Fig. 8: Astrocytic CD44 is necessary for the activation of neuroinflammatory pathways.

a Western blots of murine astrocytes transduced with viruses carrying control non-targeting (shNC) versus shRNA against murine CD44 (shmCD44-1, 3, and 4). shmCD44-3 and-4 effectively knocked down CD44 expression. shmCD44-1, 3, and 4 are independent biological replicates. The lentiviruses included a GFP-tag to label infected cells. b Representative immunofluorescence images of murine astrocytes transduced with shRNA constructs as for panel A. CD44 (red), GFP (green), and DAPI (blue) nuclei are labeled. Scale bar: 10 µM. Note complete loss of murine CD44 in GFP+ cells in sh-mCD44 3 and 4. Uninfected GFP- cells retain CD44. For a, b, the knockdown experiment was replicated twice. c Differentially expressed genes (DEGs) of PD vs control substantia nigra (SN) protoplasmic astrocytes and DEGs in murine astrocytes with CD44 knockdown (KD) vs. non-targeting shRNA control. The genes are shown by their log2 fold change (LFC) in the SN on the x-axis and CD44-KD on the y-axis. The color indicates if the genes are significantly differentially expressed in PD SN, CD44 KD, both, or none. DEGs were considered based on two-sided adjusted p-values. d Bar plot showing activation scores of select pathways in differentially expressed genes in CD44 KD astrocytes. The scores were calculated using Omnipath and decoupleR – see methods. Significantly repressed and activated pathways are shown in red. e Gene-term plot showing significantly enriched KEGG pathways derived from PathfindR analysis of CD44 DEGs, where the pathways are shown in tan nodes, the size of which corresponds to the number of genes driving the enrichment of that pathway and the genes contributing to the pathway enrichment are shown as red node (decreased DEGs) or green nodes (increased DEGs).