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Necrotizing enterocolitis induces T lymphocyte-mediated injury in the developing mammalian brain.
Necrotizing enterocolitis (NEC) causes acute intestinal necrosis in premature infants and is associated with severe neurological impairment. In NEC, Toll-like receptor 4 is activated in the intestinal epithelium, and NEC-associated brain injury is characterized by microglial activation and white matter loss through mechanisms that remain unclear. We now show that the brains of mice and humans with NEC contained CD4+ T lymphocytes that were required for the development of brain injury. Inhibition of T lymphocyte influx into the brains of neonatal mice with NEC reduced inflammation and prevented myelin loss. Adoptive intracerebroventricular delivery of gut T lymphocytes from mice with NEC into Rag1 -/- recipient mice lacking CD4+ T cells resulted in brain injury. Brain organoids derived from mice with or without NEC and from human neuronal progenitor cells revealed that IFN-γ release by CD4+ T lymphocytes induced microglial activation and myelin loss in the organoids. IFN-γ knockdown in CD4+ T cells derived from mice with NEC abrogated the induction of NEC-associated brain injury after adoptive transfer to naïve Rag1 -/- recipient mice. T cell receptor sequencing revealed that NEC mouse brain-derived T lymphocytes shared homology with gut T lymphocytes from NEC mice. Intraperitoneal injection of NEC gut-derived CD4+ T lymphocytes into naïve Rag1 -/- recipient mice induced brain injury, suggesting that gut-derived T lymphocytes could mediate neuroinflammation in NEC. These findings indicate that NEC-associated brain injury may be induced by gut-derived IFN-γ-releasing CD4+ T cells, suggesting that early management of intestinal inflammation in children with NEC could improve neurological outcomes.
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A minimal-complexity light-sheet microscope maps network activity in 3D neuronal systems.
In vitro systems mimicking brain regions, brain organoids, are revolutionizing the neuroscience field. However, characterization of their electrical activity has remained a challenge as it requires readout at millisecond timescale in 3D at single-neuron resolution. While custom-built microscopes used with genetically encoded sensors are now opening this door, a full 3D characterization of organoid neural activity has not been performed yet, limited by the combined complexity of the optical and the biological system. Here, we introduce an accessible minimalistic light-sheet microscope to the neuroscience community. Designed as an add-on to a standard inverted microscope it can be assembled within one day. In contrast to existing simplistic setups, our platform is suited to record volumetric calcium traces. We successfully extracted 4D calcium traces at high temporal resolution by using a lightweight piezo stage to allow for 5 Hz volumetric scanning combined with a processing pipeline for true 3D neuronal trace segmentation. As a proof of principle, we created a 3D connectivity map of a stem cell derived neuron spheroid by imaging its activity. Our fast, low complexity setup empowers researchers to study the formation of neuronal networks in vitro for fundamental and neurodegeneration research.
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Cilium induction triggers differentiation of glioma stem cells.
Glioblastoma multiforme (GBM) possesses glioma stem cells (GSCs) that promote self-renewal, tumor propagation, and relapse. Understanding the mechanisms of GSCs self-renewal can offer targeted therapeutic interventions. However, insufficient knowledge of GSCs' fundamental biology is a significant bottleneck hindering these efforts. Here, we show that patient-derived GSCs recruit elevated levels of proteins that ensure the temporal cilium disassembly, leading to suppressed ciliogenesis. Depleting the cilia disassembly complex components is sufficient to induce ciliogenesis in a subset of GSCs via relocating platelet-derived growth factor receptor-alpha (PDGFR-α) to a newly induced cilium. Importantly, restoring ciliogenesis enabled GSCs to switch from self-renewal to differentiation. Finally, using an organoid-based glioma invasion assay and brain xenografts in mice, we establish that ciliogenesis-induced differentiation can prevent the infiltration of GSCs into the brain. Our findings illustrate a role for cilium as a molecular switch in determining GSCs' fate and suggest cilium induction as an attractive strategy to intervene in GSCs proliferation.
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Playing Brains: The Ethical Challenges Posed by Silicon Sentience and Hybrid Intelligence in DishBrain.
The convergence of human and artificial intelligence is currently receiving considerable scholarly attention. Much debate about the resulting Hybrid Minds focuses on the integration of artificial intelligence into the human brain through intelligent brain-computer interfaces as they enter clinical use. In this contribution we discuss a complementary development: the integration of a functional in vitro network of human neurons into an in silico computing environment.To do so, we draw on a recent experiment reporting the creation of silico-biological intelligence as a case study (Kagan et al., 2022b). In this experiment, multielectrode arrays were plated with stem cell-derived human neurons, creating a system which the authors call DishBrain. By embedding the system into a virtual game-world, neural clusters were able to receive electrical input signals from the game-world and to respond appropriately with output signals from pre-assigned motor regions. Using this design, the authors demonstrate how the DishBrain self-organises and successfully learns to play the computer game 'Pong', exhibiting 'sentient' and intelligent behaviour in its virtual environment.The creation of such hybrid, silico-biological intelligence raises numerous ethical challenges. Following the neuroscientific framework embraced by the authors themselves, we discuss the arising ethical challenges in the context of Karl Friston's Free Energy Principle, focusing on the risk of creating synthetic phenomenology. Following the DishBrain's creator's neuroscientific assumptions, we highlight how DishBrain's design may risk bringing about artificial suffering and argue for a congruently cautious approach to such synthetic biological intelligence.
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Rapid Generation of Neurospheres from Hippocampal Neurons Using Extracellular-Matrix-Mimetic Scaffolds.
3D models of brain organoids represent an innovative and promising tool in neuroscience studies. However, the process of neurosphere formation in vitro remains complicated and is not always very effective. This is largely due to the lack of growth factors, guidance cues, and scaffold structures commonly found in tissues. Here we present a new, simple, and efficient method for generating neurospheres using scaffolds composed of electrospun nylon fibers with a diameter of 40-180 nm, which makes them similar to the brain extracellular matrix (ECM) components. Several main advantages of the proposed method should be highlighted. The method is fast, and the biomaterial consumption is low. Also, the resulting neurospheres are attached to the scaffold nanofibers. This not only provides the experimental convenience but also suggests that the resulting organoid models can potentially demonstrate fundamentally new properties, being closer to the nervous tissue in vivo. We demonstrate the influence of the fibrous scaffold structure on the formation, morphology, and composition of neurospheres and confirm adequate functional activity of the cellular components of these spheroids. The proposed approach can be further used for drug screening, modeling of neurodevelopmental, neurodegenerative disorders, and, potentially, therapeutic tissue engineering.
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Human brain and organoid transcriptomes reveal key receptor tyrosine kinase pathways and genetic signatures in Alzheimer's disease.
Alzheimer disease (AD) is a progressive neurodegenerative disorder marked by transcriptomic alterations affecting multiple genes. Many researchers have tried to predict major hallmarks of AD pathogenesis for diagnosis but the association between receptor tyrosine kinase (RTK) pathways and AD diagnosis is still unclear. This study aims to identify RTK-associated gene signatures crucial to AD pathogenesis and assess their potential as diagnostic biomarkers for AD. The study investigated changes in RTK pathway gene expression related to AD by analyzing brain transcriptome data from two independent public data sets (GSE84422 and GSE109887). Differentially expressed genes (DEGs) were analyzed from the GSE84422 and GSE109887 data sets and overlapping genes (oDEGs) were identified. RTK-related genes (ooDEGs) were subsequently selected through functional enrichment analysis. These were further refined into AD-related genes (disease-associated genes (DAGs)) through protein-protein interaction network analysis. Logistic regression and receiver operating characteristic analyses were conducted on the selected DAGs to evaluate their diagnostic potential, with additional gene expression validation performed in brain organoids and primary neurons. A total of 145 genes were identified as oDEGs in the above two data sets, and 18 genes were selected as ooDEGs. Six DAGs (ITGB1, AXL, GFAP, NRG1, CAV1, and RHOA) were selected. The diagnostic powers of the six DAGs for AD were 0.825 (GSE84422) and 0.884 (GSE109887). Human brain organoids and primary neuronal models were used to validate the biological relevance of these findings. AXL and ITGB1 were finally selected as key genes for RTK pathway in AD and were significantly increased in AD.
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Identification of secondary microglial formation centers in the human fetal brain.
Microglia migrate from the yolk sac and populate the developing brain. How microglia expand rapidly to meet the microglial demand in fast-expanding human fetal brains remains uncharted. Using thick sections in 5-22-gestational week (gw) brains and super-resolution scanning, we identified a large proliferative microglial aggregate (2.129 mm2) near the lateral ganglionic eminence (>12.5 gw), expanding in Down's syndrome (DS) (4.767 mm2) and Edwards syndrome (ES) (3.437 mm2) fetal brains. Ki67+ microglia within the aggregates accounted for 26.65% (DS: 38.9%; ES: 46.3%) compared with 6.32% (DS: 6.01%; ES: 5.2%) in scattered microglia. This aggregate region contained a distinct microglial population characterized by the absence of phagocytic structures and complex processes, high CSF-1R expression, abundant IL-34+ cells, and some SPP1+ bipolar microglia. We termed this structure the secondary microglial formation center (SMFC). Chimeric microglia-human cortical organoids recapitulated the SMFC in an IL-34- and CSF-1R-dependent manner, indicating that the human SMFC may compensate for the microglial shortage during the fastest expansion period.
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Spatiotemporal Changes of Cerebral Monocarboxylate Transporter 8 Expression.
Background: Mutations of monocarboxylate transporter 8 (MCT8), a thyroid hormone (TH)-specific transmembrane transporter, cause a severe neurodevelopmental disorder, the Allan-Herndon-Dudley syndrome. In MCT8 deficiency, TH is not able to reach those areas of the brain where TH uptake depends on MCT8. Currently, therapeutic options for MCT8-deficient patients are missing, as TH treatment is not successful in improving neurological deficits. Available data on MCT8 protein and transcript levels indicate complex expression patterns in neural tissue depending on species, brain region, sex, and age. However, information on human MCT8 expression is still scattered and additional efforts are needed to map sites of MCT8 expression in neurovascular units and neural tissue. This is of importance because new therapeutic strategies for this disease are urgently needed. Methods: To identify regions and time windows of MCT8 expression, we used highly specific antibodies against MCT8 to perform immunofluorescence labeling of postnatal murine brains, adult human brain tissue, and human cerebral organoids. Results: Qualitative and quantitative analyses of murine brain samples revealed stable levels of MCT8 protein expression in endothelial cells of the blood-brain barrier (BBB), choroid plexus epithelial cells, and tanycytes during postnatal development. Conversely, the neuronal MCT8 protein expression that was robustly detectable in specific brain regions of young mice strongly declined with age. Similarly, MCT8 immunoreactivity in adult human brain tissue was largely confined to endothelial cells of the BBB. Recently, cerebral organoids emerged as promising models of human neural development and our first analyses of forebrain-like organoids revealed MCT8 expression in early neuronal progenitor cell populations. Conclusions: With respect to MCT8-deficient conditions, our analyses not only strongly support the contention that the BBB presents a lifelong barrier to TH uptake but also highlight the need to decipher the TH transport role of MCT8 in early neuronal cell populations in more detail. Improving the understanding of the spatiotemporal expression in latter barriers will be critical for therapeutic strategies addressing MCT8 deficiency in the future.
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Sodium hypochlorite inactivation of human CJD prions.
Prion diseases are transmissible, fatal neurologic diseases of mammals caused by the accumulation of mis-folded, disease associated prion protein (PrPd). Creutzfeldt-Jakob Disease (CJD) is the most common human prion disease and can occur by sporadic onset (sCJD) (~85% of CJD cases), genetic mutations in the prion protein gene (10-15%) or iatrogenic transmission (rare). PrPd is difficult to inactivate and many methods to reduce prion infectivity are dangerous, caustic, expensive, or impractical. Identifying viable and safe methods for decontamination of CJD exposed materials is critically important for medical facilities and research institutions. Previous research has shown that concentrated sodium hypochlorite (bleach) was effective at inactivation of CJD prions derived from brains of mice or guinea pigs. Unfortunately, human prions adapted to rodents may mis-fold differently than in humans, and the rodent adapted prions may not have the same resistance or susceptibility to inactivation present in bona fide CJD prions. To confirm that bleach was efficacious against human sourced CJD prions, we exposed different subtypes of sCJD-infected human brain homogenates to different concentrations of bleach for increasing exposure times. Initial and residual prion seeding activity following inactivation were measured using Real-Time Quaking Induced Conversion. In addition, we tested how passage of human sCJD into either transgenic mice that expressed human prion protein, or transmission of CJD to human cerebral organoids (CO), two common laboratory practices, may affect CJD prions' susceptibility to bleach inactivation. Our results show that bleach is effective against human sourced sCJD prions, and both treatment time and concentration of bleach were important factors for CJD inactivation. CJD derived from human brains, transgenic mouse brains or CO were all susceptible to inactivation with as low as a 10 percent bleach solution with a 30-minute exposure time or a 50 percent bleach solution with as little as a 1-minute exposure time.
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11.7T Diffusion Magnetic Resonance Imaging and Tractography to Probe Human Brain Organoid Microstructure.
BACKGROUND: Human brain organoids are 3-dimensional cellular models that mimic architectural features of a developing brain. Generated from human induced pluripotent stem cells, these organoids offer an unparalleled physiologically relevant in vitro system for disease modeling and drug screening. In the current study, we sought to establish a foundation for a magnetic resonance imaging (MRI)-based, label-free imaging system that offers high-resolution capabilities for deep tissue imaging of whole organoids. METHODS: An 11.7T Bruker/89 mm microimaging system was used to collect high-resolution multishell 3-dimensional diffusion images of 2 induced pluripotent stem cell-derived human hippocampal brain organoids. The MRI features identified in the study were interpreted on the basis of similarities with immunofluorescence microscopy. RESULTS: MRI microscopy at ≤40 μm isotropic resolution provided a 3-dimensional view of organoid microstructure. T2-weighted contrast showed a rosette-like internal structure and a protruding spherical structure that correlated with immunofluorescence staining for the choroid plexus. Diffusion tractography methods can be used to model tissue microstructural features and possibly map neuronal organization. This approach complements traditional immunohistochemistry imaging methods without the need for tissue clearing. CONCLUSIONS: This proof-of-concept study shows, for the first time, the application of high-resolution diffusion MRI microscopy to image 2-mm diameter spherical human brain organoids. Application of ultrahigh-field MRI and diffusion tractography is a powerful modality for whole organoid imaging and has the potential to make a significant impact for probing microstructural changes in brain organoids used to model psychiatric disorders, neurodegenerative diseases, and viral infections of the human brain, as well as for assessing neurotoxicity in drug screening.