scholarly journals Specific Detection of Endogenous S129 Phosphorylated α-Synuclein in Tissue Using Proximity Ligation Assay

2021 ◽  
Author(s):  
Ryan Arlinghaus ◽  
Michiyo Iba ◽  
Eliezer Masliah ◽  
Mark R Cookson ◽  
Natalie Landeck
Keyword(s):  
Cell Culture ◽  
Human Brain ◽  
Brain Tissue ◽  
Mouse Brain ◽  
Lewy Bodies ◽  
Cross Reactivity ◽  
Proximity Ligation Assay ◽  
Staining Procedure ◽  
Human Brain Tissue ◽  
Proximity Ligation

BACKGROUND: Synucleinopathies are a group of neurodegenerative disorders that are pathologically characterized by the accumulation of protein aggregates called Lewy Bodies. Lewy bodies are primarily composed of α-synuclein (asyn) protein, which is phosphorylated at serine 129 (pS129) when aggregated. Currently available commercial antibodies used to stain for pS129 asyn can cross react with other proteins, thus making it difficult to specifically detect endogenous pS129 asyn and to interpret pS129 asyn staining. OBJECTIVE: To develop a staining procedure that detects pS129 asyn with high specificity and low background. METHODS: We use the fluorescent and brightfield in situ Proximity Ligation Assay (PLA) to specifically detect pS129 asyn in cell culture, mouse brain sections, and fixed human brain tissue. RESULTS: The pS129 asyn PLA specifically stained for endogenous, soluble pS129 asyn in cell culture, mouse brain sections, and human brain tissue without significant cross-reactivity or background signal. CONCLUSIONS: This PLA method can be used to specifically detect pS129 asyn in order to further explore and understand its role in health and disease.

scholarly journals The cross-reactivity of the enterovirus 71 to human brain tissue and identification of the cross-reactivity related fragments

2010 ◽  
Vol 7 (1) ◽  
pp. 47 ◽  
Author(s):  
Chun Jia ◽  
Jiang Liu ◽  
Wan Li ◽  
Chun Ma ◽  
Shu Lin ◽  
...  
Keyword(s):  
Human Brain ◽  
Brain Tissue ◽  
Enterovirus 71 ◽  
Cross Reactivity ◽  
Human Brain Tissue ◽  
The Cross

scholarly journals Immunohistochemical application of monoclonal antibodies against myelin basic protein and neurofilament triple protein subunits: advantages over antisera and technical limitations.

1983 ◽  
Vol 31 (9) ◽  
pp. 1126-1135 ◽  
Author(s):  
W F Hickey ◽  
V Lee ◽  
J Q Trojanowski ◽  
L J McMillan ◽  
T J McKearn ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Brain ◽  
Myelin Basic Protein ◽  
Brain Tissue ◽  
Mercuric Chloride ◽  
Basic Protein ◽  
Histological Study ◽  
Cross Reactivity ◽  
Human Brain Tissue ◽  
Specific Staining

Four monoclonal antibodies against guinea pig myelin basic protein (MBP), and four against subunits of bovine neurofilament triplet proteins (NF) were produced and their activity determined by enzyme-linked immunosorbant assay. The specificity and cross-reactivity of these eight monoclonal antibodies and one heterologous antiserum against each of the two central nervous system (CNS) antigens were examined in a histological study using the immunoperoxidase, antibody sandwich technique in rat and human brain tissue. Tissue sections were prepared from paraffin-embedded or fresh brain tissue that had been fixed with one of five different fixatives. The resulting immunoperoxidase labeling was then graded for intensity and examined for artifacts. One monoclonal antibody against MBP and one against NF resulted in labeling that was superior to that given by each of the antisera against their respective antigens. Of the five fixatives tested, a mercuric chloride-formalin solution gave the best preservation of these two antigens in rat and human brain tissue. The mercuric chloride-formalin solution was found to be superior to the other fixatives when immersion fixation was used, and was especially optimal when brains were perfused fixed. Three artifacts were encountered among the various antibody-fixative combinations that produced erroneous, but seemingly specific staining of Purkinje cells, neurons and axons, or astrocytes.

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

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.

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

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.

Demonstration of α1A-and α1B-adrenoceptor binding sites in human brain tissue

1989 ◽  
Vol 169 (2-3) ◽  
pp. 325-328 ◽  
Author(s):  
Gerhard Gross ◽  
Gertraud Hanft ◽  
Hubertus M. Mehdorn
Keyword(s):  
Human Brain ◽  
Brain Tissue ◽  
Binding Sites ◽  
Human Brain Tissue

The lipopigments in human brain tissue necroses

1980 ◽  
Vol 52 (2) ◽  
pp. 147-151 ◽  
Author(s):  
R. Schr�der ◽  
B. Reinartz
Keyword(s):  
Human Brain ◽  
Brain Tissue ◽  
Human Brain Tissue
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