As discussed briefly above, a major theory regarding α-synuclein is that it is prone to form aggregates (Cookson, 2005
). The aggregation process initially forms oligomeric species that are relatively soluble; these oligomers might then self-assemble into fibrillar structures that are insoluble. A major, but untested, concept is that the process of fibrillization is important to the formation of Lewy bodies as these organized structures become deposited (Mukaetova-Ladinska and McKeith, 2006
). Such inclusion bodies would represent one endpoint of the aggregation pathway but may or may not be the toxic species themselves. A possible culprit for toxicity is the transient oligomeric species formed along the way (Volles and Lansbury, 2003
). Most of this work has been developed from systems using recombinant proteins (reviewed in Uversky, 2003
What have cell models told us about aggregation? Higher order species that might be equivalent to oligomers have been seen in N27 cells expressing A53T α-synuclein (Zhou and Freed, 2005
) or in MES neurons exposed to polyunsaturated fatty acids (Sharon et al., 2003
). When aggregated species are present, they can be identified using chemical crosslinking methods and are associated with lipid membranes (Cole et al., 2002
). However, in most cell models the recovered α-synuclein is predominantly monomeric on SDS-PAGE gels and relatively soluble. This means that the species found in cells are not the types of heavily aggregated, insoluble and deposited forms seen in human brain. In turn, this limits our ability to interpret the cell models in light of aggregation pathways thought to occur in the brain and makes it hard to assess whether, for example, oligomers are the toxic species. A novel approach to this problem is to use fluorescence lifetime imaging-based techniques to probe the organization of α-synuclein within the cell, which has suggested that small oligomers are formed in a head-to-tail orientation (Klucken et al., 2006
). It is clear that developing additional novel techniques to address the toxic effects of oligomers is critical to assessing this hypothesis. For example, there are antibodies against other oligomeric proteins associated with neurodegeneration and similar reagents for α-synuclein might be extremely useful.
Some models do report the presence of α-synuclein-positive inclusion bodies, which presumably represent more aggregated and less soluble forms. In some studies, adding extra stressors increases the frequency of inclusions, such as the mitochondrial inhibitor rotenone (Lee et al., 2002
), or co-transfection with other proteins including parkin and synphilin-1 (Engelender et al., 1999
). These inclusion bodies might represent aggregated forms of α-synuclein although whether they are equivalent to mature Lewy bodies is not clear.
Perhaps looking for mature Lewy bodies in cells is too much to ask – there is scant evidence for Lewy body formation in some of the animal models (reviewed elsewhere in this volume). What these considerations mean is that we aren’t sure of the toxic species in many of the experiments that will be discussed. We know that α-synuclein can
form oligomers and larger aggregates in cells and in some circumstances these can also form inclusion bodies. However, by definition oligomers are a transient intermediate and are therefore difficult to see so it is still reasonable to question whether α-synuclein aggregation is either sufficient to explain its toxic properties or whether it is a necessary part of its toxicity. An approach that might be useful is to generate artificial mutants with radically different properties and correlate specific properties with toxicity. Such an approach has recently been tried in yeast and has indicated that the rate of synuclein fibrillization did not affect toxicity (Volles and Lansbury, 2007
). This, along with other considerations, supports the idea that the fibrillar forms themselves are not the toxic species, but leaves open the idea of whether oligomers may be critical.
Leaving aside evidence about aggregation, what would be the next immediate effect of having oligomers in the cell? Two hints are that (A) some oligomeric species are annular or pore-like and (B) α-synuclein is a lipid-associated protein at least in some structural forms (Volles and Lansbury, 2002
; Zhu et al., 2003
). Therefore, α-synuclein oligomers might form pores on intracellular membranes such as the plasma membrane. Some indirect evidence for this idea comes from observations that cells expressing α-synuclein have increased but non-specific cation permeability (Furukawa et al., 2006
). Although it has not been proven that this is due to partially aggregated synuclein species, these observations would be consistent with the formation of pores.
If pores are forming on the plasma membrane, they might also form on intracellular membranes, providing multiple targets for α-synuclein-toxicity in the cell. Some indirect evidence for this idea comes from studies of vesicles in cultured cells and isolated from the adrenal medulla (Mosharov et al., 2006
). All these conditions show evidence of catecholamine leakage from vesicles and would therefore be consistent with the formation of pores or pore-like structures. Given that α-synuclein is concentrated at synaptic vesicles, it seems reasonable to think that in a neuron there would damage to vesicles containing neurotransmitters. This point will be returned to in the section on synapses.