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Bee venom effects on ubiquitin proteasome system in hSOD1(G85R)-expressing NSC34 motor neuron cells.

Kim SH, Jung SY, Lee KW, Lee SH, Cai M, Choi SM, Yang EJ - BMC Complement Altern Med (2013)

Bottom Line: We found that GFP-hSOD1G85R overexpression induced SOD1 inclusions and reduced proteasome activity compared with the overexpression of GFP alone in NSC34 motor neuronal cells.In addition, we also observed that BV treatment restored proteasome activity and reduced the accumulation of ubiquitinated and misfolded SOD1 in GFP-hSOD1G85R-overexpressing NSC34 motor neuronal cells.However, BV treatment did not activate the autophagic pathway in these cells.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that results from a progressive loss of motor neurons. Familial ALS (fALS) is caused by missense mutations in Cu, Zn-superoxide dismutase 1 (SOD1) that frequently result in the accumulation of mutant protein aggregates that are associated with impairments in the ubiquitin-proteasome system (UPS). UPS impairment has been implicated in many neurological disorders. Bee venom (BV) extracted from honey bees has been used as a traditional medicine for treating inflammatory diseases and has been shown to attenuate the neuroinflammatory events that occur in a symptomatic ALS animal model.

Methods: NSC34 cells were transiently transfected with a WT or G85R hSOD1-GFP construct for 24 hrs and then stimulated with 2.5 μg/ml BV for 24 hrs. To determine whether a SOD1 mutation affects UPS function in NSC34 cells, we examined proteasome activity and performed western blotting and immunofluorescence using specific antibodies, such as anti-misfolded SOD1, anti-ubiquitin, anti-GRP78, anti-LC3, and anti-ISG15 antibodies.

Results: We found that GFP-hSOD1G85R overexpression induced SOD1 inclusions and reduced proteasome activity compared with the overexpression of GFP alone in NSC34 motor neuronal cells. In addition, we also observed that BV treatment restored proteasome activity and reduced the accumulation of ubiquitinated and misfolded SOD1 in GFP-hSOD1G85R-overexpressing NSC34 motor neuronal cells. However, BV treatment did not activate the autophagic pathway in these cells.

Conclusion: Our findings suggest that BV may rescue the impairment of the UPS in ALS models.

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Related in: MedlinePlus

BV treatment restores proteasome activity. NSC34 cells (2 × 106) were transfected with GFP, GFP-hSOD1 or GFP-hSOD1G85R constructs for 48 hrs and then treated with 2.5 μg/ml BV or equal volume of saline for 24 hrs prior to being lysed. The proteasome activity of the lysates was assayed using the 20S Proteasome Activity Assay kit. Proteasome activity was significantly decreased in NSC34 cells that transiently overexpressed the mutant G85R SOD1 compared with the control. BV treatment increased proteasome activity. The values shown are the means ± SEM of data obtained from three independent experiments. * p < 0.05. BV; bee venom.
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Figure 1: BV treatment restores proteasome activity. NSC34 cells (2 × 106) were transfected with GFP, GFP-hSOD1 or GFP-hSOD1G85R constructs for 48 hrs and then treated with 2.5 μg/ml BV or equal volume of saline for 24 hrs prior to being lysed. The proteasome activity of the lysates was assayed using the 20S Proteasome Activity Assay kit. Proteasome activity was significantly decreased in NSC34 cells that transiently overexpressed the mutant G85R SOD1 compared with the control. BV treatment increased proteasome activity. The values shown are the means ± SEM of data obtained from three independent experiments. * p < 0.05. BV; bee venom.

Mentions: A previous study showed that wild-type and mutant hSOD1 were degraded by macroautophagy and the proteasome [3]. To investigate whether the overexpression of hSOD1G85R in NSC34 motor neuron cells affects proteasome activity, 20S proteasome activity was examined. Consistent with previous studies, hSOD1G85R overexpression in NSC34 cells suppressed proteasome activity compared with GFP-transfected NSC34 cells (Figure 1). However, treatment with 2.5 μg/ml BV for 24 hrs significantly restored proteasome activity in hSOD1G85R-overexpressing NSC34 motor neuron cells (Figure 1). These data suggest that BV treatment improved the function of the UPS in this mutant ALS cell model.


Bee venom effects on ubiquitin proteasome system in hSOD1(G85R)-expressing NSC34 motor neuron cells.

Kim SH, Jung SY, Lee KW, Lee SH, Cai M, Choi SM, Yang EJ - BMC Complement Altern Med (2013)

BV treatment restores proteasome activity. NSC34 cells (2 × 106) were transfected with GFP, GFP-hSOD1 or GFP-hSOD1G85R constructs for 48 hrs and then treated with 2.5 μg/ml BV or equal volume of saline for 24 hrs prior to being lysed. The proteasome activity of the lysates was assayed using the 20S Proteasome Activity Assay kit. Proteasome activity was significantly decreased in NSC34 cells that transiently overexpressed the mutant G85R SOD1 compared with the control. BV treatment increased proteasome activity. The values shown are the means ± SEM of data obtained from three independent experiments. * p < 0.05. BV; bee venom.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3722004&req=5

Figure 1: BV treatment restores proteasome activity. NSC34 cells (2 × 106) were transfected with GFP, GFP-hSOD1 or GFP-hSOD1G85R constructs for 48 hrs and then treated with 2.5 μg/ml BV or equal volume of saline for 24 hrs prior to being lysed. The proteasome activity of the lysates was assayed using the 20S Proteasome Activity Assay kit. Proteasome activity was significantly decreased in NSC34 cells that transiently overexpressed the mutant G85R SOD1 compared with the control. BV treatment increased proteasome activity. The values shown are the means ± SEM of data obtained from three independent experiments. * p < 0.05. BV; bee venom.
Mentions: A previous study showed that wild-type and mutant hSOD1 were degraded by macroautophagy and the proteasome [3]. To investigate whether the overexpression of hSOD1G85R in NSC34 motor neuron cells affects proteasome activity, 20S proteasome activity was examined. Consistent with previous studies, hSOD1G85R overexpression in NSC34 cells suppressed proteasome activity compared with GFP-transfected NSC34 cells (Figure 1). However, treatment with 2.5 μg/ml BV for 24 hrs significantly restored proteasome activity in hSOD1G85R-overexpressing NSC34 motor neuron cells (Figure 1). These data suggest that BV treatment improved the function of the UPS in this mutant ALS cell model.

Bottom Line: We found that GFP-hSOD1G85R overexpression induced SOD1 inclusions and reduced proteasome activity compared with the overexpression of GFP alone in NSC34 motor neuronal cells.In addition, we also observed that BV treatment restored proteasome activity and reduced the accumulation of ubiquitinated and misfolded SOD1 in GFP-hSOD1G85R-overexpressing NSC34 motor neuronal cells.However, BV treatment did not activate the autophagic pathway in these cells.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that results from a progressive loss of motor neurons. Familial ALS (fALS) is caused by missense mutations in Cu, Zn-superoxide dismutase 1 (SOD1) that frequently result in the accumulation of mutant protein aggregates that are associated with impairments in the ubiquitin-proteasome system (UPS). UPS impairment has been implicated in many neurological disorders. Bee venom (BV) extracted from honey bees has been used as a traditional medicine for treating inflammatory diseases and has been shown to attenuate the neuroinflammatory events that occur in a symptomatic ALS animal model.

Methods: NSC34 cells were transiently transfected with a WT or G85R hSOD1-GFP construct for 24 hrs and then stimulated with 2.5 μg/ml BV for 24 hrs. To determine whether a SOD1 mutation affects UPS function in NSC34 cells, we examined proteasome activity and performed western blotting and immunofluorescence using specific antibodies, such as anti-misfolded SOD1, anti-ubiquitin, anti-GRP78, anti-LC3, and anti-ISG15 antibodies.

Results: We found that GFP-hSOD1G85R overexpression induced SOD1 inclusions and reduced proteasome activity compared with the overexpression of GFP alone in NSC34 motor neuronal cells. In addition, we also observed that BV treatment restored proteasome activity and reduced the accumulation of ubiquitinated and misfolded SOD1 in GFP-hSOD1G85R-overexpressing NSC34 motor neuronal cells. However, BV treatment did not activate the autophagic pathway in these cells.

Conclusion: Our findings suggest that BV may rescue the impairment of the UPS in ALS models.

Show MeSH
Related in: MedlinePlus