Biology subjects

Fuchtbauer, E.-M.

Publications and source records attributed to Fuchtbauer, E.-M..

4 recordsLinked to original sources

bioRxiv2021

A new transgene mouse model using an extravesicular EGFP tag to elucidate the in vivo function of extracellular vesicles

The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate. We therefore created an EV reporter using CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9-EGFP expression in cells did not affect EV size and concentration, but allowed for co-precipitation of EV markers TSG101 and ALIX from cell-conditioned medium by anti-GFP immunoprecipitation. We created a transgenic mouse where CD9-EGFP was inserted in the inverse orientation and double-floxed, ensuring Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV- Cre), in cardiomyocytes (AMHC-Cre) and kidney epithelium (Pax8-Cre), respectively. The mice showed tissue-specific EGFP expression, and plasma and urine samples were used to immunoprecipitate EVs. CD9-EGFP EVs was detected in plasma samples from CMV-Cre/CD9-EGFP and AMHC-Cre/CD9-EGFP mice, but not in PAX8-Cre/CD9-EGFP mice. On the other hand, CD9-EGFP EVs were detected in urine samples from CMV-Cre/CD9-EGFP and PAX8-Cre/CD9-EGFP mice, but not AMHC-Cre/CD9-EGFP, indicating that plasma EVs are not filtered to the urine. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to study cell-specific EVs in vivo and gain new insight into their physiological and pathophysiological function.

molecular biology↗

bioRxiv2020

SorCS1 binds the insulin receptor to enhance insulin sensitivity

Type 2 diabetes has reached endemic proportions and is a substantial burden for the affected patients and the society. Along with lifestyle factors, a number of genetic loci predisposing to type 2 diabetes have been identified, including SORCS1 that encodes the transmembrane receptor SorCS1. The ectodomain of SorCS1 (sol-SorCS1) is shed from plasma membranes but the biological function of this fragment is unknown. Here we show that sol-SorCS1 acts as a high-affinity binding partner for the insulin receptor to stabilize the receptor and increase insulin affinity, protein kinase B activation, and glucose uptake in myocytes. Sol-SorCS1 is liberated from adipocytes, and in diabetic patients the plasma concentration positively correlates with body mass index, but inversely with plasma glucose. In mouse models of insulin resistance, exogenous sol-SorCS1 restored insulin sensitivity. We conclude that sol-SorCS1 increases peripheral insulin sensitivity and propose sol-SorCS1 as a novel insulin sensitizing adipokine and potential antidiabetic agent.

physiology↗

bioRxiv2018

Expression of the miR-302/367 microRNA cluster is regulated by a conserved long non-coding host gene

MicroRNAs (miRNAs) are important regulators of cellular functions. MiR-302/367 is a polycistronic miRNA cluster including miR-302b/c/a/d (collectively termed miR-302s) and miR-367. The cluster is located in the intron of a non-coding host gene. MiR-302s have been shown to repress mRNAs required for differentiation and to induce pluripotency in somatic cells. The stem cell specific transcription factors OCT4, SOX2 and Nanog drive miR-302s expression, however, the reported expression in human and mice indicates a more complex transcriptional regulation. Here we investigate the transcriptional control and the processing of the miR-302 host gene. The murine miR-302 host gene is alternatively spliced, polyadenylated and exported from the nucleus. The regulatory sequences extend at least 2 kb upstream of the transcription start side and contain several conserved binding sites for both transcriptional activators and repressors. Reporter constructs with different upstream regions revealed a significant influence of the more distant regulatory sequences in pluripotent stem cells. The gene structure and regulatory elements like binding sites for activating and repressing transcriptional regulators, splice, and polyadenylation signals are highly conserved between mouse and human. So far, no miR-302 independent function has been annotated for the miR-302 host gene and we hypothesize that the complex and differential regulation of the miRNA transcription and processing might the reason for its conservation. Thus, regulation or micro-RNA expression might be a so far less recognized function of non-coding RNA genes.\n\nAuthor SummaryNon-coding RNAs constitute a large part of the mammalian genome. Interestingly, some long non-coding RNAs (lncRNA) are transcribed and processed in the same way as mRNAs but lack an open reading frame. Here we give evidence that a so far less recognized function of such lncRNAs could be to supply microRNAs with the complex transcriptional control and processing required for their intricate expression. As an example, we analyzed the regulatory sequences of the miR-302/367 host gene. MiR-302/367 is a microRNA cluster involved in the regulation of stem cells and cellular differentiation. We show here that the regulatory region is much more complex than anticipated, a complexity that can not be conferred alone by any of the stem cell specific transcription factors which were so far associated with the expression of miR-302/367.

genetics↗

bioRxiv2018

Isolation of cancer stem cells by selection for miR-302 expressing cells

Cancer stem cells receive increasing interest because they are believed to be a major reason for long-term therapy failure. The reason for the therapy resistance of cancer stem cells lies partially in their multi-drug resistance and partially in the ability to rest mitotically inactive in the hypoxic center of tumors. Due to their variable number and their often low proliferation rate, cancer stem cells are difficult to purify in decent quantities and to grow in cell culture systems, where they are easily outcompeted by faster growing more differentiated, i.e. less stem cell-like tumor cells. Here we present a proof of principle study based on the idea to select cancer stem cells by means of the expression of a stem cell-specific gene. We inserted a selectable egfp-neo coding sequence in the last exon of the non-coding murine miR-302 host gene. As a stem cell specific regulatory element, we used 2.1 kb of the genomic region immediately upstream of the miR-302 host gene transcription start. Stable transgenic CJ7 embryonic stem cells were used to induce teratomas. After three weeks, tumors were removed for analysis and primary cultures were established. Stem-like cells were selected from these culture based on G418 selection. When the selection was removed, stem cell morphology and miR-302 expression were rapidly lost, indicating that it were not the original ES cells that have been isolated. In conclusion, we show the possibility to use drug resistance expressed from a regulatory sequence of a stem cell-specific marker, to isolate and propagate cancer stem cells that otherwise might be hidden in the majority of tumor cells.

cancer biology↗