Here, we have examined the process of α-syn-EGFP accumulation in yeast. We have demonstrated that α-syn accumulation requires an intact N-terminus as well as the NAC domain and that clustered vesicles are integral components of α-syn accumulations similar to those observed in authentic LBs in PD. The consequences of these α-syn accumulations in yeast include the disruption of the Golgi, as well as the disruption of ER-to-Golgi and secretory vesicular trafficking, and cellular toxicity. Importantly, the overexpression of α-syn in yeast cells did not produce LB-like filamentous α-syn amyloid structures, but did recruit the accumulation of vesicles as in PD LBs. Although not a direct model for recapitulating insoluble inclusions containing amyloid α-syn, this model may provide insights into the potential function of α-syn and/or the early steps in the formation of LBs because similar vesicular accumulations are detected in human LBs of PD.
On induction with galactose, α-Syn-EGFP was first localized to the cell cortex. This interaction with membranes appears to be dependent upon the N-terminal α-helical repeats of α-syn, because deletion of this subdomain abrogates this early membrane localization. This is consistent with previous studies demonstrating that these N-terminal repeats bind phospholipids and lipid membranes and may be critical for the normal function of α-syn (Perrin et al., 2000
; Kim et al., 2006
) and that mutations in this region perturbs plasma membrane localization in yeast (Volles and Lansbury, 2007
). Hence, we propose that the binding of α-syn to the yeast plasma membrane may mimic its ability to bind synaptic vesicles in neurons and may be a useful system to study the membrane-binding function of α-syn. Furthermore, we show here that membrane localization of α-syn is required for the subsequent recruitment of vesicles, because the deletion of N-terminal repeats eliminated this membrane localization as well as vesicular clustering.
However, our data also support the view that membrane localization alone is insufficient to cause vesicle clustering because expressing the first 57 amino acids of α-syn is sufficient to cause membrane localization, but not vesicle accumulation. We also determined that deletion of the hydrophobic residues 71-82 within the NAC domain greatly reduced the ability of α-syn to induce vesicular clustering in yeast, without affecting membrane localization. Thus, the formation of vesicular accumulations may depend on the ability of α-syn to form intermolecular interactions through the hydrophobic NAC domain. Alternatively, this region may be important for α-syn to assume a conformation change that facilitates vesicle interaction.
Our EM studies revealed that α-syn-EGFP accumulations are composed of collections of vesicles. These vesicular clusters do not contain fibrils, unlike authentic human LBs. However, alterations in synaptic vesicle numbers due to manipulation of α-syn levels have been reported, including evidence that suppression of α-syn expression in cultured neurons reduced of the distal pool of synaptic vesicles (Murphy et al., 2000
), that α-syn knockout mice demonstrate an impairment in the maintenance of the reserve pool of synaptic vesicles (Cabin et al., 2002
), and that overexpression of α-syn in PC12 cells results in an accumulation of “docked” vesicles at the synapse (Larsen et al., 2006
). α-Syn–induced accumulation of vesicles in yeast may mimic the ability of α-syn to regulate synaptic vesicles in neurons. Therefore, S. cerevisiae
may be a useful system for studying the role of α-syn in the regulation and maintenance of synaptic vesicle pools.
The presence of α-syn–induced vesicular accumulations caused a severe disruption of the organization of the Golgi apparatus, but did not disrupt the ER, vacuole, or endosomes. A previous study showed that expression of α-syn caused disruption of ER-Golgi transport and cytotoxicity (Cooper et al., 2006
), whereas our data suggest that disruption of the Golgi organization may contribute to this phenotype. Golgi fragmentation has been observed in nigral neurons with α-syn–positive LBs, particularly in neurons containing pale bodies that are thought to represent an early stage of LBs formation (Fujita et al., 2006
), suggesting that the Golgi fragmentation caused by α-syn expression in yeast may be relevant to abnormalities in the human disease. Golgi fragmentation has also been observed as a consequence of prefibrillar α-syn aggregates in cell culture (Gosavi et al., 2002
). These two observations, in addition to our data, suggest that α-syn could contribute to toxicity even when it is not in a fibrillar state and that these vesicular accumulations may represent early stages of LB formation as well as PD pathogenesis. Thus, the formation of similar vesicular accumulations in neurons could serve as a concentration point or scaffold for the formation of α-syn filaments and subsequent LB formation.
One interesting observation in this yeast system is that the familial α-syn mutations, A30P and A53T, do not enhance vesicular accumulation. In fact, A30P α-syn does not form vesicular accumulations in yeast. Currently, it is unclear how A30P causes familial PD. The impaired binding to rat brain vesicles (Jensen et al., 1998
) and in yeast cells suggest that it could lead to a reduction in α-syn transport to presynaptic terminals and a loss of function in the asymmetric neuron. On the other hand, A53T α-syn was shown to enhance fibrillization in both test tube studies and in transgenic mouse models of synucleionopathies when compared with WT α-syn (Conway et al., 1998
, Giasson et al., 1999
). Thus, the lack of increased vesicle clustering in yeast cells expressing the A53T mutant suggests that this mutation does not cause disease by increasing vesicular accumulation. A previous report (Cooper et al., 2006
) showed that the A53T mutation caused a CPY trafficking block in yeast at earlier time points than WT α-synuclein. However, there was no reported difference in toxicity between WT and A53T α-synuclein expressing cells, suggesting that this CPY trafficking defect may be independent of vesicular accumulation and toxicity. Alternatively, the difference in CPY trafficking between WT and A53T α-synuclein may be insufficient to cause changes in the size and the number of vesicle clusters. Because α-syn fibrils are not detected in α-syn–expressing yeast cells, our data are consistent with enhanced fibril formation due to the A53T mutation being downstream from vesicle accumulations. Finally, these yeast models of synucleinopathies may represent unique systems to study α-syn disruption of cellular trafficking and toxicity in PD that is independent from α-syn fibril formation.
Although ER-Golgi transport-vesicle markers accumulate in yeast based on the colocalization of α-syn-EGFP with Ypt1 (Cooper et al., 2006
), our EM data suggest that there may be several other types of vesicles in the α-syn-EGFP vesicular inclusions, because vesicles of various sizes are observed. For example, we identified Sec4p, a secretory vesicle associated Rab GTPase within α-syn-EGFP accumulations, suggesting that secretory vesicles are also present. Thus, our data suggest that the α-syn-EGFP accumulations include vesicular accumulations composed of at least two different types of vesicles in the transport and secretion pathway and that α-syn is able to disrupt vesicular organization at two stages of the transport/secretion pathway. However, it cannot be ruled out that the markers themselves are mislocalized and that these are not true secretory transport vesicles. This observation may reflect an exaggeration of the normal function of α-syn, which has been hypothesized to play a role in the organization and recycling of synaptic vesicles. When α-syn is overexpressed or misregulated in humans, it may disrupt these pathways. However, in yeast there are no synaptic vesicles and α-syn may perform similar functions involving other types of vesicles, including secretory vesicles and ER-Golgi transport vesicles.
α-syn–induced vesicular accumulations, as demonstrated here in yeast, may represent an important early step in the pathogenesis of PD. Indeed, dense accumulations of vesicles concentrated in the periphery of LBs were observed in neurons in the substantia nigra of a PD patient. These vesicles may be the remains of larger vesicular accumulations that were caused by vesicle binding of α-syn as seen in the yeast model. These vesicular structures may provide a scaffold for α-syn fibrillization and therefore may be directly involved in the formation of pathological LBs. Although examination of additional cases is required to verify these findings, vesicular accumulations have been observed in neuronal pale body-like structures that have been speculated to be precursors of LBs (Hayashida et al., 1993
). Thus, accumulation of α-syn–associated vesicles may result in the eventual formation of fibrillar α-syn as LBs, which compromise neuronal survival. Alternatively, the accumulation of vesicles may directly result in neuronal toxicity. Further examination of cell and animal models is needed to explore these hypotheses.
In conclusion, we have demonstrated that the inclusions seen in S. cerevisiae expressing α-syn are comprised of clusters of vesicles. Although vesicular accumulations can be detected in LB-containing neurons in human PD, α-syn amyloid fibrils are present in authentic LBs but not in yeast cells, suggesting that vesicular clustering may be an early step in the pathogenesis of LB. Future studies in this and other model systems will provide additional insight into the exact relationship between this vesicular accumulation phenotype and α-syn fibril formation in human neurodegenerative diseases.