Aspects of protein chemistry of α-synuclein and toxicity
α-Synuclein has a strong tendency to self-associate
in vitro [
110,
111], and so a prime candidate for the underlying driving force for toxicity is the formation of aggregated species. One of the important questions about this idea is which species are present in cells/tissues. Oligomeric species can be isolated from cells [
112‐
114] and from human [
21] and mouse (both wild type and α-synuclein transgenic) brain [
115]. In both cells and brain, oligomers are particularly found in membrane-enriched fractions [
112,
115], suggesting a possible influence of the lipid environment on oligomer formation. Higher molecular weight forms have also been found in some models [
116], especially after oxidative stress [
117] or exposure to inflammatory triggers in mice [
100]. Deposited α-synuclein immunoreactivity has been seen in transgenic [
91‐
97] or viral models [
102‐
109]. However, the observation of aggregated α-synuclein by and of itself does not prove that aggregation is important; as discussed for Lewy bodies, all this proves is that deposition occurs, not that it is causal.
Some recent studies have attempted to answer this question, mainly using cell-based approaches. For example, some oligomeric forms of α-synuclein trigger calcium entry and toxicity in SY5Y cells [
118]. Interestingly, different species show differential toxicity, suggesting that not all oligomers are created equal. However, the nature of this experiment is to add α-synuclein to the outside of the cell, which may or may not be relevant to the pathophysiological situation. As α-synuclein is intracellular, it seems more likely that the protein would form aggregate inside cells. The presence of fibrils in Lewy bodies would support this contention. However, α-synuclein can end up in the extracellular media [
119] and it is possible that the conditions for aggregation might be more suitable in a milieu free of cells. The relevance of extracellular α-synuclein is an important question, raised also by the observation of Lewy bodies in grafted neurons [
41,
42] and the attendant hypothesis of 'host to graft transmission'.
Some studies have attempted to address whether intracellular aggregates of α-synuclein contribute to toxicity. For example, several imaging techniques shown that, in the context of a living cell, α-synuclein can form small oligomers, likely in an antiparallel configuration [
114,
120] and such oligomers can be associated with cell toxicity.
These approaches have been used to show that overexpression of heat shock proteins (Hsps) can mitigate both oligomer formation and toxicity [
114,
120,
121].
In vivo, Hsps can prevent toxic effects of α-synuclein in yeast [
59] and in flies [
67]. Whether these studies constitute formal proof that aggregation is required for toxicity is unclear as there are other theoretical interpretations of the data. For example, a formal possibility is that monomeric α-synuclein is toxic and, thus, any protein binding the protein directly could limit toxicity. It should be stated that the mechanism(s) by which monomers of α-synuclein could be toxic are relatively unexplored but, equally, there is an absence of proof that aggregation is absolutely required for toxicity. Alternatively, Hsps could be limiting a detrimental event downstream of the initial aggregation and thus may neither represent evidence for or against the role of aggregation in α-synuclein toxicity. Interestingly, Hsp expression in the fly model decreases neuronal toxicity without any change in the number of α-synuclein positive inclusions [
67].
Overall, these considerations show that α-synuclein is capable of protein aggregation and can be deposited into inclusion bodies of various forms
in vivo, but that there is insufficient evidence that aggregation or deposition is either necessary or sufficient for toxicity. In fact, several lines of evidence show that toxicity can be dissociated from deposition, including; the observation in cells of toxicity without deposition in some models [
81]; differential effects on toxicity and inclusions of various manipulations of α-synuclein in fly models [
66,
67]; and deposition of α-synuclein without clear toxic effects in some mouse models [e.g., [
36]]. A key challenge for the field, therefore, is to understand whether protein aggregation is at all relevant for the toxic effects of α-synuclein. One way to potentially address this is to isolate various aggregated species of the protein and to express them within a neuron. This might be extraordinarily difficult from a technical standpoint and there is always possibility that the small aggregates would seed larger ones may confound interpretation. Another potential approach would be to develop reagents that limit the biological availability of specific aggregated species and use these to probe which agents are toxic in intact cells. As an example, recombinant single chain Fv antibody fragments against aggregated α-synuclein have been described [
122,
123] that might be helpful.
α-Synuclein has many additional properties as well as the tendency to aggregate. Some of the post-translational modifications that have been reported have also been explored as possible mediators of toxicity. For example, antibodies against α-synuclein phosphorylated at Ser129 are very good at identifying Lewy pathology in the human brain [
38], suggesting either that Ser129 phosphorylation is a causal event for deposition or represents a common modification of the protein after it is deposited. Several groups have therefore made versions of α-synuclein that cannot be modified at this residue (S129A) or pseudo-phosphorylation mimics (S129D, S129E) and determined the toxic effects of expression. In
Drosophila models, S129A is less toxic but has an increased tendency to form inclusion bodies compared to wild type protein [
66]. The S129D phosphomimic has the opposite effect, i.e. increased toxicity but fewer inclusions. In contrast, similar experiments using viral overexpression in rats show the opposite result, namely that S129A greatly increases the toxic effects of expression [
124]. In mammalian cell culture, S129A has a diminished tendency to form inclusion bodies [
125].
At first glance, these results seem to suggest that the behavior of α-synuclein as it relates to toxicity is opposite in mammals compared to invertebrates where, it is important to note, the protein is not normally present. However, interpretation is complicated by several considerations. First, the expression levels of α-synuclein are critical for toxicity, which is shown by the human case where a difference in protein levels is 2-fold in the triplication cases and 1.5-fold in the duplication cases. Second, recent data suggests that the phosphomimic S129D/E α-synuclein variants have different biophysical properties compared to authentically phosphorylated wild type protein [
126]. Overall, these considerations raise some important
caveats about comparison of properties of α-synuclein in terms of concentration-dependent behaviors of the protein such as aggregation and toxicity.
One alternate approach to understand α-synuclein phosphorylation is to identify the kinase that mediates the phosphotransfer event. Casein kinase II and GRK2/5 have been shown to phosphorylate α-synuclein
in vitro or in cells and work in yeast [
64] and flies [
66] respectively shows that they are at least active
in vivo. More recently, the polo-like kinase family, specifically PLK2, have been shown to be active both
in vitro and
in vivo in generating pS129 α-synuclein [
127]. What is interesting about PLK2 is that it is known to respond to neuronal activity [
128], suggesting a possible link between neuronal phenotype and α-synuclein toxicity. However, it is not yet known in PLK2 inhibitors or gene knockout will limit the toxic effects of α-synuclein
in vivo. Such experiments are feasible in several species as PLK2 homologues are present in mice and flies, and there is at least one polo kinase in yeast.
There are a number of other modifications of α-synuclein that have been reported and some of these are found more often in pathological circumstances than under normal conditions, such as nitration or truncation. Truncation of α-synuclein is associated with a higher tendency for aggregation [
129‐
131]. Transgenic mice expressing truncated α-synuclein have substantial cell loss [
101] although in at least one line, this is a developmental and not degenerative phenotype [
132]. Again, because the window for toxicity is quite narrow, comparison between different lines is difficult. One question that arises for truncation is where such species are generated. α-Synuclein is predominantly degraded by lysosomal pathways [
133,
134], including chaperone-mediated autophagy [
135], and the lysosomal cathepsins are important in proteolysis. Therefore, some truncated species are found in the lysosomes and it seems unlikely that they would cause damage to the cell. However, α-synuclein is also a substrate for cytoplasmic calpains [
136‐
139], which are therefore more likely to generate cytoplasmic toxic truncated species. Some detail is therefore needed to prove which truncated species mediate toxicity, if any of them in fact do.
Oxidative stress, including the neurotransmitter dopamine, has been linked to increased α-synuclein aggregation [
89,
140]. Dopamine itself may contribute to the toxic effects of α-synuclein
in vitro [
89], although such a mechanism cannot explain why non-dopaminergic neurons die early in the disease process. α-Synuclein expression can enhance sensitivity to oxidative and nitrative stressors [
141,
142], although it can also be protective in some situations [
143]. In most of these situations, the role of aggregation is unclear.
In summary, α-synuclein has properties, including the potential for aggregation and post-translational modifications, which may influence its toxic effects. Whether these are required for toxicity is unclear, and some results still need to be resolved, for example for the work on S129 phosphorylation. However, there is a larger question, which is: what effects synuclein has on neurons that are responsible for its toxic effects?
Some of the relevant data from cellular systems has been reviewed previously [
144] and will be discussed here in the context of examples across multiple models.
Presumably, α-synuclein might interact with other biomolecules to mediate toxicity. Because α-synuclein can associate with lipids, membranes are one possible target.
In vitro, α-synuclein can form pore-like structures [
145,
146], and annular rings of synuclein have been isolated from the brains of patients with multiple system atrophy, a synucleinopathy [
147]. Cells expressing α-synuclein have increased cation permeability [
148] and vesicles prepared from cultured cells or isolated from the adrenal medulla show leakage of catecholamines [
149]. These events may be consistent with the formation of non-specific pores or similar structures at the plasma membrane or at a vesicle surface.
Because α-synuclein binds synaptic vesicles, it is possible that synaptic transmission would be directly or indirectly a target of synuclein toxicity. One example of this comes from work showing that A30P α-synuclein alters exocytosis of catecholamine containing vesicles in primary cells and in chromaffin cells [
150]. The effect here is probably at a late stage of the exocytosis, before vesicle membrane fusion [
150].
Further evidence for an effect of α-synuclein on vesicle function that may mediate toxicity comes from suppressor screens in yeast [
63]. In the same organism, such defects can be localized to a block in endoplasmic reticulum (ER)-golgi vesicular trafficking [
151]. Supporting this idea, there is evidence of ER stress [
87] and golgi fragmentation [
152] in mammalian cell systems.
Overexpression of Rab1, a GTPase that influences vesicle dynamics, was able to at least partially rescue the toxic effects of α-synuclein in yeast, worms and in mammalian cells [
151]. Therefore, some of the toxic effects of α-synuclein that are conserved across species involve damage to vesicular transport, which might express itself as damage to presynaptic vesicle release in a neuron.
There are also suggestions that other membranous organelles are affected by α-synuclein, including mitochondria [
87,
88,
153]. Recent data suggests that a portion of α-synuclein can localize to mitochondria, at least under some conditions [
154‐
157]. Supporting this are observations that α-synuclein expression increases cellular organismal sensitivity to rotenone, a mitochondrial complex I inhibitor [
78,
158]. Furthermore, intact mitochondrial function is required for a-synuclein toxicity in a yeast model, although it should also be noted that removal of mitochondria is also quite damaging in the same context [
57]. The mechanism by which α-synuclein interacts with and causes damage to mitochondria is not fully resolved and, given the central role of mitochondria in apoptotic pathways, perhaps such effects are secondary to the induction of apoptosis. Increased levels of α-synuclein are reported to trigger apoptosis in various cell types [
159‐
161]. Several apoptotic markers are also seen in yeast models of synuclein toxicity [
59]. α-Synuclein toxicity can be rescued by caspase inhibitors or knock down of caspase-12 [
87]. Activation of caspase-3 has been reported in transgenic mice [
162] caspase-9 has been reported in viral models in mice [
102] and rats [
106]. However, these studies show only a few caspase positive cells, and so whether apoptosis is the only way in which cells expressing α-synuclein die remains unclear.
α-Synuclein can bind to the membranes of lysosomes [
135] and inhibit lysosomal function [
163] and chaperone-mediated autophagy [
135]. Recent results suggest that CMA is implicated in the regulation of the transcription factor MEF2D and that this can be disrupted by expression of α-synuclein, leading to neuronal death [
164]. As another example of misregulated protein turnover, α-synuclein (and specifically α-synuclein oligomers) can also inhibit the proteasome [
81,
88,
163,
165‐
167], although it is not clear if the predicted altered turnover of proteasome substrates occurs
in vivo [
168].
The general principle is that multiple systems can be affected by α-synuclein expression and that if there is a common theme between them, it is likely to be that α-synuclein can binds lipids. Several lines of evidence suggest that lipid binding can promote the formation of oligomers [
115,
145,
169]. Therefore, this interpretation links a primary protein abnormality to cellular targets of the protein. As discussed elsewhere [
144], determining which events are truly primary and which are secondary remains a challenge. Although this distinction is an intellectual problem, it may also be relevant for deciding which aspects of cell death to target if we want to limit the disease process in PD.