ESR6: How does glucocerebrosidase control autophagy in the dopamine neuron?
Carmen de La Fuenta Barrigon (University College London)
Supervisor: Simon Heales

Project summary
The underlying causes of dopaminergic degeneration are unknown. However, mitochondrial impairment has been implicated as a key player in the role of neuronal cell death and reductions in mitochondrial electron transfer chain activities have been found in Parkinson’s disease (PD). Finding the relationship between oxidative stress, dysfunctional mitochondrial, energy production, lysosomal dysfunction and autophagy systems in the dopamine neuron is a key point in terms of understanding the mechanisms that underlie Parkinson’s disease.

The early stage researcher (ESR) will work with patient samples to identify the glycoanalytical profile of patient biopsies. In collaboration with the experienced researcher (ER) Dr Maria Garcia Gomez, who will utilise molecular biology techniques to introduce glucocerebrosidase mutations into neuronal cultures, the ESR will assay the effects of these mutations on autophagy and mitophagy using confocal imaging. The ESR will be trained at UCL in the area of glycolipids identification using HPLC and mass spectrometry techniques. They will also be seconded to the NIBRT SME for a 3 month period training period in high throughput glycoanalytics.

The specific aims of the project are:

  1. Set up an HPLC method for measuring the release of dopamine and its metabolites into cell culture media by the neuroblastoma cell line SH-SY5Y.
  2. Differentiate the SH-SY5Y cell line to dopaminergic neurons and treat them with rotenone (creating an oxidative stress-like environment) and CBE (to generate Gaucher disease-like lysosomes as it is a glucocerebrosidase inhibitor).
  3. Assay the effects of the Gaucher disease mutations on autophagy and mitophagy using confocal imaging.
  4. Identify the glycoanalytical profile of PD patient biopsies.
We have recently set up the HPLC method and right now we are working on the differentiation of the cell line for the further treatment.

Outputs [top]
In progress.

Training [top]

(i) Local level

    Departmental and general seminars at ICH.
  • Doctoral Skills Development Programme at UCL:
    • Literature Searching using PubMed - University College London.
    • Reference Manager & Endnote - University College London.
    • Hugh Kearns - The Seven Secrets of highly successful research students.
    • SLMS Introduction: Information Governance training and awareness event.
    • Problem Solving & Decision Taking for Research Students - University College London.

(ii) Network level
As part of the TINTIN training we have attended seminars on NMR, confocal imaging, Seahorse and Oroboros

Workshops attended: Principles of Mitochondrial Biology, Metabolism and Bioenergetics in Health and Disease MiP Spring, London 2015

Outreach [top]

    Presentations:
  • 26th June 2015: CTO-EPM Lab meeting, "The measurement of dopamine metabolites in model systems".
  • 17th April 2015: Second Inherited Metabolic Disease Research meeting, "The measurement of dopamine metabolites in model systems".

News

  • Discover Research Dublin night (September 25th 2015) An opportunity to share scientific research with the general public.
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  • Advanced Microscopy and Super Resolution (Andor) 07-09/05/15, Trinity College, Dublin: Lectures on the fundamentals of fluorescence imaging including a breakdown of different types of fluorescent probes including dyes, antibodies and fluorescent proteins were given.
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© 2017 TINTIN - A Marie Curie Initial Training Network Programme funded under Grant Agreement No. 608381