Jacob, T. C. | bioRxiv Science

bioRxiv2025

Chronic benzodiazepine treatment triggers gephyrin scaffold destabilization and GABAAR subsynaptic reorganization

Benzodiazepines (BZDs) are important clinical drugs with anxiolytic, anticonvulsant, and sedative effects mediated by potentiation of inhibitory GABA type A receptors (GABAARs). Tolerance limits the clinical utility of BZDs, yet the mechanisms underlying tolerance after chronic exposure have not been thoroughly investigated. Here, we assessed the impact of chronic (7-day) treatment with the BZD diazepam (DZP) on the dynamic plasticity and subsynaptic organization of the gephyrin scaffold and {gamma}2 subunit-containing GABAARs in primary neurons. After functional confirmation of diminished BZD sensitivity, we provide the first super-resolution analysis of inhibitory nanoscale plasticity induced by chronic BZD exposure: gephyrin subsynaptic domains were smaller and the inhibitory postsynaptic area was overall diminished by DZP treatment, resulting in a condensation of synaptic {gamma}2-GABAARs into smaller subsynaptic areas. Using a novel fluorescence-based in situ proximity ligation assay and biochemical fractionation analysis, the mechanism for gephyrin downregulation was revealed to be dependent on phosphorylation and protease cleavage. Accordingly, DZP treatment impaired gephyrin synaptic stability, demonstrated by live-imaging photobleaching experiments. Despite the loss of BZD sensitivity and stable synaptic gephyrin, 7-day DZP treatment did not reduce the surface or total protein levels of BZD-sensitive {gamma}2-GABAARs, as shown in prior short-term BZD treatment studies. Instead, chronic DZP treatment induced an accumulation of {gamma}2-GABAARs in the extrasynaptic membrane. Surprisingly, {gamma}2-GABAAR interactions with gephyrin were also enriched extrasynaptically. An identified rise in extrasynaptically-localized gephyrin cleavage fragments may function to confine receptors away from the synapse, as supported by a decrease in extrasynaptic {gamma}2-GABAAR mobility. Altogether, we find that chronic BZD treatment triggers several subtle converging plasticity events at inhibitory synapses which effectively restrict the synaptic renewal of BZD-sensitive GABAARs via mechanisms distinct from those observed with short-term treatment.

neuroscience↗

bioRxiv2019

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 {gamma}2 subunit containing GABA type A receptors (GABAARs). Here we define key molecular events impacting {gamma}2 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 {gamma}2-GABAARs. In contrast, both {gamma}2 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 {gamma}2 from cultured neurons revealed enhanced ubiquitination of this subunit following DZP exposure. To assess novel trafficking responses induced by DZP, we employed a {gamma}2 subunit containing an N terminal fluorogen-activating peptide (FAP) and pH-sensitive green fluorescent protein ({gamma}2pHFAP). Live-imaging experiments using {gamma}2pHFAP 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 2 and {gamma}2 subunits within a GABAAR in neurons, we identified reductions in synaptic clusters of this subpopulation of surface BZD sensitive receptor. Moreover, we found DZP simultaneously enhanced synaptic exchange of both {gamma}2-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.

neuroscience↗