α-Synuclein–induced Aggregation of Cytoplasmic Vesicles inSaccharomyces cerevisiae

Abstract

Aggregated ␣-synuclein (␣-syn) fibrils form Lewy bodies (LBs), the signature lesions of Parkinson's disease (PD) and related synucleinopathies, but the pathogenesis and neurodegenerative effects of LBs remain enigmatic. Recent studies have shown that when overexpressed in Saccharomyces cerevisiae, ␣-syn localizes to plasma membranes and forms cytoplasmic accumulations similar to human ␣-syn inclusions. However, the exact nature, composition, temporal evolution, and underlying mechanisms of yeast ␣-syn accumulations and their relevance to human synucleinopathies are unknown. Here we provide ultrastructural evidence that ␣-syn accumulations are not comprised of LB-like fibrils, but are associated with clusters of vesicles. Live-cell imaging showed ␣-syn initially localized to the plasma membrane and subsequently formed accumulations in association with vesicles. Imaging of truncated and mutant forms of ␣-syn revealed the molecular determinants and vesicular trafficking pathways underlying this pathological process. Because vesicular clustering is also found in LB-containing neurons of PD brains, ␣-syn-mediated vesicular accumulation in yeast represents a model system to study specific aspects of neurodegeneration in PD and related synucleinopathies. INTRODUCTIONParkinson's disease (PD) is the most prevalent neurodegenerative movement disorder and is characterized by bradykinesia, rigidity, postural instability, and resting tremor (Galvin et al., 2001;Forman et al., 2005), as well as by the loss of dopaminergic neurons and presence of neuronal inclusions, known as Lewy bodies (LBs) and Lewy neurites (Forman et al., 2005). The identification of an ␣-syn gene (SNCA) mutation associated with autosomal dominant PD (Polymeropoulos et al., 1997), and the identification of fibrillar ␣-syn as the principal component of LB pathology (Spillantini et al., 1997), indicate that the LBs formed by ␣-syn define classic PD and are implicated in disease pathogenesis. The subsequent identification of additional disease-linked SNCA mutations (Polymeropoulos et al., 1997;Kruger et al., 1998;Zarranz et al., 2004), as well as replications of the SNCA gene (Singleton et al., 2003;Chartier-Harlin et al., 2004), indicate that both mutant ␣-syn and increased expression of wildtype (WT) ␣-syn can cause neurodegeneration.In addition to PD, filamentous ␣-syn inclusions have been detected in other neurodegenerative diseases including the LB variant of Alzheimer's disease, dementia with LBs, neurodegeneration with brain iron accumulation type-1, and multiple system atrophy, which are collectively known as synucleinopathies (Spillantini et al., 1997;Duda et al., 2000). ␣-syn fibrils exhibit properties of amyloid (Spillantini et al., 1998), and ␣-syn assembles into amyloid fibrils in vitro (Han et al., 1995;Giasson et al., 1999), whereas SNCA mutations can accelerate ␣-syn fibrillization (Conway et al., 1998;Greenbaum et al., 2005). Therefore, it is plausible that ␣-syn misfolds and aggregates into amyloid fibrils that are neurotoxic and play a cau...

α-Synuclein and Its A30P Mutant Affect Actin Cytoskeletal Structure and Dynamics

“…In neurons these structures appeared as small structures, distributed especially along the neurites and at their terminal tips. These foci, attached to and rolling onto the inner face of the plasma membrane, especially near the sites of cellto-cell interaction, appear as transient but specific structures, quite different from the protein clusters close to the plasma membrane recently reported in yeast cells overexpressing ␣-synuclein-GFP (Soper et al, 2008).…”

α-Synuclein interacts with the switch region of Rab8a in a Ser129 phosphorylation-dependent manner

“…Several Rab GTPases were suggested to interact aberrantly with αS in Dementia with Lewy bodies (Soper et al, 2008(Soper et al, , 2011Sung et al, 2001). In addition, Rab proteins colocalize with glial inclusions containing αS in multiple system atrophy (Dalfo and Ferrer, 2005;Nakamura et al, 2000), Rab GTPases were found in vesicle clusters induced by αS overexpression in yeast (Gitler et al, 2008), and overexpression of the Rab GTPases Rab1, Rab3a and Rab8a decreased αS-induced neurotoxicity (Cooper et al, 2006;Gitler et al, 2008).…”

Regulation of a-Synuclein Membrane Binding and Its Implications

“…Expression of -synuclein in yeast and mammalian cells causes delays in ER-to-Golgi vesicle trafficking, an effect that was more striking with the A53T mutant (Cooper et al, 2006;Thayanidhi et al, 2010). These changes in ER-to-Golgi trafficking by -synuclein may be caused by immobilizing vesicles in the cytoplasm (Soper et al, 2008). The mechanism appears to involve rab proteins, small GTPases that regulate vesicle trafficking, as the impairment is rescued by overexpressing certain rab proteins (Gitler et al, 2008).…”

“…From a functional perspective, -synuclein appears to antagonize rab3a function. For example, over-expression of -synuclein in yeast interfered with ER-to-Golgi vesicle trafficking that was corrected by the simultaneous over-expression of the yeast homologue of rab1 (Cooper et al, 2006;Soper et al, 2008). Similarly, elevated toxicity in nematodes and rat primary neurons engineered to express wild-type or A53T -synuclein was rescued by the addition of rab8 or rab3a (Gitler et al, 2008).…”

“…Mice deficient in -synuclein are unremarkable, but exhibit increased release of dopamine under paired-pulse stimuli and reduced striatal tissue content of dopamine (Abeliovich et al, 2000). Over-expression of -synuclein in yeast leads to aggregation of vesicles, disruption in vesicular transport, and death (Gitler et al, 2008;Soper et al, 2008). Similarly, moderatesynuclein over-expression in mammalian neurons reduces transmitter release and the number of synaptic vesicles arrayed at presynaptic active zones (Nemani et al, 2010).…”