Investigators/Authors: Tim Bartels, M.Sc., Ph.D., and Dennis J. Selkoe, M.D.; Harvard Medical School and Brigham and Women’s Hospital; Boston, MA
Objective: To develop a test to detect different forms of the pathologic protein a-synuclein
Background: A hallmark of Parkinson’s is the clumping together of a protein known as a-synuclein (a-Syn) in certain cells of the brain. Although scientists don’t know exactly how or why a-Syn forms clumps, abnormally shaped forms of the protein may be to blame. The laboratory of Dr. Bartels and Dr. Selkoe recently discovered that in normal cells, four copies of the a-Syn protein associate with each other to form a structure that is called a tetramer. In people with PD, however, the a-Syn tetramer doesn’t assemble properly, which may make the protein more vulnerable to clumping. Currently, researchers do not have a method for detecting these abnormal forms of a-Syn in the blood or brains of people with PD. Therefore, the researchers plan to develop a test, known as an Enzyme-linked Immunosorbant Assay (ELISA), to detect different forms of a-Syn.
Methods/Design: The ELISA will use antibodies that can discriminate between normal and abnormal a-Syn forms to determine which forms of a-Syn are present in blood or brain tissue samples. They will use this ELISA to characterize a-Syn forms in many blood samples and brains of humans and mice with PD.
Relevance to Diagnosis/Treatment of Parkinson’s disease: Being able to do so may help scientists better understand how a-Syn forms clumps, as well as aid PD diagnosis and the search for more effective therapies.
September 2013 Project Update:
Aim 1: After screening 36 different antibody combinations under 6 different conditions, we have in principal reached our goal of detecting three different shapes or conformations that alpha-synuclein (aSyn) can assume: We can differentially measure the concentration of helically folded aSyn, unfolded aSyn, as well as total (folded and unfolded) aSyn in complex solutions. We optimized our approach to reach high sensitivities—not only in neuronal tissue and blood samples, but also in human cerebral spinal fluid (Möllenhauer, B. et al., Exp. Neurol. 2008). We validated how specifically we can detect aSyn and found that we can reliably detect aSyn regardless of the animal species, e.g., mouse aSyn is readily detected too. Our preliminary work was presented in form of a talk and poster at the PAN meeting in February in Washington DC, USA (http://www.parkinsonsaction.org/your-voice/pan-conference) as well as in poster format at the AD/PD conference in Florence, Italy in March 2013 (http://www2.kenes.com/adpd/sci/Pages/ScientificProgram.aspx).
Aim 2. When testing our approach, we have obtained preliminary data from the brains transgenic mice on the aSyn monomer to tetramer ratio. We also made some progress in obtaining 10 different brains from dementia with Lewy bodies (DLB) patients (Ian McKeith, Institute for Ageing and Health, Newcastle, UK), 17 blood samples from aSyn (SNCA gene) familial mutation carriers (John Hardy, UCL, London, UK) and sporadic PD patients (Clemens Scherzer, BWH, Boston, USA), as well as 30 cerebral spinal fluid samples of PD patients and healthy control individuals (Brit Möllenhauer, Paracelsus Klinik, Göttingen, Germany).
Our progress in establishing an conformation specific ELISA will allow us now to screen for differences in the conformation distribution of aSyn in diseased versus healthy tissue, potentially revealing correlations between disease state/progression and changes in the structure of native aSyn. Any findings there would be of great interest for the pathogenesis of DLB and PD as well as a potential biomarker for their early diagnosis. Being able to detect pathologic changes early is imperative for early and precise diagnosis of the disease.
September 2014 Project Update:
The project is now complete. Our platform is, to our knowledge, the first assay to sensitively quantify the destabilization of native a-Syn by combining conformation specific antibodies with the ELISA technique. We are still further developing the platform to make it more sensitive and are still working to improve the quality of the standards used to calibrate this assay for easier reproducibility and comparability between different labs in order to establish it as a potential PD biomarker for early diagnosis. Early diagnosis in turn would significantly improve the treatment options available to patients and hence potentially slow their disease progression.
In addition, if destabilization of a-Syn does truly correlate with disease progression, then our platform would allow for more appropriate classification of patients enrolled in clinical trials. Since a common problem of clinical trials in neurodegenerative diseases is enrolling properly diagnosed patients and enrolling patients in early, easier treatable stages of their disease, (preferably before any symptoms become apparent) an assay that detects early disease associated changes would greatly improve drug development in an indirect way.
We are using the preliminary data generated with the funding from this Parkinson’s Disease Foundation award to support continued research via a grant application to the National Institute of Neurological Disorders and Stroke (NINDS) by Dr. Bartels. Dr. Selkoe also used data from this project in his recently awarded NINDS grant to also continue this research direction. In addition to the posters presented above, the following publication was generated by this project: Dettmer U, Newman AJ, Luth ES, Bartels T, Selkoe D. (2013) “In vivo cross-linking reveals principally oligomeric forms of α-synuclein and β-synuclein in neurons and non-neural cells.” J Biol Chem. 288(9):6371-85.
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