Usefulness of a humanized tricellular static transwell blood-brain barrier model as a microphysiological system for drug development applications. - A case study based on the benchmark evaluations of blood-brain barrier microphysiological system.

Microphysiological system (MPS), a new technology for in vitro testing platforms, have been acknowledged as a strong tool for drug development. In the central nervous system (CNS), the blood‒brain barrier (BBB) limits the permeation of circulating substances from the blood vessels to the brain, thereby protecting the CNS from circulating xenobiotic compounds. At the same time, the BBB hinders drug development by introducing challenges at various stages, such as pharmacokinetics/pharmacodynamics (PK/PD), safety assessment, and efficacy assessment. To solve these problems, efforts are being made to develop a BBB MPS, particularly of a humanized type. In this study, we suggested minimal essential benchmark items to establish the BBB-likeness of a BBB MPS; these criteria support end users in determining the appropriate range of applications for a candidate BBB MPS. Furthermore, we examined these benchmark items in a two-dimensional (2D) humanized tricellular static transwell BBB MPS, the most conventional design of BBB MPS with human cell lines. Among the benchmark items, the efflux ratios of P-gp and BCRP showed high reproducibility in two independent facilities, while the directional transports meditated through Glut1 or TfR were not confirmed. We have organized the protocols of the experiments described above as standard operating procedures (SOPs). We here provide the SOPs with the flow chart including entire procedure and how to apply each SOP. Our study is important developmental step of BBB MPS towards the social acceptance, which enable end users to check and compare the performance the BBB MPSs.

Comparison of Golgi-Cox and Intracellular Loading of Lucifer Yellow for Dendritic Spine Density and Morphology Analysis in the Mouse Brain.

Dendritic spines are small protrusions on dendrites that serve as the postsynaptic site of the majority of excitatory synapses. These structures are important for normal synaptic transmission, and alterations in their density and morphology have been documented in various disease states. Over 130 years ago, Ramón y Cajal used Golgi-stained tissue sections to study dendritic morphology. Despite the array of technological advances, including iontophoretic microinjection of Lucifer yellow (LY) fluorescent dye, Golgi staining continues to be one of the most popular approaches to visualize dendritic spines. Here, we compared dendritic spine density and morphology among pyramidal neurons in layers 2/3 of the mouse medial prefrontal cortex (mPFC) and pyramidal neurons in hippocampal CA1 using three-dimensional digital reconstructions of (1) brightfield microscopy z-stacks of Golgi-impregnated dendrites and (2) confocal microscopy z-stacks of LY-filled dendrites. Analysis of spine density revealed that the LY microinjection approach enabled detection of approximately three times as many spines as the Golgi staining approach in both brain regions. Spine volume measurements were larger using Golgi staining compared to LY microinjection in both mPFC and CA1. Spine length was mostly comparable between techniques in both regions. In the mPFC, head diameter was similar for Golgi staining and LY microinjection. However, in CA1, head diameter was approximately 50% smaller on LY-filled dendrites compared to Golgi staining. These results indicate that Golgi staining and LY microinjection yield different spine density and morphology measurements, with Golgi staining failing to detect dendritic spines and overestimating spine size.

Protective Mechanisms of Liquid Formulations for Gastro-Oesophageal Reflux Disease in a Human Reconstructed Oesophageal Epithelium Model.

Purpose: A novel experimental design based on a human-reconstructed oesophageal epithelium (HO2E) model has been applied to quantitively assess the properties of a set of liquid formulations, Device A (Gerdoff® Protection), Device B (Esoxx® One), and Device C (Marial® gel) developed to form a temporary physical barrier on the oesophageal epithelium and modify epithelial permeability so to protect the oesophageal mucosa from refluxate components. Methods: The formulations were applied to a prewetted HO2E model for 15 min. Then, a 0.5% caffeine solution was applied, and its penetration kinetics was assessed at 1 h and 2 h in acidic environments (pH= 3.3) to mirror exposure of the oesophageal mucosa to acidic reflux in GORD patients. Caffeine permeated into the basolateral compartment (evaluated by HPLC-UV) and Lucifer yellow (LY) permeability were quantified 15 min after application of the caffeine in acidic environments. Results: At the 15 min timepoint, Device A reduced caffeine permeation by 77.2% and LY flux by 30.4% compared to the untreated control and with a faster mode of action than that of the other liquid formulations. Transepithelial caffeine flux was reduced, albeit with different timing and efficiency, by all three compounds up to the end of the 2 hour experiment. At 1 h, Device A reduced the caffeine flux by 79.2%; Device B, by 67.2%; and Device C, by 37%. Conclusion: These results confirm the ability of the medical devices tested to interact with the oesophageal epithelium and create a temporary physical protective film for up to 2 hours after their application. The results underline differences in the mechanism of action of the three medical devices, with Device A performing faster than the other formulations. The overall results support the relevance of the reconstructed mucosal model to investigate oesophageal epithelium-product interactions and precisely differentiate liquid formulation performance.

Monitoring Modified Lipoprotein Uptake and Macropinocytosis Associated with Macrophage Foam Cell Formation.

Macrophage foam cell formation plays a crucial role in the initiation and progression of atherosclerosis. Macrophages uptake native and modified low density lipoprotein (LDL) through either receptor-dependent or receptor-independent mechanisms to transform into lipid laden foam cells. Foam cells are involved in the formation of fatty streak that is seen during the early stages of atherosclerosis development and therefore represents a promising therapeutic target. Normal or modified lipoproteins labeled with fluorescent dyes such as 1,1'-dioctadecyl-3-3-3',3'-tetramethylindocarbocyanine perchlorate (Dil) are often used to monitor their internalization during foam cell formation. In addition, the fluorescent dye Lucifer Yellow (LY) is widely used as a marker for macropinocytosis activity. In this chapter, we describe established methods for monitoring modified lipoprotein uptake and macropinocytosis during macrophage foam cell formation.

Connexin32 ameliorates epithelial-to-mesenchymal-transition in diabetic renal tubular via inhibiting NOX4.

Renal tubulointerstitial fibrosis (RIF), characterized by epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells (TECs), is the main cause of diabetic renal fibrosis. Oxidative stress plays a pivotal role in the development of diabetic RIF. Connexin32 (Cx32), prominently expressed in renal TECs, has emerged as an important player in the regulation of oxidative stress. However, the role of Cx32 in diabetic RIF has not been explored yet. Here, we showed that adenovirus-mediated Cx32 overexpression suppressed EMT to ameliorate RIF and renal function in STZ-induced diabetic mice, while knockout (KO) of Cx32 exacerbated RIF in diabetic mice. Moreover, overexpression of Cx32 inhibited EMT and the production of extra cellular matrix (ECM) in high glucose (HG) induced NRK-52E cells, whereas knockdown of Cx32 showed the opposite effects. Furthermore, we showed that NOX4, the main source of ROS in renal tubular, was down-regulated by Cx32. Mechanistically, Cx32 down-regulated the expression of PKC alpha in a carboxyl-terminal-dependent manner, thereby inhibiting the phosphorylation at Thr147 of p22phox triggered by PKC alpha, which ultimately repressed the formation of the p22phox-NOX4 complex to reduce the protein level of NOX4. Thus, we establish Cx32 as a novel target and confirm the protection mechanism in RIF.

Re-Use of Caco-2 Monolayers in Permeability Assays-Validation Regarding Cell Monolayer Integrity.

Caco-2 monolayers are a common in vitro model used to evaluate human intestinal absorption. The reference protocol requires 21 days post-seeding to establish a stable and confluent cell monolayer, which is used in a single permeability assay during the period of monolayer stability (up to day 30). In this work, we characterize variations in the tightness of the cell monolayer over the stable time interval and evaluate the conditions required for their re-use in permeability assays. The monolayer integrity was assessed through TEER measurements and permeability of the paracellular marker Lucifer Yellow (LY), complemented with nuclei and ZO-1 staining for morphological studies and the presence of tight junctions. Over 150 permeability assays were performed, which showed that manipulation of the cell monolayer in the permeability assay may contribute significantly to the flux of LY, leading to Papp values that are dependent on the sampling duration. The assay also leads to a small decrease in the cell monolayer TEER, which is fully recovered when cell monolayers are incubated with culture media for two full days. When this procedure is followed, the cell monolayers may be used for permeability assays on days 22, 25, and 28, triplicating the throughput of this important assay.

Floral glands in myophilous and sapromyophilous species of Pleurothallidinae (Epidendroideae, Orchidaceae)-osmophores, nectaries, and a unique sticky gland.

Pleurothallidinae orchids have been the focus of many multidisciplinary studies due to their challenging systematics and taxonomy. The synapomorphies already recognized in the group are mostly related to floral characters, the last proposed being the occurrence of alkanes in the floral fragrance. The composition of the floral bouquet varied significantly among the studied species, leading us to hypothesize that the variations in volatiles emitted could be linked to the structure of osmophores, especially when comparing the myophilous and sapromyophilous pollination syndromes. Sepals and labellum at different developmental stages of seven Brazilian Pleurothallidinae species were examined using light, scanning, and transmission electron microscopy. Nectar reabsorption was assessed by Lucifer Yellow CH tracer and imaged under confocal microscopy. Nectaries were restricted to the labellum of the myophilous species, whereas osmophores occurred in the dorsal and/or lateral sepals, varying according to species. In the sapromyophilous species, floral nectaries were not detected and osmophores were restricted to the labellum. Osmophore structure was correlated with the volatiles emitted, being the trichome osmophores notably present on the sepals of both myophilous species that possess nectaries. For the first time, we demonstrated reabsorption of the released nectar in Pleurothallidinae and the occurrence of a unique gland named sticky-exudate glands, which occurred in the lateral sepals and labellum of Echinosepala aspasicensis, a sapromyophilous species, that released a heterogeneous exudate composed of polysaccharides and lipids. Similar glands have been reported in Bulbophyllum, highlighting the convergence between both groups.

Dendritic Spine Density is Increased in Arcadlin-deleted Mouse Hippocampus.

The neural network undergoes remodeling in response to neural activity and interventions, such as antidepressants. Cell adhesion molecules that link pre- and post-synaptic membranes are responsible not only for the establishment of the neural circuitry, but also for the modulation of the strength of each synaptic connection. Among the various classes of synaptic cell adhesion molecules, a non-clustered protocadherin, Arcadlin/Paraxial protocadherin/Protocadherin-8 (Acad), is unique in that it is induced quickly in response to neural activity. Although the primary structure of Arcadlin implies its cell adhesion activity, it weakens the adhesion of N-cadherin. Furthermore, Arcadlin reduces the dendritic spine density in cultured hippocampal neurons. In order to gain an insight into the function of Arcadlin in the brain, we examined the dendritic morphologies of the hippocampal neurons in Acad-/- mice. Acad-/- mice showed a higher spine density than wild-type mice. Following an electroconvulsive seizure (ECS), which strongly induces Arcadlin in the hippocampus, the spine density gradually decreased for 8 h. ECS did not reduce the spine density of CA1 apical dendrites in Acad-/- mice. Daily intraperitoneal injection of the antidepressant fluoxetine (25 mg/kg/day) for 18 days resulted in the induction of Arcadlin in the hippocampus. This treatment reduced spine density in the dentate gyrus and CA1. Chronic fluoxetine treatment did not suppress spine density in Acad-/- mice, suggesting that fluoxetine-induced decrease in spine density is largely due to Arcadlin. The present findings confirm the spine-repulsing activity of Arcadlin and its involvement in the remodeling of hippocampal neurons in response to antidepressants.

Evaluation of Human Esophageal Epithelium Permeability in Presence of Different Formulations Containing Hyaluronic Acid and Chondroitin Sulphate.

PURPOSE: New medical devices that contain hyaluronic acid (HA) and chondroitin sulphate (CS), with or without antacid components, have been developed for the treatment of gastroesophageal reflux disease (GERD) with the aim of improving oesophageal mucosal defences by creating a film on the oesophageal mucosa and acting as a mechanical barrier against the noxious components of refluxate, both acidic and basic. METHODS: The film-forming and protective efficacy of medical device A based on HA and CS plus aluminium hydroxide, device B combining HA and CS with magnesium trisilicate and device C with only the combination of HA and CS was tested on a reconstructed human oesophageal epithelium (HO2E/S/5) as a biological model in 2 different pH environments, neutral and acidic, to mimic realistic conditions. Caffeine penetration kinetics and Lucifer yellow (LY) permeability modifications induced by these products were compared to those induced by a negative control series (saline solution, code NC) and positive control series (white Vaseline, code V) under neutral and acidic pH conditions. RESULTS: Under neutral and acidic pH conditions, compared to the negative control, all the products tested reduced (>80% and 85-90%, respectively) the caffeine passage, and no significant difference was observed among the products tested. Under neutral and acidic conditions, the LY permeabilities registered with device A and device C were not different from that registered with the negative control, while an LY flux% increase was calculated after 2 hrs of treatment (21.1%) with device B under acidic conditions. CONCLUSION: These results confirm the ability of the products tested to interact with the oesophageal epithelium in order to adhere and create a stable protective film for at least 2 hours after their homogeneous distribution on the epithelium surface. Further clinical studies are needed to test these devices in the topical treatment of gastroesophageal reflux symptoms.

Intracellular Dye Microinjection in Retinal Morphological and Communication Studies.

The retina provides an excellent system to analyze neural architecture and communication mechanisms. In retinal physiological, morphological, and developmental studies, intracellular dye microinjection is an extensively applied technique. Dyes and/or chemical compounds are introduced into retinal neurons via iontophoresis injection to identify cell types, communication, and developmental changes. Here, we describe the detailed methods for ejecting the dyes Lucifer yellow and Neurobiotin into retinal neurons through micropipette and concentrate on avoiding the known pitfalls and troubleshooting.

Dedicated Setup for the Photoconversion of Fluorescent Dyes for Functional Electron Microscopy.

Here, we describe a cost-effective setup for targeted photoconversion of fluorescent signals into electron dense ones. This approach has offered invaluable insights in the morphology and function of fine neuronal structures. The technique relies on the localized oxidation of diaminobenzidine (DAB) mediated by excited fluorophores. This paper includes a detailed description of how to build a simple photoconversion setup that can increase reliability and throughput of this well-established technique. The system described here, is particularly well-suited for thick neuronal tissue, where light penetration and oxygen diffusion may be limiting DAB oxidation. To demonstrate the system, we use Correlative Light and Electron Microscopy (CLEM) to visualize functionally-labeled individual synaptic vesicles released onto an identified layer 5 neuron in an acute cortical slice. The setup significantly simplifies the photoconversion workflow, increasing the depth of photoillumination, improving the targeting of the region of interest and reducing the time required to process each individual sample. We have tested this setup extensively for the photoconversion of FM 1-43FX and Lucifer Yellow both excited at 473 nm. In principle, the system can be adapted to any dye or nanoparticle able to oxidize DAB when excited by a specific wavelength of light.

Imaging of glyphosate uptake and identification of early microscopic markers in leaves of C3 and C4 glyphosate-resistant and -susceptible species.

Details of glyphosate uptake are not fully elucidated, and although this herbicide promotes important alterations in the plant phenotype few hours after its application (early responses), a detailed description of the presumable changes in plant anatomy is still poorly assessed by now. Due to glyphosate effects over leaf permeability, the use of an inert fluorescent tracer may allow the observation of the uptake event in situ. In addition, microscopic markers might put a light on the recognition of glyphosate-resistant (GR) and -susceptible (GS) species or varieties, which may vary in C3 and C4 species due to their putative distinct leaf anatomy. Here we aimed (i) to provide a new technique to track the route of glyphosate formulation towards leaf tissues using a fluorescent tracer, and (ii) to describe the early specific microscopic alterations in GR and GS -C3 or -C4 caused by the glyphosate formulation. Roundup Transorb® was applied in seedlings cultivated in a greenhouse and response alterations in leaf anatomy were described. Lucifer Yellow CH (LYCH) was applied over the same region where glyphosate formulation was previously applied to track the alterations in leaf permeability caused by this herbicide. LYCH successfully tracked the glyphosate formulation uptake, reaching the vascular bundles of GS species, and becoming retained in leaf tissues of GR species. All species exhibited a decrease in chlorophyll content at the site of glyphosate application regardless of their photosynthetic metabolism or susceptibility. GS species showed alterations in chloroplast morphology and activity of non-enzymatic antioxidants (carotenoids and flavonoids), in addition to symptoms indicating a process of accelerated cell senescence. A specific type of cell necrosis (hypersensitive response) was observed in GR-C4 species as a way to prevent the translocation of this herbicide, while GR-C3 species accumulated phenolic compounds inside the vacuole, probably sequestrating and inactivating the glyphosate action. This study provides a reliable tool to track glyphosate formulation uptake in situ and is the first attempt to the identification of early specific microscopic markers caused by glyphosate formulation.

Investigation of Metal-Catalyzed Antibody Carbonylation With an Improved Protein Carbonylation Assay.

Protein carbonylation is a posttranslational modification referring to the occurrence of aldehydes and ketones in proteins. The current understanding of how carbonylation, in particular, metal-catalyzed carbonylation, occurs in recombinant mAbs during production and storage is very limited. To facilitate investigations into mAb carbonylation, we developed a protein carbonylation assay with improved assay robustness and precision over the conventional assays. We applied this assay to investigate mAb carbonylation under production, storage, and stress conditions and showed that iron, hydrogen peroxide, and polysorbate 20 at pharmaceutically relevant levels critically influence the extent of mAb carbonylation. In addition, we found that while carbonylation correlates with mAb aggregation in several cases, carbonylation cannot be used as a general indicator for aggregation. Furthermore, we observed that mAb carbonylation level can decrease during storage, which indicates that carbonylation products may not be stable. Finally, we report for the first time a positive correlation between carbonylation and acidic charge heterogeneity of mAbs that underwent metal-catalyzed oxidation. This finding shows that the impact of protein carbonylation on product quality for mAbs is not limited to aggregation but also extends to charge heterogeneity.

Incorporation of lipolysis in monolayer permeability studies of lipid-based oral drug delivery systems.

Lipid-based drug delivery systems, a well-tolerated class of formulations, have been evaluated extensively to enhance the bioavailability of poorly soluble drugs. However, it has been difficult to predict the in vivo performance of lipid dosage forms based on conventional in vitro techniques such as cell monolayer permeability studies because of the complexity of the gastrointestinal processing of lipid formulations. In the current study, we explored the feasibility of coupling Caco-2 and Madin-Darby canine kidney monolayer permeability studies with lipolysis, a promising in vitro technique to evaluate lipid systems. A self-emulsifying lipid delivery system was formulated using a blend of oil (castor oil), surfactant (Labrasol® or PL497), and co-surfactant (lecithin). Formulations demonstrating high drug solubility and rapid self-emulsification were selected to study the effect of lipolysis on in vitro cell permeability. Lipolysis of the formulations was carried out using pancreatin as the digestive enzyme. All the digested formulations compromised monolayer integrity as indicated by lowered trans-epithelial electrical resistance (TEER) and enhanced Lucifer yellow (LY) permeability. Further, the changes in TEER value and LY permeability were attributable to the digestion products of the formulation rather than the individual lipid excipients, drug, digestion enzyme, or the digestion buffer. The digested formulations were fractionated into pellet, oily phase, and aqueous phase, and the effect of each of these on cell viability was examined. Interestingly, the aqueous phase, which is considered important for in vivo drug absorption, was responsible for cytotoxicity. Because lipid digestion products lead to disruption of cell monolayer, it may not be appropriate to combine lipolysis with cell monolayer permeability studies. Additional in vivo studies are needed to determine any potential side effects of the lipolysis products on the intestinal permeability barrier, which could determine the suitability of lipid-based systems for oral drug delivery.

Lactobacillus gasseri SBT2055 inhibits adipose tissue inflammation and intestinal permeability in mice fed a high-fat diet.

The probiotic Lactobacillus gasseri SBT2055 (LG2055) has anti-obesity effects. Obesity is closely correlated with inflammation in adipose tissue, and maintaining adipose tissue in a less-inflamed state requires intestinal integrity or a barrier function to protect the intestine from the disruption that can be caused by a high-fat diet (HFD). Here, we examined the anti-inflammatory and intestinal barrier-protecting effects of LG2055 in C57BL/6 mice fed a normal-fat diet (NFD), HFD, or the HFD containing LG2055 (HFD-LG) for 21 weeks. HFD-LG intake significantly prevented HFD-induced increases in body weight, visceral fat mass, and the ratio of inflammatory-type macrophages to anti-inflammatory ones in adipose tissue. Mice fed the HFD showed higher intestinal permeability to a fluorescent dextran administered by oral administration and an elevated concentration of antibodies specific to lipopolysaccharides (LPS) in the blood compared with those fed the NFD, suggesting an increased penetration of the gut contents into the systemic circulation. These elevations of intestinal permeability and anti-LPS antibody levels were significantly suppressed in mice fed the HFD-LG. Moreover, treatment with LG2055 cells suppressed an increase in the cytokine-induced permeability of Caco-2 cell monolayers. These results suggest that LG2055 improves the intestinal integrity, reducing the entry of inflammatory substances like LPS from the intestine, which may lead to decreased inflammation in adipose tissue.

Scrape Loading/Dye Transfer Assay.

The scrape loading/dye transfer (SL/DT) technique is a simple functional assay for the simultaneous assessment of gap junctional intercellular communication (GJIC) in a large population of cells. The equipment needs are minimal and are typically met in standard cell biology labs, and SL/DT is the simplest and quickest of all the assays that measure GJIC. This assay has also been adapted for in vivo studies. The SL/DT assay is also conducive to a high-throughput setup with automated fluorescence microscopy imaging and analysis to elucidate more samples in shorter time, and hence can serve a broad range of in vitro pharmacological and toxicological needs.

Boron nitride nanotubes as vehicles for intracellular delivery of fluorescent drugs and probes.

AIM: To evaluate the response of cells to boron nitride nanotubes (BNNTs) carrying fluorescent probes or drugs in their inner channel by assessment of the cellular localization of the fluorescent cargo, evaluation of the in vitro release and biological activity of a drug (curcumin) loaded in BNNTs. METHODS: Cells treated with curcumin-loaded BNNTs and stimulated with lipopolysaccharide were assessed for nitric oxide release and stimulation of IL-6 and TNF-α. The cellular trafficking of two cell-permeant dyes and a non-cell-permeant dye loaded within BNNTs was imaged. RESULTS: BNNTs loaded with up to 13 wt% fluorophores were internalized by cells and controlled release of curcumin triggered cellular pathways associated with the known anti-inflammatory effects of the drug. CONCLUSION: The overall findings indicate that BNNTs can function as nanocarriers of biologically relevant probes/drugs allowing one to examine/control their local intracellular localization and biochemical effects, leading the way to applications as intracellular nanosensors.

Laminar Differences in Dendritic Structure of Pyramidal Neurons in the Juvenile Rat Somatosensory Cortex.

Pyramidal cell structure varies between different cortical areas and species, indicating that the cortical circuits that these cells participate in are likely to be characterized by different functional capabilities. Structural differences between cortical layers have been traditionally reported using either the Golgi method or intracellular labeling, but the structure of pyramidal cells has not previously been systematically analyzed across all cortical layers at a particular age. In the present study, we investigated the dendritic architecture of complete basal arbors of pyramidal neurons in layers II, III, IV, Va, Vb, and VI of the hindlimb somatosensory cortical region of postnatal day 14 rats. We found that the characteristics of basal dendritic morphologies are statistically different in each cortical layer. The variations in size and branching pattern that exist between pyramidal cells of different cortical layers probably reflect the particular functional properties that are characteristic of the cortical circuit in which they participate. This new set of complete basal dendritic arbors of 3D-reconstructed pyramidal cell morphologies across each cortical layer will provide new insights into interlaminar information processing in the cerebral cortex.

Absorption and metabolism characteristics of pristimerin as determined by a sensitive and reliable LC-MS/MS method.

In this research, a sensitive and reliable LC-MS/MS method was developed and applied to determine the concentration of pristimerin in rat plasma, cell incubation media and metabolism incubation mixtures. The absolute oral bioavailability of pristimerin is 28.4% at a dose of 1 mg·kg(-1), and the bioavailability was poor. The bidirectional transport of pristimerin across Caco-2 cells was studied in vitro. A markedly higher transport of pristimerin across Caco-2 cells was observed in the basolateral-to-apical direction and was abrogated in the presence of the P-gp inhibitor, verapamil. The result indicated that P-gp might be involved in the transport of pristimerin in intestine. The phase I and phase II metabolic stability was also investigated using human liver microsomes (HLM) and S9 fractions, respectively. Pristimerin was stable in S9 fractions but metabolized in HLM with a half-life of 20.4 min, which indicated that pristimerin could be mainly metabolized by phase I enzymes. In conclusion, the absolute oral bioavailability of pristimerin in plasma, transport across Caco-2 cell monolayers, and metabolic stability in HLM and S9 fractions were systematically investigated by using a sensitive and reliable LC-MS/MS method.

A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90.

P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmd(mdx) mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca(2+) influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome--autophagy--and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.