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1.
Aging Cell ; 22(11): e13981, 2023 11.
Article in English | MEDLINE | ID: mdl-37771191

ABSTRACT

Age-induced degeneration of the neuromuscular junction (NMJ) is associated with motor dysfunction and muscle atrophy. While the impact of aging on the NMJ presynapse and postsynapse is well-documented, little is known about the changes perisynaptic Schwann cells (PSCs), the synaptic glia of the NMJ, undergo during aging. Here, we examined PSCs in young, middle-aged, and old mice in three muscles with different susceptibility to aging. Using light and electron microscopy, we found that PSCs acquire age-associated cellular features either prior to or at the same time as the onset of NMJ degeneration. Notably, we found that aged PSCs fail to completely cap the NMJ even though they are more abundant in old compared with young mice. We also found that aging PSCs form processes that either intrude into the synaptic cleft or guide axonal sprouts to innervate other NMJs. We next profiled the transcriptome of PSCs and other Schwann cells (SCs) to identify mechanisms altered in aged PSCs. This analysis revealed that aged PSCs acquire a transcriptional pattern previously shown to promote phagocytosis that is absent in other SCs. It also showed that aged PSCs upregulate unique pro-inflammatory molecules compared to other aged SCs. Interestingly, neither synaptogenesis genes nor genes that are typically upregulated by repair SCs were induced in aged PSCs or other SCs. These findings provide insights into cellular and molecular mechanisms that could be targeted in PSCs to stave off the deleterious effects of aging on NMJs.


Subject(s)
Neuromuscular Junction , Schwann Cells , Animals , Mice , Synapses/physiology , Neuroglia , Aging
2.
ACS Infect Dis ; 3(3): 183-189, 2017 03 10.
Article in English | MEDLINE | ID: mdl-28075113

ABSTRACT

Inhibition of quorum sensing in Pseudomonas aeruginosa is of interest as a possible antivirulence strategy for this pathogenic bacterium. The LasR regulator protein is important in coordinating gene expression in response to quorum sensing signaling molecules. One predominant strategy for LasR inhibition is the development of small-molecule antagonists that mimic the native autoinducer, though the mechanism by which they inactivate LasR is not known. This work reveals that multiple antagonists function by binding to and stabilizing LasR in a conformation that renders it unable to bind DNA. Further analysis of purified LasR complexed with known antagonists indicates that DNA binding can be recovered with the addition of native autoinducer, providing insights into the reversibility of ligand binding for this transcription factor. This in vitro assay could be used to assess future promising antagonists and complements existing cell-based reporter assays.


Subject(s)
Bacterial Proteins/metabolism , Pseudomonas aeruginosa/drug effects , Quorum Sensing/drug effects , Small Molecule Libraries/pharmacology , Trans-Activators/metabolism , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/chemistry , DNA/metabolism , Gene Expression Regulation, Bacterial/drug effects , Protein Binding/drug effects , Pseudomonas aeruginosa/metabolism , Small Molecule Libraries/chemistry , Structure-Activity Relationship , Trans-Activators/chemistry , Virulence/drug effects
3.
J Biol Chem ; 291(22): 11776-86, 2016 May 27.
Article in English | MEDLINE | ID: mdl-27053110

ABSTRACT

The LasR regulator protein functions at the top of the Pseudomonas aeruginosa quorum-sensing hierarchy and is implicated in promoting bacterial virulence. Of note is recent evidence that this transcription factor may also respond to oxidative stress. Here, all cysteines in LasR were inspected to deduce their redox sensitivity and to probe the connection between stress response and LasR activity using purified LasR and individual LasR domains. Cys(79) in the ligand binding domain of LasR appears to be important for ligand recognition and folding of this domain to potentiate DNA binding but does not seem to be sensitive to oxidative stress when bound to its native ligand. Two cysteines in the DNA binding domain of LasR do form a disulfide bond when treated with hydrogen peroxide, and formation of this Cys(201)-Cys(203) disulfide bond appears to disrupt the DNA binding activity of the transcription factor. Mutagenesis of either of these cysteines leads to expression of a protein that no longer binds DNA. A cell-based reporter assay linking LasR function with ß-galactosidase activity gave results consistent with those obtained with purified LasR. This work provides a possible mechanism for oxidative stress response by LasR and indicates that multiple cysteines within the protein may prove to be useful targets for disabling its activity.


Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Oxidative Stress , Pseudomonas aeruginosa/metabolism , Quorum Sensing , Trans-Activators/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Circular Dichroism , Electrophoretic Mobility Shift Assay , Promoter Regions, Genetic/genetics , Protein Conformation , Pseudomonas aeruginosa/growth & development , Trans-Activators/chemistry , Trans-Activators/genetics , beta-Galactosidase/metabolism
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