Toshiki Kameyama

The hypothalamus is comprised of heterogenous cell populations and includes highly complex neural circuits that regulate the autonomic nerve system. Its dysfunction therefore results in severe endocrine disorders. Although recent experiments have been conducted for in vitro organogenesis of hypothalamic neurons from embryonic stem (ES) or induced pluripotent stem (iPS) cells, whether these stem cell-derived hypothalamic neurons can be useful for regenerative medicine remains unclear. We therefore performed orthotopic transplantation of mouse ES cell (mESC)-derived hypothalamic neurons into adult mouse brains. We generated electrophysiologically functional hypothalamic neurons from mESCs and transplanted them into the supraoptic nucleus of mice. Grafts extended their axons along hypothalamic nerve bundles in host brain, and some of them even projected into the posterior pituitary (PPit), which consists of distal axons of the magnocellular neurons located in hypothalamic supraoptic and paraventricular nuclei. The axonal projections to the PPit were not observed when the mESC-derived hypothalamic neurons were ectopically transplanted into the substantia nigra reticular part. These findings suggest that our stem cell-based orthotopic transplantation approach might contribute to the establishment of regenerative medicine for hypothalamic and pituitary disorders.

Human stem cell-derived organoid culture enables the in vitro analysis of the cellular function in three-dimensional aggregates mimicking native organs, and also provides a valuable source of specific cell types in the human body. We previously established organoid models of the hypothalamic-pituitary (HP) complex using human pluripotent stem cells. Although the models are suitable for investigating developmental and functional HP interactions, we consider that isolated pituitary cells are also useful for basic and translational research on the pituitary gland, such as stem cell biology and regenerative medicine. To develop a method for the purification of pituitary cells in HP organoids, we performed surface marker profiling of organoid cells derived from human induced pluripotent stem cells (iPSCs). Screening of 332 human cell surface markers and a subsequent immunohistochemical analysis identified epithelial cell adhesion molecule (EpCAM) as a surface marker of anterior pituitary cells, as well as their ectodermal precursors. EpCAM was not expressed on hypothalamic lineages; thus, anterior pituitary cells were successfully enriched by magnetic separation of EpCAM⁺ cells from iPSC-derived HP organoids. The enriched pituitary population contained functional corticotrophs and their progenitors; the former responded normally to a corticotropin-releasing hormone stimulus. Our findings would extend the applicability of organoid culture as a novel source of human anterior pituitary cells, including stem/progenitor cells and their endocrine descendants.

Hypothalamic melanin-concentrating hormone (MCH) neurons are important regulators of multiple physiological processes, such as sleep, feeding, and memory. Despite the increasing interest in their neuronal functions, the molecular mechanism underlying MCH neuron development remains poorly understood. We report that a three-dimensional culture of mouse embryonic stem cells (mESCs) can generate hypothalamic-like tissues containing MCH-positive neurons, which reproduce morphologic maturation, neuronal connectivity, and neuropeptide/neurotransmitter phenotype of native MCH neurons. Using this in vitro system, we demonstrate that Hedgehog (Hh) signaling serves to produce major neurochemical subtypes of MCH neurons characterized by the presence or absence of cocaine- and amphetamine-regulated transcript (CART). Without exogenous Hh signals, mESCs initially differentiated into dorsal hypothalamic/prethalamic progenitors and finally into MCH+CART+ neurons through a specific intermediate progenitor state. Conversely, activation of the Hh pathway specified ventral hypothalamic progenitors that generate both MCH+CART- and MCH+CART+ neurons. These results suggest that in vivo MCH neurons may originate from multiple cell lineages that arise through early dorsoventral patterning of the hypothalamus. Additionally, we found that Hh signaling supports the differentiation of mESCs into orexin/hypocretin neurons, a well-defined cell group intermingled with MCH neurons in the lateral hypothalamic area (LHA). The present study highlights and improves the utility of mESC culture in the analysis of the developmental programs of specific hypothalamic cell types.Significance StatementA growing body of literature has revealed the importance of hypothalamic melanin-concentrating hormone (MCH) neurons in energy homeostasis and the cognitive function, but their developmental biology remains relatively unknown. To establish a new approach for addressing this issue, we tested the ability of an in vitro differentiation system of mouse embryonic stem cells (mESCs) to recapitulate the development of MCH neurons. The mESC culture robustly generated MCH-positive neurons resembling native neurons in several aspects and provided evidence that Hedgehog (Hh) signaling is a key factor to produce neurochemical subtypes of MCH neurons. Our results demonstrate the suitability of mESC culture as a platform to study the molecular mechanisms underlying the development of MCH neurons and possibly of other hypothalamic cell types.

Tumor viruses gain control of cellular functions when they infect and transform host cells. Alternative splicing is one of the cellular processes exploited by tumor viruses to benefit viral replication and support oncogenesis. Epstein-Barr virus (EBV) participates in a number of cancers, as reported mostly in nasopharyngeal carcinoma (NPC) and Burkitt lymphoma (BL). Using RT-nested-PCR and Northern blot analysis in NPC and BL cells, here we demonstrate that EBV promotes specific alternative splicing of TSG101 pre-mRNA, which generates the TSG101∆154-1054 variant though the agency of its viral proteins, such as EBNA-1, Zta and Rta. The level of TSG101∆154-1054 is particularly enhanced upon EBV entry into the lytic cycle, increasing protein stability of TSG101 and causing the cumulative synthesis of EBV late lytic proteins, such as VCA and gp350/220. TSG101∆154-1054-mediated production of VCA and gp350/220 is blocked by the overexpression of a translational mutant of TSG101∆154-1054 or by the depletion of full-length TSG101, which is consistent with the known role of the TSG101∆154-1054 protein in stabilizing the TSG101 protein. NPC patients whose tumor tissues express TSG101∆154-1054 have high serum levels of anti-VCA antibodies and high levels of viral DNA in their tumors. Our findings highlight the functional importance of TSG101∆154-1054 in allowing full completion of the EBV lytic cycle to produce viral particles. We propose that targeting EBV-induced TSG101 alternative splicing has broad potential as a therapeutic to treat EBV-associated malignancies.

<title>Abstract</title>Using the TSG101 pre-mRNA, we previously discovered cancer-specific re-splicing of mature mRNA that generates aberrant transcripts/proteins. The fact that mRNA is aberrantly re-spliced in various cancer cells implies there must be an important mechanism to prevent deleterious re-splicing on the spliced mRNA in normal cells. We thus postulated that the mRNA re-splicing is controlled by specific repressors and we searched for repressor candidates by siRNA-based screening for mRNA re-splicing activity. We found that knock-down of EIF4A3, which is a core component of the exon junction complex (EJC), significantly promoted mRNA re-splicing. Remarkably, we could recapitulate cancer-specific mRNA re-splicing in normal cells by knock-down of any of the core EJC proteins, EIF4A3, MAGOH or RBM8A (Y14), implicating the EJC core as the repressor of mRNA re-splicing often observed in cancer cells. We propose that the EJC core is a critical mRNA quality control factor to prevent over-splicing of mature mRNA.

Pathogenic bacteria of the genus Bartonella can induce vasoproliferative lesions during infection. The underlying mechanisms are unclear, but involve secretion of an unidentified mitogenic factor. Here, we use functional transposon-mutant screening in Bartonella henselae to identify such factor as a pro-angiogenic autotransporter, called BafA. The passenger domain of BafA induces cell proliferation, tube formation and sprouting of microvessels, and drives angiogenesis in mice. BafA interacts with vascular endothelial growth factor (VEGF) receptor-2 and activates the downstream signaling pathway, suggesting that BafA functions as a VEGF analog. A BafA homolog from a related pathogen, Bartonella quintana, is also functional. Our work unveils the mechanistic basis of vasoproliferative lesions observed in bartonellosis, and we propose BafA as a key pathogenic factor contributing to bacterial spread and host adaptation.

TSG101 (Tumor susceptibility 101) gene and its aberrantly spliced isoform, termed TSG101∆154-1054, are tightly linked to tumorigenesis in various cancers. The aberrant TSG101∆154-1054 mRNA is generated from cancer-specific re-splicing of mature TSG101 mRNA. The TSG101∆154-1054 protein protects the full-length TSG101 protein from ubiquitin-mediated degradation, implicating TSG101∆154-1054 protein in the progression of cancer. Here, we confirmed that the presence of TSG101∆154-1054 mRNA indeed caused an accumulation of the TSG101 protein in biopsies of human nasopharyngeal carcinoma (NPC), which was recapitulated by the overexpression of TSG101∆154-1054 in the NPC cell line TW01. We demonstrate the potential function of the TSG101∆154-1054 protein in the malignancy of human NPC with scratch-wound healing and transwell invasion assays. By increasing the stability of the TSG101 protein, TSG101∆154-1054 specifically enhanced TSG101-mediated TW01 cell migration and invasion, suggesting the involvement in NPC metastasis in vivo. This finding sheds light on the functional significance of TSG101∆154-1054 generation via re-splicing of TSG101 mRNA in NPC metastasis and hints at its potential importance as a therapeutic target.

Alternative splicing (AS) contributes to the generation of distinct mRNAs from the same pre-mRNA. This mechanism is highly conserved in eukaryotes and yields proteomic diversity. Furthermore, AS can be tissue- and developmental stage-specific and is an important process in the development of the central nervous system. Although these features suggest that AS variants contribute the complexity of the brain, the characteristics of most AS variants are unclear. Therefore, it is important to identify and examine novel AS variants in neuronal tissues to fully understand neuronal functions. We have identified a novel AS variant of murine disrupted in schizophrenia 1 (Disc1), a neural developmental gene. This variant lacks exon 2 and is named 'ΔE2'. We also show differences in the expression levels and localization patterns between ΔE2 and full length Disc1 (FL) in the murine brain. Differences in the subcellular localization patterns of both isoforms in cultured SK-N-SH cells suggest that ΔE2 has different functions compared with those of FL. Further functional analysis of ΔE2 may indicate additional roles of Disc1, and may further our understanding of the developmental mechanisms for neurodevelopmental disorders such as schizophrenia.