July 2010

Document Type


Degree Name



Dept. of Molecular and Medical Genetics


Oregon Health & Science University


Neurodegeneration with brain iron accumulation (NBIA) is a heterogeneous class of disorders with the distinctive feature of axonal swellings throughout the central nervous system. Two causative NBIA genes have been identified by linkage analysis: PANK2, encoding a pantothenate kinase, and PLA2G6, encoding a group VI calciumindependent phospholipase A2 (iPLA2-VIA). Mutations in these genes are hypothesized to cause lipid dyshomeostasis and oxidative stress, leading to pantothenate kinaseassociated neurodegeneration (PKAN) and infantile and atypical neuroaxonal dystrophy. However, in order to investigate specific mechanisms of disease and develop therapies for NBIA, it is necessary to fully characterize each gene and its function. Toward that goal, my thesis research includes three projects focused on investigating the transcriptional regulation and expression of NBIA genes. First, I identified a PANK2 promoter. Historically, two nearly identical PANK2 isoforms have been described: short PANK2 and mature PANK2. However, the biological relevance of these isoforms remains unclear. I have shown that their regulation is distinct and describe a promoter for the short isoform of PANK2. In addition, I identified potential regulators of PANK2 expression, including NF-Y, FOXN4 and the human heterogeneous nuclear ribonucleoprotein A/B family. These findings validate expression of the short PANK2 isoform and enable predictions about potentially deleterious sequence variants in the PANK2 promoter region. Furthermore, I provided preliminary evidence that PANK2 transcription is not effected by iron overload and depletion, oxidative stress induced by low dosage of hydrogen peroxide or peroxisome proliferator-activated receptor (PPAR) agonists. Second, I investigated the expression and function of a family of microRNAs (miR-103/7) conserved in pantothenate kinase genes. In recent years, miR-103 and miR-107 have been proposed to play a role in multiple cellular processes, including metabolism and neurodegeneration, suggesting that their cellular role may complement pantothenate kinase activity. Interestingly, I demonstrated by qRT-PCR that mir-103 and miR-107 expression does not correlate with expression of pantothenate kinase genes in mouse tissues. In addition, the expression profile of miR-103 and its PANK2 encoded precursor, pre-miR-103, also did not correlate in mouse tissues. Therefore, miR-103-2 expression may be regulated at the level of post-transcriptional processing. Finally, in attempt to dissect miR-103/7 cellular function, I tested several predicted miR-103 targets by luciferase reporter assays and miR-103 over expression experiments; however, I was unable to confirm regulation of candidate targets. Third, I analyzed the expression of PLA2G6 and PANK2 in human fetal development. As shown by in situ hybridization histochemistry, PLA2G6 is expressed throughout the developing brain in proliferative zones, as well as in the differentiated neurons in the developing cerebral neocortex and hindbrain. Likewise, PANK2 expression is found in brain and eye, although technical difficulties prevented a more detailed study of its temporal and spatial patterning. Also, PANK2 and PLA2G6 expression were observed in basal ganglia, lateral ventricle, midbrain and thalamus of the fetal brain, as well as in spinal cord, kidney, liver, eye and lung. These results suggest that PLA2G6 and PANK2 are positioned to play a role in early neuronal development. Disruption of this process may contribute to the widespread neurological problems observed in NBIA.




School of Medicine



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