- Top of page
- Materials and methods
The aggregation of α-synuclein (α-syn) is believed to play a critical role in the pathogenesis of disorders such as dementia with Lewy bodies and Parkinson's disease. The function of α-syn remains unclear, although several lines of evidence suggest that α-syn is involved in synaptic vesicle trafficking, probably via lipid binding, and interactions with lipids have been shown to regulate α-syn aggregation. In this context, the main objective of this study was to determine whether methyl-β-cyclodextrin (MβCD), a cholesterol-extracting agent, interfered with α-syn accumulation in models of synucleinopathy. For this purpose, we studied the effects of MβCD on the accumulation of α-syn in a transfected neuronal cell line and in transgenic mice. Immunoblot analysis showed that MβCD reduced the level of α-syn in the membrane fraction and detergent-insoluble fraction of transfected cells. In agreement with the in vitro studies, treatment of mice with MβCD resulted in decreased levels of α-syn in membrane fractions and reduced accumulation of α-syn in the neuronal cell body and synapses. Taken together, these results suggest that changes in cholesterol and lipid composition using cholesterol-lowering agents may be used as a tool for the treatment of synucleinopathies.
Lewy body disease (LBD) represents a heterogeneous group of disorders, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB) (Kosaka et al. 1984; Hansen and Galasko 1992; McKeith 2000), and is characterized by degeneration of the dopaminergic system (Shastry 2001), motor alterations (Braak et al. 2002), cognitive impairment (Salmon et al. 1989) and the formation of Lewy bodies (LBs) in cortical and subcortical regions (Trojanowski and Lee 1998). The characteristic LBs in these disorders are intraneuronal inclusions composed of aggregated forms of the synaptic protein α-synuclein (α-syn) (Wakabayashi et al. 1992; Iwai et al. 1995; Irizarry et al. 1996; Spillantini et al. 1997; Takeda et al. 1998). α-Syn is capable of self-aggregating to form both oligomers and fibrillar polymers with amyloidogenic-like characteristics (Hashimoto et al. 1998; Conway et al. 2000). Under physiological conditions, α-syn remains localized to the synapses where it may play a role in neurotransmission regulation (Sudhof and Jahn 1991; Murphy et al. 2000; Sidhu et al. 2004). In solution, α-syn is a natively unfolded molecule (Weinreb et al. 1996) that may be involved in synaptic plasticity by interacting with lipids in the neuronal membrane and vesicles (Fortin et al. 2004). For example, it has been shown that α-syn has lipid-binding domains that are distributed across the N-terminal region of the protein (amino residues 1–102) (Perrin et al. 2000). Recent studies have identified additional fatty acid-binding motifs in the C-terminal region of α-syn (Sharon et al. 2001). Consistent with these structural features, α-syn binds membranes (Eliezer et al. 2001; Narayanan and Scarlata 2001; Jo et al. 2002; Chandra et al. 2003) and vesicles (Jensen et al. 1998) containing acidic phospholipids, and the binding is accompanied by a shift to a largely α-helical conformation (Perrin et al. 2000). Moreover, α-syn interacts with polyunsaturated fatty acids (PUFAs) (Perrin et al. 2001; Sharon et al. 2003b), and exposure of recombinant α-syn to PUFAs at physiological concentrations results in the formation of α-syn multimers (Perrin et al. 2001). These results suggest that PUFAs may be relevant to the physiological function of α-syn, or that α-syn may regulate neuronal levels of PUFAs.
Recent studies have shown that α-syn interacts in vivo with PUFAs and that, in brains of patients with PD or DLB, or in neuronal cells over-expressing α-syn or its mutants, the levels of PUFA are elevated (Sharon et al. 2003a). Remarkably, in other neurodegenerative disorders that overlap with LBD, such as Alzheimer's disease (AD), previous studies have suggested a potential role for PUFAs and other lipids, such as cholesterol, in the clearance and metabolism of amyloid-β protein (Aβ) (Simons et al. 1998; Wood et al. 2002; Eckert et al. 2003b). In view of these findings, drugs that may regulate lipid metabolism and lower cholesterol, such as statins (drugs that reduce de novo cholesterol synthesis) and cyclodextrins (CDs), are currently being considered as potential therapeutics for AD (Simons et al. 1998; Hutter-Paier et al. 2004; Cole et al. 2005). Cholesterol depletion with a combination of statins and methyl-β-cyclodextrin (MβCD) significantly lowers Aβ production (Simons et al. 1998), and clinical studies have indicated that there is a decreased prevalence of AD associated with the use of statins alone or in combination with MβCD to treat hypercholesterolemia (Yunomae et al. 2003).
CDs are cyclic oligosaccharides of glucopyranosyl (glycosyl) units linked in a ring formation in the α(1–4) position (Pitha et al. 1988). α-, β-, and γ-CDs are crystalline, water-soluble compounds that differ in having six, seven and eight glucose residues, respectively, forming different sizes of hydrophobic cavity that can accommodate non-polar molecules and transfer lipophilic compounds to aqueous media (Pitha et al. 1988; Loftsson and Masson 2001). CDs selectively extract membrane cholesterol by including it in their non-polar core and, unlike other cholesterol-binding agents that incorporate into membranes, CDs are strictly surface-acting. CDs have been used for many years as carriers of lipophilic drugs in pharmacological research, and recently in membrane studies and cholesterol trafficking. βCDs [including βCD, MβCD and hydroxypropyl-βCD (HPβCD)] have been shown to extract membrane cholesterol selectively from a variety of cell types (Ohtani et al. 1989; Kilsdonk et al. 1995; Klein et al. 1995), and represent unique tools for membrane studies as they neither bind nor insert into the plasma membrane.
In view of some studies suggesting that lipid intake in the diet may be a risk factor for PD (Johnson et al. 1999), and that cholesterol and α-syn may interact in lipid rafts (Fortin et al. 2004), it is possible that cholesterol-reducing agents may have a potential effect in ameliorating pathological accumulation of α-syn. In this context, the main objective of the present report was to investigate the effects of MβCD on α-syn in neuronal cell lines and transgenic (tg) mice. These studies showed that the cholesterol-extracting effects of MβCD were accompanied by reduced α-syn levels in the membrane and detergent-insoluble fractions, and reduced phosphorylated α-syn levels in the membrane fraction. In tg mice, MβCD reduced the neuronal accumulation of α-syn and ameliorated the degenerative alterations, suggesting a possible therapeutic treatment using cholesterol-lowering agents for PD and DLB.
- Top of page
- Materials and methods
The present study showed that the cholesterol-depleting agent MβCD decreased the accumulation of α-syn in the membrane and insoluble fractions of an α-syn-transfected neuronal cell line and in the brains of non-tg and hα-syn tg mice. This is of interest because, in PD, misfolded (toxic) forms of α-syn often associate with the membrane, and damage may result from the formation of abnormal pore-like structures (Lashuel et al. 2002; Quist et al. 2005). α-Syn is an abundant cytosolic molecule that concentrates in the presynaptic site (Iwai et al. 1994). Under physiological conditions, α-syn interactions with the plasma membrane are mediated by fatty acids through lipid recognition repeat domains within α-syn (Perrin et al. 2000; Sharon et al. 2001). Previous studies have shown that α-syn associates with a number of membranes, including synaptic vesicles (Maroteaux et al. 1988), lipid droplets (Jensen et al. 1998) and yeast plasma membrane (Outeiro and Lindquist 2003). Moreover, recent studies have shown that lipid rafts mediate the synaptic localization of α-syn, where it may play a role in synaptic plasticity and neurotransmission (Fortin et al. 2004). Lipid rafts are detergent-insoluble, specialized microdomains of the plasma membrane that integrate signaling pathways (Bickel 2002). Lipid rafts are enriched in cholesterol, sphingolipids and glycosylphosphatidylinositol (Simons and Ikonen 1997; Shaikh et al. 2003); however, recent studies have shown that PUFAs may also be an important component (Ma et al. 2004; Wassall et al. 2004). In addition to PUFAs, α-syn may associate with other lipids, such as cholesterol, in the membrane rafts. Consistent with this possibility, previous studies have shown that α-syn association with lipid rafts is sensitive to the cholesterol-depleting effects of MβCD (Fortin et al. 2004) and, in our models, the effects of MβCD on α-syn were paralleled by flotillin, a structural protein enriched in the lipid rafts of neurons (Kokubo et al. 2003).
The mechanisms through which MβCD may decrease α-syn accumulation are not completely clear; however, it is possible that these effects may be directly related to the cholesterol-depleting capabilities of this compound, which, in turn, results in the redistribution of α-syn aggregates from the membrane to the soluble fractions, as demonstrated by the studies with sucrose gradients. However, other indirect mechanisms, such as decreased α-syn expression or increased degradation, may play a role. Alternatively, changes in lipid levels may alter α-syn immunoreactivity in an epitope-dependent manner; however, a previous study has shown that MβCD modifies the distribution of green fluorescent protein-tagged α-syn (Fortin et al. 2004). As MβCD did not affect α-syn mRNA expression or monomeric α-syn levels in the cytosolic fractions, and we did not observe increased generation of C-terminal α-syn degradation products, it is more likely that MβCD effects on α-syn are lipid dependent. In this regard, it is possible that decreased cholesterol in lipid rafts and changes in the membrane composition and fluidity may affect the translocation and transport of α-syn to the membrane. Supporting this possibility, previous studies have shown that MβCD decreases cholesterol levels in the membrane and enhances the membrane fluidity, which, in turn, causes changes in the functional properties of the membrane and in signal transduction (Gniadecki 2004; Larbi et al. 2004). Furthermore, cholesterol extraction from the membrane may interfere with α-syn interactions with PUFAs, which participate in α-syn membrane translocation. Under basal conditions, a critical balance between PUFAs and cholesterol may regulate α-syn location to lipid rafts in synaptic membranes; however, the physiological consequences of this process on synaptic transmission are unclear.
In PD, it is possible that this lipid balance in the membrane is altered, resulting in the excessive accumulation of α-syn and the formation of toxic oligomers in the membrane. Supporting this possibility, Sharon et al. (2003a) reported the presence of elevated PUFA levels in PD and DLB brain soluble fractions, and more elevated PUFA levels in membrane fractions, accompanied by increased membrane fluidity in α-syn over-expressing neurons. However, cholesterol levels in the neurons and in PD and DLB brains are unknown. In addition to the effects on membrane cholesterol, MβCD may block α-syn accumulation by interfering with post-transcriptional modifications of α-syn, such as phosphorylation. Amongst other potential kinases, α-syn is phosphorylated at serine 129 by casein kinase II (Lee et al. 2004), and this results in increased aggregation and toxicity (Chen and Feany 2005). Consistent with this possibility, we found that treatment with MβCD also decreased the levels of phosphorylated (serine 129) α-syn in the membrane and insoluble fractions. These effects may be related to the decreased availability of α-syn to kinases because of the effects of MβCD on the membrane cholesterol levels, increased phosphatase activity or decreased kinase activity. As the effects of MβCD on phosphorylated α-syn were comparable with those of total α-syn, it is most probable that further studies will be necessary to investigate which of these potential targets is affected by MβCD and how they are affected.
In the present study, we also showed that MβCD decreased α-syn accumulation in vivo and ameliorated the neurodegenerative alterations in tg mice. These effects on α-syn tg mice are consistent with previous studies showing that CDs can rescue the disease phenotype in other in vivo models of neurological disorders, such as Niemann–Pick type C1 disease (NPC1) (Camargo et al. 2001; Yu et al. 2005). NPC1 is a disorder characterized by a defect in cholesterol trafficking and metabolism resulting in lysosomal pathology and progressive neurodegeneration (German et al. 2002; Gondre-Lewis et al. 2003; Walkley and Suzuki 2004; Chang et al. 2005; Li et al. 2005). Interestingly, recent studies have shown that α-syn accumulates in the brains of patients with Niemann–Pick disease and in NPC1 mutant mice (Mori et al. 2002; Saito et al. 2004). Together, these studies support the concept of a role of cholesterol metabolism in α-syn accumulation and of MβCD as a potential treatment for disorders with parkinsonism and α-syn accumulation (Camargo et al. 2001; Yu et al. 2005).
Notably, alterations in cholesterol levels have been associated with an increased risk for AD, mainly because cholesterol may participate in the regulation of Aβ production (McLaurin et al. 2002; Yanagisawa 2002; Burns et al. 2003; Michikawa 2003; Hartman 2005) and Aβ has been shown to accumulate in lipid rafts (Wood et al. 2002). Consequently, several cholesterol-reducing drugs, including CDs, are currently being evaluated for the treatment of AD (Simons et al. 1998; Cole et al. 2005). Similarly, it is possible that cholesterol may also play a role in LBD. The evidence in this respect is not yet conclusive; for example, some studies have indicated that α-syn associates with cholesterol-related lipid rafts (Fortin et al. 2004), and have suggested that high levels of cholesterol intake may be associated with increased risk for PD (Johnson et al. 1999). In contrast, a recent study has found that a high intake of unsaturated fatty acids may protect against PD, but no significant association has been found with saturated fat, cholesterol or trans-fat intake (de Lau et al. 2005). Furthermore, the direct effects of high cholesterol levels on α-syn accumulation and membrane structures are not entirely clear and require further investigation.
In conclusion, the present study has shown that cholesterol depletion by MβCD treatment may decrease the levels of potentially toxic, detergent-insoluble α-syn which associates with the membrane. Although further studies are necessary to elucidate the mechanism of action of CDs and the interaction between α-syn and cholesterol, cholesterol-lowering agents may be considered as potential treatments for PD.