Distinct Ultrastructural Phenotypes of Glial and Neuronal Alpha-Synuclein Inclusions in Multiple System Atrophy
Different Ultrastructural Phenotypes of α-Synuclein Inclusions in Glia and Neurons in Brain Regions of Multiple System Atrophy (MSA)
Introduction
Multiple System Atrophy (MSA) is a severe neurodegenerative disease characterized by abnormal accumulation of α-Synuclein (α-Syn) in specific brain regions, forming pathological inclusions that primarily occur in the cytoplasm of glial cells (especially oligodendrocytes), known as Glial Cytoplasmic Inclusions (GCIs). This differentiates it from other diseases characterized by abnormal α-Syn such as Parkinson’s Disease and Dementia with Lewy Bodies. These affected regions mainly include substantia nigra and putamen. However, the mechanisms behind the formation of these inclusions remain poorly understood.
Research Background
This study was conducted by researchers from the University of Basel, the Lausanne Federal Institute of Technology, the Vrije University Medical Center Amsterdam, and other institutions. It was published in the journal Brain in 2024. The researchers include Carolyn Böing, Marta di Fabrizio, Domenic Burger, John G.J.M. Bol, Evelien Huisman, Annemieke J.M. Rozemuller, Wilma D.J. van de Berg, Henning Stahlberg, and Amanda J. Lewis.
Research Methods
The research team used Correlative Light and Electron Microscopy (CLEM) to study the α-Syn pathological changes in the substantia nigra and putamen of eight brain tissue donors (all MSA-Parkinsonian variant cases). The tissue slices varied in thickness from 15 μm to 60 μm. This technique, combining optical and electron microscopy, allowed researchers to observe the pathological accumulation of α-Syn at a detailed cellular level.
Post-mortem Brain Sample Preparation
The eight donors included in the study provided explicit written consent allowing brain autopsy and the use of brain tissue for research. All samples were processed in strict adherence to ethical and legal regulations and were approved by the Institutional Review Committee of the Vrije University Medical Center. Brain samples were fixed in 4% formaldehyde for 24 hours, followed by further processing and electron microscopy imaging.
Correlative Light and Electron Microscopy (CLEM) Procedure
To study the appearance and morphology of α-Syn inclusions in different cell types in greater detail, researchers observed tissue slices under optical microscopy at up to 400x and 630x magnification and analyzed critical areas with electron microscopy. Specific pathological structures, labeled with α-Syn antibodies under optical microscopy, were further subjected to electron microscopy using CLEM to ensure that the structures observed were represented in the corresponding locations in both optical and electron imaging.
Research Results
Glial Cytoplasmic Inclusions (GCIs)
The research team observed 196 α-Syn-immunopositive inclusions, primarily occurring in the substantia nigra and putamen of the eight MSA patients. Through high-quality imaging, researchers found 128 fibrous cytoplasmic inclusions (GCIs) in oligodendrocytes. The ultrastructure of these inclusions showed long, non-branching fiber bundles mixed to varying degrees with amorphous proteins, cellular vesicles, and organelles, including mitochondria. Additionally, the study found several GCIs mixed with autophagosomes, lysosomes, and peroxisomes, providing experimental evidence for the role of the autophagy pathway in α-Syn accumulation in the course of MSA.
Neuronal Cytoplasmic Inclusions (NCIs)
In comparison to GCIs, the 20 NCIs found in neurons exhibited significant heterogeneity in ultrastructure, including both fibrous and membranous inclusions. In some inclusions, mitochondria clustered around the inclusions, consistent with recent findings in Parkinson’s Disease, but no nuclear fibers were observed.
Dark Cells
Additionally, in five patients, researchers identified 47 uniquely shaped “dark cells,” characterized by a noticeably deformed nucleus and dense nucleoplasm and cytoplasm. Two types of α-Syn aggregation were found in these cells: one similar to fibrous inclusions in GCIs, and another without apparent ultrastructure but containing large amounts of proteinaceous material and membrane fragments.
Conclusion and Significance
This study revealed the ultrastructural heterogeneity of GCIs, NCIs, and “dark cells” in MSA. This heterogeneity not only uncovers multiple mechanisms of α-Syn accumulation and transmission in different cell types but also suggests complex interactions among these structures. These findings offer new perspectives on the transmission and aggregation of α-Syn, highlighting that α-Syn may exhibit different forms and mechanisms in various cell types and stages of disease.
Highlights of the Research
First Discovery: The study first reveals the presence of α-Syn immunopositive “dark cells” in the brain of MSA patients and their potential pathological significance.
Multilayered Structural Features: Through CLEM, the research meticulously uncovers the multilayered structural features of α-Syn inclusions across different cell types (including oligodendrocytes and neurons) in MSA.
Involvement of the Autophagy Pathway: Observations of autophagy-related lysosomes and peroxisomes present in several GCIs in the MSA brain further endorse the key role of the autophagy-lysosome pathway in these diseases.
Outlook
Future research should continue to explore the cell-specific and stage-specific pathways of α-Syn aggregation through more comprehensive ultrastructure analysis, while further validating the intercellular transmission mechanisms of α-Syn through experimental models. This is not only significant for understanding the pathogenesis of MSA, but it may also provide new avenues for early diagnosis and intervention in α-Syn-associated diseases.