ESR3: The effect of glutathione on mitochondrial dynamics & function in the dopamine neuron
Natalie Adlesic (Trinity College Dublin)
Supervisor: Gavin Davey
Mitochondria are found throughout the body and their main function is to provide cells with energy. A growing body of evidence points to mitochondrial dysfunction as being a major contributor to neurodegenerative diseases such as Parkinson's disease (PD). PD is characterized by loss of synapses, neuronal cell death and protein inclusions, which mainly consist of the pre- synaptic protein α-Synuclein (α-Syn). This pathology predominantly occurs in dopaminergic neurons of the substantia nigra (SN) and later progresses to other parts of the brain. Three mutations in α-Syn, A53T, A30P and E46K, have been linked to familial PD suggesting a key role for α-Syn in the disease progression. The main aim of the project is to investigate a possible link between mitochondrial dysfunction, loss of synapses, α-Syn and reactive oxygen species (ROS). To do this, the project will start off by isolating synaptosomes and pure synaptic (SM) and non- synaptic mitochondria (NSM) from rat brains and study them for differential effects of toxins and α-Syn. The project aims to study oxidative stress in the brain by comparing generation of ROS by mitochondria located in the synapse to those found elsewhere in the brain and to expose any differences that may contribute to specific neurodegeneration in the SN and more specifically in the synapse. It will further move on to looking at dopaminergic neurons in particular and investigate what role glycosylation plays in these neurons and more specifically on ROS production.
The specific aims of the project are:
To investigate ROS production in synaptic vs non-synaptic mitochondria under different conditions
Synaptosomes and isolated synaptic and non-synaptic mitochondria were purified from whole rat brain homogenate and treated with different inhibitors to investigate how ROS production differed between in situ mitochondria in synaptosomes and isolated mitochondria as well as between synaptic and non-synaptic mitochondria. ROS production can be studied in isolated mitochondria by feeding them specific substrates, which make them respire through different complexes of the electron transport chain. It is then possible to use inhibitors of the specific complexes to study ROS production from particular places in the mitochondria. Since synapses are vulnerable in PD it is of interest to understand why and one possible mechanism is increased sensitivity to mitochondrial dysfunction. Synaptic mitochondria have previously ben found to be more vulnerable to complex I inhibition and complex I deficiencies are common in PD. It is therefore important to elucidate whether ROS production differs in the different types of mitochondria and whether this may contribute to the damage and vulnerability seen in synapses. The project has found that there are no major differences in synaptic and non-synaptic mitochondria during resting respiration however synaptic mitochondria appear to have a higher ROS production when they are compromised during active respiration suggesting a higher vulnerability to mitochondrial dysfunction while active.
To investigate α-Syn's effect on mitochondrial ROS production and the electron transport chain
The effect of the protein α-Syn on brain mitochondria has been studied before however any differential toxicity between synaptic and non-synaptic mitochondria has never been studied. Here the aim is to compare any toxicity on the individual protein complexes of the electron transport chain by α-Syn as it is a pre-synaptic protein that would mostly come into contact with synaptic mitochondria and perhaps thereby cause mitochondrial dysfunction. α-Syn toxicity was also measured on intact synaptosomes and intact isolated synaptic and non-synaptic mitochondria to assess potential increase in ROS production and changes in membrane potential. α-Syn significantly lowered the activity of complex I, complex II/III and complex IV of the electron transport chain with a preferential inhibition of complex I however there appeared to be no major differences between synaptic and non-synaptic mitochondria and the inhibition was not time dependent. It did not appear to have any effect on ROS production in synaptosomes or intact synaptic and non-synaptic mitochondria.
To investigate glycosylation in dopaminergic neurons and on α-Syn
Preliminary data has shown that keeping proteins glycosylated by inhibiting the enzyme that removes glycans from proteins leads to decreased ROS production. Similarly, inhibiting glycosylation of proteins causes an increase in ROS production in synaptosomes. The mechanism behind this is unclear and the effect of glycosylation of proteins in the brain is far from understood however remains under investigation for novel therapeutic pathways. The effect of glycosylation on dopaminergic cells will here be studied by knocking down and upregulating key enzymes responsible for glycosylation and studying the effect this has on cell morphology and function and more specifically on mitochondrial dynamics and function. α-Syn is also known to be glycosylated and it is therefore also of great interest to study the effect increased/decreased glycosylation has on α-Syn function in the dopaminergic neuron.
(i) Local level
A LAST-Ireland course was completed to obtain qualifications for animal handling.
• Organisation and Time Management — Organised by Student Learning Development at Trinity College Dublin. Attended on 20th January 2015.
• Public Speaking Skills Workshop — Organised by Student Learning Development at Trinity College Dublin. Attended on 21st January 2015.
• Thesis Writing Online Workshop — Organised by Student Learning Development at Trinity College Dublin.
(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)
- • Two oral presentations were given at Lund University, Sweden, on 21/10/2015 to undergraduate students and masters students in the Biomedicine programme to inform about Marie Curie Actions and specifically TINTIN.
- • A short interview with the communications officer for the Faculty of Medicine at Lund University, Sweden, on 6/2/2016 about Marie Curie Actions PhD programmes and specifically our TINTIN project. The interview will be made public on the Lund University webpage.