DJ-1 and neurodegeneration: its roles in mitochondria and in protein misfolding

Lead Research Organisation: University of Leicester
Department Name: Genetics

Abstract

Parkinson's disease (PD), the second most common neurodegenerative disorder after Alzheimer's disease, is characterized by loss of dopaminergic neurons and the accumulation of misfolded alpha-synuclein in the form of Lewy bodies. The clinical symptoms include muscle rigidity, resting tremor, bradykinesia and postural instability as a result of the loss of dopaminergic neurons in the substantia nigra pars compacta. At later stages, the etiology of neuronal loss in sporadic PD is unknown, although aberrant alpha-synuclein aggregation and oxidative stress may contribute to the death of specific neuronal populations. Although little is known about the pathogenesis of PD, the detailed study of several genes associated with familial PD is enhancing the understanding of the molecular mechanisms associated with the disease. Mutations in one of these genes - PARK7 - accounts for ~1-2% of the sporadic cases of early onset recessive PD. PARK7 encodes for DJ-1, a small conserved protein which is broadly expressed and primarily localized to the cytoplasm, but also found in the nucleus and associated with mitochondria. Despite the importance of this protein, the role of DJ-1 in in formation of PD remains unclear. Several functions have been suggested for DJ-1, including an oxidative stress sensor, a chaperone which deals with misfolded protein in the cells, and a regulator of mitochondrial function. In this project we seek to clarify how DJ-1 contributes to mitochondrial function and protein misfolding, and ultimately how defects in these cellular processes due to DJ-1 mutations contribute to pathogenesis in PD. For these studies we plan to implement immortalized cell lines, primary cells from mice, as well as fruit flies.

In preliminary experiments we have observed that mutations in DJ-1 alter its ability to localize to mitochondria in mammalian cells under oxidative stress conditions. We thus now seek to further characterize this effect with varied oxidants, and also in the context of alpha-synuclein overexpression. In addition, we plan to investigate the impact of these mutants on both morphology and function of mitochondria in mouse cells, as well as the effects on mitochondrial gene expression. Finally we plan to extend this approach by studying the effect of DJ-1 mutants on the structure and function of mitochondria in fruit flies.

Next we seek to further explore the role of DJ-1 in protein misfolding by investigating the relationship between DJ-1 and the Tau protein, which has been implicated in several neurodegenerative disorders. The Tau protein is involved in the pathogenesis of Alzheimer's disease and is a major component of neurofibrillary tangles present in this disorder. The gene encoding Tau has recently been genetically linked to PD. In addition, DJ-1 co-localizes with Tau inclusions in Alzheimer's disease and other brain disorders. However any possible physical/functional interactions between DJ-1 and Tau have not been studied and their relationship in the context of PD has not been explored. Thus we propose to study the interaction between DJ-1 and Tau in living mammalian cells, and to what extent, if any, this alters Tau toxicity or formation of fibers. Furthermore, we plan to extend this work to a fruit fly model of Tauopathies, to ascertain whether DJ-1 modulates disease-relevant "symptoms" in these animals. In preliminary work we have observed that DJ-1 and Tau directly interaction when expressed in mammalian cells.

Thus, our work will better define the role of DJ-1 in mitochondrial function and in protein misfolding which will help clarify its pathogenic role in PD. This added insight into DJ-1 biology may ultimately help inform therapeutic strategies for this, and other, neurodegenerative disorders.

Technical Summary

Parkinson's disease (PD), the second most common neurodegenerative disorder, presents with loss of dopaminergic neurons and the accumulation of misfolded alpha-synuclein (aSyn) in Lewy bodies. The study of genes associated with familial PD - such as PARK7 which encodes for DJ-1 - is elucidating the mechanisms underlying this disease. DJ-1 has several roles in the cell: oxidative stress sensor, protein chaperone, and a regulator of mitochondrial function. Here we seek to clarify how DJ-1 contributes to mitochondrial function and protein misfolding, and how these roles contribute to pathogenesis in PD.

In initial work we have found that mutations in DJ-1 modulate its relocalization to mitochondria in mammalian cells with paraquat treatment. We thus seek to further characterize this effect with a range of disease-causing and functional mutants, additional oxidants, and in the context of aSyn expression. We also plan to investigate how DJ-1 mutations affect function and morphology of mitochondria. Finally we plan to extend this approach by studying how DJ-1 mutants modulate the structure and function of mitochondria in fruit flies.

We will also explore the relationship between DJ-1 and the Tau protein, which is involved in the pathogenesis of Alzheimer's disease (AD), is a major component of neurofibrillary tangles, and has been recently genetically linked to PD. DJ-1 modulates misfolding/toxicity of several aggregation-prone proteins and co-localizes with Tau inclusions in AD and other brain disorders. Interactions between DJ-1 and Tau have not been studied, and their relationship in the context of PD has not been explored. Thus we propose to study the interaction between DJ-1 and Tau in living mammalian cells using bimolecular fluorescence complementation, and to what extent, if any, this alters Tau toxicity or formation of fibers. We will extend this work to a fruit fly model of Tauopathies, to ascertain whether DJ-1 modulates disease-relevant phenotypes.

Planned Impact

The common neurodegenerative disorders affect more than 700,000 people in the UK, and cases are predicted to double in the next 25 years, costing the UK economy a staggering £50 billion. Thus, in addition to the obvious patient benefits, the identification of effective treatments for neurodegenerative disorders could dramatically cut these expenditures world-wide. This proposal is focused on dissecting the cellular roles of DJ-1, a protein directly linked to familial cases of Parkinson's disease (PD), which is the second most common neurodegenerative disorder after Alzheimer's disease (AD). Futhermore, we plan to explore the relationship between DJ-1 and the Tau protein, which is involved in the pathogenesis of AD and other brain disorders, and has recently been genetically linked to PD. Thus, this proposal has implications for individuals suffering from PD, and likely AD as well, and their families. Much research has been directed toward uncovering novel therapeutics for these devastating disorders, but nevertheless, to date, the options for treatment are limited. By determining the cellular role of proteins linked to neurodegenerative disease we can enhance our understanding of the molecular mechanisms associated with pathogenesis. Ultimately, these approaches may aid in the identification of novel candidate targets and therapeutic approaches for PD and other devastating brain disorders. Therefore, this will work will directly impact upon patients suffering from PD and their families, as well as researchers and clinicians focused upon this and related disorders.

Publications

10 25 50
 
Title DNA constructs for transfection of mammalian cell lines. 
Description Generation of several normal and mutant versions of DJ-1 for use in mammalian cells for the bimolecular fluorescence assays. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact We have not yet received requests for these items, but as the related research article was only recently published we expect that we will receive requests shortly - and we will provide this research material. 
 
Description Collaboration with Dr Michael Decressac 
Organisation Telethon Foundation
Department Telethon Institute of Genetics and Medicine (TIGEM)
Country Italy 
Sector Charity/Non Profit 
PI Contribution We trained Dr Angelica Vittori in the use of Parkinson's disease fruit fly models to study the role of the insulin pathway on disease pathogenesis.
Collaborator Contribution Dr Decressac is performing parallel studies in mammalian cells and mouse models of Parkinson's./
Impact Not applicable yet.
Start Year 2014
 
Description Collaboration with Dr Miguel Martins 
Organisation Medical Research Council (MRC)
Department MRC Toxicology Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution We began collaborative work with Dr Miguel Martins with regards to the mitochondrial function of DJ-1. We are thus providing expertise on this protein and appropriate models for analysis.
Collaborator Contribution Dr Martins has given us advice on appropriate methods and approaches for mitochondrial function/dysfuction.
Impact The development of this partnership helped us win funding from the MRC. However, the proposed work is still ongoing, and we have not yet had outputs from this work.
Start Year 2013
 
Description Collaboration with Prof Charalambos Kyriacou 
Organisation University of Leicester
Country United Kingdom 
Sector Academic/University 
PI Contribution I have brought expertise on Huntington's and Parkison's disease models to this project, as well as our knowledge on the study of genetic modifiers of disease.
Collaborator Contribution Prof Kyriacou has advised on the use of fruit fly models in our work.
Impact Several of the publications listed in the portfolio have arisen from our collaboration (as indicated by the presence of Prof Kyriacou on the author list).
Start Year 2008
 
Description Collaboration with Prof Dr Tiago F Outeiro 
Organisation University of Göttingen
Department Department of Neurodegeneration and Restaurative Research
Country Germany 
Sector Academic/University 
PI Contribution The collaboration with Prof Dr Tiago Outeiro, Director of the Department of NeuroDegeneration and Restaurative Research, began in 2006, and has consisted of several projects related to Parkinson's and Huntington's disease. During this time we have co-supervised 4 PhD students and 2 postdoctoral fellows, and received funding from several grant applications. Prior to our collaboration, the research in my lab was focused primarily upon Huntington's disease, while Prof Dr Outeiro had great expertise in Parkinson's disease, but limited experience with Huntington's disease. During this collaboration my research time has provided contributions in the collaboration regarding the use and analysis of genetic models.
Collaborator Contribution We have gained from Prof Dr Outeiro's expertise in models of Parkinson's disease, which has allowed us to successfully expand our research into this area. This has been quite important, as several of our areas of interest in neurodegeneration are common between Huntington's disease and Parkinson's disease, and we are now addressing these issues in Parkinson's as well. Our collaboration has recently been strengthened and extended by work on Rab39 funded by Parkinson's UK, as well as studentship funded by the MRC through the MRC Impact DTP.
Impact PUBLICATIONS: Breda C, Nugent ML, Estranero JG, Kyriacou CP, Outeiro TF, Steinert JR, Giorgini F*. Rab11 modulates a-synuclein mediated defects in synaptic transmission and behaviour. Hum Mol Genet 2014 Oct 9. pii: ddu521. [Epub ahead of print]. Chutna O, Gonçalves S, Villar-Piqué A, Guerreiro P, Marijanovic Z, Mendes T, Ramalho J, Emmanouilidou E, Ventura S, Klucken J, Barral DC, Giorgini F, Vekrellis K, Outeiro TF. The small GTPase Rab11 co-localizes with a-synuclein in intracellular inclusions and modulates its aggregation, secretion and toxicity. Hum Mol Genet, 2014 Aug 4. pii: ddu391. [Epub ahead of print]. Zondler L, Miller-Fleming L, Repici M, Gonçalves S, Tenreiro S, Rosado-Ramos R, Betzer C, Straatman KR, Henning Jensen P, Giorgini F*, Outeiro TF*. DJ-1 interactions with a-synuclein attenuate aggregation and cellular toxicity in models of Parkinson's disease. Cell Death Dis, 2014; 5: e1350. Yin G, Lopes da Fonseca T, Eisbach SE, Martín Anduaga A, Breda C, Karpinar DP, Orcellet ML, Szego EM, Guerreiro P, Lazaro D, Braus GH , Fernandez CO, Griesinger C, Becker S, Goody RS, Itzen A, Giorgini F, Outeiro TF, Zweckstetter M. alpha-Synuclein interacts with the switch region of Rab8a in a Ser129 phosphorylation-dependent manner. Neurobiology of Disease, 2014 Jun 28. [Epub ahead of print]. Miller-Fleming L, Antas P, Faria Pais T, Smalley JL, Giorgini F*, Outeiro TF*. Yeast DJ-1 family members are required for diauxic-shift reprogramming and cell survival in stationary phase. PNAS, 2014; 111(19): 7012-7. Vittori A, Breda C, Repici M, Orth M, Roos RAC, Outeiro TF, Giorgini F*, Hollox EJ* and the REGISTRY investigators of the European Huntington's Disease Network. Copy number variation of the neuronal glucose transporter gene SLC2A3 modifies the age of onset in Huntington's disease. Hum Mol Genet, 2014; 23(12): 3129-37. Sajjad MU, Green EW, Miller-Fleming L, Hands S, Herrera F, Campesan S, Khoshnan A, Outeiro TF, Giorgini F, Wyttenbach A. DJ-1 modulates aggregation and pathogenesis in models of Huntington's disease. Hum Mol Genet, 2014; 23(3): 755-766. Amaral M, Outeiro TF, Scrutton NS, Giorgini F*. The causative role and therapeutic potential of the kynurenine pathway in neurodegenerative disease. J Mol Med, 2013; 91(6): 705-13. Amaral M, Levy C, Heyes DJ, Lafite P, Outeiro TF, Giorgini F, Leys D, Scrutton NS. Structural basis of kynurenine 3-monooxygenase inhibition. Nature, 2013; 496(7445):382-5. Repici M, Straatman KR, Enguita F, Outeiro TF, Giorgini F. Mutations in DJ-1 differentially modulate dimerization in living cells. J Mol Med, 2012 Nov 27. [Epub ahead of print]. Tauber E, Miller-Fleming L, Mason RP, Kwan W, Clapp J, Butler NJ, Outeiro TF, Muchowski PJ, Giorgini F. Functional gene expression profiling in yeast implicates translational dysfunction in mutant huntingtin toxicity. J Biol Chem 2011; 286(1):410-9. Miller-Fleming L, Giorgini F, Outeiro TF. Yeast as a Model for Studying Human Neurodegenerative Disorders. Biotechnol J 2008; 3(3): 325-38. GRANTS: 1) Project Grant - Giorgini (PI), Kyriacou (Co-PI), Outeiro (Project Partner) 01/12/2013 - 11/30/2016 Medical Research Council - MR/L003503/1 £ 460,100 "DJ-1 and neurodegeneration: its roles in mitochondria and in protein misfolding" 2) Next Step Funding - Giorgini (PI), Outeiro (Co-PI) 01/12/2012 - 31/12/2013 Parkinson's UK - G-0902 £ 19,919 "Deciphering the molecular role of DJ-1 in the etiology of Parkinson's disease-Next Step Funding" 3) Project Grant - Giorgini (PI), Outeiro (Co-PI) 01/04/2012 - 30/09/2013 Parkinson's UK - G-1203 £ 92,959 "Unravelling the pathogenic role and therapeutic potential of Rab family GTPases in Parkinson's" 4) Project Grant - Giorgini (PI), Outeiro (Co-PI) 01/10/2009 - 30/11/2012 Parkinson's UK - G-0902 £ 242,759 "Deciphering the molecular role of DJ-1 in the etiology of Parkinson's disease"
Start Year 2006
 
Description Invited seminar, Division of Neuroscience & Experimental Psychology, Faculty of Biology, Medicine and Health, University of Manchester, UK. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Research seminar.
Year(s) Of Engagement Activity 2017
 
Description 50th Anniversary for Department of Genetics, University of Leicester 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Seminar was presented, which resulted in discussion.

Discussion of interest from public and colleagues.
Year(s) Of Engagement Activity 2011,2012,2014
 
Description Brain Awareness Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact My lecture was part of a wider Brain Awareness Day event which the University of Leicester organized. It was broadly advertised and well attended. My lecture served to spark many questions from the audience how basic research using disease models can lead to advances in medicine and treatment.

The talk led to increased interest in my research, and hopefully emphasized to the audience the translational benefits of our studies.
Year(s) Of Engagement Activity 2015,2016,2017,2018
 
Description GENIE Open Day Events, University of Leicester, UK 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Open day events organized by GENIE a CETL based at the University of Leicester. ~200 school children per event learning about DNA and genetics, and the use in medicine, etc.

Generated interested in genetics amongst the school children - with greater interest in GCSE and A-level science/biology.
Year(s) Of Engagement Activity 2008,2009,2010,2011,2012
 
Description GENIE Summer School 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Talk sparked discussion/questions.

Increased interest in science/genetics amongst students.
Year(s) Of Engagement Activity 2008,2013,2014
 
Description Inaugural Lecture, University of Leicester 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact This was my inaugural lecture for promotion to professorship, which was a public lecture/event. A mix of individuals from the university and the local community attended. Estimate ~200 people attended.
Year(s) Of Engagement Activity 2017
 
Description Invited seminar, Jean-Pierre Aubert Research Centre, UMR Inser-S1172, Univ. Lille-Nord de France, Lille dedex, France 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Research seminar.
Year(s) Of Engagement Activity 2017
 
Description Invited seminar, Living Systems Institute, University of Exeter, UK. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Research seminar.
Year(s) Of Engagement Activity 2018
 
Description Parkinson's UK, East Midlands Research Support Network, First Regional Research Forum, University of Leicester, "Using genetics to understand Parkinson's" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact ~50 members of the public, as well as Parkinson's families and carers attended. Questions and discussion panel afterwards.

Requests for further Parkinson's related talks to the general public.
Year(s) Of Engagement Activity 2012
 
Description Parkinson's UK, Research Supporter's Conference, York, "Unravelling what happens inside nerve cells in Parkinson's" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact A talk to ~150 members of the public, Parkinson's families and carers. Questions and discussion panel followed.

Invitation to present at the Gretschen Amphlet Memorial Lecture - Fitzwilliam College Cambridge - Wednesday 17 April 2013.
Year(s) Of Engagement Activity 2012,2014
 
Description University Taster Days 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Undergraduate students
Results and Impact ~100 A-level students contemplating Leicester for undergraduate studies, along with parents/relatives. These talks covered my research, and the students asked several relevant questions, and were interested in how this could tie into their 3rd projects. This has been done 8 times, 7 times in Leicester and once in London.

Impacts will be determined based upon potentially increased applications to the university for study.
Year(s) Of Engagement Activity 2013,2014,2015,2016,2017