Showing papers by "Susan Lindquist published in 2008"
TL;DR: Yeast α-syn inhibits endoplasmic reticulum-to-Golgi vesicle trafficking, which is rescued by overexpression of a Rab GTPase that regulates ER→golgi trafficking, and the homologous Rab1 rescues α- syn toxicity in dopaminergic neuronal models of PD.
Abstract: alpha-Synuclein (alpha-syn), a protein of unknown function, is the most abundant protein in Lewy bodies, the histological hallmark of Parkinson's disease (PD). In yeast alpha-syn inhibits endoplasmic reticulum (ER)-to-Golgi (ER-->Golgi) vesicle trafficking, which is rescued by overexpression of a Rab GTPase that regulates ER-->Golgi trafficking. The homologous Rab1 rescues alpha-syn toxicity in dopaminergic neuronal models of PD. Here we investigate this conserved feature of alpha-syn pathobiology. In a cell-free system with purified transport factors alpha-syn inhibited ER-->Golgi trafficking in an alpha-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 alpha-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 alpha-syn and with diverse vesicle markers, suggesting that alpha-syn can impair multiple trafficking steps. Other Rabs did not ameliorate alpha-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, alpha-syn causes general defects in vesicle trafficking, to which dopaminergic neurons are especially sensitive.
478 citations
TL;DR: This work provides a mechanistic framework for investigating the toxicity of TDP-43 aggregation relevant to human disease and establishes a manipulable, high-throughput model for discovering potential therapeutic strategies.
Abstract: Protein misfolding is intimately associated with devastating human neurodegenerative diseases, including Alzheimer's, Huntington's, and Parkinson's. Although disparate in their pathophysiology, many of these disorders share a common theme, manifested in the accumulation of insoluble protein aggregates in the brain. Recently, the major disease protein found in the pathological inclusions of two of these diseases, amyotrophic lateral sclerosis (ALS) and frontal temporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U), was identified as the 43-kDa TAR-DNA-binding protein (TDP-43), providing a molecular link between them. TDP-43 is a ubiquitously expressed nuclear protein that undergoes a pathological conversion to an aggregated cytoplasmic localization in affected regions of the nervous system. Whether TDP-43 itself can convey toxicity and whether its abnormal aggregation is a cause or consequence of pathogenesis remain unknown. We report a yeast model to define mechanisms governing TDP-43 subcellular localization and aggregation. Remarkably, this simple model recapitulates several salient features of human TDP-43 proteinopathies, including conversion from nuclear localization to cytoplasmic aggregation. We establish a connection between this aggregation and toxicity. The pathological features of TDP-43 are distinct from those of yeast models of other protein-misfolding diseases, such as polyglutamine. This suggests that the yeast model reveals specific aspects of the underlying biology of the disease protein rather than general cellular stresses associated with accumulating misfolded proteins. This work provides a mechanistic framework for investigating the toxicity of TDP-43 aggregation relevant to human disease and establishes a manipulable, high-throughput model for discovering potential therapeutic strategies.
401 citations
TL;DR: It is established that impaired ER protein homeostasis is a broad and highly conserved contributor to polyQ toxicity in yeast, in PC12 cells, and, importantly, in striatal cells expressing full-length polyQ-expanded huntingtin.
Abstract: Protein misfolding, whether caused by aging, environmental factors, or genetic mutations, is a common basis for neurodegenerative diseases. The misfolding of proteins with abnormally long polyglutamine (polyQ) expansions causes several neurodegenerative disorders, such as Huntington’s disease (HD). Although many cellular pathways have been documented to be impaired in HD, the primary triggers of polyQ toxicity remain elusive. We report that yeast cells and neuron-like PC12 cells expressing polyQ-expanded huntingtin (htt) fragments display a surprisingly specific, immediate, and drastic defect in endoplasmic reticulum (ER)-associated degradation (ERAD). We further decipher the mechanistic basis for this defect in ERAD: the entrapment of the essential ERAD proteins Npl4, Ufd1, and p97 by polyQ-expanded htt fragments. In both yeast and mammalian neuron-like cells, overexpression of Npl4 and Ufd1 ameliorates polyQ toxicity. Our results establish that impaired ER protein homeostasis is a broad and highly conserved contributor to polyQ toxicity in yeast, in PC12 cells, and, importantly, in striatal cells expressing full-length polyQ-expanded huntingtin.
281 citations
TL;DR: The hypothesis that [PSI+] is a mechanism to increase survival in fluctuating environments and might function as a capacitor to promote evolvability is supported.
Abstract: Evolution depends on the manner in which genetic variation is translated into new phenotypes. There has been much debate about whether organisms might have specific mechanisms for “evolvability,” which would generate heritable phenotypic variation with adaptive value and could act to enhance the rate of evolution. Capacitor systems, which allow the accumulation of cryptic genetic variation and release it under stressful conditions, might provide such a mechanism. In yeast, the prion [PSI+] exposes a large array of previously hidden genetic variation, and the phenotypes it thereby produces are advantageous roughly 25% of the time. The notion that [PSI+] is a mechanism for evolvability would be strengthened if the frequency of its appearance increased with stress. That is, a system that mediates even the haphazard appearance of new phenotypes, which have a reasonable chance of adaptive value would be beneficial if it were deployed at times when the organism is not well adapted to its environment. In an unbiased, high-throughput, genome-wide screen for factors that modify the frequency of [PSI+] induction, signal transducers and stress response genes were particularly prominent. Furthermore, prion induction increased by as much as 60-fold when cells were exposed to various stressful conditions, such as oxidative stress (H2O2) or high salt concentrations. The severity of stress and the frequency of [PSI+] induction were highly correlated. These findings support the hypothesis that [PSI+] is a mechanism to increase survival in fluctuating environments and might function as a capacitor to promote evolvability.
256 citations
TL;DR: Hsp104 likely protects dopaminergic neurons by antagonizing toxic alpha-synuclein assemblies and might have therapeutic potential for PD and other neurodegenerative amyloidoses.
Abstract: Parkinson disease (PD) is characterized by dopaminergic neurodegeneration and intracellular inclusions of alpha-synuclein amyloid fibers, which are stable and difficult to dissolve. Whether inclusions are neuroprotective or pathological remains controversial, because prefibrillar oligomers may be more toxic than amyloid inclusions. Thus, whether therapies should target inclusions, preamyloid oligomers, or both is a critically important issue. In yeast, the protein-remodeling factor Hsp104 cooperates with Hsp70 and Hsp40 to dissolve and reactivate aggregated proteins. Metazoans, however, have no Hsp104 ortholog. Here we introduced Hsp104 into a rat PD model. Remarkably, Hsp104 reduced formation of phosphorylated alpha-synuclein inclusions and prevented nigrostriatal dopaminergic neurodegeneration induced by PD-linked alpha-synuclein (A30P). An in vitro assay employing pure proteins revealed that Hsp104 prevented fibrillization of alpha-synuclein and PD-linked variants (A30P, A53T, E46K). Hsp104 coupled ATP hydrolysis to the disassembly of preamyloid oligomers and amyloid fibers composed of alpha-synuclein. Furthermore, the mammalian Hsp70 and Hsp40 chaperones, Hsc70 and Hdj2, enhanced alpha-synuclein fiber disassembly by Hsp104. Hsp104 likely protects dopaminergic neurons by antagonizing toxic alpha-synuclein assemblies and might have therapeutic potential for PD and other neurodegenerative amyloidoses.
190 citations
TL;DR: An adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins, is demonstrated, which uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.
Abstract: Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms. Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity. However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE). This technique is finding widespread use for the detection and characterization of amyloid conformational variants. Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins. The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated. Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.
187 citations
TL;DR: It is demonstrated that HSP90-dependent alleles occur in continuously distributed, environmentally responsive traits and are amenable to quantitative genetic mapping techniques, and it is found that developmental stability is a phenotypic trait that can be affected by natural variation.
Abstract: Modulation of the activity of the molecular chaperone HSP90 has been extensively discussed as a means to alter phenotype in many traits and organisms. Such changes can be due to the exposure of cryptic genetic variation, which in some instances may also be accomplished by mild environmental alteration. Should such polymorphisms be widespread, natural selection may be more effective at producing phenotypic change in suboptimal environments. However, the frequency and identity of buffered polymorphisms in natural populations are unknown. Here, we employ quantitative genetic dissection of an Arabidopsis thaliana developmental response, hypocotyl elongation in the dark, to detail the underpinnings of genetic variation responsive to HSP90 modulation. We demonstrate that HSP90-dependent alleles occur in continuously distributed, environmentally responsive traits and are amenable to quantitative genetic mapping techniques. Furthermore, such alleles are frequent in natural populations and can have significant effects on natural phenotypic variation. We also find that HSP90 modulation has both general and allele-specific effects on developmental stability; that is, developmental stability is a phenotypic trait that can be affected by natural variation. However, effects of revealed variation on trait means outweigh effects of decreased developmental stability, and the HSP90-dependent trait alterations could be acted on by natural selection. Thus, HSP90 may centrally influence canalization, assimilation, and the rapid evolutionary alteration of phenotype through the concealment and exposure of cryptic genetic variation.
186 citations
TL;DR: This work reports how the Ssa and Ssb components of the Hsp70 chaperone system directly affect Sup35 prionogenesis and cooperate with Hsp104, and identifies the ribosome‐associated Ssb1:Zuo1:Ssz1 complex as a potent antagonist of Sup35Prionogenesis.
Abstract: Self-templating amyloid forms of Sup35 constitute the yeast prion [PSI þ ]. How the protein-remodelling factor, Hsp104, collaborates with other chaperones to regulate [PSI þ ] inheritance remains poorly delineated. Here, we report how the Ssa and Ssb components of the Hsp70 chaperone system directly affect Sup35 prionogenesis and cooperate with Hsp104. We identify the ribosomeassociated Ssb1:Zuo1:Ssz1 complex as a potent antagonist of Sup35 prionogenesis. The Hsp40 chaperones, Sis1 and Ydj1, preferentially interact with Sup35 oligomers and fibres compared with monomers, and facilitate Ssa1 and Ssb1 binding. Various Hsp70:Hsp40 pairs block prion nucleation by disassembling molten oligomers and binding mature oligomers. By binding fibres, Hsp70:Hsp40 pairs occlude prion recognition elements and inhibit seeded assembly. These inhibitory activities are partially relieved by the nucleotide exchange factor, Fes1. Low levels of Hsp104 stimulate prionogenesis and alleviate inhibition by some Hsp70:Hsp40 pairs. At high concentrations, Hsp104 eliminates Sup35 prions. This activity is reduced when Ssa1, or enhanced when Ssb1, is incorporated into nascent prions. These findings illuminate several facets of the chaperone interplay that underpins [PSI þ ] inheritance.
176 citations
TL;DR: It is established that even subtle changes in the folding homeostasis of an amyloidogenic protein can create a severe proteotoxic gain-of-function phenotype and that chaperone-mediated amyloids assembly can be cytoprotective.
Abstract: Protein conformational diseases are associated with the aberrant accumulation of amyloid protein aggregates, but whether amyloid formation is cytotoxic or protective is unclear. To address this issue, we investigated a normally benign amyloid formed by the yeast prion [RNQ+]. Surprisingly, modest overexpression of Rnq1 protein was deadly, but only when preexisting Rnq1 was in the [RNQ+] prion conformation. Molecular chaperones protect against protein aggregation diseases and are generally believed to do so by solubilizing their substrates. The Hsp40 chaperone, Sis1, suppressed Rnq1 proteotoxicity, but instead of blocking Rnq1 protein aggregation, it stimulated conversion of soluble Rnq1 to [RNQ+] amyloid. Furthermore, interference with Sis1-mediated [RNQ+] amyloid formation exacerbated Rnq1 toxicity. These and other data establish that even subtle changes in the folding homeostasis of an amyloidogenic protein can create a severe proteotoxic gain-of-function phenotype and that chaperone-mediated amyloid assembly can be cytoprotective. The possible relevance of these findings to other phenomena, including prion-driven neurodegenerative diseases and heterokaryon incompatibility in fungi, is discussed.
162 citations
TL;DR: It is demonstrated that HSP90-dependent natural variation is present at high frequencies in A. thaliana, with an expectation that at least one HSP 90-dependent polymorphism will affect nearly every quantitative trait in progeny of two different wild lines, and HSP80 is likely to occupy a central position in the translation of genotypic variation into phenotypic differences.
Abstract: HSP90 is a protein chaperone particularly important in the maturation of a diverse set of proteins that regulate key steps in a multitude of biological processes. Alterations in HSP90 function produce altered phenotypes at low penetrance in natural populations. Previous work has shown that at least some of these phenotypes are due to genetic variation that remains phenotypically cryptic until it is revealed by the impairment of HSP90 function. Exposure of such "buffered" genetic polymorphisms can also be accomplished by environmental stress, linking the appearance of new phenotypes to defects in protein homeostasis. Should such polymorphisms be widespread, natural selection may be more effective at producing phenotypic change in suboptimal environments. In evaluating this hypothesis, a key unknown factor is the frequency with which HSP90-buffered polymorphisms occur in natural populations. Here, we present Arabidopsis thaliana populations suitable for genetic mapping that have constitutively reduced HSP90 levels. We employ quantitative genetic techniques to examine the HSP90-dependent polymorphisms affecting a host of plastic plant life-history traits. Our results demonstrate that HSP90-dependent natural variation is present at high frequencies in A. thaliana, with an expectation that at least one HSP90-dependent polymorphism will affect nearly every quantitative trait in progeny of two different wild lines. Hence, HSP90 is likely to occupy a central position in the translation of genotypic variation into phenotypic differences.
134 citations
TL;DR: It is reported that the onset of prion disease is delayed by CR and in the SIRT1 KO mice fed ad libitum, suggesting a possible mechanism for the delayed onset of disease, as PrP levels are a critical determinant of how quickly mice succumb toPrion disease.
TL;DR: A chaperone-like activity of Sse1 that assists in stabilization of early folding intermediates of the Sup35 prion conformation is suggested.
Abstract: The cytosolic chaperone network of Saccharomyces cerevisiae is intimately associated with the emergence and maintenance of prion traits. Recently, the Hsp110 protein, Sse1, has been identified as a nucleotide exchange factor (NEF) for both cytosolic Hsp70 chaperone family members, Ssa1 and Ssb1. We have investigated the role of Sse1 in the de novo formation and propagation of [PSI+], the prion form of the translation termination factor, Sup35. As observed by others, we find that Sse1 is essential for efficient prion propagation. Our results suggest that the NEF activity is required for maintaining sufficient levels of substrate-free Ssa1. However, Sse1 exhibits an additional NEF-independent activity; it stimulates in vitro nucleation of Sup35NM, the prion domain of Sup35. We also observe that high levels of Sse1, but not of an unrelated NEF, very potently inhibit Hsp104-mediated curing of [PSI+]. Taken together, these results suggest a chaperone-like activity of Sse1 that assists in stabilization of early folding intermediates of the Sup35 prion conformation. This activity is not essential for prion formation under conditions of Sup35 overproduction, however, it may be relevant for spontaneous [PSI+] formation as well as for protection of the prion trait upon physiological Hsp104 induction.
TL;DR: In this article, heat shock factor 1 (HSF1) was found to play an important role in prion disease in uninoculated HSF1 knockout (KO) mice.
Abstract: Prion diseases are fatal, transmissible, neurodegenerative diseases caused by the misfolding of the prion protein (PrP). At present, the molecular pathways underlying prion-mediated neurotoxicity are largely unknown. We hypothesized that the transcriptional regulator of the stress response, heat shock factor 1 (HSF1), would play an important role in prion disease. Uninoculated HSF1 knockout (KO) mice used in our study do not show signs of neurodegeneration as assessed by survival, motor performance, or histopathology. When inoculated with Rocky Mountain Laboratory (RML) prions HSF1 KO mice had a dramatically shortened lifespan, succumbing to disease ≈20% faster than controls. Surprisingly, both the onset of home-cage behavioral symptoms and pathological alterations occurred at a similar time in HSF1 KO and control mice. The accumulation of proteinase K (PK)-resistant PrP also occurred with similar kinetics and prion infectivity accrued at an equal or slower rate. Thus, HSF1 provides an important protective function that is specifically manifest after the onset of behavioral symptoms of prion disease.
TL;DR: It is shown that 4,5-dianilinophthalimide (DAPH-1) and select derivatives are ineffective against several amyloidogenic proteins, including tau, α-synuclein, Ure2, and PrP, but antagonize the yeast prion protein, Sup35, in vitro and in vivo.
Abstract: 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 Aβ42 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, α-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.
TL;DR: It is reported that soft-tissue granulomas, a frequent feature of chronic inflammation, expressed the cellular prion protein (PrPC, encoded by Prnp) and the lymphotoxin receptor (LTbetaR), even though they lacked FDCs and did not display lymphoneogenesis.
TL;DR: Two assay formats that have been used successfully to identify small molecules that rescue asyn toxicity in yeast are described and suggest that asyn interferes with Rab function and provide new avenues for PD drug discovery.
Abstract: Recent studies implicate a disruption in Rab‐mediated protein trafficking as a possible contributing factor to neurodegeneration in Parkinson's disease (PD). Misfolding of the neuronal protein α‐synuclein (asyn) is implicated in PD. Overexpression of asyn results in cell death in a wide variety of model systems, and in several organisms, including yeast, worms, flies, and rodent primary neurons, this toxicity is suppressed by the overproduction of Rab proteins. These and other findings suggest that asyn interferes with Rab function and provide new avenues for PD drug discovery. This chapter describes two assay formats that have been used successfully to identify small molecules that rescue asyn toxicity in yeast. The 96‐well format monitors rescue by optical density and is suitable for screening thousands of compounds. A second format measures viable cells by reduction of the dye alamarBlue, a readout that is compatible with 96‐, 384‐, and 1536‐well plates allowing the screening of large libraries (>100,000 compounds). A secondary assay to eliminate mechanistically undesirable hits is also described.
01 Jan 2008
TL;DR: This chapter explores yeast cells as a discovery platform for Parkinson's disease (PD) and finding genetic factors and chemical compounds that modify the toxicity of proteins that are prone to misfoldings and producing toxic gain-of-function phenotypes in man.
Abstract: Publisher Summary This chapter explores yeast cells as a discovery platform for Parkinson's disease (PD) and finding genetic factors and chemical compounds that modify the toxicity of proteins that are prone to misfoldings and producing toxic gain-of-function phenotypes in man. The chapter briefly reviews general features of PD and associated genes to place them in context relevant to yeast models. PD is the most common neurodegenerative movement disorder. One pathological hallmark of PD is the selective loss of the DA neurons comprising the pars compacta of the substantia nigra and the presence of proteinaceous inclusion bodies, and Lewy neurites, in the affected cells. Yeast cells also have unusually high rates of homologous recombination, and yeast has progressed from being a good model to a super model. The yeast models do not simply show nonspecific toxicity due to the accumulation of misfolded proteins. The toxicity in different models is highly specific and distinct. An advantage of yeast models in drug screening is that they have a capacity to focus compound searches on the initiating events of the diseases, particularly those involved with the misfolding and aggregation of the toxic protein. Yeast cells also provide a whole collection of technical advantages for high-throughput screening—speed, robustness, low cost, genetic manipulability—making for a compelling combination.
TL;DR: In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders, and current experimental approaches to investigate the mechanism of neurodegnerative diseases based on genetic studies in model organisms.
Abstract: In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.
Patent•
17 Apr 2008
TL;DR: In this paper, the authors provide a rapid and cost-effective method to screen compounds that prevent protein misfolding and protein fibril formation and/or protein aggregation, which includes numerous neurodegenerative diseases (Parkinson's disease, Alzheimer's disease and Huntington's disease) as well as non-neuronal diseases (such as type 2 diabetes).
Abstract: PROBLEM TO BE SOLVED: To provide screening methods for identifying substances that provide therapeutic value for various diseases associated with protein misfolding. SOLUTION: Genetic screening methods and chemical screening methods, using a yeast system are provided. The methods provide a rapid and cost-effective method to screen compounds that prevent protein misfolding and/or protein fibril formation and/or protein aggregation, which includes numerous neurodegenerative diseases (Parkinson's disease, Alzheimer's disease and Huntington's disease) as well as non-neuronal diseases (such as type 2 diabetes). COPYRIGHT: (C)2009,JPO&INPIT