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Protein engineering and experiment design: JSM, LLL Hippocampal slice imaging: TPJ, DAR Visual cortex volume imaging: KP, ON Mouse epilepsy model: YS, VM, ML, DMK Mitochondria experiments: ELK, NJL Zebrafish: TK, MBA Abstract (150 words)Current techniques for monitoring GABA, the primary inhibitory neurotransmitter in vertebrates, cannot follow ephemeral transients in intact neural circuits. We applied the design principles used to create iGluSnFR, a fluorescent reporter of synaptic glutamate, to develop a GABA sensor using a protein derived from a previously unsequenced Pseudomonas fluorescens strain. Structure-guided mutagenesis and library screening led to a usable iGABASnFR (∆F/Fmax ~ 2.5, Kd ~ 9 µM, good specificity, adequate kinetics). iGABASnFR is genetically encoded, detects single action potential-evoked GABA release events in culture, and produces readily detectable fluorescence increases in vivo in mice and zebrafish. iGABASnFR enabled tracking of: (1) mitochondrial GABA content and its modulation by an anticonvulsant; (2) swimming-evoked GABAergic transmission in zebrafish cerebellum; (3) GABA release events during inter-ictal spikes and seizures in awake mice; and (4) GABAergic tone decreases during isoflurane anesthesia. iGABASnFR will permit high spatiotemporal resolution of GABA signaling in intact preparations.

The potential application of mRNA for the identification of biological fluids using molecular techniques has been a recent development in forensic serology. Constitutively expressed housekeeping genes can assess the amount of mRNA recovered from a sample, establish its suitability for downstream applications, and provide a reference point to corroborate the identity of the fluid. qPCR was utilized to compare the expression levels of housekeeping genes from forensic-like body fluid stains to establish the most appropriate assessment of human mRNA quantity prior to profiling. Although variability was observed between fluids and individuals, results indicated that beta-2 microglobulin exhibited the highest expression for all body fluids examined and across donors. A one-way analysis of variance was performed for housekeeping gene variability between donors (at the α, 0.05, significance level), and the results indicated significant differences for semen, vaginal secretions, and menstrual blood.

To develop an in vivo tool to probe brain genotoxic stress, we designed a viral proxy as a single-cell genetic sensor termed PRISM that harnesses the instability of recombinant adeno-associated virus genome processing and a hypermutable repeat sequence–dependent reporter. PRISM exploits the virus-host interaction to probe persistent neuronal DNA damage and overactive DNA damage response. A Parkinson’s disease (PD)–associated environmental toxicant, paraquat (PQ), inflicted neuronal genotoxic stress sensitively detected by PRISM. The most affected cell type in PD, dopaminergic (DA) neurons in substantia nigra, was distinguished by a high level of genotoxic stress following PQ exposure. Human alpha-synuclein proteotoxicity and propagation also triggered genotoxic stress in nigral DA neurons in a transgenic mouse model. Genotoxic stress is a prominent feature in PD patient brains. Our results reveal that PD-associated etiological factors precipitated brain genotoxic stress and detail a useful tool for probing the pathogenic significance in aging and neurodegenerative disorders.

Adrenocortical carcinoma (ACC) is a rare but deadly cancer for which few treatments exist. Here, we have undertaken a targeted bioinformatics study of The Cancer Genome Atlas (TCGA) ACC dataset focusing on the 30 genes encoding the γ-aminobutyric acid (GABA) system—an under-studied, evolutionarily-conserved system that is an emerging potential player in cancer progression. Our analysis identified a subset of ACC patients whose tumors expressed a distinct GABA system transcriptome. Transcript levels of ABAT (encoding a key GABA shunt enzyme), were upregulated in over 40% of tumors, and this correlated with several favorable clinical outcomes including patient survival; while enrichment and ontology analysis implicated two cancer-related biological pathways involved in metastasis and immune response. The phenotype associated with ABAT upregulation revealed a potential metabolic heterogeneity among ACC tumors associated with enhanced mitochondrial metabolism. Furthermore, many GABAA receptor subunit-encoding transcripts were expressed, including two (GABRB2 and GABRD) prognostic for patient survival. Transcripts encoding GABAB receptor subunits and GABA transporters were also ubiquitously expressed. The GABA system transcriptome of ACC tumors is largely mirrored in the ACC NCI-H295R cell line, suggesting that this cell line may be appropriate for future functional studies investigating the role of the GABA system in ACC cell growth phenotypes and metabolism.