Neurotoxic Microglial Activation via IFNγ-Induced Nrf2 Reduction Exacerbating Alzheimer's Disease.

Microglial neuroinflammation appears to be neuroprotective in the early pathological stage, yet neurotoxic, which often precedes neurodegeneration in Alzheimer's disease (AD). However, it remains unclear how the microglial activities transit to the neurotoxic state during AD progression, due to complex neuron-glia interactions. Here, the mechanism of detrimental microgliosis in AD by employing 3D human AD mini-brains, brain tissues of AD patients, and 5XFAD mice is explored. In the human and animal AD models, amyloid-beta (Aß)-overexpressing neurons and reactive astrocytes produce interferon-gamma (IFNγ) and excessive oxidative stress. IFNγ results in the downregulation of mitogen-activated protein kinase (MAPK) and the upregulation of Kelch-like ECH-associated Protein 1 (Keap1) in microglia, which inactivate nuclear factor erythroid-2-related factor 2 (Nrf2) and sensitize microglia to the oxidative stress and induces a proinflammatory microglia via nuclear factor kappa B (NFκB)-axis. The proinflammatory microglia in turn produce neurotoxic nitric oxide and proinflammatory mediators exacerbating synaptic impairment, phosphorylated-tau accumulation, and discernable neuronal loss. Interestingly, recovering Nrf2 in the microglia prevents the activation of proinflammatory microglia and significantly blocks the tauopathy in AD minibrains. Taken together, it is envisioned that IFNγ-driven Nrf2 downregulation in microglia as a key target to ameliorate AD pathology.

Botulinum Neurotoxin Induces Neurotoxic Microglia Mediated by Exogenous Inflammatory Responses.

Botulinum neurotoxin serotype A (BoNT/A) is widely used in therapeutics and cosmetics. The effects of multi-dosed BoNT/A treatment are well documented on the peripheral nervous system (PNS), but much less is known on the central nervous system (CNS). Here, the mechanism of multi-dosed BoNT/A leading to CNS neurodegeneration is explored by using the 3D human neuron-glia model. BoNT/A treatment reduces acetylcholine, triggers astrocytic transforming growth factor beta, and upregulates C1q, C3, and C5 expression, inducing microglial proinflammation. The disintegration of the neuronal microtubules is escorted by microglial nitric oxide, interleukin 1ß, tumor necrosis factor α, and interleukin 8. The microglial proinflammation eventually causes synaptic impairment, phosphorylated tau (pTau) aggregation, and the loss of the BoNT/A-treated neurons. Taking a more holistic approach, the model will allow to assess therapeutics for the CNS neurodegeneration under the prolonged use of BoNT/A.

Environmental aluminum oxide inducing neurodegeneration in human neurovascular unit with immunity.

Aluminum oxide nanoparticle (AlNP), a ubiquitous neurotoxin highly enriched in air pollution, is often produced as an inevitable byproduct in the manufacturing of industrial products such as cosmetics and metal materials. Meanwhile, ALNP has emerged as a significant public health concern due to its potential association with neurological diseases. However, the studies about the neurotoxic effects of AlNP are limited, partially due to the lack of physiologically relevant human neurovascular unit with innate immunity (hNVUI). Here, we employed our AlNP-treated hNVUI model to investigate the underlying mechanism of AlNP-driven neurodegeneration. First, we validated the penetration of AlNP across a blood-brain barrier (BBB) compartment and found AlNP-derived endothelial cellular senescence through the p16 and p53/p21 pathways. Our study showed that BBB-penetrating AlNP promoted reactive astrocytes, which produced a significant level of reactive oxygen species (ROS). The astrocytic neurotoxic factors caused neuronal damage, including the synaptic impairment, the accumulation of phosphoric-tau proteins, and even neuronal death. Our study suggests that AlNP could be a potential environmental risk factor of neurological disorders mediated by neuroinflammation.

Laminin-Augmented Decellularized Extracellular Matrix Ameliorating Neural Differentiation and Neuroinflammation in Human Mini-Brains.

Non-neural extracellular matrix (ECM) has limited application in humanized physiological neural modeling due to insufficient brain-specificity and safety concerns. Although brain-derived ECM contains enriched neural components, certain essential components are partially lost during the decellularization process, necessitating augmentation. Here, it is demonstrated that the laminin-augmented porcine brain-decellularized ECM (P-BdECM) is xenogeneic factor-depleted as well as favorable for the regulation of human neurons, astrocytes, and microglia. P-BdECM composition is comparable to human BdECM regarding brain-specificity through the matrisome and gene ontology-biological process analysis. As augmenting strategy, laminin 111 supplement promotes neural function by synergic effect with laminin 521 in P-BdECM. Annexin A1(ANXA1) and Peroxiredoxin(PRDX) in P-BdECM stabilized microglial and astrocytic behavior under normal while promoting active neuroinflammation in response to neuropathological factors. Further, supplementation of the brain-specific molecule to non-neural matrix also ameliorated glial cell inflammation as in P-BdECM. In conclusion, P-BdECM-augmentation strategy can be used to recapitulate humanized pathophysiological cerebral environments for neurological study.

Low-Temperature Layer-by-Layer Growth of Semiconducting Few-Layer γ-Graphyne to Exploit Robust Biocompatibility.

The sp-hybridized carbon network in single- or few-layer γ-graphyne (γ-GY) has a polarized electron distribution, which can be crucial in overcoming biosafety issues. Here, we report the low-temperature synthesis, electronic properties, and amyloid fibril nanostructures of electrostatic few-layer γ-GY. ABC stacked γ-GY is synthesized by layer-by-layer growth on a catalytic copper surface, exhibiting intrinsic p-type semiconducting properties in few-layer γ-GY. Thickness-dependent electronic properties of γ-GY elucidate interlayer interactions by electron doping between electrostatic layers and layer stacking-involved modulation of the band gap. Electrostatic few-layer γ-GY induces high electronic sensitivity and intense interaction with amyloid beta (i.e., Aß40) peptides assembling into elongated mature Aß40 fibrils. Two-dimensional biocompatible nanostructures of Aß40 fibrils/few-layer γ-GY enable excellent cell viability and high neuronal differentiation of living cells without external stimulation.

Three-dimensional human neural culture on a chip recapitulating neuroinflammation and neurodegeneration.

Neuroinflammation has either beneficial or detrimental effects, depending on risk factors and neuron-glia interactions in neurological disorders. However, studying neuroinflammation has been challenging due to the complexity of cell-cell interactions and lack of physio-pathologically relevant neuroinflammatory models. Here, we describe our three-dimensional microfluidic multicellular human neural culture model, referred to as a 'brain-on-a-chip' (BoC). This elucidates neuron-glia interactions in a controlled manner and recapitulates pathological signatures of the major neurological disorders: dementia, brain tumor and brain edema. This platform includes a chemotaxis module offering a week-long, stable chemo-gradient compared with the few hours in other chemotaxis models. Additionally, compared with conventional brain models cultured with mixed phenotypes of microglia, our BoC can separate the disease-associated microglia out of heterogeneous population and allow selective neuro-glial engagement in three dimensions. This provides benefits of interpreting the neuro-glia interactions while revealing that the prominent activation of innate immune cells is the risk factor leading to synaptic impairment and neuronal loss, validated in our BoC models of disorders. This protocol describes how to fabricate and implement our human BoC, manipulate in real time and perform end-point analyses. It takes 2 d to set up the device and cell preparations, 1-9 weeks to develop brain models under disease conditions and 2-3 d to carry out analyses. This protocol requires at least 1 month training for researchers with basic molecular biology techniques. Taken together, our human BoCs serve as reliable and valuable platforms to investigate pathological mechanisms involving neuroinflammation and to assess therapeutic strategies modulating neuroinflammation in neurological disorders.

Bright-Field and Edge-Enhanced Imaging Using an Electrically Tunable Dual-Mode Metalens.

The imaging of microscopic biological samples faces numerous difficulties due to their small feature sizes and low-amplitude contrast. Metalenses have shown great promise in bioimaging as they have access to the complete complex information, which, alongside their extremely small and compact footprint and potential to integrate multiple functionalities into a single device, allow for miniaturized microscopy with exceptional features. Here, we design and experimentally realize a dual-mode metalens integrated with a liquid crystal cell that can be electrically switched between bright-field and edge-enhanced imaging on the millisecond scale. We combine the concepts of geometric and propagation phase to design the dual-mode metalens and physically encode the required phase profiles using hydrogenated amorphous silicon for operation at visible wavelengths. The two distinct metalens phase profiles include (1) a conventional hyperbolic metalens for bright-field imaging and (2) a spiral metalens with a topological charge of +1 for edge-enhanced imaging. We demonstrate the focusing and vortex generation ability of the metalens under different states of circular polarization and prove its use for biological imaging. This work proves a method for in vivo observation and monitoring of the cell response and drug screening within a compact form factor.

Astrocytic scar restricting glioblastoma via glutamate-MAO-B activity in glioblastoma-microglia assembloid.

BACKGROUND: Glial scar formation is a reactive glial response confining injured regions in a central nervous system. However, it remains challenging to identify key factors formulating glial scar in response to glioblastoma (GBM) due to complex glia-GBM crosstalk. METHODS: Here, we constructed an astrocytic scar enclosing GBM in a human assembloid and a mouse xenograft model. GBM spheroids were preformed and then co-cultured with microglia and astrocytes in 3D Matrigel. For the xenograft model, U87-MG cells were subcutaneously injected to the Balb/C nude female mice. RESULTS: Additional glutamate was released from GBM-microglia assembloid by 3.2-folds compared to GBM alone. The glutamate upregulated astrocytic monoamine oxidase-B (MAO-B) activity and chondroitin sulfate proteoglycans (CSPGs) deposition, forming the astrocytic scar and restricting GBM growth. Attenuating scar formation by the glutamate-MAO-B inhibition increased drug penetration into GBM assembloid, while reducing GBM confinement. CONCLUSIONS: Taken together, our study suggests that astrocytic scar could be a critical modulator in GBM therapeutics.

In Vitro Tumor Models on Chip and Integrated Microphysiological Analysis Platform (MAP) for Life Sciences and High-Throughput Drug Screening.

The evolution of preclinical in vitro cancer models has led to the emergence of human cancer-on-chip or microphysiological analysis platforms (MAPs). Although it has numerous advantages compared to other models, cancer-on-chip technology still faces several challenges such as the complexity of the tumor microenvironment and integrating multiple organs to be widely accepted in cancer research and therapeutics. In this review, we highlight the advancements in cancer-on-chip technology in recapitulating the vital biological features of various cancer types and their applications in life sciences and high-throughput drug screening. We present advances in reconstituting the tumor microenvironment and modeling cancer stages in breast, brain, and other types of cancer. We also discuss the relevance of MAPs in cancer modeling and precision medicine such as effect of flow on cancer growth and the short culture period compared to clinics. The advanced MAPs provide high-throughput platforms with integrated biosensors to monitor real-time cellular responses applied in drug development. We envision that the integrated cancer MAPs has a promising future with regard to cancer research, including cancer biology, drug discovery, and personalized medicine.

Human mini-brains for reconstituting central nervous system disorders.

Neurological disorders in the central nervous system (CNS) are progressive and irreversible diseases leading to devastating impacts on patients' life as they cause cognitive impairment, dementia, and even loss of essential body functions. The development of effective medicines curing CNS disorders is, however, one of the most ambitious challenges due to the extremely complex functions and structures of the human brain. In this regard, there are unmet needs to develop simplified but physiopathologically-relevant brain models. Recent advances in the microfluidic techniques allow multicellular culture forming miniaturized 3D human brains by aligning parts of brain regions with specific cells serving suitable functions. In this review, we overview designs and strategies of microfluidics-based human mini-brains for reconstituting CNS disorders, particularly Alzheimer's disease (AD), Parkinson's disease (PD), traumatic brain injury (TBI), vascular dementia (VD), and environmental risk factor-driven dementia (ERFD). Afterward, the applications of the mini-brains in the area of medical science are introduced in terms of the clarification of pathogenic mechanisms and identification of promising biomarkers. We also present expanded model systems ranging from the CNS to CNS-connecting organ axes to study the entry pathways of pathological risk factors into the brain. Lastly, the advantages and potential challenges of current model systems are addressed with future perspectives.

Human mini-blood-brain barrier models for biomedical neuroscience research: a review.

The human blood-brain barrier (BBB) is a unique multicellular structure that is in critical demand for fundamental neuroscience studies and therapeutic evaluation. Despite substantial achievements in creating in vitro human BBB platforms, challenges in generating specifics of physiopathological relevance are viewed as impediments to the establishment of in vitro models. In this review, we provide insight into the development and deployment of in vitro BBB models that allow investigation of the physiology and pathology of neurological therapeutic avenues. First, we highlight the critical components, including cell sources, biomaterial glue collections, and engineering techniques to reconstruct a miniaturized human BBB. Second, we describe recent breakthroughs in human mini-BBBs for investigating biological mechanisms in neurology. Finally, we discuss the application of human mini-BBBs to medical approaches. This review provides strategies for understanding neurological diseases, a validation model for drug discovery, and a potential approach for generating personalized medicine.

Nanomedicine for advanced cancer immunotherapy.

Immunotherapy has emerged as a powerful strategy for liquid tumors to overcome the limitations of conventional cancer therapies. The nanomedical delivery system offers the possibility of enhancing cancer immunotherapy and expanding it to solid tumors. Here, we discuss the applications of medical nanoparticles to improve the efficacy of immunotherapy. We first focus on nanomedical particles used in cancer immunotherapy to deliver peptide and mRNA vaccines to the lymph nodes; and the exosome-based therapeutic cancer vaccine. Next, we highlight the applications of nanomedicine in immune checkpoint therapy to prolong the therapeutic effects, enhance tumor-targeting ability, and overcome drug resistance. We also evaluate the roles of nanomedical particles in oncolytic viral treatment, enabling the systemic injection of viruses or oncolytic plasmids/oncotoxic proteins; and virus entry in a receptor-independency manner. Lastly, we focus on nanoparticles in chimeric antigen receptor (CAR) T cell therapy to engineer CAR T cells, enhancing T cell proliferation and infiltration. We envision the nanomedical particles enhancing the therapeutic effects of immunotherapy and revolutionizing cancer therapy in the foreseeable future.

Engineered Human Intervertebral Disc Model Inducing Degenerative Microglial Proinflammation.

Degeneration of the intervertebral disc (IVD) is a major contributor to low back pain (LBP). IVD degeneration is characterized by abnormal production of inflammatory cytokines secreted by IVD cells. Although the underlying molecular mechanisms of LBP have not been elucidated, increasing evidence suggests that LBP is associated particularly with microglia in IVD tissues and the peridiscal space, aggravating the cascade of degenerative events. In this study, we implemented our microfluidic chemotaxis platform to investigate microglial inflammation in response to our reconstituted degenerative IVD models. The IVD models were constructed by stimulating human nucleus pulposus (NP) cells with interleukin-1ß and producing interleukin-6 (129.93 folds), interleukin-8 (18.31 folds), C-C motif chemokine ligand-2 (CCL-2) (6.12 folds), and CCL-5 (5.68 folds). We measured microglial chemotaxis (p < 0.05) toward the conditioned media of the IVD models. In addition, we observed considerable activation of neurodegenerative and deactivation of protective microglia via upregulated expression of CD11b (p < 0.001) and down-regulation of CD206 protein (p < 0.001) by soluble factors from IVD models. This, in turn, enhances the inflammatory milieu in IVD tissues, causing matrix degradation and cellular damage. Our findings indicate that degenerative IVD may induce degenerative microglial proinflammation, leading to LBP development.

Neuroinflammation in neurodegeneration via microbial infections.

Recent epidemiological studies show a noticeable correlation between chronic microbial infections and neurological disorders. However, the underlying mechanisms are still not clear due to the biological complexity of multicellular and multiorgan interactions upon microbial infections. In this review, we show the infection leading to neurodegeneration mediated by multiorgan interconnections and neuroinflammation. Firstly, we highlight three inter-organ communications as possible routes from infection sites to the brain: nose-brain axis, lung-brain axis, and gut-brain axis. Next, we described the biological crosstalk between microglia and astrocytes upon pathogenic infection. Finally, our study indicates how neuroinflammation is a critical player in pathogen-mediated neurodegeneration. Taken together, we envision that antibiotics targeting neuro-pathogens could be a potential therapeutic strategy for neurodegeneration.

High-Throughput Screening of Anti-cancer Drugs Using a Microfluidic Spheroid Culture Device with a Concentration Gradient Generator.

Tumor spheroid models are widely used for drug screening as in vitro models of the tumor microenvironment. There are various ways in which tumor spheroid models can be prepared, including the self-assembly of cells using low-adherent plates, micro-patterned plates, or hanging-drop plates. Recently, drug high-throughput screening (HTS) approaches have incorporated the use of these culture systems. These HTS culture systems, however, require complicated equipment, such as robot arms, detectors, and software for handling solutions and data processing. Here, we describe protocols that allow tumor spheroids to be tested with different concentrations of a drug in a parallel fashion using a microfluidic device that generates a gradient of anti-cancer drugs. This microfluidic spheroid culture device with a concentration gradient generator (µFSCD-CGG) enables the formation of 50 tumor spheroids and the testing of drugs at five different concentrations. First, we provide a protocol for the fabrication of the µFSCD-CGG, which has both a culture array in which tumor cells are injected and aggregate to form spheroids and a concentration gradient generator for drug testing. Second, we provide a protocol for tumor spheroid formation and HTS of anti-cancer drugs using the device. Finally, we provide a protocol for assessing the response of tumor spheroids at different drug concentrations. To address the needs of the pharmaceutical industry, this protocol can be used for various cell types, including stem cells, and the number of tumor spheroids and drug concentration ranges that can be tested in the µFSCD-CGG can be increased. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Fabrication of a microfluidic spheroid culture device with a concentration gradient generator (µFSCD-CGG) Basic Protocol 2: Seeding cells and formation of spheroids in the µFSCD-CGG Basic Protocol 3: Drug treatment and assessment of cell viability in the µFSCD-CGG.

Adipokine human Resistin promotes obesity-associated inflammatory intervertebral disc degeneration via pro-inflammatory cytokine cascade activation.

Adipokine human Resistin (hResistin), is known to be associated with insulin resistance and secrete low-grade pro-inflammatory cytokines in obesity. Although studies on low-grade inflammation of adipokine hResistin are known, studies on the effects and mechanisms of intervertebral disc degeneration (IVDD) are still lacking. Thus, we investigated the adipokine hResistin with or without pro-inflammatory cytokine IL-1ß in intervertebral disc (IVD) cells such as human annulus fibrosus (hAF) and nucleus pulposus (hNP). The protein expression changes in IL-1ß, IL-6, IL-8, MMP-1, MMP-3, and MMP-13, induced by the combined-hResistin and IL-1ß stimulation on hAF cells, was significantly greater than that of the same induced by mono-IL-1ß stimulation. Similarly, in the case of the protein expression change of inflammatory mediators induced by the combined-hResistin and IL-1ß stimulation on hNP cells was also significantly greater than that of the same induced by mono-IL-1ß stimulation. These results improve understanding of hResistin on inflammatory IVDD but also with other obesity-related inflammatory diseases.

TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia.

The membrane protein TREM2 (Triggering Receptor Expressed on Myeloid cells 2) regulates key microglial functions including phagocytosis and chemotaxis. Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer's disease (AD). Because abnormalities in Ca2+ signaling have been observed in several AD models, we investigated TREM2 regulation of Ca2+ signaling in human induced pluripotent stem cell-derived microglia (iPSC-microglia) with genetic deletion of TREM2. We found that iPSC-microglia lacking TREM2 (TREM2 KO) show exaggerated Ca2+ signals in response to purinergic agonists, such as ADP, that shape microglial injury responses. This ADP hypersensitivity, driven by increased expression of P2Y12 and P2Y13 receptors, results in greater release of Ca2+ from the endoplasmic reticulum stores, which triggers sustained Ca2+ influx through Orai channels and alters cell motility in TREM2 KO microglia. Using iPSC-microglia expressing the genetically encoded Ca2+ probe, Salsa6f, we found that cytosolic Ca2+ tunes motility to a greater extent in TREM2 KO microglia. Despite showing greater overall displacement, TREM2 KO microglia exhibit reduced directional chemotaxis along ADP gradients. Accordingly, the chemotactic defect in TREM2 KO microglia was rescued by reducing cytosolic Ca2+ using a P2Y12 receptor antagonist. Our results show that loss of TREM2 confers a defect in microglial Ca2+ response to purinergic signals, suggesting a window of Ca2+ signaling for optimal microglial motility.

An Air Particulate Pollutant Induces Neuroinflammation and Neurodegeneration in Human Brain Models.

Fine particulate matter (PM2.5), a major component among air pollutants, highlights as a global health concern. Several epidemiological studies show the correlation between chronical PM2.5 exposure and incidents of neurological disorders including Alzheimer's disease. However, the mechanisms have not been well understood, partly due to the lack of model systems that reflect the physiologically relevant innate immunity in human brains. Here, PM2.5-polluted human brain models (PMBs) are created in a 3D microfluidic platform reconstituting key aspects of human brain immunity under the PM2.5 exposure. PM2.5 penetration across a blood-brain barrier (BBB) model and accumulation in the brain tissue side of the model are first validated. Second, the PMB model shows that the BBB-penetrating PM2.5 initiates astrogliosis, resulting in slight neuronal loss and microglial infiltration. Third, it is demonstrated that the infiltrating microglia obtain M1 phenotype induced by interleukin-1ß and interferon-γ from neurons and reactive astrocytes under the PM2.5 exposure. Finally, it is observed that additional proinflammatory mediators and nitric oxide released from the M1 microglia exacerbate neuronal damages, such as synaptic impairment, phosphoric tau accumulation, and neuronal death. This study suggests that PM2.5 can be a potential environmental risk factor for dementia mediated by the detrimental neuroinflammation.

Oral Pathogenic Bacteria-Inducing Neurodegenerative Microgliosis in Human Neural Cell Platform.

Porphyromonas gingivalis is a gram-negative bacterium found in the human oral cavity and is responsible for the development of chronic periodontitis as well as neurological diseases, including Alzheimer's disease (AD). Given the significance of the roles of P. gingivalis in AD pathogenesis, it is critical to understand the underlying mechanisms of P. gingivalis-driven neuroinflammation and their contribution to neurodegeneration. Herein, we hypothesize that P. gingivalis produces secondary metabolites that may cause neurodegeneration through direct or indirect pathways mediated by microglia. To test our hypothesis, we treated human neural cells with bacterial conditioned media on our brain platforms and assessed microgliosis, astrogliosis and neurodegeneration. We found that bacteria-mediated microgliosis induced the production of nitric oxide, which causes neurodegeneration assessed with high pTau level. Our study demonstrated the elevation of detrimental protein mediators, CD86 and iNOS and the production of several pro-inflammatory markers from stimulated microglia. Through inhibition of LPS and succinate dehydrogenase in a bacterial conditioned medium, we showed a decrease in neurodegenerative microgliosis. In addition, we demonstrated the bidirectional effect of microgliosis and astrogliosis on each other exacerbating neurodegeneration. Overall, our study suggests that the mouth-brain axis may contribute to the pathogenesis of AD.