Publications by authors named "Martin L Duennwald"

35 Publications

A Quantitative Imaging-Based Protocol for Yeast Growth and Survival on Agar Plates.

STAR Protoc 2020 Dec 25;1(3):100182. Epub 2020 Nov 25.

Department of Pathology and Laboratory Medicine, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, ON N6A 3K7, Canada.

We present a detailed protocol that describes the evaluation of the growth and survival of yeast cells by quantitatively analyzing spotting assays. This simple method reproducibly detects and quantifies subtle differences in growth by measuring the density of cells within a single spot of defined size on an image of a spotting assay. Our protocol is tailored specifically for low-throughput applications, can be easily adapted for specific experimental conditions, and is accessible to yeast experts and non-experts alike. For an example of the execution of this protocol, please refer to DiGregorio et al. (Di Gregorio et al., 2020).
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http://dx.doi.org/10.1016/j.xpro.2020.100182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757406PMC
December 2020

Inclusion Formation and Toxicity of the ALS Protein RGNEF and Its Association with the Microtubule Network.

Int J Mol Sci 2020 Aug 5;21(16). Epub 2020 Aug 5.

Department of Pathology and Laboratory Medicine, University of Western Ontario, London, ON N6A 5C1, Canada.

The Rho guanine nucleotide exchange factor (RGNEF) protein encoded by the gene has been implicated in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Biochemical and pathological studies have shown that RGNEF is a component of the hallmark neuronal cytoplasmic inclusions in ALS-affected neurons. Additionally, a heterozygous mutation in has been identified in a number of familial ALS (fALS) cases that may give rise to one of two truncated variants of the protein. Little is known about the normal biological function of RGNEF or how it contributes to ALS pathogenesis. To further explore RGNEF biology we have established and characterized a yeast model and characterized RGNEF expression in several mammalian cell lines. We demonstrate that RGNEF is toxic when overexpressed and forms inclusions. We also found that the fALS-associated mutation in gives rise to an inclusion-forming and toxic protein. Additionally, through unbiased screening using the split-ubiquitin system, we have identified RGNEF-interacting proteins, including two ALS-associated proteins. Functional characterization of other RGNEF interactors identified in our screen suggest that RGNEF functions as a microtubule regulator. Our findings indicate that RGNEF misfolding and toxicity may cause impairment of the microtubule network and contribute to ALS pathogenesis.
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http://dx.doi.org/10.3390/ijms21165597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460592PMC
August 2020

Modulation of hippocampal neuronal resilience during aging by the Hsp70/Hsp90 co-chaperone STI1.

J Neurochem 2020 06 31;153(6):727-758. Epub 2019 Oct 31.

Robarts Research Institute, University of Western Ontario, London, Ontario, Canada.

Chaperone networks are dysregulated with aging, but whether compromised Hsp70/Hsp90 chaperone function disturbs neuronal resilience is unknown. Stress-inducible phosphoprotein 1 (STI1; STIP1; HOP) is a co-chaperone that simultaneously interacts with Hsp70 and Hsp90, but whose function in vivo remains poorly understood. We combined in-depth analysis of chaperone genes in human datasets, analysis of a neuronal cell line lacking STI1 and of a mouse line with a hypomorphic Stip1 allele to investigate the requirement for STI1 in aging. Our experiments revealed that dysfunctional STI1 activity compromised Hsp70/Hsp90 chaperone network and neuronal resilience. The levels of a set of Hsp90 co-chaperones and client proteins were selectively affected by reduced levels of STI1, suggesting that their stability depends on functional Hsp70/Hsp90 machinery. Analysis of human databases revealed a subset of co-chaperones, including STI1, whose loss of function is incompatible with life in mammals, albeit they are not essential in yeast. Importantly, mice expressing a hypomorphic STI1 allele presented spontaneous age-dependent hippocampal neurodegeneration and reduced hippocampal volume, with consequent spatial memory deficit. We suggest that impaired STI1 function compromises Hsp70/Hsp90 chaperone activity in mammals and can by itself cause age-dependent hippocampal neurodegeneration in mice. Cover Image for this issue: doi: 10.1111/jnc.14749.
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http://dx.doi.org/10.1111/jnc.14882DOI Listing
June 2020

A functional unfolded protein response is required for chronological aging in Saccharomyces cerevisiae.

Curr Genet 2020 Feb 25;66(1):263-277. Epub 2019 Jul 25.

Department of Anatomy and Cell Biology, The University of Western Ontario, London, N6A 5C1, Canada.

Progressive impairment of proteostasis and accumulation of toxic misfolded proteins are associated with the cellular aging process. Here, we employed chronologically aged yeast cells to investigate how activation of the unfolded protein response (UPR) upon accumulation of misfolded proteins in the endoplasmic reticulum (ER) affects lifespan. We found that cells lacking a functional UPR display a significantly reduced chronological lifespan, which contrasts previous findings in models of replicative aging. We find exacerbated UPR activation in aged cells, indicating an increase in misfolded protein burden in the ER during the course of aging. We also observed that caloric restriction, which promotes longevity in various model organisms, extends lifespan of UPR-deficient strains. Similarly, aging in pH-buffered media extends lifespan, albeit independently of the UPR. Thus, our data support a role for caloric restriction and reduced acid stress in improving ER homeostasis during aging. Finally, we show that UPR-mediated upregulation of the ER chaperone Kar2 and functional ER-associated degradation (ERAD) are essential for proper aging. Our work documents the central role of secretory protein homeostasis in chronological aging in yeast and highlights that the requirement for a functional UPR can differ between post-mitotic and actively dividing eukaryotic cells.
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http://dx.doi.org/10.1007/s00294-019-01019-0DOI Listing
February 2020

Sfp1 links TORC1 and cell growth regulation to the yeast SAGA-complex component Tra1 in response to polyQ proteotoxicity.

Traffic 2019 04;20(4):267-283

Department of Anatomy and Cell Biology, The University of Western Ontario, London, Ontario, Canada.

Chromatin remodeling regulates gene expression in response to the accumulation of misfolded polyQ proteins associated with Huntington's disease (HD). Tra1 is an essential component of both the SAGA/SLIK and NuA4 transcription co-activator complexes and is linked to multiple cellular processes, including protein trafficking and signaling pathways associated with misfolded protein stress. Cells with compromised Tra1 activity display phenotypes distinct from deletions encoding components of the SAGA and NuA4 complexes, indicating a potentially unique regulatory role of Tra1 in the cellular response to protein misfolding. Here, we employed a yeast model to define how the expression of toxic polyQ expansion proteins affects Tra1 expression and function. Expression of expanded polyQ proteins mimics deletion of SAGA/NuA4 components and results in growth defects under stress conditions. Moreover, deleting genes encoding SAGA and, to a lesser extent, NuA4 components exacerbates polyQ toxicity. Also, cells carrying a mutant Tra1 allele displayed increased sensitivity to polyQ toxicity. Interestingly, expression of polyQ proteins upregulated the expression of TRA1 and other genes encoding SAGA components, revealing a feedback mechanism aimed at maintaining Tra1 and SAGA functional integrity. Moreover, deleting the TORC1 (Target of Rapamycin) effector SFP1 abolished upregulation of TRA1 upon expression of polyQ proteins. While Sfp1 is known to adjust ribosome biogenesis and cell size in response to stress, we identified a new role for Sfp1 in the control of TRA1 expression, linking TORC1 and cell growth regulation to the SAGA acetyltransferase complex during misfolded protein stress.
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http://dx.doi.org/10.1111/tra.12637DOI Listing
April 2019

A subset of calcium-binding S100 proteins show preferential heterodimerization.

FEBS J 2019 05 21;286(10):1859-1876. Epub 2019 Feb 21.

Department of Biochemistry, The University of Western Ontario, London, Canada.

The assembly of proteins into dimers and oligomers is a necessary step for the proper function of transcription factors, muscle proteins, and proteases. In uncontrolled states, oligomerization can also contribute to illnesses such as Alzheimer's disease. The S100 protein family is a group of dimeric proteins that have important roles in enzyme regulation, cell membrane repair, and cell growth. Most S100 proteins have been examined in their homodimeric state, yet some of these important proteins are found in similar tissues implying that heterodimeric molecules can also be formed from the combination of two different S100 members. In this work, we have established co-expression methods in order to identify and quantify the distribution of homo- and heterodimers for four specific pairs of S100 proteins in their calcium-free states. The split GFP trap methodology was used in combination with other GFP variants to simultaneously quantify homo- and heterodimeric S100 proteins in vitro and in living cells. For the specific S100 proteins examined, NMR, mass spectrometry, and GFP trap experiments consistently show that S100A1:S100B, S100A1:S100P, and S100A11:S100B heterodimers are the predominant species formed compared to their corresponding homodimers. We expect the tools developed here will help establish the roles of S100 heterodimeric proteins and identify how heterodimerization might alter the specificity for S100 protein action in cells.
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http://dx.doi.org/10.1111/febs.14775DOI Listing
May 2019

RNA surveillance by uridylation-dependent RNA decay in Schizosaccharomyces pombe.

Nucleic Acids Res 2019 04;47(6):3045-3057

Department of Biochemistry, The University of Western Ontario, 1151 Richmond Street, London, ON N6A 5C1, Canada.

Uridylation-dependent RNA decay is a widespread eukaryotic pathway modulating RNA homeostasis. Terminal uridylyltransferases (Tutases) add untemplated uridyl residues to RNA 3'-ends, marking them for degradation by the U-specific exonuclease Dis3L2. In Schizosaccharomyces pombe, Cid1 uridylates a variety of RNAs. In this study, we investigate the prevalence and impact of uridylation-dependent RNA decay in S. pombe by transcriptionally profiling cid1 and dis3L2 deletion strains. We found that the exonuclease Dis3L2 represents a bottleneck in uridylation-dependent mRNA decay, whereas Cid1 plays a redundant role that can be complemented by other Tutases. Deletion of dis3L2 elicits a cellular stress response, upregulating transcription of genes involved in protein folding and degradation. Misfolded proteins accumulate in both deletion strains, yet only trigger a strong stress response in dis3L2 deficient cells. While a deletion of cid1 increases sensitivity to protein misfolding stress, a dis3L2 deletion showed no increased sensitivity or was even protective. We furthermore show that uridylyl- and adenylyltransferases cooperate to generate a 5'-NxAUUAAAA-3' RNA motif on dak2 mRNA. Our studies elucidate the role of uridylation-dependent RNA decay as part of a global mRNA surveillance, and we found that perturbation of this pathway leads to the accumulation of misfolded proteins and elicits cellular stress responses.
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http://dx.doi.org/10.1093/nar/gkz043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451125PMC
April 2019

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast.

J Vis Exp 2018 11 28(141). Epub 2018 Nov 28.

Department of Anatomy and Cell Biology, University of Western Ontario;

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington's disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.
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http://dx.doi.org/10.3791/58748DOI Listing
November 2018

ALS Yeast Models-Past Success Stories and New Opportunities.

Front Mol Neurosci 2018 30;11:394. Epub 2018 Oct 30.

Schulich School of Medicine and Dentistry, Pathology and Laboratory Medicine, Western University, London, ON, Canada.

In the past two decades, yeast models have delivered profound insights into basic mechanisms of protein misfolding and the dysfunction of key cellular pathways associated with amyotrophic lateral sclerosis (ALS). Expressing ALS-associated proteins, such as superoxide dismutase (SOD1), TAR DNA binding protein 43 (TDP-43) and Fused in sarcoma (FUS), in yeast recapitulates major hallmarks of ALS pathology, including protein aggregation, mislocalization and cellular toxicity. Results from yeast have consistently been recapitulated in other model systems and even specimens from human patients, thus providing evidence for the power and validity of ALS yeast models. Focusing on impaired ribonucleic acid (RNA) metabolism and protein misfolding and their cytotoxic consequences in ALS, we summarize exemplary discoveries that originated from work in yeast. We also propose previously unexplored experimental strategies to modernize ALS yeast models, which will help to decipher the basic pathomechanisms underlying ALS and thus, possibly contribute to finding a cure.
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http://dx.doi.org/10.3389/fnmol.2018.00394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218427PMC
October 2018

Comparative Analysis of Mutant Huntingtin Binding Partners in Yeast Species.

Sci Rep 2018 06 22;8(1):9554. Epub 2018 Jun 22.

Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth College, Hanover, New Hampshire, 03755, United States.

Huntington's disease is caused by the pathological expansion of a polyglutamine (polyQ) stretch in Huntingtin (Htt), but the molecular mechanisms by which polyQ expansion in Htt causes toxicity in selective neuronal populations remain poorly understood. Interestingly, heterologous expression of expanded polyQ Htt is toxic in Saccharomyces cerevisiae cells, but has no effect in Schizosaccharomyces pombe, a related yeast species possessing very few endogenous polyQ or Q/N-rich proteins. Here, we used a comprehensive and unbiased mass spectrometric approach to identify proteins that bind Htt in a length-dependent manner in both species. Analysis of the expanded polyQ-associated proteins reveals marked enrichment of proteins that are localized to and play functional roles in nucleoli and mitochondria in S. cerevisiae, but not in S. pombe. Moreover, expanded polyQ Htt appears to interact preferentially with endogenous polyQ and Q/N-rich proteins, which are rare in S. pombe, as well as proteins containing coiled-coil motifs in S. cerevisiae. Taken together, these results suggest that polyQ expansion of Htt may cause cellular toxicity in S. cerevisiae by sequestering endogenous polyQ and Q/N-rich proteins, particularly within nucleoli and mitochondria.
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http://dx.doi.org/10.1038/s41598-018-27900-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6015068PMC
June 2018

Yeast as a model to study protein misfolding in aged cells.

FEMS Yeast Res 2018 09;18(6)

Schulich School of Medicine and Dentistry, Western University, London, ON N6A 5C1, Canada.

Yeast models of neurodegenerative diseases associated with protein misfolding and protein aggregation have given unique insights into the underlying genetic and cellular pathomechanisms. These yeast models recapitulate central aspects of protein misfolding and the ensuing toxicity, such as interference with cellular protein quality control, concentration-dependent formation of insoluble, often amyloid-like aggregates and the associated toxicity. Advanced age is undoubtedly the highest and most common risk factor for most neurodegenerative diseases. Since yeast has served as a superb model to study cellular aspects of aging, we outline strategies to study how aging modulates protein misfolding and its toxicity, thereby opening new avenues to continue the success of yeast as powerful models to study neurodegenerative diseases.
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http://dx.doi.org/10.1093/femsyr/foy054DOI Listing
September 2018

Visualizing tRNA-dependent mistranslation in human cells.

RNA Biol 2018 9;15(4-5):567-575. Epub 2017 Nov 9.

a Department of Biochemistry , The University of Western Ontario , London , ON , Canada.

High-fidelity translation and a strictly accurate proteome were originally assumed as essential to life and cellular viability. Yet recent studies in bacteria and eukaryotic model organisms suggest that proteome-wide mistranslation can provide selective advantages and is tolerated in the cell at higher levels than previously thought (one error in 6.9 × 10 in yeast) with a limited impact on phenotype. Previously, we selected a tRNA containing a single mutation that induces mistranslation with alanine at proline codons in yeast. Yeast tolerate the mistranslation by inducing a heat-shock response and through the action of the proteasome. Here we found a homologous human tRNA (G3:U70) mutant that is not aminoacylated with proline, but is an efficient alanine acceptor. In live human cells, we visualized mistranslation using a green fluorescent protein reporter that fluoresces in response to mistranslation at proline codons. In agreement with measurements in yeast, quantitation based on the GFP reporter suggested a mistranslation rate of up to 2-5% in HEK 293 cells. Our findings suggest a stress-dependent phenomenon where mistranslation levels increased during nutrient starvation. Human cells did not mount a detectable heat-shock response and tolerated this level of mistranslation without apparent impact on cell viability. Because humans encode ∼600 tRNA genes and the natural population has greater tRNA sequence diversity than previously appreciated, our data also demonstrate a cell-based screen with the potential to elucidate mutations in tRNAs that may contribute to or alleviate disease.
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http://dx.doi.org/10.1080/15476286.2017.1379645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6103679PMC
November 2018

The Hsp70/Hsp90 Chaperone Machinery in Neurodegenerative Diseases.

Front Neurosci 2017 16;11:254. Epub 2017 May 16.

Molecular Medicine, Robarts Research Institute, University of Western OntarioLondon, ON, Canada.

The accumulation of misfolded proteins in the human brain is one of the critical features of many neurodegenerative diseases, including Alzheimer's disease (AD). Assembles of beta-amyloid (Aβ) peptide-either soluble (oligomers) or insoluble (plaques) and of tau protein, which form neurofibrillary tangles, are the major hallmarks of AD. Chaperones and co-chaperones regulate protein folding and client maturation, but they also target misfolded or aggregated proteins for refolding or for degradation, mostly by the proteasome. They form an important line of defense against misfolded proteins and are part of the cellular quality control system. The heat shock protein (Hsp) family, particularly Hsp70 and Hsp90, plays a major part in this process and it is well-known to regulate protein misfolding in a variety of diseases, including tau levels and toxicity in AD. However, the role of Hsp90 in regulating protein misfolding is not yet fully understood. For example, knockdown of Hsp90 and its co-chaperones in a model of Aβ misfolding leads to increased toxicity. On the other hand, the use of Hsp90 inhibitors in AD mouse models reduces Aβ toxicity, and normalizes synaptic function. Stress-inducible phosphoprotein 1 (STI1), an intracellular co-chaperone, mediates the transfer of clients from Hsp70 to Hsp90. Importantly, STI1 has been shown to regulate aggregation of amyloid-like proteins in yeast. In addition to its intracellular function, STI1 can be secreted by diverse cell types, including astrocytes and microglia and function as a neurotrophic ligand by triggering signaling via the cellular prion protein (PrP). Extracellular STI1 can prevent Aβ toxic signaling by (i) interfering with Aβ binding to PrP and (ii) triggering pro-survival signaling cascades. Interestingly, decreased levels of STI1 in can also increase toxicity in an amyloid model. In this review, we will discuss the role of intracellular and extracellular STI1 and the Hsp70/Hsp90 chaperone network in mechanisms underlying protein misfolding in neurodegenerative diseases, with particular focus on AD.
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http://dx.doi.org/10.3389/fnins.2017.00254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5433227PMC
May 2017

Candida albicans Is Resistant to Polyglutamine Aggregation and Toxicity.

G3 (Bethesda) 2017 01 5;7(1):95-108. Epub 2017 Jan 5.

Department of Molecular Genetics, University of Toronto, Ontario M5S 1A8, Canada

Disruption of protein quality control can be detrimental, having toxic effects on single cell organisms and contributing to neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's in humans. Here, we examined the effects of polyglutamine (polyQ) aggregation in a major fungal pathogen of humans, Candida albicans, with the goal of identifying new approaches to disable this fungus. However, we discovered that expression of polyQ stretches up to 230Q had no effect on C. albicans ability to grow and withstand proteotoxic stress. Bioinformatics analysis demonstrates that C. albicans has a similarly glutamine-rich proteome to the unicellular fungus Saccharomyces cerevisiae, which exhibits polyQ toxicity with as few as 72Q. Surprisingly, global transcriptional profiles indicated no significant change upon induction of up to 230Q. Proteomic analysis highlighted two key interactors of 230Q, Sis1 and Sgt2; however, loss of either protein had no additional effect on C. albicans toxicity. Our data suggest that C. albicans has evolved powerful mechanisms to overcome the toxicity associated with aggregation-prone proteins, providing a unique model for studying polyQ-associated diseases.
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http://dx.doi.org/10.1534/g3.116.035675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217127PMC
January 2017

Polyglutamine toxicity in yeast uncovers phenotypic variations between different fluorescent protein fusions.

Traffic 2017 01 1;18(1):58-70. Epub 2016 Nov 1.

Department of Anatomy and Cell Biology, The University of Western Ontario, London, Canada.

The palette of fluorescent proteins (FPs) available for live-cell imaging contains proteins that strongly differ in their biophysical properties. FPs cannot be assumed to be equivalent and in certain cases could significantly perturb the behavior of fluorescent reporters. We employed Saccharomyces cerevisiae to comprehensively study the impact of FPs on the toxicity of polyglutamine (polyQ) expansion proteins associated with Huntington's disease. The toxicity of polyQ fusion constructs is highly dependent on the sequences flanking the polyQ repeats. Thus, they represent a powerful tool to study the impact of fluorescent fusion partners. We observed significant differences on polyQ aggregation and toxicity between commonly used FPs. We generated a novel series of vectors with latest yeast-optimized FPs for investigation of Htt toxicity, including a newly optimized blue FP for expression in yeast. Our study highlights the importance of carefully choosing the optimal FPs when designing tagging strategies.
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http://dx.doi.org/10.1111/tra.12453DOI Listing
January 2017

CAG Expansions Are Genetically Stable and Form Nontoxic Aggregates in Cells Lacking Endogenous Polyglutamine Proteins.

mBio 2016 Sep 27;7(5). Epub 2016 Sep 27.

Department of Biochemistry, Geisel School of Medicine at Dartmouth College, Hanover, New Hampshire, USA Department of Medicine, Geisel School of Medicine at Dartmouth College, Hanover, New Hampshire, USA

Proteins containing polyglutamine (polyQ) regions are found in almost all eukaryotes, albeit with various frequencies. In humans, proteins such as huntingtin (Htt) with abnormally expanded polyQ regions cause neurodegenerative diseases such as Huntington's disease (HD). To study how the presence of endogenous polyQ aggregation modulates polyQ aggregation and toxicity, we expressed polyQ expanded Htt fragments (polyQ Htt) in Schizosaccharomyces pombe In stark contrast to other unicellular fungi, such as Saccharomyces cerevisiae, S. pombe is uniquely devoid of proteins with more than 10 Q repeats. We found that polyQ Htt forms aggregates within S. pombe cells only with exceedingly long polyQ expansions. Surprisingly, despite the presence of polyQ Htt aggregates in both the cytoplasm and nucleus, no significant growth defect was observed in S. pombe cells. Further, PCR analysis showed that the repetitive polyQ-encoding DNA region remained constant following transformation and after multiple divisions in S. pombe, in contrast to the genetic instability of polyQ DNA sequences in other organisms. These results demonstrate that cells with a low content of polyQ or other aggregation-prone proteins can show a striking resilience with respect to polyQ toxicity and that genetic instability of repetitive DNA sequences may have played an important role in the evolutionary emergence and exclusion of polyQ expansion proteins in different organisms.

Importance: Polyglutamine (polyQ) proteins encoded by repetitive CAG DNA sequences serve a variety of normal biological functions. Yet some proteins with abnormally expanded polyQ regions cause neurodegeneration through unknown mechanisms. To study how distinct cellular environments modulate polyQ aggregation and toxicity, we expressed CAG-expanded huntingtin fragments in Schizosaccharomyces pombe In stark contrast to many other eukaryotes, S. pombe is uniquely devoid of proteins containing long polyQ tracts. Our results show that S. pombe cells, despite their low content of endogenous polyQ proteins, exhibit striking and unexpected resilience with respect to polyQ toxicity and that genetic instability of repetitive DNA sequences may have played an important role in the emergence and expansion of polyQ domains in eukaryotic evolution.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5040113PMC
http://dx.doi.org/10.1128/mBio.01367-16DOI Listing
September 2016

Saccharomyces cerevisiae Tti2 Regulates PIKK Proteins and Stress Response.

G3 (Bethesda) 2016 06 1;6(6):1649-59. Epub 2016 Jun 1.

Department of Biochemistry, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, N6A5C1 Canada

The TTT complex is composed of the three essential proteins Tel2, Tti1, and Tti2 The complex is required to maintain steady state levels of phosphatidylinositol 3-kinase-related kinase (PIKK) proteins, including mTOR, ATM/Tel1, ATR/Mec1, and TRRAP/Tra1, all of which serve as regulators of critical cell signaling pathways. Due to their association with heat shock proteins, and with newly synthesized PIKK peptides, components of the TTT complex may act as cochaperones. Here, we analyze the consequences of depleting the cellular level of Tti2 in Saccharomyces cerevisiae We show that yeast expressing low levels of Tti2 are viable under optimal growth conditions, but the cells are sensitive to a number of stress conditions that involve PIKK pathways. In agreement with this, depleting Tti2 levels decreased expression of Tra1, Mec1, and Tor1, affected their localization and inhibited the stress responses in which these molecules are involved. Tti2 expression was not increased during heat shock, implying that it does not play a general role in the heat shock response. However, steady state levels of Hsp42 increase when Tti2 is depleted, and tti2L187P has a synthetic interaction with exon 1 of the human Huntingtin gene containing a 103 residue polyQ sequence, suggesting a general role in protein quality control. We also find that overexpressing Hsp90 or its cochaperones is synthetic lethal when Tti2 is depleted, an effect possibly due to imbalanced stoichiometry of a complex required for PIKK assembly. These results indicate that Tti2 does not act as a general chaperone, but may have a specialized function in PIKK folding and/or complex assembly.
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http://dx.doi.org/10.1534/g3.116.029520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4889661PMC
June 2016

A Toolbox for Rapid Quantitative Assessment of Chronological Lifespan and Survival in Saccharomyces cerevisiae.

Traffic 2016 06 1;17(6):689-703. Epub 2016 Apr 1.

Department of Anatomy and Cell Biology, The University of Western Ontario, London, N6A 5C1, Canada.

Saccharomyces cerevisiae is a well-established model organism to study the mechanisms of longevity. One of the two aging paradigms studied in yeast is termed chronological lifespan (CLS). CLS is defined by the amount of time non-dividing yeast cells can survive at stationary phase. Here, we propose new approaches that allow rapid and efficient quantification of survival rates in aging yeast cultures using either a fluorescent cell counter or microplate imaging. We have generated a software called analysr (Analytical Algorithm for Yeast Survival Rates) that allows automated and highly reproducible analysis of cell survival in aging yeast cultures using fluorescent data. To demonstrate the efficiency of our new experimental tools, we tested the previously characterized ability of caloric restriction to extend lifespan. Interestingly, we found that this process is independent of the expression of three central yeast heat shock proteins (Hsp26, Hsp42, Hsp104). Finally, our new assay is easily adaptable to other types of toxicity studies. Here, we assessed the toxicity of various concentrations of acetic acid, a known contributor of yeast chronological aging. These assays provide researchers with cost-effective, low- and high-content assays that can serve as an efficient complement to the time-consuming colony forming unit assay usually used in CLS studies.
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http://dx.doi.org/10.1111/tra.12391DOI Listing
June 2016

Cellular stress responses in protein misfolding diseases.

Future Sci OA 2015 Sep 1;1(2):FSO42. Epub 2015 Sep 1.

Department of Pathology & Laboratory Medicine, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada.

Many human diseases, particularly neurodegenerative diseases, are associated with protein misfolding. Cellular protein quality control includes all processes that ensure proper protein folding and thus prevent the toxic consequences of protein misfolding. The heat shock response (HSR) and the unfolded protein response (UPR) are major stress response pathways within protein quality control that antagonize protein misfolding in the cytosol and the endoplasmic reticulum, respectively. Huntington's disease is an inherited neurodegenerative disease caused by the misfolding of an abnormally expanded polyglutamine (polyQ) region in the protein huntingtin (Htt), polyQHtt. Using Huntington's disease as a paradigm, I review here the central role of both the HSR and the UPR in defining the toxicity associated with polyQHtt in Huntington's disease. These findings may begin to unravel a previously unappreciated cooperation between different stress response pathways in cells expressing misfolded proteins and consequently in neurodegenerative diseases.
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http://dx.doi.org/10.4155/fso.15.42DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5137871PMC
September 2015

Yeast as a platform to explore polyglutamine toxicity and aggregation.

Methods Mol Biol 2013 ;1017:153-61

Department of Pathology, Schulich School of Medicine and Dentistry, University of Western Ontario, Ontario, Canada.

Protein misfolding is associated with many neurodegenerative diseases, including neurodegenerative diseases caused by polyglutamine expansion proteins, such as Huntington's disease. The model organism baker's yeast (Saccharomyces cerevisiae) has provided important general insights into the basic cellular mechanisms underlying protein misfolding. Furthermore, experiments in yeast have identified cellular factors that modulate the toxicity and the aggregation associated with polyglutamine expansion proteins. Notably, many features discovered in yeast have been proven to be highly relevant in other model organisms and in human pathology. The experimental protocols depicted here serve to reliably determine polyglutamine toxicity and polyglutamine aggregation in yeast.
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http://dx.doi.org/10.1007/978-1-62703-438-8_11DOI Listing
December 2013

Poly-glutamine expanded huntingtin dramatically alters the genome wide binding of HSF1.

J Huntingtons Dis 2012 ;1(1):33-45

Department of Biological Engineering, Massachusetts Institute of Technology, 77 Mass Ave., Cambridge, MA, USA.

In Huntington's disease (HD), polyglutamine expansions in the huntingtin (Htt) protein cause subtle changes in cellular functions that, over-time, lead to neurodegeneration and death. Studies have indicated that activation of the heat shock response can reduce many of the effects of mutant Htt in disease models, suggesting that the heat shock response is impaired in the disease. To understand the basis for this impairment, we have used genome-wide chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) to examine the effects of mutant Htt on the master regulator of the heat shock response, HSF1. We find that, under normal conditions, HSF1 function is highly similar in cells carrying either wild-type or mutant Htt. However, polyQ-expanded Htt severely blunts the HSF1-mediated stress response. Surprisingly, we find that the HSF1 targets most affected upon stress are not directly associated with proteostasis, but with cytoskeletal binding, focal adhesion and GTPase activity. Our data raise the intriguing hypothesis that the accumulated damage from life-long impairment in these stress responses may contribute significantly to the etiology of Huntington's disease.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3537492PMC
May 2016

Small heat shock proteins potentiate amyloid dissolution by protein disaggregases from yeast and humans.

PLoS Biol 2012 19;10(6):e1001346. Epub 2012 Jun 19.

Boston Biomedical Research Institute, Watertown, Massachusetts, United States of America.

How small heat shock proteins (sHsps) might empower proteostasis networks to control beneficial prions or disassemble pathological amyloid is unknown. Here, we establish that yeast sHsps, Hsp26 and Hsp42, inhibit prionogenesis by the [PSI+] prion protein, Sup35, via distinct and synergistic mechanisms. Hsp42 prevents conformational rearrangements within molten oligomers that enable de novo prionogenesis and collaborates with Hsp70 to attenuate self-templating. By contrast, Hsp26 inhibits self-templating upon binding assembled prions. sHsp binding destabilizes Sup35 prions and promotes their disaggregation by Hsp104, Hsp70, and Hsp40. In yeast, Hsp26 or Hsp42 overexpression prevents [PSI+] induction, cures [PSI+], and potentiates [PSI+]-curing by Hsp104 overexpression. In vitro, sHsps enhance Hsp104-catalyzed disaggregation of pathological amyloid forms of α-synuclein and polyglutamine. Unexpectedly, in the absence of Hsp104, sHsps promote an unprecedented, gradual depolymerization of Sup35 prions by Hsp110, Hsp70, and Hsp40. This unanticipated amyloid-depolymerase activity is conserved from yeast to humans, which lack Hsp104 orthologues. A human sHsp, HspB5, stimulates depolymerization of α-synuclein amyloid by human Hsp110, Hsp70, and Hsp40. Thus, we elucidate a heretofore-unrecognized human amyloid-depolymerase system that could have applications in various neurodegenerative disorders.
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http://dx.doi.org/10.1371/journal.pbio.1001346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3378601PMC
October 2012

Pharmacological tuning of heat shock protein 70 modulates polyglutamine toxicity and aggregation.

ACS Chem Biol 2012 Sep 22;7(9):1556-64. Epub 2012 Jun 22.

Boston Biomedical Research Institute, Watertown, MA 04272-2829, USA.

Nine neurodegenerative disorders are caused by the abnormal expansion of polyglutamine (polyQ) regions within distinct proteins. Genetic and biochemical evidence has documented that the molecular chaperone, heat shock protein 70 (Hsp70), modulates polyQ toxicity and aggregation, yet it remains unclear how Hsp70 might be used as a potential therapeutic target in polyQ-related diseases. We have utilized a pair of membrane-permeable compounds that tune the activity of Hsp70 by either stimulating or by inhibiting its ATPase functions. Using these two pharmacological agents in both yeast and PC12 cell models of polyQ aggregation and toxicity, we were surprised to find that stimulating Hsp70 solubilized polyQ conformers and simultaneously exacerbated polyQ-mediated toxicity. By contrast, inhibiting Hsp70 ATPase activity protected against polyQ toxicity and promoted aggregation. These findings clarify the role of Hsp70 as a possible drug target in polyQ disorders and suggest that Hsp70 uses ATP hydrolysis to help partition polyQ proteins into structures with varying levels of proteotoxicity. Our results thus support an emerging concept in which certain kinds of polyQ aggregates may be protective, while more soluble polyQ species are toxic.
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http://dx.doi.org/10.1021/cb300166pDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3448832PMC
September 2012

Impaired heat shock response in cells expressing full-length polyglutamine-expanded huntingtin.

PLoS One 2012 23;7(5):e37929. Epub 2012 May 23.

Regenerative Biology Program, Boston Biomedical Research Institute, Watertown, Massachusetts, United States of America.

The molecular mechanisms by which polyglutamine (polyQ)-expanded huntingtin (Htt) causes neurodegeneration in Huntington's disease (HD) remain unclear. The malfunction of cellular proteostasis has been suggested as central in HD pathogenesis and also as a target of therapeutic interventions for the treatment of HD. We present results that offer a previously unexplored perspective regarding impaired proteostasis in HD. We find that, under non-stress conditions, the proteostatic capacity of cells expressing full length polyQ-expanded Htt is adequate. Yet, under stress conditions, the presence of polyQ-expanded Htt impairs the heat shock response, a key component of cellular proteostasis. This impaired heat shock response results in a reduced capacity to withstand the damage caused by cellular stress. We demonstrate that in cells expressing polyQ-expanded Htt the levels of heat shock transcription factor 1 (HSF1) are reduced, and, as a consequence, these cells have an impaired a heat shock response. Also, we found reduced HSF1 and HSP70 levels in the striata of HD knock-in mice when compared to wild-type mice. Our results suggests that full length, non-aggregated polyQ-expanded Htt blocks the effective induction of the heat shock response under stress conditions and may thus trigger the accumulation of cellular damage during the course of HD pathogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0037929PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3359295PMC
October 2012

Growth assays to assess polyglutamine toxicity in yeast.

J Vis Exp 2012 Mar 5(61):e3461. Epub 2012 Mar 5.

Boston Biomedical Research Institute, USA.

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. Huntington's disease (HD) is caused by the abnormal expansion of a polyglutamine (polyQ) region within the protein huntingtin. The polyQ-expanded huntingtin protein attains an aberrant conformation (i.e. it misfolds) and causes cellular toxicity. At least eight further neurodegenerative diseases are caused by polyQ-expansions, including the Spinocerebellar Ataxias and Kennedy's disease. The model organism yeast has facilitated significant insights into the cellular and molecular basis of polyQ-toxicity, including the impact of intra- and inter-molecular factors of polyQ-toxicity, and the identification of cellular pathways that are impaired in cells expressing polyQ-expansion proteins. Importantly, many aspects of polyQ-toxicity that were found in yeast were reproduced in other experimental systems and to some extent in samples from HD patients, thus demonstrating the significance of the yeast model for the discovery of basic mechanisms underpinning polyQ-toxicity. A direct and relatively simple way to determine polyQ-toxicity in yeast is to measure growth defects of yeast cells expressing polyQ-expansion proteins. This manuscript describes three complementary experimental approaches to determine polyQ-toxicity in yeast by measuring the growth of yeast cells expressing polyQ-expansion proteins. The first two experimental approaches monitor yeast growth on plates, the third approach monitors the growth of liquid yeast cultures using the BioscreenC instrument. Furthermore, this manuscript describes experimental difficulties that can occur when handling yeast polyQ models and outlines strategies that will help to avoid or minimize these difficulties. The protocols described here can be used to identify and to characterize genetic pathways and small molecules that modulate polyQ-toxicity. Moreover, the described assays may serve as templates for accurate analyses of the toxicity caused by other disease-associated misfolded proteins in yeast models.
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http://dx.doi.org/10.3791/3461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460590PMC
March 2012

Polyglutamine misfolding in yeast: toxic and protective aggregation.

Prion 2011 Oct-Dec;5(4):285-90. Epub 2011 Oct 1.

Boston Biomedical Research Institute, Watertown, MA, USA.

Protein misfolding is associated with many human diseases, including neurodegenerative diseases, such as Alzheimer disease, Parkinson disease and Huntington disease. Protein misfolding often results in the formation of intracellular or extracellular inclusions or aggregates. Even though deciphering the role of these aggregates has been the object of intense research activity, their role in protein misfolding diseases is unclear. Here, I discuss the implications of studies on polyglutamine aggregation and toxicity in yeast and other model organisms. These studies provide an excellent experimental and conceptual paradigm that contributes to understanding the differences between toxic and protective trajectories of protein misfolding. Future studies like the ones discussed here have the potential to transform basic concepts of protein misfolding in human diseases and may thus help to identify new therapeutic strategies for their treatment.
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http://dx.doi.org/10.4161/pri.18071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4012402PMC
April 2013

Ordered assembly of heat shock proteins, Hsp26, Hsp70, Hsp90, and Hsp104, on expanded polyglutamine fragments revealed by chemical probes.

J Biol Chem 2011 Nov 3;286(47):40486-93. Epub 2011 Oct 3.

Department of Pathology, University of Michigan, Ann Arbor, Michigan 48109, USA.

In Saccharomyces cerevisae, expanded polyglutamine (polyQ) fragments are assembled into discrete cytosolic aggregates in a process regulated by the molecular chaperones Hsp26, Hsp70, Hsp90, and Hsp104. To better understand how the different chaperones might cooperate during polyQ aggregation, we used sequential immunoprecipitations and mass spectrometry to identify proteins associated with either soluble (Q25) or aggregation-prone (Q103) fragments at both early and later times after induction of their expression. We found that Hsp26, Hsp70, Hsp90, and other chaperones interact with Q103, but not Q25, within the first 2 h. Further, Hsp70 and Hsp90 appear to be partially released from Q103 prior to the maturation of the aggregates and before the recruitment of Hsp104. To test the importance of this seemingly ordered process, we used a chemical probe to artificially enhance Hsp70 binding to Q103. This treatment retained both Hsp70 and Hsp90 on the polyQ fragment and, interestingly, limited subsequent exchange for Hsp26 and Hsp104, resulting in incomplete aggregation. Together, these results suggest that partial release of Hsp70 may be an essential step in the continued processing of expanded polyQ fragments in yeast.
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http://dx.doi.org/10.1074/jbc.M111.284448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220478PMC
November 2011

Monitoring polyglutamine toxicity in yeast.

Methods 2011 Mar 6;53(3):232-7. Epub 2010 Dec 6.

Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472, USA.

Experiments in yeast have significantly contributed to our understanding of general aspects of biochemistry, genetics, and cell biology. Yeast models have also delivered deep insights in to the molecular mechanism underpinning human diseases, including neurodegenerative diseases. Many neurodegenerative diseases are associated with the conversion of a protein from a normal and benign conformation into a disease-associated and toxic conformation - a process called protein misfolding. The misfolding of proteins with abnormally expanded polyglutamine (polyQ) regions causes several neurodegenerative diseases, such as Huntington's disease and the Spinocerebellar Ataxias. Yeast cells expressing polyQ expansion proteins recapitulate polyQ length-dependent aggregation and toxicity, which are hallmarks of all polyQ-expansion diseases. The identification of modifiers of polyQ toxicity in yeast revealed molecular mechanisms and cellular pathways that contribute to polyQ toxicity. Notably, several of these findings in yeast were reproduced in other model organisms and in human patients, indicating the validity of the yeast polyQ model. Here, we describe different expression systems for polyQ-expansion proteins in yeast and we outline experimental protocols to reliably and quantitatively monitor polyQ toxicity in yeast.
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http://dx.doi.org/10.1016/j.ymeth.2010.12.001DOI Listing
March 2011

Countering amyloid polymorphism and drug resistance with minimal drug cocktails.

Prion 2010 Oct-Dec;4(4):244-51. Epub 2010 Oct 12.

Boston Biomedical Research Institute, Watertown, MA, USA.

Several fatal, progressive neurodegenerative diseases, including various prion and prion-like disorders, are connected with the misfolding of specific proteins. These proteins misfold into toxic oligomeric species and a spectrum of distinct self-templating amyloid structures, termed strains. Hence, small molecules that prevent or reverse these protein-misfolding events might have therapeutic utility. Yet it is unclear whether a single small molecule can antagonize the complete repertoire of misfolded forms encompassing diverse amyloid polymorphs and soluble oligomers. We have begun to investigate this issue using the yeast prion protein Sup35 as an experimental paradigm. We have discovered that a polyphenol, (-)epigallocatechin-3-gallate (EGCG), effectively inhibited the formation of infectious amyloid forms (prions) of Sup35 and even remodeled preassembled prions. Surprisingly, EGCG selectively modulated specific prion strains and even selected for EGCG-resistant prion strains with novel structural and biological characteristics. Thus, treatment with a single small molecule antagonist of amyloidogenesis can select for novel, drug-resistant amyloid polymorphs. Importantly, combining EGCG with another small molecule, 4,5-bis-(4-methoxyanilino)phthalimide, synergistically antagonized and remodeled a wide array of Sup35 prion strains without producing any drug-resistant prions. We suggest that minimal drug cocktails, small collections of drugs that collectively antagonize all amyloid polymorphs, should be identified to besiege various neurodegenerative disorders.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3268956PMC
http://dx.doi.org/10.4161/pri.4.4.13597DOI Listing
June 2011

A synergistic small-molecule combination directly eradicates diverse prion strain structures.

Nat Chem Biol 2009 Dec 1;5(12):936-46. Epub 2009 Nov 1.

Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, USA.

Safely eradicating prions, amyloids and preamyloid oligomers may ameliorate several fatal neurodegenerative disorders. Yet whether small-molecule drugs can directly antagonize the entire spectrum of distinct amyloid structures or 'strains' that underlie distinct disease states is unclear. Here, we investigated this issue using the yeast prion protein Sup35. We have established how epigallocatechin-3-gallate (EGCG) blocks synthetic Sup35 prionogenesis, eliminates preformed Sup35 prions and disrupts inter- and intramolecular prion contacts. Unexpectedly, these direct activities were strain selective, altered the repertoire of accessible infectious forms and facilitated emergence of a new prion strain that configured original, EGCG-resistant intermolecular contacts. In vivo, EGCG cured and prevented induction of susceptible, but not resistant strains, and elicited switching from susceptible to resistant forms. Importantly, 4,5-bis-(4-methoxyanilino)phthalimide directly antagonized EGCG-resistant prions and synergized with EGCG to eliminate diverse Sup35 prion strains. Thus, synergistic small-molecule combinations that directly eradicate complete strain repertoires likely hold considerable therapeutic potential.
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http://dx.doi.org/10.1038/nchembio.246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909773PMC
December 2009