Abstract
Canavan disease (CD) is an autosomal recessive neurodegenerative disorder that begins in infancy and causes death by early childhood. CD is typified by a loss of white matter and astrogliosis in the brain, which are caused by inactivating mutations in the gene that encodes aspartoacylase (ASPA). Normally, ASPA metabolizes N-acetyl-L-aspartic acid (NAA). Research indicates the consequent increase in NAA that occurs in the absence of functional ASPA causes the white matter degeneration and astrogliosis observed in CD. Another neurological phenotype caused by high levels of NAA in CD is dysmyelination of the CNS and increased oligodendrocyte death. Because the molecular etiology of CD phenotypes is increased NAA, the most effective treatment for afflicted infants would likely be to permanently reduce the levels of NAA in the CNS and, thereby, help promote remyelination. One major goal of this research project was to generate ASPA-expressing astroglia from human induced pluripotent stem cells (hiPSCs) to determine if they can be used as a therapeutic tool for reducing elevated NAA levels. To remyelinate the CNS of CD patients, it will also be important to use oligodendrocytes because they are the only myelinating glial cell in the brain. It is therefore critical to understand the mechanisms used by oligodendrocyte precursor cells (OPCs) to differentiate into terminally differentiated oligodendrocytes. The transcription factor SOX2 maintains stem cell populations and promotes neurogenesis; however, the specific function of SOX2 within the oligodendrocyte lineage is not well understood. Therefore, the second major goal of this research was to elucidate the role SOX2 plays in oligodendrocyte development. First, to determine a pure population of hiPSC-derived astroglia, immunocytochemistry (ICC) was used to identify cells expressing the astroglia markers S100B, CD44, and GFAP. These cells were then transduced with a lentiviral vector carrying the ASPA gene under the control of a constitutive promoter and puromycin was used to specifically select ASPA-expressing astroglia. Subsequent analysis using quantitative RT/PCR indicated that ASPA expression in transduced astroglia was not significantly different from non-transduced astroglia. This result suggested that our ASPA-expressing astroglia were likely not expressing ASPA efficiently enough to be a valuable therapy for reducing NAA levels for CD patients. To determine the function of Sox2 in the oligodendrocyte lineage, Cre-loxP-mediated Sox2 gene deletion was used to eliminate Sox2 expression in PDGFRα+ OPCs in postnatal mice. Postnatal spinal cords and brains were then analyzed. Ablating Sox2 in the PDGFRα+ OPCs led to decline in OPCs, pre-myelinating oligodendrocytes, and mature oligodendrocyte. This decline was due to a lack of proliferative OPCs. There was no difference found in the level of cell apoptosis after Sox2 gene ablation. In addition, myelin basic protein (MBP), and 2', 3’-Cyclic-nucleotide 3'-phosphodiesterase (CNPase), proteins critical for myelination, were not impacted after Sox2 deletion. These findings indicate a vital role of Sox2 in oligodendrocyte development. Future research will determine if overexpression of Sox2 in mice is sufficient to drive oligodendrocyte development.