Our data show that overexpression of α-synuclein impairs macroautophagy. This provides a mechanism whereby copy-number mutations of α-synuclein contribute to PD. Because sporadic PD is also associated with α-synuclein accumulation, our data may have much wider implications.
Our findings appear specific to wild-type α-synuclein, as the PD-associated point mutants A53T and A30P had no effect on LC3-II levels (Fig. S1 A). This may be because either A53T and A30P have no effects or have different effects on autophagy compared with the wild-type protein. Alternatively, these mutants do impair autophagy in a similar way to the wild-type protein, but their effects are masked by their known inhibition of CMA (Cuervo et al., 2004
), which leads to a compensatory increase in autophagosome formation.
We have found that overexpression of wild-type α-synuclein in vitro and in vivo inhibits autophagosome synthesis, as determined by a range of different specific assays. These assays reveal a decrease in omegasome formation, a decrease in LC3-II lipidation, impaired LC3 vesicle formation, and accumulation of autophagy substrates (mutant huntingtin and p62). When viewed in the context of previous data showing that α-synuclein affects Rab1a function (Cooper et al., 2006
), we were interested in determining whether α-synuclein acted via Rab1a on autophagy. We found that knockdown of Rab1a mimicked the effects seen by α-synuclein overexpression, by decreasing omegasome formation, decreasing LC3-II levels, and increasing autophagy substrates. Importantly, Rab1a expression was able to rescue the inhibitory effects of α-synuclein on both omegasome and LC3 vesicle formation.
We believe that α-synuclein and Rab1a exert their control on autophagy via a very early step in the initiation of autophagosome synthesis, as Atg9 was found to be mislocalized with α-synuclein overexpression and Rab1a knockdown. Atg9 knockdown itself reduced both omegasome formation and LC3-II levels, indicating that Atg9 controls a step in autophagosome formation upstream of omegasome formation.
These findings are important for understanding PD molecular pathogenesis and further understanding the regulation of autophagy. Our data identify a new role for Rab1a in the regulation of autophagy. The effects we observe are not simply caused by a generic impairment of ER–Golgi transport because other Rabs which mediate this step do not impair autophagy when knocked down. Furthermore, the robust autophagy inducer trehalose (Sarkar et al., 2007
) appears to decrease secretion (Fig. S2 C), suggesting that bulk secretion can be uncoupled from autophagy induction. Similarly, the absence of changes in Atg9 glycosylation with Rab1a knockdown suggests that Atg9 transport, at least from the ER to the Golgi, is not overtly impaired. Our data are consistent with a model whereby other molecules trafficked via Rab1a (but not Rab2 or Rab1b) modulate Atg9 trafficking, which, in turn regulates omegasome formation.
Interestingly, in yeast, the Golgi-associated COG (conserved oligomeric Golgi) complex is important for autophagosome assembly and appears to also influence Atg9 trafficking (Yen et al., 2010
). It will be informative to assess this process in mammalian cells and to also test whether specific perturbation of later steps in the secretory pathway affects autophagy in mammalian cells. However, it is important to reiterate that our data suggest that certain secretory routes (e.g., Rab1b and Rab2) can be impaired without decreasing autophagosome synthesis, whereas pathways regulated specifically by Rab1a and its effectors are critical for autophagy. Thus, either Rab1a influences autophagy via a specific Rab1a-related secretory route or may be acting via a novel, secretory-independent function of Rab1a.
Although our data suggest that specific secretion-associated components regulate autophagy, recent publications have identified an interesting situation in which autophagy regulates a specialized form of secretion. The autophagosome participates in the secretion of acyl coenzyme A–binding protein (AcbA) in starvation conditions in various species of yeast by bypassing the vacuole and fusing with the plasma membrane. This process involves the Golgi-associated proteins (GRASPs), autophagy proteins, plasma membrane SNARES, the endosomal compartments, and peroxisome activity (Duran et al., 2010
; Manjithaya et al., 2010
). These studies raise the possibility of a reciprocal interplay of the secretory pathway and the nonvacuole–lysosomal pathway. Although our findings involve the autophagosome–lysosome pathway and a secretory-independent function of Rab1a, it will be important to further elucidate the interaction of the secretory and autophagy pathways within mammalian cells and how a new Rab1a-specific regulation of autophagy fits into the intersection of these pathways.
The impairment of Rab1a function by overexpression of α-synuclein will have cumulative effects on both the secretory pathway, as shown in and previously (Cooper et al., 2006
), and on autophagy. Although the effects we observe of α-synuclein on secretion within mammalian cells are reproducible and consistent, these effects appear to be relatively minor compared with the effects we observe of α-synuclein and Rab1a on autophagy. Cumulatively, these assays show a 30–50% decrease in autophagosome synthesis. Although this does not represent a complete block of autophagy (which would be lethal; Tsukamoto et al., 2008
), a partial block of the magnitude we observed, or smaller, would be likely to manifest significant consequences over many decades in postmitotic neurons. It is of interest to note that reduction of α-synuclein levels enhances autophagy. Thus, it is likely that even small increases (or decreases) in the levels of this protein will modulate autophagy. The cumulative effects of a partial inhibition of both the secretory pathway and autophagy may explain dopamine secretion defects and proteinaceous accumulation and cell death, respectively.
Overall, this situation appears to represent a specific example of how the proteostasis of the whole cell can be disrupted by a specific aggregate-prone protein, a model which was proposed by Gidalevitz et al. (2006)
in another disease context. In this case, the culprit, α-synuclein, impairs autophagy, a major route for the clearance of aggregate-prone intracytoplasmic proteins, and this in turn increases the concentration of such proteins and increases their probability of aggregation. However, the predicted outcome of reduced autophagic clearance will have pleiotropic effects. In addition to the accumulation of aggregate-prone proteins, the cells will not be as effective in clearing dysfunctional mitochondria (Twig et al., 2008
) and will have increased susceptibility to certain apoptotic insults (Ravikumar et al., 2006
); these are all processes that have been implicated in PD (; Cookson and van der Brug, 2008
). Thus, autophagy inhibition caused by wild-type α-synuclein may provide a unifying mechanism for many of the apparently disconnected cellular pathologies in PD. It is interesting to consider that there may be compensatory regulation of CMA and macroautophagy when one of these pathways is perturbed (Martinez-Vicente et al., 2008
). However, if one considers that both macroautophagy and CMA are inhibited by wild-type α-synuclein (Martinez-Vicente et al., 2008
), this is likely to be particularly deleterious.
Importantly, our proposed mechanism for the inhibitory role of α-synuclein on autophagy furthers our understanding of the disease (). Although the inhibition of early secretion by α-synuclein may explain the dopaminergic defects seen in PD, it does not sufficiently explain the formation of inclusions and cell death characteristic of PD. In principle, multiplications of the α-synuclein locus leading to increased protein levels of α-synuclein will inhibit autophagy. The inhibition of autophagy increases accumulation of aggregate-prone proteins and sensitizes the cell to proapoptotic assaults. Increased aggregation and apoptosis are characteristic of PD, and therefore, inhibition of autophagy by α-synuclein overexpression may further explain the pathologies characteristic of PD. Collectively, the ability of α-synuclein to inhibit both autophagy and secretion may act as a potent impetus for neurodegeneration.
Figure 8. Cumulative molecular effects of α-synuclein overexpression. Diagram illustrating the pleiotropic effects of increased intracellular levels of α-synuclein on vital intracellular pathways. The effects of α-synuclein on macroautophagy (more ...)