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Supervisors: Professor Philippa Mills, Dr Karin Tuschl
Background:
Our research group aims to identify new treatments for neurodegenerative childhood disorders associated with metal dyshomeostasis. One such metal is manganese that is essential for normal brain function, however, in excess it becomes neurotoxic and leads to dystonia-parkinsonism, psychiatric and intellectual disability. The manganese transporters SLC30A10 and SLC39A14 act in conjunction to mediate manganese excretion. We have previously shown that loss-of-function mutations in either gene lead to manganese neurotoxicity, termed hypermanganesaemia with dystonia 1 (HMNDYT1) and 2 (HMNDYT2). Chelation therapy with intravenous EDTACaNa2 has some effect but causes significant adverse effects. Therefore, there is a great need to identify novel therapeutic strategies to reduce morbidity and mortality associated with these disorders and improve patients’ quality of life.
Aims/Objectives:
This project aims to develop a novel treatment approach using antisense oligonucleotides (AONs) to reduce manganese overload. The key objectives are to show that:
1. AONs can be used to limit manganese uptake by targeting the manganese uptake transporter SLC39A8.
2. Silencing of the manganese uptake transporter prevents manganese accumulation and neurotoxicity in Slc30a10 knockout mice.
Methods:
The student will have the exciting opportunity to acquire a wide range of molecular and cell biology techniques and high-resolution imaging skills. They will gain experience in disease modelling using cell and mouse models of manganese neurotoxicity. The student will learn how to design AONs and test their efficacy in cell culture using qPCR, Western blotting and immunofluorescence staining. They will gain experience in mouse husbandry, histopathology and behavioural analysis. The student is expected to complete their PhD thesis, publish their data in peer-reviewed journals and present their findings at international scientific meetings.
Timeline:
Month 1-6: Design of AONs that target SLC39A8 mRNA and assess their efficacy in human fibroblasts and murine 3T3 cells using qPCR, Western blotting and immunofluorescence.
Month 1-12: Determine the phenotype of SLC30A10 deficient SH-SY5Y cells using immunofluorescence, qPCR and ICP-MS. Assess the efficacy of AONs on manganese levels/tolerance and metal distribution.
Month 13-21: Define the manganese neurotoxicity phenotype of Slc30a10 knockout mice through neuro-histopathology studies, locomotor behaviour (automated gait analysis) and metal distribution analysis (ICP-MS and LA-ICP-MS).
Month 22-30: Assess the effect of the two AONs with greatest knockdown efficiency in cell culture in Slc30a10 knockout mice and determine whether SLC39A8 silencing can prevent cerebral manganese overload and prolong survival.
Month 30-36: Completion of PhD thesis and publication of research findings.
References:
1. Tuschl K et al. Maintaining Translational Relevance in Animal Models of Manganese Neurotoxicity. J Nutr. 2020;150:1360-1369.
2. Aguti S et al. Exon-Skipping Oligonucleotides Restore Functional Collagen VI by Correcting a Common COL6A1 Mutation in Ullrich CMD. Mol Ther Nucleic Acids. 2020;21:205-216.
3. S. Anagianni, K. Tuschl, Genetic Disorders of Manganese Metabolism, Curr Neurol Neurosci Rep, 19 (2019) 33.
4. Tuschl K et al. Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia. Nat Comms. 2016. 7:11601.
5. Tuschl K et al. Syndrome of hepatic cirrhosis, dystonia, polycythaemia and hypermanganesaemia – caused by mutations in SLC30A10, a manganese transporter in man. Am J Hum Genet. 2012. 90:457-466.
