Limits...
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.

Show MeSH

Related in: MedlinePlus

Lack of laforin enhances cell sensitivity to endoplasmic reticulum stress in human neuroblastoma SH-SY5Y cells.SH-SY5Y cells were stably transfected with empty pSuper.neoGFP vector or pSUPER-laforin expressing a shRNA against laforin, as described in Materials and Methods. A) Stable transfectants were treated with 1 µM thapsigargin for 18 h and analyzed by western-blot using anti-BIP/Grp78, anti-laforin and anti-tubulin antibodies. B) The expression of additional ER-stress markers such as CHOP and pEIF2α (phospho-EIF2alpha) was analyzed in the stable transfectants at different times after the treatment with 1 µM thapsigargin (see Supplementary Fig. S1 for time course appearance of different ER-stress markers); right panel shows quantification (mean±SEM) of different blots expressed as percentage respect to tubulin lebels. C) Quantitative real time PCR analysis of the expression of BIP/Grp78 in stable transfectants treated with 1 µM thapsigargin for 18 h; expression of target gene was normalized using GAPDH as an internal control; data are expressed as fold induction over untreated control (mean±SEM) of four independent measurements. D) Proteasome activity was measured in extracts from transfectants used in A) treated with 1 µM thapsigargin. E) Growth of control and laforin depleted transfectants was assessed by flow cytometry as described in Materials and Methods. The percentage of apoptotic cells in the sub-G1 population was measured in cells treated or not with 1 µM thapsigargin for 18 h. The left panel shows a representative analysis of three independent experiments of cells treated with thapsigargin; the right panel shows the corresponding mean±SEM. F) Extracts from laforin-depleted and control cells treated or not with 1 µM thapsigargin for 18 h were analyzed by western-blotting using anti-caspase 3 and anti-activated caspase 3 antibodies; tubulin was used as a loading control. G) Cell sensitivity of laforin depleted and non-depleted cells to thapsigargin treatment. Stable laforin depleted SH-SY5Y and control cells were cultured in 96-well plates and treated with different amounts of thapsigargin for 24 hr. Then, cell viability was assessed using the AlamarBlue assay as described in Materials and Methods. Each point represents mean±SEM of three independent measurements and expresses the percentage of viability respect to the corresponding untreated cells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2692001&req=5

pone-0005907-g002: Lack of laforin enhances cell sensitivity to endoplasmic reticulum stress in human neuroblastoma SH-SY5Y cells.SH-SY5Y cells were stably transfected with empty pSuper.neoGFP vector or pSUPER-laforin expressing a shRNA against laforin, as described in Materials and Methods. A) Stable transfectants were treated with 1 µM thapsigargin for 18 h and analyzed by western-blot using anti-BIP/Grp78, anti-laforin and anti-tubulin antibodies. B) The expression of additional ER-stress markers such as CHOP and pEIF2α (phospho-EIF2alpha) was analyzed in the stable transfectants at different times after the treatment with 1 µM thapsigargin (see Supplementary Fig. S1 for time course appearance of different ER-stress markers); right panel shows quantification (mean±SEM) of different blots expressed as percentage respect to tubulin lebels. C) Quantitative real time PCR analysis of the expression of BIP/Grp78 in stable transfectants treated with 1 µM thapsigargin for 18 h; expression of target gene was normalized using GAPDH as an internal control; data are expressed as fold induction over untreated control (mean±SEM) of four independent measurements. D) Proteasome activity was measured in extracts from transfectants used in A) treated with 1 µM thapsigargin. E) Growth of control and laforin depleted transfectants was assessed by flow cytometry as described in Materials and Methods. The percentage of apoptotic cells in the sub-G1 population was measured in cells treated or not with 1 µM thapsigargin for 18 h. The left panel shows a representative analysis of three independent experiments of cells treated with thapsigargin; the right panel shows the corresponding mean±SEM. F) Extracts from laforin-depleted and control cells treated or not with 1 µM thapsigargin for 18 h were analyzed by western-blotting using anti-caspase 3 and anti-activated caspase 3 antibodies; tubulin was used as a loading control. G) Cell sensitivity of laforin depleted and non-depleted cells to thapsigargin treatment. Stable laforin depleted SH-SY5Y and control cells were cultured in 96-well plates and treated with different amounts of thapsigargin for 24 hr. Then, cell viability was assessed using the AlamarBlue assay as described in Materials and Methods. Each point represents mean±SEM of three independent measurements and expresses the percentage of viability respect to the corresponding untreated cells.

Mentions: Because LD is a neurological disorder, we sought to replicate the experiments in a human neuroblastoma cell line (SH-SY5Y). In addition, we decided to study the effect of long-term laforin depletion. To this end, we constructed a plasmid expressing a shRNA based on oligo #2108 (pSUPER-Laf) that was introduced in SH-SY5Y cells to select stable transfectants. These stable transfectants were grown in selective media for five days and, as shown in Fig. 2A, they presented undetectable levels of laforin. We then measured the amount of BIP/Grp78 under untreated and ER-stress conditions (induced by thapsigargin). As shown in Fig. 2A, we found a significant increase in the levels of this marker in comparison to stable transfectants obtained with an empty plasmid, but only when the cells were under conditions of ER-stress (thapsigargin treatment). The higher levels of expression of BIP/Grp78 in laforin-depleted cells treated with thapsigargin were confirmed by quantitative real time PCR (Fig. 2C). In addition, we measured the levels of additional ER-stress markers such as CHOP and phospho-EIF2alpha. As shown in Fig. 2B, higher levels of these two markers were found in laforin-depleted cells treated with thapsigargin. We also measured the activity of the proteasome in these cells and found a significant decrease in its activity in SH-SY5Y laforin-depleted cells subjected to conditions of ER-stress (thapsigargin treatment) (Fig. 2D). Similar results were obtained in two independent stable transfectants; not shown.


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)

Lack of laforin enhances cell sensitivity to endoplasmic reticulum stress in human neuroblastoma SH-SY5Y cells.SH-SY5Y cells were stably transfected with empty pSuper.neoGFP vector or pSUPER-laforin expressing a shRNA against laforin, as described in Materials and Methods. A) Stable transfectants were treated with 1 µM thapsigargin for 18 h and analyzed by western-blot using anti-BIP/Grp78, anti-laforin and anti-tubulin antibodies. B) The expression of additional ER-stress markers such as CHOP and pEIF2α (phospho-EIF2alpha) was analyzed in the stable transfectants at different times after the treatment with 1 µM thapsigargin (see Supplementary Fig. S1 for time course appearance of different ER-stress markers); right panel shows quantification (mean±SEM) of different blots expressed as percentage respect to tubulin lebels. C) Quantitative real time PCR analysis of the expression of BIP/Grp78 in stable transfectants treated with 1 µM thapsigargin for 18 h; expression of target gene was normalized using GAPDH as an internal control; data are expressed as fold induction over untreated control (mean±SEM) of four independent measurements. D) Proteasome activity was measured in extracts from transfectants used in A) treated with 1 µM thapsigargin. E) Growth of control and laforin depleted transfectants was assessed by flow cytometry as described in Materials and Methods. The percentage of apoptotic cells in the sub-G1 population was measured in cells treated or not with 1 µM thapsigargin for 18 h. The left panel shows a representative analysis of three independent experiments of cells treated with thapsigargin; the right panel shows the corresponding mean±SEM. F) Extracts from laforin-depleted and control cells treated or not with 1 µM thapsigargin for 18 h were analyzed by western-blotting using anti-caspase 3 and anti-activated caspase 3 antibodies; tubulin was used as a loading control. G) Cell sensitivity of laforin depleted and non-depleted cells to thapsigargin treatment. Stable laforin depleted SH-SY5Y and control cells were cultured in 96-well plates and treated with different amounts of thapsigargin for 24 hr. Then, cell viability was assessed using the AlamarBlue assay as described in Materials and Methods. Each point represents mean±SEM of three independent measurements and expresses the percentage of viability respect to the corresponding untreated cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2692001&req=5

pone-0005907-g002: Lack of laforin enhances cell sensitivity to endoplasmic reticulum stress in human neuroblastoma SH-SY5Y cells.SH-SY5Y cells were stably transfected with empty pSuper.neoGFP vector or pSUPER-laforin expressing a shRNA against laforin, as described in Materials and Methods. A) Stable transfectants were treated with 1 µM thapsigargin for 18 h and analyzed by western-blot using anti-BIP/Grp78, anti-laforin and anti-tubulin antibodies. B) The expression of additional ER-stress markers such as CHOP and pEIF2α (phospho-EIF2alpha) was analyzed in the stable transfectants at different times after the treatment with 1 µM thapsigargin (see Supplementary Fig. S1 for time course appearance of different ER-stress markers); right panel shows quantification (mean±SEM) of different blots expressed as percentage respect to tubulin lebels. C) Quantitative real time PCR analysis of the expression of BIP/Grp78 in stable transfectants treated with 1 µM thapsigargin for 18 h; expression of target gene was normalized using GAPDH as an internal control; data are expressed as fold induction over untreated control (mean±SEM) of four independent measurements. D) Proteasome activity was measured in extracts from transfectants used in A) treated with 1 µM thapsigargin. E) Growth of control and laforin depleted transfectants was assessed by flow cytometry as described in Materials and Methods. The percentage of apoptotic cells in the sub-G1 population was measured in cells treated or not with 1 µM thapsigargin for 18 h. The left panel shows a representative analysis of three independent experiments of cells treated with thapsigargin; the right panel shows the corresponding mean±SEM. F) Extracts from laforin-depleted and control cells treated or not with 1 µM thapsigargin for 18 h were analyzed by western-blotting using anti-caspase 3 and anti-activated caspase 3 antibodies; tubulin was used as a loading control. G) Cell sensitivity of laforin depleted and non-depleted cells to thapsigargin treatment. Stable laforin depleted SH-SY5Y and control cells were cultured in 96-well plates and treated with different amounts of thapsigargin for 24 hr. Then, cell viability was assessed using the AlamarBlue assay as described in Materials and Methods. Each point represents mean±SEM of three independent measurements and expresses the percentage of viability respect to the corresponding untreated cells.
Mentions: Because LD is a neurological disorder, we sought to replicate the experiments in a human neuroblastoma cell line (SH-SY5Y). In addition, we decided to study the effect of long-term laforin depletion. To this end, we constructed a plasmid expressing a shRNA based on oligo #2108 (pSUPER-Laf) that was introduced in SH-SY5Y cells to select stable transfectants. These stable transfectants were grown in selective media for five days and, as shown in Fig. 2A, they presented undetectable levels of laforin. We then measured the amount of BIP/Grp78 under untreated and ER-stress conditions (induced by thapsigargin). As shown in Fig. 2A, we found a significant increase in the levels of this marker in comparison to stable transfectants obtained with an empty plasmid, but only when the cells were under conditions of ER-stress (thapsigargin treatment). The higher levels of expression of BIP/Grp78 in laforin-depleted cells treated with thapsigargin were confirmed by quantitative real time PCR (Fig. 2C). In addition, we measured the levels of additional ER-stress markers such as CHOP and phospho-EIF2alpha. As shown in Fig. 2B, higher levels of these two markers were found in laforin-depleted cells treated with thapsigargin. We also measured the activity of the proteasome in these cells and found a significant decrease in its activity in SH-SY5Y laforin-depleted cells subjected to conditions of ER-stress (thapsigargin treatment) (Fig. 2D). Similar results were obtained in two independent stable transfectants; not shown.

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