Neuroregeneration Improved by Sodium-D,L-Beta-Hydroxybutyrate in Primary Neuronal Cultures.

Ketone bodies are considered alternative fuels for the brain when glucose availability is limited. To determine the neuroregenerative potential of D,L-sodium-beta-hydroxybutyrate (D/L-BHB), Sprague Dawley rat primary cortical neurons were exposed to simulated central nervous system injury using a scratch assay. The neuronal cell migration, cell density and degree of regeneration in the damaged areas (gaps) in the absence (control) and presence of BHB (2 mM) were documented with automated live-cell imaging by the CytoSMART system over 24 h, which was followed by immunocytochemistry, labeling synapsin-I and ß3-tubulin. The cell density was significantly higher in the gaps with BHB treatment after 24 h compared to the control. In the control, only 1.5% of the measured gap areas became narrower over 24 h, while in the BHB-treated samples 49.23% of the measured gap areas became narrower over 24 h. In the control, the gap expanded by 63.81% post-injury, while the gap size decreased by 10.83% in response to BHB treatment, compared to the baseline. The cell density increased by 97.27% and the gap size was reduced by 74.64% in response to BHB, compared to the control. The distance travelled and velocity of migrating cells were significantly higher with BHB treatment, while more synapsin-I and ß3-tubulin were found in the BHB-treated samples after 24 h, compared to the control. The results demonstrate that D/L-BHB enhanced neuronal migration and molecular processes associated with neural regeneration and axonogenesis. These results may have clinical therapeutic applications in the future for nervous system injuries, such as for stroke, concussion and TBI patients.

All-Trans Retinoic Acid Increases DRP1 Levels and Promotes Mitochondrial Fission.

In the heart, mitochondrial homeostasis is critical for sustaining normal function and optimal responses to metabolic and environmental stressors. Mitochondrial fusion and fission are thought to be necessary for maintaining a robust population of mitochondria, and disruptions in mitochondrial fission and/or fusion can lead to cellular dysfunction. The dynamin-related protein (DRP1) is an important mediator of mitochondrial fission. In this study, we investigated the direct effects of the micronutrient retinoid all-trans retinoic acid (ATRA) on the mitochondrial structure in vivo and in vitro using Western blot, confocal, and transmission electron microscopy, as well as mitochondrial network quantification using stochastic modeling. Our results showed that ATRA increases DRP1 protein levels, increases the localization of DRP1 to mitochondria in isolated mitochondrial preparations. Our results also suggested that ATRA remodels the mitochondrial ultrastructure where the mitochondrial area and perimeter were decreased and the circularity was increased. Microscopically, mitochondrial network remodeling is driven by an increased rate of fission over fusion events in ATRA, as suggested by our numerical modeling. In conclusion, ATRA results in a pharmacologically mediated increase in the DRP1 protein. It also results in the modulation of cardiac mitochondria by promoting fission events, altering the mitochondrial network, and modifying the ultrastructure of mitochondria in the heart.

Site-specific opening of the blood-brain barrier by extracellular histones.

BACKGROUND: Increased extracellular histones in the bloodstream are known as a biomarker for vascular dysfunction associated with severe trauma or sepsis. There is limited information regarding the pathogenic role of circulating histones in neuroinflammation and cerebrovascular endothelial injury. Particularly, it remains unclear whether histones affect the blood-brain barrier (BBB) permeability function. METHODS: The direct effects of unfractionated histones on endothelial barrier properties were first assessed in brain microvascular endothelial cell monolayers by measuring transendothelial electrical resistance and solute flux. This was followed by in vivo mouse experiments, where BBB function was assessed by quantifying brain tissue accumulation of intravenously injected tracers of different molecular sizes, and comparison was made in mice receiving a sublethal dose of histones versus sterile saline. In parallel, the endothelial barrier ultrastructure was examined in histone- and saline-injected animals under transmission electron microscopy, corresponding to the expression of tight junction and adherens junction proteins. RESULTS: Histones increased paracellular permeability to sodium fluorescein and reduced barrier resistance at 100 µg/mL; these responses were accompanied by discontinuous staining of the tight junction proteins claudin-5 and zona ocludens-1. Interestingly, the effects of histones did not seem to result from cytotoxicity, as evidenced by negative propidium iodide staining. In vivo, histones increased the paracellular permeability of the BBB to small tracers of < 1-kDa, whereas tracers larger than 3-kDa remained impermeable across brain microvessels. Further analysis of different brain regions showed that histone-induced tracer leakage and loss of tight junction protein expression mainly occurred in the hippocampus, but not in the cerebral cortex. Consistently, opening of tight junctions was found in hippocampal capillaries from histone-injected animals. Protein expression levels of GFAP and iBA1 remained unchanged in histone-injected mice indicating that histones did not affect reactive gliosis. Moreover, cell membrane surface charge alterations are involved in histone-induced barrier dysfunction and tight junction disruption. CONCLUSIONS: Extracellular histones cause a reversible, region-specific increase in BBB permeability to small molecules by disrupting tight junctions in the hippocampus. We suggest that circulating histones may contribute to cerebrovascular injury or brain dysfunction by altering BBB structure and function.

Endothelial-Stromal Communication in Murine and Human Corneas.

The purpose of this study is to identify and characterize interactions of corneal endothelial cells with the posterior stroma. Corneal endothelial-stromal interactions were examined in developing postnatal day 3 (P3) and mature postnatal day 30 (P30) C57BL/6 mice and adult human corneas. Flat mounts and cross-sections were studied using immunofluorescence microscopy. F-actin was labeled with phalloidin to evaluate cell processes traversing Descemet's membrane (DM). Dynamic cell-cell communication was evaluated with fluorescence recovery after photobleaching (FRAP) using calcein acetoxymethyl dye. Endothelial-stromal interactions were observed across the whole cornea transversing DM during early postnatal development (P3), while these interactions became restricted to the periphery in the mature murine cornea (P30). In adult human corneas, endothelial extensions through the DM were observed in the peripheral cornea. The pattern of FRAP in both mature mice and human central corneas demonstrated endothelial-endothelial cell communication. In contrast, in the human cornea 2, distinct patterns were observed consistent with endothelial-endothelial and stromal-endothelial communication. Endothelial-stromal interactions were observed in the entire cornea during early postnatal mouse corneas. This evidence of endothelial-posterior stromal contact contradicts the hypothesis that corneal endothelial cells are isolated from the stroma by the DM and provides direct data to support endothelial-stromal comunication that may directly influence posterior corneal structure and function. Anat Rec, 2020. © 2020 American Association for Anatomy.

AKT2 maintains brain endothelial claudin-5 expression and selective activation of IR/AKT2/FOXO1-signaling reverses barrier dysfunction.

Inflammation-induced blood-brain barrier (BBB) dysfunction and microvascular leakage are associated with a host of neurological disorders. The tight junction protein claudin-5 (CLDN5) is a crucial protein necessary for BBB integrity and maintenance. CLDN5 is negatively regulated by the transcriptional repressor FOXO1, whose activity increases during impaired insulin/AKT signaling. Owing to an incomplete understanding of the mechanisms that regulate CLDN5 expression in BBB maintenance and dysfunction, therapeutic interventions remain underdeveloped. Here, we show a novel isoform-specific function for AKT2 in maintenance of BBB integrity. We identified that AKT2 during homeostasis specifically regulates CLDN5-dependent barrier integrity in brain microvascular endothelial cells (BMVECs) and that intervention with a selective insulin-receptor (IR) agonist, demethylasterriquinone B1 (DMAQ-B1), rescued IL-1ß-induced AKT2 inactivation, FOXO1 nuclear accumulation, and loss of CLDN5-dependent barrier integrity. Moreover, DMAQ-B1 attenuated preclinical CLDN5-dependent BBB dysfunction in mice subjected to experimental autoimmune encephalomyelitis. Taken together, the data suggest a regulatory role for IR/AKT2/FOXO1-signaling in CLDN5 expression and BBB integrity during neuroinflammation.

A mouse model of renal ischemia-reperfusion injury solely induced by cold ischemia.

Transplanted kidneys usually experience several episodes of ischemia, including cold ischemia during allograft storage in preservation solution. However, previous studies focusing on cold renal ischemia were only carried out in vitro or ex vivo. In the present study, we developed and characterized an in vivo mouse model of renal ischemia-reperfusion injury (IRI) induced exclusively by cold ischemia. C57BL/6 mice underwent right kidney nephrectomy, and the left kidney was kept cool with circulating cold saline in a kidney cup, while body temperature was maintained at 37°C. We clamped the renal pedicle and flushed out the blood inside the kidney with cold saline via an opening on the renal vein. The severity of renal IRI was examined with different ischemic durations. We found that the mice with <2 h of cold ischemia exhibited no significant changes in renal function or histopathology; animals with 3 or 4 h of cold ischemia developed into mild to moderate acute kidney injury with characteristic features, including the elevation in plasma creatinine concentration and reduction in glomerular filtration rate and tubular necrosis, followed by a subsequent recovery. However, mice with 5 h of cold ischemia died in a few days with severe acute kidney injury. In summary, we generated a mouse model of renal IRI induced exclusively by cold ischemia, which mimics graft cold storage in preservation solution, and renal function can be evaluated in vivo.

Palmitoyl acyltransferase DHHC21 mediates endothelial dysfunction in systemic inflammatory response syndrome.

Endothelial dysfunction is a hallmark of systemic inflammatory response underlying multiple organ failure. Here we report a novel function of DHHC-containing palmitoyl acyltransferases (PATs) in mediating endothelial inflammation. Pharmacological inhibition of PATs attenuates barrier leakage and leucocyte adhesion induced by endothelial junction hyperpermeability and ICAM-1 expression during inflammation. Among 11 DHHCs detected in vascular endothelium, DHHC21 is required for barrier response. Mice with DHHC21 function deficiency (Zdhhc21dep/dep) exhibit marked resistance to injury, characterized by reduced plasma leakage, decreased leucocyte adhesion and ameliorated lung pathology, culminating in improved survival. Endothelial cells from Zdhhc21dep/dep display blunted barrier dysfunction and leucocyte adhesion, whereas leucocytes from these mice did not show altered adhesiveness. Furthermore, inflammation enhances PLCß1 palmitoylation and signalling activity, effects significantly reduced in Zdhhc21dep/dep and rescued by DHHC21 overexpression. Likewise, overexpression of wild-type, not mutant, PLCß1 augments barrier dysfunction. Altogether, these data suggest the involvement of DHHC21-mediated PLCß1 palmitoylation in endothelial inflammation.

Thrombomodulin Induces a Quiescent Phenotype and Inhibits Migration in Vascular Smooth Muscle Cells In Vitro.

BACKGROUND: Loss of critical endothelial cell function and subsequent vascular smooth muscle cell (VSMC) migration is central to the pathology of injury-induced neointimal hyperplasia and recurrent stenosis. Thrombomodulin (TM), well known for its function as an endothelial surface anticoagulant, may have an unknown direct effect on VSMC physiology that would be lost after injury. Here, we examined a novel effect of TM on VSMC by testing the hypothesis that direct application of TM induces favorable changes to the morphology of VSMC and inhibits their migration. METHODS: Primary human VSMC were harvested using the explant technique and used in early passage (1-4) for all experiments. Laser-scanning confocal fluorescent imaging was performed to assess the effect of soluble TM on VSMC morphology. In vitro, migration of VSMC was measured using: (1) a 4-hr modified Boyden chemotaxis assay and (2) a 24-hr electric cell-substrate impedance sensing injury migration assay. Migration experiments were conducted with VSMC exposed to increasing doses of soluble recombinant TM. Recombinant thrombin served as a positive control and serum-free media as a negative control for all experimentation. Data were analyzed using a Student's t-test or repeated measures analysis of variance where appropriate (α < 0.05). RESULTS: VSMC exposed to TM clearly demonstrated a quiescent morphology with organized stress fibers consistent with a quiescent, differentiated, contractile phenotype; whereas, thrombin stimulation led to an activated, dedifferentiated, synthetic phenotype. VSMC demonstrated a low, baseline level of migration in unstimulated serum-free conditions. Thrombin significantly stimulated VSMC migration as expected. TM, independent of thrombin, significantly inhibited baseline VSMC migration in a dose-response fashion. The maximal inhibition was observed at (5 µg/mL) with 70% reduction (56 ± 1.7 vs. 18 ± 3.5 cells/5 high-power fields, P = 0.0005). CONCLUSIONS: TM has a direct effect on VSMC resulting in a quiescent, differentiated and contractile phenotype, and inhibition of migration. This effect is independent of the presence of thrombin. These findings provide new knowledge in understanding the pathophysiology of vascular injury and support a strategy focused on restoring key endothelial function to prevent intimal hyperplasia.

Functional Expression of Aquaporin-2 Tagged with Photoconvertible Fluorescent Protein in mpkCCD Cells.

BACKGROUND: Vasopressin induced trafficking of aquaporin-2 (AQP2) containing vesicles has been studied in kidney cell lines using conventional fluorescent proteins as tags. However, trafficking of fluorescent tagged AQP2, which resembles the vectorial translocation of native AQP2 from cytoplasm to apical membrane has not been demonstrated at real time. Using a photoconvertible fluorescent protein tag on AQP2 might allow the simultaneous tracking of two separate populations of AQP2 vesicle after subcellular local photoconversion. METHODS: A spacer was used to link a photoconvertible fluorescent protein (mEos2) to the amino-terminus of AQP2. The DNA constructs were expressed in mpkCCD cells. The trafficking of chimeric protein was visualized with high speed confocal microscopy in 4 dimensions. RESULTS: Chimeric AQP2 expressed in mpkCCD cell conferred osmotic water permeability to the cells. Subcellular photoconversion with a 405 nm laser pulse converted green chimeras to red chimeras locally. Forskolin stimulation triggered chimeric AQP2 to translocate from acidic organelles to apical plasma membrane. By serendipity, the rate of apical accumulation was found to increase when mEos2 was tagged to the carboxyl-terminus in at least one of the AQP2 molecules within the tetramer. CONCLUSION: Functional photoconvertible chimeric AQP2 was successfully expressed in mpkCCD cells, in which forskolin induced apical trafficking and accumulation of chimeric AQP2. The proof-of-concept to monitor two populations of AQP2 vesicle simultaneously was demonstrated.

Zebrafish embryo model of Bartonella henselae infection.

Bartonella henselae (Bh) is an emerging zoonotic pathogen that has been associated with a variety of human diseases, including bacillary angiomatosis that is characterized by vasoproliferative tumor-like lesions on the skin of some immunosuppressed individuals. The study of Bh pathogenesis has been limited to in vitro cell culture systems due to the lack of an animal model. Therefore, we wanted to investigate whether the zebrafish embryo could be used to model human infection with Bh. Our data showed that Tg(fli1:egfp)(y1) zebrafish embryos supported a sustained Bh infection for 7 days with >10-fold bacterial replication when inoculated in the yolk sac. We showed that Bh recruited phagocytes to the site of infection in the Tg(mpx:GFP)uwm1 embryos. Infected embryos showed evidence of a Bh-induced angiogenic phenotype and an increase in the expression of genes encoding pro-inflammatory factors and pro-angiogenic markers. However, infection of zebrafish embryos with a deletion mutant in the major adhesin (BadA) resulted in little or no bacterial replication and a diminished host response, providing the first evidence that BadA is critical for in vivo infection. Thus, the zebrafish embryo provides the first practical model of Bh infection that will facilitate efforts to identify virulence factors and define molecular mechanisms of Bh pathogenesis.

Non-muscle Mlck is required for ß-catenin- and FoxO1-dependent downregulation of Cldn5 in IL-1ß-mediated barrier dysfunction in brain endothelial cells.

Aberrant elevation in the levels of the pro-inflammatory cytokine interleukin-1ß (IL-1ß) contributes to neuroinflammatory diseases. Blood-brain barrier (BBB) dysfunction is a hallmark phenotype of neuroinflammation. It is known that IL-1ß directly induces BBB hyperpermeability but the mechanisms remain unclear. Claudin-5 (Cldn5) is a tight junction protein found at endothelial cell-cell contacts that are crucial for maintaining brain microvascular endothelial cell (BMVEC) integrity. Transcriptional regulation of Cldn5 has been attributed to the transcription factors ß-catenin and forkhead box protein O1 (FoxO1), and the signaling molecules regulating their nuclear translocation. Non-muscle myosin light chain kinase (nmMlck, encoded by the Mylk gene) is a key regulator involved in endothelial hyperpermeability, and IL-1ß has been shown to mediate nmMlck-dependent barrier dysfunction in epithelia. Considering these factors, we tested the hypothesis that nmMlck modulates IL-1ß-mediated downregulation of Cldn5 in BMVECs in a manner that depends on transcriptional repression mediated by ß-catenin and FoxO1. We found that treating BMVECs with IL-1ß induced barrier dysfunction concomitantly with the nuclear translocation of ß-catenin and FoxO1 and the repression of Cldn5. Most importantly, using primary BMVECs isolated from mice null for nmMlck, we identified that Cldn5 repression caused by ß-catenin and FoxO1 in IL-1ß-mediated barrier dysfunction was dependent on nmMlck.

In vivo live-analysis of cell cycle checkpoints in Drosophila early embryos.

Live-imaging of cells has been an excellent technique to provide us with highly accurate and valuable information about cell cycle checkpoint regulation and DNA damage responses. Early stage Drosophila embryos have several advantages to be studied by live-imaging. Fly embryos are much tougher than cultured cells and stand up to relatively rough manipulation, such as protein/chemical microinjection followed by time-lapse imaging. Cell cycles in the embryonic cleavage stage progress rapidly (9-20 min/cycle) and nuclear divisions are synchronous, allowing observation of multiple nuclei/cell cycles in a short period of time. Somatic precursor nuclei form a monolayer at the cortex of the embryo during the syncytial blastoderm stage (cell cycles 10-13). Thus the nuclei in this stage are particularly accessible by various microscopic techniques (Sullivan and Theurkauf, 1995, Curr. Opin. Cell Biol. 7, 18-22). Live-imaging of embryos complements the versatility of the Drosophila embryonic system, in which we can utilize various approaches, including genetics and biochemistry, to obtain comprehensive understanding of biological processes. In this chapter, we will describe basic methods of microinjection and live-imaging during early embryogenesis by differential interference contrast (DIC) or confocal microscopy, and the use of such methods to study cell cycle checkpoints.

Recognition of a bicoid mRNA localization signal by a protein complex containing Swallow, Nod, and RNA binding proteins.

Localization of mRNAs, a process essential for embryonic body patterning in Drosophila, requires recognition of cis-acting signals by cellular components responsible for movement and anchoring. We have purified a large multiprotein complex that binds a minimal form of the bicoid mRNA localization signal in a manner both specific and sensitive to inactivating mutations. Identified complex components include the RNA binding proteins Modulo, PABP, and Smooth, the known localization factor Swallow, and the kinesin family member Nod. We demonstrate that localization of bcd mRNA is defective in modulo mutants. The presence of three required localization components (Swallow, Modulo, and specific RNA binding activity) within the recognition complex strongly implicates it in mRNA localization.