Our research plan:
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| Click to enlarge |
(1) Zebrafish as a model organism for understaning Peroxisomal disease pathogenesis
We use zebrafish as a model organism to study human peroxisomal diseases. The embryo of zebrafish is transparent so that its developmental process is clearly seen and able to analyze under a microscope. Their fundamental body plan and organ structure are similar with that of the human therefore the study of zebrafish's development and physiology can be applied to human for understanding our body and health.
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| We use zebrafish! |
We are making knock-out fish that have mutated genes that are pathogenic causing human peroxisomal diseases, which for example are PEX genes. To target a specific gene for knocking-out, we use TALEN (TAL-effecter endonuclease). TALEN works as a pair of synthetic protein, which can find and bind to the target DNA with a sequence specific manner, and then cut the DNA double helix placed in between two TALENs. Imprecise double helix repairing results in a deletorious mutaion of the target genes. We are now examining the phenotype of the knock-out fish to understand human pathology.
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| TALENs |
(2) In vitro study of patint's neural cells differentiated from iPS cells
Peroxisomal disease patients develop a symptom in their brain. In peroxisomal biogeneis disorder (PBD) patients, the brain development is severely affected. Heterotopia of the cortical layer and microgyrus is often seen in the petient's brain, and also in the mice model. Such phenomena would have been caused by aberrant migration of neuronal precursor cells so that the cells halt at the ectopic position during brain development. The other symptom of the human patinents is the neuronal degeneration, which is often seen in ALD patients (cerebrum form) and also in the milder form of PBD. The myelin sheath degeneration (demyelination) is considered as the primary cause of this symptom.
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| A colony of iPS cells |
To study the process of the symptom development in the brain of the patients, examining patient’s neural cells is required, which has been practically impossible until recently. Introduction of iPS cells and subsequent in vitro differentiation into neuronal lineage enabled us to examine patient-derived neuronal cells. We are establishing iPS cells that are induced from patient's fibroblasts, which is done by applying the defined four genes (the Yamanaka factors; OCT4, SOX2, KLF4 and c-MYC). Subsequently the cells are converted into neural lineage for detailed analysis aiming to find the primary sign and study the progress of the neural degeneration.
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| Neurons differentiated from iPS cells |
(3) Study of PBD fibroblasts
In PBD patients, the formation of peroxisomes is extremely disturbed. The peroxisomes show specific phenotype based on the affected genes. There are 13 causal genes which lead to PBD when mutated and they are all the members of the PEX genes. Each PEX gene has a function either of translocating peroxisomal membrane proteins or of introducing peroxisomal matrix proteins into the peroxisome during peroxisomal biogenesis.
We study the peroxisomal phenotype of fibroblasts obtained from patients who show different severity of the symptom, focusing on the intracellular movement and heterogenity of the peroxisome and affected metabolic agents such as fatty acids and lipids. We use immunohistochemistry (antibody staining against peroxosome-localizing proteins), live imaging, and LCMS to elucidate these cellular characteristecs of the patient's cells.
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| Fibroblasts |
(4) Metabolome analysis by LCMS and GCMS
The metabolic alteration is the direct result from peroxisomal defects. In PBD patient the biogenesis of the peroxisome is severely affected so that the function of the peroxisome as a field of the metabolic activity is lost or severely restricted. The metabolic activity includes catabolism of very long chain fatty acid (VLCFA), phytanic acid, pristanic acid, and anabolism of bile acid and ether lipids including plasmalogens. Other metabolic agents affected in patients might be present because of our lack of comprehensive understanding of peroxisomal function.
We are developing the method for measuring above metabolites as well as others using liquid chromatography-mass spectrometry (LC-MS) aiming to apply this method for experimental and clinical uses. Our established method so far can measure VLCFAs, pytanic acid, pristanic acid and ether lipid derivatives in a single analysis. By comparing their amount between patient and control samples, we are able to detect several novel metabolites affected in patients. Investigating these new metabolic markers would help us to find out the metabolic pathway that would lead to the disease symptoms when affected so that could be the target of the clinical intervention.
