Retrograde transport of masseter muscle-derived neprilysin to hippocampus

•Trigeminal N contained high neprilysin (NEP) protein without the corresponding mRNA.•The discrepancy between mRNA-protein suggested the transport of NEP to trigeminal N.•C2C12 myotube released exosomal NEP (eNEP) in response to cholinergic carbachol.•Masseter exported eNEP to hippocampus via trigeminal N upon muscle contraction.•Masseter derived NEP may contribute to the clearance of amyloid β in hippocampus. Although the effects of neprilysin (NEP), also called CD10, on the clearance of Alzheimer’s disease (AD)-associated amyloid-β (Aβ) have been reported, NEP is not made in the brain, and the mechanism for the transport of NEP to the brain has not been investigated. Our hypothesis is that muscle packages NEP in exosomes in response to a neuromuscular signal and sends it to the brain via retrograde axonal transport. The masseter muscle (MM) and the trigeminal nerve (TGN) are good candidates for this mechanism by virtue of their proximity to the brain. The aim of this study was to trace the NEP protein from the MM, through the TGN, and to the hippocampus (HPC) in muscle contraction models in vitro and in vivo. NEP expression in mouse tissue lysates was analyzed by RT-PCR and Western blot. Four-week-old mice were perfused to remove blood NEP contamination. The MM expressed substantial levels of NEP protein and mRNA. On the other hand, a remarkably high level of NEP protein was measured in the TGN in the absence of mRNA. NEP protein, without the corresponding mRNA, was also detected in the HPC. These results suggested that the MM derived NEP was taken up by the TGN, which in turn permitted NEP access to the central nervous system and within it the HPC. When the MM was induced to contract by electric stimulation in freshly euthanized mice, NEP protein decreased in the MM in a stimulus time-dependent manner, while that in the TGN and the HPC increased sequentially. Furthermore, NIR-labeled exosomes tracked along the same route. Finally, carbachol induced secretion of exosomal NEP in C2C12-derived myotube cells. These results support our hypothesis that MM-derived NEP is transported along the TGN to reach the HPC following electrical or cholinergic stimulation.