Publications by authors named "Thomas R Jahn"

25 Publications

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Autophagy inhibition rescues structural and functional defects caused by the loss of mitochondrial chaperone in .

Autophagy 2021 Jan 25:1-15. Epub 2021 Jan 25.

Research Group Synaptic Plasticity, Hertie Institute for Clinical Brain Research, University of Tübingen , Tübingen, Germany.

We investigated in larval and adult models whether loss of the mitochondrial chaperone is sufficient to cause pathological alterations commonly observed in Parkinson disease. At affected larval neuromuscular junctions, no effects on terminal size, bouton size or number, synapse size, or number were observed, suggesting that we studied an early stage of pathogenesis. At this stage, we noted a loss of synaptic vesicle proteins and active zone components, delayed synapse maturation, reduced evoked and spontaneous excitatory junctional potentials, increased synaptic fatigue, and cytoskeleton rearrangements. The adult model displayed ATP depletion, altered body posture, and susceptibility to heat-induced paralysis. Adult phenotypes could be suppressed by knockdown of , and . The knockdown of components of the macroautophagy/autophagy machinery or overexpression of human broadly rescued larval and adult phenotypes, while disease-associated variants did not. Overexpression of or promotion of autophagy exacerbated defects. AEL: after egg laying; AZ: active zone; brp: bruchpilot; Csp: cysteine string protein; dlg: discs large; eEJPs: evoked excitatory junctional potentials; GluR: glutamate receptor; HO: hydrogen peroxide; mEJP: miniature excitatory junctional potentials; MT: microtubule; NMJ: neuromuscular junction; PD: Parkinson disease; : PTEN-induced putative kinase 1; PSD: postsynaptic density; SSR: subsynaptic reticulum; SV: synaptic vesicle; VGlut: vesicular glutamate transporter.
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http://dx.doi.org/10.1080/15548627.2020.1871211DOI Listing
January 2021

Disassembly of Tau fibrils by the human Hsp70 disaggregation machinery generates small seeding-competent species.

J Biol Chem 2020 07 28;295(28):9676-9690. Epub 2020 May 28.

Center for Molecular Biology of Heidelberg University (ZMBH) and German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany

The accumulation of amyloid Tau aggregates is implicated in Alzheimer's disease (AD) and other tauopathies. Molecular chaperones are known to maintain protein homeostasis. Here, we show that an ATP-dependent human chaperone system disassembles Tau fibrils We found that this function is mediated by the core chaperone HSC70, assisted by specific cochaperones, in particular class B J-domain proteins and a heat shock protein 110 (Hsp110)-type nucleotide exchange factor (NEF). The Hsp70 disaggregation machinery processed recombinant fibrils assembled from all six Tau isoforms as well as Sarkosyl-resistant Tau aggregates extracted from cell cultures and human AD brain tissues, demonstrating the ability of the Hsp70 machinery to recognize a broad range of Tau aggregates. However, the chaperone activity released monomeric and small oligomeric Tau species, which induced the aggregation of self-propagating Tau conformers in a Tau cell culture model. We conclude that the activity of the Hsp70 disaggregation machinery is a double-edged sword, as it eliminates Tau amyloids at the cost of generating new seeds.
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http://dx.doi.org/10.1074/jbc.RA120.013478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363153PMC
July 2020

N368-Tau fragments generated by legumain are detected only in trace amount in the insoluble Tau aggregates isolated from AD brain.

Acta Neuropathol Commun 2019 11 13;7(1):177. Epub 2019 Nov 13.

Neuroscience Discovery, AbbVie Deutschland GmbH & Co. KG, Knollstrasse, 67061, Ludwigshafen, Germany.

Intraneuronal insoluble inclusions made of Tau protein are neuropathological hallmarks of Alzheimer Disease (AD). Cleavage of Tau by legumain (LGMN) has been proposed to be crucial for aggregation of Tau into fibrils. However, it remains unclear if LGMN-cleaved Tau fragments accumulate in AD Tau inclusions.Using an in vitro enzymatic assay and non-targeted mass spectrometry, we identified four putative LGMN cleavage sites at Tau residues N167-, N255-, N296- and N368. Cleavage at N368 generates variously sized N368-Tau fragments that are aggregation prone in the Thioflavin T assay in vitro. N368-cleaved Tau is not detected in the brain of legumain knockout mice, indicating that LGMN is required for Tau cleavage in the mouse brain in vivo. Using a targeted mass spectrometry method in combination with tissue fractionation and biochemical analysis, we investigated whether N368-cleaved Tau is differentially produced and aggregated in brain of AD patients and control subjects. In brain soluble extracts, despite reduced uncleaved Tau in AD, levels of N368-cleaved Tau are comparable in AD and control hippocampus, suggesting that LGMN-mediated cleavage of Tau is not altered in AD. Consistently, levels of activated, cleaved LGMN are also similar in AD and control brain extracts. To assess the potential accumulation of N368-cleaved Tau in insoluble Tau aggregates, we analyzed sarkosyl-insoluble extracts from AD and control hippocampus. Both N368-cleaved Tau and uncleaved Tau were significantly increased in AD as a consequence of pathological Tau inclusions accumulation. However, the amount of N368-cleaved Tau represented only a very minor component (< 0.1%) of insoluble Tau.Our data indicate that LGMN physiologically cleaves Tau in the mouse and human brain generating N368-cleaved Tau fragments, which remain largely soluble and are present only in low proportion in Tau insoluble aggregates compared to uncleaved Tau. This suggests that LGMN-cleaved Tau has limited role in the progressive accumulation of Tau inclusions in AD.
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http://dx.doi.org/10.1186/s40478-019-0831-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6854719PMC
November 2019

Changes in Glutathione Redox Potential Are Linked to Aβ-Induced Neurotoxicity.

Cell Rep 2018 08;24(7):1696-1703

Schaller Research Group Proteostasis in Neurodegenerative Disease, University of Heidelberg and the DKFZ, INF 581, 69120 Heidelberg, Germany. Electronic address:

Glutathione is the major low-molecular weight thiol of eukaryotic cells. It is central to one of the two major NADPH-dependent reducing systems and is likely to play a role in combating oxidative stress, a process suggested to play a key role in Alzheimer's disease (AD). However, the nature and relevance of redox changes in the onset and progression of AD are still uncertain. Here, we combine genetically encoded redox sensors with our Drosophila models of amyloid-beta (Aβ) aggregation. We find that changes in glutathione redox potential (E) closely correlate with disease onset and progression. We observe this redox imbalance specifically in neurons, but not in glia cells. E changes and Aβ deposition are also accompanied by increased JNK stress signaling. Furthermore, pharmacologic and genetic manipulation of glutathione synthesis modulates Aβ-mediated neurotoxicity, suggesting a causal relationship between disturbed glutathione redox homeostasis and early AD pathology.
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http://dx.doi.org/10.1016/j.celrep.2018.07.052DOI Listing
August 2018

Unconventional Secretion Mediates the Trans-cellular Spreading of Tau.

Cell Rep 2018 05;23(7):2039-2055

Schaller Research Group at the University of Heidelberg and the DKFZ, Proteostasis in Neurodegenerative Disease (B180), INF 581, 69120 Heidelberg, Germany. Electronic address:

The progressive deposition of misfolded hyperphosphorylated tau is a pathological hallmark of tauopathies, including Alzheimer's disease. However, the underlying molecular mechanisms governing the intercellular spreading of tau species remain elusive. Here, we show that full-length soluble tau is unconventionally secreted by direct translocation across the plasma membrane. Increased secretion is favored by tau hyperphosphorylation, which provokes microtubule detachment and increases the availability of free protein inside cells. Using a series of binding assays, we show that free tau interacts with components enriched at the inner leaflet of the plasma membrane, finally leading to its translocation across the plasma membrane mediated by sulfated proteoglycans. We provide further evidence that secreted soluble tau species spread trans-cellularly and are sufficient for the induction of intracellular tau aggregation in adjacent cells. Our study demonstrates the mechanistic details of tau secretion and provides insights into the initiation and progression of tau pathology.
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http://dx.doi.org/10.1016/j.celrep.2018.04.056DOI Listing
May 2018

Seed-induced acceleration of amyloid-β mediated neurotoxicity in vivo.

Nat Commun 2017 09 11;8(1):512. Epub 2017 Sep 11.

Proteostasis in Neurodegenerative Disease (B180), German Cancer Research Center, Schaller Research Group at the University of Heidelberg and DKFZ, Heidelberg, 69120, Germany.

Seeded propagation of amyloid-beta (Aβ) pathology is suggested to contribute to the progression of Alzheimer's disease. Local overproduction of aggregation-prone Aβ variants could explain the focal initiation of a seeding cascade that subsequently triggers widespread pathology. Several animal models support this seeding concept by demonstrating accelerated Aβ deposition following inoculation with Aβ-containing homogenates, however its role in progressive neurodegeneration remains unclear. Here, we present a non-invasive approach to study Aβ seeding processes in vivo using Drosophila models. We show that small amounts of aggregation-competent Aβ seeds, generated in selected neuronal clusters, can induce the deposition of the pan-neuronally expressed and otherwise soluble Aβ. Moreover, our models visualize the accelerated formation and propagation of amyloid pathology throughout the brain, which correlates with severe neurotoxicity. Taken together, these in vivo models provide mechanistic insights into disease-related processes and represent versatile genetic tools to determine novel modifiers of the Aβ seeding cascade.Seeding of amyloid beta from one brain region to another is thought to contribute to the progression of Alzheimer's disease, although to date most studies have depended on inoculation of animals with exogenous amyloid. Here the authors describe a genetic seed and target system in Drosophila which may be useful for the mechanistic study of seeding of amyloid in vivo.
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http://dx.doi.org/10.1038/s41467-017-00579-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5594032PMC
September 2017

Bin1 directly remodels actin dynamics through its BAR domain.

EMBO Rep 2017 11 11;18(11):2051-2066. Epub 2017 Sep 11.

Proteostasis in Neurodegenerative Disease (B180), Schaller Research Group at the University of Heidelberg and DKFZ, Heidelberg, Germany

Endocytic processes are facilitated by both curvature-generating BAR-domain proteins and the coordinated polymerization of actin filaments. Under physiological conditions, the N-BAR protein Bin1 has been shown to sense and curve membranes in a variety of cellular processes. Recent studies have identified Bin1 as a risk factor for Alzheimer's disease, although its possible pathological function in neurodegeneration is currently unknown. Here, we report that Bin1 not only shapes membranes, but is also directly involved in actin binding through its BAR domain. We observed a moderate actin bundling activity by human Bin1 and describe its ability to stabilize actin filaments against depolymerization. Moreover, Bin1 is also involved in stabilizing tau-induced actin bundles, which are neuropathological hallmarks of Alzheimer's disease. We also provide evidence for this effect , where we observed that downregulation of Bin1 in a model of tauopathy significantly reduces the appearance of tau-induced actin inclusions. Together, these findings reveal the ability of Bin1 to modify actin dynamics and provide a possible mechanistic connection between Bin1 and tau-induced pathobiological changes of the actin cytoskeleton.
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http://dx.doi.org/10.15252/embr.201744137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666605PMC
November 2017

The KIF1A homolog Unc-104 is important for spontaneous release, postsynaptic density maturation and perisynaptic scaffold organization.

Sci Rep 2017 03 27;7:38172. Epub 2017 Mar 27.

Junior Research Group Synaptic Plasticity, Hertie-Institute for Clinical Brain Research, University of Tübingen, Otfried-Müller-Str. 27, 72076 Tübingen 72076, Germany.

The kinesin-3 family member KIF1A has been shown to be important for experience dependent neuroplasticity. In Drosophila, amorphic mutations in the KIF1A homolog unc-104 disrupt the formation of mature boutons. Disease associated KIF1A mutations have been associated with motor and sensory dysfunctions as well as non-syndromic intellectual disability in humans. A hypomorphic mutation in the forkhead-associated domain of Unc-104, unc-104, impairs active zone maturation resulting in an increased fraction of post-synaptic glutamate receptor fields that lack the active zone scaffolding protein Bruchpilot. Here, we show that the unc-104mutation causes defects in synaptic transmission as manifested by reduced amplitude of both evoked and miniature excitatory junctional potentials. Structural defects observed in the postsynaptic compartment of mutant NMJs include reduced glutamate receptor field size, and altered glutamate receptor composition. In addition, we observed marked loss of postsynaptic scaffolding proteins and reduced complexity of the sub-synaptic reticulum, which could be rescued by pre- but not postsynaptic expression of unc-104. Our results highlight the importance of kinesin-3 based axonal transport in synaptic transmission and provide novel insights into the role of Unc-104 in synapse maturation.
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http://dx.doi.org/10.1038/srep38172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5366810PMC
March 2017

The Drosophila KIF1A Homolog unc-104 Is Important for Site-Specific Synapse Maturation.

Front Cell Neurosci 2016 5;10:207. Epub 2016 Sep 5.

Junior Research Group Synaptic Plasticity, Hertie-Institute for Clinical Brain Research, University of TübingenTübingen, Germany; Schaller Research Group at the University of Heidelberg and DKFZ, Proteostasis in Neurodegenerative Disease (B180), German Cancer Research CenterHeidelberg, Germany.

Mutations in the kinesin-3 family member KIF1A have been associated with hereditary spastic paraplegia (HSP), hereditary and sensory autonomic neuropathy type 2 (HSAN2) and non-syndromic intellectual disability (ID). Both autosomal recessive and autosomal dominant forms of inheritance have been reported. Loss of KIF1A or its homolog unc-104 causes early postnatal or embryonic lethality in mice and Drosophila, respectively. In this study, we use a previously described hypomorphic allele of unc-104, unc-104(bris) , to investigate the impact of partial loss-of-function of kinesin-3 on synapse maturation at the Drosophila neuromuscular junction (NMJ). Unc-104(bris) mutants exhibit structural defects where a subset of synapses at the NMJ lack all investigated active zone (AZ) proteins, suggesting a complete failure in the formation of the cytomatrix at the active zone (CAZ) at these sites. Modulating synaptic Bruchpilot (Brp) levels by ectopic overexpression or RNAi-mediated knockdown suggests that the loss of AZ components such as Ca(2+) channels and Liprin-α is caused by impaired kinesin-3 based transport rather than due to the absence of the key AZ organizer protein, Brp. In addition to defects in CAZ assembly, unc-104(bris) mutants display further defects such as depletion of dense core and synaptic vesicle (SV) markers from the NMJ. Notably, the level of Rab3, which is important for the allocation of AZ proteins to individual release sites, was severely reduced at unc-104(bris) mutant NMJs. Overexpression of Rab3 partially ameliorates synaptic phenotypes of unc-104(bris) larvae, suggesting that lack of presynaptic Rab3 contributes to defects in synapse maturation.
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http://dx.doi.org/10.3389/fncel.2016.00207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5011141PMC
September 2016

Cellular and molecular modifier pathways in tauopathies: the big picture from screening invertebrate models.

J Neurochem 2016 Apr 11;137(1):12-25. Epub 2016 Feb 11.

Schaller Research Group at the University of Heidelberg and DKFZ, Proteostasis in Neurodegenerative Disease (B180), German Cancer Research Center, Heidelberg, Germany.

Abnormal tau accumulations were observed and documented in post-mortem brains of patients affected by Alzheimer's disease (AD) long before the identification of mutations in the Microtubule-associated protein tau (MAPT) gene, encoding the tau protein, in a different neurodegenerative disease called Frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). The discovery of mutations in the MAPT gene associated with FTDP-17 highlighted that dysfunctions in tau alone are sufficient to cause neurodegeneration. Invertebrate models have been diligently utilized in investigating tauopathies, contributing to the understanding of cellular and molecular pathways involved in disease etiology. An important discovery came with the demonstration that over-expression of human tau in Drosophila leads to premature mortality and neuronal dysfunction including neurodegeneration, recapitulating some key neuropathological features of the human disease. The simplicity of handling invertebrate models combined with the availability of a diverse range of experimental resources make these models, in particular Drosophila a powerful invertebrate screening tool. Consequently, several large-scale screens have been performed using Drosophila, to identify modifiers of tau toxicity. The screens have revealed not only common cellular and molecular pathways, but in some instances the same modifier has been independently identified in two or more screens suggesting a possible role for these modifiers in regulating tau toxicity. The purpose of this review is to discuss the genetic modifier screens on tauopathies performed in Drosophila and C. elegans models, and to highlight the common cellular and molecular pathways that have emerged from these studies. Here, we summarize results of tau toxicity screens providing mechanistic insights into pathological alterations in tauopathies. Key pathways or modifiers that have been identified are associated with a broad range of processes including, but not limited to, phosphorylation, cytoskeleton organization, axonal transport, regulation of cellular proteostasis, transcription, RNA metabolism, cell cycle regulation, and apoptosis. We discuss the utility and application of invertebrate models in elucidating the cellular and molecular functions of novel and uncharacterized disease modifiers identified in large-scale screens as well as for investigating the function of genes identified as risk factors in genome-wide association studies from human patients in the post-genomic era. In this review, we combined and summarized several large-scale modifier screens performed in invertebrate models to identify modifiers of tau toxicity. A summary of the screens show that diverse cellular processes are implicated in the modification of tau toxicity. Kinases and phosphatases are the most predominant class of modifiers followed by components required for cellular proteostasis and axonal transport and cytoskeleton elements.
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http://dx.doi.org/10.1111/jnc.13532DOI Listing
April 2016

Reticulon-like-1, the Drosophila orthologue of the hereditary spastic paraplegia gene reticulon 2, is required for organization of endoplasmic reticulum and of distal motor axons.

Hum Mol Genet 2012 Aug 27;21(15):3356-65. Epub 2012 Apr 27.

Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK.

Several causative genes for hereditary spastic paraplegia encode proteins with intramembrane hairpin loops that contribute to the curvature of the endoplasmic reticulum (ER), but the relevance of this function to axonal degeneration is not understood. One of these genes is reticulon2. In contrast to mammals, Drosophila has only one widely expressed reticulon orthologue, Rtnl1, and we therefore used Drosophila to test its importance for ER organization and axonal function. Rtnl1 distribution overlapped with that of the ER, but in contrast to the rough ER, was enriched in axons. The loss of Rtnl1 led to the expansion of the rough or sheet ER in larval epidermis and elevated levels of ER stress. It also caused abnormalities specifically within distal portions of longer motor axons and in their presynaptic terminals, including disruption of the smooth ER (SER), the microtubule cytoskeleton and mitochondria. In contrast, proximal axon portions appeared unaffected. Our results provide direct evidence for reticulon function in the organization of the SER in distal longer axons, and support a model in which spastic paraplegia can be caused by impairment of axonal the SER. Our data provide a route to further understanding of both the role of the SER in axons and the pathological consequences of the impairment of this compartment.
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http://dx.doi.org/10.1093/hmg/dds167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3392112PMC
August 2012

Expression in drosophila of tandem amyloid β peptides provides insights into links between aggregation and neurotoxicity.

J Biol Chem 2012 Jun 29;287(24):20748-54. Epub 2012 Mar 29.

Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, United Kingdom.

The generation and subsequent aggregation of amyloid β (Aβ) peptides play a crucial initiating role in the pathogenesis of Alzheimer disease (AD). The two main isoforms of these peptides have 40 (Aβ(40)) or 42 residues (Aβ(42)), the latter having a higher propensity to aggregate in vitro and being the main component of the plaques observed in vivo in AD patients. We have designed a series of tandem dimeric constructs of these Aβ peptides to probe the manner in which changes in the aggregation kinetics of Aβ affect its deposition and toxicity in a Drosophila melanogaster model system. The levels of insoluble aggregates were found to be substantially elevated in flies expressing the tandem constructs of both Aβ(40) and Aβ(42) compared with the equivalent monomeric peptides, consistent with the higher effective concentration, and hence increased aggregation rate, of the peptides in the tandem repeat. A unique feature of the Aβ(42) constructs, however, is the appearance of high levels of soluble oligomeric aggregates and a corresponding dramatic increase in their in vivo toxicity. The toxic nature of the Aβ(42) peptide in vivo can therefore be attributed to the higher kinetic stability of the oligomeric intermediate states that it populates relative to those of Aβ(40) rather than simply to its higher rate of aggregation.
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http://dx.doi.org/10.1074/jbc.M112.350124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3370257PMC
June 2012

Ovine PrP transgenic Drosophila show reduced locomotor activity and decreased survival.

Biochem J 2012 Jun;444(3):487-95

Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 OES, U.K.

Drosophila have emerged as a model system to study mammalian neurodegenerative diseases. In the present study we have generated Drosophila transgenic for ovine PrP (prion protein) to begin to establish an invertebrate model of ovine prion disease. We generated Drosophila transgenic for polymorphic variants of ovine PrP by PhiC31 site-specific germ-line transformation under expression control by the bi-partite GAL4/UAS (upstream activating sequence) system. Site-specific transgene insertion in the fly genome allowed us to test the hypothesis that single amino acid codon changes in ovine PrP modulate prion protein levels and the phenotype of the fly when expressed in the Drosophila nervous system. The Arg(154) ovine PrP variants showed higher levels of PrP expression in neuronal cell bodies and insoluble PrP conformer than did His(154) variants. High levels of ovine PrP expression in Drosophila were associated with phenotypic effects, including reduced locomotor activity and decreased survival. Significantly, the present study highlights a critical role for helix-1 in the formation of distinct conformers of ovine PrP, since expression of His(154) variants were associated with decreased survival in the absence of high levels of PrP accumulation. Collectively, the present study shows that variants of the ovine PrP are associated with different spontaneous detrimental effects in ovine PrP transgenic Drosophila.
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http://dx.doi.org/10.1042/BJ20112141DOI Listing
June 2012

The iFly tracking system for an automated locomotor and behavioural analysis of Drosophila melanogaster.

Integr Biol (Camb) 2011 Jul 23;3(7):755-60. Epub 2011 Jun 23.

Department of Chemistry, University of Cambridge, Cambridge, UK.

The use of animal models in medical research provides insights into molecular and cellular mechanisms of human disease, and helps identify and test novel therapeutic strategies. Drosophila melanogaster--the common fruit fly--is one of the most well-established model organisms, as its study can be performed more readily and with far less expense than for other model animal systems, such as mice, fish, or primates. In the case of fruit flies, standard assays are based on the analysis of longevity and basic locomotor functions. Here we present the iFly tracking system, which enables to increase the amount of quantitative information that can be extracted from these studies, and to reduce significantly the duration and costs associated with them. The iFly system uses a single camera to simultaneously track the trajectories of up to 20 individual flies with about 100 μm spatial and 33 ms temporal resolution. The statistical analysis of fly movements recorded with such accuracy makes it possible to perform a rapid and fully automated quantitative analysis of locomotor changes in response to a range of different stimuli. We anticipate that the iFly method will reduce very considerably the costs and the duration of the testing of genetic and pharmacological interventions in Drosophila models, including an earlier detection of behavioural changes and a large increase in throughput compared to current longevity and locomotor assays.
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http://dx.doi.org/10.1039/c0ib00149jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5011414PMC
July 2011

Modeling serpin conformational diseases in Drosophila melanogaster.

Methods Enzymol 2011 ;499:227-58

Department of Genetics, University of Cambridge, Cambridge, United Kingdom.

Transgenic Drosophila melanogaster have been used to model both the physiological and pathological behavior of serpins. The ability to generate flies expressing serpins and to rapidly assess associated phenotypes contributes to the power of this paradigm. While providing a whole-organism model of serpinopathies the powerful toolkit of genetic interventions allows precise molecular dissection of important biological pathways. In this chapter, we summarize the contribution that flies have made to the serpin field and then describe some of the experimental methods that are employed in these studies. In particular, we will describe the generation of transgenic flies, the assessment of phenotypes, and the principles of how to perform a genetic screen.
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http://dx.doi.org/10.1016/B978-0-12-386471-0.00012-2DOI Listing
October 2011

Detection of early locomotor abnormalities in a Drosophila model of Alzheimer's disease.

J Neurosci Methods 2011 Apr 18;197(1):186-9. Epub 2011 Feb 18.

Department of Genetics, University of Cambridge, UK; Department of Chemistry, University of Cambridge, UK.

Behavioural assays represent sensitive methods for detecting neuronal dysfunction in model organisms. A number of manual methods have been established for Drosophila, however these are time-consuming and generate parameter-poor phenotype descriptors. Here, we have developed an automated computer vision system to monitor accurately the three-dimensional locomotor trajectories of flies. This approach allows the quantitative description of fly trajectories, using small fly cohorts and short acquisition times. The application of this approach to a Drosophila model of Alzheimer's disease enables the early detection of progressive locomotor deficits and the quantitative assessment of phenotype severity. The approach can be widely applied to different disease models in a number of model organisms.
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http://dx.doi.org/10.1016/j.jneumeth.2011.01.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712187PMC
April 2011

The common architecture of cross-beta amyloid.

J Mol Biol 2010 Jan 23;395(4):717-27. Epub 2009 Sep 23.

Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.

Amyloid fibril deposition is central to the pathology of more than 30 unrelated diseases including Alzheimer's disease and Type 2 diabetes. It is generally accepted that amyloid fibrils share common structural features despite each disease being characterised by the deposition of an unrelated protein or peptide. The structure of amyloid fibrils has been studied using X-ray fibre diffraction and crystallography, solid-state NMR and electron paramagnetic resonance, and many different, sometimes opposing, models have been suggested. Many of these models are based on the original interpretation of the cross-beta diffraction pattern for cross-beta silk in which beta-strands run perpendicular to the fibre axis, although alternative models include beta-helices and natively structured proteins. Here, we have analysed opposing model structures and examined the necessary structural elements within the amyloid core structure, as well as producing idealised models to test the limits of the core conformation. Our work supports the view that amyloid fibrils share a number of common structural features, resulting in characteristic diffraction patterns. This pattern may be satisfied by structures in which the strands align close to perpendicular to the fibre axis and are regularly arranged to form beta-sheet ribbons. Furthermore, the fibril structure contains several beta-sheets that associate via side-chain packing to form the final protofilament structure.
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http://dx.doi.org/10.1016/j.jmb.2009.09.039DOI Listing
January 2010

Globular tetramers of beta(2)-microglobulin assemble into elaborate amyloid fibrils.

J Mol Biol 2009 May 5;389(1):48-57. Epub 2009 Apr 5.

Department of Crystallography and Institute of Structural and Molecular Biology, Birkbeck College, London, UK.

Amyloid fibrils are ordered polymers in which constituent polypeptides adopt a non-native fold. Despite their importance in degenerative human diseases, the overall structure of amyloid fibrils remains unknown. High-resolution studies of model peptide assemblies have identified residues forming cross-beta-strands and have revealed some details of local beta-strand packing. However, little is known about the assembly contacts that define the fibril architecture. Here we present a set of three-dimensional structures of amyloid fibrils formed from full-length beta(2)-microglobulin, a 99-residue protein involved in clinical amyloidosis. Our cryo-electron microscopy maps reveal a hierarchical fibril structure built from tetrameric units of globular density, with at least three different subunit interfaces in this homopolymeric assembly. These findings suggest a more complex superstructure for amyloid than hitherto suspected and prompt a re-evaluation of the defining features of the amyloid fold.
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http://dx.doi.org/10.1016/j.jmb.2009.03.066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726924PMC
May 2009

HDX-ESI-MS reveals enhanced conformational dynamics of the amyloidogenic protein beta(2)-microglobulin upon release from the MHC-1.

J Am Soc Mass Spectrom 2009 Feb 17;20(2):278-86. Epub 2008 Oct 17.

Astbury Centre for Structural Molecular Biology, Institute of Molecular and Cellular Biology, University of Leeds, Leeds, United Kingdom.

The light chain of the major histocompatibility complex class 1 (MHC-1), the protein beta(2)-microglobulin (beta(2)m), has amyloidogenic properties that arise only upon its dissociation from the MHC-1. Here hydrogen/deuterium exchange electrospray ionization mass spectrometry (HDX-ESI-MS) has been used to compare the solution dynamics of beta(2)m in its MHC-1 bound state compared with those of beta(2)m as a free monomer. The capability of tandem mass spectrometry to dissociate the MHC-1 into its individual constituents in the gas phase following deuterium incorporation in solution has permitted the direct observation of the exchange properties of MHC-1 bound beta(2)m for the first time. The HDX-ESI-MS data show clearly that the H-->D exchange of MHC-1 bound beta(2)m follows EX2 kinetics and that about 20 protons remain protected from exchange after 17 days. Free from the MHC-1, monomeric beta(2)m exhibits significantly different HDX behavior, which encompasses both EX1 and EX2 kinetics. The EX2 kinetics indicate a tenfold increase in the rate of exchange compared with MHC-1 bound beta(2)m, with just 10 protons remaining protected from EX2 exchange and therefore exchanging only via the EX1 mechanism. The EX1 kinetics observed for unbound beta(2)m are consistent with unfolding of its exchange-protected core with a t(1/2) of 68 min (pH 7, 37 degrees C). Thus, upon dissociation from the stabilizing influence of the MHC-1, free beta(2)m becomes highly dynamic and undergoes unfolding transitions that result in an aggregation-competent protein.
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http://dx.doi.org/10.1016/j.jasms.2008.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2642988PMC
February 2009

A common beta-sheet architecture underlies in vitro and in vivo beta2-microglobulin amyloid fibrils.

J Biol Chem 2008 Jun 18;283(25):17279-86. Epub 2008 Apr 18.

Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.

Misfolding and aggregation of normally soluble proteins into amyloid fibrils and their deposition and accumulation underlies a variety of clinically significant diseases. Fibrillar aggregates with amyloid-like properties can also be generated in vitro from pure proteins and peptides, including those not known to be associated with amyloidosis. Whereas biophysical studies of amyloid-like fibrils formed in vitro have provided important insights into the molecular mechanisms of amyloid generation and the structural properties of the fibrils formed, amyloidogenic proteins are typically exposed to mild or more extreme denaturing conditions to induce rapid fibril formation in vitro. Whether the structure of the resulting assemblies is representative of their natural in vivo counterparts, thus, remains a fundamental unresolved issue. Here we show using Fourier transform infrared spectroscopy that amyloid-like fibrils formed in vitro from natively folded or unfolded beta(2)-microglobulin (the protein associated with dialysis-related amyloidosis) adopt an identical beta-sheet architecture. The same beta-strand signature is observed whether fibril formation in vitro occurs spontaneously or from seeded reactions. Comparison of these spectra with those of amyloid fibrils extracted from patients with dialysis-related amyloidosis revealed an identical amide I' absorbance maximum, suggestive of a characteristic and conserved amyloid fold. Our results endorse the relevance of biophysical studies for the investigation of the molecular mechanisms of beta(2)-microglobulin fibrillogenesis, knowledge about which may inform understanding of the pathobiology of this protein.
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http://dx.doi.org/10.1074/jbc.M710351200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2427364PMC
June 2008

Folding versus aggregation: polypeptide conformations on competing pathways.

Arch Biochem Biophys 2008 Jan 8;469(1):100-17. Epub 2007 Jun 8.

Astbury Centre for Structural and Molecular Biology, Institute of Molecular and Cellular Biology, University of Leeds, Mount Preston Street, Leeds LS2 9JT, UK.

Protein aggregation has now become recognised as an important and generic aspect of protein energy landscapes. Since the discovery that numerous human diseases are caused by protein aggregation, the biophysical characterisation of misfolded states and their aggregation mechanisms has received increased attention. Utilising experimental techniques and computational approaches established for the analysis of protein folding reactions has ensured rapid advances in the study of pathways leading to amyloid fibrils and amyloid-related aggregates. Here we describe recent experimental and theoretical advances in the elucidation of the conformational properties of dynamic, heterogeneous and/or insoluble protein ensembles populated on complex, multidimensional protein energy landscapes. We discuss current understanding of aggregation mechanisms in this context and describe how the synergy between biochemical, biophysical and cell-biological experiments are beginning to provide detailed insights into the partitioning of non-native species between protein folding and aggregation pathways.
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http://dx.doi.org/10.1016/j.abb.2007.05.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2706318PMC
January 2008

Direct observation of oligomeric species formed in the early stages of amyloid fibril formation using electrospray ionisation mass spectrometry.

J Mol Biol 2006 Nov 1;364(1):9-19. Epub 2006 Sep 1.

Astbury Centre for Structural Molecular Biology, Garstang and Astbury Buildings, University of Leeds, Leeds, UK.

Numerous debilitating human disorders result from protein misfolding and amyloid formation. Despite the grave nature of these maladies, our understanding of the structural mechanism of fibril assembly is limited. Of paramount importance is the need to identify and characterize oligomeric species formed early during fibril assembly, so that the nature of the initiating assembly mechanism can be revealed and species that may be toxic to cells identified. However, the transient nature of early oligomeric species, combined with their heterogeneity and instability, has precluded detailed analysis to date. Here, we have used electrospray ionisation mass spectrometry (ESI-MS), complemented by analytical ultracentrifugation (AUC) and measurements of thioflavin-T fluorescence, to monitor the early stages of assembly of amyloid-like fibrils formed from human beta-2-microglobulin (beta2m) in vitro. We show that worm-like fibrils that form with nucleation-independent kinetics assemble by a mechanism consistent with monomer addition, with species ranging from monomer to > or = 13-mer being identified directly and uniquely as transient assembly intermediates. By contrast, only monomers, dimers, trimers and tetramers are observed during nucleated growth, which leads to the formation of long straight fibrils. The results highlight the unique power of non-covalent ESI-MS to identify protein assembly intermediates in complex heterogeneous systems and demonstrate its great potential to identify and characterise individual species formed early during amyloid assembly.
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http://dx.doi.org/10.1016/j.jmb.2006.08.081DOI Listing
November 2006

Amyloid formation under physiological conditions proceeds via a native-like folding intermediate.

Nat Struct Mol Biol 2006 Mar 19;13(3):195-201. Epub 2006 Feb 19.

Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.

Although most proteins can assemble into amyloid-like fibrils in vitro under extreme conditions, how proteins form amyloid fibrils in vivo remains unresolved. Identifying rare aggregation-prone species under physiologically relevant conditions and defining their structural properties is therefore an important challenge. By solving the folding mechanism of the naturally amyloidogenic protein beta-2-microglobulin at pH 7.0 and 37 degrees C and correlating the concentrations of different species with the rate of fibril elongation, we identify a specific folding intermediate, containing a non-native trans-proline isomer, as the direct precursor of fibril elongation. Structural analysis using NMR shows that this species is highly native-like but contains perturbation of the edge strands that normally protect beta-sandwich proteins from self-association. The results demonstrate that aggregation pathways can involve self-assembly of highly native-like folding intermediates, and have implications for the prevention of this, and other, amyloid disorders.
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http://dx.doi.org/10.1038/nsmb1058DOI Listing
March 2006

A systematic study of the effect of physiological factors on beta2-microglobulin amyloid formation at neutral pH.

Biochemistry 2006 Feb;45(7):2311-21

Astbury Centre for Structural Molecular Biology, Garstang Building, University of Leeds, Leeds LS2 9JT, United Kingdom.

Beta(2)-microglobulin (beta(2)m) forms amyloid fibrils that deposit in the musculo-skeletal system in patients undergoing long-term hemodialysis. How beta(2)m self-assembles in vivo is not understood, since the monomeric wild-type protein is incapable of forming fibrils in isolation in vitro at neutral pH, while elongation of fibril-seeds made from recombinant protein has only been achieved at low pH or at neutral pH in the presence of detergents or cosolvents. Here we describe a systematic study of the effect of 11 physiologically relevant factors on beta(2)m fibrillogenesis at pH 7.0 without denaturants. By comparing the results obtained for the wild-type protein with those of two variants (DeltaN6 and V37A), the role of protein stability in fibrillogenesis is explored. We show that DeltaN6 forms low yields of amyloid-like fibrils at pH 7.0 in the absence of seeds, suggesting that this species could initiate fibrillogenesis in vivo. By contrast, high yields of amyloid-like fibrils are observed for all proteins when assembly is seeded with fibril-seeds formed from recombinant protein at pH 2.5 stabilized by the addition of heparin, serum amyloid P component (SAP), apolipoprotein E (apoE), uremic serum, or synovial fluid. The results suggest that the conditions within the synovium facilitate fibrillogenesis of beta(2)m and show that different physiological factors may act synergistically to promote fibril formation. By comparing the behavior of wild-type beta(2)m with that of DeltaN6 and V37A, we show that the physiologically relevant factors enhance fibrillogenesis by stabilizing fibril-seeds, thereby allowing fibril extension by rare assembly competent species formed by local unfolding of native monomers.
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http://dx.doi.org/10.1021/bi052434iDOI Listing
February 2006

The Yin and Yang of protein folding.

FEBS J 2005 Dec;272(23):5962-70

Astbury Centre for Structural Molecular Biology and Institute of Molecular and Cellular Biology, Gerstang Building, University of Leeds, UK.

The study of protein aggregation saw a renaissance in the last decade, when it was discovered that aggregation is the cause of several human diseases, making this field of research one of the most exciting frontiers in science today. Building on knowledge about protein folding energy landscapes, determined using an array of biophysical methods, theory and simulation, new light is now being shed on some of the key questions in protein-misfolding diseases. This review will focus on the mechanisms of protein folding and amyloid fibril formation, concentrating on the role of partially folded states in these processes, the complexity of the free energy landscape, and the potentials for the development of future therapeutic strategies based on a full biophysical description of the combined folding and aggregation free-energy surface.
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http://dx.doi.org/10.1111/j.1742-4658.2005.05021.xDOI Listing
December 2005