Engineered GABA oxidase for GABA sensing in vivo

Project Summary The goal of this project is to engineer a g-aminobutyric acid (GABA) oxidase suitable for the creation of a high- performance, implantable, electroenzymatic GABA microsensor. GABA is the most important inhibitory neurotransmitter in the brain, yet technology to monitor GABA transients at the cellular level and at the near-

real-time, millisecond time-scale has been slow to emerge, especially for the deep brain. The need is acute, as glutamate and GABA dominate the excitatory/inhibitory (E/I) ratio in the brain, dysregulation of which has been associated with Alzheimer’s disease, seizures, autism, schizophrenia and bipolar disorder, which affect

nearly 25M people in the US. Since GABA itself is not electroactive, a proven approach to creation of a high- performance sensor entails the immobilization on an electrode of a H2O2-producing oxidase selective for the analyte of interest. The H2O2 produced by the enzyme-catalyzed oxidation of the analyte is electrooxidized at

the underlying electrode thereby giving rise to a current that may be correlated to the analyte concentration. However, a GABA oxidase is not commercially available, and other biosensing strategies have significant drawbacks. Since GABA is ubiquitous in the environment, there is evidence for enzyme activity closely related

to that necessary for sensor development. In fact, an enzyme with native oxidase activity for closely related methyl-GABA that shows modest activity with GABA has been identified. Our team has cloned and expressed this enzyme, demonstrated its activity with GABA and constructed a biosensor showing modest ability to sense

the target. Our aims are to employ directed protein evolution to create a highly active and selective GABA oxidase using advanced ultrahigh throughput techniques and to demonstrate its utility for the construction of a high-performance GABA microbiosensor. Promising prototype biosensors will be tested as they are created in

the striatum of anesthetized rats. This project will leverage the proven expertise of the team in the areas of protein engineering; of neuroscience; and of implantable, electroenzymatic microsensor construction to create technology that will help further illuminate the function of exceedingly important neural circuitry.