Propofol reduces microglia activation and neurotoxicity through inhibition of extracellular vesicle release.

Propofol is an established anesthetic widely used for induction and maintenance of anesthesia. We investigated propofol for its anti-inflammatory effects on microglia and found that propofol treatment is associated with substantial lower levels of extracellular vesicles (EVs) in immune activated microglia. Importantly, EVs collected from immune activated microglia reversed propofol-mediated anti-inflammatory and neuroprotective effects, suggesting that propofol reduces proinflammatory microglia activation and microglia-mediated neurotoxicity through inhibition of EV release. These data shed new insight into a novel molecular mechanism of propofol-mediated neuroprotective and immunomodulatory effects through inhibition of EV release.

Zika virus propagation and release in human fetal astrocytes can be suppressed by neutral sphingomyelinase-2 inhibitor GW4869.

Zika virus (ZIKV) is a neurotrophic flavivirus that is capable of infecting humans, leading to brain abnormalities during fetal development. The ZIKV infectivity in neural target cells remains poorly understood. Here, we found that ZIKV specifically infected glial fibrillary acidic protein- and S100B-positive primary human astrocytes derived from fetal brains. In contrast, neuron-specific Class III ß-tubulin (TuJ1)-positive neurons in the astrocyte cultures and SOX2-positive neural progenitor cells derived from the fetal brains were less susceptible to ZIKV infection compared with astrocytes. The infected astrocytes released competent viral particles and manifested programmed cell death with a progressive cytopathic effect. Interestingly, ZIKV infection in human fetal astrocytes induced a significant increase of extracellular vesicles (EVs). Treatment with GW4869, a specific inhibitor of neutral sphingomyelinase-2, decreased EV levels, suppressed ZIKV propagation, and reduced the release of infectious virions in astrocytes. Therefore, ZIKV infects primary human fetal astrocytes and the infection can be suppressed by neutral sphingomyelinase-2 inhibitor GW4869. Further investigation into sphingomyelin metabolism and EVs may provide insights to the therapeutic treatment of ZIKV infection.

Glutaminase 1 regulates the release of extracellular vesicles during neuroinflammation through key metabolic intermediate alpha-ketoglutarate.

BACKGROUND: Extracellular vesicles (EVs) are important in the intercellular communication of the central nervous system, and their release is increased during neuroinflammation. Our previous data demonstrated an increased release of EVs during HIV-1 infection and immune activation in glial cells. However, the molecular mechanism by which infection and inflammation increase EV release remains unknown. In the current study, we investigated the role of glutaminase 1 (GLS1)-mediated glutaminolysis and the production of a key metabolic intermediate α-ketoglutarate on EV release. METHODS: Human monocyte-derived macrophage primary cultures and a BV2 microglia cell line were used to represent the innate immune cells in the CNS. Transmission electron microscopy, nanoparticle tracking analysis, and Western blots were used to determine the EV regulation. GLS1 overexpression was performed using an adenovirus vector in vitro and transgenic mouse models in vivo. Data were evaluated statistically by ANOVA, followed by the Bonferroni post-test for paired observations. RESULTS: Our data revealed an increased release of EVs in GLS1-overexpressing HeLa cells. In HIV-1-infected macrophages and immune-activated microglia BV2 cells, treatment with bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) or CB839, two specific GLS inhibitors, significantly decreased EV release, suggesting a critical role of GLS1 in EV release. Furthermore, addition of α-ketoglutarate or ceramide rescued EV release during BPTES treatment, implicating α-ketoglutarate and ceramide as critical downstream effectors for GLS inhibitors. These findings were further corroborated with the investigation of brain tissues in GLS1-transgenic mice. The EV levels were significantly higher in GLS1 transgenic mice than those in control mice, suggesting that GLS1 increases EV release in vivo. CONCLUSIONS: These findings suggest that GLS1-mediated glutaminolysis and its downstream production of α-ketoglutarate are essential in regulating EV release during HIV-1 infection and immune activation. These new mechanistic regulations may help understand how glutamine metabolism shapes EV biogenesis and release during neuroinflammation.

Glutaminase C overexpression in the brain induces learning deficits, synaptic dysfunctions, and neuroinflammation in mice.

Glutaminolysis, a metabolic process that converts glutamine to glutamate, is particularly important for the central nervous system since glutamate is the major transmitter of excitatory synapses. Glutaminase is the mitochondrial enzyme that catalyzes the first step of glutaminolysis. Two genes encode at least four isoforms of glutaminase in humans. Gls1 gene encodes isoforms kidney-type glutaminase (KGA) and glutaminase C (GAC) through alternative splicing, whereas Gls2 gene encodes liver-type glutaminase isoforms. KGA and GAC have been associated with several neurological diseases. However, it remains unclear whether changes in their expressions can directly cause brain abnormalities. Using a transgenic approach, we generated mice that overexpressed GAC in the brain. The resulting transgenic mice had severe impairments in spatial and fear learning compared with littermate controls. The learning deficits were consistent with diminished hippocampal long-term potentiation in the hippocampal slices of the GAC transgenic mice. Furthermore, we found increases in astrocyte and microglia markers, inflammatory factors, and a decrease in synapse marker synaptophysin, suggesting neuroinflammation and synaptic changes in the GAC transgenic mouse brains. In conclusion, these findings provide the first evidence that GAC overexpression in the brain has deleterious effects on learning and synaptic integrity in vivo.

Glutaminase-containing microvesicles from HIV-1-infected macrophages and immune-activated microglia induce neurotoxicity.

BACKGROUND: HIV-1-infected and/or immune-activated microglia and macrophages are pivotal in the pathogenesis of HIV-1-associated neurocognitive disorders (HAND). Glutaminase, a metabolic enzyme that facilitates glutamate generation, is upregulated and may play a pathogenic role in HAND. Our previous studies have demonstrated that glutaminase is released to the extracellular fluid during HIV-1 infection and neuroinflammation. However, key molecular mechanisms that regulate glutaminase release remain unknown. Recent advances in understanding intercellular trafficking have identified microvesicles (MVs) as a novel means of shedding cellular contents. We posit that during HIV-1 infection and immune activation, microvesicles may mediate glutaminase release, generating excessive and neurotoxic levels of glutamate. RESULTS: MVs isolated through differential centrifugation from cell-free supernatants of monocyte-derived macrophages (MDM) and BV2 microglia cell lines were first confirmed in electron microscopy and immunoblotting. As expected, we found elevated number of MVs, glutaminase immunoreactivities, as well as glutaminase enzyme activity in the supernatants of HIV-1 infected MDM and lipopolysaccharide (LPS)-activated microglia when compared with controls. The elevated glutaminase was blocked by GW4869, a neutral sphingomyelinase inhibitor known to inhibit MVs release, suggesting a critical role of MVs in mediating glutaminase release. More importantly, MVs from HIV-1-infected MDM and LPS-activated microglia induced significant neuronal injury in rat cortical neuron cultures. The MV neurotoxicity was blocked by a glutaminase inhibitor or GW4869, suggesting that the neurotoxic potential of HIV-1-infected MDM and LPS-activated microglia is dependent on the glutaminase-containing MVs. CONCLUSIONS: These findings support MVs as a potential pathway/mechanism of excessive glutamate generation and neurotoxicity in HAND and therefore MVs may serve as a novel therapeutic target.

Novel polymer linkers for single molecule AFM force spectroscopy.

Flexible polymer linkers play an important role in various imaging and probing techniques that require surface immobilization, including atomic force microscopy (AFM). In AFM force spectroscopy, polymer linkers are necessary for the covalent attachment of molecules of interest to the AFM tip and the surface. The polymer linkers tether the molecules and provide their proper orientation in probing experiments. Additionally, the linkers separate specific interactions from nonspecific short-range adhesion and serve as a reference point for the quantitative analysis of single molecule probing events. In this report, we present our results on the synthesis and testing of a novel polymer linker and the identification of a number of potential applications for its use in AFM force spectroscopy experiments. The synthesis of the linker is based on the well-developed phosphoramidate (PA) chemistry that allows the routine synthesis of linkers with predetermined lengths and PA composition. These linkers are homogeneous in length and can be terminated with various functional groups. PA linkers with different functional groups were synthesized and tested in experimental systems utilizing different immobilization chemistries. We probed interactions between complementary DNA oligonucleotides; DNA and protein complexes formed by the site-specific binding protein SfiI; and interactions between amyloid peptide (Aß42). The results of the AFM force spectroscopy experiments validated the feasibility of the proposed approach for the linker design and synthesis. Furthermore, the properties of the tether (length, functional groups) can be adjusted to meet the specific requirements for different force spectroscopy experiments and system characteristics, suggesting that it could be used for a large number of various applications.

Development of a flow-based ultrafast immunoextraction and reverse displacement immunoassay: analysis of free drug fractions.

A flow-based method employing a reverse displacement immunoassay was combined with ultrafast immunoextraction and near-infrared fluorescence detection for the analysis of free drug fractions, using phenytoin as a model analyte. Factors considered in the design of this method included the sample application conditions, the design of the immobilized drug analog column, the utilization of antibodies or F(ab) fragments as labeled binding agents, and the label application and column regeneration conditions. In the final method, sample injections led to the displacement of labeled binding agents from an immobilized phenytoin analog column. This displacement peak appeared within 20-30 s of sample injection and was proportional in size to the free phenytoin concentration in the sample. It was possible with this method to regenerate the column using only the application of additional label between sample injections. This method was used to measure clinically relevant concentrations of free phenytoin in serum and drug/protein mixtures and gave good correlation with ultrafiltration, while also being faster to perform and requiring significantly less sample. This technique was not limited to free phenytoin measurements but could be adapted for other drugs or analytes through the use of appropriate columns and binding agents.

Characterization of interaction kinetics between chiral solutes and human serum albumin by using high-performance affinity chromatography and peak profiling.

Peak profiling and high-performance columns containing immobilized human serum albumin (HSA) were used to study the interaction kinetics of chiral solutes with this protein. This approach was tested using the phenytoin metabolites 5-(3-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) as model analytes. HSA columns provided some resolution of the enantiomers for each phenytoin metabolite, which made it possible to simultaneously conduct kinetic studies on each chiral form. The dissociation rate constants for these interactions were determined by using both the single flow rate and multiple flow rate peak profiling methods. Corrections for non-specific interactions with the support were also considered. The final estimates obtained at pH 7.4 and 37°C for the dissociation rate constants of these interactions were 8.2-9.6 s(-1) for the two enantiomers of m-HPPH and 3.2-4.1 s(-1) for the enantiomers of p-HPPH. These rate constants agreed with previous values that have been reported for other drugs and solutes that have similar affinities and binding regions on HSA. The approach used in this report was not limited to phenytoin metabolites or HSA but could be applied to a variety of other chiral solutes and proteins. This method could also be adopted for use in the rapid screening of drug-protein interactions.

Detection of heterogeneous drug-protein binding by frontal analysis and high-performance affinity chromatography.

This study examined the use of frontal analysis and high-performance affinity chromatography for detecting heterogeneous binding in biomolecular interactions, using the binding of acetohexamide with human serum albumin (HSA) as a model. It was found through the use of this model system and chromatographic theory that double-reciprocal plots could be used more easily than traditional isotherms for the initial detection of binding site heterogeneity. The deviations from linearity that were seen in double-reciprocal plots as a result of heterogeneity were a function of the analyte concentration, the relative affinities of the binding sites in the system and the amount of each type of site that was present. The size of these deviations was determined and compared under various conditions. Plots were also generated to show what experimental conditions would be needed to observe these deviations for general heterogeneous systems or for cases in which some preliminary information was available on the extent of binding heterogeneity. The methods developed in this work for the detection of binding heterogeneity are not limited to drug interactions with HSA but could be applied to other types of drug-protein binding or to additional biological systems with heterogeneous binding.

Characterization of drug interactions with serum proteins by using high-performance affinity chromatography.

The binding of drugs with serum proteins can affect the activity, distribution, rate of excretion, and toxicity of pharmaceutical agents in the body. One tool that can be used to quickly analyze and characterize these interactions is high-performance affinity chromatography (HPAC). This review shows how HPAC can be used to study drug-protein binding and describes the various applications of this approach when examining drug interactions with serum proteins. Methods for determining binding constants, characterizing binding sites, examining drug-drug interactions, and studying drug-protein dissociation rates will be discussed. Applications that illustrate the use of HPAC with serum binding agents such as human serum albumin, α(1)-acid glycoprotein, and lipoproteins will be presented. Recent developments will also be examined, such as new methods for immobilizing serum proteins in HPAC columns, the utilization of HPAC as a tool in personalized medicine, and HPAC methods for the high-throughput screening and characterization of drug-protein binding.

Kinetic studies of drug-protein interactions by using peak profiling and high-performance affinity chromatography: examination of multi-site interactions of drugs with human serum albumin columns.

Carbamazepine and imipramine are drugs that have significant binding to human serum albumin (HSA), the most abundant serum protein in blood and a common transport protein for many drugs in the body. Information on the kinetics of these drug interactions with HSA would be valuable in understanding the pharmacokinetic behavior of these drugs and could provide data that might lead to the creation of improved assays for these analytes in biological samples. In this report, an approach based on peak profiling was used with high-performance affinity chromatography to measure the dissociation rate constants for carbamazepine and imipramine with HSA. This approach compared the elution profiles for each drug and a non-retained species on an HSA column and control column over a board range of flow rates. Various approaches for the corrections of non-specific binding between these drugs and the support were considered and compared in this process. Dissociation rate constants of 1.7 (±0.2) s(-1) and 0.67 (±0.04) s(-1) at pH 7.4 and 37°C were estimated by this approach for HSA in its interactions with carbamazepine and imipramine, respectively. These results gave good agreement with rate constants that have determined by other methods or for similar solute interactions with HSA. The approach described in this report for kinetic studies is not limited to these particular drugs or HSA but can also be extended to other drugs and proteins.

CHROMATOGRAPHIC ANALYSIS OF DRUG INTERACTIONS IN THE SERUM PROTEOME.

The binding of drugs with serum proteins and binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins is an important process in determining the activity and fate of many pharmaceuticals in the body. A variety of techniques have been used to study drug interactions with serum proteins, but there is still a need for faster or better methods for such work. High-performance liquid chromatography (HPLC) is one tool that has been utilized in many formats for these types of measurements. Advantages of using HPLC for this application include its speed and precision, its ability to be automated, its good limits of detection, and its compatibility with a wide range of assay formats and detectors. This review will discuss various approaches in which HPLC can be employed for the study of drug-protein interactions. These techniques include the use of soluble proteins in zonal elution and frontal analysis methods or vacancy techniques such as the Hummel-Dreyer method. Zonal elution and frontal analysis methods that make use of immobilized proteins and high-performance affinity chromatography will also be presented. A variety of applications will be examined, ranging from the determination of free drug fractions to the measurement of the strength or rate of a drug-protein interaction. Newer developments that will be discussed include recent work in the creation of novel mathematical approaches for HPLC studies of drug-protein binding, the use of HPLC methods for the high-throughput screening of drug-protein binding, and the creation and use of affinity monoliths or affinity microcolumns for examining drug-protein systems.

Studies by biointeraction chromatography of binding by phenytoin metabolites to human serum albumin.

Biointeraction studies based on high performance affinity chromatography were used to investigate the binding of human serum albumin (HSA) to two major phenytoin metabolites: 5-(3-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH). This was initially examined by conducting self-competition zonal elution experiments in which m-HPPH or p-HPPH were placed in both the mobile phase and injected sample. It was found that each metabolite had a single major binding site on HSA. Competitive zonal elution experiments using l-tryptophan, warfarin, digitoxin, and cis-clomiphene as site-selective probes indicated that m-HPPH and p-HPPH were interacting with the indole-benzodiazepine site of HSA. The estimated association equilibrium constants for m-HPPH and p-HPPH at this site were 3.2 (+/-1.2)x10(3) and 5.7 (+/-0.7)x10(3)M(-1), respectively, at pH 7.4 and 37 degrees C. Use of these metabolites as competing agents for injections of phenytoin demonstrated that m-HPPH and p-HPPH had direct competition with this drug at the indole-benzodiazepine site. However, the use of phenytoin as a competing agent indicated that this drug had additional negative allosteric interactions on the binding of these metabolites to HSA. These results agreed with previous studies on the binding of phenytoin to HSA and its effects on the interactions of HSA with site-selective probes for the indole-benzodiazepine site.