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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 toxicity. a. Enhanced CD expression protects against α-syn overexpression-induced cell death. GFP was visualized under the fluorescence microscope and demonstrated more survival cells after co-transfection of GFP-α-syn and CD compared to transfection with GFP-α-syn alone. Viable cells were counted by trypan blue exclusion method. b. mRNA of α-syn is unchanged by CD transfection. SHSY5Y cells were transfected with vector alone, CD, α-syn, or α-syn+CD. Paired Student t-tests were conducted on RQ values for each group to determine significance. c. Western blot analyses indicate that CD transfection results in truncation of α-syn-GFP (the appearance of a band below the full-length 37 kDa band), and a reduction of endogenous 17 kDa α-syn monomers. CD is synthesized as a prepropeptide (53 kDa); the signal peptide is cleaved upon CD insertion into the endoplasmic reticulum (47 kDa). The CD zymogen is then activated in the acidic lysosomal environment to produce the 32 and 14 kDa products [13,47]. d. Enhanced CD expression reduces A53T and A30P mutant α-syn-induced cell death, but does not reduce Y125A mutant α-syn-, 10 μM chloroquine-, or 2 μM staurosporine-induced cell death. For a-d, *p < 0.05 compared to control (CTL); †p < 0.05 compared to otherwise identical transfection except without CD. n = 3 transfection for each experimental conditions. Student t-test was used. e. Increased protein levels correlate with transfection of respective cDNAs. SHSY5Y cells were transfected with control vector, or respective cDNA in each lane. The respective antibodies used for the immunoblots are at the left side of the gel images. Mutated α-syn-GFP were produced. CD transfection did not produce significant truncation intermediate products on the mutated α-syn-GFP, suggesting that the protection against A53T and A30P may be via alteration of their intracellular targeting rather than direct cleavage. CD 53 kDa and 47 kDa precursors and the 32 kDa mature product are shown. Actin immunoblot was used as a loading control.
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Figure 5: CD reduces α-syn toxicity. a. Enhanced CD expression protects against α-syn overexpression-induced cell death. GFP was visualized under the fluorescence microscope and demonstrated more survival cells after co-transfection of GFP-α-syn and CD compared to transfection with GFP-α-syn alone. Viable cells were counted by trypan blue exclusion method. b. mRNA of α-syn is unchanged by CD transfection. SHSY5Y cells were transfected with vector alone, CD, α-syn, or α-syn+CD. Paired Student t-tests were conducted on RQ values for each group to determine significance. c. Western blot analyses indicate that CD transfection results in truncation of α-syn-GFP (the appearance of a band below the full-length 37 kDa band), and a reduction of endogenous 17 kDa α-syn monomers. CD is synthesized as a prepropeptide (53 kDa); the signal peptide is cleaved upon CD insertion into the endoplasmic reticulum (47 kDa). The CD zymogen is then activated in the acidic lysosomal environment to produce the 32 and 14 kDa products [13,47]. d. Enhanced CD expression reduces A53T and A30P mutant α-syn-induced cell death, but does not reduce Y125A mutant α-syn-, 10 μM chloroquine-, or 2 μM staurosporine-induced cell death. For a-d, *p < 0.05 compared to control (CTL); †p < 0.05 compared to otherwise identical transfection except without CD. n = 3 transfection for each experimental conditions. Student t-test was used. e. Increased protein levels correlate with transfection of respective cDNAs. SHSY5Y cells were transfected with control vector, or respective cDNA in each lane. The respective antibodies used for the immunoblots are at the left side of the gel images. Mutated α-syn-GFP were produced. CD transfection did not produce significant truncation intermediate products on the mutated α-syn-GFP, suggesting that the protection against A53T and A30P may be via alteration of their intracellular targeting rather than direct cleavage. CD 53 kDa and 47 kDa precursors and the 32 kDa mature product are shown. Actin immunoblot was used as a loading control.

Mentions: Excessive α-syn induces neuron death in cell cultures, and in a variety of genetic and viral delivery based animal models [42-45]. To examine the potential of elevating CD level as a means to reduce α-syn-induced cell death, we transfected human neuroblastoma SHSY5Y cells with α-syn-GFP, in the absence or presence of increased CD expression (Fig. 5a). Similar to previous studies of α-syn overexpression in yeast, worms and rat neurons[44], we found that overexpression of α-syn-GFP induced significant cell death in SHSY5Y cells. Co-transfection of human CD provided significant protection against α-syn-GFP overexpression-induced cell death (Fig. 5a). Quantitative real-time RT-PCR confirmed the up-regulation of α-syn mRNA by α-syn transfection, and lack of effect of CD transfection on α-syn mRNA level (Fig. 5b). Furthermore, co-expression of CD with α-syn-GFP in human neuroblastoma SHSY5Y cells produced a cleavage product of α-syn-GFP and reduced endogenous monomeric 17 kDa α-syn (Fig. 5c).


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 toxicity. a. Enhanced CD expression protects against α-syn overexpression-induced cell death. GFP was visualized under the fluorescence microscope and demonstrated more survival cells after co-transfection of GFP-α-syn and CD compared to transfection with GFP-α-syn alone. Viable cells were counted by trypan blue exclusion method. b. mRNA of α-syn is unchanged by CD transfection. SHSY5Y cells were transfected with vector alone, CD, α-syn, or α-syn+CD. Paired Student t-tests were conducted on RQ values for each group to determine significance. c. Western blot analyses indicate that CD transfection results in truncation of α-syn-GFP (the appearance of a band below the full-length 37 kDa band), and a reduction of endogenous 17 kDa α-syn monomers. CD is synthesized as a prepropeptide (53 kDa); the signal peptide is cleaved upon CD insertion into the endoplasmic reticulum (47 kDa). The CD zymogen is then activated in the acidic lysosomal environment to produce the 32 and 14 kDa products [13,47]. d. Enhanced CD expression reduces A53T and A30P mutant α-syn-induced cell death, but does not reduce Y125A mutant α-syn-, 10 μM chloroquine-, or 2 μM staurosporine-induced cell death. For a-d, *p < 0.05 compared to control (CTL); †p < 0.05 compared to otherwise identical transfection except without CD. n = 3 transfection for each experimental conditions. Student t-test was used. e. Increased protein levels correlate with transfection of respective cDNAs. SHSY5Y cells were transfected with control vector, or respective cDNA in each lane. The respective antibodies used for the immunoblots are at the left side of the gel images. Mutated α-syn-GFP were produced. CD transfection did not produce significant truncation intermediate products on the mutated α-syn-GFP, suggesting that the protection against A53T and A30P may be via alteration of their intracellular targeting rather than direct cleavage. CD 53 kDa and 47 kDa precursors and the 32 kDa mature product are shown. Actin immunoblot was used as a loading control.
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Figure 5: CD reduces α-syn toxicity. a. Enhanced CD expression protects against α-syn overexpression-induced cell death. GFP was visualized under the fluorescence microscope and demonstrated more survival cells after co-transfection of GFP-α-syn and CD compared to transfection with GFP-α-syn alone. Viable cells were counted by trypan blue exclusion method. b. mRNA of α-syn is unchanged by CD transfection. SHSY5Y cells were transfected with vector alone, CD, α-syn, or α-syn+CD. Paired Student t-tests were conducted on RQ values for each group to determine significance. c. Western blot analyses indicate that CD transfection results in truncation of α-syn-GFP (the appearance of a band below the full-length 37 kDa band), and a reduction of endogenous 17 kDa α-syn monomers. CD is synthesized as a prepropeptide (53 kDa); the signal peptide is cleaved upon CD insertion into the endoplasmic reticulum (47 kDa). The CD zymogen is then activated in the acidic lysosomal environment to produce the 32 and 14 kDa products [13,47]. d. Enhanced CD expression reduces A53T and A30P mutant α-syn-induced cell death, but does not reduce Y125A mutant α-syn-, 10 μM chloroquine-, or 2 μM staurosporine-induced cell death. For a-d, *p < 0.05 compared to control (CTL); †p < 0.05 compared to otherwise identical transfection except without CD. n = 3 transfection for each experimental conditions. Student t-test was used. e. Increased protein levels correlate with transfection of respective cDNAs. SHSY5Y cells were transfected with control vector, or respective cDNA in each lane. The respective antibodies used for the immunoblots are at the left side of the gel images. Mutated α-syn-GFP were produced. CD transfection did not produce significant truncation intermediate products on the mutated α-syn-GFP, suggesting that the protection against A53T and A30P may be via alteration of their intracellular targeting rather than direct cleavage. CD 53 kDa and 47 kDa precursors and the 32 kDa mature product are shown. Actin immunoblot was used as a loading control.
Mentions: Excessive α-syn induces neuron death in cell cultures, and in a variety of genetic and viral delivery based animal models [42-45]. To examine the potential of elevating CD level as a means to reduce α-syn-induced cell death, we transfected human neuroblastoma SHSY5Y cells with α-syn-GFP, in the absence or presence of increased CD expression (Fig. 5a). Similar to previous studies of α-syn overexpression in yeast, worms and rat neurons[44], we found that overexpression of α-syn-GFP induced significant cell death in SHSY5Y cells. Co-transfection of human CD provided significant protection against α-syn-GFP overexpression-induced cell death (Fig. 5a). Quantitative real-time RT-PCR confirmed the up-regulation of α-syn mRNA by α-syn transfection, and lack of effect of CD transfection on α-syn mRNA level (Fig. 5b). Furthermore, co-expression of CD with α-syn-GFP in human neuroblastoma SHSY5Y cells produced a cleavage product of α-syn-GFP and reduced endogenous monomeric 17 kDa α-syn (Fig. 5c).

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