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Multimodal Imaging of Brain Metastasis-Derived Extracellular Vesicles Using Superparamagnetic Iron Oxide Nanoparticle Labeling.
BACKGROUND: Extracellular vesicles (EVs) are crucial mediators in brain metastasis (BM), facilitating pre-metastatic niche formation and metastatic progression. However, tracking their distribution and interactions in vivo remains challenging. OBJECTIVE: To develop and validate a method for labeling BM-derived EVs using superparamagnetic iron oxide nanoparticles (SPIONs) that enables their visualization and tracking through magnetic resonance imaging (MRI). METHODS: Three SPION variants with different coatings and sizes were evaluated using two patient-derived BM cell lines. The labeled EVs were characterized using transmission electron microscopy (TEM), colorimetric iron assays, dynamic light scattering and nanoparticle tracking analysis (NTA) in Nanospacer devices. The functionality and visualization of SPION-labeled EVs were assessed in fetal rat brain organoids (FRBOs) using Prussian blue staining, TEM, and MRI. Detection sensitivity was determined using agar phantoms, and in vivo tracking was validated through intramuscular injections in mice. RESULTS: Uncoated 5 nm SPIONs demonstrated superior labeling efficiency, successfully marking over 90% of cells within 24 hours without significantly affecting cell growth. These SPIONs were effectively incorporated into BM-derived EVs while maintaining their original size distribution. The labeled EVs were successfully internalized by FRBOs and could be visualized using multiple imaging modalities. Agar phantom studies revealed significant changes in T2 and T2* relaxation times, which was further confirmed through in vivo MRI following intramuscular injections. CONCLUSION: This study establishes a reliable protocol for labeling BM-derived EVs with SPIONs, enabling their visualization across various biological contexts, from subcellular to tissue levels. This proposed model facilitates a valuable tool for spatially tracking BM-EVs in vivo, identifying specific target cells, and investigating their functional role in metastatic progression.
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The ultrastructure of the developing medial preoptic nucleus in the postnatal rat.
The postnatal maturation of the medial preoptic area (MPOA) of male and female rats was studied by means of light and electron microscopy. In Golgi-Kopsch preparations, bi- and multipolar cells were present at birth in both sexes; the perikarya of most of these cells, as seen with the electron microscope, exhibited an immature appearance characterized by a sparse organelle content. During the next 5-10 days, an increase in cell size and cytoplasmic differentiation occurred in each sex. By 10 days, many of the MPOA neurons displayed an increased organelle density with the most prominent change being a proliferation of rough endoplasmic reticulum. Further neuronal development was also evidenced by a greater frequency of dendritic spines than seen at either birth or 5 days. Lastly, the number of synapses per unit area increased rapidly in each sex during the first two weeks of postnatal life. Thus, a period of increasing cytological maturation and synaptic organization closely paralleled previously demonstrated stages of functional differentiation of gonadotrophin regulatory mechanisms in the rat brain.
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Tubular profiles do not form transendothelial channels through the blood-brain barrier.
The contribution of tubular profiles within the mammalian cerebral endothelium to the formation of transcellular channels was analysed following exposure of the endothelium to native horseradish peroxidase (HRP) dissolved in saline or dimethyl sulphoxide (DMSO) administered intravenously in mice. Within 5-15 min, but not at 30 min to 2 h postinjection, peroxidase-positive extravasations were evident within the parenchyma of the forebrain and brainstem of mice exposed and not exposed to DMSO. The extravasations may be associated with the rupture of interendothelial tight junctions at the level of arterioles as a consequence of the perfusion-fixation process. Ultrastructural inspection of endothelia within and away from areas of peroxidase extravasation revealed the following intraendothelial, peroxidase-positive organelles: presumptive endocytic vesicles, endosomes (a prelysosomal compartment), multivesicular and dense bodies, and tubular profiles. Statistical analysis of the concentration of HRP-labelled presumptive endocytic vesicles, which may coalesce to form tubules, within endothelia from mice injected intravenously with HRP-DMSO compared to mice receiving HRP-saline revealed no significant difference. HRP-positive tubular profiles were blunt-ended, variable in length and width, and appeared free in the cytoplasm or in continuity with dense bodies. Labelled tubules free in the cytoplasm were positioned parallel to the luminal and abluminal plasma membranes and were less frequently oblique or perpendicular to these membranes. Tubular profiles analysed in serial thin sections or with a goniometer tilt stage did not establish membrane continuities with the luminal and abluminal plasma membranes. Peroxidase-positive tubular profiles were similar morphologically to those exhibiting acid hydrolase activity but did not share morphological and enzyme cytochemical similarities with the endoplasmic reticulum that stained for glucose-6-phosphatase (G6Pase) activity. G6Pase-positive profiles of endoplasmic reticulum were not observed to contribute to a transendothelial canalicular network. Our results suggest that: (i) peroxidase-labelled tubules, acid hydrolase-positive tubules, and G6Pase-positive endoplasmic reticulum do not form transcellular channels through the cerebral endothelium; (ii) tubular profiles labelled with blood-borne HRP in the cerebral endothelium are associated with the endosome apparatus and/or the lysosomal system of organelles; and (iii) DMSO does not appear to alter the permeability of the blood-brain barrier to blood-borne protein.
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Elucidating the role of DEAF1 in neurodevelopment and shared molecular pathways in high-risk autism genes using cortical organoids.
Neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD) and intellectual disability (ID), are genetically heterogeneous. DEAF1 has emerged as a key NDD risk gene, with pathogenic variants linked to DEAF1-associated neurodevelopmental disorder (DAND), but its role in human neurodevelopment remains unclear. Human cortical organoids (hCOs) provide a physiologically relevant model that recapitulates fetal brain development with an authentic human genetic background. Here, we show that a DEAF1 mutation in human embryonic stem cells disrupts chromatin accessibility at neuronal gene loci, leading to significant transcriptional alterations. In hCOs, this mutation results in aberrant progenitor proliferation, disrupted cortical lamination, and impaired neuronal differentiation. Furthermore, we identify WNT signaling, TGFβ superfamily signaling, and cell cycle regulation as commonly dysregulated pathways across multiple ASD-associated genetic perturbations. Pharmacological inhibition of WNT signaling with a Porcupine inhibitor partially rescues phenotypic defects in DEAF1-mutant hCOs. Our findings identify DEAF1 as a critical regulator of neurodevelopment and support pathway-targeted, mutation-independent therapeutic strategies for ASD and related disorders.
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Quantification of glioma cell invasion by confocal laser scanning microscopy in an in vitro co-culture system.
Malignant invasion in co-cultures of spheroids from the glioma cell line GaMg into brain cell aggregates (BA) was determined by two different techniques: by confocal laser scanning microscopy (CLSM) and by conventional light microscopic observations of semi-thin sections obtained from co-cultures. The remaining BA volumes were detected by CLSM in vital dye-fluorescent-stained co-cultures. The same specimens were fixed and embedded in Epon, and cut for histologic and morphometric analyses. The results show that CLSM can be used for continuous determination of progressive glioma invasion. Compared to the light microscopic observations, the degree of invasion appeared slightly lower when analyzed by CLSM. We conclude that the CLSM provides the possibility for continuous studies on interaction between normal and malignant cells. Therefore it considerably improves existing methods for studying tumor cell invasion in vitro.
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On the mechanism of enhanced release of [14C]glutamate in hippocampal long-term potentiation.
K+-induced release of [14C]glutamate was studied in slices of dentate gyrus prepared from control rats and rats in which long-term potentiation (LTP) had been induced in vivo. At all concentrations of Ca2+ studied, release from potentiated slices was greater than from control slices. In the same preparations both Ruthenium Red and caffeine enhanced basal release but in potentiated tissue the Ruthenium Red-induced release was significantly greater than in control tissue. These results are discussed in the light of our recent finding that enhanced transmitter release is associated with LTP.
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Effects of Sevoflurane on the Development of a Human Brain Microphysiological System.
Animal studies have shown that early life exposure to general anesthetics may impair brain development. However, the implications of this phenomenon in human patients remain unclear. In this study, we use an induced pluripotent stem cell (iPSC)-derived human brain microphysiological system (bMPS) to investigate the effects of early sevoflurane (SEV) exposure on human brain development. Human iPSCs were cultured and differentiated into neural progenitor cells (NPCs) and then into bMPS. At week 8, bMPSs were exposed to 2.4% SEV for 4 h. Four weeks after exposure, immunofluorescence (IF), Western blotting (WB), and quantitative real-time polymerase chain reaction (qPCR) were conducted to evaluate the alteration of nerve cells in bMPS. After SEV exposure, the number of apoptotic cells increases, and the level of neural differentiation markers decreases. The ratios of mature neurons over NPCs and mature oligodendrocytes over oligodendrocyte progenitor cells (OPCs) are reduced, which leads to a reduction in myelination. SEV also impedes the development of astrocytes and synaptogenesis, especially the formation of excitatory synapses. Meanwhile, SEV increases the expression of molecules in the mammalian target of rapamycin (mTOR) signal pathway. In conclusion, early SEV exposure substantially disrupts the development of human brain tissue, and the mTOR signal pathway is likely to be involved in this alteration.
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Assembloid CRISPR screens reveal impact of disease genes in human neurodevelopment.
The assembly of cortical circuits involves the generation and migration of interneurons from the ventral to the dorsal forebrain1-3, which has been challenging to study at inaccessible stages of late gestation and early postnatal human development4. Autism spectrum disorder and other neurodevelopmental disorders (NDDs) have been associated with abnormal cortical interneuron development5, but which of these NDD genes affect interneuron generation and migration, and how they mediate these effects remains unknown. We previously developed a platform to study interneuron development and migration in subpallial organoids and forebrain assembloids6. Here we integrate assembloids with CRISPR screening to investigate the involvement of 425 NDD genes in human interneuron development. The first screen aimed at interneuron generation revealed 13 candidate genes, including CSDE1 and SMAD4. We subsequently conducted an interneuron migration screen in more than 1,000 forebrain assembloids that identified 33 candidate genes, including cytoskeleton-related genes and the endoplasmic reticulum-related gene LNPK. We discovered that, during interneuron migration, the endoplasmic reticulum is displaced along the leading neuronal branch before nuclear translocation. LNPK deletion interfered with this endoplasmic reticulum displacement and resulted in abnormal migration. These results highlight the power of this CRISPR-assembloid platform to systematically map NDD genes onto human development and reveal disease mechanisms.
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ELAVL4, splicing, and glutamatergic dysfunction precede neuron loss in MAPT mutation cerebral organoids.
Frontotemporal dementia (FTD) because of MAPT mutation causes pathological accumulation of tau and glutamatergic cortical neuronal death by unknown mechanisms. We used human induced pluripotent stem cell (iPSC)-derived cerebral organoids expressing tau-V337M and isogenic corrected controls to discover early alterations because of the mutation that precede neurodegeneration. At 2 months, mutant organoids show upregulated expression of MAPT, glutamatergic signaling pathways, and regulators, including the RNA-binding protein ELAVL4, and increased stress granules. Over the following 4 months, mutant organoids accumulate splicing changes, disruption of autophagy function, and build-up of tau and P-tau-S396. By 6 months, tau-V337M organoids show specific loss of glutamatergic neurons as seen in individuals with FTD. Mutant neurons are susceptible to glutamate toxicity, which can be rescued pharmacologically by the PIKFYVE kinase inhibitor apilimod. Our results demonstrate a sequence of events that precede neurodegeneration, revealing molecular pathways associated with glutamate signaling as potential targets for therapeutic intervention in FTD.
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TREX1 is required for microglial cholesterol homeostasis and oligodendrocyte terminal differentiation in human neural assembloids.
Three Prime Repair Exonuclease 1 (TREX1) gene mutations have been associated with Aicardi-Goutières Syndrome (AGS) - a rare, severe pediatric autoimmune disorder that primarily affects the brain and has a poorly understood etiology. Microglia are brain-resident macrophages indispensable for brain development and implicated in multiple neuroinflammatory diseases. However, the role of TREX1 - a DNase that cleaves cytosolic nucleic acids, preventing viral- and autoimmune-related inflammatory responses - in microglia biology remains to be elucidated. Here, we leverage a model of human embryonic stem cell (hESC)-derived engineered microglia-like cells, bulk, and single-cell transcriptomics, optical and transmission electron microscopy, and three-month-old assembloids composed of microglia and oligodendrocyte-containing organoids to interrogate TREX1 functions in human microglia. Our analyses suggest that TREX1 influences cholesterol metabolism, leading to an active microglial morphology with increased phagocytosis in the absence of TREX1. Notably, regulating cholesterol metabolism with an HMG-CoA reductase inhibitor, FDA-approved atorvastatin, rescues these microglial phenotypes. Functionally, TREX1 in microglia is necessary for the transition from gliogenic intermediate progenitors known as pre-oligodendrocyte precursor cells (pre-OPCs) to precursors of the oligodendrocyte lineage known as OPCs, impairing oligodendrogenesis in favor of astrogliogenesis in human assembloids. Together, these results suggest routes for therapeutic intervention in pathologies such as AGS based on microglia-specific molecular and cellular mechanisms.