AbstractIntroduction: Parkinson’s disease (PD) is the second most common neurodegenerative disorder in the world1. Whilst the majority of PD patients have no known cause of the disease (‘sporadic’), around 5-10% of PD patients have genetic, familial forms of the disease2. LRRK2 and PINK1 are two independent genes that respectively cause autosomal dominant and autosomal recessive forms of PD. They both encode protein kinases, which control signal transduction pathways through reversible phosphorylation of target proteins3. Current research aims to better understand and visualise the activation of both proteins and their associated pathways, to help uncover their role in PD pathogenesis.
Aims: The work detailed within this thesis is divided into two main projects ‘A’ and ‘B’, which respectively concern experiments I conducted in the LRRK2 and PINK1 signal transduction pathways.
The main aim of Project A was to explore the activation of LRRK2 during infection of immune cells, in which I specifically focused on human peripheral blood neutrophils and mouse bone marrow derived macrophages (BMDMs). All infection experiments were conducted using P. aeruginosa, a Gram-negative opportunistic bacterium. In addition, I aimed to explore LRRK2 activation within neutrophils isolated from the sputum samples of cystic fibrosis (CF) patients, who are frequently infected with strains of P. aeruginosa.
The main aim of Project B was to investigate if activation of the PINK1/Parkin pathway could be detected within peripheral blood neutrophils, which would provide a quickly accessible and valuable human bio-source to interrogate PINK1 activity.
Methods: For Project A, I directly isolated peripheral blood neutrophils from whole blood donated by healthy volunteers, and extracted BMDMs from the femurs of sacrificed mice. I subsequently cultured and infected both cell types with standardised (PAO1) or uncharacterised clinical isolate strains of P. aeruginosa, over a time-course of 0-4 hours. In subsequent immunoblot analysis of cell lysates, I measured LRRK2 activity through use of the highly specific MJFF-pRab10 antibody, which detects Rab10 phosphorylated by LRRK2 at Thr73. I also directly isolated neutrophils from the sputum of CF patients with chronic P. aeruginosa infection, and similarly analysed them for LRRK2 activity, using the MJFF-pRab10 antibody.
For Project B, I isolated peripheral blood neutrophils and treated them with the mitochondrial uncoupler CCCP for durations of 3-20 hours, and analysed neutrophil lysates for PINK1 stabilisation through immunoblotting with the PINK1 (Novus) antibody. I also conducted PINK1 immuno-precipitation (IP) experiments to confirm the presence of endogenous PINK1 in peripheral blood neutrophils.
Results: My results in Project A revealed an increase in LRRK2-dependent Rab10 phosphorylation during infection of human peripheral blood neutrophils with P. aeruginosa infection. Furthermore, sputum neutrophils isolated from CF patients revealed markedly elevated levels of LRRK2-dependent Rab10 phosphorylation compared to controls, suggesting that LRRK2 activity may play an important role in human neutrophils during infection. In contrast, infection of mouse BMDMs with P. aeruginosa resulted in a progressive increase in LRRK2 Ser935 phosphorylation, but did not result in any observable LRRK2-dependent Rab10 phosphorylation. Further investigation into LRRK2 Ser935 phosphorylation in BMDMs following P. aeruginosa infection was shown to be mediated by the IKK family of kinases.
My results in Project B revealed that endogenous PINK1 could not be defected in stimulated peripheral blood neutrophils with CCCP between 3-20 hour time-courses.
Conclusions: The results I obtained in Project A collectively indicated that the LRRK2 kinase is influenced by P. aeruginosa infection, of which key differences in its activation and phosphorylation of serine residues exist depending on immune cell type and human/mouse species. My results support future work which aims to explore the mechanisms behind P. aeruginosa infection and LRRK2 activity. Furthermore, my preliminary findings of LRRK2 activation within sputum neutrophils of CF patients with P. aeruginosa infection provides the basis for a future clinical study to assess the natural course of LRRK2 activation during CF infective exacerbations.
Whilst my results in Project B did not demonstrate that PINK1 could be activated within peripheral blood neutrophils, future work could consider the use of different mitochondrial uncouplers, fractionation experiments, or more sensitive antibody readouts of the PINK1/Parkin pathway, to validate the current findings.
|Date of Award||2019|
|Supervisor||Dario Alessi (Supervisor) & Miratul Muqit (Supervisor)|
- Parkinson's disease
- Immune system
- Pseudomonas aeruginosa
- Protein kinases
- Rab biology