2016. no clear function/mechanism is known. A possible function for the short form has not been described. Many questions remain about C9 protein function and its possible involvement in ALS/FTD. To address this issue, we examined the effects of C9 KD in different brain-derived cell models. This revealed unexpected effects on cell morphology as well as on expression of multiple genes, including many relevant to ALS. Among these, a number of endothelin (e.g., 0.001; ****, 0.0001. Significance was assessed via the unpaired test. (I) Phase-contrast image Rabbit polyclonal to Noggin (40) of NHAs treated with control siRNA, taken with an inverted phase-contrast microscope. Bar, 10 m. (J) Phase-contrast image (40) of NHAs treated with C9 siRNA, taken with an inverted phase-contrast microscope. Black arrows indicate vacuole formation, and the box shows a zoomed-in image, with the white arrow showing vacuoles. (K) Western blot showing p62 and C9 protein levels after C9 siRNA (siC9) treatment of NHAs compared to those in control siRNA-treated cells (siCtrl). A feature of C9 ALS is usually a cerebral pathology of p62-positive inclusions BMS-191095 (44). Using an immunofluorescence (IF) assay with anti-p62 antibodies, we found that C9 KD led to extensive accumulation of p62 aggregates (Fig. 1D to ?toF),F), and BMS-191095 Western blots revealed an overall increase in p62 levels. We also observed increases in nuclear and cell sizes of 1 1.9- and 5.2-fold, respectively (Fig. 1G and ?andH;H; Fig. S2). p62 aggregation was also observed recently following C9 KD in mouse cortical neurons (36) and in C9 KO mice (34). Use of a second, impartial siRNA confirmed both the vacuolization/cell size and p62 phenotypes (Fig. S3). To determine if the morphological changes observed in U87 cells occurred in normal glial cells, we knocked down C9 in normal human astrocytes (NHAs) and detected a similar vacuole formation phenotype and increased cell size (Fig. 1I and ?andJ)J) as well as increased p62 levels (Fig. 1K). C9 KD results in broad changes in gene expression. We next investigated whether the above results reflect changes in gene expression induced by reduced C9 protein levels. To this end, we used genome-wide RNA sequencing (RNA-seq) to identify genes that undergo changes in expression following C9 KD in U87 cells. Reads were mapped using Bowtie (45), and differential gene expression was decided using GFOLD (46). We used a 2-fold cutoff to identify genes that were differentially expressed. Unexpectedly, our analysis revealed that upon C9 KD, 2,650 genes were differentially expressed relative to those in cells treated with control siRNA (siCtrl) (see Table S1). While possible mechanisms for this dysregulation are described below, among these genes BMS-191095 were many known to be expressed differentially in ALS patient brains and in ALS patient-derived iPS cells, such as in C9 and control siRNA-treated U87 cells (Fig. 2A). Since were all significantly upregulated (3.3-, 2.6-, and 3.5-fold, respectively) upon C9 KD, while EDN2 mRNA levels were slightly increased (1.4-fold) (Fig. 2A). These results were all confirmed with a second C9 siRNA (Fig. S4). We also examined expression of the genes upon C9 KD in NHAs. EDN1 and EDNRA mRNA levels were both increased, by 5.2- and 3.1-fold, respectively (Fig. 2B); EDNRB mRNA, however, was not expressed (data not shown). To extend these results to a neuronal cell line, we also determined if C9 depletion caused EDN upregulation in SH-SY5Y neuroblastoma cells. RT-qPCR analysis showed that EDN1 mRNA levels were elevated 4.5-fold following C9 depletion (Fig. 2C). EDNRA and EDNRB mRNA levels, however, were not significantly affected (data not shown). Open in a separate window FIG 2 C9 KD leads to altered expression of numerous genes relevant to ALS. (A) RT-qPCR analysis of IL-8, NEAT1, EDN1, EDN2, EDNRA, and EDNRB mRNA levels in U87 cells treated with control and C9 siRNAs. Experiments were performed as three biological replicates (= 3), and error bars represent standard errors (SE). (B) RT-qPCR (= 3).