Nancy J. Leidenheimer

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 GABA_A receptor subunit-encoding transcripts were expressed, including two (GABRB2 and GABRD) prognostic for patient survival. Transcripts encoding GABA_B 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.

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 (ΔF/F _max ~ 2.5, K _d ~ 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 functional unit for inter-neuronal communication in the central nervous system is the neuronal synapse. The number of postsynaptic neurotransmitter receptors at the cell surface is an important determinant of synaptic efficacy and plasticity. A diverse array of post-translational processes regulate postsynaptic receptor number, including receptor exocytosis, lateral diffusion, surface stabilization, endocytosis, and recycling, thus highlighting the importance of mechanisms that control postsynaptic receptor levels. Another putative post-translational mechanism for regulating receptor surface expression is cognate ligand chaperoning. It has been proposed that neurotransmitters function as cognate ligand chaperones by binding, within the endoplasmic reticulum (ER) lumen, to their nascent neurotransmitter receptors and facilitating receptor biogenesis. Here we discuss proof-of-concept evidence that small molecules can selectively facilitate the biogenesis of their targets and examine the specific evidence in support of cognate ligand chaperoning of neurotransmitter receptor biogenesis.

Pharmacological chaperones (PCs) are small molecules that bind to nascent protein targets to facilitate their biogenesis. The ability of PCs to assist in the folding and subsequent forward trafficking of disease-causative protein misfolding mutants has opened new avenues for the treatment of conformational diseases such as cystic fibrosis and lysosomal storage disorders. In this chapter, an overview of the use of PCs for the treatment of conformational disorders is provided. Beyond the therapeutic application of PCs for the treatment of these disorders, pharmacological chaperoning of wild-type integral membrane proteins is discussed. Central to this discussion is the notion that the endoplasmic reticulum is a reservoir of viable but inefficiently processed wild-type protein folding intermediates whose biogenesis can be facilitated by PCs to increase functional pools. To date, the potential therapeutic use of PCs to enhance the biogenesis of wild-type proteins has received little attention. Here the rationale for the development of PCs that target WT proteins is discussed. Also considered is the likelihood that some commonly used therapeutic agents may exert unrecognized pharmacological chaperoning activity on wild-type targets in patient populations.

GABAA receptors mediate fast inhibitory neurotransmission in the brain. Dysfunction of these receptors is associated with various psychiatric/neurological disorders and drugs targeting this receptor are widely used therapeutic agents. Both the efficacy and plasticity of GABAA receptor-mediated neurotransmission depends on the number of surface GABAA receptors. An understudied aspect of receptor cell surface expression is the post-translational regulation of receptor biogenesis within the endoplasmic reticulum (ER). We have previously shown that exogenous GABA can act as a ligand chaperone of recombinant GABAA receptors in the early secretory pathway leading us to now investigate whether endogenous GABA facilitates the biogenesis of GABAA receptors in primary cerebral cortical cultures. In immunofluorescence labeling experiments, we have determined that neurons expressing surface GABAA receptors contain both GABA and its degradative enzyme GABA transaminase (GABA-T). Treatment of neurons with GABA-T inhibitors, a treatment known to increase intracellular GABA levels, decreases the interaction of the receptor with the ER quality control protein calnexin, concomittantly increasing receptor forward-trafficking and plasma membrane insertion. The effect of GABA-T inhibition on the receptor/calnexin interaction is not due to the activation of surface GABAA or GABAB receptors. Consistent with our hypothesis that GABA acts as a cognate ligand chaperone in the ER, immunogold-labeling of rodent brain slices reveals the presence of GABA within the rough ER. The density of this labeling is similar to that present in mitochondria, the organelle in which GABA is degraded. Lastly, the effect of GABA-T inhibition on the receptor/calnexin interaction was prevented by pretreatment with a GABA transporter inhibitor. Together, these data indicate that endogenous GABA acts in the rough ER as a cognate ligand chaperone to facilitate the biogenesis of neuronal GABAA receptors.

Neuronal inhibition is of paramount importance in maintaining the delicate and dynamic balance between excitatory and inhibitory influences in the central nervous system. GABA (γ-aminobutyric acid), the primary inhibitory neurotransmitter in brain, exerts its fast inhibitory effects through ubiquitously expressed GABA_A receptors. Activation of these heteropentameric receptors by GABA results in the gating of an integral chloride channel leading to membrane hyperpolarization and neuronal inhibition. To participate in neurotransmission, the receptor must reside on the cell surface. The trafficking of nascent receptors to the cell surface involves posttranslational modification and the interaction of the receptor with proteins that reside within the secretory pathway. The subsequent insertion of the receptor into specialized regions of the plasma membrane is dictated by receptor composition and other factors that guide insertion at synaptic or perisynaptic/extrasynaptic sites, where phasic and tonic inhibition are mediated, respectively. Once at the cell surface, the receptor is laterally mobile and subject to both constitutive and regulated endocytosis. Following endocytosis the receptor undergoes either recycling to the plasma membrane or degradation. These dynamic processes profoundly affect the strength of GABAergic signaling, neuronal inhibition, and presumably synaptic plasticity. Heritable channelopathies that affect receptor trafficking have been recently recognized and compelling evidence exists that mechanisms underlying acquired epilepsy involve GABA_A receptor internalization. Additionally, GABA_A receptor endocytosis has been identified as an early event in the ischemic response that leads to excitotoxicity and cell death. This chapter summarizes what is known regarding the regulation of receptor trafficking and cell surface expression and its impact on nervous system function from both cell biology and disease perspectives.

Little is known regarding the mechanism(s) by which glycine receptors are endocytosed. Here we examined the endocytosis of homomeric α1 glycine receptors expressed in HEK 293 cells using immunofluorescence/confocal microscopy and whole-cell patch-clamp recordings. Our studies demonstrate that constitutive endocytosis of glycine receptors is blocked by the dominant negative dynamin construct K44A and that intracellular dialysis with peptide P4, a dynamin/amphiphysin-disrupting peptide, increased whole-cell glycine-gated chloride currents. To examine whether receptor endocytosis could be regulated by PKC, experiments with the PKC activator PMA (phorbol 12-myristate 13-acetate) were performed. PMA, but not its inactive analogue PMM (phorbol 12-monomyristate), stimulated receptor endocytosis and inhibited glycine-gated chloride currents. Similar to constitutive endocytosis, PKC-stimulated endocytosis was blocked by dynamin K44A. Mutation of a putative AP2 adaptin dileucine motif (L314A, L315A) present in the receptor cytoplasmic loop blocked PMA-stimulated receptor endocytosis and also prevented PMA inhibition of glycine receptor currents. In patch-clamp experiments, intracellular dialysis of a 12-amino acid peptide corresponding to the region of the receptor containing the dileucine motif prevented PKC modulation of wild-type glycine receptors. Unlike PKC modulation of the receptor, constitutive endocytosis was not affected by mutation of this dileucine motif. These results demonstrate that PKC activation stimulates glycine receptor endocytosis, that both constitutive endocytosis and PKC-stimulated endocytosis are dynamin-dependent, and that PKC-stimulated endocytosis, but not constitutive endocytosis, occurs via the dileucine motif (L314A, L315A) within the cytoplasmic loop of the receptor.