[1] 6w a-Synuclein oligomerization: a role for lipids?

[1] 97w a-Synuclein is a core component of the proteinaceous aggregates observed in several neurodegenerative diseases. A central role of a-synuclein in neurodegeneration was demonstrated by the discovery of missense a-synuclein mutations in familial Parkinson's disease. However, the specific mechanism by which a-synuclein contributes to these diseases remains unclear. A recent study by Sharon et al. linked the presence of specific fatty acids to the appearance of a-synuclein oligomers in vivo. a-Synuclein oligomers might be a first step in the formation of a-synuclein aggregates present in a number of neurodegenerative diseases, although their cytotoxicity remains to be directly demonstrated.

[1] 193w Although the possible existence of a-synuclein oligomers in vivo is intriguing, we still do not know how cytotoxic they are or whether they form protofibrils in the same way as those described in in vitro systems [2,6,7]. The possible toxicity of a-synuclein oligomers does not preclude a toxic insult from monomeric a-synuclein, a larger a-synuclein homomultimer, or from a-synuclein in complex with other proteins. The presence of a toxic protein complex of a-synuclein and 14-3-3 has been suggested by Xu et al. [20], who described dopaminedependent a-synuclein-induced apoptosis in dopaminergic neurons. No a-synuclein protofibrils were observed in the presence, or absence, of dopamine in their cell lines. It might be interesting to re-investigate the possible presence of a-synuclein oligomers with Lipidex 1000 treatment in the system used by Xu et al. [20] -this might allow examination of functional roles of a-synuclein oligomers and the impact of dopamine metabolism on the potential cytotoxicity of a-synuclein oligomers. It is interesting to speculate that the selective death of dopaminergic neurons in Parkinson's disease is caused by a combination of cellular stress from dopamine metabolism and a-synuclein oligomers that might include other protein complexes as constitutive components.

[1] 74w a-Synuclein has notoriety for being implicated in many neurodegenerative diseases. A central role for this protein in Parkinson's disease has been established with the discovery of two Parkinson's disease familial mutations. a-Synuclein is a primary component of Lewy bodies, as well as of abnormal proteinaceous aggregates present in Parkinson's disease, dementia with Lewy bodies, multiple system atrophy and other diseases [1]. However, the specific mechanism by which a-synuclein contributes to these diseases remains unclear.

[2] 94w Oligomerization of a-synuclein a-Synuclein has been found in insoluble aggregates in neurodegenerative diseases. One notion gaining support is that the primary insult leading to the deaths of relevant cell populations is formation of a-synuclein prefibrillar oligomers, rather than formation of the insoluble fibrils that are the hallmarks of many neurodegenerative diseases [2]. This phenomenon might not be confined to a-synuclein but, rather, common to soluble oligomers in general: for example, b-amyloid (Ab) soluble oligomers in Alzheimer's disease [3], and even soluble oligomers formed by non-disease-associated proteins, have been shown to be inherently cytotoxic [4].

[3] 196w Evidence supporting this notion comes mostly from in vitro experiments. The formation of oligomeric species of a-synuclein in vitro was found to parallel that of Ab, nonfibrillar oligomers of which are toxic in cell culture [2] and can disrupt cellular processes in vivo [5]. Further in vitro experiments showed that the Parkinson's-disease-related a-synuclein mutations (A30P and A53T) promote the formation of fibrillar and non-fibrillar aggregates, and 20 -25 molecules of a-synuclein were found to form oligomers with pore-like morphologies [6]. Noting that the A30P and A53T mutations cause an earlier appearance of these structures than that seen with wild-type a-synuclein, Volles and Lansbury [7] posit that such a structure might be responsible for permeabilization of membranes by prefibrillar a-synuclein, in in vitro assays and possibly in in vivo conditions leading to the disease. Although this is intriguing, the formation of a-synuclein pre-fibrillar aggregates resembling pores has yet to be demonstrated in a cellular context. Gosavi et al. [8] provide correlative evidence of the disruption of Golgi complex with the appearance of a-synuclein aggregates, but the nature of the a-synuclein aggregates is unclear and the role of monomeric a-synuclein, or larger a-synuclein aggregates, cannot be ruled out.

[4] 115w The formation of a-synuclein oligomers can be affected by neurological injuries. Oxidative injuries, which are linked to many neurodegenerative diseases, can affect a-synuclein in several ways, including the nitration of tyrosine residues (nitrated a-synuclein has been observed in Lewy bodies) or the formation of a dopaminea-synuclein adduct [9,10]. Consistently, both of these phenomena have effects on the formation of a-synuclein oligomers in vitro. Nitration induces the formation of a-synuclein oligomers and stabilizes a-synuclein polymers [9], although it can lead to the accumulation of oligomerized but pre-fibrillar species of a-synuclein [11], whereas dopamine reduces the amount of fibrillar a-synuclein in a cell-free system, possibly stabilizing a pre-fibrillar species of a-synuclein by forming a dopaminea-synuclein adduct [10].

[5] 312w The in vitro association between a-synuclein and lipid membranes, and particularly small vesicles, is well established. Although a-synuclein is generally cytosolic in its distribution, it is enriched in synaptosomal fractions of mouse and human brain, and similarities between the N terminus of a-synuclein and the lipid-binding domains of some apolipoproteins suggest a role for a-synuclein interactions with lipid membranes [12]. Further data in support of a lipida-synuclein interaction were observed by Sharon et al. [13], who reported lipid-binding proteinlike domains in the N-terminal and C-terminal regions of a-synuclein, and observed high-molecular-weight a-synuclein complexes following delipidating treatments of cytosolic fractions from mouse brain and from a mesencephalic neuronal cell line (MES cells). Sharon et al. [14] have provided evidence that these high-molecular-weight reactive bands are in fact a-synuclein multimers, rather than a-synuclein in a heterogeneous protein complex, using 2D gel electrophoresis and size-exclusion chromatography of extracts from a-synuclein-expressing MES cells and mouse brains. Furthermore, in a comparison of MES cells stably expressing wild-type and A53T a-synuclein, Sharon et al. [14] found that the A53T mutation caused the appearance of oligomeric a-synuclein significantly earlier than the appearance of equivalent amounts of a-synuclein multimers in cells expressing wild-type a-synuclein. The apparent larger multimers they observed are strongly reminiscent of the high-molecular-weight a-synuclein-reactive bands seen in brain tissue of individuals afflicted with synucleinopathies such as Parkinson's disease, multiple system atrophy and possibly Alzheimer's disease [15,16]. Sharon et al. concluded that delipidation revealed pre-existing a-synuclein oligomers, although it remains to be rigorously ruled out that removal of lipids did not promote oligomerization by exposing previously hidden epitopes. The requirement of high temperature (378 C) treatment with Lipidex 1000 is intriguing because such treatment at 08 C is sufficient to bind free lipids, whereas at 378 C Lipidex 1000 binds both free and proteinbound lipids [17]. The presence of other proteins in these high-molecular-weight complexes cannot be discounted.

[6] 165w Multimerization of recombinant a-synuclein in vitro can be promoted by the treatment of long-chain polyunsaturated fatty acids (PUFAs) [18]. In keeping with the observations of Perrin et al. [18], Sharon et al. [14] cultured MES cells stably expressing human wild-type and mutant a-synuclein in the presence of a variety of fatty acids, and found that the formation of a-synuclein multimers correlated well with the length and degree of saturation of fatty acids in a time-dependent manner. They observed that long chain PUFAs promoted a-synuclein multimerization, whereas saturated fatty acids decreased the levels of a-synuclein multimers; mono-unsaturated fatty acids had no discernable effect. In what could be a capsule summary of a-synuclein aggregation in Parkinson's disease, the authors observed the formation of a-synuclein oligomers as early as one hour after adding PUFAs to MES cell culture media, with amounts of oligomers increasing in a time-dependent fashion, until at later time-points the levels of a-synuclein oligomers diminished again, as very high-molecular-weight and gelexcluded material began to appear.

[7] 126w How might PUFAs promote a-synuclein oligomerization? In the study by Perrin et al. [18], a concentration of arachidonic acid in excess of the critical micelle concentration (CMC) was necessary to detect a significant increase in a-synuclein multimerization. Possibly a-synuclein requires a micellar or vesicular surface to provide a focal point for the initiation of oligomerization. However, Sharon et al. [14] observed a-synuclein multimerization in the presence of PUFA concentrations as low as half the CMC, questioning a requirement for a micellar or vesicular surface for a-synuclein oligomerization. How a-synuclein oligomerization might be mediated by free fatty acids is unclear. Longer and increasingly unsaturated fatty acids, even in free and non-micellar form, might promote an a-synuclein conformation that is more favorable for oligomerization with other a-synuclein molecules.

[8] 83w What is the significance of the effect of PUFA on a-synuclein oligomerization? If the accumulation of a-synuclein oligomers correlates with cytosolic PUFA levels in brains of patients with synucleinopathy, we could speculate that PUFA-induced formation of a-synuclein oligomers might be a key nucleation step that leads to the subsequent formation of pre-fibrillar and fibrillar a-synuclein. If this is so, it might be possible to manipulate fatty acids in the brain in such a way as to reduce the formation of toxic a-synuclein species.

[9] 115w Consistent with their in vitro observation, Sharon et al. [14] report that the expression of human a-synuclein in transgenic mice leads to a-synuclein multimerization in older animals. The age-related accumulation of human a-synuclein multimers exposed by delipidation in transgenic mice is suggestive of a role for a-synuclein in neurodegeneration. It is tempting to interpret this agerelated accumulation of a-synuclein oligomers as recapitulating human Parkinson's disease. However, the presence of a-synuclein multimers in normal mice as young as two-months old has been demonstrated using a commercial lysis buffer [19]. It will be interesting to compare the effects of these different extraction buffers side by side and determine crucial factors that affect the detection of a-synuclein oligomers.

[10] 117w Finally, Sharon et al. [14] detected multimeric a-synuclein in normal human brain and in human brain samples from individuals afflicted with Parkinson's disease or dementia with Lewy bodies. As was the case for a-synuclein transgenic mice, the detection of a-synuclein oligomers was enhanced by delipidating treatment. Furthermore, Sharon et al. detected a twofold increase in the ratio of a-synuclein dimers to monomers in brain samples from individuals with Parkinson's disease or multiple system atrophy relative to the ratio in control brains. These data suggest that some of the wild-type soluble a-synuclein in the brains of the diseased individuals might have already oligomerized, or might have a higher tendency to oligomerize than the a-synuclein of normal human brain.