PROJECT SUMMARY/ ABSTRACT This exploratory bioengineering project is aimed at designing, building and testing a light-activated glutamate decarboxylase (GAD) as a novel optogenetic tool for noninvasive inhibition of neuronal activity. GAD produces gamma-aminoburyric acid, GABA, a central neurotransmitter involved in reducing neuronal excitability. Enzy- matic activity of the engineered enzyme will be controlled by light in the near-infrared window (NIRW) of the spectrum (670-900 nm) that penetrates through the skull and brain tissue much better than visible light. We expect the NIRW light-activated GAD (NIRW-GAD) expressed in specific brain regions to be activated via ex- tracorporeal light (e.g., transcranially). NIRW-GAD will represent a unique optogenetic research tool for nonin- vasive, spatiotemporally controlled long-term inhibition of neuronal activity in deep or surface brain regions. In the future, it may be developed into a gene therapy for neurological and psychiatric disorders involving hyper- active brain, such as epilepsy, schizophrenia, anxiety and autism spectrum disorder. The NIRW-GAD protein will be engineered using the homodimeric bacteriophytochrome engineering approach, where bacteriophyto- chromes are a class of photoreceptors that sense light within the NIRW spectrum. In Aim 1, the NIRW-GAD prototypes will be designed and screened for light-inducible GAD activity in yeast. The dynamic range and back- ground activity of the enzyme will be optimized using mutagenesis. In Aim 2, we will examine the NIRW-GAD expression and the effects of NIRW treatment in the somatosensory cortex neurons of mice. We will also quantify the effect of NIRW-GAD on GABAergic synapses and GABA quantal content in cortical neurons in vitro. Upon completion of this project, we expect to have engineered and tested a unique optogenetic tool for noninvasive control of the major inhibitory neurotransmitter in silencing of neuronal activity in mammals. 0

PROJECT NARRATIVE This exploratory project intends to engineer a novel optogenetic tool to boost GABA synthesis in interneurons using external light sources. This tool is expected to rescue deficits caused by interneuronpathies associated with a variety of debilitating neurological and psychiatric disorders, such as epilepsy, schizophrenia, anxiety and autism spectrum disorder. 0