Abstract
Conditional expression of diphtheria toxin receptor (DTR) is widely used for tissue-specific ablation of cells. However, diphtheria toxin (DT) crosses the blood–brain barrier, which limits its utility for ablating peripheral cells using Cre drivers that are also expressed in the central nervous system (CNS). Here we report the development of a brain-sparing DT, termed BRAINSPAReDT, for tissue-specific genetic ablation of cells outside the CNS. We prevent blood–brain barrier passage of DT through PEGylation, which polarizes the molecule and increases its size. We validate BRAINSPAReDT with regional genetic sympathectomy: BRAINSPAReDT ablates peripheral but not central catecholaminergic neurons, thus avoiding the Parkinson-like phenotype associated with full dopaminergic depletion. Regional sympathectomy compromises adipose tissue thermogenesis, and renders mice susceptible to obesity. We provide a proof of principle that BRAINSPAReDT can be used for Cre/DTR tissue-specific ablation outside the brain using CNS drivers, while consolidating the link between adiposity and the sympathetic nervous system.
Highlights
Conditional expression of diphtheria toxin receptor (DTR) is widely used for tissue-specific ablation of cells
We found that PEGylated DT (PEGyDT)-regionally sympathectomized mice have defective thermogenesis, which is a key mechanism of energy dissipation from brown and beige adipose tissues
The same analysis was performed in other brain areas containing tyrosine hydroxylase (TH) þ neurons (Supplementary Fig. 3 for area postrema, locus coeruleus, hypothalamus and sagittal plane of substantia nigra), and the results indicate that PEGyDT does not ablate catecholaminergic neurons in the brain when injected systemically
Summary
Conditional expression of diphtheria toxin receptor (DTR) is widely used for tissue-specific ablation of cells. Diphtheria toxin (DT) crosses the blood–brain barrier, which limits its utility for ablating peripheral cells using Cre drivers that are expressed in the central nervous system (CNS). Conditional expression of the diphtheria toxin receptor (DTR) using the Cre/lox system is a widely used tool for tissue-specific ablation of cells This tool is of limited utility for targeted genetic ablation of peripheral cells because diphtheria toxin (DT) crosses the blood–brain barrier (BBB) and many peripheral Cre drivers have additional expression in the central nervous system (CNS), driving ablation in the brain. This chemical modification increases the size and polarity of DT and spares DTR-expressing cells in the brain To validate this tool in vivo, we implemented a genetic method for regional sympathectomy using BRAINSPAReDT. This tool should enable systematic molecular study of the SNS as well as a plethora of peripheral neurons or cells that may be labelled by CNS markers
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