Cathepsin D expression level affects α-synuclein processing, aggregation, and toxicity in vivo

Center for Neurologic Diseases, Department of Neurology, Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Molecular Brain (Impact Factor: 4.9). 03/2009; 2(1):5. DOI: 10.1186/1756-6606-2-5
Source: PubMed


Elevated SNCA gene expression and intracellular accumulation of the encoded alpha-synuclein (aSyn) protein are associated with the development of Parkinson disease (PD). To date, few enzymes have been examined for their ability to degrade aSyn. Here, we explore the effects of CTSD gene expression, which encodes the lysosomal protease cathepsin D (CathD), on aSyn processing.
Over-expression of human CTSD cDNA in dopaminergic MES23.5 cell cultures induced the marked proteolysis of exogenously expressed aSyn proteins in a dose-dependent manner. Unexpectedly, brain extractions, Western blotting and ELISA quantification revealed evidence for reduced levels of soluble endogenous aSyn in ctsd knock-out mice. However, these CathD-deficient mice also contained elevated levels of insoluble, oligomeric aSyn species, as detected by formic acid extraction. In accordance, immunohistochemical studies of ctsd-mutant brain from mice, sheep and humans revealed selective synucleinopathy-like changes that varied slightly among the three species. These changes included intracellular aSyn accumulation and formation of ubiquitin-positive inclusions. Furthermore, using an established Drosophila model of human synucleinopathy, we observed markedly enhanced retinal toxicity in ctsd-null flies.
We conclude from these complementary investigations that: one, CathD can effectively degrade excess aSyn in dopaminergic cells; two, ctsd gene mutations result in a lysosomal storage disorder that includes microscopic and biochemical evidence of aSyn misprocessing; and three, CathD deficiency facilitates aSyn toxicity. We therefore postulate that CathD promotes 'synucleinase' activity, and that enhancing its function may lower aSyn concentrations in vivo.

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    • "In transgenic models, mutant GBA leads to α-synuclein accumulation in ubiquitinated inclusions whereas virallymediated overexpression of wild-type GBA is protective (Sardi et al., 2011). Similarly, knockout of the lysosomal protease cathepsin D in mice causes accumulation of α-synuclein in brain whereas cathepsin D overexpression is protective (Cullen et al., 2009). In this context, Nedd4, a ubiquitin ligase that targets proteins to lysosomes , provides an enzymatic link between this striking association of α-synucleinopathy and lysosomal degradation, and given its specificity for only a few protein-substrates, an opportunity for a targeted therapy aimed at preventing the accumulation of α-synuclein in brain. "
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    ABSTRACT: Parkinson's disease is a neurodegenerative disorder, characterised by accumulation and misfolding of α-synuclein. Although the level of α-synuclein in neurons is fundamentally linked to the onset of neurodegeneration, multiple pathways have been implicated in its degradation, and it remains unclear which are the critical ubiquitination enzymes that protect against α-synuclein accumulation in vivo. The ubiquitin ligase Nedd4 targets α-synuclein to the endosomal-lysosomal pathway in cultured cells. Here we asked whether Nedd4-mediated degradation protects against α-synuclein-induced toxicity in the Drosophila and rodent models of Parkinson's disease. We show that overexpression of Nedd4 can rescue the degenerative phenotype from ectopic expression of α-synuclein in the Drosophila eye. Overexpressed Nedd4 in the Drosophila brain, prevented the α-synuclein-induced locomotor defect whereas reduction in endogenous Nedd4 by RNAi led to worsening motor function and increased loss of dopaminergic neurons. Accordingly, AAV-mediated expression of wild-type but not the catalytically inactive Nedd4 decreased the α-synuclein-induced dopaminergic cell loss in the rat substantia nigra and reduced α-synuclein accumulation. Collectively, our data in two evolutionarily distant model organisms strongly suggest that Nedd4 is a modifier of α-synuclein pathobiology and thus a potential target for small molecule activators.
    Full-text · Article · Dec 2013 · Neurobiology of Disease
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    • "Cathepsins are a class of lysosomal proteases that play important roles in proteolysis during physiological processes. They are reportedly involved in a number of diseases, such as cancer [1], [2], [3], atherosclerosis [4], arthritis [5] and neurodegenerative diseases [6], [7]. Several cathepsins can function outside of cells. "
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    ABSTRACT: Cathepsin D (CD) plays an important role in both biological and pathological processes, although the cleavage characteristics and substrate selection of CD have yet to be fully explored. We employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the CD cleavage sites in bovine serum albumin (BSA). We found that the hydrophobic residues at P1 were not only a preferential factor for CD cleavage but that the hydrophobicity at P1' also contributed to CD recognition. The concept of hydrophobic scores of neighbors (HSN) was proposed to describe the hydrophobic microenvironment of CD recognition sites. The survey of CD cleavage characteristics in several proteins suggested that the HSN was a sensitive indicator for judging the favorable sites in peptides for CD cleavage, with HSN values of 0.5-1.0 representing a likely threshold. Ovalbumin (OVA), a protein resistant to CD cleavage in its native state, was easily cleaved by CD after denaturation, and the features of the cleaved peptides were quite similar to those found in BSA, where a higher HSN value indicated greater cleavability. We further conducted two-dimensional gel electrophoresis (2DE) to find more proteins that were insensitive to CD cleavage in CD-knockdown cells. Based on an analysis of secondary and three-dimensional structures, we postulated that intact proteins with a structure consisting of all α-helices would be relatively accessible to CD cleavage.
    Full-text · Article · Jun 2013 · PLoS ONE
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    • "Due to the role of iron in oxidative stress, iron chelators have been used as a neuroprotective strategy in different in vitro and in vivo neurotoxic models of PD. Deferoxamine (DFO), one such chelator, acts by binding Fe 3+ and thereby preventing iron ions from catalyzing redox reactions that lead to free radical formation (Cullen et al., 2009). DFO was first used for the treatment of disorders related to iron overload and has been shown to protect neurons in the 6-OHDA and MPTP animal models of PD (Ben-Shachar et al., 1992; Kaur et al., 2003; Shachar et al., 2004). "
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    ABSTRACT: Parkinson's disease is characterized by neuronal death in the substantia nigra and the presence of intracellular inclusions of α-synuclein in the Lewy bodies. Several lines of data support a role for iron in Parkinson's disease: iron is present in Lewy bodies, iron accumulates in the dopaminergic neurons in the substantia nigra, and Parkinson's disease is correlated with polymorphisms of several genes implicated in iron metabolism. Furthermore, iron can compromise the solubility of α-synuclein through direct interaction and can induce neurotoxicity in vitro. Here, we investigate the possible neuroprotective effect of the iron chelator deferoxamine in vivo to elucidate whether iron chelation can provide meaningful therapy for Parkinson's disease. Hence, we used a Parkinson's disease animal model based on unilateral injection of a recombinant adeno-associated viral vector encoding α-synuclein in the rat midbrain. Rats were treated with a novel deferoxamine delivery approach: 6 mg of the compound was administered intranasally three times a week for 3 or 7 weeks. The behavior of the animals and histopathological changes in the brain were analyzed. Our data show that although intranasal administration of deferoxamine in rats did not protect them from dopaminergic cell death, it did decrease the number of the pathological α-synuclein formations at the terminal level. In addition, this treatment resulted in changes in the immune response and an overall partial improvement in motor behavior. Taken together, our data show that in vivo iron chelation can modulate α-synuclein-induced pathology in the central nervous system. Our data suggest that chronic administration of intranasal deferoxamine may be a valid approach to limiting the mishandling of α-synuclein in the central nervous system observed in Parkinson's disease and slowing disease progression.
    Full-text · Article · Mar 2013 · Experimental Neurology
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