Archives of oral biology · 2020
Trans-trigeminal transport of masseter-derived neprilysin to hippocampus.
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Kuwajima Yukinori, Nagai Manavi S, Lee Cliff, Galaburda Albert M, Kobayashi Takuya, Nakasato Ayaka, Da Silva John D, Nagai Shigemi Ishikawa, Nagai Masazumi
Аннотация
OBJECTIVE: To show the possible occurrence of exosomal transport of neprilysin from masseter muscle to hippocampus via trigeminal nerve in the living mouse.
DESIGN: Mouse C2C12 myotube-derived exosomes were labeled with near-infrared (NIR) dye and injected into the masseter muscle to track their fluorescence from masseter muscle to hippocampus via trigeminal nerve. A plasmid vector encoding green fluorescent protein (GFP)-tagged neprilysin (GFP-neprilysin) was transfected into masseter muscle of C57BL/6 J mice. Expression of mRNA and encoded protein of the transgene was identified in masseter muscle, trigeminal nerve and hippocampus by RT-PCR and Western blot, respectively.
RESULTS: Peak of exosomal NIR in masseter muscle at time 0 rapidly reduced at 3 h and 6 h along with the subsequent increases in trigeminal nerve and hippocampus. Expression of GFP-neprilysin mRNA was detected in masseter muscle, but not trigeminal nerve and hippocampus. On the other hand, the corresponding protein of GFP-neprilysin was identified in the three tissues on day 3 after transfection into masseter muscle as a single band on Western blots with anti-GFP and anti-neprilysin antibodies.
CONCLUSION: The appearance of GFP-neprilysin protein in trigeminal nerve and hippocampus without a corresponding mRNA expression indicated the protein's origin from the masseter muscle. Concomitant migration of NIR-exosomes from masseter muscle to hippocampus via trigeminal nerve suggested the possible occurrence of exosomal transport of neprilysin.
DESIGN: Mouse C2C12 myotube-derived exosomes were labeled with near-infrared (NIR) dye and injected into the masseter muscle to track their fluorescence from masseter muscle to hippocampus via trigeminal nerve. A plasmid vector encoding green fluorescent protein (GFP)-tagged neprilysin (GFP-neprilysin) was transfected into masseter muscle of C57BL/6 J mice. Expression of mRNA and encoded protein of the transgene was identified in masseter muscle, trigeminal nerve and hippocampus by RT-PCR and Western blot, respectively.
RESULTS: Peak of exosomal NIR in masseter muscle at time 0 rapidly reduced at 3 h and 6 h along with the subsequent increases in trigeminal nerve and hippocampus. Expression of GFP-neprilysin mRNA was detected in masseter muscle, but not trigeminal nerve and hippocampus. On the other hand, the corresponding protein of GFP-neprilysin was identified in the three tissues on day 3 after transfection into masseter muscle as a single band on Western blots with anti-GFP and anti-neprilysin antibodies.
CONCLUSION: The appearance of GFP-neprilysin protein in trigeminal nerve and hippocampus without a corresponding mRNA expression indicated the protein's origin from the masseter muscle. Concomitant migration of NIR-exosomes from masseter muscle to hippocampus via trigeminal nerve suggested the possible occurrence of exosomal transport of neprilysin.