ER1: The mechanisms that control mitochondrial dynamics & function in the dopamine neuron
Maria Garcia Gomez (University College London)
Supervisor: Simon Heales
Project summary
Several major neurological illnesses such as Parkinson’s disease have been associated with a selective loss of dopaminergic neurons, and certain metabolic defects have been seen to potentiate dopaminergic cell death in the brain, interfering with normal neurotransmission. However, the molecular and biochemical mechanisms that underlie these pathological processes are still not well understood. Hence, we have chosen Gaucher disease as a model of metabolic disease to study the link between metabolic defects and neurodegeneration that ultimately will make possible the development of novel therapeutics that modify the dopaminergic system. My role as ER in this project is therefore to develop an in vitro model of Gaucher disease through downregulation of GBA gene expression to further study its implication in neuronal function.
The specific aims of the project are:
- Choose suitable target cell lines. In addition to the SHSY5Y neuroblastoma cell line that is able to differentiate to dopaminergic neurons after retinoic acid treatment, we have measured by RT-qPCR the basal expression of the gene that we intend to silence (GBA) in different human cell lines, both adherent (HeLa and 293T) and monocytic (THP1 and U937) in order to choose those with a GBA basal expression similar to the neroblastoma cells. Thereby we will validate the gene silencing tools in different cell types to demonstrate their efficacy. To investigate ROS production in synaptic vs non-synaptic mitochondria under different conditions.
- Design and develop interference RNAs (shRNA) to silence the human GBA gene. So far, we have designed 8 different shRNAs (provided by The Cancer Institute, UK) binding different sequences of the GBA mRNA. These shRNAs were cloned into a lentiviral backbone harboring different sequences from the miR30 to increase their efficacy and two different reporter genes (EGFP and Puromycin resistance gene) to track the shRNA expression once inside the cells and to select the cells expressing the shRNAs respectively. All bacteria clones carrying the shRNAs were sequenced using SnapGene software and the sequencing service from Source Bioscience, and a blast alignment of each shRNA to the human genome was carried out to determine their potential off-target effects. In addition, a scramble shRNA binding no sequence of the human genome was used as negative control in all experiments.
-
Check silencing efficiency of developed shRNAs. Bacteria clones containing each shRNAs were amplified and DNA was used to transfect 293T and HeLa cells. Two days after transfection, cells were harvested and the RNA isolated to analyzed GBA expression by RT- qPCR and determine which shRNA was the most efficient.
- Lentiviral vector production. The three most efficient shRNAs and the scramble shRNA were packaged in lentiviral particles using the PEI-based second generation packaging method in 293T cells and lentiviral supernatants were titrated by transduction of HT1080 cells and subsequent EGFP expression analysis by flow cytometry using the FACS Calibur equipment and FlowJo software, obtaining titers around 108 infective viral particles per ml (ivp/mL).
- Transduction experiments and functional analysis. In order to determine the most efficient shRNA-viral supernatant leading to gene silencing without affecting cell survival we assayed five different multiplicities of infection (MOI from 0.5 to 30) in SHSY5Y cells with one of the shRNAs, obtaining transduction percentages from 74.5 to 98.8%. We also observed that MOIs above 10 were toxic for the cells and 15 days post-transduction survival was more reduced in GBA-interfered cells that in those treated with the scramble virus. In addition, after analyzing GBA gene expression by RT-qPCR and GBA enzymatic activity by biochemical analysis we determined that MOIs of 3 and 10ivp/mL were the highly efficient for GBA silencing in non-differentiated SHSY5Y cells.
Results [top]
In progress.
Outputs [top]
Meeting abstracts
Our preliminary results have been presented in three different symposiums in a poster format under the title
"Modeling Gaucher Disease Through interference RNA technology".
- British Society for Gene and Cell Therapy Annual Conference 2015. 9th-11th June, 2015, Glasgow (UK).
- Annual Symposium for Postdocs, Genetics and Genomic Medicine Department, Institute of Child Health, University College London (UK).
- 3rd International Symposium of the Society of Spanish Researchers in the United Kingdom, 4th July, 2015, London (UK).
Work supported by TINTIN European Project and Marie Curie Initial Training Network
Training [top]
(i) Local level
During this last 10 months I have done a training course at UCL to use the EndNote program more efficiently given its utility to manage scientific bibliography, and also I got induction courses of the core facilities that I am using to carry out our research, such as flow cytometry and fluorescence microscopy facilities.
(ii) Network level
Neuronal Cell Metabolism, TCD organized by Seahorse. Course focused on the state-of- the-art Seahorse analysis to study alterations in fundamental metabolism.
- Training in Mitochondrial and cellular respiratory physiology, TCD organized by Oroboros. Course focused on how to measure oxygen consumption rates and transmembrane potential differences to determine hydrogen peroxide production, membrane potential , ATP production and Ca2+ production.
- NMR Mini Boot Camp of BioBank Analyses and Metabolomic Transformation, TCD organized by Agilent. This course included lectures introducing the underlying principles and practices of modern NMR spectroscopy, and hands-on experience to be able to run and analyse biomolecular samples.
- Fluorescence and electron microscopy imaging of cells, TCD organized by Andor.
Outreach [top]
During the last ten months the following meetings were attended:
- Gene, Cell and Molecular Therapies for Inherited Metabolic Diseases. 11th-12th June, 2015, Glasgow (UK).
- Centre for Inborn Errors of Metabolism, (one-day symposium, November 2014 and May 1015) celebrated at Institute of Child Health, University College London (UK).
- British Society for Gene and Cell Therapy Annual Conference 2015. 9th-11th June, 2015, Glasgow (UK).
- Annual Symposium for Postdocs, Genetics and Genomic Medicine Department, Institute of Child Health, University College London (UK).
- 3rd International Symposium of the Society of Spanish Researchers in the United
Kingdom, 4th July, 2015, London (UK).
Recently I have also presented my work in the Centre for Translational Omics meeting of the Genetics and Genomic Medicine Department (Institute of Child Health, University College London) in a 30 min oral talk, where I had the opportunity to explain my project and discuss my results and future ideas with other researchers of the department.
