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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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Proteasome inhibitors induce accumulation of insoluble, untranslocated PrP in primary neurons.(A) Cerebellar granule neurons (CGN), cortical (Cx) and hippocampal (Hipp) neurons from C57BL/6J mice were treated for 24h with 5 μM epoxomicin (Epoxo), 100 μM ALLN, 5 μM MG132, or the vehicle alone (CT). Cell lysates were centrifuged at 186,000 x g for 40 min, and PrP in the supernatants (S) and pellets (P) was visualized by immunoblotting with antibody P45-66. The asterisks and arrows indicate bands corresponding to untranslocated PrP and mature, unglycosylated PrP, respectively. (B) Cortical neurons from Tg(WT-E1) mice were exposed to 5 μM MG132, epoxomicin (Epoxo), lactacystin β-lactone (β-Lac) or the vehicle alone (CT). After 24 hours, cells were lysed and centrifuged at 186,000 x g for 40 min. PrP was visualized by immunoblotting with antibody 3F4 (upper panel), and with an antibody against the N-terminal signal peptide (α-SP) (lower panel). Molecular mass markers are in kilodaltons.
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pone-0013725-g001: Proteasome inhibitors induce accumulation of insoluble, untranslocated PrP in primary neurons.(A) Cerebellar granule neurons (CGN), cortical (Cx) and hippocampal (Hipp) neurons from C57BL/6J mice were treated for 24h with 5 μM epoxomicin (Epoxo), 100 μM ALLN, 5 μM MG132, or the vehicle alone (CT). Cell lysates were centrifuged at 186,000 x g for 40 min, and PrP in the supernatants (S) and pellets (P) was visualized by immunoblotting with antibody P45-66. The asterisks and arrows indicate bands corresponding to untranslocated PrP and mature, unglycosylated PrP, respectively. (B) Cortical neurons from Tg(WT-E1) mice were exposed to 5 μM MG132, epoxomicin (Epoxo), lactacystin β-lactone (β-Lac) or the vehicle alone (CT). After 24 hours, cells were lysed and centrifuged at 186,000 x g for 40 min. PrP was visualized by immunoblotting with antibody 3F4 (upper panel), and with an antibody against the N-terminal signal peptide (α-SP) (lower panel). Molecular mass markers are in kilodaltons.

Mentions: We investigated whether cytosolic PrP was detectable in primary neurons cultured from the neocortex, hippocampus and cerebellum of newborn mice. Because cytosolic PrP is rapidly degraded by the proteasomes [14], [15], cells were treated with a panel of different proteasome inhibitors. Lactacystin-β-lactone, MG132 (Z-Leu-Leu-Leu-al), ALLN (Ac-Leu-Leu-NorLeu-al) or epoxomycin caused accumulation of an insoluble form of PrP of approximately 27 kDa in cortical and hippocampal neurons (Fig. 1A, middle and bottom panels, and Fig. 1B top panel). This form had a larger molecular mass then mature, unglycosylated PrP in the soluble fractions, and was recognized by an antibody (α-SP), which selectively reacts with the N-terminal signal peptide of PrP [30] (Fig. 1B, lower panel), indicating that it corresponded to the untranslocated form of cytosolic PrP previously described in transfected cells (hereafter referred to as SP-PrP) [14], [15], [16], [17]. In cortical and hippocampal cells SP-PrP was first detected after 2 h treatment, and reached a maximum within 8 h (data not shown) which, based on quantitative evaluation of Western blots, corresponded to approximately 10% of total PrP. Consistent with previous findings [14], [15], SP-PrP was not detected in proteasome inhibitor-treated CGN (Fig. 1A, top panel). PrP levels were similar in the different neuronal cultures, ruling out that the failure to detect SP-PrP in CGN was due to lower PrP expression.


Cell type-specific neuroprotective activity of untranslocated prion protein.

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

Proteasome inhibitors induce accumulation of insoluble, untranslocated PrP in primary neurons.(A) Cerebellar granule neurons (CGN), cortical (Cx) and hippocampal (Hipp) neurons from C57BL/6J mice were treated for 24h with 5 μM epoxomicin (Epoxo), 100 μM ALLN, 5 μM MG132, or the vehicle alone (CT). Cell lysates were centrifuged at 186,000 x g for 40 min, and PrP in the supernatants (S) and pellets (P) was visualized by immunoblotting with antibody P45-66. The asterisks and arrows indicate bands corresponding to untranslocated PrP and mature, unglycosylated PrP, respectively. (B) Cortical neurons from Tg(WT-E1) mice were exposed to 5 μM MG132, epoxomicin (Epoxo), lactacystin β-lactone (β-Lac) or the vehicle alone (CT). After 24 hours, cells were lysed and centrifuged at 186,000 x g for 40 min. PrP was visualized by immunoblotting with antibody 3F4 (upper panel), and with an antibody against the N-terminal signal peptide (α-SP) (lower panel). Molecular mass markers are in kilodaltons.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2965675&req=5

pone-0013725-g001: Proteasome inhibitors induce accumulation of insoluble, untranslocated PrP in primary neurons.(A) Cerebellar granule neurons (CGN), cortical (Cx) and hippocampal (Hipp) neurons from C57BL/6J mice were treated for 24h with 5 μM epoxomicin (Epoxo), 100 μM ALLN, 5 μM MG132, or the vehicle alone (CT). Cell lysates were centrifuged at 186,000 x g for 40 min, and PrP in the supernatants (S) and pellets (P) was visualized by immunoblotting with antibody P45-66. The asterisks and arrows indicate bands corresponding to untranslocated PrP and mature, unglycosylated PrP, respectively. (B) Cortical neurons from Tg(WT-E1) mice were exposed to 5 μM MG132, epoxomicin (Epoxo), lactacystin β-lactone (β-Lac) or the vehicle alone (CT). After 24 hours, cells were lysed and centrifuged at 186,000 x g for 40 min. PrP was visualized by immunoblotting with antibody 3F4 (upper panel), and with an antibody against the N-terminal signal peptide (α-SP) (lower panel). Molecular mass markers are in kilodaltons.
Mentions: We investigated whether cytosolic PrP was detectable in primary neurons cultured from the neocortex, hippocampus and cerebellum of newborn mice. Because cytosolic PrP is rapidly degraded by the proteasomes [14], [15], cells were treated with a panel of different proteasome inhibitors. Lactacystin-β-lactone, MG132 (Z-Leu-Leu-Leu-al), ALLN (Ac-Leu-Leu-NorLeu-al) or epoxomycin caused accumulation of an insoluble form of PrP of approximately 27 kDa in cortical and hippocampal neurons (Fig. 1A, middle and bottom panels, and Fig. 1B top panel). This form had a larger molecular mass then mature, unglycosylated PrP in the soluble fractions, and was recognized by an antibody (α-SP), which selectively reacts with the N-terminal signal peptide of PrP [30] (Fig. 1B, lower panel), indicating that it corresponded to the untranslocated form of cytosolic PrP previously described in transfected cells (hereafter referred to as SP-PrP) [14], [15], [16], [17]. In cortical and hippocampal cells SP-PrP was first detected after 2 h treatment, and reached a maximum within 8 h (data not shown) which, based on quantitative evaluation of Western blots, corresponded to approximately 10% of total PrP. Consistent with previous findings [14], [15], SP-PrP was not detected in proteasome inhibitor-treated CGN (Fig. 1A, top panel). PrP levels were similar in the different neuronal cultures, ruling out that the failure to detect SP-PrP in CGN was due to lower PrP expression.

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