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Access Type

WSU Access

Date of Award

January 2017

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

Miriam L. Greenberg

Abstract

Cardiolipin (CL) is a unique phospholipid that is primarily localized within the inner mitochondrial membrane. Newly synthesized CL undergoes acyl remodeling to produce CL species enriched with unsaturated acyl groups. The tafazzin gene (TAZ) encodes a transacylase that remodels CL. Deficiencies in CL remodeling cause Barth syndrome (BTHS), an X-linked genetic disorder resulting from TAZ mutations that lead to reduced total CL content and an accumulation of monolysocardiolipin (MLCL), an intermediate of the CL remodeling pathway. However, which of these biochemical outcomes contributes to the physiological defects is not fully understood.

Deletion of yeast CL phospholipase rescues the taz1growth phenotype. We concluded that an increased MLCL/CL, but not decreased CL unsaturation, is likely the primary cause of decreased respiratory growth and chronological life span observed in taz1. This suggests that the physiological defects of BTHS patients is possibly due to increased MLCL/CL ratio instead of decreased unsaturated CL, and that attenuation of CL phospholipases may potentially treat BTHS.

Furthermore, our findings suggested a possibility that CLD1 expression is upregulated in response to oxidative stress. CL peroxidation, resulting from oxidative stress, has been described in mammalian cells. Based on this, one of the physiological roles of CL remodeling is to remove peroxidized CL. Liquid chromatography−mass-spectrometry-based phospholipidomics was combined with genetic and nutritional manipulations to assay CL incorporation of PUFA during the CL biosynthetic and post-synthetic remodeling processes in yeast. Our results demonstrated that yeast readily incorporate PUFA to synthesize oxidizable CL. Although multiple CL-hydroperoxides and CL-dihydroperoxides were readily detected in these cells, cell growth and life span were not impacted. cld1Δ cells expressing ∆12-desaturase were utilized to determine the effect of peroxidation on CL remodeling. Using this novel yeast model, in which cells expressed ∆12-desaturase, the specificity of Cld1 to peroxidized CL, and its role in deacylating peroxidized CL, was determined. In cells expressing desaturase, loss of CLD1 led to increased peroxidized CL species, as well as decreased cell growth and life span. The findings from this study may contribute to our understanding of CL remodeling and its mechanistic roles in mitigating oxidative stress.

To probe defects resulting from CL deficiency in mammalian cells, I constructed a tafazzin knockout C2C12 cell line, which exhibits an increased MLCL/CL ratio, decreased respiration capacity, increased ROS generation and decreased membrane potential. Although WT and TAZ-KO C2C12 cells can differentiate into myotubes, differentiation was significantly decreased in TAZ-KO C2C12 cells under certain conditions. Taken together, these findings indicate that CL remodeling plays a role in myotube differentiation.

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