Aggregation state determines the localization and function of M1- and M23-aquaporin-4 in astrocytes.
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ABSTRACT:
SUBMITTER: Smith AJ
PROVIDER: S-EPMC3926963 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Autoantibodies against astrocyte water channel aquaporin-4 (AQP4) are highly specific for the neuroinflammatory disease neuromyelitis optica (NMO). We measured the binding of NMO autoantibodies to AQP4 in human astrocyte-derived U87MG cells expressing M1 and/or M23 AQP4, or M23 mutants that do not form orthogonal array of particles (OAPs). Binding affinity was quantified by two-color fluorescence ratio imaging of cells stained with NMO serum or a recombinant monoclonal NMO autoantibody (NMO-rAb), together with a C terminus anti-AQP4 antibody. NMO-rAb titrations showed binding with dissociation constants down to 44 ± 7 nm. Different NMO-rAbs and NMO patient sera showed a wide variation in NMO-IgG binding to M1 versus M23 AQP4. Differences in binding affinity rather than stoichiometry accounted for M1 versus M23 binding specificity, with consistently greater affinity of NMO-IgG binding to M23 than M1 AQP4. Binding and OAP measurements in cells expressing different M1:M23 ratios or AQP4 mutants indicated that the differential binding of NMO-IgG to M1 versus M23 was due to OAP assembly rather than to differences in the M1 versus M23 N termini. Purified Fab fragments of NMO-IgG showed similar patterns of AQP4 isoform binding, indicating that structural changes in the AQP4 epitope upon array assembly, and not bivalent cross-linking of whole IgG, result in the greater binding affinity to OAPs. Our study establishes a quantitative assay of NMO-IgG binding to AQP4 and indicates remarkable, OAP-dependent heterogeneity in NMO autoantibody binding specificity.
| S-EPMC3091256 | biostudies-literature
Project description:Tetramers of aquaporin-4 (AQP4) water channels form supramolecular assemblies in cell membranes called orthogonal arrays of particles (OAPs). We previously reported evidence that a short (M23) AQP4 isoform produced by alternative splicing forms OAPs by an intermolecular N-terminus interaction, whereas the full-length (M1) AQP4 isoform does not by itself form OAPs but can coassemble with M23 in OAPs as heterotetramers. Here, we developed a model to predict number distributions of OAP size, shape, and composition as a function M23:M1 molar ratio. Model specifications included: random tetrameric assembly of M1 with M23; intertetramer associations between M23 and M23, but not between M1 and M23 or M1; and a free energy constraint limiting OAP size. Model predictions were tested by total internal reflection fluorescence microscopy of AQP4-green-fluorescent protein chimeras and native gel electrophoresis of cells expressing different M23:M1 ratios. Experimentally validated model predictions included: 1), greatly increased OAP size with increasing M23:M1 ratio; 2), marked heterogeneity in OAP size at fixed M23:M1, with increased M23 fraction in larger OAPs; and 3), preferential M1 localization at the periphery of OAPs. The model was also applied to test predictions about binding to AQP4 OAPs of a pathogenic AQP4 autoantibody found in the neuroinflammatory demyelinating disease neuromyelitis optica. Our model of AQP4 OAPs links a molecular-level interaction of AQP4 with its supramolecular assembly in cell membranes.
| S-EPMC3123972 | biostudies-literature
Project description:Rationale: Reducing cardiomyocyte death and enhancing their proliferation after myocardial infarction is perhaps the single largest challenge for cardiac tissue regeneration. Survivin (SVV) is the smallest member of the inhibitor of apoptosis (IAP) family but plays two important roles; inhibiting caspase-9 activation in the intrinsic apoptosis pathway, and regulating microtubule dynamics and chromosome segregation during cell division. Genetic depletion of cardiac SVV leads to incomplete cardiomyocyte division and abnormal heart development. However, the function of SVV in adult hearts after myocardial infarction remains unclear. Methods: A homozygous inducible cardiomyocyte-specific SVV knockout transgenic mouse model was established through crossbreeding SVVflox/flox and ?MHC-MCM transgenic mice. Adult mice received consecutive intraperitoneal injection of tamoxifen to induce genetic removal of SVV in cardiomyocytes. A SVV overexpressing model was established via local delivery of SVV in wild-type mouse hearts. Results: We found that 30.82% of cardiomyocytes in the peri-infarct region of SVV knockout mice were apoptotic, significantly higher than the 22.18% in control mice. In addition, ejection fraction was 29.00±0.40% in knockout mice compared to 38.04±0.50% in control mice 21 days after myocardial infarction. On the contrary, locally overexpressing SVV in the heart improved cardiac functions. Unexpectedly, we found that altering the subcellular localization of SVV overexpression produced different outcomes. Overexpression of SVV in the cytoplasm decreased cardiomyocyte apoptosis, whereas overexpression of SVV in the nucleus enhanced cardiac regeneration. The ejection fraction of mice overexpressing SVV was 36.58±0.91%, significantly higher than 28.18±1.70% in the GFP control group. Apoptotic cardiomyocytes were only 4.63% in mouse overexpressing cytosolic SVV, compared to 9.31% in the GFP group, and activation of caspase-3 was also reduced. Moreover, mice overexpressing NLS-SVV exhibited a better ejection fraction (36.19±1.02%,) than GFP controls (26.69±0.75%). NLS-SVV enhanced H3P-positive cardiomyocytes in the border zone to 0.28%, compared to only 0.08% in GFP group, through interacting with Aurora B. Conclusions: We demonstrate the importance of SVV subcellular localization in regulating post-MI cardiac repair and regeneration. We hope that this will open new translational approaches through targeted delivery of SVV.
| S-EPMC5695150 | biostudies-other
Project description:In astrocytes, unknown mechanisms regulate the expression of M1 and M23 isoforms of water channel aquaporin-4 (M1-AQP4 and M23-AQP4). The ratio between these two isoforms controls the AQP4 assembly state in the plasma membrane known as orthogonal arrays of particles (OAPs). To give new insights into these mechanisms, here, we explore the regulation of AQP4 expression in the spinal cord of a CRISPR/Cas9 M23-null mouse model (M23-null). In the M23-null spinal cord OAP assembly, the perivascular localization of AQP4 and M1-AQP4 protein were drastically reduced. In heterozygous, M1-AQP4 was proportionally reduced with M23-AQP4, maintaining the isoform ratio unaffected. We hypothesize a role of the M23-AQP4 in the regulation of M1-AQP4 expression. M1-AQP4 transcription, splicing and M1-AQP4 protein degradation were found to be unaffected in M23-null spinal cord and in M23-null astrocyte primary culture. The translational control was investigated by mRNA-protein pull down and quantitative mass spectrometry, to isolate and quantify AQP4 mRNA binding proteins (AQP4-RBPs). Compared to WT, in M23-null spinal cord, the interaction between AQP4 mRNA and polypyrimidine tract binding protein 1, a positive regulator of AQP4 translation, was higher, while interaction with the RNA helicase DDX17 was lower. In astrocyte primary cultures, DDX17 knockdown upregulated AQP4 protein expression and increased cell swelling, leaving AQP4 mRNA levels unchanged. Here, we identify AQP4-RBPs and provide evidence that in mouse spinal cord M23-AQP4 deletion changes the interaction between AQP4 mRNA and some RBPs involved in AQP4 translation. We describe for the first time the RNA helicase DDX17 as a regulator of AQP4 expression in astrocytes.
| S-EPMC8361696 | biostudies-literature
Project description:Aquaporins (AQPs) are transmembrane water channels ubiquitously expressed in mammalian tissues. They play prominent roles in maintaining cellular fluid balance. Although expression of AQP1, -3, -4, -5, -8, -9, and -11 has been reported in the central nervous system, it is AQP4 that is predominately expressed. Its importance in fluid regulation in cerebral edema conditions has been highlighted in several studies, and we have also shown that translational regulation of AQP4 by miR-320a could prove to be useful in infarct volume reduction in middle cerebral artery occluded rat brain. There is evidence for the existence of two AQP4 transcripts (M1 and M23) in the brain arising from two alternative promoters. Because the AQP4 M1 isoform exhibits greater water permeability, in this study, we explored the possibility of microRNA-based transcriptional regulation of the AQP4 M1 promoter. Using RegRNA software, we identified 34 microRNAs predicted to target the AQP4 M1 promoter region. MicroRNA profiling, quantitative stem-loop PCR, and luciferase reporter assays revealed that miR-130a, -152, -668, -939, and -1280, which were highly expressed in astrocytes, could regulate the promoter activity. Of these, miR-130a was identified as a strong transcriptional repressor of the AQP4 M1 isoform. In vivo studies revealed that LNA(TM) anti-miR-130a could up-regulate the AQP4 M1 transcript and its protein to bring about a reduction in cerebral infarct and promote recovery.
| S-EPMC3320947 | biostudies-literature
Project description:Aquaporin 0 (AQP0) formerly known as membrane intrinsic protein (MIP), is expressed exclusively in the lens during terminal differentiation of fiber cells. AQP0 plays an important role not only in the regulation of water content but also in cell-to-cell adhesion of the lens fiber cells. We have investigated the thermal stress-induced structural alterations of detergent (octyl glucoside)-solubilized calf lens AQP0. The results show an increase in the amount of AQP0 that aggregated as the temperature increased from 40°C to 65°C. ?-Crystallin, molecular chaperone abundantly present in the eye lens, completely prevented the AQP0 aggregation at a 1?1 (weight/weight) ratio. Since ?-crystallin consists of two gene products namely ?A- and ?B-crystallins, we have tested the recombinant proteins on their ability to prevent thermal-stress induced AQP0 aggregation. In contrast to the general observation made with other target proteins, ?A-crystallin exhibited better chaperone-like activity towards AQP0 compared to ?B-crystallin. Neither post-translational modifications (glycation) nor C-terminus truncation of AQP0 have any appreciable effect on its thermal aggregation properties. ?-Crystallin offers similar protection against thermal aggregation as in the case of the unmodified AQP0, suggesting that ?crystallin may bind to either intracellular loops or other residues of AQP0 that become exposed during thermal stress. Far-UV circular dichroism studies indicated a loss of ?helical structures when AQP0 was subjected to temperatures above 45°C, and the presence of ?-crystallin stabilized these secondary structures. We report here, for the first time, that ?-crystallin protects AQP0 from thermal aggregation. Since stress-induced structural perturbations of AQP0 may affect the integrity of the lens, presence of the molecular chaperone, ?-crystallin (particularly ?A-crystallin) in close proximity to the lens membrane is physiologically relevant.
| S-EPMC3842347 | biostudies-literature
Project description:In aged humans and mice, aggregates of hypobranched glycogen molecules called polyglucosan bodies (PGBs) accumulate in hippocampal astrocytes. PGBs are known to drive cognitive decline in neurological diseases but remain largely unstudied in the context of typical brain aging. Here, we show that PGBs arise in autophagy-dysregulated astrocytes of the aged C57BL/6J mouse hippocampus. To map the genetic cause of age-related PGB accumulation, we quantified PGB burden in 32 fully sequenced BXD-recombinant inbred mouse strains, which display a 400-fold variation in hippocampal PGB burden at 16-18 months of age. A major modifier locus was mapped to chromosome 1 at 72–75 Mb, which we defined as the Pgb1 locus. To evaluate candidate genes and downstream mechanisms by which Pgb1 controls the aggregation of glycogen, extensive hippocampal transcriptomic and proteomic datasets were produced for aged mice of the BXD family. We utilized these datasets to identify Smarcal1 and Usp37 as potential regulators of PGB accumulation. To assess the effect of PGB burden on age-related cognitive decline, we performed phenome-wide association scans, transcriptomic analyses as well as conditioned fear memory and Y-maze testing. Importantly, we did not find any evidence suggesting a negative impact of PGBs on cognition. Taken together, our study demonstrates that the Pgb1 locus controls glycogen aggregation in astrocytes of the aged hippocampus without affecting age-related cognitive decline.
2024-08-13 | GSE274733 | GEO
Project description:In recent years, the transient receptor potential melastatin member 8 (TRPM8) channel has emerged as a promising prognostic marker and putative therapeutic target in prostate cancer (PCa). However, the mechanisms of prostate-specific regulation and functional evolution of TRPM8 during PCa progression remain unclear. Here we show, for the first time to our knowledge, that only secretory mature differentiated human prostate primary epithelial (PrPE) luminal cells expressed functional plasma membrane TRPM8 ((PM)TRPM8) channels. Moreover, PCa epithelial cells obtained from in situ PCa were characterized by a significantly stronger (PM)TRPM8-mediated current than that in normal cells. This (PM)TRPM8 activity was abolished in dedifferentiated PrPE cells that had lost their luminal secretory phenotype. However, we found that in contrast to (PM)TRPM8, endoplasmic reticulum TRPM8 ((ER)TRPM8) retained its function as an ER Ca(2+) release channel, independent of cell differentiation. We hypothesize that the constitutive activity of (ER)TRPM8 may result from the expression of a truncated TRPM8 splice variant. Our study provides insight into the role of TRPM8 in PCa progression and suggests that TRPM8 is a potentially attractive target for therapeutic intervention: specific inhibition of either (ER)TRPM8 or (PM)TRPM8 may be useful, depending on the stage and androgen sensitivity of the targeted PCa.
| S-EPMC1866249 | biostudies-literature
Project description:Environmental conditions during early mammalian embryo development are critical and some adaptational phenomena are observed. However, the mechanisms underlying them remain largely masked. Previously, we reported that AQP5 expression is modified by the environmental condition without losing the developmental potency. In this study, AQP11 was examined instead. To compare expression pattern between in vivo and in vitro, we conducted quantitative RT-PCR and analyzed localization of the AQP11 by whole mount immunofluorescence. When the fertilized embryos were developed in the maternal tracts, the level of Aqp11 transcripts was decreased devrepamatically until 2-cell stage. Its level increased after 2-cell stage and peaked at 4-cell stage, but decreased again devrepamatically until morula stage. Its transcript level increased again at blastocyst stage. In contrast, the levels of Aqp11 transcript in embryos cultured in vitro were as follows. The patterns of expression were similar but the overall levels were low compared with those of embryos grown in the maternal tracts. AQP11 proteins were localized in submembrane cytoplasm of embryos collected from maternal reproductive tracts. The immune-reactive signals were detected in both trophectoderm and inner cell mass. However, its localization was altered in in vitro culture condition. It was localized mainly in the plasma membrane of the blastocysts contacting with external environment. The present study suggests that early stage embryo can develop successfully by themselves adapting to their environmental condition through modulation of the expression level and localization of specific genes like AQP11.
| S-EPMC4415664 | biostudies-literature
Project description:The astrocytic aquaporin-4 (AQP4) water channel is the target of pathogenic antibodies in a spectrum of relapsing autoimmune inflammatory central nervous system disorders of varying severity that is unified by detection of the serum biomarker neuromyelitis optica (NMO)-IgG. Neuromyelitis optica is the most severe of these disorders. The two major AQP4 isoforms, M1 and M23, have identical extracellular residues. This report identifies two novel properties of NMO-IgG as determinants of pathogenicity. First, the binding of NMO-IgG to the ectodomain of astrocytic AQP4 has isoform-specific outcomes. M1 is completely internalized, but M23 resists internalization and is aggregated into larger-order orthogonal arrays of particles that activate complement more effectively than M1 when bound by NMO-IgG. Second, NMO-IgG binding to either isoform impairs water flux directly, independently of antigen down-regulation. We identified, in nondestructive central nervous system lesions of two NMO patients, two previously unappreciated histopathological correlates supporting the clinical relevance of our in vitro findings: (i) reactive astrocytes with persistent foci of surface AQP4 and (ii) vacuolation in adjacent myelin consistent with edema. The multiple molecular outcomes identified as a consequence of NMO-IgG interaction with AQP4 plausibly account for the diverse pathological features of NMO: edema, inflammation, demyelination, and necrosis. Differences in the nature and anatomical distribution of NMO lesions, and in the clinical and imaging manifestations of disease documented in pediatric and adult patients, may be influenced by regional and maturational differences in the ratio of M1 to M23 proteins in astrocytic membranes.
| S-EPMC3268278 | biostudies-literature