Fig. 9
Mitophagy gene signature and mitochondrial impairments in ADNP patient-derived cell lines. (A) Expression levels of mitophagy-related genes in ADNP patient LCLs compared to age and sex-matched controls. mRNA sequencing demonstrated an upregulated mitophagy gene signature in patient LCLs as compared to controls. (B) RT-PCR showing a significant increase of mitophagy-related genes in ADNP patients versus control LCLs. Expression values were normalized using the housekeeping genes GAPDH, RPL13A and SDHA. Data was subsequently analyzed with an unpaired student T-test assuming unequal variances with a Welch’s multiple testing correction. (C) Subcellular mitochondrial distribution using the MitoTracker® Red CM-H2XRos fluorescent staining of control subjects (A-B) and ADNP patient (C-D) fibroblasts. The white arrow indicated the unstained nucleus of the fibroblast. (D) MitoTracker® red fluorescent signal (RFU) was normalized to brightfield cell count and quantified in patient and control fibroblasts using a multimode microplate reader (Tecan Spark™) at an excitation of 485 nm. A significant decrease (p = 0.01, student T-test) in red fluorescent signal, corresponding to the mitochondrial activity, was observed in patient-derived fibroblasts (blue) compared to the control cells (red). (E) The Seahorse Cell Mito Stress Assay was used to measure changes in oxygen consumption rate (OCR) in patient-derived (blue) and control (red) fibroblasts after different triggers that inhibit or activate mitochondrial respiration. Oligo = oligomycin, a complex V inhibitor to decrease the electron flow through the electron transport chain (ETC); FCCP = Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, the uncoupling agent to promote maximum electro flow through of the ETC; Rot/AA = rotenone and antimycin, complex I and complex II inhibitors respectively, to shut down the mitochondrial-related respiration. (F) Based on the changes in OCR, several aspects of the mitochondrial respiration could be quantified using the Agilent Seahorse analytics software. Statistical significance was calculated using an unpaired student T-test assuming equal variances. Data is shown as mean ± sd. The basal respiration (p = 0.04, *), proton leak (p = 0.21; ns), ATP-linked respiration (p = 0.06, ns), maximal respiration (p = 0.28, ns), spare capacity (p = 0.84, ns), and non-mitochondrial respiration (p = 0.79; ns) were indicated for patients (blue) compared controls (red)