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  Treatment and biomarker development for inherited manganese transporter defects using cell models of SLC30A10 deficiency


   BRC Translational Research (Non-Clinical) PhD Studentships

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  Dr Karin Tuschl, Dr P Mills, Dr Wendy Heywood  No more applications being accepted  Funded PhD Project (UK Students Only)

About the Project

Manganese is essential to human health and development. However, excess manganese is neurotoxic causing manganism, a neurodegenerative disorder characterised by dystonia-parkinsonism. We have identified two inherited childhood disorders of manganese overload due to loss-of-function mutations in either the efflux transporter SLC30A10 (Hypermanganesaemia with Dystonia 1, HMNDYT1) or the influx transporter SLC39A14 (HMNDYT2).1-2 Both transporters are essential for the liver to action its role as the “housekeeper” of manganese homeostasis. Impaired hepatic excretion of manganese results in liver disease and accumulation of manganese in the basal ganglia (Figure 1). Manganese toxicity has also been implicated in common neurodegenerative disorders (e.g. Parkinson’s, Alzheimer’s disease) and in end-stage liver disease causing impaired biliary manganese excretion.3 A better understanding of the molecular and biological consequences of manganese toxicity has the potential to benefit multiple disease groups that share cellular manganese dyshomeostasis.

Current treatment of manganese overload is limited to chelation with calcium disodium edetate (EDTA).1-2 While life-long therapy can improve motor symptoms and halt liver disease, the risk of liver decompensation and neurological sequelae persist. The need for monthly intravenous administration over five days and associated adverse effects make EDTA a poor drug for clinical application, aggravating the disease burden. Blood manganese levels poorly correlate with disease severity and treatment efficacy. Therefore, there is an immense clinical need to identify suitable disease biomarkers and develop alternative therapeutic approaches.

We have demonstrated the crucial role of SLC30A10 in manganese homeostasis across vertebrates with loss-of-function leading to manganese overload in the liver and brain with associated neurotoxicity.

Observations across disease models suggest that manganese toxicity causes oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress and Ca2+ dysregulation, leading to altered resting-state neuronal activity (Figure 3).4 However, the key molecular event(s) and the hierarchical interactions between them remain unknown.    

Aims/Objectives:

This project aims to better understand the mechanisms underlying manganese homeostasis with the view to identifying novel disease biomarkers and therapeutic approaches for inherited manganese transporter defects and neurodegenerative disorders associated with manganese overload.

•  Objective 1: Characterisation of the manganese overload phenotypes of SLC30A10 loss-of-function cell models to determine key players in manganese toxicity and identify readouts for drug testing.

•  Objective 2: Determine whether SLC30A10 mRNA therapy can rescue manganese overload phenotypes identified in Objective 1. 

•  Objective 3: Identification of novel regulators of manganese homeostasis and disease biomarkers using cell models from objective 1 and patient serum.

Methods:

Objective 1 (month 1-12):

Hep3B (shared by Prof Bartnikas, Brown University, US)5   and SH-SY5Y cells (GenScript) that carry loss-of-function mutations in SLC30A10 will undergo multi-scale phenotypical characterisation to identify the molecular and biological pathways affected by manganese toxicity. The chosen cell lines are the most relevant to examine manganese dyshomeostasis since both liver and brain (dopaminergic neurotransmission) are affected in HMNDYT1. Phenotypical characterisation at baseline and upon MnCl2 exposure will include:

- Survival analysis.

- Multi-element Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) analysis (Mn, Ca, Fe, Zn).

- 52Mn uptake and efflux studies.

- Manganese distribution: Fura-2, M1 Mn2+ sensor.

- Calcium homeostasis: Ca-NPEGTA, Rhod-5n, Fura-2.

- Oxidative stress: probes for ROS, OH·, lipid peroxidation, glutathione.

- Mitochondrial function: Δψm, respiratory chain activity, oxygen consumption, seahorse analysis.

Milestone 1 (month 12): Readouts for drug testing identified.

Cell models will be used for proteomic studies in objective 3.

Objective 2 (month 13-18):

Phenotypical read-outs identified under objective 1 will be used to assess the pharmacokinetics and efficacy of SLC30A10 mRNA therapy (unformulated and LNP encapsulated provided by commercial partner Moderna):

- Protein expression and subcellular localisation will be confirmed longitudinally over 7 days using Western blot and immunohistochemistry. 

- Manganese concentration and flux will be determined by ICP-MS and 52Mn flux studies (collaborator Prof Blower, KCL).

Milestone 2 (month 18): Functional rescue of Mn overload phenotypes and duration of effect of mRNA therapy confirmed in vitro.

Objective 3 (month 13-30):

Proteomic analysis will be performed on control, SLC30A10 KO and mRNA treated Hep3B/SH-SY5Y cells using label free proteomic techniques:

1. with fractionated lysate digests for deep proteome coverage.

2. after enrichment of manganese binding proteins using Immobilized Metal Chelate Affinity Chromatography (column charged with Mn2+).

This will identify changes in protein expression upon manganese overload. Identified candidate proteins or metabolites will be validated in control and patient serum using multiplex targeted LC-MS/MS assays with the view to developing novel biomarkers for disease monitoring.

Protein candidates and hierarchical interactions between them will be further explored in cell models established in objective 1 (e.g. using inhibitors of biological pathways/Ca2+ flux). 

Milestone 3 (month 24): Lead candidates identified. 

Milestone 4 (month 30): Verification of candidates in control/patient serum.

Milestone 5 (month 30): Possible hierarchical interactions of manganese toxicity effects determined. 

Biological Sciences (4)

 About the Project