REGULATION OF GABAAR SIGNALING AND NEUROADAPTATIONS IN RESPONSE TO DIAZEPAM

Despite 50+ years of use as anxiolytics, anti-convulsants, and sedative/hypnotic agents, the mechanisms underlying benzodiazepine (BZD) tolerance are poorly understood. BZDs potentiate the actions of GABA, the primary inhibitory neurotransmitter in the adult brain, through positive allosteric modulation of γ2 subunit containing GABA type A receptors (GABAARs). Sustained treatment with BZD drugs is intimately associated with the development of tolerance, dependence, withdrawal and addiction. BZD efficacy diminishes after prolonged or high dose acute exposure, with tolerance to the sedative/hypnotic effects forming most quickly. We investigated the adaptive mechanisms occurring during initial exposure to the classical BZD, Diazepam (DZP), and the molecular signature of the mouse brain during established sedative tolerance. We found cultured neurons treated 24 h with DZP presented no change in surface or synaptic levels of γ2-GABAARs. In contrast, both γ2 and the key inhibitory synaptic scaffolding protein gephyrin levels were decreased after a single DZP treatment in vitro and in vivo. Live-imaging and label-free quantitative proteomics further revealed alterations in γ2 subunit surface trafficking, internalization and lysosomal targeting. In comparison, mice treated seven days with DZP had altered GABAAR subunit composition, reduced responsiveness to DZP, and tonic inhibition was diminished. Furthermore, DZP increased excitatory NMDA receptor subunit levels and function. State of the art mass spectrometry experiments revealed increased CaMKII subunits, which are positive regulators of NMDA receptors and involved in tolerance to other drugs. Downstream bioinformatics analysis confirmed robust synaptic plasticity after DZP. Together, we describe a time-dependent downregulation of synaptic GABAAR function after initial DZP exposure followed by an adaptive increase in excitatory neurotransmission, neuronal remodeling and altered synaptic GABAAR composition

Diazepam Accelerates GABAAR Synaptic Exchange and Alters Intracellular Trafficking

Despite 50+ years of clinical use as anxiolytics, anti-convulsants, and sedative/hypnotic agents, the mechanisms underlying benzodiazepine (BZD) tolerance are poorly understood. BZDs potentiate the actions of gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, through positive allosteric modulation of γ2 subunit containing GABA type A receptors (GABAARs). Here we define key molecular events impacting γ2 GABAAR and the inhibitory synapse gephyrin scaffold following initial sustained BZD exposure in vitro and in vivo. Using immunofluorescence and biochemical experiments, we found that cultured cortical neurons treated with the classical BZD, diazepam (DZP), presented no substantial change in surface or synaptic levels of γ2-GABAARs. In contrast, both γ2 and the postsynaptic scaffolding protein gephyrin showed diminished total protein levels following a single DZP treatment in vitro and in mouse cortical tissue. We further identified DZP treatment enhanced phosphorylation of gephyrin Ser270 and increased generation of gephyrin cleavage products. Selective immunoprecipitation of γ2 from cultured neurons revealed enhanced ubiquitination of this subunit following DZP exposure. To assess novel trafficking responses induced by DZP, we employed a γ2 subunit containing an N terminal fluorogen-activating peptide (FAP) and pH-sensitive green fluorescent protein (γ2pHFAP). Live-imaging experiments using γ2pHFAP GABAAR expressing neurons identified enhanced lysosomal targeting of surface GABAARs and increased overall accumulation in vesicular compartments in response to DZP. Using fluorescence resonance energy transfer (FRET) measurements between α2 and γ2 subunits within a GABAAR in neurons, we identified reductions in synaptic clusters of this subpopulation of surface BZD sensitive receptor. Additional time-series experiments revealed the gephyrin regulating kinase ERK was inactivated by DZP at multiple time points. Moreover, we found DZP simultaneously enhanced synaptic exchange of both γ2-GABAARs and gephyrin using fluorescence recovery after photobleaching (FRAP) techniques. Finally we provide the first proteomic analysis of the BZD sensitive GABAAR interactome in DZP vs. vehicle treated mice. Collectively, our results indicate DZP exposure elicits down-regulation of gephyrin scaffolding and BZD sensitive GABAAR synaptic availability via multiple dynamic trafficking processes

Retrotransposons Are the Major Contributors to the Expansion of the \u3ci\u3eDrosophila ananassae\u3c/i\u3e Muller F Element

The discordance between genome size and the complexity of eukaryotes can partly be attributed to differences in repeat density. The Muller F element (∼5.2 Mb) is the smallest chromosome in Drosophila melanogaster, but it is substantially larger (\u3e18.7 Mb) in D. ananassae. To identify the major contributors to the expansion of the F element and to assess their impact, we improved the genome sequence and annotated the genes in a 1.4-Mb region of the D. ananassae F element, and a 1.7-Mb region from the D element for comparison. We find that transposons (particularly LTR and LINE retrotransposons) are major contributors to this expansion (78.6%), while Wolbachia sequences integrated into the D. ananassae genome are minor contributors (0.02%). Both D. melanogaster and D. ananassae F-element genes exhibit distinct characteristics compared to D-element genes (e.g., larger coding spans, larger introns, more coding exons, and lower codon bias), but these differences are exaggerated in D. ananassae. Compared to D. melanogaster, the codon bias observed in D. ananassae F-element genes can primarily be attributed to mutational biases instead of selection. The 5′ ends of F-element genes in both species are enriched in dimethylation of lysine 4 on histone 3 (H3K4me2), while the coding spans are enriched in H3K9me2. Despite differences in repeat density and gene characteristics, D. ananassae F-element genes show a similar range of expression levels compared to genes in euchromatic domains. This study improves our understanding of how transposons can affect genome size and how genes can function within highly repetitive domains

γ2 GABAAR Trafficking and the Consequences of Human Genetic Variation

GABA type A receptors (GABAARs) mediate the majority of fast inhibitory neurotransmission in the central nervous system (CNS). Most prevalent as heteropentamers composed of two α, two β, and a γ2 subunit, these ligand-gated ionotropic chloride channels are capable of extensive genetic diversity (α1-6, β1-3, γ1-3, δ, , , π, ρ1-3). Part of this selective GABAAR assembly arises from the critical role for γ2 in maintaining synaptic receptor localization and function. Accordingly, mutations in this subunit account for over half of the known epilepsy-associated genetic anomalies identified in GABAARs. Fundamental structure–function studies and cellular pathology investigations have revealed dynamic GABAAR trafficking and synaptic scaffolding as critical regulators of GABAergic inhibition. Here, we introduce in vitro and in vivo findings regarding the specific role of the γ2 subunit in receptor trafficking. We then examine γ2 subunit human genetic variation and assess disease related phenotypes and the potential role of altered GABAAR trafficking. Finally, we discuss new-age imaging techniques and their potential to provide novel insight into critical regulatory mechanisms of GABAAR function