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Evidence that coated vesicles isolated from brain are calcium-sequestering organelles resembling sarcoplasmic reticulum.
Coated vesicles from the brain have been purified to near morphological homogeneity by a modification of the method of Pearse. These vesicles resemble sarcoplasmic reticulum fragments isolated from skeletal muscle. They contain proteins with 100,000- and 55,000-dalton mol wt which co-migrate on polyacrylamide gels, in the presence of sodium dodecyl sulfate, with the two major proteins of the sarcoplasmic reticulum fragment. These vesicles contain adenosine triphosphatase (ATPase) activity which is stimulated by calcium ions in the presence of Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.), displaying maximal activity at 8 x 10(-7) M Ca ++. They take up calcium ions from the medium, and this uptake is stimulated by ATP and by potassium oxalate, a calcium-trapping agent. The 100,000-dalton protein of the coated vesicles displays immunological reactivity with an antiserum directed against the 100,000-dalton, calcium-stimulated ATPase of the sarcoplasmic reticulum. As with the sarcoplasmic reticulum fragment, this protein becomes radiolabeled when coated vesicles are briefly incubated with gamma-labeled [32P]ATP. The possible functions of coated vesicles as calcium-sequestering organelles are discussed.
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[Infectious activity of cell fractions isolated from the brain of mice with scrapie].
The agent of scrapie was found to induce clinically similar diseases in BALB/c and NIH mice but differing in the duration of the incubation period (155-180 days and 110-140 days, respectively) and pathomorphological manifestations. Differentiated examinations of a brain cell pool from mice with scrapie revealed more infectious activity not neutralized with nucleases and pronasa in membrane-free cell fractions than in fractions of slowly sedimenting membranes.
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Protocol for generating human cerebral organoids from two-dimensional cultures of pluripotent stem cells bypassing embryoid body aggregation.
Human cerebral organoids (hCOs) provide an excellent model for the study of human brain development and disease. Here, we present a protocol to obtain hCOs directly from two-dimensional (2D) pluripotent stem cell (PSC) cultures, avoiding cell dissociation and posterior embryoid body (EB) aggregation. We describe steps for subjecting 2D cultures to a neural fate and subsequently developing hCOs. We then detail the evaluation of different cellular types. For complete details on the use and execution of this protocol, please refer to González-Sastre et al.1.
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The anuran Mauthner cell and its synaptic bed.
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Isolation and characterization of endothelial cells from rat and cow brain white matter.
A procedure is described for obtaining pure cultures of endothelial cells from rat and cow brain white matter. The morphology of the tissue-cultured cells was studied by both light and transmission electron microscopy. Weibel-Palade bodies, described in the literature as specific for endothelial cells, appeared in small numbers in our cultured cells. Autoradiographic, scintillation counting, immunological and histochemical studies were performed. The usefulness of pure endothelial cell cultures is discussed.
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Single cell transcriptomics reveals distinct transcriptional responses to oxycodone and buprenorphine by iPSC-derived brain organoids from patients with opioid use disorder.
The opioid epidemic represents a national crisis. Oxycodone is one of the most prescribed opioid medications in the United States, whereas buprenorphine is currently the most prescribed medication for opioid use disorder (OUD) pharmacotherapy. Given the extensive use of prescription opioids and the global opioid epidemic, it is essential to understand how opioids modulate brain cell type function at the single-cell level. We performed single nucleus RNA-seq (snRNA-seq) using iPSC-derived forebrain organoids from three male OUD subjects in response to oxycodone, buprenorphine, or vehicle for seven days. We utilized the snRNA-seq data to identify differentially expressed genes following drug treatment using the Seurat integrative analysis pipeline. We utilized iPSC-derived forebrain organoids and single-cell sequencing technology as an unbiased tool to study cell-type-specific and drug-specific transcriptional responses. After quality control filtering, we analyzed 25787 cells and identified sixteen clusters using unsupervised clustering analysis. Our results reveal distinct transcriptional responses to oxycodone and buprenorphine by iPSC-derived brain organoids from patients with OUD. Specifically, buprenorphine displayed a significant influence on transcription regulation in glial cells. However, oxycodone induced type I interferon signaling in many cell types, including neural cells in brain organoids. Finally, we demonstrate that oxycodone, but not buprenorphine activated STAT1 and induced the type I interferon signaling in patients with OUD. These data suggest that elevation of STAT1 expression associated with OUD might play a role in transcriptional regulation in response to oxycodone. In summary, our results provide novel mechanistic insight into drug action at single-cell resolution.
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Identification of minor components of coated vesicles by use of permeation chromatography.
Coated vesicles are thought to be vehicles for the intracellular transport of membranes. Clathrin is the major protein component of coated vesicles. Minor components of these organelles can be identified in highly purified preparations if they can be shown to copurify with clathrin. To show copurification we have made use of the relatively uniform diameter of coated vesicles (50-150 nm) to fractionate conventionally purified coated vesicles according to size in glass bead columns of 200-nm pore size. We have found that bovine brain coated vesicles prepared by the standard procedure of Pearse can be contaminated with large membrane fragments that are removed by permeation chromatography on such glass bead columns. Gel electrophoretic analysis of column fractions shows that only three major polypeptide chains, and a family of polypeptides with molecular weights close to 100,000 are always in constant ratio to clathrin, and are unique to fractions containing coated vesicles. Two other major polypeptides that appear to be components of coated vesicles are also present in other membrane fractions. We have also used permeation chromatography to monitor artifactual membrane trapping during vesicle isolation. Pure radiolabeled synaptic vesicle membranes were added to bovine brain tissue before homogenization. Considerable amounts of the added radioactivity could be recovered in the fractions conventionally pooled in the preparation of coated vesicles. After permeation chromatography, the radioactivity in the coated vesicle peak was reduced essentially to background.
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Three-dimensional molecular cartography of human cerebral organoids revealed by double-barcoded spatial transcriptomics.
Spatially resolved transcriptomics is revolutionizing our understanding of complex tissues, but scaling these approaches to multiple tissue sections and three-dimensional tissue reconstruction remains challenging and cost prohibitive. In this work, we present a low-cost strategy for manufacturing molecularly double-barcoded DNA arrays, enabling large-scale spatially resolved transcriptomics studies. We applied this technique to spatially resolve gene expression in several human brain organoids, including the reconstruction of a three-dimensional view from multiple consecutive sections, revealing gene expression heterogeneity throughout the tissue.
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Recent advances in cell-based in vitro models for predicting drug permeability across brain, intestinal, and pulmonary barriers.
INTRODUCTION: Recent years have witnessed remarkable progress in the development of cell-based in vitro models aimed at predicting drug permeability, particularly focusing on replicating the barrier properties of the blood-brain barrier (BBB), intestinal epithelium, and lung epithelium. AREA COVERED: This review provides an overview of 2D in vitro platforms, including monocultures and co-culture systems, highlighting their respective advantages and limitations. Additionally, it discusses tools and techniques utilized to overcome these limitations, paving the way for more accurate predictions of drug permeability. Furthermore, this review delves into emerging technologies, particularly microphysiological systems (MPS), encompassing static platforms such as organoids and dynamic platforms like microfluidic devices. Literature searches were performed using PubMed and Google Scholar. We focus on key terms such as in vitro permeability models, MPS, organoids, intestine, BBB, and lungs. EXPERT OPINION: The potential of these MPS to mimic physiological conditions more closely offers promising avenues for drug permeability assessment. However, transitioning these advanced models from bench to industry requires rigorous validation against regulatory standards. Thus, there is a pressing need to validate MPS to industry and regulatory agency standards to exploit their potential in drug permeability prediction fully. This review underscores the importance of such validation processes to facilitate the translation of these innovative technologies into routine pharmaceutical practice.
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Citizen and Patient Participation in Precision Medicine: Epilepsy Treatment Using Brain Organoids Derived from iPS Cells.