The Biochemistry, Ultrastructure, and Subunit Assembly Mechanism of AMPA Receptors

The AMPA-type ionotropic glutamate receptors (AMPA-Rs) are tetrameric ligand-gated ion channels that play crucial roles in synaptic transmission and plasticity. Our knowledge about the ultrastructure and subunit assembly mechanisms of intact AMPA-Rs was very limited. However, the new studies using single particle EM and X-ray crystallography are revealing important insights. For example, the tetrameric crystal structure of the GluA2cryst construct provided the atomic view of the intact receptor. In addition, the single particle EM structures of the subunit assembly intermediates revealed the conformational requirement for the dimer-to-tetramer transition during the maturation of AMPA-Rs. These new data in the field provide new models and interpretations. In the brain, the native AMPA-R complexes contain auxiliary subunits that influence subunit assembly, gating, and trafficking of the AMPA-Rs. Understanding the mechanisms of the auxiliary subunits will become increasingly important to precisely describe the function of AMPA-Rs in the brain. The AMPA-R proteomics studies continuously reveal a previously unexpected degree of molecular heterogeneity of the complex. Because the AMPA-Rs are important drug targets for treating various neurological and psychiatric diseases, it is likely that these new native complexes will require detailed mechanistic analysis in the future. The current ultrastructural data on the receptors and the receptor-expressing stable cell lines that were developed during the course of these studies are useful resources for high throughput drug screening and further drug designing. Moreover, we are getting closer to understanding the precise mechanisms of AMPA-R-mediated synaptic plasticity

Identification of Inhibitors of Triacylglyceride Accumulation in Muscle Cells: Comparing HTS Results from 1536-Well Plate-Based and High-Content Platforms

Excess caloric consumption leads to triacylglyceride (TAG) accumulation in tissues that do not typically store fat, such as skeletal muscle. This ectopic accumulation alters cells, contributing to the pathogenesis of metabolic syndrome, a major health problem worldwide. We developed a 1536-well assay to measure intracellular TAG accumulation in differentiating H9c2 myoblasts. For this assay, cells were incubated with oleic acid to stimulate TAG accumulation prior to adding compounds. We used Nile red as a fluorescent dye to quantify TAG content with a microplate reader. The cell nuclei were counterstained with DAPI nuclear stain to assess cell count and filter cytotoxic compounds. In parallel, we developed an image-based assay in H9c2 cells to measure lipid accumulation levels via high-content analysis, exploiting the dual-emission spectra characteristic of Nile red staining of neutral and phospholipids. Using both approaches, we successfully screened ~227,000 compounds from the National Institutes of Health library. The screening data from the plate reader and IC50 values correlated with that from the Opera QEHS cell imager. The 1536-well plate reader assay is a powerful high-throughout screening platform to identify potent inhibitors of TAG accumulation to better understand the molecular pathways involved in lipid metabolism that lead to lipotoxicity

MondoA coordinately regulates skeletal myocyte lipid homeostasis and insulin signaling

Intramuscular lipid accumulation is a common manifestation of chronic caloric excess and obesity that is strongly associated with insulin resistance. The mechanistic links between lipid accumulation in myocytes and insulin resistance are not completely understood. In this work, we used a high-throughput chemical biology screen to identify a small-molecule probe, SBI-477, that coordinately inhibited triacylglyceride (TAG) synthesis and enhanced basal glucose uptake in human skeletal myocytes. We then determined that SBI-477 stimulated insulin signaling by deactivating the transcription factor MondoA, leading to reduced expression of the insulin pathway suppressors thioredoxin-interacting protein (TXNIP) and arrestin domain-containing 4 (ARRDC4). Depleting MondoA in myocytes reproduced the effects of SBI-477 on glucose uptake and myocyte lipid accumulation. Furthermore, an analog of SBI-477 suppressed TXNIP expression, reduced muscle and liver TAG levels, enhanced insulin signaling, and improved glucose tolerance in mice fed a high-fat diet. These results identify a key role for MondoA-directed programs in the coordinated control of myocyte lipid balance and insulin signaling and suggest that this pathway may have potential as a therapeutic target for insulin resistance and lipotoxicity

Inhibitors of Myocyte Triacylglyceride Accumulation

Obesity is associated with a wide range of public health and economic problems throughout diverse demographic groups. As there is a high incidence of insulin resistance and type-2 diabetes among the obese, a mechanistic understanding of the relationship between insulin resistance and caloric consumption / obesity is needed. In cases of obesity and insulin resistance, triacylglycerides (TAG) accumulate within skeletal muscle cell; however, the mechanism of this relationship is not well understood. The research program described herein therefore sought small molecule probes that inhibit TAG accumulation. Toward this end, screening of 227,000 compounds resulted in the identification of MLS-0308942, which inhibited TAG accumulation with an IC50 of 1.7 μM. Subsequent medicinal chemistry follow up identified MLS-0472732 (ML377), which had similar potency (IC50 1.1 μM) with some improved ADME/T properties. Data were confirmed in the H9c2 high content assay (IC50 = 0.84 and 0.79 μM, respectively, with full response) and in a human primary cell assay. Furthermore, the compounds tested negatively for DGAT inhibition and cytotoxicity. Several additional analogs with comparable potency were observed, and the established SAR suggests the synthesis of new analogs in the future, which may address the poor microsomal stability exhibited thus far with the new series of compounds

Biomass composition of Arthrospira platensis during cultivation on industrial process water and harvesting

Microalgae have the ability to utilize nutrients from wastewater and use it for biomass production. The effluent from a biogas process was tested as a nutrient source for blue-green microalga Arthrospira platensis cultivation and compared with conventional synthetic medium. Cultivation was carried out in four different concentrations of industrial process water (25, 50, 75, and 100%). The biomass was then harvested by microfiltration, and centrifugation followed by freeze drying. Variations in biomass composition were studied, in order to investigate effects of industrial process water on A. platensis over 30 days of cultivation. Applied harvesting techniques were evaluated for their effect on physiochemical properties of the biomass. Arthrospira platensis was able to grow in all tested wastewater concentrations except 100%, however, increase of wastewater concentration in medium resulted in a decreased growth rate. Partial substitution of synthetic Zarrouk medium with 25% of wastewater showed no adverse effect on chemical composition of the biomass including high protein content (45–58% dry weight) and favorable fatty acid composition (42–45% PUFAs of total fatty acids). Evaluation by optical microscopy showed that microfiltration caused cell rupture at the moderate level while centrifugation had more severe effect on A. platensis. Effect of centrifugal forces and shear stress on A. platensis cells was confirmed by detecting lower lipid content in samples after applying both microfiltration and centrifugation due to cell content leakage