Autophagy, 11(3), 487C502

Autophagy, 11(3), 487C502. BBB integrity. To decipher how miR\195 promoted BBB integrity, we first exhibited that TJ proteins were metabolized via autophagicClysosomal pathway and the autophagic adaptor p62 was necessary to promote TJ protein degradation in cerebral ECs. Next, proteomic analysis of exosomes revealed miR\195\suppressed thrombospondin\1 (TSP1) as a major contributor to BBB disruption. Moreover, TSP1 was demonstrated to activate selective autophagy of TJ proteins by increasing the formation of claudin\5\p62 and ZO1\p62 complexes in cerebral ECs while TSP1 impaired general autophagy. Delivering TSP1 antibody into the circulation showed dose\dependent reduction of BBB leakage by 20%C40% in 25\month\aged mice. Intravenous or intracerebroventricular injection of miR\195 rescued TSP1\induced BBB leakage. Dementia patients with BBB damage had higher levels of serum TSP1 compared to those without BBB damage (from three impartial experiments, and each experiment was performed in triplicate. *from three impartial experiments. *from three impartial experiments. *test. #from three impartial experiments. **from three impartial experiments. *alleles (promoter to generate mice. Both mice and mice were generated by the National Laboratory Animal Center, Taiwan. Genomic DNA isolated from tail biopsies was used to confirm the genotype of the mice. Cre recombinase is usually expressed in brain, eyes, heart, spleen, skeleton muscle, small intestine, appendix, and skin. Since the mice were used to study BBB integrity in the present study, mice were denoted as brain\specific KO mice in the present study. 4.2. Cell culture Mouse astrocyte cell line ALT (BCRC60581) and mouse cerebral endothelial cells bEnd.3 (BCRC60515) were obtained from Bioresource Collection and Research Center, Taiwan. ALT and bEnd.3 cells were maintained in DMEM TBK1/IKKε-IN-5 supplemented with 10% FBS (Invitrogen, Waltham, MA, USA), 1% penicillin and streptomycin (Biowest, Loire Valley, France), and 1% l\glutamine (Invitrogen) in a humidified incubator under an atmosphere of 5% CO2 at 37 C. 4.3. BBB permeability determined by the Evans blue assay BBB disruption was assessed by measuring the amount of Evans blue extravasation. Briefly, mice were intravenously injected TBK1/IKKε-IN-5 with 0.1?ml 4% Evans blue (Sigma\Aldrich). After 60?min, the animals were perfused transcardially with phosphate\buffered saline (PBS) to remove intravascular Evans Blue. The brains were homogenized in formamide (Sigma\Aldrich) and incubated in the dark at 60C for 24?h. The absorbance of eluted Evans blue dye in formamide answer was measured using a spectrophotometer at 610?nm and normalized to plasma levels. 4.4. BBB integrity measured by TBK1/IKKε-IN-5 MRI 4.4.1. Animals Male C57BL/6 WT mice and the age\ and sex\matched miR\195a KO mice were used for MRI assessments. To perform MRI measurement, TBK1/IKKε-IN-5 mice were tail\vein\injected with Resovist (5?mg/kg, Schering AG), a clinically approved Plau superparamagnetic iron oxide (Fe3O4) developed for contrast\enhanced magnetic resonance imaging (MRI) (Reimer & Balzer, 2003), under the anesthesia with isoflurane (Proane?, Aesica Queenborough, Lte.). MR Data Acquisition MRI experiments were performed with a 9.4 T horizontal\bore animal MR scanning system (Biospec 94/20) equipped with a mouse\imaging cryocoil (MRI CryoProbe). The imaging parameters used for the studies were as follows: field of view =19.2??12.8??6.4?mm3, matrix dimension?=?384??256??128, voxel size =50??50??50?m3, repetition time (TR)?=?50?ms, echo time (TE)?=?13.5?ms, slice thickness?=?0.1?mm, bandwidth?=?25?kHz, and total scan time?=?27?min and 18?s. The large\scale B0 inhomogeneity was minimized by region of interest (ROI)\based shimming (provided with the system). MR Data Processing Multichannel MR images were reconstructed using MATLAB (The MathWorks) and then separated into magnitude and phase images. The magnitude images of the individual channels of the coil array were combined using the sum\of\squares method, and the phase images were assembled using complex summation. Subsequently, the combined magnitude and phase images were used for quantitative susceptibility mapping (QSM) reconstruction. For quantitative analysis of QSM images, images were processed using ImageJ to analyze the area of Resovist extravasation. The BBB integrity was determined by the difference of QSM quantified at 1?h to that at time 0. 4.5. FITCCdextran permeability assay The permeability of in vitro BBB model was assessed using FITCCdextran fluorescein. The ECs were seeded onto 6\well collagen\coated Transwell inserts (0.4?m pore size) (Millipore) at 5??105 cells per well. ECs were produced to confluence to mimic the cellCcell environment in the vasculature, and ECs were subsequently treated with exosomes or TSP1 depends.

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