Mito-FUNCAT-FACS reveals cellular heterogeneity in mitochondrial translation.

Mitochondria possess their own genome that encodes components of oxidative phosphorylation (OXPHOS) complexes, and mitochondrial ribosomes within the organelle translate the mRNAs expressed from the mitochondrial genome. Given the differential OXPHOS activity observed in diverse cell types, cell growth conditions, and other circumstances, cellular heterogeneity in mitochondrial translation can be expected. Although individual protein products translated in mitochondria have been monitored, the lack of techniques that address the variation in overall mitochondrial protein synthesis in cell populations poses analytic challenges. Here, we adapted mitochondrial-specific fluorescent noncanonical amino acid tagging (FUNCAT) for use with fluorescence-activated cell sorting (FACS) and developed mito-FUNCAT-FACS. The click chemistry-compatible methionine analog L-homopropargylglycine (HPG) enabled the metabolic labeling of newly synthesized proteins. In the presence of cytosolic translation inhibitors, HPG was selectively incorporated into mitochondrial nascent proteins and conjugated to fluorophores via the click reaction (mito-FUNCAT). The application of in situ mito-FUNCAT to flow cytometry allowed us to separate changes in net mitochondrial translation activity from those of the organelle mass and detect variations in mitochondrial translation in cancer cells. Our approach provides a useful methodology for examining mitochondrial protein synthesis in individual cells.

Tri-culture of spatially organizing human skeletal muscle cells, endothelial cells, and fibroblasts enhances contractile force and vascular perfusion of skeletal muscle tissues.

Constructing engineered human skeletal muscle tissues that resemble the function and microstructure of human skeletal muscles is key to utilizing them in a variety of applications such as drug development, disease modeling, regenerative medicine, and engineering biological machines. However, current in vitro skeletal muscle tissues are far inferior to native muscles in terms of contractile function and lack essential cues for muscle functions, particularly heterotypic cell-cell interactions between myoblasts, endothelial cells, and fibroblasts. Here, we develop an engineered muscle tissue with a coaxial three-layered tubular structure composed of an inner endothelial cell layer, an endomysium-like layer with fibroblasts in the middle, and an outer skeletal muscle cell layer, similar to the architecture of native skeletal muscles. Engineered skeletal muscle tissues with three spatially organized cell types produced thicker myotubes and lowered Young's modulus through extracellular matrix remodeling, resulting in 43% stronger contractile force. Furthermore, we demonstrated that fibroblasts localized in the endomysium layer induced angiogenic sprouting of endothelial cells into the muscle layer more effectively than fibroblasts homogeneously distributed in the muscle layer. This layered tri-culture system enables a structured spatial configuration of the three main cell types of skeletal muscle and promotes desired paracrine signaling, resulting in improved angiogenesis and increased contractile force. This research offers new insights to efficiently obtain new human skeletal muscle models, transplantable tissues, and actuators for biological machines.

A widespread family of heat-resistant obscure (Hero) proteins protect against protein instability and aggregation.

Proteins are typically denatured and aggregated by heating at near-boiling temperature. Exceptions to this principle include highly disordered and heat-resistant proteins found in extremophiles, which help these organisms tolerate extreme conditions such as drying, freezing, and high salinity. In contrast, the functions of heat-soluble proteins in non-extremophilic organisms including humans remain largely unexplored. Here, we report that heat-resistant obscure (Hero) proteins, which remain soluble after boiling at 95°C, are widespread in Drosophila and humans. Hero proteins are hydrophilic and highly charged, and function to stabilize various "client" proteins, protecting them from denaturation even under stress conditions such as heat shock, desiccation, and exposure to organic solvents. Hero proteins can also block several different types of pathological protein aggregations in cells and in Drosophila strains that model neurodegenerative diseases. Moreover, Hero proteins can extend life span of Drosophila. Our study reveals that organisms naturally use Hero proteins as molecular shields to stabilize protein functions, highlighting their biotechnological and therapeutic potential.

Advances in construction and modeling of functional neural circuits in vitro.

Over the years, techniques have been developed to culture and assemble neurons, which brought us closer to creating neuronal circuits that functionally and structurally mimic parts of the brain. Starting with primary culture of neurons, preparations of neuronal culture have advanced substantially. Development of stem cell research and brain organoids has opened a new path for generating three-dimensional human neural circuits. Along with the progress in biology, engineering technologies advanced and paved the way for construction of neural circuit structures. In this article, we overview research progress and discuss perspective of in vitro neural circuits and their ability and potential to acquire functions. Construction of in vitro neural circuits with complex higher-order functions would be achieved by converging development in diverse major disciplines including neuroscience, stem cell biology, tissue engineering, electrical engineering and computer science.

Polyploidy of MDA-MB-231 cells drives increased extravasation with enhanced cell-matrix adhesion.

Metastasis, the leading cause of cancer-related deaths, involves a complex cascade of events, including extravasation. Despite extensive research into metastasis, the mechanisms underlying extravasation remain unclear. Molecular targeted therapies have advanced cancer treatment, yet their efficacy is limited, prompting exploration into novel therapeutic targets. Here, we showed the association of polyploidy in MDA-MB-231 breast cancer cells and their extravasation, using microfluidic systems to reproduce the in vivo microvascular environment. We observed enhanced extravasation in polyploid cells alongside upregulated expression of genes involved in cell-substrate adhesion and cell mechanical dynamics. These findings offer insights into the relationship between polyploidy and extravasation, highlighting potential targets for cancer therapy.

A simple and inexpensive laser dissection of fasciculated axons from motor nerve organoids.

Motor nerve organoids could be generated by culturing a spheroid of motor neurons differentiated from human induced pluripotent stem (iPS) cells within a polydimethylsiloxane (PDMS) chip which guides direction and fasciculation of axons extended from the spheroid. To isolate axon bundles from motor nerve organoids, we developed a rapid laser dissection method based on localized photothermal combustion. By illuminating a blue laser on a black mark on the culture device using a dry-erase marker, we induced highly localized heating near the axon bundles. Moving the laser enabled spatial control over the local heating and severing of axon bundles. This laser dissection requires a black mark, as other colors did not produce the same localized heating effect. A CO2 laser destroyed the tissue and the device and could not be used. With this simple, economical laser dissection technique, we could rapidly collect abundant pure axon samples from motor nerve organoids for biochemical analysis. Extracted axonal proteins and RNA were indistinguishable from manual dissection. This method facilitates efficient axon isolation for further analyses.

Early differential impact of MeCP2 mutations on functional networks in Rett syndrome patient-derived human cerebral organoids.

Human cerebral organoids derived from induced pluripotent stem cells can recapture early developmental processes and reveal changes involving neurodevelopmental disorders. Mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene are associated with Rett syndrome, and disease severity varies depending on the location and type of mutation. Here, we focused on neuronal activity in Rett syndrome patient-derived organoids, analyzing two types of MeCP2 mutations - a missense mutation (R306C) and a truncating mutation (V247X) - using calcium imaging with three-photon microscopy. Compared to isogenic controls, we found abnormal neuronal activity in Rett organoids and altered network function based on graph theoretic analyses, with V247X mutations impacting functional responses and connectivity more severely than R306C mutations. These changes paralleled EEG data obtained from patients with comparable mutations. Labeling DLX promoter-driven inhibitory neurons demonstrated differences in activity and functional connectivity of inhibitory and excitatory neurons in the two types of mutation. Transcriptomic analyses revealed HDAC2-associated impairment in R306C organoids and decreased GABAA receptor expression in excitatory neurons in V247X organoids. These findings demonstrate mutation-specific mechanisms of vulnerability in Rett syndrome and suggest targeted strategies for their treatment.

Complex activity and short-term plasticity of human cerebral organoids reciprocally connected with axons.

An inter-regional cortical tract is one of the most fundamental architectural motifs that integrates neural circuits to orchestrate and generate complex functions of the human brain. To understand the mechanistic significance of inter-regional projections on development of neural circuits, we investigated an in vitro neural tissue model for inter-regional connections, in which two cerebral organoids are connected with a bundle of reciprocally extended axons. The connected organoids produced more complex and intense oscillatory activity than conventional or directly fused cerebral organoids, suggesting the inter-organoid axonal connections enhance and support the complex network activity. In addition, optogenetic stimulation of the inter-organoid axon bundles could entrain the activity of the organoids and induce robust short-term plasticity of the macroscopic circuit. These results demonstrated that the projection axons could serve as a structural hub that boosts functionality of the organoid-circuits. This model could contribute to further investigation on development and functions of macroscopic neuronal circuits in vitro.

The A53T mutation in α-synuclein enhances pro-inflammatory activation in human microglia upon inflammatory stimulus.

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disease, following Alzheimer's. It is characterized by the aggregation of α-synuclein into Lewy bodies and Lewy neurites in the brain. Microglia-driven neuroinflammation may contribute to neuronal death in PD, however the exact role of microglia remains unclear and has been understudied. The A53T mutation in the gene coding for α-synuclein has been linked to early-onset PD, and exposure to A53T-mutant human α-synuclein increases the potential for inflammation of murine microglia. To date, its effect has not been studied in human microglia. METHODS: Here, we used 2-dimensional cultures of human iPSC-derived microglia and transplantation of these cells into the mouse brain to assess the cell-autonomous effects of the A53T mutation on human microglia. RESULTS: We found that A53T-mutant human microglia had an intrinsically increased propensity towards pro-inflammatory activation upon inflammatory stimulus. Additionally, transplanted A53T mutant microglia showed a strong decrease in catalase expression in non-inflammatory conditions, and increased oxidative stress. CONCLUSIONS: Our results indicate that A53T mutant human microglia display cell-autonomous phenotypes that may worsen neuronal damage in early-onset PD.

High-throughput Assessment of Mitochondrial Protein Synthesis in Mammalian Cells Using Mito-FUNCAT FACS.

In addition to cytosolic protein synthesis, mitochondria also utilize another translation system that is tailored for mRNAs encoded in the mitochondrial genome. The importance of mitochondrial protein synthesis has been exemplified by the diverse diseases associated with in organello translation deficiencies. Various methods have been developed to monitor mitochondrial translation, such as the classic method of labeling newly synthesized proteins with radioisotopes and the more recent ribosome profiling. However, since these methods always assess the average cell population, measuring the mitochondrial translation capacity in individual cells has been challenging. To overcome this issue, we recently developed mito-fluorescent noncanonical amino acid tagging (FUNCAT) fluorescence-activated cell sorting (FACS), which labels nascent peptides generated by mitochondrial ribosomes with a methionine analog, L-homopropargylglycine (HPG), conjugates the peptides with fluorophores by an in situ click reaction, and detects the signal in individual cells by FACS equipment. With this methodology, the hidden heterogeneity of mitochondrial translation in cell populations can be addressed.

The A53T mutation in α-synuclein enhances pro-inflammatory activation in human microglia.

Parkinson's disease (PD) is characterized by the aggregation of α-synuclein into Lewy bodies and Lewy neurites in the brain. Microglia-driven neuroinflammation may contribute to neuronal death in PD, however the exact role of microglia remains unclear and has been understudied. The A53T mutation in the gene coding for α-synuclein has been linked to early-onset PD, and exposure to A53T-mutant human α-synuclein increases the potential for inflammation of murine microglia. To date, its effect has not been studied in human microglia. Here, we used 2-dimensional cultures of human iPSC-derived microglia and transplantation of these cells into the mouse brain to assess the effects of the A53T mutation on human microglia. We found that A53T-mutant human microglia had an intrinsically increased propensity towards pro-inflammatory activation upon inflammatory stimulus. Additionally, A53T mutant microglia showed a strong decrease in catalase expression in non-inflammatory conditions, and increased oxidative stress. Our results indicate that A53T mutant human microglia display cell-autonomous phenotypes that may worsen neuronal damage in early-onset PD.

Processing of nanolitre liquid plugs for microfluidic cell-based assays.

Plugs, i.e. droplets formed in a microchannel, may revolutionize microfluidic cell-based assays. This study describes a microdevice that handles nanolitre-scale liquid plugs for the preparation of various culture setups and subsequent cellular assays. An important feature of this mode of liquid operation is that the recirculation flow generated inside the plug promotes the rapid mixing of different solutions after plugs are merged, and it keeps cell suspensions homogeneous. Thus, serial dilutions of reagents and cell suspensions with different cell densities and cell types were rapidly performed using nanolitres of solution. Cells seeded through the plug processing grew well in the microdevice, and subsequent plug processing was used to detect the glucose consumption of cells and cellular responses to anticancer agents. The plug-based microdevice may provide a useful platform for cell-based assay systems in various fields, including fundamental cell biology and drug screening applications.

In vitro modelling of human proprioceptive sensory neurons in the neuromuscular system.

Proprioceptive sensory neurons (pSN) are an essential and undervalued part of the neuromuscular circuit. A protocol to differentiate healthy and amyotrophic lateral sclerosis (ALS) human neural stem cells (hNSC) into pSN, and their comparison with the motor neuron (MN) differentiation process from the same hNSC sources, facilitated the development of in vitro co-culture platforms. The obtained pSN spheroids cultured interact with human skeletal myocytes showing the formation of annulospiral wrapping-like structures between TrkC + neurons and a multinucleated muscle fibre, presenting synaptic bouton-like structures in the contact point. The comparative analysis of the genetic profile performed in healthy and sporadic ALS hNSC differentiated to pSN suggested that basal levels of ETV1, critical for motor feedback from pSN, were much lower for ALS samples and that the differences between healthy and ALS samples, suggest the involvement of pSN in ALS pathology development and progression.

Multiscale engineered human skeletal muscles with perfusable vasculature and microvascular network recapitulating the fluid compartments.

Creating a vasculature in engineered human skeletal muscle tissues (ehSMTs) enables us to create thick tissues, increase cell survival in implantation, provide models of blood-organ barriers for drug testing, and enhance muscle differentiation through paracrine signaling. Here, contractile ehSMTs with a central perfusable vascular channel and microvascular networks growing from this central vasculature into the surrounding skeletal muscle tissue were newly demonstrated. Because coculturing muscle cells and endothelial cells requires incompatible media, we recapitulated thein vivoextracellular fluid compartments between blood plasma and interstitial fluid by creating anin vitroperfusable vasculature running through skeletal muscle tissue with a physiologic cell density. By using this model, we constructed large vascularized ehSMTs and showed the potential to be utilized for drug testing platforms. Also, we found that coculturing with two separate media from an early stage of muscle differentiation led to increased contractile force, thicker myotubes, and improved muscle differentiation.

Human sensory neurons modulate melanocytes through secretion of RGMB.

Melanocytes are surrounded by diverse cells, including sensory neurons in our skin, but their interaction and functional importance have been poorly investigated. In this study, we find that melanocytes and nociceptive neurons contact more in human skin color patch tissue than control. Co-culture with human iPSC-derived sensory neurons significantly induces morphogenesis and pigmentation of human melanocytes. To reveal melanocyte-stimulating factors secreted from neurons, we perform proteomic analyses and identify RGMB in the sensory neuron-conditioned medium. RGMB protein induces morphogenesis and melanin production of melanocytes, demonstrating that RGMB is a melanocyte-stimulating factor released from sensory neurons. Transcriptome analysis suggests that the melanosome transport machinery can be controlled by RGMB, leading us to identify the vesicle production response of melanocytes upon RGMB treatment. This study discovers a role of sensory neurons in modulating multiple aspects of human melanocytes through secretion of a key factor: RGMB.

MPS1 inhibition primes immunogenicity of KRAS-LKB1 mutant lung cancer.

KRAS-LKB1 (KL) mutant lung cancers silence STING owing to intrinsic mitochondrial dysfunction, resulting in T cell exclusion and resistance to programmed cell death (ligand) 1 (PD-[L]1) blockade. Here we discover that KL cells also minimize intracellular accumulation of 2'3'-cyclic GMP-AMP (2'3'-cGAMP) to further avoid downstream STING and STAT1 activation. An unbiased screen to co-opt this vulnerability reveals that transient MPS1 inhibition (MPS1i) potently re-engages this pathway in KL cells via micronuclei generation. This effect is markedly amplified by epigenetic de-repression of STING and only requires pulse MPS1i treatment, creating a therapeutic window compared with non-dividing cells. A single course of decitabine treatment followed by pulse MPS1i therapy restores T cell infiltration in vivo, enhances anti-PD-1 efficacy, and results in a durable response without evidence of significant toxicity.

eIF2B-capturing viral protein NSs suppresses the integrated stress response.

Various stressors such as viral infection lead to the suppression of cap-dependent translation and the activation of the integrated stress response (ISR), since the stress-induced phosphorylated eukaryotic translation initiation factor 2 [eIF2(αP)] tightly binds to eIF2B to prevent it from exchanging guanine nucleotide molecules on its substrate, unphosphorylated eIF2. Sandfly fever Sicilian virus (SFSV) evades this cap-dependent translation suppression through the interaction between its nonstructural protein NSs and host eIF2B. However, its precise mechanism has remained unclear. Here, our cryo-electron microscopy (cryo-EM) analysis reveals that SFSV NSs binds to the α-subunit of eIF2B in a competitive manner with eIF2(αP). Together with SFSV NSs, eIF2B retains nucleotide exchange activity even in the presence of eIF2(αP), in line with the cryo-EM structures of the eIF2B•SFSV NSs•unphosphorylated eIF2 complex. A genome-wide ribosome profiling analysis clarified that SFSV NSs expressed in cultured human cells attenuates the ISR triggered by thapsigargin, an endoplasmic reticulum stress inducer. Furthermore, SFSV NSs introduced in rat hippocampal neurons and human induced-pluripotent stem (iPS) cell-derived motor neurons exhibits neuroprotective effects against the ISR-inducing stress. Since ISR inhibition is beneficial in various neurological disease models, SFSV NSs may be a promising therapeutic ISR inhibitor.

On-chip 3D neuromuscular model for drug screening and precision medicine in neuromuscular disease.

This protocol describes the design, fabrication and use of a 3D physiological and pathophysiological motor unit model consisting of motor neurons coupled to skeletal muscles interacting via the neuromuscular junction (NMJ) within a microfluidic device. This model facilitates imaging and quantitative functional assessment. The 'NMJ chip' enables real-time, live imaging of axonal outgrowth, NMJ formation and muscle maturation, as well as synchronization of motor neuron activity and muscle contraction under optogenetic control for the study of normal physiological events. The proposed protocol takes ~2-3 months to be implemented. Pathological behaviors associated with various neuromuscular diseases, such as regression of motor neuron axons, motor neuron death, and muscle degradation and atrophy can also be recapitulated in this system. Disease models can be created by the use of patient-derived induced pluripotent stem cells to generate both the motor neurons and skeletal muscle cells used. This is demonstrated by the use of cells from a patient with sporadic amyotrophic lateral sclerosis but can be applied more generally to models of neuromuscular disease, such as spinal muscular atrophy, NMJ disorder and muscular dystrophy. Models such as this hold considerable potential for applications in precision medicine, drug screening and disease risk assessment.

Three-Dimensional Motor Nerve Organoid Generation.

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and neurodegenerative diseases. Although numerous studies of axons have been conducted, our understanding of formation and dysfunction of axon fascicles is still limited due to the lack of robust three-dimensional in vitro models. Here, we describe a step-by-step protocol for the rapid generation of a motor nerve organoid (MNO) from human induced pluripotent stem (iPS) cells in a microfluidic-based tissue culture chip. First, fabrication of chips used for the method is described. From human iPS cells, a motor neuron spheroid (MNS) is formed. Next, the differentiated MNS is transferred into the chip. Thereafter, axons spontaneously grow out of the spheroid and assemble into a fascicle within a microchannel equipped in the chip, which generates an MNO tissue carrying a bundle of axons extended from the spheroid. For the downstream analysis, MNOs can be taken out of the chip to be fixed for morphological analyses or dissected for biochemical analyses, as well as calcium imaging and multi-electrode array recordings. MNOs generated with this protocol can facilitate drug testing and screening and can contribute to understanding of mechanisms underlying development and diseases of axon fascicles.

Modeling Nanocarrier Transport across a 3D In Vitro Human Blood-Brain-Barrier Microvasculature.

Polymer nanoparticles (NPs), due to their small size and surface functionalization potential have demonstrated effective drug transport across the blood-brain-barrier (BBB). Currently, the lack of in vitro BBB models that closely recapitulate complex human brain microenvironments contributes to high failure rates of neuropharmaceutical clinical trials. In this work, a previously established microfluidic 3D in vitro human BBB model, formed by the self-assembly of human-induced pluripotent stem cell-derived endothelial cells, primary brain pericytes, and astrocytes in triculture within a 3D fibrin hydrogel is exploited to quantify polymer NP permeability, as a function of size and surface chemistry. Microvasculature are perfused with commercially available 100-400 nm fluorescent polystyrene (PS) NPs, and newly synthesized 100 nm rhodamine-labeled polyurethane (PU) NPs. Confocal images are taken at different timepoints and computationally analyzed to quantify fluorescence intensity inside/outside the microvasculature, to determine NP spatial distribution and permeability in 3D. Results show similar permeability of PS and PU NPs, which increases after surface-functionalization with brain-associated ligand holo-transferrin. Compared to conventional transwell models, the method enables rapid analysis of NP permeability in a physiologically relevant human BBB set-up. Therefore, this work demonstrates a new methodology to preclinically assess NP ability to cross the human BBB.