ARID1B controls transcriptional programs of axon projection in an organoid model of the human corpus callosum.

Mutations in ARID1B, a member of the mSWI/SNF complex, cause severe neurodevelopmental phenotypes with elusive mechanisms in humans. The most common structural abnormality in the brain of ARID1B patients is agenesis of the corpus callosum (ACC), characterized by the absence of an interhemispheric white matter tract that connects distant cortical regions. Here, we find that neurons expressing SATB2, a determinant of callosal projection neuron (CPN) identity, show impaired maturation in ARID1B+/- neural organoids. Molecularly, a reduction in chromatin accessibility of genomic regions targeted by TCF-like, NFI-like, and ARID-like transcription factors drives the differential expression of genes required for corpus callosum (CC) development. Through an in vitro model of the CC tract, we demonstrate that this transcriptional dysregulation impairs the formation of long-range axonal projections, causing structural underconnectivity. Our study uncovers new functions of the mSWI/SNF during human corticogenesis, identifying cell-autonomous axonogenesis defects in SATB2+ neurons as a cause of ACC in ARID1B patients.

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.

dCas13-mediated translational repression for accurate gene silencing in mammalian cells.

Current gene silencing tools based on RNA interference (RNAi) or, more recently, clustered regularly interspaced short palindromic repeats (CRISPR)‒Cas13 systems have critical drawbacks, such as off-target effects (RNAi) or collateral mRNA cleavage (CRISPR‒Cas13). Thus, a more specific method of gene knockdown is needed. Here, we develop CRISPRδ, an approach for translational silencing, harnessing catalytically inactive Cas13 proteins (dCas13). Owing to its tight association with mRNA, dCas13 serves as a physical roadblock for scanning ribosomes during translation initiation and does not affect mRNA stability. Guide RNAs covering the start codon lead to the highest efficacy regardless of the translation initiation mechanism: cap-dependent, internal ribosome entry site (IRES)-dependent, or repeat-associated non-AUG (RAN) translation. Strikingly, genome-wide ribosome profiling reveals the ultrahigh gene silencing specificity of CRISPRδ. Moreover, the fusion of a translational repressor to dCas13 further improves the performance. Our method provides a framework for translational repression-based gene silencing in eukaryotes.

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.

MeCP2 represses the activity of topoisomerase IIß in long neuronal genes.

A unique signature of neurons is the high expression of the longest genes in the genome. These genes have essential neuronal functions, and disruption of their expression has been implicated in neurological disorders. DNA topoisomerases resolve DNA topological constraints and facilitate neuronal long gene expression. Conversely, the Rett syndrome protein, methyl-CpG-binding protein 2 (MeCP2), can transcriptionally repress long genes. How these factors regulate long genes is not well understood, and whether they interact is not known. Here, we identify and map a functional interaction between MeCP2 and topoisomerase IIß (TOP2ß) in mouse neurons. We profile neuronal TOP2ß activity genome wide, detecting enrichment at regulatory regions and gene bodies of long genes, including MeCP2-regulated genes. We show that loss and overexpression of MeCP2 alter TOP2ß activity at MeCP2-regulated genes. These findings uncover a mechanism of TOP2ß inhibition by MeCP2 in neurons and implicate TOP2ß dysregulation in disorders caused by MeCP2 disruption.

yaaJ, the tRNA-Specific Adenosine Deaminase, Is Dispensable in Bacillus subtilis.

Post-transcriptional modifications of tRNA are crucial for their core function. The inosine (I; 6-deaminated adenosine) at the first position in the anticodon of tRNAArg(ICG) modulates the decoding capability and is generally considered essential for reading CGU, CGC, and CGA codons in eubacteria. We report here that the Bacillus subtilis yaaJ gene encodes tRNA-specific adenosine deaminase and is non-essential for viability. A ß-galactosidase reporter assay revealed that the translational activity of CGN codons was not impaired in the yaaJ-deletion mutant. Furthermore, tRNAArg(CCG) responsible for decoding the CGG codon was dispensable, even in the presence or absence of yaaJ. These results strongly suggest that tRNAArg with either the anticodon ICG or ACG has an intrinsic ability to recognize all four CGN codons, providing a fundamental concept of non-canonical wobbling mediated by adenosine and inosine nucleotides in the anticodon. This is the first example of the four-way wobbling by inosine nucleotide in bacterial cells. On the other hand, the absence of inosine modification induced +1 frameshifting, especially at the CGA codon. Additionally, the yaaJ deletion affected growth and competency. Therefore, the inosine modification is beneficial for translational fidelity and proper growth-phase control, and that is why yaaJ has been actually conserved in B. subtilis.

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.

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.

Two-photon, red light uncaging of alkyl radicals from organorhodium(III) phthalocyanine complexes.

A stepwise two-photon, red light excitation of organorhodium(III) phthalocyanine complexes was found to induce the activation of the axial metal-carbon bond to generate alkyl radicals/aldehydes. The cooperative action of the photouncaging reaction and the photochemical generation of reactive oxygen species were indicated to induce the cell deaths.

From real-time single to multicompartmental Hodgkin-Huxley neurons on FPGA for bio-hybrid systems.

Modeling biological neural networks has been a field opening to major advances in our understanding of the mechanisms governing the functioning of the brain in normal and pathological conditions. The emergence of real-time neuromorphic platforms has been leading to a rising significance of bio-hybrid experiments as part of the development of neuromorphic biomedical devices such as neuroprosthesis. To provide a new tool for the neurological disorder characterization, we design real-time single and multicompartmental Hodgkin-Huxley neurons on FPGA. These neurons allow biological neural network emulation featuring improved accuracy through compartment modeling and show integration in bio-hybrid system thanks to its real-time dynamics.

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.

Modeling Axonal Degeneration Using Motor Nerve Organoids.

Degeneration of axons is characteristic of many devastating diseases including amyotrophic lateral sclerosis (ALS). However, lack of an in vitro neuronal culture system that mimics damages on nerves and axonal tracts hampered development of effective treatments. Here, we describe a method to model degeneration of motor neuron axons using motor nerve organoids that are formed with human induced pluripotent stem cells. In this protocol, motor neuron axon degeneration can be rapidly induced with chemical damages. Neuroprotective effects of compounds can be examined using the degenerated axons. This motor neuron axon bundle degeneration model should facilitate future screening for drugs against diseases affecting axon fascicles.

Localised light delivery on melanoma cells using optical microneedles.

Light-based therapy is an emerging treatment for skin cancer, which has received increased attention due to its drug-free and non-invasive approach. However, the limitation of current light therapy methods is the inability for light to penetrate the skin and reach deep lesions. As such, we have developed a polylactic acid (PLA) microneedles array as a novel light transmission platform to perform in vitro evaluation regarding the effect of light therapy on skin cancer. For the first time, we designed and fabricated a microneedle array system with a height fixation device that can be installed in a cell culture dish and an LED array for blue light irradiation. The effect of the blue light combined with the microneedles on cell apoptosis was evaluated using B16F10 melanoma cells and analyzed by Hoechst staining. Our results demonstrate that blue light can be transmitted by microneedles to skin cells and effectively affect cell viability.

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.

Light-Induced Differentiation of Forebrain Organoids by NVOC-SAG.

Hedgehog signaling pathway shapes our body by regulating proliferation and differentiation of cells. The spatial and temporal distribution pattern of its ligands finely controls the activity of the Hedgehog pathway during development. To mimic the active regulation of Hedgehog pathway, we have developed a light-inducible Hedgehog signaling activator 6-nitroveratryloxy-carbonyl Smoothened agonist (NVOC-SAG). Here we describe a method to selectively induce ventral differentiation of human iPS cell-derived forebrain organoids in a light-dependent manner. This article describes preparation of NVOC-SAG, culture of iPS cell-derived forebrain organoids, light irradiation, and downstream analyses.

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.

Reduced PHOX2B stability causes axonal growth impairment in motor neurons with TARDBP mutations.

Amyotrophic lateral sclerosis (ALS) is an adult-onset incurable motor neuron (MN) disease. The reasons for selective MN vulnerability in ALS are unknown. Axonal pathology is among the earliest signs of ALS. We searched for novel modulatory genes in human MN axon shortening affected by TARDBP mutations. In transcriptome analysis of RNA present in the axon compartment of human-derived induced pluripotent stem cell (iPSC)-derived MNs, PHOX2B (paired-like homeobox protein 2B) showed lower expression in TARDBP mutant axons, which was consistent with axon qPCR and in situ hybridization. PHOX2B mRNA stability was reduced in TARDBP mutant MNs. Furthermore, PHOX2B knockdown reduced neurite length in human MNs. Finally, phox2b knockdown in zebrafish induced short spinal axons and impaired escape response. PHOX2B is known to be highly express in other types of neurons maintained after ALS progression. Collectively, TARDBP mutations induced loss of axonal resilience, which is an important ALS-related phenotype mediated by PHOX2B downregulation.

Light-inducible control of cellular proliferation and differentiation by a Hedgehog signaling inhibitor.

The Hedgehog (Hh) signaling pathway is a major regulator of cell differentiation and proliferation. Aberrant activation of the Hh pathway has been implicated in several types of cancer. To understand the Hedgehog pathway and fight against related diseases, it is important to inhibit Hedgehog signaling in a targeted manner. However, no tools are available for the precise inhibition of Hh signaling in a spatiotemporal manner. In this study, we synthesized and evaluated the bioactivity of a light-inducible Hh pathway inhibitor (NVOC-SANT-75). NVOC-SANT-75 inhibits transcription factor Gli1 in NIH3T3 cells and controls proliferation and differentiation of primary cultured mouse cerebellar neurons in a light-irradiation-dependent manner. The light-inducible Hedgehog signaling inhibitors may be a new candidate for light-mediated cancer treatment.