Canavan Disease (CD) is a devastating disease, which is caused by a recessive mutation in the enzyme aspartoacylase (ASPA). ASPA hydrolyzes W-acetylaspartate (NAA) to generate L-aspartate and acetate. In mammalian tissues, NAA is one of the most abundant metabolites and its level is detected highest in the brain as compared to other organs (1, 2). It is found mostly concentrated in the cerebral cortex area, while much lower concentration can be detected in the medulla. For more than a decade, NAA has served as an important tool in clinical application due to its high resonance property, which is captured by NMR to examine the status of neurons and underlying pathology in CNS. In the CNS, the substrate NAA and its converting enzyme, ASPA, have recently gained much attention due to their implication in CD. The lack of functional ASPA causes an accumulation of NAA and leukodystrophy that can be visualized in the white matter regions of the CD brain (3, 4). The finding of ASPA deficiency as the basic biochemical defect in CD, had, for the first time, suggested that hydrolysis of NAA by ASPA is crucial for maintenance of functionally intact white matter in brain. It was also reported that the ASPA activity occurs predominantly in the white matter (5, 6). Further work in this area has validated both localization of ASPA protein and enzyme activity (79) and NAA (10, 11) in cells of oligodendrocyte lineage. A recent study showed that ASPA activity appears at a high level in purified myelin and that neuronal NAA contributes acetyl groups for myelin lipid biosynthesis, perhaps via transaxonal movement in the optic system (12). A subsequent study reported the presence of fatty acid synthesizing enzymes necessary for the production of acyl chain within myelin and
* Mental Retardation Research Center, Department of Neurobiology and Psychiatry, Brain Research Institute, David Geffen School of Medicine, University of California, Los Angeles. Sankar Surendran and Reuben Matalon; Division of Genetics, Department of Pediatrics, University of Texas Medical Branch, Galveston.
thus supported the transaxonal movement on NAA in neurons (13). It is not clearly understood, however, if increase in the subtrate NAA or the lack of individual product, aspartate and actetate, or combination of all, are responsible for observed neuropathology of CD. And most importantly, the casual target gene functions and temporal onset of events during development are not understood at this time.
In this preliminary study we report preparation of primary cultures of mixed glia from the newborn rat and ASPA knockout (KO) mice cerebral cortex for characterization of glial cell types on the basis of cell and lineage specific marker gene expressions. The generation of ASPA KO mice strain and its characterization has been well described by Matalon et al., (14). The ASPA KO mouse serves as a model for CD to address metabolic and developmental targets in the brain that can also throw light into ASPA mutationmediated events in CD. The ASPA KO mouse characterization had revealed a lack of ASPA enzyme activity and spongy degeneration in cerebral regions with neuropathology similar to CD (14). While the underlying relationship of oligodendrocyte development to ASPA deficiency has not been reported at this time, a profound decrease in expression of GABA-A receptor has been seen in ASPA KO mice (15). In addition, a reduced level of GABA and glutamate, an over-expression of Spi1 transcription factor has been observed in adult ASPA KO brain (15). For the purpose of establishing primary cell culture as a tool to study developmental events in ASPA mutation, a characterization of ASPA gene expression, protein and enzyme levels were examined along with glial lineage-specific markers in mixed glial cultures from normal newborn rat cortex. Since the subventricular zone gives rise to early glial progenitors, we cultured cortical tissue for analysis. The primary cell cultures of ASPA wild type (wt) and KO newborn mice were similarly prepared and characterized for early development of oligodendrocytes.
Our laboratory has pioneered the preparation of cerebral cortex mixed glial cell cultures, pure cultures of oligodendrocytes and astrocytes from newborn rodent brains (16, 17). Utilizing these cultures, a wide range of developmental studies have been possible based on cellular, biochemical and molecular approach. The oligodendroglial development procedes through several stages from progenitor cell to the mature, myelin-synthesizing oligodendrocyte (reviewed in 18, 19). The onset of oligodendrogenesis, its survival and maturation is a multi-step process, which is dependent upon availability of several essential factors.
The postnatal genesis of oligodendrocyte progenitors in newborn cultures can be identified by immunocytochemical staining for the early oligodendrocyte markers such as platelet-derived growth factor receptor alpha (PDGFRa, 20, 21), the proteoglycan NG2, GD3 and A2B5 (Reviewed: 22, 23). Working with cyclic nucleotide phosphohydrolase (CNP)-enhanced green fluorescent protein (EGFP) transgenic mice, Aguirre and Gallo (24) demonstrated that postnatal subventricular zone NG2+ progenitors can migrate distances and develop into interneurons and oligodendrocytes in a region-specific manner. The study also shows that the cortical, olfactory bulb and cerebellar NG2+ cells have limited migration ability and they give rise to glia. Morphologically, oligodendrocyte progenitors appear as bipolar or cells with bright soma, developing more processes and becoming less motile with maturity. These early oligodendrocytes also are responsive to mitogens, PDGF-AA, NT3 and bFGF. Expression of functional TrkC receptors have been characterized in these early progenitors and are responsive to NT-3 via PTK pathways to promote cell proliferation, i.e., transition of cell-cycle from G1 to S phase, and in cell survival by preventing PARP fragmentation (25, 26). The NT3 KO
mice show a severe reduction in oligodendrocyte development in their spinal cord at birth and are still born (27). The next step in oligodendrocyte progenitor differentiation occurs when they adopt a phenotype referred to as "pre-oligodendrocytes" (preOL). These cells are multipolar and still express PDGFaR. At this stage, these cells stop the expression of the A2B5 marker and acquired immunoreactivity for sulfatides recognized by the mab O4. PreOLs become immature, non-mitotic oligodendocytes that express galacto-cerebroside (GalC), CNP and some isoforms of myelin basic proteins (MBPs) but still do not synthesize myelin membranes (28, 29). The final stage of oligodendrocyte differentiation is characterized by the appearance of the myelinating oligodendrocyte, which expresses 4 MBP isoforms, MAG, PLP/DM20 proteins, and MOG (30, 31). We have begun characterizing these stages in ASPA wt and KO mice cultures, to delineate if observed myelin deficiency is the result of a disruption at any one of these stages.
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