani S, Airaghi S, Forloni G, Chiesa R - PLoS ONE (2010)

Bottom Line: However, it is not clear how cytosolic PrP localization influences neuronal viability, with either cytotoxic or anti-apoptotic effects reported in different studies.Untranslocated PrP boosted the resistance of cortical and hippocampal neurons to apoptotic insults but had no effect on cerebellar cells.These results indicate cell type-dependent differences in the efficiency of PrP translocation, and argue that cytosolic PrP targeting might serve a physiological neuroprotective function.

View Article: PubMed Central - PubMed

Affiliation: Dulbecco Telethon Institute, Milan, Italy.

ABSTRACT

Background: A key pathogenic role in prion diseases was proposed for a cytosolic form of the prion protein (PrP). However, it is not clear how cytosolic PrP localization influences neuronal viability, with either cytotoxic or anti-apoptotic effects reported in different studies. The cellular mechanism by which PrP is delivered to the cytosol of neurons is also debated, and either retrograde transport from the endoplasmic reticulum or inefficient translocation during biosynthesis has been proposed. We investigated cytosolic PrP biogenesis and effect on cell viability in primary neuronal cultures from different mouse brain regions.

Principal findings: Mild proteasome inhibition induced accumulation of an untranslocated form of cytosolic PrP in cortical and hippocampal cells, but not in cerebellar granules. A cyclopeptolide that interferes with the correct insertion of the PrP signal sequence into the translocon increased the amount of untranslocated PrP in cortical and hippocampal cells, and induced its synthesis in cerebellar neurons. Untranslocated PrP boosted the resistance of cortical and hippocampal neurons to apoptotic insults but had no effect on cerebellar cells.

Significance: These results indicate cell type-dependent differences in the efficiency of PrP translocation, and argue that cytosolic PrP targeting might serve a physiological neuroprotective function.

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Related in: MedlinePlus

Untranslocated PrP shows cytosolic localization.Hippocampal neurons from C57BL/6J mice were transfected with a plasmid encoding PrP-EGFP (green). Twelve days after transfection cells were exposed to the vehicle (A) or treated with 10 μM CAM741 for 24 h plus 5 μM MG132 during the last 6 h (B–D). Cells were then fixed and reacted with DAPI (blue) to stain the nuclei. Cells in C and D were also immunostained with an anti-PDI or anti-golgin antibody (red) to visualize the ER and Golgi, respectively. Scale bar  = 10 μm in A (also applicable to B), and 5 μm in C (also applicable to D).
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pone-0013725-g003: Untranslocated PrP shows cytosolic localization.Hippocampal neurons from C57BL/6J mice were transfected with a plasmid encoding PrP-EGFP (green). Twelve days after transfection cells were exposed to the vehicle (A) or treated with 10 μM CAM741 for 24 h plus 5 μM MG132 during the last 6 h (B–D). Cells were then fixed and reacted with DAPI (blue) to stain the nuclei. Cells in C and D were also immunostained with an anti-PDI or anti-golgin antibody (red) to visualize the ER and Golgi, respectively. Scale bar  = 10 μm in A (also applicable to B), and 5 μm in C (also applicable to D).

Mentions: To determine the cellular localization of SP-PrP, we transfected hippocampal neurons with a plasmid encoding a PrP-enhanced green fluorescent protein (PrP-EGFP) fusion molecule [33], and induced robust synthesis of untranslocated PrP-EGFP by treating the cells with an inhibitor of PrP translocation (see below). We imaged PrP-EGFP in fixed, DAPI-stained cells by confocal microscopy to visualize its localization in relation to the nucleus. Consistent with previous immunolocalization of a non-fluorescent version of PrP in cultured neurons [15], PrP-EGFP distributed on the cell soma and along the neurites of untreated cells (Fig. 3A). There was also a fraction in intracellular compartments that co-localized with the ER and Golgi (not shown), as expected for proteins in transit towards the cell surface. In treated neurons, PrP-EGFP showed a fine punctate cytoplasmic fluorescence (Fig. 3B–D), the majority of which did not co-localize with ER or Golgi markers (Fig. 3C and D, respectively).


Cell type-specific neuroprotective activity of untranslocated prion protein.

Restelli E, Fioriti L, Mantovani S, Airaghi S, Forloni G, Chiesa R - PLoS ONE (2010)

Untranslocated PrP shows cytosolic localization.Hippocampal neurons from C57BL/6J mice were transfected with a plasmid encoding PrP-EGFP (green). Twelve days after transfection cells were exposed to the vehicle (A) or treated with 10 μM CAM741 for 24 h plus 5 μM MG132 during the last 6 h (B–D). Cells were then fixed and reacted with DAPI (blue) to stain the nuclei. Cells in C and D were also immunostained with an anti-PDI or anti-golgin antibody (red) to visualize the ER and Golgi, respectively. Scale bar  = 10 μm in A (also applicable to B), and 5 μm in C (also applicable to D).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2965675&req=5

pone-0013725-g003: Untranslocated PrP shows cytosolic localization.Hippocampal neurons from C57BL/6J mice were transfected with a plasmid encoding PrP-EGFP (green). Twelve days after transfection cells were exposed to the vehicle (A) or treated with 10 μM CAM741 for 24 h plus 5 μM MG132 during the last 6 h (B–D). Cells were then fixed and reacted with DAPI (blue) to stain the nuclei. Cells in C and D were also immunostained with an anti-PDI or anti-golgin antibody (red) to visualize the ER and Golgi, respectively. Scale bar  = 10 μm in A (also applicable to B), and 5 μm in C (also applicable to D).
Mentions: To determine the cellular localization of SP-PrP, we transfected hippocampal neurons with a plasmid encoding a PrP-enhanced green fluorescent protein (PrP-EGFP) fusion molecule [33], and induced robust synthesis of untranslocated PrP-EGFP by treating the cells with an inhibitor of PrP translocation (see below). We imaged PrP-EGFP in fixed, DAPI-stained cells by confocal microscopy to visualize its localization in relation to the nucleus. Consistent with previous immunolocalization of a non-fluorescent version of PrP in cultured neurons [15], PrP-EGFP distributed on the cell soma and along the neurites of untreated cells (Fig. 3A). There was also a fraction in intracellular compartments that co-localized with the ER and Golgi (not shown), as expected for proteins in transit towards the cell surface. In treated neurons, PrP-EGFP showed a fine punctate cytoplasmic fluorescence (Fig. 3B–D), the majority of which did not co-localize with ER or Golgi markers (Fig. 3C and D, respectively).

Bottom Line: However, it is not clear how cytosolic PrP localization influences neuronal viability, with either cytotoxic or anti-apoptotic effects reported in different studies.Untranslocated PrP boosted the resistance of cortical and hippocampal neurons to apoptotic insults but had no effect on cerebellar cells.These results indicate cell type-dependent differences in the efficiency of PrP translocation, and argue that cytosolic PrP targeting might serve a physiological neuroprotective function.

View Article: PubMed Central - PubMed

Affiliation: Dulbecco Telethon Institute, Milan, Italy.

ABSTRACT

Background: A key pathogenic role in prion diseases was proposed for a cytosolic form of the prion protein (PrP). However, it is not clear how cytosolic PrP localization influences neuronal viability, with either cytotoxic or anti-apoptotic effects reported in different studies. The cellular mechanism by which PrP is delivered to the cytosol of neurons is also debated, and either retrograde transport from the endoplasmic reticulum or inefficient translocation during biosynthesis has been proposed. We investigated cytosolic PrP biogenesis and effect on cell viability in primary neuronal cultures from different mouse brain regions.

Principal findings: Mild proteasome inhibition induced accumulation of an untranslocated form of cytosolic PrP in cortical and hippocampal cells, but not in cerebellar granules. A cyclopeptolide that interferes with the correct insertion of the PrP signal sequence into the translocon increased the amount of untranslocated PrP in cortical and hippocampal cells, and induced its synthesis in cerebellar neurons. Untranslocated PrP boosted the resistance of cortical and hippocampal neurons to apoptotic insults but had no effect on cerebellar cells.

Significance: These results indicate cell type-dependent differences in the efficiency of PrP translocation, and argue that cytosolic PrP targeting might serve a physiological neuroprotective function.

Show MeSH
Related in: MedlinePlus