• obesity
• sympathetic-neuron associated macrophage
• slc6a2
• melanocortin receptor
• PEG-amphetamine
• SAM

The identification of sympathetic neuron-associated macrophages (SAMs) changed the way we think the communications between sympathetic neurons and adipocytes.
SAMs possess the machinery for uptaking and degrading the norepinephrine (NE) released by the neuron, a NE transporter (Slc6a2 or NET) and the monoamine oxidase A (MAOA) respectively.
In the adipocytes NE starts a cascade of events, which leads to lipolysis and thermogenesis. SAMs play a big role in the modulation of adipocyte activation, subtracting the available NE from the extracellular space. SAMs are recruited in obesity and they switch to a pro-inflammatory state, which also upregulates the machinery for NE clearance, decreasing the adipocyte activation. Genetically ablating Slc6a2 was shown to have a protective phenotype against obesity.
Amphetamine can inhibit NE uptake and it is already known for ameliorating obesity symptoms, so it was chosen to reproduce pharmacologically the knock out. The downside of this drug is its potent stimulatory and addictive effect on the central nervous system. To overcome these side effects, we chose a PEGylation strategy to increase the molecule hydrodynamic size, in order to prevent its passage through the brain blood barrier.
Before proceeding with in vivo test, we had to demonstrate that pegylated amphetamine was still functional. We addressed this question with an ex-vivo assay on superior cervical ganglia (SCG). We incubated explanted SCGs with a negative control, amphetamine and pegylated amphetamine and we compared their ability to block NET, analyzing the total content of NE in the medium and inside sorted SAMs.

Nevertheless, inhibiting SAMs’ uptake of NE only mitigates obesity symptoms and doesn’t remove any of the causes of inflammation. Our RNAseq data suggested the expression of melanocortin receptors (MCRs) in SAMs. MCRs are G-Protein Coupled Receptors (GPCRs) known to influence fundamental macrophage functions: antagonizing the release of pro-inflammatory cytokines and chemokines, inhibiting leukocyte chemoattraction, inducing release of anti-inflammatory cytokines and augmenting phagocytosis and efferocytosis. The melanocortin receptors in SAMs seemed good candidates for developing drug therapies to decrease the inflammatory state typical of obesity.
To address this proposition, we tried to confirm the RNAseq results by quantitative PCR of SAMs sorted from nerve fibers innervating the subcutaneous fat pads.
To the best of our knowledge, and based on our preliminary results, we cannot conclusively determine if MCRs are indeed expressed in SAMs.