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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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Deficits of proteasomes in CD deficient mice. a. α-syn (Snca) mRNA is down-regulated in CD deficient brains compared to wildtype control brains. Ctsb, Ctsl, Ctsf, Ctsh, Atg7, UCHL1, Park2, and Psmb7 mRNA levels are up-regulated. Atg12 and Psmb6 mRNA levels appear to be normal. b. Western blot analyses show an increase of steady state GAPDH, a CMA substrate. n = 3 p25 brain. *p < 0.05 by Student t-test, compared between Ctsd+/+ and Ctsd-/- brains. c. Extracts from Ctsd-/- cortex exhibit reduced proteasome activities compared to Ctsd+/+ as indicated by assays with trypsin-like fluorigenic substrate (VGR-AMC, reaching maximum at 60 min), chymotrypsin-like fluorigenic substrate (Z-GGL-AMC, reaching maximum at 120 min), and peptidylglutamyl peptide-like fluorigenic substrate (Suc-LLVY-AMC, reaching maximum at 120 min). The activities that are inhibitable by the proteasome inhibitor lactacystin were quantified. n = 3 mice each genotype. *p < 0.05 by Student t-test. d. Normal expression of proteins involved in UPS. Western blot analyses of UCHL1, Usp14, Rpt3, α4 and β1 indicate that these UPS factors are expressed normally in Ctsd+/+ and Ctsd-/- cortical extracts. Actin immunoblotting was used as a loading control. n = 3 mice each genotype. e. A diagram regarding onset of relative pathologies.
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Figure 3: Deficits of proteasomes in CD deficient mice. a. α-syn (Snca) mRNA is down-regulated in CD deficient brains compared to wildtype control brains. Ctsb, Ctsl, Ctsf, Ctsh, Atg7, UCHL1, Park2, and Psmb7 mRNA levels are up-regulated. Atg12 and Psmb6 mRNA levels appear to be normal. b. Western blot analyses show an increase of steady state GAPDH, a CMA substrate. n = 3 p25 brain. *p < 0.05 by Student t-test, compared between Ctsd+/+ and Ctsd-/- brains. c. Extracts from Ctsd-/- cortex exhibit reduced proteasome activities compared to Ctsd+/+ as indicated by assays with trypsin-like fluorigenic substrate (VGR-AMC, reaching maximum at 60 min), chymotrypsin-like fluorigenic substrate (Z-GGL-AMC, reaching maximum at 120 min), and peptidylglutamyl peptide-like fluorigenic substrate (Suc-LLVY-AMC, reaching maximum at 120 min). The activities that are inhibitable by the proteasome inhibitor lactacystin were quantified. n = 3 mice each genotype. *p < 0.05 by Student t-test. d. Normal expression of proteins involved in UPS. Western blot analyses of UCHL1, Usp14, Rpt3, α4 and β1 indicate that these UPS factors are expressed normally in Ctsd+/+ and Ctsd-/- cortical extracts. Actin immunoblotting was used as a loading control. n = 3 mice each genotype. e. A diagram regarding onset of relative pathologies.

Mentions: While bulk protein degradation appears to be normal in Ctsd-/- mice[27], we found that α-syn mRNA is down-regulated in Ctsd-/- brains at p25 when α-syn accumulation occurs (Fig. 3a). This is consistent with the finding that α-syn mRNA is either unchanged or down-regulated in the majority of sporadic PD cases[39], indicating that α-syn accumulation is unlikely to be the consequence of elevated gene expression. Prior studies reported that CB but not CL protein is up-regulated in Ctsd-/- brains at p23[28]. To better understand the role of CD in selective protein degradation and PD, we analyzed the expression of genes encoding other brain-enriched lysosomal proteases, autophagy-associated factors, proteasome subunits, and genes linked to familial PD. Interestingly, genes encoding other lysosomal cathepsins B, L, F and H (Ctsb, Ctsl, Ctsf, and Ctsh) mRNAs are all up-regulated at p25 (Fig. 3a). This result may suggest a common transcription regulatory mechanism for these cathepsins in response to CD deficiency. Alternatively, the influx of macrophages or microglia into the Ctsd-/- brain at this age may lead to an increase in cathepsin mRNA expression[40]. We also determined that accumulation of autophagosomes in Ctsd-/- neurons is accompanied with transcriptional up-regulation of Atg7 but not Atg12 (Fig. 3a). Both Atg7 and Atg12 are involved in an ubiquitin-like activity important for autophagosome expansion. Up-regulation of Atg7 may indicate an increase of autophagosome production in addition to a blockade of autophagy completion [28,38,40]. mRNA of Park2 which encodes Parkin, mutation of which has been found in a subset of autosomal recessive PD;UCHL1, mutation of which has been found in a subset of autosomal dominant PD; and Psmb7 which encodes proteasome 20S core β2 subunit are also modestly up-regulated in response to CD deficiency, indicating a compensatory response to CD deficiency at the level of gene transcription (Fig. 3a).


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)

Deficits of proteasomes in CD deficient mice. a. α-syn (Snca) mRNA is down-regulated in CD deficient brains compared to wildtype control brains. Ctsb, Ctsl, Ctsf, Ctsh, Atg7, UCHL1, Park2, and Psmb7 mRNA levels are up-regulated. Atg12 and Psmb6 mRNA levels appear to be normal. b. Western blot analyses show an increase of steady state GAPDH, a CMA substrate. n = 3 p25 brain. *p < 0.05 by Student t-test, compared between Ctsd+/+ and Ctsd-/- brains. c. Extracts from Ctsd-/- cortex exhibit reduced proteasome activities compared to Ctsd+/+ as indicated by assays with trypsin-like fluorigenic substrate (VGR-AMC, reaching maximum at 60 min), chymotrypsin-like fluorigenic substrate (Z-GGL-AMC, reaching maximum at 120 min), and peptidylglutamyl peptide-like fluorigenic substrate (Suc-LLVY-AMC, reaching maximum at 120 min). The activities that are inhibitable by the proteasome inhibitor lactacystin were quantified. n = 3 mice each genotype. *p < 0.05 by Student t-test. d. Normal expression of proteins involved in UPS. Western blot analyses of UCHL1, Usp14, Rpt3, α4 and β1 indicate that these UPS factors are expressed normally in Ctsd+/+ and Ctsd-/- cortical extracts. Actin immunoblotting was used as a loading control. n = 3 mice each genotype. e. A diagram regarding onset of relative pathologies.
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Figure 3: Deficits of proteasomes in CD deficient mice. a. α-syn (Snca) mRNA is down-regulated in CD deficient brains compared to wildtype control brains. Ctsb, Ctsl, Ctsf, Ctsh, Atg7, UCHL1, Park2, and Psmb7 mRNA levels are up-regulated. Atg12 and Psmb6 mRNA levels appear to be normal. b. Western blot analyses show an increase of steady state GAPDH, a CMA substrate. n = 3 p25 brain. *p < 0.05 by Student t-test, compared between Ctsd+/+ and Ctsd-/- brains. c. Extracts from Ctsd-/- cortex exhibit reduced proteasome activities compared to Ctsd+/+ as indicated by assays with trypsin-like fluorigenic substrate (VGR-AMC, reaching maximum at 60 min), chymotrypsin-like fluorigenic substrate (Z-GGL-AMC, reaching maximum at 120 min), and peptidylglutamyl peptide-like fluorigenic substrate (Suc-LLVY-AMC, reaching maximum at 120 min). The activities that are inhibitable by the proteasome inhibitor lactacystin were quantified. n = 3 mice each genotype. *p < 0.05 by Student t-test. d. Normal expression of proteins involved in UPS. Western blot analyses of UCHL1, Usp14, Rpt3, α4 and β1 indicate that these UPS factors are expressed normally in Ctsd+/+ and Ctsd-/- cortical extracts. Actin immunoblotting was used as a loading control. n = 3 mice each genotype. e. A diagram regarding onset of relative pathologies.
Mentions: While bulk protein degradation appears to be normal in Ctsd-/- mice[27], we found that α-syn mRNA is down-regulated in Ctsd-/- brains at p25 when α-syn accumulation occurs (Fig. 3a). This is consistent with the finding that α-syn mRNA is either unchanged or down-regulated in the majority of sporadic PD cases[39], indicating that α-syn accumulation is unlikely to be the consequence of elevated gene expression. Prior studies reported that CB but not CL protein is up-regulated in Ctsd-/- brains at p23[28]. To better understand the role of CD in selective protein degradation and PD, we analyzed the expression of genes encoding other brain-enriched lysosomal proteases, autophagy-associated factors, proteasome subunits, and genes linked to familial PD. Interestingly, genes encoding other lysosomal cathepsins B, L, F and H (Ctsb, Ctsl, Ctsf, and Ctsh) mRNAs are all up-regulated at p25 (Fig. 3a). This result may suggest a common transcription regulatory mechanism for these cathepsins in response to CD deficiency. Alternatively, the influx of macrophages or microglia into the Ctsd-/- brain at this age may lead to an increase in cathepsin mRNA expression[40]. We also determined that accumulation of autophagosomes in Ctsd-/- neurons is accompanied with transcriptional up-regulation of Atg7 but not Atg12 (Fig. 3a). Both Atg7 and Atg12 are involved in an ubiquitin-like activity important for autophagosome expansion. Up-regulation of Atg7 may indicate an increase of autophagosome production in addition to a blockade of autophagy completion [28,38,40]. mRNA of Park2 which encodes Parkin, mutation of which has been found in a subset of autosomal recessive PD;UCHL1, mutation of which has been found in a subset of autosomal dominant PD; and Psmb7 which encodes proteasome 20S core β2 subunit are also modestly up-regulated in response to CD deficiency, indicating a compensatory response to CD deficiency at the level of gene transcription (Fig. 3a).

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