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Lysosomal enzyme cathepsin D protects against alpha-synuclein aggregation and toxicity.

Qiao L, Hamamichi S, Caldwell KA, Caldwell GA, Yacoubian TA, Wilson S, Xie ZL, Speake LD, Parks R, Crabtree D, Liang Q, Crimmins S, Schneider L, Uchiyama Y, Iwatsubo T, Zhou Y, Peng L, Lu Y, Standaert DG, Walls KC, Shacka JJ, Roth KA, Zhang J - Mol Brain (2008)

Bottom Line: In addition to impaired macroautophagy, CD deficiency reduced proteasome activity, suggesting an essential role for lysosomal CD function in regulating multiple proteolytic pathways that are important for α-syn metabolism.Conversely, CD overexpression reduces α-syn aggregation and is neuroprotective against α-syn overexpression-induced cell death in vitro.Our data identify a conserved CD function in α-syn degradation and identify CD as a novel target for LB disease therapeutics.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Alabama at Birmingham, USA. lqiao@uab.edu

ABSTRACT
α-synuclein (α-syn) is a main component of Lewy bodies (LB) that occur in many neurodegenerative diseases, including Parkinson's disease (PD), dementia with LB (DLB) and multi-system atrophy. α-syn mutations or amplifications are responsible for a subset of autosomal dominant familial PD cases, and overexpression causes neurodegeneration and motor disturbances in animals. To investigate mechanisms for α-syn accumulation and toxicity, we studied a mouse model of lysosomal enzyme cathepsin D (CD) deficiency, and found extensive accumulation of endogenous α-syn in neurons without overabundance of α-syn mRNA. In addition to impaired macroautophagy, CD deficiency reduced proteasome activity, suggesting an essential role for lysosomal CD function in regulating multiple proteolytic pathways that are important for α-syn metabolism. Conversely, CD overexpression reduces α-syn aggregation and is neuroprotective against α-syn overexpression-induced cell death in vitro. In a C. elegans model, CD deficiency exacerbates α-syn accumulation while its overexpression is protective against α-syn-induced dopaminergic neurodegeneration. Mutated CD with diminished enzymatic activity or overexpression of cathepsins B (CB) or L (CL) is not protective in the worm model, indicating a unique requirement for enzymatically active CD. Our data identify a conserved CD function in α-syn degradation and identify CD as a novel target for LB disease therapeutics.

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CD reduces α-syn aggregation. a. CD reduces α-syn aggregation in an aggregation assay. 50% of the cells transfected with α-syn-GFP, synphilin and empty vector exhibited visible α-syn aggregates. Co-transfection of CD together with α-syn-GFP and synphilin reduced the number of cells with visible α-syn aggregates to 20%. n = 3 independent transfection, each in quadruplicate. *p < 0.05 compared to absence of exogenous CD by Student t-test. b. Double immunostaining for CD and α-syn in the aggregation assay after transfection of H4 cells with α-syn+synphilin+vector and α-syn+synphilin+CD. Many more cells transfected with synphilin+α-syn+vector have α-syn aggregates than cells transfected with synphilin+α-syn+CD. α-syn = red; CD = green. Higher magnification images of the boxed areas are shown at the left-most panel. α-syn aggregates tended to be found in cellular areas with low levels of CD staining, in H4 cells with either α-syn+synphilin+vector or α-syn+synphilin+CD. Scale bar = 25 micron. Arrows point to α-syn aggregates. c. Double immunostaining for CD and the V5 tag of synphilin in the aggregation assay after transfection of H4 cells with α-syn+synphilin+vector and α-syn+synphilin+CD. Many more cells transfected with synphilin+α-syn+vector have α-syn aggregates than cells transfected with synphilin+α-syn+CD. Synphilin = red; CD = green. The levels of synphilin immunostaining were indistinguishable in cells transfected with synphilin+α-syn+vector versus cells transfected with synphilin+α-syn+CD. This is true in cells with or without aggregates. Scale bar = 25 micron. Arrows point to α-syn aggregates.
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Figure 4: CD reduces α-syn aggregation. a. CD reduces α-syn aggregation in an aggregation assay. 50% of the cells transfected with α-syn-GFP, synphilin and empty vector exhibited visible α-syn aggregates. Co-transfection of CD together with α-syn-GFP and synphilin reduced the number of cells with visible α-syn aggregates to 20%. n = 3 independent transfection, each in quadruplicate. *p < 0.05 compared to absence of exogenous CD by Student t-test. b. Double immunostaining for CD and α-syn in the aggregation assay after transfection of H4 cells with α-syn+synphilin+vector and α-syn+synphilin+CD. Many more cells transfected with synphilin+α-syn+vector have α-syn aggregates than cells transfected with synphilin+α-syn+CD. α-syn = red; CD = green. Higher magnification images of the boxed areas are shown at the left-most panel. α-syn aggregates tended to be found in cellular areas with low levels of CD staining, in H4 cells with either α-syn+synphilin+vector or α-syn+synphilin+CD. Scale bar = 25 micron. Arrows point to α-syn aggregates. c. Double immunostaining for CD and the V5 tag of synphilin in the aggregation assay after transfection of H4 cells with α-syn+synphilin+vector and α-syn+synphilin+CD. Many more cells transfected with synphilin+α-syn+vector have α-syn aggregates than cells transfected with synphilin+α-syn+CD. Synphilin = red; CD = green. The levels of synphilin immunostaining were indistinguishable in cells transfected with synphilin+α-syn+vector versus cells transfected with synphilin+α-syn+CD. This is true in cells with or without aggregates. Scale bar = 25 micron. Arrows point to α-syn aggregates.

Mentions: To further understand how CD activity influences α-syn homeostasis, we examined whether enhancing CD expression can reduce α-syn aggregation. We adopted the simple culture system developed by McLean and colleagues in which an α-syn-green fluorescent protein (GFP) fusion protein (α-syn-GFP) forms visible aggregates in cells when co-expressed with synphilin[41]. We transfected H4 neuroglioma cells with α-syn-GFP, synphilin, and CD. As a control, cells were transfected with α-syn-GFP, synphilin and empty vector pcDNA3.1. Approximately 50% of control transfected cells exhibited α-syn aggregates, consistent with published findings[41]. Remarkably, transfection of CD together with α-syn-GFP and synphilin led to less than 20% of transfected cells exhibiting α-syn aggregates (Fig. 4a). To address the issue of whether CD could affect synphilin levels, we performed immunocytochemistry experiments to compare α-syn+synphilin+vector transfected cells and α-syn+synphilin+CD transfected cells. We chose this approach because very low transfection efficiency was achievable (less than 5%) in this aggregation assay. Thus, we cannot perform western blot analyses or quantitative RT-PCR assays for this experiment. Double immunostaining for CD and the V5 epitope tag on synphilin and double immunostaining for CD and α-syn were performed (Figs. 4b and 4c). We quantified the number of transfected cells with α-syn aggregates and those without α-syn aggregates. Many more cells that were transfected with synphilin+α-syn+vector exhibited α-syn aggregates in comparison to cells transfected with synphilin+α-syn+CD. Nonetheless, the levels of synphilin immunostaining were indistinguishable in cells transfected with synphilin+α-syn+vector versus cells transfected with synphilin+α-syn+CD. This is true in cells with or without aggregates. We conclude that CD reduction of α-syn aggregation is unlikely to be secondary to a reduction in synphilin levels.


Lysosomal enzyme cathepsin D protects against alpha-synuclein aggregation and toxicity.

Qiao L, Hamamichi S, Caldwell KA, Caldwell GA, Yacoubian TA, Wilson S, Xie ZL, Speake LD, Parks R, Crabtree D, Liang Q, Crimmins S, Schneider L, Uchiyama Y, Iwatsubo T, Zhou Y, Peng L, Lu Y, Standaert DG, Walls KC, Shacka JJ, Roth KA, Zhang J - Mol Brain (2008)

CD reduces α-syn aggregation. a. CD reduces α-syn aggregation in an aggregation assay. 50% of the cells transfected with α-syn-GFP, synphilin and empty vector exhibited visible α-syn aggregates. Co-transfection of CD together with α-syn-GFP and synphilin reduced the number of cells with visible α-syn aggregates to 20%. n = 3 independent transfection, each in quadruplicate. *p < 0.05 compared to absence of exogenous CD by Student t-test. b. Double immunostaining for CD and α-syn in the aggregation assay after transfection of H4 cells with α-syn+synphilin+vector and α-syn+synphilin+CD. Many more cells transfected with synphilin+α-syn+vector have α-syn aggregates than cells transfected with synphilin+α-syn+CD. α-syn = red; CD = green. Higher magnification images of the boxed areas are shown at the left-most panel. α-syn aggregates tended to be found in cellular areas with low levels of CD staining, in H4 cells with either α-syn+synphilin+vector or α-syn+synphilin+CD. Scale bar = 25 micron. Arrows point to α-syn aggregates. c. Double immunostaining for CD and the V5 tag of synphilin in the aggregation assay after transfection of H4 cells with α-syn+synphilin+vector and α-syn+synphilin+CD. Many more cells transfected with synphilin+α-syn+vector have α-syn aggregates than cells transfected with synphilin+α-syn+CD. Synphilin = red; CD = green. The levels of synphilin immunostaining were indistinguishable in cells transfected with synphilin+α-syn+vector versus cells transfected with synphilin+α-syn+CD. This is true in cells with or without aggregates. Scale bar = 25 micron. Arrows point to α-syn aggregates.
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Figure 4: CD reduces α-syn aggregation. a. CD reduces α-syn aggregation in an aggregation assay. 50% of the cells transfected with α-syn-GFP, synphilin and empty vector exhibited visible α-syn aggregates. Co-transfection of CD together with α-syn-GFP and synphilin reduced the number of cells with visible α-syn aggregates to 20%. n = 3 independent transfection, each in quadruplicate. *p < 0.05 compared to absence of exogenous CD by Student t-test. b. Double immunostaining for CD and α-syn in the aggregation assay after transfection of H4 cells with α-syn+synphilin+vector and α-syn+synphilin+CD. Many more cells transfected with synphilin+α-syn+vector have α-syn aggregates than cells transfected with synphilin+α-syn+CD. α-syn = red; CD = green. Higher magnification images of the boxed areas are shown at the left-most panel. α-syn aggregates tended to be found in cellular areas with low levels of CD staining, in H4 cells with either α-syn+synphilin+vector or α-syn+synphilin+CD. Scale bar = 25 micron. Arrows point to α-syn aggregates. c. Double immunostaining for CD and the V5 tag of synphilin in the aggregation assay after transfection of H4 cells with α-syn+synphilin+vector and α-syn+synphilin+CD. Many more cells transfected with synphilin+α-syn+vector have α-syn aggregates than cells transfected with synphilin+α-syn+CD. Synphilin = red; CD = green. The levels of synphilin immunostaining were indistinguishable in cells transfected with synphilin+α-syn+vector versus cells transfected with synphilin+α-syn+CD. This is true in cells with or without aggregates. Scale bar = 25 micron. Arrows point to α-syn aggregates.
Mentions: To further understand how CD activity influences α-syn homeostasis, we examined whether enhancing CD expression can reduce α-syn aggregation. We adopted the simple culture system developed by McLean and colleagues in which an α-syn-green fluorescent protein (GFP) fusion protein (α-syn-GFP) forms visible aggregates in cells when co-expressed with synphilin[41]. We transfected H4 neuroglioma cells with α-syn-GFP, synphilin, and CD. As a control, cells were transfected with α-syn-GFP, synphilin and empty vector pcDNA3.1. Approximately 50% of control transfected cells exhibited α-syn aggregates, consistent with published findings[41]. Remarkably, transfection of CD together with α-syn-GFP and synphilin led to less than 20% of transfected cells exhibiting α-syn aggregates (Fig. 4a). To address the issue of whether CD could affect synphilin levels, we performed immunocytochemistry experiments to compare α-syn+synphilin+vector transfected cells and α-syn+synphilin+CD transfected cells. We chose this approach because very low transfection efficiency was achievable (less than 5%) in this aggregation assay. Thus, we cannot perform western blot analyses or quantitative RT-PCR assays for this experiment. Double immunostaining for CD and the V5 epitope tag on synphilin and double immunostaining for CD and α-syn were performed (Figs. 4b and 4c). We quantified the number of transfected cells with α-syn aggregates and those without α-syn aggregates. Many more cells that were transfected with synphilin+α-syn+vector exhibited α-syn aggregates in comparison to cells transfected with synphilin+α-syn+CD. Nonetheless, the levels of synphilin immunostaining were indistinguishable in cells transfected with synphilin+α-syn+vector versus cells transfected with synphilin+α-syn+CD. This is true in cells with or without aggregates. We conclude that CD reduction of α-syn aggregation is unlikely to be secondary to a reduction in synphilin levels.

Bottom Line: In addition to impaired macroautophagy, CD deficiency reduced proteasome activity, suggesting an essential role for lysosomal CD function in regulating multiple proteolytic pathways that are important for α-syn metabolism.Conversely, CD overexpression reduces α-syn aggregation and is neuroprotective against α-syn overexpression-induced cell death in vitro.Our data identify a conserved CD function in α-syn degradation and identify CD as a novel target for LB disease therapeutics.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Alabama at Birmingham, USA. lqiao@uab.edu

ABSTRACT
α-synuclein (α-syn) is a main component of Lewy bodies (LB) that occur in many neurodegenerative diseases, including Parkinson's disease (PD), dementia with LB (DLB) and multi-system atrophy. α-syn mutations or amplifications are responsible for a subset of autosomal dominant familial PD cases, and overexpression causes neurodegeneration and motor disturbances in animals. To investigate mechanisms for α-syn accumulation and toxicity, we studied a mouse model of lysosomal enzyme cathepsin D (CD) deficiency, and found extensive accumulation of endogenous α-syn in neurons without overabundance of α-syn mRNA. In addition to impaired macroautophagy, CD deficiency reduced proteasome activity, suggesting an essential role for lysosomal CD function in regulating multiple proteolytic pathways that are important for α-syn metabolism. Conversely, CD overexpression reduces α-syn aggregation and is neuroprotective against α-syn overexpression-induced cell death in vitro. In a C. elegans model, CD deficiency exacerbates α-syn accumulation while its overexpression is protective against α-syn-induced dopaminergic neurodegeneration. Mutated CD with diminished enzymatic activity or overexpression of cathepsins B (CB) or L (CL) is not protective in the worm model, indicating a unique requirement for enzymatically active CD. Our data identify a conserved CD function in α-syn degradation and identify CD as a novel target for LB disease therapeutics.

Show MeSH
Related in: MedlinePlus