Glycoengineering artificial receptors for microglia to phagocytose Aβ aggregates

Oligomeric and fibrillar amyloid-β (Aβ) are principally internalized via receptor-mediated endocytosis (RME) by microglia, the main scavenger of Aβ in the brain. Nevertheless, the inflammatory cascade will be evoked after vast Aβ aggregate binding to pattern recognition receptors on the cell membrane, which then significantly decreases the expression of these receptors and further deteriorate Aβ deposition. This vicious circle will weaken the ability of microglia for Aβ elimination. Herein, a combination of metabolic glycoengineering and self-triggered click chemistry is utilized to engineer microglial membranes with ThS as artificial Aβ receptors to promote microglia to phagocytose Aβ aggregates. Additionally, to circumvent the undesirable immune response during the process of the bioorthogonal chemistry reaction and Aβ-microglial interaction, Mn-porphyrin metal–organic frameworks (Mn-MOFs) with superoxide dismutase (SOD) and catalase (CAT) mimic activity are employed to carry N-azidoacetylmannosamine (AcManNAz) and eradicate over-expressed reactive oxygen species (ROSs). The artificial Aβ receptors independent of a signal pathway involved in immunomodulation as well as Mn-MOFs with antioxidant properties can synergistically promote the phagocytosis and clearance of Aβ with significantly enhanced activity and negligible adverse effects. The present study will not only provide valuable insight into the rational design of the microglial surface engineering strategy via bioorthogonal chemistry, but also hold great potential for other disease intervention associated with receptor starvation.

3 spectrometer. Fluorescence microscopy images were obtained using a JASCO FP-6500 spectrofluorometer with the slit width for the excitation and emission of 3 nm. Electrospray ionization (ESI) mass spectra (MS) were acquired using the Quattro Premier XE mass spectrometer (Waters, USA).

Synthesis
of 5,10,15,20-Tetrakis(4-methoxycarbonylphenyl)porphyrin (TPPCOOMe, 1): 100 mL of propionic acid in a 500 mL three necked flask were added 3.0 g of pyrrole and 6.9 g of methyl p-formylbenzoate, and the solution was refluxed at 140 ℃ for 12 h in darkness. After the reaction mixture was cooled to room temperature, crystals were collected by suction-filtration to afford purple crystals.

Synthesis of 5,10,15,20-Tetrakis(4-methoxycarbonylphenyl)porphyrin-Mn
(Mn-TPPCOOMe, 2): 0.854 g of 1 and 2.5 g of MnCl2·4H2O in 100 mL of DMF was refluxed for 6 h in darkness. After the mixture was cooled to room temperature, 150 mL of H2O was added. The resultant precipitate was filtered and washed with 50 mL of H2O for two times. The obtained solid was dissolved in CHCl3, followed by washing three times with water. The organic layer was dried over anhydrous magnesium sulfate and evaporated to afford quantitative dark green crystals.
mOC78. The sample was prepared as previously described. Briefly, the powdered Aβ42 was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol solution at a concentration of 1 mg mL -1 (240 µM). The solution was shaken in a 4 °C freezer for 4 h for sufficient dissolution and then stored at -20 ℃ as a stock solution. Before use, the solvent 1,1,1,3,3,3-hexafluoro-2-propanol was removed by evaporation with a gentle stream of nitrogen. The peptide was dissolved in Milli-Q water. Aggregates formation was carried out at 37 ℃ incubator with or without Cu. The presence of aggregates in these preparations has been previously confirmed and characterized with CD spectrum.
Cell Culture: BV2 cells, obtained from American Type Culture Collection, were grown on plates in MEM media containing 10% fetal bovine serum, 1% serum L-glutamate and 1% streptomycin in a humidified atmosphere of 5% CO2 and 95% 5 air. The media were changed every three days, and the cells were passaged by trypsinization before confluence. Immunofluorescence images were acquired with a Nikon Confocal Microscope (A1R MP).

Measurement of Inflammatory Cytokines Level: After the BV2 cells were labeled
with ThS, BV-2 cells were stimulated with different Aβ aggregates for overnight. The supernatants were collected and stored at -20 °C until assays for TNF-α and IL-1β were performed. TNF-α and IL-1β levels were detected by mouse TNF-α ELISA kits and IL-1β ELISA kits according to the procedures provided by the manufacturers.

Measurement of Level of CD11b:
After treatment with Aβ42 aggregates, BV2 cells were blocked in 0.1% triton-X and 10% BSA in PBS for 1 h and then incubated in primary antibody CD11b 1:500 (Rat polyclonal) in PBS overnight at 4 °C. Cells were rinsed 3 times in PBS for 5 min before incubation in secondary goat anti-rat (495 nm) at a 1:1,000 dilution of for 1 h. Glass coverslips were rinsed 3 times in PBS prior to adhesion to microscope slides. Immunofluorescence images were acquired with a Nikon Confocal Microscope (A1R MP) and flow cytometry results were obtained by BD LSRFortessa™ Cell Analyzer.

Handling of mice:
All animal experiments were performed according to the NIH guidelines for the care and use of laboratory animals (NIH Publication No. 85-23 Rev. 1985) and approved by the Jilin University Animal Care and Use Committee. Eightto 12-week-old Kunming mice (sex-randomized) were used as test animals. The data was expressed as mean ± standard deviation (SD) and performed in at least 3 specimens (n=3). W Kunming mice stereotactically injected bilaterally with 5 μL of Mn-MOF (5mg/mL) at a rate of 1 μL/3 min (RWD Life Science Co. Ltd Shenzhen).
The injection coordinates, relative to Bregma, were anteroposterior, −1.70 mm; mediolateral, 1.50 mm; and dorsoventral, −1.50 mm. After anesthesia and exposure of the cisterna magna, CSF was obtained using a glass-pulled micropipette at 24 h or two weeks post-injection, ensuring that the CSF was not contaminated with blood. About 7 10 ml of the CSF was obtained. The CSF was immediately diluted 10-fold in 1%

CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) in PBS with
protease inhibitors (Roche Diagnostics). The Cu, Mn, and Zr content of the samples was measured by ICP-MS (Varian 720-ES). At the same time, the whole brains and major organs including heart, liver, spleen, lung and kidney were harvested and fixed in 10% formalin, embedded in paraffin, and sectioned at two weeks post-injection.
Terminal deoxynucleotidyltransferase-mediated nick-end labeling (TUNEL) staining of brain slices and hematoxylin and eosin (H/E) of brain and major organs was conducted.