For the characterization of mixed glial cells from newborn rat cerebral cortex, we examined ASPA gene expression by in situ hybridization with digoxigenin (DIG)-labeled ASPA sense and antisense probes (Fig. 1A, B). A full-length mouse ASPA cDNA was cloned in Bluescript (SKII) riboprobe vector and was labeled in the opposite direction using a kit and hybridization was carried out as per manufacturer's (Roche Molecular Biochemicals, Germany) protocol. Sense riboprobe was used as a negative control. The expression of ASPA is visible with anti-sense probe within round cell bodies (Fig. 1B arrows) on the bed layer of astrocytes not stained for ASPA. The ASPA activity was determined in cell lysates prepared from rat cortical cultures of mixed glial cells, neurons, and pure cultures of oligodendrocytes and astrocytes as described (14). The highest level of ASPA activity, 1.3mU/mg protein, was observed in oligodendrocyte cell lysates, approximately 6-times higher than that reported in a whole brain tissue lysate for wt mouse (14). The astrocytes and neuronal cultures showed less than 0.01mU/mg protein level of ASPA activity (Fig.1C). The cultures of mixed glial cells exhibited nearly 29% ASPA activity of pure cultures of oligodendrocytes.

The mixed glial cultures from normal rat brain cortex were immunostained, examined for expression and localization of ASPA protein. A rabbit polyclonal antibody generated against mouse ASPA in the laboratory of Dr. Matalon was IgG purified with a kit (Pierce, Rockford, IL) and used at 1:1000 dilution. The oligodendrocyte cells showed co-localization of ASPA protein with oligodendrocyte specific cell markers, A2B5 (Fig. 1D), and cyclic nucleotide phosphohydrolase (CNP) (Fig. 1F) in a mixed glial cell culture. The double immunostaining of ASPA with glial fibrillary acidic protein (GFAP), a marker for astrocyte, showed a discrete staining for ASPA and GFAP in mixed glial cultures (Fig. 1G).

Our preliminary observations show an early arrival of ASPA KO (-/-) oligodendrocyte cells on the bedlayer of astrocytes by 1-2 days, as compared to the wt (+/+) oligodendrocyte cells. In some instances upto 50% more oligodendrocyte-like cells can be seen in the KO cultures within 3-4 days of plating. Once arrived, ASPA KO oligodendrocyte cells appear to be less adherent to the bedlayer of astrocytes and become unattached.

We have analyzed a few early (NG2, GD3, A2B5, GPDH), mid (O4) and late (MBP, PLP) oligodendrocyte cell markers and GFAP in mixed glial cultures (Figure 2). The preliminary results show a robust staining for proteoglycan, NG2 and GD3, characterized as progenitor oligodendrocyte cell marker (Fig. 2A, B). A strong signal for A2B5 and cytosolic glycerol-3- phosphate dehydrogenase (GPDH) expressed early in development

DIG Anti-Sense

DIG Anti-Sense

Astro Oligo Neuron Mixed Cell Type Qia

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