Sex-Dependent Differences in the Ischemia/Reperfusion-Induced Expression of AMPA Receptors.

Following ischemia/reperfusion, AMPA receptors (AMPARs) mediate pathologic delayed neuronal death through sustained expression of calcium-permeable AMPARs, leading to excitotoxicity. Preventing the surface removal of GluA2-containing AMPARs may yield new therapeutic targets for the treatment of ischemia/reperfusion. This study utilized acute organotypic hippocampal slices from aged male and female Sprague Dawley rats and subjected them to oxygen-glucose deprivation/reperfusion (OGD/R) to examine the mechanisms underlying the internalization and degradation of GluA2-containing AMPARs. We determined the effect of OGD/R on AMPAR subunits at the protein and mRNA transcript levels utilizing Western blot and RT-qPCR, respectively. Hippocampal slices from male and female rats responded to OGD/R in a paradoxical manner with respect to AMPARs. GluA1 and GluA2 AMPAR subunits were degraded following OGD/R in male rats but were increased in female rats. There was a rapid decrease in GRIA1 (GluA1) and GRIA2 (GluA2) mRNA levels in the male hippocampus following ischemic insult, but this was not observed in females. These data indicate a sex-dependent difference in how AMPARs in the hippocampus respond to ischemic insult, and may help explain, in part, why premenopausal women have a lower incidence/severity of ischemic stroke compared with men of the same age.

Liposomal Encapsulated FSC231, a PICK1 Inhibitor, Prevents the Ischemia/Reperfusion-Induced Degradation of GluA2-Containing AMPA Receptors.

Strokes remain one of the leading causes of disability within the United States. Despite an enormous amount of research effort within the scientific community, very few therapeutics are available for stroke patients. Cytotoxic accumulation of intracellular calcium is a well-studied phenomenon that occurs following ischemic stroke. This intracellular calcium overload results from excessive release of the excitatory neurotransmitter glutamate, a process known as excitotoxicity. Calcium-permeable AMPA receptors (AMPARs), lacking the GluA2 subunit, contribute to calcium cytotoxicity and subsequent neuronal death. The internalization and subsequent degradation of GluA2 AMPAR subunits following oxygen-glucose deprivation/reperfusion (OGD/R) is, at least in part, mediated by protein-interacting with C kinase-1 (PICK1). The purpose of the present study is to evaluate whether treatment with a PICK1 inhibitor, FSC231, prevents the OGD/R-induced degradation of the GluA2 AMPAR subunit. Utilizing an acute rodent hippocampal slice model system, we determined that pretreatment with FSC231 prevented the OGD/R-induced association of PICK1-GluA2. FSC231 treatment during OGD/R rescues total GluA2 AMPAR subunit protein levels. This suggests that the interaction between GluA2 and PICK1 serves as an important step in the ischemic/reperfusion-induced reduction in total GluA2 levels.

Oxidative Stress Underlies the Ischemia/Reperfusion-Induced Internalization and Degradation of AMPA Receptors.

Stroke is the fifth leading cause of death annually in the United States. Ischemic stroke occurs when a blood vessel supplying the brain is occluded. The hippocampus is particularly susceptible to AMPA receptor-mediated delayed neuronal death as a result of ischemic/reperfusion injury. AMPA receptors composed of a GluA2 subunit are impermeable to calcium due to a post-transcriptional modification in the channel pore of the GluA2 subunit. GluA2 undergoes internalization and is subsequently degraded following ischemia/reperfusion. The subsequent increase in the expression of GluA2-lacking, Ca2+-permeable AMPARs results in excitotoxicity and eventually delayed neuronal death. Following ischemia/reperfusion, there is increased production of superoxide radicals. This study describes how the internalization and degradation of GluA1 and GluA2 AMPAR subunits following ischemia/reperfusion is mediated through an oxidative stress signaling cascade. U251-MG cells were transiently transfected with fluorescently tagged GluA1 and GluA2, and different Rab proteins to observe AMPAR endocytic trafficking following oxygen glucose-deprivation/reperfusion (OGD/R), an in vitro model for ischemia/reperfusion. Pretreatment with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a superoxide dismutase mimetic, ameliorated the OGD/R-induced, but not agonist-induced, internalization and degradation of GluA1 and GluA2 AMPAR subunits. Specifically, MnTMPyP prevented the increased colocalization of GluA1 and GluA2 with Rab5, an early endosomal marker, and with Rab7, a late endosomal marker, but did not affect the colocalization of GluA1 with Rab11, a marker for recycling endosomes. These data indicate that oxidative stress may play a vital role in AMPAR-mediated cell death following ischemic/reperfusion injury.

Scattering from layered seafloors: Comparisons between theory and integral equations.

Acoustic scattering from layered seafloors exhibits dependence on both the mean geoacoustic layering, as well as the roughness properties of each layer. Several theoretical treatments of this environment exist, including the small roughness perturbation approximation, the Kirchhoff approximation, and three different versions of the small slope approximation. All of these models give different results for the scattering cross section and coherent reflection coefficient, and there is currently no way to distinguish which model is the most correct. In this work, an integral equation for scattering from a layered seafloor with rough interfaces is presented, and compared with small roughness perturbation method, and two of the small slope approximations. It is found that the most recent small slope approximation by Jackson and Olson [J. Acoust. Soc. Am. 147(1), 56-73 (2020)] is the most accurate when the root-mean-square (rms) roughness is large, and some models are in close agreement with each other when the rms roughness is small.

The small-slope approximation for layered, fluid seafloors.

The small-slope approximation (SSA) for rough-interface scattering is most commonly applied to the upper boundary of either impenetrable media or uniform half-space media, but has been recently developed for layered media in the acoustic and electromagnetic cases. The present work gives an overview of three forms of the SSA for layered media. The first has been previously presented in the acoustics literature. The second is from the electromagnetics literature and in the present work is converted to the fluid-sediment problem. A missing proof is supplied of a key consistency condition demanded of the small-slope ansatz. As is usual, these small-slope results are expressed in k-space. A third SSA for layered seafloors follows from conversion of the usual half-space formulation from k-space to coordinate space. This form turns out to be useful for reverberation simulations. The three different approaches are compared with respect to scattering strength and the coherent reflection coefficient, but an assessment of their relative merits will require comparison with exact calculations.

The mutual scattering cross section.

A generalization of the conventional interface scattering cross section is introduced. This new object will be called the mutual scattering cross section, and, like the conventional cross section, can be used in narrow-band sonar applications. It can treat both sea-surface and seafloor scattering and is useful in cases where large arrays are employed as well as in multipath environments. The application to large arrays with uniform half-space water column and seafloor is examined briefly, but the bulk of this article is devoted to multipathing in the ocean waveguide. Comparisons with more accurate, but more numerically intensive, approaches in range-independent environments show that the mutual cross section can provide an efficient solution for the reverberation intensity time series. The mutual cross section incorporates interference effects causing oscillations in the reverberation time series. Such oscillations have been reported in the literature, but previous modeling efforts have been ad hoc, not based on scattering physics. The mutual cross section is shown to model backscattering enhancement due to multipathing, another phenomenon not seen in simpler models. Expressions for the mutual cross section are derived for seafloor roughness scattering and sediment volume scattering.

A time-domain model for seafloor scattering.

Bottom scattering is important for a number of underwater applications: it is a source of noise in target detection and a source of information for sediment classification and geoacoustic inversion. While current models can predict the effective interface scattering strength for layered sediments, these models cannot directly compute the ensemble averaged mean-square pressure. A model for bottom scattering due to a point source is introduced which provides a full-wave solution for mean-square scattered pressure as a function of time under first-order perturbation theory. Examples of backscatter time series from various types of seafloors will be shown, and the advantages and limitations of this model will be discussed.

The relative effect of particles and turbulence on acoustic scattering from deep sea hydrothermal vent plumes revisited.

The relative importance of suspended particles and turbulence as backscattering mechanisms within a hydrothermal plume located on the Endeavour Segment of the Juan de Fuca Ridge is determined by comparing acoustic backscatter measured by the Cabled Observatory Vent Imaging Sonar (COVIS) with model calculations based on in situ samples of particles suspended within the plume. Analysis of plume samples yields estimates of the mass concentration and size distribution of particles, which are used to quantify their contribution to acoustic backscatter. The result shows negligible effects of plume particles on acoustic backscatter within the initial 10-m rise of the plume. This suggests turbulence-induced temperature fluctuations are the dominant backscattering mechanism within lower levels of the plume. Furthermore, inversion of the observed acoustic backscatter for the standard deviation of temperature within the plume yields a reasonable match with the in situ temperature measurements made by a conductivity-temperature-depth instrument. This finding shows that turbulence-induced temperature fluctuations are the dominant backscattering mechanism and demonstrates the potential of using acoustic backscatter as a remote-sensing tool to measure the temperature variability within a hydrothermal plume.

Identification of NADPH oxidase as a key mediator in the post-ischemia-induced sequestration and degradation of the GluA2 AMPA receptor subunit.

A hallmark of ischemic/reperfusion injury is a change in subunit composition of synaptic 2-amino-3-(3-hydroxy-5-methylisoazol-4-yl)propionic acid receptors (AMPARs). This change in AMPAR subunit composition leads to an increase in surface expression of GluA2-lacking Ca(2+) /Zn(2+) permeable AMPARs. These GluA2-lacking AMPARs play a key role in promoting delayed neuronal death following ischemic injury. At present, the mechanism(s) responsible for the ischemia/reperfusion-induced subunit composition switch and degradation of the GluA2 subunit remain unclear. In this study, we investigated the role of NADPH oxidase, and its importance in mediating endocytosis and subsequent degradation of the GluA2 AMPAR subunit in adult rat hippocampal slices subjected to oxygen-glucose deprivation/reperfusion (OGD/R) injury. In hippocampal slices pre-treated with the NADPH oxidase inhibitor apocynin attenuated OGD/R-mediated sequestration of GluA2 and GluA1 as well as prevent the degradation of GluA2. We provide compelling evidence that NADPH oxidase mediated sequestration of GluA1- and GluA2- involved activation of p38 MAPK. Furthermore, we demonstrate that inhibition of NADPH oxidase blunts the OGD/R-induced association of GluA2 with protein interacting with C kinase-1. In summary, this study identifies a novel mechanism that may underlie the ischemia/reperfusion-induced AMPAR subunit composition switch and a potential therapeutic target.

Attenuation of sound in sand sediments due to porosity fluctuations.

At high frequencies, the attenuation measured in sand sediments is larger than that predicted by Biot theory. To account for this discrepancy, perturbation theory is used to incorporate losses due to scattering by porosity variations into both Biot's poroelastic equations and the effective density fluid model. While previous results showed that fluctuations in the bulk frame modulus were insufficient to produce significant attenuation in a sand sediment, modest levels of fluctuations in the porosity produce significant scattering loss. By using the sediment parameters and the heterogeneity power spectrum measured during the Sediment Acoustics Experiment in 2004, the perturbation theory result shows good agreement with the sound speed and attenuation data without any free parameters.

NADPH oxidase mediates the oxygen-glucose deprivation/reperfusion-induced increase in the tyrosine phosphorylation of the N-methyl-D-aspartate receptor NR2A subunit in retinoic acid differentiated SH-SY5Y Cells.

BACKGROUND: Evidence exists that oxidative stress promotes the tyrosine phosphorylation of N-methyl-D-aspartate receptor (NMDAR) subunits during post-ischemic reperfusion of brain tissue. Increased tyrosine phosphorylation of NMDAR NR2A subunits has been reported to potentiate receptor function and exacerbate NMDAR-induced excitotoxicity. Though the effect of ischemia on tyrosine phosphorylation of NMDAR subunits has been well documented, the oxidative stress signaling cascades mediating the enhanced tyrosine phosphorylation of NR2A subunits remain unclear. RESULTS: We report that the reactive oxygen species (ROS) generator NADPH oxidase mediates an oxidative stress-signaling cascade involved in the increased tyrosine phosphorylation of the NR2A subunit in post-ischemic differentiated SH-SY5Y neuroblastoma cells. Inhibition of NADPH oxidase attenuated the increased tyrosine phosphorylation of the NMDAR NR2A subunit, while inhibition of ROS production from mitochondrial or xanthine oxidase sources failed to dampen the post-ischemic increase in tyrosine phosphorylation of the NR2A subunit. Additionally, inhibition of NADPH oxidase blunted the interaction of activated Src Family Kinases (SFKs) with PSD-95 induced by ischemia/reperfusion. Lastly, inhibition of NADPH oxidase also markedly reduced cell death in post-ischemic SH-SY5Y cells stimulated by NMDA. CONCLUSIONS: These data indicate that NADPH oxidase has a key role in facilitating NMDAR NR2A tyrosine phosphorylation via SFK activation during post-ischemic reperfusion.

Application of small-roughness perturbation theory to reverberation in range-dependent waveguides.

A rough-interface reverberation model is developed for range-dependent environments. First-order perturbation theory is employed, and the unperturbed background medium can be layered and heterogeneous with arbitrary range dependence. To calculate the reverberation field, two-way forward scatter due to the slowly changing unperturbed environment is handled by fast numerical methods. Backscatter due to small roughness superimposed on any of the slowly varying interfaces is handled efficiently using a Monte Carlo approach. Numerical examples are presented to demonstrate the application of the model. The primary purpose of the model is to incorporate relevant physics while improving computational speed.

Mid-frequency geoacoustic inversion using bottom loss data from the Shallow Water 2006 Experiment.

Geoacoustic inversion work has typically been carried out at frequencies below 1 kHz, assuming flat, horizontally stratified bottom models. Despite the relevance to Navy sonar systems many of which operate at mid-frequencies (1-10 kHz), limited inversion work has been carried out in this frequency band. This paper is an effort to demonstrate the viability of geoacoustic inversion using bottom loss data between 2 and 5 kHz. The acoustic measurements were taken during the Shallow Water 2006 Experiment off the coast of New Jersey. A half-space bottom model, with three parameters density, compressional wave speed, and attenuation, was used for inversion by fitting the model to data in the least-square sense. Inverted sediment sound speed and attenuation were compared with direct measurements and with inversion results using different techniques carried out in SW06. Inverted results of the present work are consistent with other measurements, considering the known spatial variability in this area. The observations and modeling results demonstrate that forward scattering from topographical changes is important at mid-frequencies and should be taken into account in sound propagation predictions and geoacoustic inversion. To cope with fine-scale topographic variability, measurement technique such as averaging over tracks may be necessary.

Dispersion and attenuation due to scattering from heterogeneities of the frame bulk modulus of a poroelastic medium.

While Biot theory can successfully account for the dispersion observed in sand sediments, the attenuation at high frequencies has been observed to increase more rapidly than Biot theory would predict. In an effort to account for this additional loss, perturbation theory is applied to Biot's poroelastic equations to model the loss due to the scattering of energy from heterogeneities in the sediment. A general theory for propagation loss is developed and applied to a medium with a randomly varying frame bulk modulus. The theory predicts that these heterogeneities produce an overall softening of the medium as well as scattering of energy from the mean fast compressional wave into incoherent fast and slow compressional waves. This theory is applied to two poroelastic media: a weakly consolidated sand sediment and a consolidated sintered glass bead pack. The random variations in the frame modulus do not have significant effects on the propagation through the sand sediment but do play an important role in the propagation through the consolidated medium.

Differential role of beta-arrestin ubiquitination in agonist-promoted down-regulation of M1 vs M2 muscarinic acetylcholine receptors.

BACKGROUND: Sustained agonist-promoted ubiquitination of beta-arrestin has been correlated with increased stability of the GPCR - beta-arrestin complex. Moreover, abrogation of beta-arrestin ubiquitination has been reported to inhibit receptor internalization with minimal effects on receptor degradation. RESULTS: Herein we report that agonist activation of M1 mAChRs produces a sustained beta-arrestin ubiquitination but no stable co-localization with beta-arrestin. In contrast, sustained ubiquitination of beta-arrestin by activation of M2 mAChRs does result in stable co-localization between the M2 mAChR and beta-arrestin. Internalization of receptors was unaffected by proteasome inhibitors, but down-regulation was significantly reduced, suggesting a role for the ubiquitination machinery in promoting down-regulation of the receptors. Given the ubiquitination status of beta-arrestin following agonist treatment, we sought to determine the effects of beta-arrestin ubiquitination on M1 and M2 mAChR down-regulation. A constitutively ubiquitinated beta-arrestin 2 chimera in which ubiquitin is fused to the C-terminus of beta-arrestin 2 (YFP-beta-arrestin 2-Ub) significantly increased agonist-promoted down-regulation of both M1 and M2 mAChRs, with the effect substantially higher on the M2 mAChR. Based on this observation, we were interested in examining the effects of disruption of potential ubiquitination sites in the beta-arrestin sequence on receptor down-regulation. Agonist-promoted internalization of the M2 mAChR was not affected by expression of beta-arrestin lysine mutants lacking putative ubiquitination sites, beta-arrestin 2K18R, K107R, K108R, K207R, K296R, while down-regulation and stable co-localiztion of the receptor with this beta-arrestin lysine mutant were significantly reduced. Interestingly, expression of beta-arrestin 2K18R, K107R, K108R, K207R, K296R increased the agonist-promoted down-regulation of the M1 mAChR but did not result in a stable co-localiztion of the receptor with this beta-arrestin lysine mutant. CONCLUSION: These findings indicate that ubiquitination of beta-arrestin has a distinct role in the differential trafficking and degradation of M1 and M2 mAChRs.

Transient hypoxia induces sequestration of M1 and M2 muscarinic acetylcholine receptors.

Oxidative stress has been implicated in impairing muscarinic acetylcholine receptor (mAChR) signaling activity. It remains unclear, however, whether alterations in the cell surface distribution of mAChRs following oxidative stress contribute to the diminished mAChR signaling activity. We report here that M1 and M2 mAChRs, stably expressed in Chinese hamster ovary cells, undergo sequestration following transient hypoxic-induced oxidative stress (2% O2). Sequestration of M1 and M2 mAChRs following transient hypoxia was associated with an increase in phosphorylation of these receptors. Over-expression of a catalytically inactive G protein-coupled receptor kinase 2 (GRK2 K220R) blocked the increased phosphorylation and sequestration of the M2, but not M1, mAChRs following transient hypoxia. Hypoxia induced phosphorylation and sequestration of the M1 mAChR was, however, blocked by over-expression of a catalytically inactive casein kinase 1 alpha (CK1alpha K46R). These results are the first demonstration that M1 and M2 mAChRs undergo sequestration following transient hypoxia. The data suggest that increased phosphorylation of M1 and M2 mAChRs underlies the mechanism responsible for sequestration of these receptors following transient hypoxia. We report here that distinct pathways involving CK1alpha and GRK2 mediated sequestration of M1 and M2 mAChRs following transient hypoxic-induced oxidative stress.

Agonist mediated internalization of M2 mAChR is beta-arrestin-dependent.

BACKGROUND: Muscarinic acetylcholine receptors (mAChRs) undergo agonist-promoted internalization, but evidence suggesting that the mechanism of internalization is beta-arrestin dependent has been contradictory and unclear. Previous studies using heterologous over-expression of wild type or dominant-negative forms of beta-arrestins have reported that agonist-promoted internalization of M2 mAChRs is a beta-arrestin- and clathrin-independent phenomenon. In order to circumvent the complications associated with the presence of endogenous beta-arrestin that may have existed in these earlier studies, we examined agonist-promoted internalization of the M2 mAChR in mouse embryonic fibroblasts (MEFs) derived from beta-arrestin knockout mice that lack expression of either one or both isoforms of beta-arrestin (beta-arrestin 1 and 2). RESULTS: In wild type MEF cells transiently expressing M2 mAChRs, 40% of surface M2 mAChRs underwent internalization and sorted into intracellular compartments following agonist stimulation. In contrast, M2 mAChRs failed to undergo internalization and sorting into intracellular compartments in MEF beta-arrestin double knockout cells following agonist stimulation. In double knockout cells, expression of either beta-arrestin 1 or 2 isoforms resulted in rescue of agonist-promoted internalization. Stimulation of M2 mAChRs led to a stable co-localization with GFP-tagged beta-arrestin within endocytic structures in multiple cell lines; the compartment to which beta-arrestin localized was determined to be the early endosome. Agonist-promoted internalization of M2 mAChRs was moderately rescued in MEF beta-arrestin 1 and 2 double knockout cells expressing exogenous arrestin mutants that were selectively defective in interactions with clathrin (beta-arrestin 2 DeltaLIELD), AP-2 (beta-arrestin 2-F391A), or both clathrin/AP-2. Expression of a truncated carboxy-terminal region of beta-arrestin 1 (319-418) completely abrogated agonist-promoted internalization of M2 mAChRs in wild type MEF cells. CONCLUSION: In summary, this study demonstrates that agonist-promoted internalization of M2 mAChRs is beta-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with beta-arrestin in early endosomal vesicles.