TDP-43 · Taming TDP43: High-throughput screening for compounds to reduce aggregation of the new player in MND

Taming TDP43: high-throughput screening for compounds to reduce aggregation of the new player in MND

Motor neuron disease (MND) - also known as amyotrophic lateral sclerosis - is a degenerative disorder of the upper and lower motor neurons which has a global incidence of 2 in 100 000 per annum. Typically, onset is in mid to late life and the disease is fatal within 2 - 5 years. Professor Shaw's group was the first to identify mutations in the transactivation response deoxyribonucleic acid (TAR DNA) binding protein (TDP-43) in familial and sporadic MND / amyotrophic lateral sclerosis (ALS). Mutations have been subsequently reported in 1-4 % of all MND cases. Even in the absence of mutation, non-mutant (wildtype) TDP-43 is found to be abnormally clumped together (aggregated) in greater than 90 % of MND patients and in 60 % of fronto-temporal lobar dementia with ubiquitin (FTLD-U) patients. This project sought to develop a cellular model of TDP-43 pathology to screen a drug library for disease-modifying properties before testing these drugs in neurons generated from stem cells from a patient with ALS. Resources generated To develop a cell model of TDP-43 pathology which was amenable to screening, various plasmids and cell lines were generated and validated (protein made continuously / or only upon addition of doxycycline; various tags to distinguish added TDP-43 from the cells own TDP-43; various mutations; human kidney (HEK293) and neuroblastoma SHSY-5Y cell lines). Characterisation of cell lines Cell lines making wildtype, deleted nuclear localising sequence (DNLS) or C-terminal fragment (CTF) TDP-43 were characterised. They were shown to have TDP-43 in the nucleus (wildtype) or in mainly the cytoplasm (?NLS and CTF) as expected. The level of TDP-43 protein made was tightly regulated by the levels of the doxycycline inducer in these cells, and protein production was reversible upon washout of doxycycline, allowing for measurement of the rate at which the cell degrades TDP-43. Validation of epitope tags To be confident in our TDP-43-based cellular models of ALS and FTLD, we sought to determine the effects of adding detection tags to TDP-43 on its function. We found that most functions of TDP-43 were retained even when TDP-43 was fused to a large green fluorescent protein (GFP) tag, however, GFP-tagged TDP-43 showed a tendency to be trapped in the nucleus and to clump (aggregate) under conditions of stress or overexpression. We determined that TDP-43 with small tags should be employed preferentially over TDP-43 fused to GFP or other large tags in studies of ALS, FTD and related pathologies. Stable cell lines overexpressing wildtype or mutant TDP-43 do not recapitulate MND pathology basally Inducing cells to make more TDP-43, either wildtype or mutant, did not result in TDP-43 aggregation or cell death, therefore we sought ways to enhance TDP-43-dependent toxicity, or aggregation, using paradigms relevant to disease. Protein aggregates and / or cytotoxicity Chronic application of the oxidative stressor arsenite caused the formation of TDP-43 aggregates, as did MG132, which inhibits one of the cells degradation systems- the proteasome. Interestingly, TDP-43 aggregation did not enhance or diminish the toxicity of these stressors. The large, single, aggregates of TDP-43 which formed when the cell degradation system was impaired chemically with MG132 were reminiscent of aggregates seen in patient tissue, and were positive for signals which direct the cell to degrade proteins (ubiquitin, ubiquilin 2, p62). This suggests that proteasome impairment may be a relevant way to model ALS and FTLD in cells to conduct a drug screen. However, due to the major cell death associated with MG132 treatment, this model was unsuitable for screening large numbers of drugs- leaving insufficient numbers of cells in assay plates. In addition, MG132 treatment introduces unacceptable levels of variability. We have been working therefore with a commercial partner to develop an assay which can cope with these limitations. This collaboration aims to screen a library of 300,000 drugs and will continue the aims and objectives of the current Marie Curie proposal. Characterisation of turnover pathways for TDP-43 Concurrent to efforts to optimise a screening system, the cellular models generated to date have been used to explore the ways in which TDP-43 protein is degraded by the cell. We showed that a short fragment of TDP-43, which is presumed to be important in 'seeding' the formation of TDP-43 aggregates, is actually degraded more rapidly than full-length TDP-43. There is a strong involvement of two different protein degradation systems in clearance of TDP-43, with inhibition of either system slowing TDP-43 degradation. Interestingly, despite slowing TDP-43 degradation to a similar degree, inhibitors of the proteasome caused TDP-43 aggregate formation while inhibitors of a parallel system (autophagy) did not. Currently ongoing work is examining the role of these two systems in degrading TDP-43 which is already aggregated. Before large-scale screening of drugs can be conducted, targeted approaches which examine the way the cell handles aggregated proteins are of great interest. Drug screening Our lab has now optimised methods for growing stem cells from patients with ALS, and of making them into neurons which show features of disease. Once we have screened our drug library of 300 000 drugs in the simple cells discussed above, we will test the most promising drugs for their ability to rescue these neurons from disease. Conclusions This project has established cell models with which to study aspects of cell pathology in ALS and FTLD. The project has characterised the way in which TDP-43, the major protein implicated in these diseases, is degraded by the cell, and shown that impairment of this degradative process generates the TDP-43 aggregates which are characteristic of disease. Socio-economic impacts of the project This project is the start of a comprehensive screening initiative by the Shaw Lab. It has attracted the interest of an international industry partner, and has the potential to attract funding and researchers to the European Economic Area, as will drug candidates that emerge from screening. Contact details: Prof. Christopher Shaw, email: christopher.shaw@kcl.ac.uk

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