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Increased endoplasmic reticulum stress and decreased proteasomal function in lafora disease models lacking the phosphatase laforin.

Vernia S, Rubio T, Heredia M, Rodríguez de Córdoba S, Sanz P - PLoS ONE (2009)

Bottom Line: Here, we demonstrate that siRNA silencing of laforin in Hek293 and SH-SY5Y cells increases their sensitivity to agents triggering ER-stress, which correlates with impairment of the ubiquitin-proteasomal pathway and increased apoptosis.We demonstrate that, in addition to regulating glycogen synthesis, laforin and malin play a role protecting cells from ER-stress, likely contributing to the elimination of unfolded proteins.These data suggest that proteasomal dysfunction and ER-stress play an important role in the pathogenesis of LD, which may offer novel therapeutic approaches for this fatal neurodegenerative disorder.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biomedicina de Valencia, CSIC and CIBER de Enfermedades Raras, Valencia, Spain.

ABSTRACT

Background: Lafora progressive myoclonus epilepsy (Lafora disease; LD) is a fatal autosomal recessive neurodegenerative disorder caused by loss-of-function mutations in either the EPM2A gene, encoding the dual specificity phosphatase laforin, or the EPM2B gene, encoding the E3-ubiquitin ligase malin. Previously, we and others have shown that both proteins form a functional complex that regulates glycogen synthesis by a novel mechanism involving ubiquitination and proteasomal degradation of at least two proteins, glycogen synthase and R5/PTG. Since laforin and malin localized at the endoplasmic reticulum (ER) and their regulatory role likely extend to other proteins unrelated to glycogen metabolism, we postulated that their absence may also affect the ER-unfolded protein response pathway.

Methodology/principal findings: Here, we demonstrate that siRNA silencing of laforin in Hek293 and SH-SY5Y cells increases their sensitivity to agents triggering ER-stress, which correlates with impairment of the ubiquitin-proteasomal pathway and increased apoptosis. Consistent with these findings, analysis of tissue samples from a LD patient lacking laforin, and from a laforin knockout (Epm2a-/-) mouse model of LD, demonstrates constitutive high expression levels of ER-stress markers BIP/Grp78, CHOP and PDI, among others.

Conclusions/significance: We demonstrate that, in addition to regulating glycogen synthesis, laforin and malin play a role protecting cells from ER-stress, likely contributing to the elimination of unfolded proteins. These data suggest that proteasomal dysfunction and ER-stress play an important role in the pathogenesis of LD, which may offer novel therapeutic approaches for this fatal neurodegenerative disorder.

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Laforin deletion results in increased ER stress in the liver of Epm2a-/- mice.A and B) Western blot analyses of laforin and ER-stress markers (BIP/Grp78, CHOP, SOD2) in liver (A) and whole brain (B) biopsies of two Epm2a+/+ (A and B) and two Epm2a-/- mice (1 and 2). A representative blot of four different animals of each type is shown. Right panels show normalized intensities (mean±SEM; n: 4) of different markers expressed as a percentage with respect to control mice. Tubulin was used as a loading control. * In brain samples, a band of 94 kDa is recognized by the anti-BIP/Grp78 antibody instead of the corresponding 78 kDa band.
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pone-0005907-g003: Laforin deletion results in increased ER stress in the liver of Epm2a-/- mice.A and B) Western blot analyses of laforin and ER-stress markers (BIP/Grp78, CHOP, SOD2) in liver (A) and whole brain (B) biopsies of two Epm2a+/+ (A and B) and two Epm2a-/- mice (1 and 2). A representative blot of four different animals of each type is shown. Right panels show normalized intensities (mean±SEM; n: 4) of different markers expressed as a percentage with respect to control mice. Tubulin was used as a loading control. * In brain samples, a band of 94 kDa is recognized by the anti-BIP/Grp78 antibody instead of the corresponding 78 kDa band.

Mentions: To confirm the results obtained with the cell line models (see above) in laforin knockout (Epm2a-/-) mice, several markers of ER-stress were analyzed in extracts from different tissues of 9-month old mice. As expected RT-PCR analysis indicated that the Epm2a-/- mice, kindly provided by Dr. Delgado-Escueta, lacked laforin expression because of the described deletion of the EPM2A gene [21] (not shown), and antibodies against laforin were unable to detect any band in crude extracts from different tissues of these animals (Fig. 3). In agreement with data obtained with Hek293 and SH-SY5Y cells, extracts from the liver of Epm2a-/- mice contained higher levels of BIP/Grp78 and CHOP compared to age and sex matched C57BL6 control mice (Fig. 3A). In addition, we also detected higher levels of SOD2 (superoxide dismutase 2), another ER-stress marker related to oxidative stress (Fig. 3A). These data indicated that the liver of Epm2a-/- animals, lacking laforin, was under permanent conditions of ER-stress. In contrast, analysis of whole brain extracts from these animals failed to detect differences in the levels of the ER-stress markers between Epm2a-/- and control animals (Fig. 3B). We also measured the activity of the proteasome in mouse liver and whole brain extracts. As shown in Fig. 4A, we observed a significant decrease in the activity of the proteasome in liver extracts from Epm2a-/- mice in comparison to liver extracts from control mice. However, we were not able to observe differences in the activity of the proteasome in whole brain extracts, which correlated with the absence of differences in the amount of ER-stress markers in extracts from this tissue (see above). Western blot analyses of two different subunits of the 26S proteasome complex in these liver samples indicated that the observed changes in the activity of the proteasome were not due to differences in proteasome levels (Fig. 4B). These results supported the idea that loss of laforin correlated with induction of ER-stress markers and impaired proteasomal function, at least in mouse liver.


Increased endoplasmic reticulum stress and decreased proteasomal function in lafora disease models lacking the phosphatase laforin.

Vernia S, Rubio T, Heredia M, Rodríguez de Córdoba S, Sanz P - PLoS ONE (2009)

Laforin deletion results in increased ER stress in the liver of Epm2a-/- mice.A and B) Western blot analyses of laforin and ER-stress markers (BIP/Grp78, CHOP, SOD2) in liver (A) and whole brain (B) biopsies of two Epm2a+/+ (A and B) and two Epm2a-/- mice (1 and 2). A representative blot of four different animals of each type is shown. Right panels show normalized intensities (mean±SEM; n: 4) of different markers expressed as a percentage with respect to control mice. Tubulin was used as a loading control. * In brain samples, a band of 94 kDa is recognized by the anti-BIP/Grp78 antibody instead of the corresponding 78 kDa band.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2692001&req=5

pone-0005907-g003: Laforin deletion results in increased ER stress in the liver of Epm2a-/- mice.A and B) Western blot analyses of laforin and ER-stress markers (BIP/Grp78, CHOP, SOD2) in liver (A) and whole brain (B) biopsies of two Epm2a+/+ (A and B) and two Epm2a-/- mice (1 and 2). A representative blot of four different animals of each type is shown. Right panels show normalized intensities (mean±SEM; n: 4) of different markers expressed as a percentage with respect to control mice. Tubulin was used as a loading control. * In brain samples, a band of 94 kDa is recognized by the anti-BIP/Grp78 antibody instead of the corresponding 78 kDa band.
Mentions: To confirm the results obtained with the cell line models (see above) in laforin knockout (Epm2a-/-) mice, several markers of ER-stress were analyzed in extracts from different tissues of 9-month old mice. As expected RT-PCR analysis indicated that the Epm2a-/- mice, kindly provided by Dr. Delgado-Escueta, lacked laforin expression because of the described deletion of the EPM2A gene [21] (not shown), and antibodies against laforin were unable to detect any band in crude extracts from different tissues of these animals (Fig. 3). In agreement with data obtained with Hek293 and SH-SY5Y cells, extracts from the liver of Epm2a-/- mice contained higher levels of BIP/Grp78 and CHOP compared to age and sex matched C57BL6 control mice (Fig. 3A). In addition, we also detected higher levels of SOD2 (superoxide dismutase 2), another ER-stress marker related to oxidative stress (Fig. 3A). These data indicated that the liver of Epm2a-/- animals, lacking laforin, was under permanent conditions of ER-stress. In contrast, analysis of whole brain extracts from these animals failed to detect differences in the levels of the ER-stress markers between Epm2a-/- and control animals (Fig. 3B). We also measured the activity of the proteasome in mouse liver and whole brain extracts. As shown in Fig. 4A, we observed a significant decrease in the activity of the proteasome in liver extracts from Epm2a-/- mice in comparison to liver extracts from control mice. However, we were not able to observe differences in the activity of the proteasome in whole brain extracts, which correlated with the absence of differences in the amount of ER-stress markers in extracts from this tissue (see above). Western blot analyses of two different subunits of the 26S proteasome complex in these liver samples indicated that the observed changes in the activity of the proteasome were not due to differences in proteasome levels (Fig. 4B). These results supported the idea that loss of laforin correlated with induction of ER-stress markers and impaired proteasomal function, at least in mouse liver.

Bottom Line: Here, we demonstrate that siRNA silencing of laforin in Hek293 and SH-SY5Y cells increases their sensitivity to agents triggering ER-stress, which correlates with impairment of the ubiquitin-proteasomal pathway and increased apoptosis.We demonstrate that, in addition to regulating glycogen synthesis, laforin and malin play a role protecting cells from ER-stress, likely contributing to the elimination of unfolded proteins.These data suggest that proteasomal dysfunction and ER-stress play an important role in the pathogenesis of LD, which may offer novel therapeutic approaches for this fatal neurodegenerative disorder.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biomedicina de Valencia, CSIC and CIBER de Enfermedades Raras, Valencia, Spain.

ABSTRACT

Background: Lafora progressive myoclonus epilepsy (Lafora disease; LD) is a fatal autosomal recessive neurodegenerative disorder caused by loss-of-function mutations in either the EPM2A gene, encoding the dual specificity phosphatase laforin, or the EPM2B gene, encoding the E3-ubiquitin ligase malin. Previously, we and others have shown that both proteins form a functional complex that regulates glycogen synthesis by a novel mechanism involving ubiquitination and proteasomal degradation of at least two proteins, glycogen synthase and R5/PTG. Since laforin and malin localized at the endoplasmic reticulum (ER) and their regulatory role likely extend to other proteins unrelated to glycogen metabolism, we postulated that their absence may also affect the ER-unfolded protein response pathway.

Methodology/principal findings: Here, we demonstrate that siRNA silencing of laforin in Hek293 and SH-SY5Y cells increases their sensitivity to agents triggering ER-stress, which correlates with impairment of the ubiquitin-proteasomal pathway and increased apoptosis. Consistent with these findings, analysis of tissue samples from a LD patient lacking laforin, and from a laforin knockout (Epm2a-/-) mouse model of LD, demonstrates constitutive high expression levels of ER-stress markers BIP/Grp78, CHOP and PDI, among others.

Conclusions/significance: We demonstrate that, in addition to regulating glycogen synthesis, laforin and malin play a role protecting cells from ER-stress, likely contributing to the elimination of unfolded proteins. These data suggest that proteasomal dysfunction and ER-stress play an important role in the pathogenesis of LD, which may offer novel therapeutic approaches for this fatal neurodegenerative disorder.

Show MeSH
Related in: MedlinePlus