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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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Neurons synthesizing SP-PrP are more resistant to proteasome inhibitors.Cerebellar granule neurons (CGN), cortical (Cx) and hippocampal (Hipp) neurons prepared from C57BL/6J (Prnp+/+) and PrP knockout (Prnp0/0) mice were exposed to 1 μM epoxomicin (A), 100 μM ALLN (B) or 5 μM MG132 (C). Cell survival was quantified after 24 h by MTT assay and expressed as a percentage of the values for cells treated with the vehicle. Data are the mean ± SEM of 12–24 replicates from 2–4 independent experiments; **p<0.01 by Tukey-Kramer test. (D) Cortical neurons were prepared from Prnp0/0 (PrP level: 0X), Prnp+/+ (PrP level: 1X), and Tg(WT-E1)+/−/Prnp+/0 (PrP level: ∼2.5X) littermates obtained by crossing Tg(WT-E1)+/−/Prnp+/0 and Prnp+/0 mice. Cell viability was analyzed after 24-h treatment with 5 μM MG132 and expressed as a percentage of the values for cells treated with the vehicle. Data are the mean ± SEM of 8-23 replicates from 2 independent experiments; *p<0.05, ***p<0.001 by Dunnett's test.
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pone-0013725-g004: Neurons synthesizing SP-PrP are more resistant to proteasome inhibitors.Cerebellar granule neurons (CGN), cortical (Cx) and hippocampal (Hipp) neurons prepared from C57BL/6J (Prnp+/+) and PrP knockout (Prnp0/0) mice were exposed to 1 μM epoxomicin (A), 100 μM ALLN (B) or 5 μM MG132 (C). Cell survival was quantified after 24 h by MTT assay and expressed as a percentage of the values for cells treated with the vehicle. Data are the mean ± SEM of 12–24 replicates from 2–4 independent experiments; **p<0.01 by Tukey-Kramer test. (D) Cortical neurons were prepared from Prnp0/0 (PrP level: 0X), Prnp+/+ (PrP level: 1X), and Tg(WT-E1)+/−/Prnp+/0 (PrP level: ∼2.5X) littermates obtained by crossing Tg(WT-E1)+/−/Prnp+/0 and Prnp+/0 mice. Cell viability was analyzed after 24-h treatment with 5 μM MG132 and expressed as a percentage of the values for cells treated with the vehicle. Data are the mean ± SEM of 8-23 replicates from 2 independent experiments; *p<0.05, ***p<0.001 by Dunnett's test.

Mentions: To test the effect of SP-PrP on the viability of cultured neurons we used a previously described experimental paradigm [11], [15]. Neurons cultured from PrP knockout (Prnp0/0) and wild-type (Prnp+/+) mice were exposed to proteasome inhibitors and their viability was evaluated after 24 h. There was no difference in viability between Prnp0/0 and Prnp+/+ CGN; in contrast, cortical and hippocampal neurons from Prnp+/+ mice were significantly more resistant to the inhibitors than their Prnp0/0 counterparts (Fig. 4A–C). Supraphysiological PrP expression further increased cortical cell resistance to the inhibitors, indicating a dose-dependent effect of PrP expression on neuronal survival (Fig. 4D). The fact that the cells that survived best were those synthesizing SP-PrP suggested that this isoform could have cytoprotective activity.


Cell type-specific neuroprotective activity of untranslocated prion protein.

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

Neurons synthesizing SP-PrP are more resistant to proteasome inhibitors.Cerebellar granule neurons (CGN), cortical (Cx) and hippocampal (Hipp) neurons prepared from C57BL/6J (Prnp+/+) and PrP knockout (Prnp0/0) mice were exposed to 1 μM epoxomicin (A), 100 μM ALLN (B) or 5 μM MG132 (C). Cell survival was quantified after 24 h by MTT assay and expressed as a percentage of the values for cells treated with the vehicle. Data are the mean ± SEM of 12–24 replicates from 2–4 independent experiments; **p<0.01 by Tukey-Kramer test. (D) Cortical neurons were prepared from Prnp0/0 (PrP level: 0X), Prnp+/+ (PrP level: 1X), and Tg(WT-E1)+/−/Prnp+/0 (PrP level: ∼2.5X) littermates obtained by crossing Tg(WT-E1)+/−/Prnp+/0 and Prnp+/0 mice. Cell viability was analyzed after 24-h treatment with 5 μM MG132 and expressed as a percentage of the values for cells treated with the vehicle. Data are the mean ± SEM of 8-23 replicates from 2 independent experiments; *p<0.05, ***p<0.001 by Dunnett's test.
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Related In: Results  -  Collection

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pone-0013725-g004: Neurons synthesizing SP-PrP are more resistant to proteasome inhibitors.Cerebellar granule neurons (CGN), cortical (Cx) and hippocampal (Hipp) neurons prepared from C57BL/6J (Prnp+/+) and PrP knockout (Prnp0/0) mice were exposed to 1 μM epoxomicin (A), 100 μM ALLN (B) or 5 μM MG132 (C). Cell survival was quantified after 24 h by MTT assay and expressed as a percentage of the values for cells treated with the vehicle. Data are the mean ± SEM of 12–24 replicates from 2–4 independent experiments; **p<0.01 by Tukey-Kramer test. (D) Cortical neurons were prepared from Prnp0/0 (PrP level: 0X), Prnp+/+ (PrP level: 1X), and Tg(WT-E1)+/−/Prnp+/0 (PrP level: ∼2.5X) littermates obtained by crossing Tg(WT-E1)+/−/Prnp+/0 and Prnp+/0 mice. Cell viability was analyzed after 24-h treatment with 5 μM MG132 and expressed as a percentage of the values for cells treated with the vehicle. Data are the mean ± SEM of 8-23 replicates from 2 independent experiments; *p<0.05, ***p<0.001 by Dunnett's test.
Mentions: To test the effect of SP-PrP on the viability of cultured neurons we used a previously described experimental paradigm [11], [15]. Neurons cultured from PrP knockout (Prnp0/0) and wild-type (Prnp+/+) mice were exposed to proteasome inhibitors and their viability was evaluated after 24 h. There was no difference in viability between Prnp0/0 and Prnp+/+ CGN; in contrast, cortical and hippocampal neurons from Prnp+/+ mice were significantly more resistant to the inhibitors than their Prnp0/0 counterparts (Fig. 4A–C). Supraphysiological PrP expression further increased cortical cell resistance to the inhibitors, indicating a dose-dependent effect of PrP expression on neuronal survival (Fig. 4D). The fact that the cells that survived best were those synthesizing SP-PrP suggested that this isoform could have cytoprotective activity.

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