Design of neural organoids engineered by mechanical forces.

Neural organoids consist of three-dimensional tissue derived from pluripotent stem cells that could recapitulate key features of the human brain. During the past decade, organoid technology has evolved in the field of human brain science by increasing the quality and applicability of its products. Among them, a novel approach involving the design of neural organoids engineered by mechanical forces has emerged. This review describes previous approaches for the generation of neural organoids, the engineering of neural organoids by mechanical forces, and future challenges for the application of mechanical forces in the design of neural organoids.

Induction of inverted morphology in brain organoids by vertical-mixing bioreactors.

Organoid technology provides an opportunity to generate brain-like structures by recapitulating developmental steps in the manner of self-organization. Here we examined the vertical-mixing effect on brain organoid structures using bioreactors and established inverted brain organoids. The organoids generated by vertical mixing showed neurons that migrated from the outer periphery to the inner core of organoids, in contrast to orbital mixing. Computational analysis of flow dynamics clarified that, by comparison with orbital mixing, vertical mixing maintained the high turbulent energy around organoids, and continuously kept inter-organoid distances by dispersing and adding uniform rheological force on organoids. To uncover the mechanisms of the inverted structure, we investigated the direction of primary cilia, a cellular mechanosensor. Primary cilia of neural progenitors by vertical mixing were aligned in a multidirectional manner, and those by orbital mixing in a bidirectional manner. Single-cell RNA sequencing revealed that neurons of inverted brain organoids presented a GABAergic character of the ventral forebrain. These results suggest that controlling fluid dynamics by biomechanical engineering can direct stem cell differentiation of brain organoids, and that inverted brain organoids will be applicable for studying human brain development and disorders in the future.

Gene expression of PLAT and ATS3 proteins increases plant resistance to insects.

MAIN CONCLUSION: Genes of the PLAT protein family, including PLAT and ATS3 subfamilies of higher plants and homologs of liverwort, are involved in plant defense against insects. Laticifer cells in plants contain large amounts of anti-microbe or anti-insect proteins and are involved in plant defense against biotic stresses. We previously found that PLAT proteins accumulate in laticifers of fig tree (Ficus carica) at comparable levels to those of chitinases, and the transcript level of ATS3, another PLAT domain-containing protein, is highest in the transcriptome of laticifers of Euphorbia tirucalli. In this study, we investigated whether the PLAT domain-containing proteins are involved in defense against insects. Larvae of the lepidopteran Spodoptera litura showed retarded growth when fed with Nicotiana benthamiana leaves expressing F. carica PLAT or E. tirucalli ATS3 genes, introduced by agroinfiltration using expression vector pBYR2HS. Transcriptome analysis of these leaves indicated that ethylene and jasmonate signaling were activated, leading to increased expression of genes for PR-1, ß-1,3-glucanase, PR5 and trypsin inhibitors, suggesting an indirect mechanism of PLAT- and ATS3-induced resistance in the host plant. Direct cytotoxicity of PLAT and ATS3 to insects was also possible because heterologous expression of the corresponding genes in Drosophila melanogaster caused apoptosis-mediated cell death in this insect. Larval growth retardation of S. litura occurred when they were fed radish sprouts, a good host for agroinfiltration, expressing any of nine homologous genes of dicotyledon Arabidopsis thaliana, monocotyledon Brachypodium distachyon, conifer Picea sitchensis and liverwort Marchantia polymorpha. Of these nine genes, the heterologous expression of A. thaliana AT5G62200 and AT5G62210 caused significant increases in larval death. These results indicated that the PLAT protein family has largely conserved anti-insect activity in the plant kingdom (249 words).

Neuron-specific knockdown of Drosophila PDHB induces reduction of lifespan, deficient locomotive ability, abnormal morphology of motor neuron terminals and photoreceptor axon targeting.

Pyruvate dehydrogenase complex deficiency (PDCD) is a common primary cause of defects in mitochondrial function and also can lead to peripheral neuropathy. Pyruvate dehydrogenase E1 component subunit beta (PDHB) is a subunit of pyruvate dehydrogenase E1, which is a well-known component of PDC. In Drosophila melanogaster, the CG11876 (dPDHB) gene is a homolog of human PDHB. In this study, we established a Drosophila model with neuron-specific knockdown of dPDHB to investigate its role in neuropathy pathogenesis. Knockdown of dPDHB in pan-neurons induced locomotor defects in both larval and adult stages, which were consistent with abnormal morphology of the motor neuron terminals at neuromuscular junctions and mitochondrial fragmentation in brains. Moreover, neuron-specific knockdown of dPDHB also shortened the lifespan of adult flies. In addition, flies with knockdown of dPDHB manifested a rough eye phenotype and aberrant photoreceptor axon targeting. These results with the Drosophila model suggest the involvement of PDHB in peripheral neuropathy.

Overexpression of dJmj differentially affects intestinal stem cells and differentiated enterocytes.

Jumonji (Jmj)/Jarid2 is a DNA-binding transcriptional repressor mediated via histone methylation. Nevertheless, the well-known function of Jmj is as a scaffold for the recruitment of various complexes including Polycomb repressive complex 2 (PRC2), and required for mouse embryonic stem cell development. However, PRC2 independent function is suggested for Drosophila Jumonji (dJmj). To clarify the function of dJmj during cell differentiation, we used Drosophila adult intestinal stem cell system that allows to follow stem cell behaviors in vivo. Overexpression of dJmj in intestinal stem cells/enteroblasts (ISCs/EBs) induces cell-autonomous ISC proliferation followed by differentiation, that is controlled by the Notch and EGFR pathway. In contrast, overexpression of dJmj in enterocytes (ECs) resulted in activation of the JNK pathway in ECs followed by the induction of apoptosis. Activated JNK increased the level of Yorkie in ECs and induced the reduction of Upd proteins and EGFR ligands, which activated the JAK/STAT and EGFR pathway in both ISCs and EBs to promote ISC proliferation. The Notch signaling pathway appears to be highly activated to support the differentiation of EBs to ECs. Thus, the combination of these signaling pathways caused by ECs-specific dJmj-overexpression induced non-cell-autonomous ISC proliferation and differentiation. Surprisingly, these effects did not relate to H3K27me3 status, likely represented PRC2 activity, in cells that overexpressed dJmj. Instead of this, the disappearance of H3K27me3 in ISC/EB-specific overexpressed dJmj suggested a possible PRC2-independent role of dJmj in regulating chromatin structure.

Polycomb-dependent nucleolus localization of Jumonji/Jarid2 during Drosophila spermatogenesis.

Drosophila Jumonji/Jarid2 (dJmj) has been identified as a component of Polycomb repressive complex 2. However, it is suggested that dJmj has both PRC-dependent and -independent roles. Subcellular localization of dJmj during spermatogenesis is unknown. We therefore performed immunocytochemical analyses with specific antibodies to dJmj and tri-methylation at lysine 27 on histone H3 (H3K27me3). Interestingly, dJmj exclusively localizes at nucleolus in the late growth stage. Examination of the dJmj localization in various Polycomb group (PcG) mutant lines at the late growth stage allowed identification of some PcG genes, including Polycomb (Pc), to be responsible for dJmj recruitment to nucleolus. In addition, we found that size of nucleolus was decreased in some of these mutant lines. In a mutant of testis-specific TAF homolog (tTAF) that is responsible for nucleolus localization of Pc, dJmj signals were detected not only at nucleolus but also on the condensed chromatin in the late growth stage. Duolink In situ Proximity ligation assay clarified that Pc interacts with dJmj at nucleolus in the late growth stage. Furthermore, the level of H3K27me3 decreased in nuclei at this stage. Taken together, we conclude that tTAF is responsible for recruitments of dJmj to nucleolus in the late growth stage that appears to be mediated by Pc. Compartmentalization of dJmj in nucleolus together with some of PcG may be necessary to de-repress the expression of genes required to cellular growth and proliferation in the following meiotic divisions.

Ubiquitin carboxyl hydrolase L1 significance for human diseases.

Ubiquitin carboxyl hydrolase L1 (UCH-L1) is an abundant multifunctional neuron protein. It plays an important role in maintaining the ubiquitin proteasome system (UPS), vital for recognizing and degrading dysfunctional proteins in organisms. In recent decades, UCH-L1 has been implicated in the pathogenesis of many diseases, including neurodegenerative disorders, cancer and diabetes. However, the mechanisms of UCH-L1 involvement have yet to be revealed in detail. Since UCH-L1 contributes many different functions to cell metabolism, its role and regulation might be more complex than previously thought and it has become a research target in many laboratories. In this review, we summarize recent findings related to the actions of UCH-L1 in several human diseases.