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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 accumulates in neuronal cell bodies in p25 Ctsd-/- cortex. a. Immunohistochemical detection of α-syn and ubiquitin in p25 Ctsd+/+ (+/+) and Ctsd-/- (-/-) cortex. Scale bar = 20 micron. Arrows point to intense α-syn and ubiquitin immunoreactive cells. b. α-syn accumulation occurs in NeuN+ neuronal cell bodies. α-syn = Cy3 (red), NeuN = FITC (green). Wildtype (+/+) brains exhibit diffuse α-syn staining consistent with a synaptic distribution. Ctsd-/- brains showed neurons with cytoplasmic accumulation of α-syn immunoreactivity. Scale bar = 10 micron. c. α-syn does not exhibit pronounced accumulation in GFAP+ cells. α-syn = Cy3 (red). GFAP = FITC (green). Scale bar = 10 micron. d. Accumulation of high molecular weight α-syn and ubiquitinated proteins in both the TritonX-100 soluble and the insoluble fractions of the Ctsd-/- mice. Intensity of α-syn monomer, α-syn oligomers, and Ub-positive smears were quantified and compared between Ctsd+/+ and Ctsd-/- extracts. Truncated 12 kDa and 10 kDa α-syn fragments are reduced in Ctsd-/- extracts. e. Quantification of the western results. n = 3 mice each genotype. *p < 0.05 compared to Ctsd+/+ by Student t-test. S = TritonX-100 soluble. IS = TritonX-100 insoluble. f. A small fraction of accumulated α-syn in Ctsd-/- brains being mono-ubiquitinated. Western blot analysis of TritonX-100 soluble Ctsd-/- brain extract (Input), pulldown product by a polyclonal α-syn antibody C-20 (Santa Cruz) (middle lane), and pulldown product by a same-isotype control antibody (IgG). Immunoblot was probed with mAb1510 against ubiquitin. Shown are the 25 kDa bands of ubiquitin immunoreactivity that were pulled down by the C-20 anti-α-syn antibody. g. Western blot analysis of ubiquitin with p21 Ctsd+/+ and Ctsd-/- cortical extracts. The intensity of each lane was quantified and shown in the bar graph. *p < 0.05 Student t-test. h. Western blot analyses of Ctsd+/+ and Ctsd-/- cortical extracts, together with human DLB brain extracts using anti-phospho-α-syn antibody provided by Dr. Iwatsubo [33,36,38]. Arrows indicate positions of immunoreactive bands that are increased in Ctsd-/- mice and the position of α-syn monomer.
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Figure 1: α-syn accumulates in neuronal cell bodies in p25 Ctsd-/- cortex. a. Immunohistochemical detection of α-syn and ubiquitin in p25 Ctsd+/+ (+/+) and Ctsd-/- (-/-) cortex. Scale bar = 20 micron. Arrows point to intense α-syn and ubiquitin immunoreactive cells. b. α-syn accumulation occurs in NeuN+ neuronal cell bodies. α-syn = Cy3 (red), NeuN = FITC (green). Wildtype (+/+) brains exhibit diffuse α-syn staining consistent with a synaptic distribution. Ctsd-/- brains showed neurons with cytoplasmic accumulation of α-syn immunoreactivity. Scale bar = 10 micron. c. α-syn does not exhibit pronounced accumulation in GFAP+ cells. α-syn = Cy3 (red). GFAP = FITC (green). Scale bar = 10 micron. d. Accumulation of high molecular weight α-syn and ubiquitinated proteins in both the TritonX-100 soluble and the insoluble fractions of the Ctsd-/- mice. Intensity of α-syn monomer, α-syn oligomers, and Ub-positive smears were quantified and compared between Ctsd+/+ and Ctsd-/- extracts. Truncated 12 kDa and 10 kDa α-syn fragments are reduced in Ctsd-/- extracts. e. Quantification of the western results. n = 3 mice each genotype. *p < 0.05 compared to Ctsd+/+ by Student t-test. S = TritonX-100 soluble. IS = TritonX-100 insoluble. f. A small fraction of accumulated α-syn in Ctsd-/- brains being mono-ubiquitinated. Western blot analysis of TritonX-100 soluble Ctsd-/- brain extract (Input), pulldown product by a polyclonal α-syn antibody C-20 (Santa Cruz) (middle lane), and pulldown product by a same-isotype control antibody (IgG). Immunoblot was probed with mAb1510 against ubiquitin. Shown are the 25 kDa bands of ubiquitin immunoreactivity that were pulled down by the C-20 anti-α-syn antibody. g. Western blot analysis of ubiquitin with p21 Ctsd+/+ and Ctsd-/- cortical extracts. The intensity of each lane was quantified and shown in the bar graph. *p < 0.05 Student t-test. h. Western blot analyses of Ctsd+/+ and Ctsd-/- cortical extracts, together with human DLB brain extracts using anti-phospho-α-syn antibody provided by Dr. Iwatsubo [33,36,38]. Arrows indicate positions of immunoreactive bands that are increased in Ctsd-/- mice and the position of α-syn monomer.

Mentions: To investigate the involvement of lysosomal functions in α-syn clearance, we analyzed mice deficient in CD, previously generated by a targeted insertion of the neo marker in exon 4[27]. CD-deficient (Ctsd-/-) mice die at approximately postnatal day 26 (p26) due to a combination of nervous system and systemic abnormalities. Extensive neuron death resulting from activation of both apoptotic and non-apoptotic pathways has been observed in these mice [27-31]. We examined brains from p21 and p25 Ctsd-/- mice and found significant α-syn accumulation in neuronal cell bodies in p25 Ctsd-/- but not wildtype cortex (Fig. 1a). In contrast to the brains of human lipidoses patients[32] where α-syn aggregates are found in both neurons and glia and co-localize with lipids, in Ctsd-/- brains α-syn accumulations do not co-localize with autofluorescent lipofuscin (data not shown).


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 accumulates in neuronal cell bodies in p25 Ctsd-/- cortex. a. Immunohistochemical detection of α-syn and ubiquitin in p25 Ctsd+/+ (+/+) and Ctsd-/- (-/-) cortex. Scale bar = 20 micron. Arrows point to intense α-syn and ubiquitin immunoreactive cells. b. α-syn accumulation occurs in NeuN+ neuronal cell bodies. α-syn = Cy3 (red), NeuN = FITC (green). Wildtype (+/+) brains exhibit diffuse α-syn staining consistent with a synaptic distribution. Ctsd-/- brains showed neurons with cytoplasmic accumulation of α-syn immunoreactivity. Scale bar = 10 micron. c. α-syn does not exhibit pronounced accumulation in GFAP+ cells. α-syn = Cy3 (red). GFAP = FITC (green). Scale bar = 10 micron. d. Accumulation of high molecular weight α-syn and ubiquitinated proteins in both the TritonX-100 soluble and the insoluble fractions of the Ctsd-/- mice. Intensity of α-syn monomer, α-syn oligomers, and Ub-positive smears were quantified and compared between Ctsd+/+ and Ctsd-/- extracts. Truncated 12 kDa and 10 kDa α-syn fragments are reduced in Ctsd-/- extracts. e. Quantification of the western results. n = 3 mice each genotype. *p < 0.05 compared to Ctsd+/+ by Student t-test. S = TritonX-100 soluble. IS = TritonX-100 insoluble. f. A small fraction of accumulated α-syn in Ctsd-/- brains being mono-ubiquitinated. Western blot analysis of TritonX-100 soluble Ctsd-/- brain extract (Input), pulldown product by a polyclonal α-syn antibody C-20 (Santa Cruz) (middle lane), and pulldown product by a same-isotype control antibody (IgG). Immunoblot was probed with mAb1510 against ubiquitin. Shown are the 25 kDa bands of ubiquitin immunoreactivity that were pulled down by the C-20 anti-α-syn antibody. g. Western blot analysis of ubiquitin with p21 Ctsd+/+ and Ctsd-/- cortical extracts. The intensity of each lane was quantified and shown in the bar graph. *p < 0.05 Student t-test. h. Western blot analyses of Ctsd+/+ and Ctsd-/- cortical extracts, together with human DLB brain extracts using anti-phospho-α-syn antibody provided by Dr. Iwatsubo [33,36,38]. Arrows indicate positions of immunoreactive bands that are increased in Ctsd-/- mice and the position of α-syn monomer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: α-syn accumulates in neuronal cell bodies in p25 Ctsd-/- cortex. a. Immunohistochemical detection of α-syn and ubiquitin in p25 Ctsd+/+ (+/+) and Ctsd-/- (-/-) cortex. Scale bar = 20 micron. Arrows point to intense α-syn and ubiquitin immunoreactive cells. b. α-syn accumulation occurs in NeuN+ neuronal cell bodies. α-syn = Cy3 (red), NeuN = FITC (green). Wildtype (+/+) brains exhibit diffuse α-syn staining consistent with a synaptic distribution. Ctsd-/- brains showed neurons with cytoplasmic accumulation of α-syn immunoreactivity. Scale bar = 10 micron. c. α-syn does not exhibit pronounced accumulation in GFAP+ cells. α-syn = Cy3 (red). GFAP = FITC (green). Scale bar = 10 micron. d. Accumulation of high molecular weight α-syn and ubiquitinated proteins in both the TritonX-100 soluble and the insoluble fractions of the Ctsd-/- mice. Intensity of α-syn monomer, α-syn oligomers, and Ub-positive smears were quantified and compared between Ctsd+/+ and Ctsd-/- extracts. Truncated 12 kDa and 10 kDa α-syn fragments are reduced in Ctsd-/- extracts. e. Quantification of the western results. n = 3 mice each genotype. *p < 0.05 compared to Ctsd+/+ by Student t-test. S = TritonX-100 soluble. IS = TritonX-100 insoluble. f. A small fraction of accumulated α-syn in Ctsd-/- brains being mono-ubiquitinated. Western blot analysis of TritonX-100 soluble Ctsd-/- brain extract (Input), pulldown product by a polyclonal α-syn antibody C-20 (Santa Cruz) (middle lane), and pulldown product by a same-isotype control antibody (IgG). Immunoblot was probed with mAb1510 against ubiquitin. Shown are the 25 kDa bands of ubiquitin immunoreactivity that were pulled down by the C-20 anti-α-syn antibody. g. Western blot analysis of ubiquitin with p21 Ctsd+/+ and Ctsd-/- cortical extracts. The intensity of each lane was quantified and shown in the bar graph. *p < 0.05 Student t-test. h. Western blot analyses of Ctsd+/+ and Ctsd-/- cortical extracts, together with human DLB brain extracts using anti-phospho-α-syn antibody provided by Dr. Iwatsubo [33,36,38]. Arrows indicate positions of immunoreactive bands that are increased in Ctsd-/- mice and the position of α-syn monomer.
Mentions: To investigate the involvement of lysosomal functions in α-syn clearance, we analyzed mice deficient in CD, previously generated by a targeted insertion of the neo marker in exon 4[27]. CD-deficient (Ctsd-/-) mice die at approximately postnatal day 26 (p26) due to a combination of nervous system and systemic abnormalities. Extensive neuron death resulting from activation of both apoptotic and non-apoptotic pathways has been observed in these mice [27-31]. We examined brains from p21 and p25 Ctsd-/- mice and found significant α-syn accumulation in neuronal cell bodies in p25 Ctsd-/- but not wildtype cortex (Fig. 1a). In contrast to the brains of human lipidoses patients[32] where α-syn aggregates are found in both neurons and glia and co-localize with lipids, in Ctsd-/- brains α-syn accumulations do not co-localize with autofluorescent lipofuscin (data not shown).

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