SCA28 is an autosomal dominant ataxia associated with oculomotor anomalies, due to AFG3L2 gene missense mutations. The encoded protein is a member of the mitochondrial m-AAA protease (ATPases Associated with a variety of cellular Activities), and forms an hexameric complex in the Inner Mitochondrial Membrane, which exerts proteolytic activity and protein quality surveillance functions. We performed a whole genome expression profiling (based on Affymetrix Human Genome U133A 2.0 Chip Array) using lymphoblastoid cell lines (LCLs) from four SCA28 patients (mutations p.T654I, p.M666V, p. M666T and p.G671R), and six unrelated healthy controls matched for sex and age. We found 117 probes whose expression was statistically different, 60 of which up-regulated (Fold Change - FC = 1.5-16) and 57 down-regulated (FC = 0.7-0.1).The differentially expressed genes (n=88) were clustered, in five functional categories: (1) Cell Growth and Metabolism; (2) Apoptosis activation; (3) Oxidative Stress Response; (4) Cell Adhesion and Membrane Components; (5) Calcium Binding Proteins. To verify these pathways, we performed functional experiments on seven patients’ LCLs which showed: 1) a delayed growth compared to controls (p<0.005), and an increased number of cells (> 15%) in G0/G1 phase (p<0.001); 2) an increased mortality of patients’ cells due to apoptosis (AnnexinV/Propidium Iodide test) (p<0.05); 3) normal intracellular ROS levels in basal conditions (DCFH-DA test at FACS analysis). We also evaluated the respiratory chain activity in LCLs mitochondria, measuring ATP synthesis after treatment with specific respiratory chain blocking agents. No significant difference compared to controls was measurable, likely because an impairment in ATP production is absent or below detectable levels. We did not find mitochondrial DNA large deletions by long-range PCR. In conclusion, whole genome expression profiling in SCA28 LCLs allowed to identify several altered pathways that may be related to the disease. It is conceivable that the activation of the G1-S checkpoint and apoptosis is due to a cell cycle checkpoint surveyor responding to an abnormal cellular stress coming from the mitochondria. In more sensible cell types or tissues, such as Purkinje cells in cerebellum, this may result in a detectable alteration leading to the disease.
A Genome-wide Expression profiling to unravel effect of missense mutations in SCA28 patients
MANCINI, CECILIA;LO BUONO, NICOLA;BRUSSINO, Alessandro;CAGNOLI, CLAUDIA;Limongi T;FUNARO, Ada;MIGONE, Nicola;BRUSCO, Alfredo
2011-01-01
Abstract
SCA28 is an autosomal dominant ataxia associated with oculomotor anomalies, due to AFG3L2 gene missense mutations. The encoded protein is a member of the mitochondrial m-AAA protease (ATPases Associated with a variety of cellular Activities), and forms an hexameric complex in the Inner Mitochondrial Membrane, which exerts proteolytic activity and protein quality surveillance functions. We performed a whole genome expression profiling (based on Affymetrix Human Genome U133A 2.0 Chip Array) using lymphoblastoid cell lines (LCLs) from four SCA28 patients (mutations p.T654I, p.M666V, p. M666T and p.G671R), and six unrelated healthy controls matched for sex and age. We found 117 probes whose expression was statistically different, 60 of which up-regulated (Fold Change - FC = 1.5-16) and 57 down-regulated (FC = 0.7-0.1).The differentially expressed genes (n=88) were clustered, in five functional categories: (1) Cell Growth and Metabolism; (2) Apoptosis activation; (3) Oxidative Stress Response; (4) Cell Adhesion and Membrane Components; (5) Calcium Binding Proteins. To verify these pathways, we performed functional experiments on seven patients’ LCLs which showed: 1) a delayed growth compared to controls (p<0.005), and an increased number of cells (> 15%) in G0/G1 phase (p<0.001); 2) an increased mortality of patients’ cells due to apoptosis (AnnexinV/Propidium Iodide test) (p<0.05); 3) normal intracellular ROS levels in basal conditions (DCFH-DA test at FACS analysis). We also evaluated the respiratory chain activity in LCLs mitochondria, measuring ATP synthesis after treatment with specific respiratory chain blocking agents. No significant difference compared to controls was measurable, likely because an impairment in ATP production is absent or below detectable levels. We did not find mitochondrial DNA large deletions by long-range PCR. In conclusion, whole genome expression profiling in SCA28 LCLs allowed to identify several altered pathways that may be related to the disease. It is conceivable that the activation of the G1-S checkpoint and apoptosis is due to a cell cycle checkpoint surveyor responding to an abnormal cellular stress coming from the mitochondria. In more sensible cell types or tissues, such as Purkinje cells in cerebellum, this may result in a detectable alteration leading to the disease.
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