MRC DiMeN Doctoral Training Partnership: Investigating α-synuclein aggregation in microglia using human microglia transplanted into the mouse brain and cryo-electron microscopy

Background 

Traditional cell culture models fail to mimic human microglial physiology 1. In contrast, human microglia transplanted into mouse brains retain their human identity, and more closely resemble ex vivo human microglia, providing a physiologically-relevant model for assessing microglial dysfunction in neurodegeneration 2,3.  

α-synuclein aggregation is central to Parkinson’s disease. The A53T mutation in α-synuclein, linked to early-onset Parkinson’s, accelerates its aggregation 4. In a recent study, Dr. Krzisch transplanted A53T-mutant human microglia in a young, healthy mouse brain and found cell-autonomous increased pro-inflammatory activation upon inflammatory stimulus (lipopolysaccharide) compared to isogenic controls 5.  

While α-synuclein expression was similar in both A53T-mutant and control microglia 5, it remains unclear whether mutant α-synuclein forms aggregates within A53T-mutant microglia and whether these aggregates drive their hyperactivation. Another question is how the A53T mutation affects the internalization and response of human microglia to alpha-synuclein aggregates. 

Objectives 

Combining the microglia transplantation model with cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET), we will study α-synuclein aggregation in A53T-mutant human microglia and their internalization of alpha-synuclein aggregates.   

Novelty and timeliness 

This project will provide mechanistic insights on the impact of the A53T mutation in α-synuclein on microglial function, and better our understanding of how human microglia internalize α-synuclein aggregates. While many studies have shown that extracellular α-synuclein aggregates get internalized and activate microglia 6, few have used human microglia, and none have used transplanted human microglia. Many recent studies have suggested a pivotal role of microglia in neurodegeneration 7. This study will further our understanding of the impact of alpha-synuclein aggregation on microglial function using cutting-edge technologies.  

Experimental approach 

The student will first use non-denaturing gel electrophoresis to detect α-synuclein aggregates in A53T-mutant and isogenic control human microglia, both transplanted and cultured. Next, the student will collaborate with Dr. Rene Frank’s laboratory to investigate the subcellular localization of these aggregates in transplanted microglia using super-resolution fluorescence microscopy and cryoEM. These experiments will be performed with or without inflammatory stimulus (lipopolysaccharide).  

In parallel, the student will inject α-synuclein aggregates from Parkinson’s patients’ brains in the substantia nigra of mice transplanted with A53T-mutant or isogenic control human microglia and assess their effect on microglial function using immunofluorescence and functional assays. They will characterize their uptake by transplanted microglia using non-denaturing gel electrophoresis, super-resolution fluorescence microscopy and cryoEM. Finally, they will determine the structure of α-synuclein aggregates within transplanted human microglia using cryoET. 

 This project combines cutting-edge methods in human pluripotent stem cell biology (Krzisch Lab) and cryo-electron microscopy/tomography (Frank Lab) to further our understanding of a disease of ageing, Parkinson’s disease. In this project, the PhD student will use advanced technologies to determine how α-synuclein aggregation impacts human microglia.  This research will open new avenues of research into other neurodegenerative disorders involving protein aggregation, such as Huntington’s and Alzheimer’s. Additionally, mastering these sought-after technologies will enhance the student’s employment prospects in both academia and industry. 

Marine Krzisch’s lab website and social media pages:  

Website: https://biologicalsciences.leeds.ac.uk/biological-sciences/staff/3272/dr-marine-krzisch 

LinkedIn: https://www.linkedin.com/in/marine-krzisch-a193867/ 

X: https://x.com/mkrzisch 

ResearchGate: https://www.researchgate.net/profile/Marine-Krzisch 

Rene Frank’s lab website and social media pages:  

Website: https://sites.google.com/view/renefrankgroup/ren%C3%A9-frank-group 

 X: https://x.com/drrenefrank 

Benefits of being in the DiMeN DTP: 

This project is part of the Discovery Medicine North Doctoral Training Partnership (DiMeN DTP), a diverse community of PhD students across the North of England researching the major health problems facing the world today. Our partner institutions (Universities of Leeds, Liverpool, Newcastle, York and Sheffield) are internationally recognised as centres of research excellence and can offer you access to state-of-the-art facilities to deliver high impact research. 

We are very proud of our student-centred ethos and committed to supporting you throughout your PhD. As part of the DTP, we offer bespoke training in key skills sought after in early career researchers, as well as opportunities to broaden your career horizons in a range of non-academic sectors. 

Being funded by the MRC means you can access additional funding for research placements, training opportunities or internships in science policy, science communication and beyond. Further information on the programme and how to apply can be found on our website: 

https://www.dimen.org.uk/