ESR5: The effect of glutathione on mitochondrial dynamics & function in the dopamine neuron
Cécile Crès (Trinity College Dublin)
Supervisor: Gavin Davey
The specific aims of the project are:
- Study the effects of mitochondrial dysfunction on lysosomes As mitochondrial impairment is one of the features of PD, we want to analyse how it affects lysosomes using confocal microscopy. For this purpose, we study lysosomes and mitochondria in PC- 12 cells treated or not with rotenone, a specific inhibitor of the mitochondrial complex I. While, both organelles can be seen in the cell body of untreated PC-12 cells, their respective distribution is more striking in the neurites: lysosomes are mainly restricted at the distal ends of the neurites whereas mitochondria are more closed to the cell body. After the addition of rotenone, the intensity of the fluorescent signals associated with mitochondria and lysosomes decreases rapidly overtime. However, the deficiency of the mitochondrial complex I does not appear to have an effect on the distribution of the lysosomes. As we have been dealing with cells issues and poor transfection efficiency, the experiments to assess the effect of rotenone on lysosomes at lower concentrations have been delayed. These experiments in PC-12 cells as well as in primary rat neurons, will help us determine the order of appearance of the different effects induced by rotenone (mitochondrial deficiency, increase of the release of mitochondrial-derived vesicles, lysosomal membrane permeabilization).
- Study the effects of glucocerebrosidase inactivation on mitochondria Mutations in the GBA1 gene encoding the lysosomal form of glucocerebrosidase are high risk factors for PD. Therefore we want to study how the inactivation of glucocerebrosidase affects mitochondria in PC-12 cells. The cells are treated with CBE (conduritol-beta-epoxide), a specific inhibitor of GBA1 and we assess the effect on the mitochondrial membrane potential using TMRM, a fluorescent dye that accumulates in healthy mitochondria. While CBE has been shown to decrease significantly the activity of the lysosomal form of glucocerebrosidase in PC-12 cells, we have not been able to measure the mitochondrial membrane potential with TMRM as this technique still needs to be improved.
- Investigate the existence of a communication pathway between lysosomes and mitochondria It was shown recently that KIFC1, a c-terminal kinesin, transports material from lysosomes to mitochondria in a unicellular parasite. In view of the existence of three different forms of this motor protein in mammalians (KIFC1, KIFC2 and KIFC3), we decided to investigate if this communication pathway occurs also in PC-12 cells and primary rat neurons. Amongst the three kinesins, KIFC3 is the one with the highest expression level, both in PC-12 cells and primary rat neurons. We also noticed that KIFC1 and KIFC2 expression levels are significantly higher in primary neurons compared to PC-12 cells. The next step will be to knockdown KIFC1, KIFC2 and KIFC3 and to study if this has any effect on lysosomes and mitochondria in normal conditions but also when cells are treated with rotenone or CBE.
(i) Local level
Training in mammalian cell culture, DNA and siRNA transfections, confocal microscopy, quantitative PCR and the application of spectrophotometry to enzymology. Training in western blotting and animal laboratory techniques will shortly be taken.
(ii) Network level
NMR spectroscopy training camp (3rd- 5th November 2014)
Confocal microscopy training (6th-7th November 2014 and 7th-8th May 2015)
Flow cytometry training (6th-7th November 2014)
Respirometry training (5th-6th May 2015)
A presentation at the Parkinson's Association of Ireland is planned. An article will be written in the college's newsletter in collaboration with two other TINTIN fellows based in Dublin.