The pellet was then solubilized in cold PBS and centrifuged at 100,000 for 70 min
April 21, 2022
The pellet was then solubilized in cold PBS and centrifuged at 100,000 for 70 min. protein that is enriched in astrocytes and mediates exosome secretion, by reducing the association of Sp1 with the enhancer of the gene. Importantly, overexpression of B-crystallin rescues defective exosome launch from HD astrocytes as well as mHtt aggregates in the striatum of HD140Q KI mice. Our results demonstrate that mHtt reduces the manifestation of B-crystallin in astrocytes to decrease exosome secretion in the HD brains, contributing to nonCcell-autonomous neurotoxicity in HD. SIGNIFICANCE STATEMENT Huntington’s disease (HD) is definitely characterized by selective neurodegeneration that preferentially happens in the striatal P7C3 medium spiny neurons. Recent studies in different HD mouse models shown that dysfunction of astrocytes, a major type of glial cell, prospects to neuronal vulnerability. Growing evidence demonstrates exosomes secreted from astrocytes consist of neuroprotective cargoes that could support the P7C3 survival of neighboring neurons. We found that mHtt in astrocytes impairs exosome secretion by reducing B-crystallin, a protein that is indicated primarily in glial cells and mediates exosome secretion. Overexpression of B-crystallin could alleviate the deficient exosome launch and neuropathology in HD mice. Our results exposed a new pathological pathway that affects the essential support of glial cells to neurons in the HD mind. mice (Hickey et Esam al., 2008) were maintained in the Emory University or college Animal facility. Both male and female pups from these mice were utilized for main cultures. Male and female adult mice at different age groups were utilized for viral injection and mind cells isolation. This study purely followed the recommendations in the National Institutes of Health before collecting the brains for further analysis. Exosomes from WT or KI astrocytes tradition resuspended in PBS for exosome injection. A total of 8 l of PBS, WT, or KI astrocytic exosomes were injected into two sites of striatum in one hemisphere (0.6/0.4 mm anterior to bregma, 2.0 mm lateral to the midline, 3.5 mm ventral to dura) in 9-month-old P7C3 KI mice with 4 l for each site. PBS injection served like a control. Seven days after injection, the mouse brains were dissected and prepared for immunocytochemistry. Immunofluorescent staining. Cultured astrocytes were fixed with 4% PFA for 8C10 min. Mouse brains were sectioned at 10 m thickness using a cryostat at ?20C. Sections were mounted onto gelatin-coated sides and fixed with 4% PFA for 10 min. Fixed sections were clogged with 3% BSA/0.2% Triton X-100 for 30 min at space temperature. Following incubation of fixed sections with main antibodies and washes at 4C over night, fluoro-conjugated secondary antibodies and Hoechst nuclear dye were added to the samples for staining. An Olympus FV1000 inverted microscope with a digital video camera system captured astrocyte sample and mind section images. Quantitative analysis of GFAP staining occurred using the method in our earlier studies (Hong et al., 2016). Briefly, National Institutes of Health ImageJ software was used to measure GFAP immunostaining intensity. Color images acquired having a 63 objective were converted to 8-bit black-and-white images. The Threshold function was used to adjust the background to focus on GFAP-specific staining. The same threshold was applied to all images captured for analysis. P7C3 Finally, the Measure function quantified GFAP staining intensity in each image. Each group experienced 7C10 images per section and 8 sections per group were examined. Purification of exosomes. Exosomes were prepared from tradition medium as explained previously (Thry et al., 2006; Takeuchi et al., 2015; Iguchi et al., 2016; Westergard et al., 2016). Briefly, astrocytes were cultured in 100 mm dish (2 106). The astrocyte tradition medium was replaced with the tradition medium comprising 10% exosome-free FBS (EXO-FBS-50A-1, SBI). After 24 h incubation, astrocyte tradition medium was transferred to a centrifuge tube and centrifuged sequentially at 300 for 10 min, 2000 for 10 min, 10,000 for 30 min, and 100,000 for 70 min. The pellet was resuspended in chilly PBS and centrifuged at 100,000 for an additional 70 min. The exosome-containing pellet was resuspended in appropriate buffers. All centrifugations occurred at 4C. After eliminating cell tradition medium, astrocytes were lysed in NP40 buffer. The purity of exosomes was recognized by Western blotting using exosome marker antibodies to Alix, flotillin-1, and HSC70 P7C3 as explained in earlier studies.