Isolated astrocytes from young BMAL1aKO and wildtype mice
Purpose: The core clock protein BMAL1 serves as the primary positive circadian transcriptional regulator and its depletion in astrocytes not only disrupts circadian function, but also leads to a unique cell-autonomous activation phenotype. We have undertaken RNAsequencing of isolated astrocytes from young wildtype and BMAL1 astrocyte-specific knockout mice (Bmal1fl/fl, ALDH1L1-CreERT2, termed BMAL1 aKO) to uncover changes to gene expression as a result of BMAL1 disruption in astrocytes. Methods: BMAL1 aKO mice and wildtype littermates were given tamoxifen at 2 months of age and harvested around 7 months of age and brains were collected. After collagenase digestion and debris removal, astrocytes were isolated from the rest of the brain cells (termed "flowthrough") using Miltenyi MACS Anti-ACSA-2 magnetic beads. RNA was extracted in Trizol reagent and sent for sequencing. Samples from BMAL1 aKO and wildtype astrocytes were compared and flowthrough samples were compared to astrocytes to confirm cell-type purity. Results: BMAL1 aKO results in both up- and down-regulation of many astrocyte genes. GO pathway analysis points to lysosomal pathways as dysregulated by knockout of BMAL1 in astrocytes. While astrocytes are enriched by our isolation methods, there is some degree of contamination from neurons and oligodendrocytes. Conclusions: While our astrocyte isolation method has some caveats in terms of purity, BMAL1 aKO appears to dysregulate lysosomal pathways, which will be investigated by further experiments. Overall design: 3 wildtype astrocyte samples compared to 5 BMAL1 aKO astrocyte samples, 4 flowthrough samples of 2 wildtype and 2 BMAL1 aKO brains each.