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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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α-syn accumulation does not co-localize with LC3 or CB immunoreactivity and is independent of active caspase-3 immunoreactivity. a. Immunostaining with Atg8/LC3 and α-syn antibodies shows that LC3 staining is increased in Ctsd-/- mice compared to Ctsd+/+ mice, and intense α-syn immunoreactivity does not colocalize with LC3 immunoreactivity (arrows, p25). LC3 = FITC (green). α-syn = Cy3 (red). Arrowheads point to cells with high LC3 immunoreactivity but without intense α-syn immunostaining. b. α-syn accumulation does not overlap with CB immunoreactivity. CB = FITC (green). α-syn = Cy3 (red). Arrow points to α-syn intense immunoreactive staining adjacent to CB staining. c. Neurons with active caspase-3 immunoreactivity do not exhibit intense α-syn immunoreactivity in Ctsd-/- brains. α-syn = FITC (green). Active caspase-3 = Cy3 (red). Arrows point to active caspase-3 immunoreactivity. Scale bar = 10 micron. n = 3 mice each genotype.
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Figure 2: α-syn accumulation does not co-localize with LC3 or CB immunoreactivity and is independent of active caspase-3 immunoreactivity. a. Immunostaining with Atg8/LC3 and α-syn antibodies shows that LC3 staining is increased in Ctsd-/- mice compared to Ctsd+/+ mice, and intense α-syn immunoreactivity does not colocalize with LC3 immunoreactivity (arrows, p25). LC3 = FITC (green). α-syn = Cy3 (red). Arrowheads point to cells with high LC3 immunoreactivity but without intense α-syn immunostaining. b. α-syn accumulation does not overlap with CB immunoreactivity. CB = FITC (green). α-syn = Cy3 (red). Arrow points to α-syn intense immunoreactive staining adjacent to CB staining. c. Neurons with active caspase-3 immunoreactivity do not exhibit intense α-syn immunoreactivity in Ctsd-/- brains. α-syn = FITC (green). Active caspase-3 = Cy3 (red). Arrows point to active caspase-3 immunoreactivity. Scale bar = 10 micron. n = 3 mice each genotype.

Mentions: Prior studies found autophagosomes start to accumulate in Ctsd-/- brains as early as p1, compared to Ctsd+/+ age-matched controls [28,30]. Furthermore, CB immunostaining as well as enzymatic activities are increased as early as p21 in Ctsd-/- brains [28,30]. Electron microscopy studies demonstrated that CB was associated with irregularly shaped and membrane-bound structures containing electron-dense materials, characterizing them as lysosomes in Ctsd-/- brains [28,30]. We found that α-syn accumulation does not become prominent until near p25 in Ctsd-/- brains. α-syn accumulation occurs in only a sub-population of neurons that exhibit enhanced Atg8 (AuTophaGy8)/LC3 (light chain 3) staining, indicating that AV accumulation precedes α-syn accumulation (Fig. 2a). We also found that α-syn accumulations are adjacent to, but do not overlap with, immunoreactivity for the AV-associated protein Atg8/LC3 or the lysosomal associated protein CB, suggesting that α-syn accumulation formed outside of autophagosomes and lysosomes (Fig. 2b). Neuronal populations immunoreactive for the apoptotic marker, cleaved caspase-3, are distinct from those with intense α-syn accumulation (Fig. 2c).


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)

α-syn accumulation does not co-localize with LC3 or CB immunoreactivity and is independent of active caspase-3 immunoreactivity. a. Immunostaining with Atg8/LC3 and α-syn antibodies shows that LC3 staining is increased in Ctsd-/- mice compared to Ctsd+/+ mice, and intense α-syn immunoreactivity does not colocalize with LC3 immunoreactivity (arrows, p25). LC3 = FITC (green). α-syn = Cy3 (red). Arrowheads point to cells with high LC3 immunoreactivity but without intense α-syn immunostaining. b. α-syn accumulation does not overlap with CB immunoreactivity. CB = FITC (green). α-syn = Cy3 (red). Arrow points to α-syn intense immunoreactive staining adjacent to CB staining. c. Neurons with active caspase-3 immunoreactivity do not exhibit intense α-syn immunoreactivity in Ctsd-/- brains. α-syn = FITC (green). Active caspase-3 = Cy3 (red). Arrows point to active caspase-3 immunoreactivity. Scale bar = 10 micron. n = 3 mice each genotype.
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Figure 2: α-syn accumulation does not co-localize with LC3 or CB immunoreactivity and is independent of active caspase-3 immunoreactivity. a. Immunostaining with Atg8/LC3 and α-syn antibodies shows that LC3 staining is increased in Ctsd-/- mice compared to Ctsd+/+ mice, and intense α-syn immunoreactivity does not colocalize with LC3 immunoreactivity (arrows, p25). LC3 = FITC (green). α-syn = Cy3 (red). Arrowheads point to cells with high LC3 immunoreactivity but without intense α-syn immunostaining. b. α-syn accumulation does not overlap with CB immunoreactivity. CB = FITC (green). α-syn = Cy3 (red). Arrow points to α-syn intense immunoreactive staining adjacent to CB staining. c. Neurons with active caspase-3 immunoreactivity do not exhibit intense α-syn immunoreactivity in Ctsd-/- brains. α-syn = FITC (green). Active caspase-3 = Cy3 (red). Arrows point to active caspase-3 immunoreactivity. Scale bar = 10 micron. n = 3 mice each genotype.
Mentions: Prior studies found autophagosomes start to accumulate in Ctsd-/- brains as early as p1, compared to Ctsd+/+ age-matched controls [28,30]. Furthermore, CB immunostaining as well as enzymatic activities are increased as early as p21 in Ctsd-/- brains [28,30]. Electron microscopy studies demonstrated that CB was associated with irregularly shaped and membrane-bound structures containing electron-dense materials, characterizing them as lysosomes in Ctsd-/- brains [28,30]. We found that α-syn accumulation does not become prominent until near p25 in Ctsd-/- brains. α-syn accumulation occurs in only a sub-population of neurons that exhibit enhanced Atg8 (AuTophaGy8)/LC3 (light chain 3) staining, indicating that AV accumulation precedes α-syn accumulation (Fig. 2a). We also found that α-syn accumulations are adjacent to, but do not overlap with, immunoreactivity for the AV-associated protein Atg8/LC3 or the lysosomal associated protein CB, suggesting that α-syn accumulation formed outside of autophagosomes and lysosomes (Fig. 2b). Neuronal populations immunoreactive for the apoptotic marker, cleaved caspase-3, are distinct from those with intense α-syn accumulation (Fig. 2c).

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