Method Development for a Brain Neprilysin Enzymatic Activity Assay and Validation in Two Rodent Models of Cognition Katherine Lundeen1, Anne T. Kanta1, Joseph McLaughlin1, Daniela Radl1, Erica L. Bradshaw-Pierce1, Melinda Manuel1 1Takeda California, San Diego, CA, USA
Neprilysin (NEP) is a membrane metallo-endopeptidase responsible for the cleavage and inactivation of several neuropeptides, and has been shown to play an important role in cognition. NEP knock-out mice have been observed to have significant cognitive impairment. We describe a protocol for rapid and reproducible measurement of neprilysin enzymatic activity in brain homogenate, and a subsequent study of brain NEP in two rodent models of cognition. Brain NEP is very sensitive to the method of brain isolation and homogenization, and enzyme activity can be severely affected by handling, heat, foaming, and shear-force. The best results were previously obtained with a Dounce homogenizer or a hand-held rotor-stator device on ice. However, this manual method is very time and labor-intensive and can affect the results of a large study where time is of the essence. We developed an automated method for brain homogenization that can process up to 12 rat brains at once in only 17 seconds using an Omni Beadruptor instrument with novel modifications to the standard protocol. The enzyme activity results were equal or superior to those obtained with manual methods. Rat brains were thawed on ice, and cold PBS containing protease inhibitors and an anti-foaming reagent was added at a volume/weight ratio of 2:1. The brains were homogenized in 7ml Omni tubes without beads at a setting of 5 M/s for 17 seconds in an Omni Beadruptor. Homogenate was normalized according to total protein concentration, and clarified supernatant was diluted in 50mM MES pH6.5, with a final concentration of 25µM substrate (Suc-Ala-Ala-Phe-AMC, Bachem). The liberated fluorescence signal was detected with an excitation of 360nm and emission of 450nm after a 60-minute incubation. Specific NEP activity was determined by calculating the difference between the total activity and samples that were pre-incubated with 10µM Thiorphan, a selective NEP inhibitor. Two models of rat cognition were evaluated for NEP activity: the cysteamine model, and aged rats. We observed a significant inhibition of enzyme activity in the cysteamine model within one hour. In the aged rats, we observed a similar inhibition of brain NEP activity at 12 and 24 months.
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