Understanding the mechanisms of manganese neurotoxicity and deficiency using slc39a14 loss-of-functi

Understanding the mechanisms of manganese neurotoxicity and deficiency using slc39a14 loss-of-function zebrafish

Background:

Our research groups work together complementary with the aim to better understand the disease pathobiology of neurometabolic disorders characterised by metal dyshomeostasis, ultimately, to develop new treatment strategies for affected children. One such metal is manganese that is essential for normal brain function, however, both overload and deficiency lead to neurological disease.

We and others have recently identified a new group of disorders, the so called “inherited manganese transporter defects”, that are caused by mutations in genes required for the uptake and excretion of manganese (SLC39A8, SLC39A14, SLC30A10). Impairment of manganese homeostasis within the brain leads to a disabling movement disorder and neurodevelopmental disease of childhood.

In order to study the molecular effects of manganese dyshomeostasis in vivo, we have generated a zebrafish slc39a14 loss-of-function mutant using CRISPR/Cas9 genome editing that resembles the human disease with impaired locomotor activity that is associated with abnormal neuronal activity. Transcriptome analysis of slc39a14^-/- zebrafish demonstrates that loss of slc39a14 leads to concurrent manganese neurotoxicity and deficiency within different tissues or subcellular compartments, both associated with calcium dyshomeostasis.

As a next step, it is imperative to delineate the neuronal subtypes involved in order to dissect the molecular consequences of manganese dyshomeostasis, a prerequisite for identifying novel treatment targets.

Aims/Objectives:

This project aims to improve our understanding of how manganese is involved in disease processes underlying inherited manganese transporter defects.

The key objectives are to:

1. Determine the neuronal subtypes and their transcriptomic signatures affected by manganese dyshomeostasis using single cell RNA sequencing (scRNAseq).

2. Relate the identified transcriptomic changes to alterations in brain activity and neurochemistry.

Methods:

The student will have the exciting opportunity to join an exceptional research environment at UCL (http://zebrafishucl.org) and work closely with the Busch lab at Queen Mary’s University who will share their expertise in bioinformatic analysis. The student will acquire a wide range of techniques including zebrafish husbandry, embryology and manipulation, bioinformatic analysis of scRNAseq data, high-resolution imaging, computational and programming approaches, molecular biology (in situ hybridisation, immunohistochemistry, CRISPR/Cas9 genome editing). The student will have the opportunity to attend an EMBL bioinformatics course on scRNAseq at the start of the project. 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 1-18: Determine the effects of manganese toxicity and deficiency on single cell level using scRNAseq on brains from unexposed and MnCl₂ exposed slc39a14^-/- larvae. Bioinformatic analysis will be supported by the Busch lab.

- Month 13-30: Follow up transcriptomic changes with whole brain activity, neuroanatomical and neurochemical analysis using high-resolution imaging as well as CRISPR/Cas9 genome editing with the view to delineate the mechanisms underlying manganese neurotoxicity and deficiency.

- Month 19-33: Assess brain activity in vivo using calcium imaging of transgenic zebrafish (Tg(elavl3:GCaMP6s)) to assess specific circuit dysfunctions. 

- Month 30-36: Completion of PhD thesis and publication of research findings.

References:

1. Tuschl K et al. Loss of slc39a14 causes simultaneous manganese hypersensitivity and deficiency in zebrafish. Dis Model Mech. 2022. doi: 10.1242/dmm.044594.
2. Tuschl K et al. Maintaining Translational Relevance in Animal Models of Manganese Neurotoxicity. J Nutr. 2020;150:1360-1369.
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