Sgk1 inhibition in glia ameliorates pathologies and symptoms of Parkinson's disease (2021)
Figure 4

Fig. 4. SGK1 inhibition protects glia from mitochondrial damage and cell senescence.

A and B, Ppargc1a (PGC1α) mRNA expression upregulated in VM-glia treated with the SGK1 inhibitors in the RNA-seq data (A) and q-PCR analysis (B). Data are represented as mean ± SEM. n=3; Student's t-test. Significantly different at p=0.0011#, 0.0499* in graph B.

C, Immunoblot for PGC1α protein expression. Data are represented as mean ± SEM. n=3. Student's t-test. Significantly different at p=0.0077* in graph C.

D, Mitochondrial biogenesis estimated by Mitotimer reporter gene expression in cultured VM-glia. Green fluorescence indicates newly synthesized mitochondria, and red fluorescence indicates old or damaged mitochondria. MFIs were estimated in 3 independent cultures (>5 randomly selected microscopic fields/culture). Data are represented as mean ± SEM. One-way ANOVA. Significantly different at p=3.78E-08*, 0.00003** in graph D.

E, Mitochondrial ROS levels estimated using the MitoSox red probe. Fluorescence intensity was measured using ImageJ, and ROS levels are presented as mean fluorescent intensity (MFI). Mitochondrial damage was induced by treatment with the mitochondrial toxins CCCP (2µM)+H2O2(500 µM) for 4hr. MFIs were estimated in 3 independent cultures (>5 randomly selected microscopic fields/culture). Data are represented as mean ± SEM. One-way ANOVA. Significantly different at p=0.0013*, 0.0273# in graph E.

F, Mitochondrial membrane potential (JC-1). The red fluorescence (JC-1 Aggregate) indicates disrupted mitochondrial membrane potential, and green fluorescence (JC-1 Monomer, insets) indicates intact potential. The inset images were taken from the same microscopic fields of the respective JC-1 aggregate images. Mitochondrial damage was induced by treatment with the mitochondrial toxins CCCP (2µM)+H2O2(500 µM) for 4hr. MFIs were estimated in 3 independent cultures (>5 randomly selected microscopic fields/culture). Data are represented as mean ± SEM. One-way ANOVA. Significantly different at p=0.0006*, 0.0093# in graph F.

G, Intracellular levels of ATP, an indicator of mitochondrial oxidative phosphorylation. Data are expressed as mean ± SEM. n=3 cultures; one-way ANOVA. Significantly different at p= 0.0009*, 0.00004**, 0.04913#, 0.0024+in graph G.

H-M, Glial cell senescence rescued by SGK1 inhibition. (H) Expression of genes associated with cell senescence in the RNA-seq data (VM-glia with vs. without GSK-650394 treatment). (I) Intracellular ROS levels estimated by DCF-DA. Data are represented as mean ± SEM. n=3; one-way ANOVA. Significantly different at p=0.0074*, 0.012# in graph I. (J) Senescence-associated β-galactosidase (SA-β-gal) activity. Data are represented as mean ± SEM. n=3; one-way ANOVA. Significantly different at p=2.05E-06*, 0.0042#, 0.0006+ in graph J. (K) Immunoblots for proteins associated with cell senescence. Significantly different at p=0.008*, 0.001# (TNFA), 0.01*, 0.009# (HMGB1), 7.32E-10*, 1.23E-8# (IL6), 9.62E-11*, 1.01E-5# (MMP3), 0.001*, 5.72E-4# (LAMIN B) in graph K. (L) IL-6 levels secreted from cultured VM-glia. The cytokine levels were estimated using ELISA of the media from the VM-glia with and without the SGK1 inhibitor. Data are represented as mean ± SEM. n=3 cultures. One-way ANOVA. Significantly different at p=0.0028*, 0.0062# in graph L. (M) Glial cell viability following treatment with senolytic compounds (azithromycin, fisetin). Data are represented as mean ± SEM. n=3; Student's t-test. Significantly different at p=0.0073*,0.0018**, 0.0286#, 0.0269##, 0.0045### in graph M.

Data information: Scale bar, 20µm.