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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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Expression of ER-stress markers is increased in brain necropsies of a LD patient.A) Quantitative-real time PCR analysis of ER-stress markers in brain necropsies from both control and LD patient. Expression of target genes was normalized using GAPDH as an internal control. Data are expressed as fold induction over control (mean±SEM) of four independent measurements. B) Extracts from brain necropsies from both control and LD patient were analyzed by western blot using the indicated antibodies. Membranes were probed with anti-tubulin antibodies to ensure equal protein loading. * non-specific band. C) Quantitative-real time PCR analysis of ER-stress markers in skeletal muscle necropsies from both control and LD patient. Expression of target genes was normalized using GAPDH as an internal control. Data are expressed as fold induction over control (mean±SEM) of four independent measurements.
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pone-0005907-g005: Expression of ER-stress markers is increased in brain necropsies of a LD patient.A) Quantitative-real time PCR analysis of ER-stress markers in brain necropsies from both control and LD patient. Expression of target genes was normalized using GAPDH as an internal control. Data are expressed as fold induction over control (mean±SEM) of four independent measurements. B) Extracts from brain necropsies from both control and LD patient were analyzed by western blot using the indicated antibodies. Membranes were probed with anti-tubulin antibodies to ensure equal protein loading. * non-specific band. C) Quantitative-real time PCR analysis of ER-stress markers in skeletal muscle necropsies from both control and LD patient. Expression of target genes was normalized using GAPDH as an internal control. Data are expressed as fold induction over control (mean±SEM) of four independent measurements.

Mentions: We also analyzed the levels of different ER-stress markers in a brain necropsy from an LD patient with mutations in the EPM2A gene (compound heterozygous with R241Stop and ex1-33bpdel mutations). Using quantitative real time PCR, we detected higher levels of mRNAs corresponding to BIP/Grp78 and CHOP, in comparison to samples obtained from necropsies of an age-matched control subject (Fig. 5A). In addition, we also detected higher levels of mRNAs corresponding to SOD2 (superoxide dismutase 2), PDI (protein disulphide isomerase) and TTAse1 (thioltransferase 1), three ER-markers related to oxidative stress (Fig. 5A). In agreement with these results, western blot analysis confirmed the presence of higher levels of BIP/Grp78, PDI and SOD2 in brain tissue samples from the LD patient (Fig. 5B). The expression of ER-stress markers (i.e., BIP/Grp78 and CHOP) was also increased in skeletal muscle samples from the same patient in comparison to a control subject (Fig. 5C).


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)

Expression of ER-stress markers is increased in brain necropsies of a LD patient.A) Quantitative-real time PCR analysis of ER-stress markers in brain necropsies from both control and LD patient. Expression of target genes was normalized using GAPDH as an internal control. Data are expressed as fold induction over control (mean±SEM) of four independent measurements. B) Extracts from brain necropsies from both control and LD patient were analyzed by western blot using the indicated antibodies. Membranes were probed with anti-tubulin antibodies to ensure equal protein loading. * non-specific band. C) Quantitative-real time PCR analysis of ER-stress markers in skeletal muscle necropsies from both control and LD patient. Expression of target genes was normalized using GAPDH as an internal control. Data are expressed as fold induction over control (mean±SEM) of four independent measurements.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2692001&req=5

pone-0005907-g005: Expression of ER-stress markers is increased in brain necropsies of a LD patient.A) Quantitative-real time PCR analysis of ER-stress markers in brain necropsies from both control and LD patient. Expression of target genes was normalized using GAPDH as an internal control. Data are expressed as fold induction over control (mean±SEM) of four independent measurements. B) Extracts from brain necropsies from both control and LD patient were analyzed by western blot using the indicated antibodies. Membranes were probed with anti-tubulin antibodies to ensure equal protein loading. * non-specific band. C) Quantitative-real time PCR analysis of ER-stress markers in skeletal muscle necropsies from both control and LD patient. Expression of target genes was normalized using GAPDH as an internal control. Data are expressed as fold induction over control (mean±SEM) of four independent measurements.
Mentions: We also analyzed the levels of different ER-stress markers in a brain necropsy from an LD patient with mutations in the EPM2A gene (compound heterozygous with R241Stop and ex1-33bpdel mutations). Using quantitative real time PCR, we detected higher levels of mRNAs corresponding to BIP/Grp78 and CHOP, in comparison to samples obtained from necropsies of an age-matched control subject (Fig. 5A). In addition, we also detected higher levels of mRNAs corresponding to SOD2 (superoxide dismutase 2), PDI (protein disulphide isomerase) and TTAse1 (thioltransferase 1), three ER-markers related to oxidative stress (Fig. 5A). In agreement with these results, western blot analysis confirmed the presence of higher levels of BIP/Grp78, PDI and SOD2 in brain tissue samples from the LD patient (Fig. 5B). The expression of ER-stress markers (i.e., BIP/Grp78 and CHOP) was also increased in skeletal muscle samples from the same patient in comparison to a control subject (Fig. 5C).

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