Critical changes in hypothalamic gene networks in response to pancreatic cancer as found by single-cell RNA sequencing

OBJECTIVE: Cancer cachexia is a devastating chronic condition characterized by involuntary weight loss, muscle wasting, abnormal fat metabolism, anorexia, and fatigue. However, the molecular mechanisms underlying this syndrome remain poorly understood. In particular, the hypothalamus may play a central role in cachexia, given that it has direct access to peripheral signals because of its anatomical location and attenuated blood–brain barrier. Furthermore, this region has a critical role in regulating appetite and metabolism. METHODS: To provide a detailed analysis of the hypothalamic response to cachexia, we performed single-cell RNA-seq combined with RNA-seq of the medial basal hypothalamus (MBH) in a mouse model for pancreatic cancer. RESULTS: We found many cell type-specific changes, such as inflamed endothelial cells, stressed oligodendrocyes and both inflammatory and moderating microglia. Lcn2, a newly discovered hunger suppressing hormone, was the highest induced gene. Interestingly, cerebral treatment with LCN2 not only induced many of the observed molecular changes in cachexia but also affected gene expression in food-intake decreasing POMC neurons. In addition, we found that many of the cachexia-induced molecular changes found in the hypothalamus mimic those at the primary tumor site. CONCLUSION: Our data reveal that multiple cell types in the MBH are affected by tumor-derived factors or host factors that are induced by tumor growth, leading to a marked change in the microenvironment of neurons critical for behavioral, metabolic, and neuroendocrine outputs dysregulated during cachexia. The mechanistic insights provided in this study explain many of the clinical features of cachexia and will be useful for future therapeutic development

Expression of 8-oxoguanine DNA glycosylase (Ogg1) in mouse retina

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Functional analysis of the mouse CBP gene in the adult central nervous system

CBP (CREB-binding protein) was originally characterized as a transcriptional coactivator of CREB (cAMP response element-binding protein) which is implicated in many processes including the formation of long-term memory. CBP itself is regulated by nuclear Ca2+ and Ca2+/Calmodulin kinase IV, suggesting a crucial role in synaptic plasticity. Mutations in human CBP gene are associated with the Rubinstein-Taybi syndrome (RTS) characterized by mental retardation and patterning abnormalities. These features suggest that CBP plays a central role in cAMP-mediated gene expression, which in turn is implicated in the formation of long-term memory. The expression patterns of CBP and its homologue p300 were analyzed. Both are expressed in most of forebrain areas, with particularly high expression in the hippocampus. Parts of the mouse CBP gene were characterized. Six exons encode the CREB-binding domain (CBD). Two gene targeting experiments were performed to generate mouse lines with mutations in the CBP gene. The first was a deletion of the CBD exon 2, resulting in a frameshift and, thus, a truncated CBP protein (CBPstop523). In the second experiment, a point mutation was aimed to be introduced in the CBD exon 5 (CBPTyr658Ala). After germline transmission, three mouse lines were obtained from CBPstop523 allele: CBD2+/-, a general deletion of the CBD exon 2; CBD2.floxed, which will be crossed with different Cre mouse lines for tissue-specific mutants; and CBD2.floxed.neo, which might be a hypomorphic allele due to the Neo cassette. For the construct CBPTyr658Ala none of the chimeras gave germline transmission, as apparently the splicing of the inverted allele was incorrect, possibly leading to strong reduction of vitality. Meantime, six CBP mutants were generated to elucidate functional domains of CBP in vitro: a deletion mutation CBPACBD2-5; CBPllOOaa, which was reported in mutant mice displaying RTS-like phenotypes; and a point mutation CBP658Ala, which was shown to be critical for interaction with phospho-CREB. The other three mutants retain different sizes of truncated N-terminal CBP and similar proteins were observed in RTS. Both CBP658Ala and CBPACBD2-5 interferred with the cAMP pathway, but not with nuclear receptor-mediated functions. RTS mutants showed strong inhibitory effects on the cAMP pathway

Phosphatidylinositol 3 kinase modulation of trophoblast cell differentiation

Abstract Background The trophoblast lineage arises as the first differentiation event during embryogenesis. Trophoblast giant cells are one of several end-stage products of trophoblast cell differentiation in rodents. These cells are located at the maternal-fetal interface and are capable of invasive and endocrine functions, which are necessary for successful pregnancy. Rcho-1 trophoblast stem cells can be effectively used as a model for investigating trophoblast cell differentiation. In this report, we evaluated the role of the phosphatidylinositol 3-kinase (PI3K) signaling pathway in the regulation of trophoblast cell differentiation. Transcript profiles from trophoblast stem cells, differentiated trophoblast cells, and differentiated trophoblast cells following disruption of PI3K signaling were generated and characterized. Results Prominent changes in gene expression accompanied the differentiation of trophoblast stem cells. PI3K modulated the expression of a subset of trophoblast cell differentiation-dependent genes. Among the PI3K-responsive genes were those encoding proteins contributing to the invasive and endocrine phenotypes of trophoblast giant cells. Conclusions Genes have been identified with differential expression patterns associated with trophoblast stem cells and trophoblast cell differentiation; a subset of these genes are regulated by PI3K signaling, including those impacting the differentiated trophoblast giant cell phenotype.Peer Reviewe