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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

SP-PrP has no effect on viability of cerebellar granule neurons.(A) Hippocampal neurons from C57BL/6J mice were treated with 5 μM MG132 alone or with 10 μM CAM741 for 18 h, and PrP was analyzed by Western blot as described in the legend to Fig. 1. Note the higher level of SP-PrP in the presence of CAM741 (compare lanes 4 and 6). (B) Cerebellar granule neurons from C57BL/6J mice were treated with 10 μM CAM741, 10 μM MG132 or with the two drugs simultaneously for 24 h, before Western blot analysis with antibody 12B2. The PrP band in lane 8 also reacted with the α-SP and α-GP antibodies (on the right). (C) Cell survival was quantified by MTT assay and expressed as a percentage of the values for cells treated with the vehicle. Data are the mean ± SEM of 18–20 replicates from two independent experiments. The Bonferroni test did not find any difference between MG132 and CAM+MG groups.
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pone-0013725-g006: SP-PrP has no effect on viability of cerebellar granule neurons.(A) Hippocampal neurons from C57BL/6J mice were treated with 5 μM MG132 alone or with 10 μM CAM741 for 18 h, and PrP was analyzed by Western blot as described in the legend to Fig. 1. Note the higher level of SP-PrP in the presence of CAM741 (compare lanes 4 and 6). (B) Cerebellar granule neurons from C57BL/6J mice were treated with 10 μM CAM741, 10 μM MG132 or with the two drugs simultaneously for 24 h, before Western blot analysis with antibody 12B2. The PrP band in lane 8 also reacted with the α-SP and α-GP antibodies (on the right). (C) Cell survival was quantified by MTT assay and expressed as a percentage of the values for cells treated with the vehicle. Data are the mean ± SEM of 18–20 replicates from two independent experiments. The Bonferroni test did not find any difference between MG132 and CAM+MG groups.

Mentions: Transgenic mice engineered to express PrP in the cytosol show massive degeneration of CGN, suggesting that cytosolic PrP may be selectively toxic to these cells [11]. To test this we sought ways to induce cytosolic PrP localization in cultured CGN. CAM741, a cyclopeptolide that inhibits co-translational translocation by interfering with the correct insertion of the signal peptide into the translocon [36], [37], increased the amount of SP-PrP in hippocampal and cortical neurons (Fig. 6A, compare lanes 4 and 6, and data not shown). CAM741 induced an unglycosylated PrP species in CGN (Fig. 6B, lanes 7 and 8), which was confirmed identical to SP-PrP by reactivity with the α-SP antibody and an antibody directed against the C-terminal GPI-anchoring signal (α-GP) [17] (Fig. 6B, right). When we analyzed how SP-PrP accumulation affected CGN viability, we found no significant difference between cells treated with MG132 alone or in combination with CAM741 (Fig. 6C), even though the latter accumulated SP-PrP (Fig. 6B, lanes 7 and 8).


Cell type-specific neuroprotective activity of untranslocated prion protein.

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

SP-PrP has no effect on viability of cerebellar granule neurons.(A) Hippocampal neurons from C57BL/6J mice were treated with 5 μM MG132 alone or with 10 μM CAM741 for 18 h, and PrP was analyzed by Western blot as described in the legend to Fig. 1. Note the higher level of SP-PrP in the presence of CAM741 (compare lanes 4 and 6). (B) Cerebellar granule neurons from C57BL/6J mice were treated with 10 μM CAM741, 10 μM MG132 or with the two drugs simultaneously for 24 h, before Western blot analysis with antibody 12B2. The PrP band in lane 8 also reacted with the α-SP and α-GP antibodies (on the right). (C) Cell survival was quantified by MTT assay and expressed as a percentage of the values for cells treated with the vehicle. Data are the mean ± SEM of 18–20 replicates from two independent experiments. The Bonferroni test did not find any difference between MG132 and CAM+MG groups.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2965675&req=5

pone-0013725-g006: SP-PrP has no effect on viability of cerebellar granule neurons.(A) Hippocampal neurons from C57BL/6J mice were treated with 5 μM MG132 alone or with 10 μM CAM741 for 18 h, and PrP was analyzed by Western blot as described in the legend to Fig. 1. Note the higher level of SP-PrP in the presence of CAM741 (compare lanes 4 and 6). (B) Cerebellar granule neurons from C57BL/6J mice were treated with 10 μM CAM741, 10 μM MG132 or with the two drugs simultaneously for 24 h, before Western blot analysis with antibody 12B2. The PrP band in lane 8 also reacted with the α-SP and α-GP antibodies (on the right). (C) Cell survival was quantified by MTT assay and expressed as a percentage of the values for cells treated with the vehicle. Data are the mean ± SEM of 18–20 replicates from two independent experiments. The Bonferroni test did not find any difference between MG132 and CAM+MG groups.
Mentions: Transgenic mice engineered to express PrP in the cytosol show massive degeneration of CGN, suggesting that cytosolic PrP may be selectively toxic to these cells [11]. To test this we sought ways to induce cytosolic PrP localization in cultured CGN. CAM741, a cyclopeptolide that inhibits co-translational translocation by interfering with the correct insertion of the signal peptide into the translocon [36], [37], increased the amount of SP-PrP in hippocampal and cortical neurons (Fig. 6A, compare lanes 4 and 6, and data not shown). CAM741 induced an unglycosylated PrP species in CGN (Fig. 6B, lanes 7 and 8), which was confirmed identical to SP-PrP by reactivity with the α-SP antibody and an antibody directed against the C-terminal GPI-anchoring signal (α-GP) [17] (Fig. 6B, right). When we analyzed how SP-PrP accumulation affected CGN viability, we found no significant difference between cells treated with MG132 alone or in combination with CAM741 (Fig. 6C), even though the latter accumulated SP-PrP (Fig. 6B, lanes 7 and 8).

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