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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.

The GABA (γ-aminobutyric acid) shunt, a bypass mechanism for a portion of the tricarboxylic acid (TCA) cycle, is responsible in the mammalian central nervous system for the synthesis and degradation of the inhibitory neurotransmitter GABA. Glutamic acid decarboxylase (GAD67 and GAD65; gene names GAD1 and GAD2, respectively) synthesizes GABA from glutamate, while GABA-transaminase (GABA-T; gene name ABAT) metabolizes GABA into succinic semialdehyde, which is dehydrogenated into succinate. Although poorly studied in mammals, the GABA shunt plays a protective role against pH, thermo-, and oxidative stresses in plants and bacteria. We hypothesize that both cell non-autonomous GABA from the tumor microenvironment (TME), and cell-autonomous production of GABA contribute to cancer progression. Cell types within the TME such as endothelial and T-cells, are known to secrete GABA. To investigate the utilization of cell non-autonomous GABA by prostate cancer cells, we determined via LC-MS/MS that although the prostate adenocarcinoma LNCaP cell line does not contain significant amounts of GABA, it is capable of taking up exogenous GABA. The effect of exogenous GABA treatment on the metabolic capacity of LNCaP cells was examined using the SEAhorse XF24 Bioanalyzer. GABA treatment increased oxygen consumption rate while decreasing the rate of glycolysis and glycolytic reserve. This effect was blocked by co-treatment with GABA-T inhibitors, suggesting the ability of GABA to increase mitochondrial respiration occurs through the GABA shunt. GABA treatment did not affect the metabolic phenotype of a normal epithelial cell line control. Regarding cell-autonomous production of GABA, recent studies show dysregulation of genes encoding GABA shunt enzymes in various cancers; specifically relevant to this study, GAD1 is upregulated in castration-resistant prostate cancer cell lines (Ippolito 2014). Consistent with the involvement of GAD1 in aggressive prostate cancers, our analysis of the TCGA Provisional prostate cancer dataset reveals that GAD1 mRNA expression level increases with increasing Gleason Score (Pearson r = 0.19; p < 0.00005). Additionally, analysis of the Beltran (2016) data set reveals that GAD1 transcripts are upregulated in treatment-induced neuroendocrine prostate cancer compared to prostate adenocarcinoma (unpaired t-test, p < 0.0005). We hypothesize that both cell non-autonomous and autonomous GABA play a role in cancer progression. Uptake of GABA from the TME may support metabolic reprogramming of primary tumor cells, while upregulation of GAD1 and subsequent increase in cell-autonomous GABA production may provide a malignant advantage to late-stage cancer. The GABA shunt may offer a novel therapeutic target for prostate cancer treatment by exploiting a poorly understood metabolic pathway linked to the TCA cycle. We thank the LSUHSC-S Feist-Weiller Cancer Center for financial support. Citation Format: Erika L. Knott, Sumitra Miriyala, Hyung Nam, Manikandan Panchatcharam, Nancy Leidenheimer. The role of the GABA shunt in prostate cancer metabolic reprogramming and aggressive phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5486.

Although an upregulation of aerobic glycolysis (the Warburg effect) is a canonical hallmark of cancer, it is now recognized that most cancerous cells continue to rely on the TCA cycle as a source of both biosynthetic precursors (DeBarardinis & Chandel, 2016) and the majority of ATP production (Zu & Guppy, 2004). Drug-resistant cancers have been associated with metabolic reprogramming (Rahman & Hasan, 2015) involving an upregulation in oxidative phosphorylation (Wolf, 2014). Two advanced, treatment-resistant variants of prostate cancer are castration-resistant adenocarcinoma (CRPC-adeno) and neuroendocrine prostate cancer (NEPC). Emerging evidence suggests that the γ-aminobutyric acid (GABA) shunt—an evolutionarily conserved pathway for energy production, biosynthetic precursors, and ROS management—may be used by both CRPC-adeno and NEPC. The GABA shunt is a bypass for two steps of the TCA cycle, wherein GABA is synthesized from glutamate by a GAD1-encoded decarboxylase, transaminated into succinic semialdehyde, and metabolized into the TCA cycle intermediate succinate. Analysis of publicly available transcript datasets (via cBioPortal) indicates that GAD1 mRNA expression increases with increasing Gleason score (p < 0.00005, prostate adenocarcinoma TCGA provisional dataset), with higher expression in both CRPC-adeno compared to primary adenocarcinoma (Ippolito and Piwnica-Worms, 2014), and NEPC compared to CRPC-adeno (p < 0.005, Beltran, et al., 2016). Expression of GAD1 transcripts appears to be contingent on low levels of AR transcripts (Trento/Cornell/Broad data set), consistent with the AR independence of the NEPC phenotype. Similarly, GABA levels increase as AR expression is lost during transdifferentiation to NEPC (Solorzano, et al., 2018). GAD67 protein levels in prostate tumors are a more accurate predictor of patient risk for metastasis than Gleason score (Azuma, et al., 2003), and flux through the GABA shunt is critical for the growth of brain tumors in mouse xenograft models (Schnepp, et al., 2017; Neman, et al., 2014). Our experiments show that treatment of an early-stage prostate cancer cell line model (LNCaP) with GABA (100 µM, 24h) induces a shift from aerobic glycolysis to oxidative metabolism. This metabolic reprogramming toward increased oxidative phosphorylation requires at least 6 hours to emerge, and is maximal by 18 hours. During this time period, the presence of GABA in the mitochondrial matrix steadily increases, as shown by our newly-developed GABA-sensing fluorescent receptor, mitoGABASnFR. The observed increases in oxygen consumption are accompanied by GABA-induced decreases in extracellular acidification and lactate levels. In light of these findings, we propose that the GABA shunt represents an understudied pathway for inducing a metabolic shift towards oxidative phosphorylation in advanced prostate cancer, and therefore may be a target for future anticancer therapies. Citation Format: Erika L. Knott, Sumitra Miriyala, Manikandan Panchatcharam, Nancy Leidenheimer. Prostate cancer cells utilize the GABA shunt to enhance oxidative metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4379.

The gamma-aminobutyric acid (GABA) system is evolutionarily conserved and, in mammals, is comprised of 30 genes encoding 4 enzymes, 21 receptor subunits, and 5 transporters. The GABA system has primarily been studied for its role in inhibitory neurotransmission in the brain; however, the physiological importance of this system in peripheral tissues is less well-recognized. Although a few GABA system components have been linked to cancer progression, this information is disparate, limited, and largely unsupervised. Here, we undertook a systematic bioinformatics investigation. To this end, we accessed RNA-seq / microarray transcript data and mass spectrometry / immunohistochemistry protein data from patient tumor datasets within The Cancer Genome Atlas (TCGA), NCBI's Gene Expression Omnibus (GEO), and the NCI's Clinical Proteomic Tumor Analysis Consortium (CPTAC). In order to visualize / analyze data, bioinformatics user-interface platforms were used, including cBioPortal, the Human Protein Atlas, UALCAN, and KM-Plotter. Kaplan-Meier survival curves, differential gene expression between tumor vs. normal samples (across cancer subtypes and stages), and targeted gene set enrichment / correlations were assessed. Several GABA system genes - such as ABAT, GABRP, and GABBR1 - were commonly altered across the >13 cancer types examined (including reproductive, digestive, and secretory and solid organ tumors). Unexpectedly, other transcripts such as ALDH5A1 were highly expressed across all cancers (RNA-seq log2 RSEM median value ~9). Based on Kaplan-Meier survival curve analysis, 11 GABA system genes were identified as prognostic markers for at least one type of cancer (log-rank P value < 0.001 when analyzed using optimized cutoff values; average FKPM > 1). Furthermore, 19 of the 30 investigated genes showed significant alterations in transcript levels between normal adjacent vs. tumor tissues (p < 0.05; minimum median expression = 1 TPM) in at least one cancer type. Interestingly, unique transcript expression patterns of GABA system genes were found among cancer subtypes, such as between triple-negative vs. ER+ breast cancers. Our efforts seek both to identify cancers that leverage the GABA system for their progression, and also to provide a basis for subsequent hypothesis-driven experimentation. Additionally, the data analyzed herein may reveal opportunities to repurpose drugs for cancer treatment, as there are a variety of GABA system-targeting therapeutic agents used to treat neurological and psychiatric disorders. We thank the LSU Health School of Graduate Studies and Feist-Weiller Cancer Center for support. Citation Format: Erika Lynn Knott, Nancy J. Leidenheimer. GABA system expression level changes persist across cancers of many organ systems [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4401.