Publications by authors named "Aaron Voigt"

31 Publications

Posthypoxic behavioral impairment and mortality of Drosophila melanogaster are associated with high temperatures, enhanced predeath activity and oxidative stress.

Exp Mol Med 2021 Feb 9;53(2):264-280. Epub 2021 Feb 9.

Department of Neurology, Medical Faculty, RWTH Aachen University, 52074, Aachen, Germany.

Hypoxia is an underlying pathophysiological condition of a variety of devastating diseases, including acute ischemic stroke (AIS). We are faced with limited therapeutic options for AIS patients, and even after successful restoration of cerebral blood flow, the poststroke mortality is still high. More basic research is needed to explain mortality after reperfusion and to develop adjunct neuroprotective therapies. Drosophila melanogaster (D.m.) is a suitable model to analyze hypoxia; however, little is known about the impacts of hypoxia and especially of the subsequent reperfusion injury on the behavior and survival of D.m. To address this knowledge gap, we subjected two wild-type D.m. strains (Canton-S and Oregon-R) to severe hypoxia (<0.3% O) under standardized environmental conditions in a well-constructed hypoxia chamber. During posthypoxic reperfusion (21% O), we assessed fly activity (evoked and spontaneous) and analyzed molecular characteristics (oxidative stress marker abundance, reactive oxygen species (ROS) production, and metabolic activity) at various timepoints during reperfusion. First, we established standard conditions to induce hypoxia in D.m. to guarantee stable and reproducible experiments. Exposure to severe hypoxia under defined conditions impaired the climbing ability and reduced the overall activity of both D.m. strains. Furthermore, a majority of the flies died during the early reperfusion phase (up to 24 h). Interestingly, the flies that died early exhibited elevated activity before death compared to that of the flies that survived the entire reperfusion period. Additionally, we detected increases in ROS and stress marker (Catalase, Superoxide Dismutase and Heat Shock Protein 70) levels as well as reductions in metabolic activity in the reperfusion phase. Finally, we found that changes in environmental conditions impacted the mortality rate. In particular, decreasing the temperature during hypoxia or the reperfusion phase displayed a protective effect. In conclusion, our data suggest that reperfusion-dependent death might be associated with elevated temperatures, predeath activity, and oxidative stress.
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http://dx.doi.org/10.1038/s12276-021-00565-3DOI Listing
February 2021

An engineered monomer binding-protein for α-synuclein efficiently inhibits the proliferation of amyloid fibrils.

Elife 2019 08 21;8. Epub 2019 Aug 21.

Institut für Physikalische Biologie, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.

Removing or preventing the formation of [Formula: see text]-synuclein aggregates is a plausible strategy against Parkinson's disease. To this end, we have engineered the [Formula: see text]-wrapin AS69 to bind monomeric [Formula: see text]-synuclein with high affinity. In cultured cells, AS69 reduced the self-interaction of [Formula: see text]-synuclein and formation of visible [Formula: see text]-synuclein aggregates. In flies, AS69 reduced [Formula: see text]-synuclein aggregates and the locomotor deficit resulting from [Formula: see text]-synuclein expression in neuronal cells. In biophysical experiments in vitro, AS69 highly sub-stoichiometrically inhibited both primary and autocatalytic secondary nucleation processes, even in the presence of a large excess of monomer. We present evidence that the AS69-[Formula: see text]-synuclein complex, rather than the free AS69, is the inhibitory species responsible for sub-stoichiometric inhibition of secondary nucleation. These results represent a new paradigm that high affinity monomer binders can lead to strongly sub-stoichiometric inhibition of nucleation processes.
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http://dx.doi.org/10.7554/eLife.46112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721797PMC
August 2019

Quantification of Protein Aggregates Using Bimolecular Fluorescence Complementation.

Methods Mol Biol 2019 ;1873:183-193

Department of Neurology, University Medical Center, RWTH Aachen University, Aachen, 52074, Germany.

Neuronal accumulations of the presynaptic protein α-synuclein represent a hallmark of Parkinson's disease (PD). Multiple system atrophy and dementia with Lewy bodies are other neurodegenerative diseases in which α-synuclein aggregates are the main pathological characteristic. This is why these diseases are summarized as synucleinopathies. The abnormal α-Synuclein accumulation eventually results in the formation of Lewy bodies and Lewy neurites in surviving neurons of the brain. α-Synuclein aggregation has been widely researched in vitro but little is known about α-synuclein aggregation in vivo. This is why it is still unclear whether α-synuclein accumulation and/or which of the resulting aggregates (soluble oligomers or insoluble fibrils) actually trigger neurotoxic events. In our study, we employed the robust genetic makeup of Drosophila melanogaster in combination with bimolecular fluorescence complementation and developed an in vivo assay allowing to determine abundance of soluble α-synuclein aggregates (most likely oligomers). Insoluble α-synuclein aggregates (most likely fibrils) are detected by filter retardation assay. We provide an experimental outline to verify the reported assay system. The described method is easy, inexpensive, fast, and sensitive. Altogether, the described assay system is ideal for large-scale screening approaches aiming to test/identify compounds/conditions that affect α-synuclein aggregation in vivo.Moreover, the experimental outline reported here could serve as a template to analyze aggregation of proteins other than α-synuclein. As accumulation of specific proteins is a hallmark of almost every neurodegenerative disease, only minor changes to our protocol should be necessary for a similar analysis. We have put special focus on critical evaluation steps to consider when adapting our protocol to other aggregate-forming proteins.
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http://dx.doi.org/10.1007/978-1-4939-8820-4_11DOI Listing
May 2019

Monitoring α-synuclein multimerization in vivo.

FASEB J 2019 02 25;33(2):2116-2131. Epub 2018 Sep 25.

Department of Neurology, University Medical Center, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany.

The pathophysiology of Parkinson's disease is characterized by the abnormal accumulation of α-synuclein (α-Syn), eventually resulting in the formation of Lewy bodies and neurites in surviving neurons in the brain. Although α-Syn aggregation has been extensively studied in vitro, there is limited in vivo knowledge on α-Syn aggregation. Here, we used the powerful genetics of Drosophila melanogaster and developed an in vivo assay to monitor α-Syn accumulation by using a bimolecular fluorescence complementation assay. We found that both genetic and pharmacologic manipulations affected α-Syn accumulation. Interestingly, we also found that alterations in the cellular protein degradation mechanisms strongly influenced α-Syn accumulation. Administration of compounds identified as risk factors for Parkinson's disease, such as rotenone or heavy metal ions, had only mild or even no impact on α-Syn accumulation in vivo. Finally, we show that increasing phosphorylation of α-Syn at serine 129 enhances the accumulation and toxicity of α-Syn. Altogether, our study establishes a novel model to study α-Syn accumulation and illustrates the complexity of manipulating proteostasis in vivo.-Prasad, V., Wasser, Y., Hans, F., Goswami, A., Katona, I., Outeiro, T. F., Kahle, P. J., Schulz, J. B., Voigt, A. Monitoring α-synuclein multimerization in vivo.
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http://dx.doi.org/10.1096/fj.201800148RRRDOI Listing
February 2019

Linking amyotrophic lateral sclerosis and spinal muscular atrophy through RNA-transcriptome homeostasis: a genomics perspective.

J Neurochem 2017 04 28;141(1):12-30. Epub 2017 Feb 28.

Cancer Research Center (CiC-IBMCC, CSIC/USAL/IBSAL), Consejo Superior de Investigaciones Científicas (CSIC) and Universidad de Salamanca (USAL), Salamanca, Spain.

In this review, we present our most recent understanding of key biomolecular processes that underlie two motor neuron degenerative disorders, amyotrophic lateral sclerosis, and spinal muscular atrophy. We focus on the role of four multifunctional proteins involved in RNA metabolism (TDP-43, FUS, SMN, and Senataxin) that play a causal role in these diseases. Recent results have led to a novel scenario of intricate connections between these four proteins, bringing transcriptome homeostasis into the spotlight as a common theme in motor neuron degeneration. We review reported functional and physical interactions between these four proteins, highlighting their common association with nuclear bodies and small nuclear ribonucleoprotein particle biogenesis and function. We discuss how these interactions are turning out to be particularly relevant for the control of transcription and chromatin homeostasis, including the recent identification of an association between SMN and Senataxin required to ensure the resolution of DNA-RNA hybrid formation and proper termination by RNA polymerase II. These connections strongly support the existence of common pathways underlying the spinal muscular atrophy and amyotrophic lateral sclerosis phenotype. We also discuss the potential of genome-wide expression profiling, in particular RNA sequencing derived data, to contribute to unravelling the underlying mechanisms. We provide a review of publicly available datasets that have addressed both diseases using these approaches, and highlight the value of investing in cross-disease studies to promote our understanding of the pathways leading to neurodegeneration.
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http://dx.doi.org/10.1111/jnc.13945DOI Listing
April 2017

α-Synuclein enhances histone H3 lysine-9 dimethylation and H3K9me2-dependent transcriptional responses.

Sci Rep 2016 11 3;6:36328. Epub 2016 Nov 3.

Laboratory of Functional Neurogenetics, Department of Neurodegeneration, Hertie Institute for Clinical Brain Research, Faculty of Medicine, University of Tübingen, 72076 Tübingen, Germany.

α-Synuclein (αS) is a protein linked to Parkinson's disease (PD) and related neurodegenerative disorders. It is mostly localized within synapses, but αS has also been suggested to play a role in the nucleus. We used transgenic Drosophila and inducible SH-SY5Y neuroblastoma cells to investigate the effects of αS on chromatin with a particular focus on histone modifications. Overexpression of αS in male flies as well as in retinoic acid pre-treated neuroblastoma cells led to an elevation of histone H3K9 methylations, mostly mono- (H3K9me1) and di- (H3K9me2). The transient increase of H3K9 methylation in αS-induced SH-SY5Y cells was preceded by mRNA induction of the euchromatic histone lysine N-methyltransferase 2 (EHMT2). EHMT2 and H3K9me2 can function within the REST complex. Chromatin immunoprecipitation (ChIP) analyses of selected candidate, REST regulated genes showed significantly increased H3K9me2 promoter occupancy of genes encoding the L1CAM cell adhesion molecule and the synaptosomal-associated protein SNAP25, whose reduced expression levels were confirmed by RT-qPCR in αS induced cells. Treatment with EHMT inhibitor UNC0638 restored the mRNA levels of L1CAM and SNAP25. Thus, αS overexpression enhances H3K9 methylations via ΕΗΜΤ2 resulting in elevated H3K9me2 at the SNAP25 promoter, possibly affecting SNARE complex assembly and hence synaptic vesicle fusion events regulated by αS.
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http://dx.doi.org/10.1038/srep36328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5093762PMC
November 2016

Rab7 induces clearance of α-synuclein aggregates.

J Neurochem 2016 09 4;138(5):758-74. Epub 2016 Aug 4.

Department of Neurology, RWTH University Aachen, Aachen, Germany.

Parkinson's disease can be caused by mutations in the α-synuclein gene and is characterized by aggregates of α-synuclein protein. Aggregates are degraded by the autophago-lysosomal pathway. Since Rab7 has been shown to regulate trafficking of late endosomes and autophagosomes, we hypothesized that over-expressing Rab7 might be beneficial in Parkinson's disease. To test this hypothesis, we expressed the pathogenic A53T mutant of α-synuclein in HEK293 cells and Drosophila melanogaster. In HEK293 cells, EGFP-Rab7-decorated vesicles contain α-synuclein. Rab7 over-expression reduced the percentage of cells with α-synuclein particles and the amount of α-synuclein protein. Time-lapse microscopy confirmed that particles frequently disappeared with Rab7 over-expression. Clearance of α-synuclein is explained by the increased occurrence of acidified α-synuclein vesicles with Rab7 over-expression, presumably representing autolysosomes. Rab7 over-expression reduced apoptosis and the percentage of dead cells in trypan blue staining. In the fly model, Rab7 rescued the locomotor deficit induced by neuronal expression of A53T-α-synuclein. These beneficial effects were not produced by Rab7 missense mutations causing Charcot Marie Tooth neuropathy, or by the related GTPases Rab5, Rab9, or Rab23. Using mass spectrometry, we identified Rab7 in neuromelanin granules purified from human substantia nigra, indicating that Rab7 might be involved in the biogenesis of these possibly protective, autophagosome-like organelles in dopaminergic neurons. Taken together, Rab7 increased the clearance of α-synuclein aggregates, reduced cell death, and rescued the phenotype in a fly model of Parkinson's disease. These findings indicate that Rab7 is rate-limiting for aggregate clearance, and that Rab7 activation may offer a therapeutic strategy for Parkinson's disease. Cells over-expressing aggregation-prone A53T alpha-synuclein develop cytoplasmic aggregates mimicking changes observed in Parkinson's disease. When following cells in time-lapse microscopy, some few cells are able to remove these aggregates (Opazo et al. 2008). We now show that the percentage of cells clearing all aggregates from their cytosol is greatly increased with Rab7 over-expression, indicating that availability of Rab7 is rate-limiting for autophagic clearance of aggregates. The functional significance of this effect in neurons was confirmed in a Drosophila melanogaster model of Parkinson's disease.
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http://dx.doi.org/10.1111/jnc.13712DOI Listing
September 2016

The mitochondrial kinase PINK1: functions beyond mitophagy.

J Neurochem 2016 10 2;139 Suppl 1:232-239. Epub 2016 Jun 2.

Molecular Cell Biology, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Germany.

Mutations in the genes encoding the mitochondrial kinase PINK1 and the E3 ubiquitin ligase Parkin cause autosomal recessive Parkinson's disease (PD). Pioneering work in Drosophila melanogaster revealed that the loss of PINK1 or Parkin function causes similar phenotypes including dysfunctional mitochondria. Further research showed that PINK1 can act upstream of Parkin in a mitochondrial quality control pathway to induce removal of damaged mitochondria in a process called mitophagy. Albeit the PINK1/Parkin-induced mitophagy pathway is well established and has recently been elucidated in great detail, its pathophysiological relevance is being debated. Mounting evidence indicates that PINK1 has additional functions, for example, in regulating complex I activity and maintaining neuronal viability in response to stress. Here, we discuss mitophagy-dependent and -independent functions of PINK1 and their possible role in PD pathogenesis. Mutations in the PINK1 gene, encoding a mitochondrial kinase, are associated with autosomal recessive Parkinson's disease. In this review, we summarize and discuss the functional roles of PINK1 in maintaining mitochondrial integrity, eliminating damaged mitochondria, and promoting cell survival. This article is part of a special issue on Parkinson disease.
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http://dx.doi.org/10.1111/jnc.13655DOI Listing
October 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

Quantitative interaction proteomics of neurodegenerative disease proteins.

Cell Rep 2015 May 7;11(7):1134-46. Epub 2015 May 7.

Max Delbrück Center for Molecular Medicine, Robert-Rössle-Straße 10, 13092 Berlin, Germany. Electronic address:

Several proteins have been linked to neurodegenerative disorders (NDDs), but their molecular function is not completely understood. Here, we used quantitative interaction proteomics to identify binding partners of Amyloid beta precursor protein (APP) and Presenilin-1 (PSEN1) for Alzheimer's disease (AD), Huntingtin (HTT) for Huntington's disease, Parkin (PARK2) for Parkinson's disease, and Ataxin-1 (ATXN1) for spinocerebellar ataxia type 1. Our network reveals common signatures of protein degradation and misfolding and recapitulates known biology. Toxicity modifier screens and comparison to genome-wide association studies show that interaction partners are significantly linked to disease phenotypes in vivo. Direct comparison of wild-type proteins and disease-associated variants identified binders involved in pathogenesis, highlighting the value of differential interactome mapping. Finally, we show that the mitochondrial protein LRPPRC interacts preferentially with an early-onset AD variant of APP. This interaction appears to induce mitochondrial dysfunction, which is an early phenotype of AD.
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http://dx.doi.org/10.1016/j.celrep.2015.04.030DOI Listing
May 2015

Impaired retrograde transport by the Dynein/Dynactin complex contributes to Tau-induced toxicity.

Hum Mol Genet 2015 Jul 20;24(13):3623-37. Epub 2015 Mar 20.

Department of Neurology, University Hospital, RWTH Aachen, Germany,

The gene mapt codes for the microtubule-associated protein Tau. The R406W amino acid substitution in Tau is associated with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) characterized by Tau-positive filamentous inclusions. These filamentous Tau inclusions are present in a group of neurodegenerative diseases known as tauopathies, including Alzheimer's disease (AD). To gain more insights into the pathomechanism of tauopathies, we performed an RNAi-based large-scale screen in Drosophila melanogaster to identify genetic modifiers of Tau[R406W]-induced toxicity. A collection of RNAi lines, putatively silencing more than 7000 genes, was screened for the ability to modify Tau[R406W]-induced toxicity in vivo. This collection covered more than 50% of all protein coding fly genes and more than 90% of all fly genes known to have a human ortholog. Hereby, we identified 62 genes that, when silenced by RNAi, modified Tau-induced toxicity specifically. Among these 62 modifiers were three subunits of the Dynein/Dynactin complex. Analysis on segmental nerves of fly larvae showed that pan neural Tau[R406W] expression and concomitant silencing of Dynein/Dynactin complex members synergistically caused strong pathological changes within the axonal compartment, but only minor changes at synapses. At the larval stage, these alterations did not cause locomotion deficits, but became evident in adult flies. Our data suggest that Tau-induced detrimental effects most likely originate from axonal rather than synaptic dysfunction and that impaired retrograde transport intensifies detrimental effects of Tau in axons. In conclusion, our findings contribute to the elucidation of disease mechanisms in tauopathies like FTDP-17 or AD.
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http://dx.doi.org/10.1093/hmg/ddv107DOI Listing
July 2015

Drosophila gene tao-1 encodes proteins with and without a Ste20 kinase domain that affect cytoskeletal architecture and cell migration differently.

Open Biol 2015 Jan;5(1):140161

Abteilung Molekulare Entwicklungsbiologie, Max Planck Institut für Biophysikalische Chemie, Göttingen, Germany

Tao-1, the single representative of the Sterile 20 kinase subfamily in Drosophila, is best known for destabilizing microtubules at the actin-rich cortex, regulating the cytoskeletal architecture of cells. More recently, Tao-1 was shown to act in the Salvador-Warts-Hippo pathway by phosphorylating Hippo, regulating cell growth as well as cell polarity. Here, we show that tao-1 encodes two proteins, one with the Sterile 20 kinase domain (Tao-L) and one without it (Tao-S), and that they act in an antagonistic manner. Tao-L expression causes lamellipodia-like cell protrusions, whereas Tao-S expression results in filopodia-like structures that make cells stick to the surface they attach to. Ectopic Tao-1 expression in the anterior region of Drosophila embryos results in pole cell formation as normally observed at the posterior end. Tao-S expression causes primordial germ cells (PGCs) to adhere to the inner wall of the gut primordia and prevents proper transepithelial migration to the gonads. Conversely, RNAi knockdowns of Tao-1 cause disordered migration of PGCs out of the gut epithelium, their dispersal within the embryo and cell death. The results reveal a novel function of Tao-1 in cell migration, which is based on antagonistic activities of two proteins encoded by a single gene.
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http://dx.doi.org/10.1098/rsob.140161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4313371PMC
January 2015

Nuclear import factor transportin and arginine methyltransferase 1 modify FUS neurotoxicity in Drosophila.

Neurobiol Dis 2015 Feb 8;74:76-88. Epub 2014 Nov 8.

German Center for Neurodegenerative Diseases (DZNE) Tübingen, Germany; Hertie Institute for Clinical Brain Research, Laboratory of Functional Neurogenetics, Tübingen, Germany. Electronic address:

Inclusions containing Fused in Sarcoma (FUS) are found in familial and sporadic cases of the incurable progressive motor neuron disease amyotrophic lateral sclerosis and in a common form of dementia, frontotemporal dementia. Most disease-associated mutations are located in the C-terminal proline-tyrosine nuclear localization sequence (PY-NLS) of FUS and impair its nuclear import. It has been shown in cell culture that the nuclear import of FUS is mediated by transportin, which binds the PY-NLS and the last arginine/glycine/glycine-rich (RGG) domain of FUS. Methylation of this last RGG domain by protein arginine methyltransferases (PRMTs) weakens transportin binding and therefore impairs nuclear translocation of FUS. To investigate the requirements for the nuclear import of FUS in an in vivo model, we generated different transgenic Drosophila lines expressing human FUS wild type (hFUS wt) and two disease-related variants P525L and R495X, in which the NLS is mutated or completely absent, respectively. To rule out effects caused by heterologous hFUS expression, we analysed the corresponding variants for the Drosophila FUS orthologue Cabeza (Caz wt, P398L, Q349X). Expression of these variants in eyes and motor neurons confirmed the PY-NLS-dependent nuclear localization of FUS/Caz and caused neurodegenerative effects. Surprisingly, FUS/Caz toxicity was correlated to the degree of its nuclear localization in this overexpression model. High levels of nuclear FUS/Caz became insoluble and reduced the endogenous Caz levels, confirming FUS autoregulation in Drosophila. RNAi-mediated knockdown of the two transportin orthologues interfered with the nuclear import of FUS/Caz and also enhanced the eye phenotype. Finally, we screened the Drosophila PRMT proteins (DART1-9) and found that knockdown of Dart1 led to a reduction in methylation of hFUS P525L and aggravated its phenotype. These findings show that the molecular mechanisms controlling the nuclear import of FUS/Caz and FUS autoregulation are conserved between humans and Drosophila. In addition to the well-known neurodegenerative effects of FUS loss-of function, our data suggest toxic potential of overexpressed FUS in the nucleus and of insoluble FUS.
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http://dx.doi.org/10.1016/j.nbd.2014.11.003DOI Listing
February 2015

Analysis of dopaminergic neuronal dysfunction in genetic and toxin-induced models of Parkinson's disease in Drosophila.

J Neurochem 2014 Nov 31;131(3):369-82. Epub 2014 Jul 31.

Institute of Zoology, University of Regensburg, Regensburg, Germany.

Drosophila melanogaster has contributed significantly to the understanding of disease mechanisms in Parkinson's disease (PD) as it is one of the very few PD model organisms that allow the study of age-dependent behavioral defects, physiology and histology, and genetic interactions among different PD-related genes. However, there have been contradictory results from a number of recent reports regarding the loss of dopaminergic neurons in different PD fly models. In an attempt to re-evaluate and clarify this issue, we have examined three different genetic (α-synuclein, Pink1, parkin) and two toxin-based (rotenone and paraquat) models of the disease for neuronal cell loss. Our results showed no dopaminergic neuronal loss in all models tested. Despite this surprising result, we found additional phenotypes showing the dysfunctional status of the dopaminergic neurons in most of the models analyzed. A common feature found in most models is a quantifiable decrease in the fluorescence of a green-fluorescent protein reporter gene in dopaminergic neurons that correlates well with other phenotypes found for these models and can be reliably used as a hallmark of the neurodegenerative process when modeling diseases affecting the dopaminergic system in Drosophila. Analyzing three genetic and two toxin-based Drosophila models of Parkinson's disease (PD) through green fluorescent protein reporter and α-tyrosine hydroxylase staining, we have found the number of dopaminergic neurons to remain unchanged. Despite the lack of neuronal loss, we have detected a remarkable decrease in a reporter green-fluorescent protein (GFP) signal in dopaminergic neurons, suggesting an abnormal neuronal status that correlates with the phenotypes associated with those PD fly models.
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http://dx.doi.org/10.1111/jnc.12818DOI Listing
November 2014

UBE2E ubiquitin-conjugating enzymes and ubiquitin isopeptidase Y regulate TDP-43 protein ubiquitination.

J Biol Chem 2014 Jul 13;289(27):19164-79. Epub 2014 May 13.

From the Graduate School of Cellular and Molecular Neuroscience, University of Tübingen, Tübingen 72076, Germany, Laboratory of Functional Neurogenetics, Department of Neurodegeneration, German Center for Neurodegenerative Diseases (DZNE), Tübingen 72076, Germany, Laboratory of Functional Neurogenetics, Department of Neurodegeneration and

Trans-activation element DNA-binding protein of 43 kDa (TDP-43) characterizes insoluble protein aggregates in distinct subtypes of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. TDP-43 mediates many RNA processing steps within distinct protein complexes. Here we identify novel TDP-43 protein interactors found in a yeast two-hybrid screen using an adult human brain cDNA library. We confirmed the TDP-43 interaction of seven hits by co-immunoprecipitation and assessed their co-localization in HEK293E cells. As pathological TDP-43 is ubiquitinated, we focused on the ubiquitin-conjugating enzyme UBE2E3 and the ubiquitin isopeptidase Y (UBPY). When cells were treated with proteasome inhibitor, ubiquitinated and insoluble TDP-43 species accumulated. All three UBE2E family members could enhance the ubiquitination of TDP-43, whereas catalytically inactive UBE2E3(C145S) was much less efficient. Conversely, silencing of UBE2E3 reduced TDP-43 ubiquitination. We examined 15 of the 48 known disease-associated TDP-43 mutants and found that one was excessively ubiquitinated. This strong TDP-43(K263E) ubiquitination was further enhanced by proteasomal inhibition as well as UBE2E3 expression. Conversely, UBE2E3 silencing and expression of UBPY reduced TDP-43(K263E) ubiquitination. Moreover, wild-type but not active site mutant UBPY reduced ubiquitination of TDP-43 C-terminal fragments and of a nuclear import-impaired mutant. In Drosophila melanogaster, UBPY silencing enhanced neurodegenerative TDP-43 phenotypes and the accumulation of insoluble high molecular weight TDP-43 and ubiquitin species. Thus, UBE2E3 and UBPY participate in the regulation of TDP-43 ubiquitination, solubility, and neurodegeneration.
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http://dx.doi.org/10.1074/jbc.M114.561704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4081952PMC
July 2014

CDK5 protects from caspase-induced Ataxin-3 cleavage and neurodegeneration.

J Neurochem 2014 Jun 24;129(6):1013-23. Epub 2014 Mar 24.

Department of Neurology, University of Göttingen, Göttingen, Germany; Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Goettingen, Germany.

Spinocerebellar ataxia type 3 (SCA3) is one of at least nine inherited neurodegenerative diseases caused by an expansion of a polyglutamine tract within corresponding disease-specific proteins. In case of SCA3, mutation of Ataxin-3 results in aggregation of misfolded protein, formation of intranuclear as well as cytosolic inclusion bodies and cell death in distinct neuronal populations. Since cyclin-dependent kinase-5 (CDK5) has been shown to exert beneficial effects on aggregate formation and cell death in various polyglutamine diseases, we tested its therapeutic potential for SCA3. Our data show increased caspase-dependent Ataxin-3 cleavage, aggregation, and neurodegeneration in the absence of sufficient CDK5 activity. This disease-propagating effect could be reversed by mutation of the caspase cleavage site in Ataxin-3. Moreover, reduction of CDK5 expression levels by RNAi in vivo enhances SCA3 toxicity as assayed in a Drosophila model for SCA3. In summary, we present CDK5 as a potent neuroprotectant, regulating cleavage and thereby toxicity of Ataxin-3 and other polyglutamine proteins. We propose that increased caspase-dependent cleavage of mutated Ataxin-3, because of missing CDK5 shielding, leads to aggregation and cell death. Moreover, reduction of CDK5 expression levels by RNAi in vivo enhances SCA3 toxicity as assayed in a Drosophila model for SCA3. We think that CDK5 functions as a shield against cleavage-induced toxification and thereby is an interesting target for therapeutic intervention in polyQ disease in general.
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http://dx.doi.org/10.1111/jnc.12684DOI Listing
June 2014

A global in vivo Drosophila RNAi screen identifies a key role of ceramide phosphoethanolamine for glial ensheathment of axons.

PLoS Genet 2013 12;9(12):e1003980. Epub 2013 Dec 12.

Max Planck Institute for Experimental Medicine, Göttingen, Germany ; Department of Neurology, University of Göttingen, Göttingen, Germany.

Glia are of vital importance for all complex nervous system. One of the many functions of glia is to insulate and provide trophic and metabolic support to axons. Here, using glial-specific RNAi knockdown in Drosophila, we silenced 6930 conserved genes in adult flies to identify essential genes and pathways. Among our screening hits, metabolic processes were highly represented, and genes involved in carbohydrate and lipid metabolic pathways appeared to be essential in glia. One critical pathway identified was de novo ceramide synthesis. Glial knockdown of lace, a subunit of the serine palmitoyltransferase associated with hereditary sensory and autonomic neuropathies in humans, resulted in ensheathment defects of peripheral nerves in Drosophila. A genetic dissection study combined with shotgun high-resolution mass spectrometry of lipids showed that levels of ceramide phosphoethanolamine are crucial for axonal ensheathment by glia. A detailed morphological and functional analysis demonstrated that the depletion of ceramide phosphoethanolamine resulted in axonal defasciculation, slowed spike propagation, and failure of wrapping glia to enwrap peripheral axons. Supplementing sphingosine into the diet rescued the neuropathy in flies. Thus, our RNAi study in Drosophila identifies a key role of ceramide phosphoethanolamine in wrapping of axons by glia.
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http://dx.doi.org/10.1371/journal.pgen.1003980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3861124PMC
August 2014

Drosophila melanogaster as a model organism for Alzheimer's disease.

Mol Neurodegener 2013 Nov 22;8:35. Epub 2013 Nov 22.

Department of Neurology, University Medical Center, RWTH Aachen, Pauwelsstrasse 30, D-52074 Aachen, Germany.

Drosophila melanogaster provides an important resource for in vivo modifier screens of neurodegenerative diseases. To study the underlying pathogenesis of Alzheimer's disease, fly models that address Tau or amyloid toxicity have been developed. Overexpression of human wild-type or mutant Tau causes age-dependent neurodegeneration, axonal transport defects and early death. Large-scale screens utilizing a neurodegenerative phenotype induced by eye-specific overexpression of human Tau have identified several kinases and phosphatases, apoptotic regulators and cytoskeleton proteins as determinants of Tau toxicity in vivo. The APP ortholog of Drosophila (dAPPl) shares the characteristic domains with vertebrate APP family members, but does not contain the human Aβ42 domain. To circumvent this drawback, researches have developed strategies by either direct secretion of human Aβ42 or triple transgenic flies expressing human APP, β-secretase and Drosophila γ-secretase presenilin (dPsn). Here, we provide a brief overview of how fly models of AD have contributed to our knowledge of the pathomechanisms of disease.
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http://dx.doi.org/10.1186/1750-1326-8-35DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4222597PMC
November 2013

Drosophila as a screening tool to study human neurodegenerative diseases.

J Neurochem 2013 Nov 21;127(4):453-60. Epub 2013 Oct 21.

Department of Neurology, University Medical Center, RWTH Aachen, Aachen, Germany.

In an aging society, research involving neurodegenerative disorders is of paramount importance. Over the past few years, research on Alzheimer's and Parkinson's diseases has made tremendous progress. Experimental studies, however, rely mostly on transgenic animal models, preferentially using mice. Although experiments on mice have enormous advantages, they also have some inherent limitations, some of which can be overcome by the use of Drosophila melanogaster as an experimental animal. Among the major advantages of using the fly is its small genome, which can also be modified very easily. The fact that its genome lends itself to diverse alterations (e. g. mutagenesis, transposons) has made the fly a useful organism to perform large-scale and genome-wide screening approaches. This has opened up an entirely new field of experimental research aiming to elucidate genetic interactions and screen for modifiers of disease processes in vivo. Here, we provide a brief overview of how flies can be used to analyze molecular mechanisms underlying human neurodegenerative diseases.
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http://dx.doi.org/10.1111/jnc.12446DOI Listing
November 2013

TRAP1 rescues PINK1 loss-of-function phenotypes.

Hum Mol Genet 2013 Jul 21;22(14):2829-41. Epub 2013 Mar 21.

Department of Neurology, University Medical Center, RWTH Aachen, Aachen D-52074, Germany.

PTEN-induced kinase 1 (PINK1) is a serine/threonine kinase that is localized to mitochondria. It protects cells from oxidative stress by suppressing mitochondrial cytochrome c release, thereby preventing cell death. Mutations in Pink1 cause early-onset Parkinson's disease (PD). Consistently, mitochondrial function is impaired in Pink1-linked PD patients and model systems. Previously, in vitro analysis implied that the protective effects of PINK1 depend on phosphorylation of the downstream factor, TNF receptor-associated protein 1 (TRAP1). Furthermore, TRAP1 has been shown to mitigate α-Synuclein-induced toxicity, linking α-Synuclein directly to mitochondrial dysfunction. These data suggest that TRAP1 seems to mediate protective effects on mitochondrial function in pathways that are affected in PD. Here we investigated the potential of TRAP1 to rescue dysfunction induced by either PINK1 or Parkin deficiency in vivo and in vitro. We show that overexpression of human TRAP1 is able to mitigate Pink1 but not parkin loss-of-function phenotypes in Drosophila. In addition, detrimental effects observed after RNAi-mediated silencing of complex I subunits were rescued by TRAP1 in Drosophila. Moreover, TRAP1 was able to rescue mitochondrial fragmentation and dysfunction upon siRNA-induced silencing of Pink1 but not parkin in human neuronal SH-SY5Y cells. Thus, our data suggest a functional role of TRAP1 in maintaining mitochondrial integrity downstream of PINK1 and complex I deficits but parallel to or upstream of Parkin.
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http://dx.doi.org/10.1093/hmg/ddt132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3690968PMC
July 2013

Large-scale screen for modifiers of ataxin-3-derived polyglutamine-induced toxicity in Drosophila.

PLoS One 2012 5;7(11):e47452. Epub 2012 Nov 5.

Department of Neurology, University Medical Center, RWTH Aachen, Aachen, Germany.

Polyglutamine (polyQ) diseases represent a neuropathologically heterogeneous group of disorders. The common theme of these disorders is an elongated polyQ tract in otherwise unrelated proteins. So far, only symptomatic treatment can be applied to patients suffering from polyQ diseases. Despite extensive research, the molecular mechanisms underlying polyQ-induced toxicity are largely unknown. To gain insight into polyQ pathology, we performed a large-scale RNAi screen in Drosophila to identify modifiers of toxicity induced by expression of truncated Ataxin-3 containing a disease-causing polyQ expansion. We identified various unknown modifiers of polyQ toxicity. Large-scale analysis indicated a dissociation of polyQ aggregation and toxicity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0047452PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3489908PMC
April 2013

The mitochondrial chaperone protein TRAP1 mitigates α-Synuclein toxicity.

PLoS Genet 2012 Feb 2;8(2):e1002488. Epub 2012 Feb 2.

Department of Neurology, University Medical Center, RWTH Aachen, Germany.

Overexpression or mutation of α-Synuclein is associated with protein aggregation and interferes with a number of cellular processes, including mitochondrial integrity and function. We used a whole-genome screen in the fruit fly Drosophila melanogaster to search for novel genetic modifiers of human [A53T]α-Synuclein-induced neurotoxicity. Decreased expression of the mitochondrial chaperone protein tumor necrosis factor receptor associated protein-1 (TRAP1) was found to enhance age-dependent loss of fly head dopamine (DA) and DA neuron number resulting from [A53T]α-Synuclein expression. In addition, decreased TRAP1 expression in [A53T]α-Synuclein-expressing flies resulted in enhanced loss of climbing ability and sensitivity to oxidative stress. Overexpression of human TRAP1 was able to rescue these phenotypes. Similarly, human TRAP1 overexpression in rat primary cortical neurons rescued [A53T]α-Synuclein-induced sensitivity to rotenone treatment. In human (non)neuronal cell lines, small interfering RNA directed against TRAP1 enhanced [A53T]α-Synuclein-induced sensitivity to oxidative stress treatment. [A53T]α-Synuclein directly interfered with mitochondrial function, as its expression reduced Complex I activity in HEK293 cells. These effects were blocked by TRAP1 overexpression. Moreover, TRAP1 was able to prevent alteration in mitochondrial morphology caused by [A53T]α-Synuclein overexpression in human SH-SY5Y cells. These results indicate that [A53T]α-Synuclein toxicity is intimately connected to mitochondrial dysfunction and that toxicity reduction in fly and rat primary neurons and human cell lines can be achieved using overexpression of the mitochondrial chaperone TRAP1. Interestingly, TRAP1 has previously been shown to be phosphorylated by the serine/threonine kinase PINK1, thus providing a potential link of PINK1 via TRAP1 to α-Synuclein.
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http://dx.doi.org/10.1371/journal.pgen.1002488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3271059PMC
February 2012

Targeted ablation of oligodendrocytes triggers axonal damage.

PLoS One 2011 27;6(7):e22735. Epub 2011 Jul 27.

Max-Planck-Institute of Experimental Medicine, Göttingen, Germany.

Glial dysfunction has been implicated in a number of neurodegenerative diseases. In this study we investigated the consequences of glial and oligodendrocyte ablation on neuronal integrity and survival in Drosophila and adult mice, respectively. Targeted genetic ablation of glia was achieved in the adult Drosophila nervous system using the GAL80-GAL4 system. In mice, oligodendrocytes were depleted by the injection of diphtheria toxin in MOGi-Cre/iDTR double transgenic animals. Acute depletion of oligodendrocytes induced axonal injury, but did not cause neuronal cell death in mice. Ablation of glia in adult flies triggered neuronal apoptosis and resulted in a marked reduction in motor performance and lifespan. Our study shows that the targeted depletion of glia triggers secondary neurotoxicity and underscores the central contribution of glia to neuronal homeostasis. The models used in this study provide valuable systems for the investigation of therapeutic strategies to prevent axonal or neuronal damage.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0022735PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3144945PMC
December 2011

Drosophila tao controls mushroom body development and ethanol-stimulated behavior through par-1.

J Neurosci 2011 Jan;31(3):1139-48

Department of Anatomy, University of California, San Francisco, San Francisco, California 94143, USA.

In both mammalian and insect models of ethanol-induced behavior, low doses of ethanol stimulate locomotion. However, the mechanisms of the stimulant effects of ethanol on the CNS are mostly unknown. We have identified tao, encoding a serine-threonine kinase of the Ste20 family, as a gene necessary for ethanol-induced locomotor hyperactivity in Drosophila. Mutations in tao also affect behavioral responses to cocaine and nicotine, making flies resistant to the effects of both drugs. We show that tao function is required during the development of the adult nervous system and that tao mutations cause defects in the development of central brain structures, including the mushroom body. Silencing of a subset of mushroom body neurons is sufficient to reduce ethanol-induced hyperactivity, revealing the mushroom body as an important locus mediating the stimulant effects of ethanol. We also show that mutations in par-1 suppress both the mushroom body morphology and behavioral phenotypes of tao mutations and that the phosphorylation state of the microtubule-binding protein Tau can be altered by RNA interference knockdown of tao, suggesting that tao and par-1 act in a pathway to control microtubule dynamics during neural development.
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http://dx.doi.org/10.1523/JNEUROSCI.4416-10.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3045818PMC
January 2011

Fas/CD95 regulatory protein Faim2 is neuroprotective after transient brain ischemia.

J Neurosci 2011 Jan;31(1):225-33

Department of Neurology, University Hospital and Medical School, RWTH Aachen University, D-52074 Aachen, Germany.

Death receptor (DR) signaling has a major impact on the outcome of numerous neurological diseases, including ischemic stroke. DRs mediate not only cell death signals, but also proinflammatory responses and cell proliferation. Identification of regulatory proteins that control the switch between apoptotic and alternative DR signaling opens new therapeutic opportunities. Fas apoptotic inhibitory molecule 2 (Faim2) is an evolutionary conserved, neuron-specific inhibitor of Fas/CD95-mediated apoptosis. To investigate its role during development and in disease models, we generated Faim2-deficient mice. The ubiquitous null mutation displayed a viable and fertile phenotype without overt deficiencies. However, lack of Faim2 caused an increase in susceptibility to combined oxygen-glucose deprivation in primary neurons in vitro as well as in caspase-associated cell death, stroke volume, and neurological impairment after cerebral ischemia in vivo. These processes were rescued by lentiviral Faim2 gene transfer. In summary, we provide evidence that Faim2 is a novel neuroprotective molecule in the context of cerebral ischemia.
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http://dx.doi.org/10.1523/JNEUROSCI.2188-10.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6622730PMC
January 2011

TDP-43-mediated neuron loss in vivo requires RNA-binding activity.

PLoS One 2010 Aug 18;5(8):e12247. Epub 2010 Aug 18.

Department of Neurology, University Medical Center, RWTH Aachen, Aachen, Germany.

Alteration and/or mutations of the ribonucleoprotein TDP-43 have been firmly linked to human neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The relative impacts of TDP-43 alteration, mutation, or inherent protein function on neural integrity, however, remain less clear--a situation confounded by conflicting reports based on transient and/or random-insertion transgenic expression. We therefore performed a stringent comparative investigation of impacts of these TDP-43 modifications on neural integrity in vivo. To achieve this, we systematically screened ALS/FTLD-associated and synthetic TDP-43 isoforms via same-site gene insertion and neural expression in Drosophila; followed by transposon-based motor neuron-specific transgenesis in a chick vertebrate system. Using this bi-systemic approach we uncovered a requirement of inherent TDP-43 RNA-binding function--but not ALS/FTLD-linked mutation, mislocalization, or truncation--for TDP-43-mediated neurotoxicity in vivo.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0012247PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2923622PMC
August 2010

Dopamine-dependent neurodegeneration in Drosophila models of familial and sporadic Parkinson's disease.

Neurobiol Dis 2010 Oct 6;40(1):113-9. Epub 2010 Mar 6.

Institute of Zoology, Universitaetsstr. 31, University of Regensburg, D-93040 Regensburg, Germany.

Parkinson's disease has been found to be caused by both, genetic and environmental factors. Despite the diversity of causes involved, a convergent pathogenic mechanism might underlie the special vulnerability of dopaminergic neurons in different forms of Parkinsonism. In recent years, a number of reports have proposed dopamine as a common player responsible in the loss of dopaminergic neurons independent of its etiology. Using RNAi lines we were able to generate flies with drastically reduced dopamine levels in the dopaminergic neurons. Combining these flies with a chemically induced Parkinson model (rotenone) and a familial form of Parkinson (mutant alpha-synuclein) we were able to show a strong reduction of neurotoxicity and a protection of the dopaminergic neurons when cellular dopamine levels were reduced. These results show that dopamine homeostasis plays a central role for the susceptibility of dopaminergic neurons to environmental and genetic factors in in vivo models of Parkinson disease.
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http://dx.doi.org/10.1016/j.nbd.2010.02.012DOI Listing
October 2010

Knockdown of transactive response DNA-binding protein (TDP-43) downregulates histone deacetylase 6.

EMBO J 2010 Jan 12;29(1):209-21. Epub 2009 Nov 12.

Laboratory of Functional Neurogenetics, Department of Neurodegeneration, Hertie Institute for Clinical Brain Research, Tübingen, Germany.

TDP-43 is an RNA/DNA-binding protein implicated in transcriptional repression and mRNA processing. Inclusions of TDP-43 are hallmarks of frontotemporal dementia and amyotrophic lateral sclerosis. Besides aggregation of TDP-43, loss of nuclear localization is observed in disease. To identify relevant targets of TDP-43, we performed expression profiling. Thereby, histone deacetylase 6 (HDAC6) downregulation was discovered on TDP-43 silencing and confirmed at the mRNA and protein level in human embryonic kidney HEK293E and neuronal SH-SY5Y cells. This was accompanied by accumulation of the major HDAC6 substrate, acetyl-tubulin. HDAC6 levels were restored by re-expression of TDP-43, dependent on RNA binding and the C-terminal protein interaction domains. Moreover, TDP-43 bound specifically to HDAC6 mRNA arguing for a direct functional interaction. Importantly, in vivo validation in TDP-43 knockout Drosophila melanogaster confirmed the specific downregulation of HDAC6. HDAC6 is necessary for protein aggregate formation and degradation. Indeed, HDAC6-dependent reduction of cellular aggregate formation and increased cytotoxicity of polyQ-expanded ataxin-3 were found in TDP-43 silenced cells. In conclusion, loss of functional TDP-43 causes HDAC6 downregulation and might thereby contribute to pathogenesis.
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http://dx.doi.org/10.1038/emboj.2009.324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2808372PMC
January 2010

Pre-fibrillar alpha-synuclein variants with impaired beta-structure increase neurotoxicity in Parkinson's disease models.

EMBO J 2009 Oct 10;28(20):3256-68. Epub 2009 Sep 10.

Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany.

The relation of alpha-synuclein (alphaS) aggregation to Parkinson's disease (PD) has long been recognized, but the mechanism of toxicity, the pathogenic species and its molecular properties are yet to be identified. To obtain insight into the function different aggregated alphaS species have in neurotoxicity in vivo, we generated alphaS variants by a structure-based rational design. Biophysical analysis revealed that the alphaS mutants have a reduced fibrillization propensity, but form increased amounts of soluble oligomers. To assess their biological response in vivo, we studied the effects of the biophysically defined pre-fibrillar alphaS mutants after expression in tissue culture cells, in mammalian neurons and in PD model organisms, such as Caenorhabditis elegans and Drosophila melanogaster. The results show a striking correlation between alphaS aggregates with impaired beta-structure, neuronal toxicity and behavioural defects, and they establish a tight link between the biophysical properties of multimeric alphaS species and their in vivo function.
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http://dx.doi.org/10.1038/emboj.2009.257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2771093PMC
October 2009

BAG1 modulates huntingtin toxicity, aggregation, degradation, and subcellular distribution.

J Neurochem 2009 Nov 27;111(3):801-7. Epub 2009 Aug 27.

Department of Neurology, University Hospital Göttingen, Waldweg, Göttingen, Germany.

Bcl-2-associated athanogene-1 (BAG1) is a multifunctional protein delivering chaperone-recognized unfolded substrates to the proteasome for degradation. It has been shown to be essential for proper CNS development in vivo, playing a crucial role in neuronal survival and differentiation. With regard to Huntington's disease, a sequestration of BAG1 into inclusion bodies and a neuroprotective effect in double transgenic mice have been reported. Here, we show that BAG1 reduces aggregation and accelerates degradation of mutant huntingtin (htt-mut). Moreover, it reduces nuclear levels of htt-mut. This effect can be overcome by over-expression of seven in absentia homolog 1, an E3 ligase negatively regulated by BAG1 and known to be involved in nuclear import of htt-mut. In vivo, BAG1 proved to be protective in a Drosophila melanogaster Huntington's disease model, preventing photoreceptor cell loss induced by htt-mut. In summary, we present BAG1 as a therapeutic tool modulating key steps in htt toxicity in vitro and ameliorating htt toxicity in vivo.
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http://dx.doi.org/10.1111/j.1471-4159.2009.06363.xDOI Listing
November 2009