A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in
            <i>Drosophila</i>

Apostol, Barbara L.; Kazantsev, Alexsey; Raffioni, Simona; Illés, K.; Pallos, Judit; Bodai, László; Slepko, Natalia; Bear, James E.; Gertler, Frank B.; Hersch, Steven M.; Housman, David E.; Marsh, J. Lawrence; Thompson, Leslie M.

doi:10.1073/pnas.2628045100

Cited by 163 publications

(181 citation statements)

References 52 publications

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“…Our approach has enabled the direct observation of protein production and aggregation in real time in bacterial cell cultures and therefore opens the door to exploration of how cellular factors, such as molecular chaperones, or environmental variables, such as temperature, affect the rate and amount of aggregation and to the testing of potential therapeutic agents that might modulate aggregation (24,25). Our results complement previous efforts to follow protein aggregation in vivo (26)(27)(28)(29)(30).…”

Section: Discussionsupporting

confidence: 56%

Monitoring protein stability and aggregationin vivoby real-time fluorescent labeling

Ignatova

Gierasch

2003

Proc. Natl. Acad. Sci. U.S.A.

224

239

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In vivo fluorescent labeling of an expressed protein has enabled the observation of its stability and aggregation directly in bacterial cells. Mammalian cellular retinoic acid-binding protein I (CRABP I) was mutated to incorporate in a surface-exposed omega loop the sequence Cys-Cys-Gly-Pro-Cys-Cys, which binds specifically to a biarsenical fluorescein dye (FlAsH). Unfolding of labeled tetra-Cys CRABP I is accompanied by enhancement of FlAsH fluorescence, which made it possible to determine the free energy of unfolding of this protein by urea titration in cells and to follow in real time the formation of inclusion bodies by a slow-folding, aggregationprone mutant (FlAsH-labeled P39A tetra-Cys CRABP I). Aggregation in vivo displayed a concentration-dependent apparent lag time similar to observations of protein aggregation in purified in vitro model systems.protein aggregation ͉ protein folding ͉ fluorescence P rotein folding is an exquisitely optimized process that normally succeeds in producing functional molecules in vivo, despite physicochemical conditions that are very challenging. The heteropolymeric nature of the polypeptide chain encodes a great diversity of complex architectures of native proteins but also presents many side chain groups that are marginally soluble in aqueous solution. Not surprisingly, aggregation of newly synthesized proteins emerges as a process that competes with folding in vivo. In bacterial cells, aggregation of partially folded intermediates manifests itself in the production of insoluble inclusion bodies and often occurs when an exogenous protein is overexpressed (1). Several human diseases, including Alzheimer's, Huntington's, and spongiform encephalopathies, are characterized by the formation of insoluble protein aggregates (2). These distinctive aggregated states seem to be formed from partially folded, partially unfolded, or other nonnative intermediates and not from the native state. Thus, considerable research has been dedicated to the goal of achieving a fundamental understanding of protein aggregation, and much progress has been achieved in several systems in vitro (3). By contrast, relatively little is known about how this process occurs in vivo. Complicating research on protein folding and aggregation in vivo is the need to study these processes in the complex and concentrated environment of the cell. It is therefore highly desirable to develop methods to observe the fates of proteins directly in cells, including their thermodynamic stabilities, their kinetics of folding, the nature of folding intermediates and the energy landscape of folding, and the effects of mutations. Here, we have taken advantage of new approaches to labeling protein molecules in the cell with fluorescent dyes (4) to monitor the synthesis, folding, and aggregation of a protein whose in vitro folding has been well characterized.Cellular retinoic acid-binding protein I (CRABP I) is a 136-residue member of the large family of intracellular lipidbinding proteins, which fold into ␤-barrel structures (Fi...

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Section: Discussionsupporting

confidence: 56%

Monitoring protein stability and aggregationin vivoby real-time fluorescent labeling

Ignatova

Gierasch

2003

Proc. Natl. Acad. Sci. U.S.A.

224

239

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“…Despite the increasing evidence that htt inclusions are not toxic, aggregation assays are used to identify potential therapeutic compounds (8,24), and inhibition of inclusion formation is viewed as a positive outcome in therapeutic preclinical mouse trials (10,25). The presence of inclusions without pathology in the shortstop mouse suggests that primary screening for compounds that selectively inhibit inclusions may not identify agents that will be beneficial in clinical trials.…”

Section: Discussionmentioning

confidence: 99%

“…It is still controversial whether htt inclusions are pathogenic (2), benign biomarkers (5), or neuroprotective (4, 6). The distinction between these hypotheses is clinically relevant, because much therapeutic research has focused on screening compounds for their ability to inhibit inclusion formation (7,8). A decrease in inclusion formation has been interpreted as a positive outcome in preclinical therapeutic trials with mouse models (9, 10).…”

mentioning

confidence: 99%

Absence of behavioral abnormalities and neurodegenerationin vivodespite widespread neuronal huntingtin inclusions

Slow

Graham

Osmand

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

250

201

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We have serendipitously established a mouse that expresses an N-terminal human huntingtin (htt) fragment with an expanded polyglutamine repeat (Ϸ120) under the control of the endogenous human promoter (shortstop). Frequent and widespread htt inclusions occur early in shortstop mice. Despite these inclusions, shortstop mice display no clinical evidence of neuronal dysfunction and no neuronal degeneration as determined by brain weight, striatal volume, and striatal neuronal count. These results indicate that htt inclusions are not pathogenic in vivo. In contrast, the full-length yeast artificial chromosome (YAC) 128 model with the identical polyglutamine length and same level of transgenic protein expression as the shortstop demonstrates significant neuronal dysfunction and loss. In contrast to the YAC128 mouse, which demonstrates enhanced susceptibility to excitotoxic death, the shortstop mouse is protected from excitotoxicity, providing in vivo evidence suggesting that neurodegeneration in Huntington disease is mediated by excitotoxic mechanisms.Huntington disease ͉ mouse models ͉ excitotoxicity ͉ aggregates ͉ fragment H untingtin (htt), the protein product encoded by the gene mutated in Huntington disease (HD), forms aggregates and inclusion bodies in the presence of a pathogenic expanded polyglutamine (polyQ) repeat. Htt protein inclusions are a hallmark of HD and are present in brains of human patients (1), in HD mouse models (2, 3), and in cell culture models of HD (4). It is still controversial whether htt inclusions are pathogenic (2), benign biomarkers (5), or neuroprotective (4, 6). The distinction between these hypotheses is clinically relevant, because much therapeutic research has focused on screening compounds for their ability to inhibit inclusion formation (7,8). A decrease in inclusion formation has been interpreted as a positive outcome in preclinical therapeutic trials with mouse models (9, 10).Increasing evidence in vitro in cell culture models supports the hypothesis that htt inclusions are not pathogenic (5, 11). In a recent study, Arrasate et al. (4) discovered that in their cell culture system, neurons with inclusions had an increased likelihood of survival compared with neurons without inclusions. However, because these results were obtained in a cell culture system, the question of whether htt inclusions are toxic in vivo during the lifespan of an organism and therefore clinically relevant for patients with HD remains unanswered.Examinations of inclusions in brains from HD patients are limited due to the inability to sample inclusions over the natural history of the disease, and, therefore, studies of mouse models of HD can be useful in determining the role of htt inclusions in vivo. The yeast artificial chromosome (YAC) 128 model of HD, which expresses full-length mutant htt, forms intranuclear inclusions 12 months after the onset of behavioral changes measured by rotarod and 6 months after striatal neuronal degeneration (3).During the development of the full-length YAC mouse models, a m...

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“…44 Following the success of this forward-genetic approach, reverse genetics have been used extensively more recently (that is, start with a gene of interest and investigate whether mutations or manipulations compromise a phenotype in D. melanogaster, such as learning and memory). This approach has been used to draw connections between fly phenotypes and genes involved in psychiatric disorders in humans, such as Alzheimer's disease and other neurodegenerative disorders, [45][46][47][48][49][50] autism [51][52][53] and attention deficit and hyperactivity disorder. 54 A specific example of reverse genetic approaches in this model involves expressing DISC1, a gene associated with an increased risk of schizophrenia, and finding alterations in sleep architecture as a consequence.…”

Section: Drosophila Melanogastermentioning

confidence: 99%

Big ideas for small brains: what can psychiatry learn from worms, flies, bees and fish?

et al. 2010

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While the research community has accepted the value of rodent models as informative research platforms, there is less awareness of the utility of other small vertebrate and invertebrate animal models. Neuroscience is increasingly turning to smaller, non-rodent models to understand mechanisms related to neuropsychiatric disorders. Although they can never replace clinical research, there is much to be learnt from 'small brains'. In particular, these species can offer flexible genetic 'tool kits' that can be used to explore the expression and function of candidate genes in different brain regions. Very small animals also offer efficiencies with respect to high-throughput screening programs. This review provides a concise overview of the utility of models based on worm, fruit fly, honeybee and zebrafish. Although these species may have small brains, they offer the neuropsychiatric research community opportunities to explore some of the most important research questions in our field.

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A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in Drosophila

Cited by 163 publications

References 52 publications

Monitoring protein stability and aggregationin vivoby real-time fluorescent labeling

Monitoring protein stability and aggregationin vivoby real-time fluorescent labeling

Absence of behavioral abnormalities and neurodegenerationin vivodespite widespread neuronal huntingtin inclusions

Big ideas for small brains: what can psychiatry learn from worms, flies, bees and fish?

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