While Cre-dependent viral systems permit the manipulation of many neuron types, some cell populations cannot be targeted by a single DNA recombinase. Although the combined use of Flp and Cre recombinases can overcome this limitation, insufficient recombinase activity can reduce the efficacy of existing Cre+Flp-dependent viral systems. We developed a sensitive dual recombinase-activated viral approach: tTA-driven Recombinase-Guided Intersectional Targeting (tTARGIT) AAVs. tTARGIT AAVs utilize a Flp-dependent tetracycline transactivator (tTA) 'Driver' AAV and a tetracycline response element (TRE)-driven, Cre-dependent 'Payload' AAV to express the transgene of interest. We employed this system in Slc17a6FlpO;LeprCre mice to manipulate LepRb neurons of the ventromedial hypothalamus (VMH; LepRbVMH neurons) while omitting neighboring LepRb populations. We defined the circuitry of LepRbVMH neurons and roles for these cells in the control of food intake and energy expenditure. Thus, the tTARGIT system mediates robust recombinase-sensitive transgene expression, permitting the precise manipulation of previously intractable neural populations.
All data generated or analysed during this study are included in the manuscript and supporting files
- Paul V Sabatini
- Jine Wang
- David P Olson
- Martin G Myers Jr
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Richard D Palmiter, Howard Hughes Medical Institute, University of Washington, United States
© 2021, Sabatini et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Individuals recently diagnosed with a cardiovascular disease are at higher risk of developing a mental illness, with mortality increasing when both conditions are present.
MicroRNAs (miRNA) and other components contained in extracellular vesicles may reflect the presence of a disease. Lung tissue, sputum, and sera of individuals with idiopathic pulmonary fibrosis (IPF) show alterations in miRNA expression. We designed this study to test whether urine and/or tissue derived exosomal miRNAs from individuals with IPF carry cargo that can promote fibrosis.
Exosomes were isolated from urine (U-IPFexo), lung tissue myofibroblasts (MF-IPFexo), serum from individuals with IPF (n=16) and age/sex-matched controls without lung disease (n=10). We analyzed microRNA expression of isolated exosomes and their in vivo bio-distribution. We investigated the effect on ex vivo skin wound healing and in in vivo mouse lung models.
U-IPFexo or MF-IPFexo expressed miR-let-7d, miR-29a-5p, miR-181b-3p and miR-199a-3p consistent with previous reports of miRNA expression obtained from lung tissue/sera from patients with IPF. In vivo bio-distribution experiments detected bioluminescent exosomes in the lung of normal C57Bl6 mice within 5 min after intravenous infusion, followed by distribution to other organs irrespective of exosome source. Exosomes labeled with gold nanoparticles and imaged by transmission electron microscopy were visualized in alveolar epithelial type I and type II cells. Treatment of human and mouse lung punches obtained from control, non-fibrotic lungs with either U-IPFexo or MF-IPFexo produced a fibrotic phenotype. A fibrotic phenotype was also induced in a human ex vivo skin model and in in vivo lung models.
Our results provide evidence of a systemic feature of IPF whereby exosomes contain pro-fibrotic miRNAs when obtained from a fibrotic source and interfere with response to tissue injury as measured in skin and lung models.
This work was supported in part by Lester and Sue Smith Foundation and The Samrick Family Foundation and NIH grants R21 AG060338 (SE and MKG), U01 DK119085 (IP, RS, MTC).