Limits...
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.

Show MeSH

Related in: MedlinePlus

Increased CD expression reduces α-syn-toxicity in C. elegans. a. RNAi knockdown of a C. elegans Ctsd ortholog worsens aggregation of human α-syn in vivo. Isogenic worm strain expressing α-syn::GFP alone (a) or with TOR-2 (b), in body wall muscle cells of C. elegans. The presence of TOR-2, a protein with chaperone activity, attenuates the misfolded α-syn protein (b). When worms expressing α-syn::GFP + TOR-2 are exposed to CD RNAi, the misfolded α-syn::GFP returns (c). d-f. Overexpression of CD protects DA neurons from α-syn-induced degeneration. Worm DA neurons degenerate as animals age. At the 7 d stage, most worms are missing anterior DA neurons of the CEP (cephalic) and/or ADE (anterior deirid) classes. d. Note the presence of 3 of 4 CEP DA neurons (arrows) and the absence of the 2 ADE neurons. e. Overexpression of CD protects worms from neurodegeneration whereby worms display all 4 CEP (arrows) and both ADE (arrowheads) neurons. f. RNAi knock down of asp-4 does not reduce tor-2 expression level. Semi-quantitative RT-PCR was performed by using primers to amplify cdk-5 (loading control) and tor-2. For all strains analyzed, normal (non-RNAi) condition was used as a negative control, and tor-2 RNAi was used as a positive control.g. The percentage of worms exhibiting the wildtype neuronal complement of all 6 anterior DA neurons (30%) was significantly greater than animals without CD overexpression (15%). CD mutants (D295R and F229I), CB and CL, in transgenic worms overexpressing human cDNAs encoding these mutated CD or the representative lysosomal cysteine proteases, do not have the same effect as the wildtype CD in reducing α-syn toxicity. *p < 0.001 compared to α-syn alone, by Fisher Exact Test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2600785&req=5

Figure 6: Increased CD expression reduces α-syn-toxicity in C. elegans. a. RNAi knockdown of a C. elegans Ctsd ortholog worsens aggregation of human α-syn in vivo. Isogenic worm strain expressing α-syn::GFP alone (a) or with TOR-2 (b), in body wall muscle cells of C. elegans. The presence of TOR-2, a protein with chaperone activity, attenuates the misfolded α-syn protein (b). When worms expressing α-syn::GFP + TOR-2 are exposed to CD RNAi, the misfolded α-syn::GFP returns (c). d-f. Overexpression of CD protects DA neurons from α-syn-induced degeneration. Worm DA neurons degenerate as animals age. At the 7 d stage, most worms are missing anterior DA neurons of the CEP (cephalic) and/or ADE (anterior deirid) classes. d. Note the presence of 3 of 4 CEP DA neurons (arrows) and the absence of the 2 ADE neurons. e. Overexpression of CD protects worms from neurodegeneration whereby worms display all 4 CEP (arrows) and both ADE (arrowheads) neurons. f. RNAi knock down of asp-4 does not reduce tor-2 expression level. Semi-quantitative RT-PCR was performed by using primers to amplify cdk-5 (loading control) and tor-2. For all strains analyzed, normal (non-RNAi) condition was used as a negative control, and tor-2 RNAi was used as a positive control.g. The percentage of worms exhibiting the wildtype neuronal complement of all 6 anterior DA neurons (30%) was significantly greater than animals without CD overexpression (15%). CD mutants (D295R and F229I), CB and CL, in transgenic worms overexpressing human cDNAs encoding these mutated CD or the representative lysosomal cysteine proteases, do not have the same effect as the wildtype CD in reducing α-syn toxicity. *p < 0.001 compared to α-syn alone, by Fisher Exact Test.

Mentions: To further investigate the role of CD activity in α-syn clearance in vivo, we generated transgenic C. elegans expressing a human α-syn and GFP fusion protein in body wall muscle cells. In these worms, human α-syn::GFP forms aggregates as worms develop and age (Fig. 6a). As in mammalian cells[46], co-expression of the worm TOR-2 protein chaperone ameliorated the formation of α-syn::GFP aggregates (Fig. 6b). Importantly, this established a genetic background within which enhancement of α-syn aggregation could be more readily visualized by RNA interference (RNAi). Using bacterial RNAi feeding to specifically target the C. elegans ortholog of Ctsd, we knocked down Ctsd in α-syn::GFP + TOR-2 transgenic worms. RNAi targeting of Ctsd led to a return of fluorescent aggregates over time (Fig. 6c), and RNAi of Ctsd did not affect tor-2 mRNA levels as examined by real-time RT-PCR (Fig. 6f). Thus, Ctsd deficiency led to α-syn aggregation in both mice and worms.


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)

Increased CD expression reduces α-syn-toxicity in C. elegans. a. RNAi knockdown of a C. elegans Ctsd ortholog worsens aggregation of human α-syn in vivo. Isogenic worm strain expressing α-syn::GFP alone (a) or with TOR-2 (b), in body wall muscle cells of C. elegans. The presence of TOR-2, a protein with chaperone activity, attenuates the misfolded α-syn protein (b). When worms expressing α-syn::GFP + TOR-2 are exposed to CD RNAi, the misfolded α-syn::GFP returns (c). d-f. Overexpression of CD protects DA neurons from α-syn-induced degeneration. Worm DA neurons degenerate as animals age. At the 7 d stage, most worms are missing anterior DA neurons of the CEP (cephalic) and/or ADE (anterior deirid) classes. d. Note the presence of 3 of 4 CEP DA neurons (arrows) and the absence of the 2 ADE neurons. e. Overexpression of CD protects worms from neurodegeneration whereby worms display all 4 CEP (arrows) and both ADE (arrowheads) neurons. f. RNAi knock down of asp-4 does not reduce tor-2 expression level. Semi-quantitative RT-PCR was performed by using primers to amplify cdk-5 (loading control) and tor-2. For all strains analyzed, normal (non-RNAi) condition was used as a negative control, and tor-2 RNAi was used as a positive control.g. The percentage of worms exhibiting the wildtype neuronal complement of all 6 anterior DA neurons (30%) was significantly greater than animals without CD overexpression (15%). CD mutants (D295R and F229I), CB and CL, in transgenic worms overexpressing human cDNAs encoding these mutated CD or the representative lysosomal cysteine proteases, do not have the same effect as the wildtype CD in reducing α-syn toxicity. *p < 0.001 compared to α-syn alone, by Fisher Exact Test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2600785&req=5

Figure 6: Increased CD expression reduces α-syn-toxicity in C. elegans. a. RNAi knockdown of a C. elegans Ctsd ortholog worsens aggregation of human α-syn in vivo. Isogenic worm strain expressing α-syn::GFP alone (a) or with TOR-2 (b), in body wall muscle cells of C. elegans. The presence of TOR-2, a protein with chaperone activity, attenuates the misfolded α-syn protein (b). When worms expressing α-syn::GFP + TOR-2 are exposed to CD RNAi, the misfolded α-syn::GFP returns (c). d-f. Overexpression of CD protects DA neurons from α-syn-induced degeneration. Worm DA neurons degenerate as animals age. At the 7 d stage, most worms are missing anterior DA neurons of the CEP (cephalic) and/or ADE (anterior deirid) classes. d. Note the presence of 3 of 4 CEP DA neurons (arrows) and the absence of the 2 ADE neurons. e. Overexpression of CD protects worms from neurodegeneration whereby worms display all 4 CEP (arrows) and both ADE (arrowheads) neurons. f. RNAi knock down of asp-4 does not reduce tor-2 expression level. Semi-quantitative RT-PCR was performed by using primers to amplify cdk-5 (loading control) and tor-2. For all strains analyzed, normal (non-RNAi) condition was used as a negative control, and tor-2 RNAi was used as a positive control.g. The percentage of worms exhibiting the wildtype neuronal complement of all 6 anterior DA neurons (30%) was significantly greater than animals without CD overexpression (15%). CD mutants (D295R and F229I), CB and CL, in transgenic worms overexpressing human cDNAs encoding these mutated CD or the representative lysosomal cysteine proteases, do not have the same effect as the wildtype CD in reducing α-syn toxicity. *p < 0.001 compared to α-syn alone, by Fisher Exact Test.
Mentions: To further investigate the role of CD activity in α-syn clearance in vivo, we generated transgenic C. elegans expressing a human α-syn and GFP fusion protein in body wall muscle cells. In these worms, human α-syn::GFP forms aggregates as worms develop and age (Fig. 6a). As in mammalian cells[46], co-expression of the worm TOR-2 protein chaperone ameliorated the formation of α-syn::GFP aggregates (Fig. 6b). Importantly, this established a genetic background within which enhancement of α-syn aggregation could be more readily visualized by RNA interference (RNAi). Using bacterial RNAi feeding to specifically target the C. elegans ortholog of Ctsd, we knocked down Ctsd in α-syn::GFP + TOR-2 transgenic worms. RNAi targeting of Ctsd led to a return of fluorescent aggregates over time (Fig. 6c), and RNAi of Ctsd did not affect tor-2 mRNA levels as examined by real-time RT-PCR (Fig. 6f). Thus, Ctsd deficiency led to α-syn aggregation in both mice and worms.

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