Alcatl Regulate Mitochondrial Morpholoty And Biological Function

CL is a polyglycerophospholipid exclusively localized in the mitochondria, where itregulates mitochondrial function and oxidative phosphorylation in species from yeast tomammals. Oxidative damage to CL from pathological remodeling is implicated in theetiology of mitochondrial dysfunction commonly associated with diabetes, obesity, and othermetabolic diseases.To investigate the fundamental mechanisms of these pathological process,ALCAT1, a lyso-CL acyltransferase was introduced. The results of this researchdemonstrated this gene was upregulated by oxidative stress, catalyzes the synthesis of CLspecies that are highly sensitive to oxidative damage, leading to mitochondrial morphologychange, dysfunction and ROS production. Consequently, ALCAT1deficiency prevented thedamage of ROS and significantly improved mitochondrial mtDNA content and kept theirfidelity. Thus, the activities of the enzymes that remodel CL perform vital action inmaintaining the structural and functional mitochondrial hence may regulate the cellularprocesses from energy metabolism to apoptosis. These results emphasize the close interplaybetween CL and MFN, demonstrate the functional importance of CL remodeling withALCAT1, and provide insights into the pathogenesis of ALCAT1-dependent disease. Mythesis is organized into the following three parts.1. ALCAT1regulates mitochondrial morphology and function in vitro.To explore ALCAT1gene regulation in vitro and in vivo, ALCAT1gene wassubcloned to p-BABEpuro plasmid. The virus produced from this system was transfected inC2C12cells and highly expressing ALCAT1was screened by puromycin. Also, we generatedALCAT1disrupted C57/BL6mice. Analysis of cells revealed that more than95%mitochondria of ALCAT1stably overexpressed cells were dramatically fragmented andexhibit a smaller spherical structure, while the vector cells were characterized by a network offilamentous, thread-like mitochondria. Results also showed ALCAT1incredibly reducedmtDNA copy number and increased mtDNA point mutation rate in ALCAT1stably expressedcells compare to the vector control, which was reversed by ALCAT1deficiency on MEFsfrom ALCAT1KO mice. Moreover, the defects on ALCAT1stably expressed cells werefurther confirmed using Western blot analysis. ALCAT1induced a dramatic mitochondrialprotein prohibitin loss in total fraction, associated with dramatic mitochondrial respiratory complexes content deficiency.Finally, we found that ALCAT1overexpression in C2C12cellscaused a40%reduction in total CL levels by selectively decreasing the content of C16-C18fatty acids in CL, including oleic acid and linoleic acid, the dominant acyl forms of CL inmetabolically active tissues.2. ALCAT1overexpression linked to oxidative stress.To provide further evidence for a causative role of ALCAT1in oxidative stress, weanalyzed the expression of genes involved in ROS production and oxidative response by real-time RT-PCR analysis. ALCAT1overexpression significantly increased expression of genesthat cause oxidative stress, such as NAD(P)H quinone oxidoreductase-1(Nqo1) and NADPHoxidase-4(Nox4), and decreased expression of genes encoding antioxidative enzymes,including glutathione peroxidases (Gpx), peroxiredoxins (Prdx), thioredoxin reductases(Txnrd) in C2C12cells. Consistent with increased ROS in C2C12cells overexpressingALCAT1, the MDA level was significantly lower in liver of ALCAT1ko mice relative tothe wild-type mice. Furthermore, treatment with H2O2resulted in significant production ofoxidant reagent DCF and MDA,2folds higher in C2C12cells overexpressing ALCAT1relative to the vector control cells. Also mtDNA depletion was further exacerbated aftertreatment with H2O2. The expression of ALCAT1was inducible with hydrogen peroxide, tert-butylhydroperoxide (T-Bu) in vitro and high-fat diet in vivo. ALCAT1mRNA expressionwas significantly increased in isolated cardiomyocytes in culture by T-Bu, and by H2O2treatment. Supportively, ALCAT1mRNA expression in liver, heart, and skeletal muscle wereall upregulated in response to high-fat diet in C56BL/6mice.3. ALCAT1regulates mitochondrial biogenesis through modulation of MFN2.MFN2, MFN1and OPA1mRNA and protein level in ALCAT1stably transfected celland ALCAT1KO mice model were tested. All these three mitochondrial fusion related genes,had lower mRNA levels in ALCAT1overexpression cell line. On the protein level, MFN1and MFN2level were dramatically reduced by74.6%and38.0%. Conversely, the mRNAlevels of MFN1and MFN2elevated on MEFs isolated from ALCAT1KO mice. MFN2protein level was also significantly upregulated, relative to these from the WT control.Moreover, we conducted polyethylene glycol-(PEG) mediated mitochonrial fusion assay.Cells overexpression ALCAT1caused fusion defect as the deficiency of MFN2. Then wefurther transient transfected the expression vector of MFN1, MFN2and OPA1into ALCAT1overexpression cells, respectively, to see if the morphology could be restored. Disruptedmitochondria morphology was partially rescued by MFN1and MFN2, but not OPA1.Additionally, MFN2rescue the mitochondrial respiratory capacity defect on ComplexI and Vof ALCAT1stably overexpressed cells, which suggest MFN2could be the downstream target of ALCAT1. Given the above observations, we next sought to assess whether continuousoxidative stress was evident in MFN2deficiency ALCAT1overexpression cells.When treatedwith H2O2, we observed that the gradually increased ROS were accompanied by attenuatedmitochondrial MFN2. Besides, a2h preincubation of ALCAT1cells withdiphenyleneiodonium (DPI) dramaticlly recovered the impairment of MFN2in a dose-dependent mannor. Taken together, these results suggest that the ALCAT1overexpressioncell line characterized of higher H2O2production and more sensitive to H2O2treatment, weredue to the MFN2deficiency mediated by ROS.