- #ABLATION AND WEIGHT GAIN 2017 DRIVERS#
- #ABLATION AND WEIGHT GAIN 2017 DRIVER#
- #ABLATION AND WEIGHT GAIN 2017 FULL#
Conversion of DT to PEGyDT was nearly complete (relative 95% conversion) and resulted in a heterogeneous mixture differing in both the copy number of PEG units and conjugation sites. Within a margin of variation, these values are in agreement with the expected mass additions (218 Da) for PEGylation. DT (G 2–S 536) has a molecular weight of 58,336 Da, and matrix-assisted laser desorption/ionization–time-of-flight (MALDI–TOF) analysis showed the best-resolved signal as 2+ peak at 29,169 m/ z. The modification state of DT was analysed by mass spectrometry to estimate how many PEG molecules were attached to DT ( Fig. 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. In particular, BRAINSPAReDT may now be used for genetic loss of function of subsets of SNS neurons. We provide a proof-of-principle demonstration that BRAINSPAReDT can generally be used for Cre/DTR ablation in the periphery, thus circumventing off-target cell ablation in the brain. These results are consistent with the role of the SNS in lipolysis and thermogenesis. Thus, mice regionally sympathectomized with BRAINSPAReDT are prone to obesity, despite having normal eating behaviour. 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. In these mice, BRAINSPAReDT successfully ablates peripheral catecholaminergic neurons, but not dopaminergic neurons in the brain, and thus avoids the Parkinson-like phenotype that is a consequence of dopaminergic depletion.
#ABLATION AND WEIGHT GAIN 2017 DRIVER#
Using a catecholaminergic Cre driver ( TH-Cre), we conditionally express the DTR in the SNS and dopaminergic neurons in the brain. Thus, genetic ablation via expression of Cre-inducible DTR and diphtheria toxin injection would be a feasible strategy to achieve a regional sympathectomy and tissue specificity 6. Moreover, neither a surgical nor a chemical approach is amenable to specifically sympathectomize organs such as the adipose tissue, which covers a large body region. However, surgical sympathectomy damages nerves of unknown identity that are mixed within the SNS bundles 5. Unlike the chemical ablation, which is systemic, surgical sympathectomy is a localized intervention that affects the sympathetic nervous system (SNS) innervation of a confined anatomical region or organ 2, 3, 4. General sympathectomy has been traditionally performed with systemic injection of chemicals such as guanethidine or 6-hydroxydopamine 1. To validate this tool in vivo, we implemented a genetic method for regional sympathectomy using BRAINSPAReDT. This chemical modification increases the size and polarity of DT and spares DTR-expressing cells in the brain.
To limit DT action and prevent it from entering the brain, we chemically modified DT by PEGylation to develop a brain-sparing DT (BRAINSPAReDT). Small uncharged molecules tend to cross the BBB, whereas those that are large and/or polarized tend to have limited permeability. The ability of a molecule to cross the BBB depends on two critical molecular properties: polarity and size.
#ABLATION AND WEIGHT GAIN 2017 DRIVERS#
However, 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), thus driving ablation in the brain. 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.Ĭonditional expression of the diphtheria toxin receptor (DTR) using the Cre/lox system is a widely used tool for tissue-specific ablation of cells. Regional sympathectomy compromises adipose tissue thermogenesis, and renders mice susceptible to obesity.
#ABLATION AND WEIGHT GAIN 2017 FULL#
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. We prevent blood–brain barrier passage of DT through PEGylation, which polarizes the molecule and increases its size. Here we report the development of a brain-sparing DT, termed BRAINSPAReDT, for tissue-specific genetic ablation of cells outside the CNS. 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). Conditional expression of diphtheria toxin receptor (DTR) is widely used for tissue-specific ablation of cells.