Interactions Between Mitochondrial Transporters and the Protein Synthesis Machinery in 22q11.2 Deletion Syndrome Público
Lee, Chelsea (Spring 2018)
Abstract
Schizophrenia is a chronic and complex neuropsychiatric disorder. Schizophrenia’s polygenic nature makes it difficult to study and pinpoint causative disease mechanisms. To circumvent these challenges, our lab chose to focus on 22q11.2 Deletion Syndrome (22q11.2DS). 22q11.2DS is the highest genetic risk factor for schizophrenia aside from having two parents or an identical twin diagnosed with schizophrenia. Our lab used quantitative proteomic analysis to compare fibroblasts from 22q11.2DS patients and their unaffected family members. The proteome revealed that the mitochondria was one of the central organelles affected in 22q11.2DS. This lead our lab to propose the following overarching hypothesis: defects in mitochondrial function and composition caused by the 22q11.2 microdeletion syndrome contribute to behavioral and synaptic phenotypes shared by schizophrenia and other neurodevelopmental disorders. The proteome showed hits on the Electron Transport Chain and the inner mitochondrial matrix. Immunoblots showed altered levels of SLC25A1 (a mitochondrial citrate transporter) and proteins encoding for mitochondrial ribosome subunits in 22q11.2DS patients. The SLC25A1 gene is found in the core deletion region of 22q11.2DS. These findings were central to my thesis, leading to the hypothesis that: SLC25A1 null cells possess altered mitochondrial protein synthesis leaving them more susceptible to mitochondrial ribosome protein synthesis inhibiting drugs. To test this, I used Agilent Seahorse Mitochondrial Stress Test assays to measure mitochondrial respiration rates in HAP1 SLC25A1 KO cells and HAP1 control cells treated with varying antibiotics that inhibit mitochondrial ribosome protein synthesis or proteostasis (minocycline, doxycycline, chloramphenicol, linezolid, and actinonin). I predicted that SLC25A1 null cells should have differential susceptibility to agents that selectively block mitochondrial ribosome protein synthesis as compared to wild type cells. Antibiotics used varied in structural chemistry and mechanisms of action. In cells lacking SLC25A1, I found that treatment with antibiotics targeting the mitochondrial ribosome led to a decrease in oxygen consumption rates. HAP1 control cells showed no decrease in oxygen consumption rates. To ensure that the decrease in mitochondrial respiration in SLC25A1 null cells was due to mitochondrial ribosome dysfunction, cells were also treated with carbenicillin, a beta lactamase inhibitor. Neither the HAP1 control cells or HAP1 SLC25A1 KO cells showed a significant decrease in mitochondrial respiration when treated with carbenicillin. These results support my hypothesis that SLC25A1 null cells possess altered mitochondrial protein synthesis and are more susceptible to drugs inhibiting mitochondrial ribosome protein synthesis.
Table of Contents
Introduction………………………………………………………………………………………1
Methods…………………………………………………………………………………………18
Results…………………………………………………………………………………………..23
Discussion………………………………………………………………………………………35
References………………………………………………………………………………………42
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Interactions Between Mitochondrial Transporters and the Protein Synthesis Machinery in 22q11.2 Deletion Syndrome () | 2018-04-09 12:53:11 -0400 |
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