␣-Synuclein (␣-syn), a protein of unknown function, is the most abundant protein in Lewy bodies, the histological hallmark of Parkinson's disease (PD). In yeast ␣-syn inhibits endoplasmic reticulum (ER)-to-Golgi (ER3 Golgi) vesicle trafficking, which is rescued by overexpression of a Rab GTPase that regulates ER3 Golgi trafficking. The homologous Rab1 rescues ␣-syn toxicity in dopaminergic neuronal models of PD. Here we investigate this conserved feature of ␣-syn pathobiology. In a cell-free system with purified transport factors ␣-syn inhibited ER3 Golgi trafficking in an ␣-syn dose-dependent manner. Vesicles budded efficiently from the ER, but their docking or fusion to Golgi membranes was inhibited. Thus, the in vivo trafficking problem is due to a direct effect of ␣-syn on the transport machinery. By ultrastructural analysis the earliest in vivo defect was an accumulation of morphologically undocked vesicles, starting near the plasma membrane and growing into massive intracellular vesicular clusters in a dose-dependent manner. By immunofluorescence/immunoelectron microscopy, these clusters were associated both with ␣-syn and with diverse vesicle markers, suggesting that ␣-syn can impair multiple trafficking steps. Other Rabs did not ameliorate ␣-syn toxicity in yeast, but RAB3A, which is highly expressed in neurons and localized to presynaptic termini, and RAB8A, which is localized to post-Golgi vesicles, suppressed toxicity in neuronal models of PD. Thus, ␣-syn causes general defects in vesicle trafficking, to which dopaminergic neurons are especially sensitive. endoplasmic reticulum ͉ Rab GTPase ͉ yeasts ͉ vesicle trafficking ͉ Golgi

Cells respond to stimuli by changes in various processes, including signaling pathways and gene expression. Efforts to identify components of these responses increasingly depend on mRNA profiling and genetic library screens, yet the functional roles of the genes identified by these assays often remain enigmatic. By comparing the results of these two assays across various cellular responses, we found that they are consistently distinct. Moreover, genetic screens tend to identify response regulators, while mRNA profiling frequently detects metabolic responses. We developed an integrative approach that bridges the gap between these data using known molecular interactions, thus highlighting major response pathways. We harnessed this approach to reveal cellular pathways related to alpha-synuclein, a small lipid-binding protein implicated in several neurodegenerative disorders including Parkinson disease. For this we screened an established yeast model for alphasynuclein toxicity to identify genes that when overexpressed alter cellular survival. Application of our algorithm to these data and data from mRNA profiling provided functional explanations for many of these genes and revealed novel relations between alpha-synuclein toxicity and basic cellular pathways.Cells live in a dynamic environment in which they confront various perturbations such as sudden environmental changes, toxins, and mutations. The response to such perturbations is #To whom correspondence should be addressed. E-mail: lindquist_admin@wi.mit.edu (S. L.); fraenkel-admin@mit.edu (E.F.). 7 Present Address: Department of Cell and Developmental Biology, The University of Pennsylvania, Philadelphia, PA, USA 8 Present Address: Medical College of Georgia, Augusta, GA, USA 9 Present Address: Boston Biomedical Research Institute, Watertown, MA, USA. * These authors contributed equally to this work + These authors contributed equally to this work Summary: A novel approach that integrates genetic hits, differentially expressed genes and known molecular interactions reveals a dramatically enhanced view of cellular responses and was used to create the first cellular map of alpha-synuclein toxicity. NIH Public Access Author ManuscriptNat Genet. Author manuscript; available in PMC 2009 September 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript typically complex and comprises signaling and metabolic changes, as well as changes in gene expression. Revealing the cellular mechanisms responding to a specific perturbation may unravel its nature, thus illuminating disease mechanisms 1 or a drug's mode of action 2 ,3 , and identify points of intervention with potential therapeutic value 4 .High-throughput experimental techniques including mRNA profiling and genetic screening are commonly used for revealing components of these response pathways because they provide a genome-and proteome-wide view of molecular changes. mRNA profiling experiments rapidly identify genes that are differentially expressed following stimuli. Genetic screening...

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SUMMARY α-Synuclein (α-syn) is a small lipid-binding protein involved in vesicle trafficking whose function is poorly characterized. It is of great interest to human biology and medicine because α-syn dysfunction is associated with several neurodegenerative disorders, including Parkinson’s disease (PD). We previously created a yeast model of α-syn pathobiology, which established vesicle trafficking as a process that is particularly sensitive to α-syn expression. We also uncovered a core group of proteins with diverse activities related to α-syn toxicity that is conserved from yeast to mammalian neurons. Here, we report that a yeast strain expressing a somewhat higher level of α-syn also exhibits strong defects in mitochondrial function. Unlike our previous strain, genetic suppression of endoplasmic reticulum (ER)-to-Golgi trafficking alone does not suppress α-syn toxicity in this strain. In an effort to identify individual compounds that could simultaneously rescue these apparently disparate pathological effects of α-syn, we screened a library of 115,000 compounds. We identified a class of small molecules that reduced α-syn toxicity at micromolar concentrations in this higher toxicity strain. These compounds reduced the formation of α-syn foci, re-established ER-to-Golgi trafficking and ameliorated α-syn-mediated damage to mitochondria. They also corrected the toxicity of α-syn in nematode neurons and in primary rat neuronal midbrain cultures. Remarkably, the compounds also protected neurons against rotenone-induced toxicity, which has been used to model the mitochondrial defects associated with PD in humans. That single compounds are capable of rescuing the diverse toxicities of α-syn in yeast and neurons suggests that they are acting on deeply rooted biological processes that connect these toxicities and have been conserved for a billion years of eukaryotic evolution. Thus, it seems possible to develop novel therapeutic strategies to simultaneously target the multiple pathological features of PD.

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Mechanisms to safely eliminate amyloids and preamyloid oligomers associated with many devastating diseases are urgently needed. Biophysical principles dictate that small molecules are unlikely to perturb large intermolecular protein-protein interfaces, let alone extraordinarily stable amyloid interfaces. Yet 4,5-dianilinophthalimide (DAPH-1) reverses A␤42 amyloidogenesis and neurotoxicity, which is associated with Alzheimer's disease. Here, we show that DAPH-1 and select derivatives are ineffective against several amyloidogenic proteins, including tau, ␣-synuclein, Ure2, and PrP, but antagonize the yeast prion protein, Sup35, in vitro and in vivo. This allowed us to exploit several powerful new tools created for studying the conformational transitions of Sup35 and decipher the mechanisms by which DAPH-1 and related compounds antagonize the prion state. During fibrillization, inhibitory DAPHs alter the folding of Sup35's amyloidogenic core, preventing amyloidogenic oligomerization and specific recognition events that nucleate prion assembly. Select DAPHs also are capable of attacking preformed amyloids. They remodel Sup35 prion-specific intermolecular interfaces to create morphologically altered aggregates with diminished infectivity and self-templating activity. Our studies provide mechanistic insights and reinvigorate hopes for small-molecule therapies that specifically disrupt intermolecular amyloid contacts.

Most RNA molecules collapse rapidly and reach the native state through a pathway that contains numerous traps and unproductive intermediates. The D135 group II intron ribozyme is unusual in that it can fold slowly and directly to the native state, despite its large size and structural complexity. Here we use hydroxyl radical footprinting and native gel analysis to monitor the timescale of tertiary structure collapse and to detect the presence of obligate intermediates along the folding pathway of D135. We find that structural collapse and native folding of Domain 1 precede assembly of the entire ribozyme, indicating that D1 contains an on-pathway intermediate to folding of the D135 ribozyme. Subsequent docking of Domains 3 and 5, for which D1 provides a preorganized scaffold, appears to be very fast and independent of one another. In contrast to other RNAs, the D135 ribozyme undergoes slow tertiary collapse to a compacted state, with a rate constant that is also limited by the formation D1. These findings provide a new paradigm for RNA folding and they underscore the diversity of RNA biophysical behaviors.

Group II introns are well recognized for their remarkable catalytic capabilities, but little is known about their three-dimensional structures. In order to obtain a global view of an active enzyme, hydroxyl radical cleavage was used to de®ne the solvent accessibility along the backbone of a ribozyme derived from group II intron ai5g. These studies show that a highly homogeneous ribozyme population folds into a catalytically compact structure with an extensively internalized catalytic core. In parallel, a model of the intron core was built based on known tertiary contacts. Although constructed independently of the footprinting data, the model implicates the same elements for involvement in the catalytic core of the intron.