Biology subjects

Johnson, J. W.

Publications and source records attributed to Johnson, J. W..

5 recordsLinked to original sources

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↗

bioRxiv2025

The insidious degeneration of white matter and cognitive decline in Fabry disease

Fabry disease is a rare X-linked deficiency of lysosomal alpha-galactosidase that causes glycolipid accumulation in tissues, including the brain. The most common neurologic sequelae of Fabry are cognitive decline and white matter lesions (WMLs) on brain magnetic resonance imaging (MRI). In the at-large population, however, WMLs are non-specific, highly prevalent, and most are clinically silent. Thus, we compared Fabry to typical brain aging to identify factors unique to Fabry-related cognitive decline. Twenty adult Fabry patients (75% female; median age 36.4 yrs, range: 19.8-63.2 yrs; 95% on enzyme replacement therapy) without a history of stroke or other neurologic diseases and 20 age/sex-matched healthy controls were enrolled in a case-control study. All participants underwent a neurocognitive assessment and a 3.0 T MRI study of the brain that used structural MRI (e.g., fluid-attenuated inversion recovery, FLAIR), semi-quantitative MRI (e.g., normalized FLAIR signal intensity), and quantitative MRI (diffusion tensor imaging, bound-pool fraction imaging). During a blinded review of structural MRIs, a neuroradiologists categorization of case-control status did not correspond to disease status (Fishers test, P>0.99) but rather to age (P=0.004), indicating qualitative changes associated with Fabry were similar to normal age-related brain alterations. Using quantitative MRI, however, we detected evidence of microstructural damage in the white matter of younger Fabry adults (<40 yrs). With age, WML severity increased and the corpus callosum atrophied in Fabry, phenomena absent in controls and consistent with progressive tissue damage. Neurocognitive assessments identified trends for lower verbal intelligence quotient and executive function in the younger Fabry participants, which became statistically significant in the older Fabry cohort. Our data indicate that the early onset of microstructural damage in Fabry drives the insidious degeneration of white matter, leading to reduced cognition. Aging Fabry patients may benefit from serial cognitive assessments to identify unmet therapeutic needs.

genetics↗

bioRxiv2024

State-specific inhibition of NMDA receptors by memantine depends on intracellular calcium and provides insights into NMDAR channel blocker tolerability

NMDA receptors (NMDARs) are key mediators of neuronal Ca2+ influx. NMDAR-mediated Ca2+ influx plays a central role in synaptogenesis, synaptic plasticity, dendritic integration, and neuronal survival. However, excessive NMDAR-mediated Ca2+ influx initiates cellular signaling pathways that result in neuronal death and is broadly associated with neurological disease. Drugs targeting NMDARs are of great clinical interest, but widespread alteration of NMDAR activity can generate negative side effects. The NMDAR channel blocker memantine is a well-tolerated Alzheimers disease medication that shows promise in treatment of other neurological disorders. Memantine enhances desensitization of NMDARs in a subtype- and Ca2+-dependent manner, thereby more effectively inhibiting NMDARs on neurons that experience increased buildup of intracellular Ca2+. However, little is known about the properties or implications of the interaction between intracellular Ca2+ and NMDAR inhibition by memantine or other NMDAR channel blockers. Utilizing customized Ca2+ buffering solutions and whole-cell patch-clamp recordings, we demonstrated that memantine inhibition of both recombinant and native NMDARs increases with increasing intracellular Ca2+ and that the effect of intracellular Ca2+ on memantine action depends on NMDAR subtype. Neuroprotection assays and recordings of postsynaptic currents revealed that memantine preferentially inhibits NMDARs under neurotoxic conditions whereas ketamine, a clinically useful NMDAR channel blocker with strong side effects, inhibits strongly across contexts. Our results present a previously unexamined form of state-specific antagonism, Ca2+-dependent NMDAR channel block, that could have a profound impact on the design of drugs that selectively target NMDAR subpopulations involved in disease.

neuroscience↗

bioRxiv2020

Identification and characterization of distinct murine brown adipocyte lineages

Brown adipose tissue (BAT) plays an important role in the regulation of body weight and glucose homeostasis. While increasing evidence supports white adipose tissue heterogeneity, little is known about heterogeneity within murine BAT. Using single cell RNA sequencing of the stromal vascular fraction of murine BAT and analysis of 67 brown preadipocyte and adipocyte clones we unravel heterogeneity within brown preadipocytes. Statistical analysis of gene expression profiles from these clones identifies markers distinguishing brown adipocyte lineages. We confirm the presence of distinct brown adipocyte populations in vivo using three identified markers; Eif5, Tcf25, and Bin1. Functionally, we demonstrate that loss of Bin1 enhances UCP1 expression and mitochondrial respiration, suggesting that Bin1 marks a dormant brown adipocyte type. The existence of multiple brown adipocyte lineages suggests distinct functional properties of BAT depending on its cellular composition, with potentially distinct function in thermogenesis and the regulation of whole body energy homeostasis.

cell biology↗

bioRxiv2018

A macrophage-based screen identifies antibacterial compounds selective for intracellular Salmonella Typhimurium

Salmonella Typhimurium (S. Tm) evades the innate immune response by residing within host phagocytes. To identify inhibitors of intracellular S. Tm growth, we performed parallel chemical screens against S. Tm growing in macrophage-mimicking media and within macrophages. These screens identified novel antibacterials, and revealed that antibiotics with limited Gram-negative coverage are active against intracellular S. Tm. Screening of a S. Tm deletion library in the presence of one compound, metergoline, revealed that outer membrane perturbation enhanced activity in vitro. Combined with our observation of atypical cell surface characteristics of intracellular S. Tm, our work indicates that the bacterial outer membrane is permeabilized within macrophages. We show that metergoline targets the bacterial cytoplasmic membrane, and prolongs animal survival during a systemic S. Tm infection. This work highlights the predictive nature of intracellular screens for in vivo efficacy, and uncovers new aspects of bacterial physiology of intracellular S. Tm.

microbiology↗