Abstract
Fatty acid oxidation is an important energy source during early development and periods of starvation. Energy production is deficient when fatty acid β-oxidation is disrupted in humans, and a wide range of clinical presentations may develop. Carnitine Palmitoyltransferase (CPT) 2 is involved in the carnitine shuttle pathway and allows long-chain fatty acids (LCFAs) to undergo β-oxidation for ATP production. CPT2 deficiency is a disorder of fatty acid transport across the mitochondrial membrane. CPT2 deficiencies are associated with neurological deficits and our recent case study confirmed a proband with CPT2 deficiency and schizophrenia. The association between CPT2 deficiency and schizophrenia has not previously been reported. As such, this thesis hypothesizes that reduced CPT2 function leads to abnormal embryonic development that influences brain function by disrupting brain morphology, network formation, and gene expression associated with schizophrenia. To address the hypothesis, a zebrafish CPT2 deficient model system was developed to investigate CPT2 deficiency during early vertebrate development. Two knockdown morpholino oligonucleotides (MO), a translation blocker (TB) and a splice blocker (SB), were used to reduce CPT2 expression at the onset of embryonic development and were compared to scrambled, non-specific control (CTRL) MO-injected fish and uninjected wildtype (WT) fish. Liquid chromatography in tandem with mass spectrometry (LC-MS/MS) analysis was performed for acyl-carnitine species and showed a significant increase in total acyl-carnitine levels in CPT2 knockdown larvae when compared to controls. Lipid staining indicated improper lipid utilization. The morphological assessment showed significant phenotypic differences between knockdown larvae and controls. SB larvae exhibited significantly reduced body and brain size compared to controls. Behavioral assays using ViewPoint ZebraBox showed significant changes in CPT2 knockdown fish swimming behavior compared to controls. Immunofluorescence (IF) for acetyl-tubulin demonstrated a significant change in the pattern of axonal projections throughout the brain. Anti-tyrosine hydroxylase (TH) IF demonstrated increased TH immunoreactivity in the hypothalamic region in SB fish. Reverse transcription–quantitative polymerase chain reaction (RT-qPCR) of neurotransmitter, metabolic, and schizophrenia-related genes indicate a wide spectrum of gene expression changes in knockdown larvae when compared to control larvae. A seizure assay using a multi-electrode array dish with 64 electrodes (MED64) system showed SB larvae exhibited a significant increase in electrical hyperactivity during the addition of a convulsant drug pentylenetetrazol (PTZ) compared to control larvae. These data suggest that CPT2 deficiency can be effectively modeled in the vertebrate zebrafish model system and that the inability to properly utilize β-oxidation during early brain development heightens seizure-like activity and leads to an upregulation of genes associated with schizophrenia and other neurodegenerative diseases.