Super-resolution microscopy informs on the molecular architecture of alpha-synuclein inclusions in model systems and in the human brain

Lewy bodies (LBs) and Lewy neurites are pathological hallmarks of Parkinsons disease and other progressive neurodegenerative disorders known as Lewy body diseases (LBD). These proteinaceous deposits are immunopositive for alpha-synuclein (aSyn) and several other proteins, as neurofilament components. The structural organization and composition of aSyn inclusions is still unclear and needs to be addressed in greater detail, as this may open novel avenues for our understanding of the disease-relevant pathological events. In this study, we investigated the molecular architecture of aSyn inclusions, both in cell models and in human brain tissue, using state-of-art super resolution X10 Expansion microscopy (ExM). This approach physically expands specimens embedded into a swellable gel, preserving their biological information. Then, the specimen can be analyzed using standard epifluorescence microscopes, thereby obtaining nanoscale information. The combination of different cell models, mouse and human brain tissue enabled us to distinguish different types aSyn assemblies (e.g. ring shape or tubular structures), and a conserved pattern of aSyn inclusions surrounded/encaged by intermediate filament proteins. Overall, X10 ExM enabled us to gain insight into the architecture and biology of aSyn inclusions and constitutes a powerful tool in the quest to understanding underlying disease mechanisms in synucleinopathies.

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RBIO-06. IMPLEMENTATION OF HUMAN INDUCED PLURIPOTENT STEM CELL DERIVED CEREBRAL ORGANOIDS TO MODEL NORMAL TISSUE RADIORESPONSE

Neuro-Oncology ◽

10.1093/neuonc/noaa215.806 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii193-ii193

Author(s):

Lawrence Bronk ◽

Sanjay Singh ◽

Riya Thomas ◽

Luke Parkitny ◽

Mirjana Maletic-Savatic ◽

...

Keyword(s):

Stem Cell ◽

Human Brain ◽

Brain Tissue ◽

Human Tissue ◽

Model Systems ◽

Human Brain Tissue ◽

Post Irradiation ◽

Mature Neurons ◽

Induced Pluripotent ◽

Effects Of Radiation

Abstract Treatment-related sequelae following cranial irradiation have life changing impacts for patients and their caregivers. Characterization of the basic response of human brain tissue to irradiation has been difficult due to a lack of preclinical models. The direct study of human brain tissue in vitro is becoming possible due to advances in stem cell biology, neuroscience, and tissue engineering with the development of organoids as novel model systems which enable experimentation with human tissue models. We sought to establish a cerebral organoid (CO) model to study the radioresponse of normal human brain tissue. COs were grown using human induced pluripotent stem cells and a modified Lancaster protocol. Compositional analysis during development of the COs showed expected populations of neurons and glia. We confirmed a population of microglia-like cells within the model positive for the makers Iba1 and CD68. After 2-months of maturation, COs were irradiated to 0, 10, and 20 Gy using a Shepard Mark-II Cs-137 irradiator and returned to culture. Subsets of COs were prepared for immunostaining at 30- and 70-days post-irradiation. To examine the effect of irradiation on the neural stem cell (NSC) population, sections were stained for SOX2 and Ki-67 expression denoting NSCs and proliferation respectively. Slides were imaged and scored using the CellProfiler software package. The percentage of proliferating NSCs 30-days post-irradiation was found to be significantly reduced for irradiated COs (5.7% (P=0.007) and 3.4% (P=0.001) for 10 and 20 Gy respectively) compared to control (12.7%). The reduction in the proliferating NSC population subsequently translated to a reduced population of NeuN-labeled mature neurons 70 days post-irradiation. The loss of proliferating NSCs and subsequent reduction in mature neurons demonstrates the long-term effects of radiation. Our initial results indicate COs will be a valuable model to study the effects of radiation therapy on normal and diseased human tissue.

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Pathogenic LRRK2 Mutations Do Not Alter Gene Expression in Cell Model Systems or Human Brain Tissue

PLoS ONE ◽

10.1371/journal.pone.0022489 ◽

2011 ◽

Vol 6 (7) ◽

pp. e22489 ◽

Cited By ~ 25

Author(s):

Michael J. Devine ◽

Alice Kaganovich ◽

Mina Ryten ◽

Adamantios Mamais ◽

Daniah Trabzuni ◽

...

Keyword(s):

Gene Expression ◽

Human Brain ◽

Brain Tissue ◽

Cell Model ◽

Model Systems ◽

Human Brain Tissue

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TMOD-12. THE ROLE OF INTEGRIN α V AND CD44 IN GBM MIGRATION USING HUMAN ORGANOTYPIC SLICES

Neuro-Oncology ◽

10.1093/neuonc/noz175.1111 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi265-vi265

Author(s):

Zev Binder ◽

Sarah Hyun Ji Kim ◽

Pei-Hsun Wu ◽

Anjil Giri ◽

Gary Gallia ◽

...

Keyword(s):

Human Brain ◽

Cell Motility ◽

Brain Tissue ◽

Brain Slices ◽

Model System ◽

Model Systems ◽

In Vivo Model ◽

Human Brain Tissue

Abstract Current model systems used for GBM research include traditional in vitro cell line-based assays and in vivo animal studies. In vitro model systems offer the advantages of being easy to use, relatively inexpensive, and fast growing. However, these models lack key elements of the pathology they are attempting to model, including the biochemical and biophysical microenvironment and three-dimensional structure inherent to human brain tissue. In vivo model systems address these limitations, but have restrictions of their own. Species differences may result in non-applicable results and animal experiments are often not designed like clinical trials. Evidence of the limitations of current GBM models is found in the disparity between basic research findings and successful new treatments for GBMs in the clinic. Here we present an alternative model system for the study of human GBM cell motility and invasion, which features advantages of both in vitro and in vivo model systems. Using human organotypic brain slices as scaffolding for tumor growth, we explored the dynamic process of GBM cell invasion within human brain tissue. To demonstrate the utility of the model system, we investigated the effects of depletion of integrin α V (ITGAV) and CD44 on GBM cell motility. These two cell-surface proteins have been identified to have key functions in GBM cell motility. However, knockdown of ITGAV had little effect on tumor cell motility in organotypics while CD44 knockdown significantly reduced cell movement. Finally, we compare motility results from cells in human brain slices to those from cells growing on standard Matrigel and in mouse brain organotypics. We found significant differences in motility depending on the substrate in which the cells were moving. Our findings highlight the physiologic characteristics of human brain organotypics and demonstrate the use of real-time imaging in the ex vivo system.

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From Cultured Rodent Neurons to Human Brain Tissue: Model Systems for Pharmacological and Translational Neuroscience

ACS Chemical Neuroscience ◽

10.1021/acschemneuro.8b00098 ◽

2018 ◽

Vol 9 (8) ◽

pp. 1975-1985 ◽

Cited By ~ 5

Author(s):

Joel Wellbourne-Wood ◽

Jean-Yves Chatton

Keyword(s):

Human Brain ◽

Brain Tissue ◽

Model Systems ◽

Human Brain Tissue ◽

Translational Neuroscience ◽

Tissue Model

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Alpha-synuclein is present in the nucleus in human brain tissue and is pathologically modified in Dementia with Lewy Bodies

10.1101/2021.10.20.465125 ◽

2021 ◽

Author(s):

David J Koss ◽

Daniel Erskine ◽

Andrew Porter ◽

Marta Leite ◽

Johannes Attems ◽

...

Keyword(s):

Human Brain ◽

Brain Tissue ◽

Cortical Neurons ◽

Dementia With Lewy Bodies ◽

Neuronal Cells ◽

Lewy Bodies ◽

Disease Process ◽

Alpha Synuclein ◽

Dna Integrity ◽

Human Brain Tissue

Background Dementia with Lewy bodies (DLB) is pathologically-defined by the cytoplasmic accumulation of alpha-synuclein (aSyn) within neuronal cells in the brain. aSyn is predominately pre-synaptic, but has been reported present in various subcellular compartments in cell and animal models. In particular, nuclear aSyn (aSynNuc) is evident in-vitro and in disease models and has been associated with altered DNA integrity, gene transcription, nuclear homeostasis. However, owing to various factors, the presence of aSynNuc in the human brain remains controversial, as does its role in synucleinopathies. Methods Here, we close this gap and provide a unique demonstration confirming the presence of aSynNuc in control (Con) and in DLB post-mortem brain tissue via immunohistochemistry, immunoblot, and label-free mass-spectrometry (MS). Results Discrete intra-nuclear aSyn puncta reactive against phosphorylated serine 129-aSyn (pS129-aSyn) and pan-aSyn antibodies were observed in cortical neurons and non-neuronal cells in fixed brain sections and isolated nuclear preparations from Con and DLB cases. Subsequent biochemical analysis of subcellular fractionated tissue confirmed aSynNuc as present at levels ~10-fold lower than in the cytoplasm. Critically, however, an increase in monomeric pS129-aSyn was observed in DLB cases alongside higher molecular weight pan- and pS129-reactive aSyn species, consistent with the formation of intranuclear phosphorylated aSynNuc oligomers. Furthermore, the occurrence of aSynNuc was confirmed via MS, with 6 unique aSyn derived peptide sequences identified in nuclear fractions (71.4% aSyn sequence coverage). Conclusions Collectively, our data confirm the presence of aSynNuc in human brain tissue and describe DLB-associated nuclear pathology. These findings address a major controversy in the synucleinopathy field by confirming the presence of aSynNuc in autoptic human brain tissue and, for the first time, identify that aSyn is aggregated into novel and potentially pathological assemblies in the nucleus as part of the DLB disease process and thus may contribute to the DLB phenotype.

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