Alterations in lysosomal and proteasomal markers in Parkinson's disease: Relationship to alpha-synuclein inclusions

Abstract

We explored the relationship between ubiquitin proteasome system (UPS) and lysosomal markers and the formation of α-synuclein (α-syn) inclusions in nigral neurons in Parkinson disease (PD). Lysosome Associated Membrane Protein 1(LAMP1), Cathepsin D (CatD), and Heat Shock Protein73 (HSP73) immunoreactivity were significantly decreased within PD nigral neurons when compared to age-matched controls. This decrease was significantly greater in nigral neurons that contained α-syn inclusions. Immunoreactivity for 20S proteasome was similarly reduced in PD nigral neurons, but only in cells that contained inclusions. In aged control brains, there is staining for α-syn protein, but it is non-aggregated and there is no difference in LAMP1, CatD, HSP73 or 20S proteasome immunoreactivity between α-syn positive or negative neuromelanin-laden nigral neurons. Targeting over-expression of mutant human α-syn in the rat substantia nigra using viral vectors revealed that lysosomal and proteasomal markers were significantly decreased in the neurons that displayed α-syn-ir inclusions. These findings suggest that α-syn aggregation is a key feature associated with decline of proteasome and lysosome and support the hypothesis that cell degeneration in PD involves proteosomal and lysosomal dysfunction, impaired protein clearance, and protein accumulation and aggregation leading to cell death.

Introduction

In recent years, converging evidence suggests that the accumulation of unwanted and misfolded proteins, due to increased protein production and/or impaired clearance, plays a central role in the pathogenesis in PD (Olanow, 2007). PD is characterized by the accumulation of aggregated proteins in Lewy bodies and Lewy neurites. Genetic studies (see review: Bonifati, 2007) have identified familial cases that are associated with mutations that could cause excess production of mutant or wildtype proteins (e.g. α-synuclein) (Polymeropoulos et al., 1997, Singleton et al., 2003) or impairment in the capacity of the UPS or lysosomal systems to clear unwanted proteins (e.g. Parkin, LRRK2, DJ-1, and PINK1 mutation) (Abou-Sleiman et al., 2006, Wang et al., 1999, Chung et al., 2004). Further, pathological studies demonstrate that there are alterations in proteasomal structure and function in the substantia nigra pars compacta (SNc) in patients with sporadic PD (McNaught et al., 2003, Bedford et al., 2008).

Much of the interest in the role of misfolded proteins in PD has focused on alpha-synuclein (α-syn). α-syn is a major component of Lewy bodies in sporadic forms of PD (Spillantini et al., 1997) and mutations or multiplication of the gene are associated with rare familial forms of the disorder (Krüger et al., 1998, Polymeropoulos et al., 1997, Singleton et al., 2003, Nishioka et al., 2006). Over-expression of mutant or wildtype α-syn in transgenic (tg) mice (Masliah et al., 2000) and Drosophila (Feany and Bender, 2000) can cause alterations in nigrostriatal function and in some instances dopamine neuronal degeneration (e.g. Thiruchelvam et al., 2004) with inclusion bodies. Similarly targeted over-expression of α-syn in the nigrostriatal system of rats and nonhuman primates (Kirik et al., 2002, Kirik et al., 2003) induces neuronal degeneration and mimics several of the pathological and behavioral features of PD. The link between α-syn and neurodegeneration is further strengthened by the finding that accumulation of α-syn is associated with down-regulation of dopamine phenotypes in the SNc with aging of normal humans and monkeys (Chu and Kordower, 2007) and in PD (Chu et al., 2006). Down-regulation of alpha-synuclein expression can rescue dopaminergic cells from cell death in the SNc of Parkinson's disease model (Hayashita-Kinoh et al., 2006).

Although the precise mechanism of degradation is not precisely understood, clearance of α-syn appears to involve both the UPS and lysosome systems (Pan et al., 2008, Vogiatzi et al., 2008, Bennett et al., 2005, Olanow and McNaught, 2006). Lysosomes are cellular organelles that contain a wide variety of proteases (e.g. cathepsins and hydrolases) whose activity maintains the turnover of membrane proteins and other cellular macromolecules. Lysosomal processing capacity diminishes progressively over the lifespan of an animal (Kiffin et al., 2007, Terman, 2006), and lysosomal malfunction is associated with age-related neurodegenerative disorders (Nixon et al., 2000, Rubinsztein, 2006, Terman, 2006, Bandhyopadhyay and Cuervo, 2007, Pan et al., 2008). The connection between lysosomal dysfunction and neurodegeneration is further demonstrated by the finding that inhibition of lysosomal enzymes leads to abnormal protein accumulation and aggregation, synaptic loss, and neuronal demise in laboratory models (Ivy et al., 1984, Koike et al., 2000, Bendiske and Bahr, 2003, Felbor et al., 2002). The UPS is the primary system for clearing unwanted intracellular proteins from eukaryocytes (Goldberg, 1999, Tanaka et al., 2001a). Misfolded proteins are poly-ubiquitinated at lysine residues which signal for their transfer to, and degradation by, the proteasome. The proteasome is a multicatalytic protease with trypsin, chymotrypsin, and PHPG activities that degrade the unwanted proteins into their constituent amino acids and small peptides. In the PD nigra, abnormalities in the expression of the alpha subunit of the proteasome, and in each of its enzyme activities, have been observed (McNaught et al., 2003). Further, proteasomal inhibition in laboratory models leads to degeneration of dopamine neurons coupled with inclusions that stain for α-syn (McNaught et al., 2006, Rideout et al., 2005, Lim, 2007). The purpose of the present study was to assess the interrelationships between α-syn accumulation, aggregation and markers of the lysosome and UPS systems in PD. To do this, we evaluated 1) whether lysosomal and UPS markers are altered in remaining substantia nigra neurons in PD; 2) whether the formation of α-syn inclusions in PD is specifically associated with disturbances in markers of the lysosome and UPS; and 3) whether viral over-expression of α-syn is, by itself, sufficient to induce changes in markers of lysosomal and UPS function in the rodent substantia nigra. To accomplish these aims, the relative levels of proteins involved in UPS and lysosomal functional pathways were analyzed by immunostaining and with the use of quantitative immunofluorescence intensity measurements within the substantia nigra of PD cases and in rats with targeted over-expression of α-syn. To determine the specificity of any findings, the data obtained in these cases were compared with findings seen in age-matched controls.