Unveiling Parkinson's Disease Through The Microscopic Lens
Parkinson’s disease (PD) is a neurodegenerative disorder that affects millions of people worldwide. This disease is caused by the loss of neurons in a specific area of the brain called Substantia nigra pars compact, which leads to a deficit in dopamine (neurotransmitter) production. Due to the lack of dopamine, motor symptoms are commonly associated with PD, such as tremors, rigidity, and slowness of movement (bradykinesia). However, today, PD is associated with a broad spectrum of non-motor symptoms as well. These include depression, cognitive dysfunction, hallucinations, hyposmia, and rapid eye movement sleep behavior disorder (RBD). Besides the loss of dopaminergic neurons, surviving neurons present abnormal protein clumps, known as Lewy bodies (LBs), and they are mainly composed of alpha-synuclein (aSyn). In my work, I have been seeking to understand how aSyn and other proteins form these protein clumps within the cells.
Early in my career, I developed a strong interest in microscopy. Whenever I step into the microscope room, it never fails to ignite my curiosity. However, epifluorescence microscopes have limited resolution, making it difficult to study processes that occur at the nanoscopic level. More advanced types of microscopes, like confocal or even Stimulated Emission Depletion (STED) microscopes, exist, but they are not always widely available and require specialized training. Not to mention, they are expensive equipment. A groundbreaking imaging technique developed by Dr. Edward Boyden and his team at MIT (Chen F et al. Science, 2015) offers a simple but revolutionary solution to overcome these resolution limitations. The resolution is achieved by embedding a biological specimen in a swellable polymer and subsequently expanding it. This new super-resolution technique is called Expansion Microscopy (ExM), and it provides nanoscale precision while preserving biological information. Furthermore, it does not require intense computational processing like other super-resolution techniques.
At the WPC 2023 in Barcelona, Spain, I presented a collaborative study where we used the X10 ExM protocol (Truckenbrod S et al. EMBO reports, 2018) to investigate the molecular architecture of aSyn in cells and human brain tissue. Using well-established cellular models, we have observed that aSyn can accumulate in assemblies with different morphologies, ranging from nanoscale clusters to ring- or even tubular-shaped structures. We have identified that these aSyn ring-like structures colocalize with mitochondria and not with lysosomes. Our results align with previous observations where the authors of that study, using STED microscopy to study the morphology of human LBs, reported that aSyn localizes with mitochondria (Moors TE et al. Acta Neuropathol, 2021). These findings not only validate our cellular model as a useful tool to understand aSyn biology in a simplified system but also demonstrate the power of this technique. One of the most frequently asked questions at the WPC meeting was about the application of ExM to human brain tissue. Many attendees mentioned that they have been trying to implement ExM in human brain tissue, but not very successfully. It has taken us a while to develop a protocol that works effectively with aSyn antibodies. We are currently studying the distribution of aSyn and phosphorylated aSyn (a hallmark of PD pathology) within the LBs and Lewy neurites. Our goal is to continue optimizing aSyn antibody protocols and to characterize the distribution and structure not only of aSyn but of other organelles too.
ExM stands as a powerful tool for studying PD. By overcoming the limitations of traditional imaging methods, this innovative approach holds the potential to unravel the intricate details of cellular and molecular changes associated with PD. With this widely accessible technique, the examination of aSyn aggregation will facilitate a deeper understanding of their formation and distribution within the brain, ultimately paving the way for more targeted therapeutic interventions and improved patient outcomes. Further integration of ExM with other advanced technologies, such as single-cell RNA sequencing, could significantly enhance our understanding of the molecular alterations occurring in PD and potentially lead to the identification of novel therapeutic targets and treatment strategies.
Diana Lázaro, PhD rer. nat. She is a postdoctoral researcher at the Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania. She was a poster presenter at the WPC 2023.
𝕏: @df_lazaro
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