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Summary of lipid and protein quantifications of KO and normal brain and myelin. KO mice gave decreased myelin yield based on myelin protein per brain and brain protein. Myelin phospholipid phosphorus relative to myelin protein was decreased in KO myelin. Cerebrosides were decreased in KO brain relative to brain protein and in KO myelin relative to myelin protein

Summary of lipid and protein quantifications of KO and normal brain and myelin. KO mice gave decreased myelin yield based on myelin protein per brain and brain protein. Myelin phospholipid phosphorus relative to myelin protein was decreased in KO myelin. Cerebrosides were decreased in KO brain relative to brain protein and in KO myelin relative to myelin protein

Myelin Whole Brain

Figure 1. Thin-layer chromatography of lipids from brain and myelin of normal and ASPA KO mice. A: Neutral and zwitterionic lipids were subjected to TLC on silica gel 60 plates as described. The main observed difference was significant depletion of the upper cerebroside band (Cereb 1) and moderate reduction of the lower band (Cereb 2) in the KO samples (see Table). Chol, cholesterol; Cer, ceramide; EPG, ethanolamine phosphoglycerides; CPG, choline phosphoglycerides; SM, sphingomyelin. B: Standard curves obtained with known amounts of brain cerebrosides; Cereb 1 and Cereb 2 were separately quantified.

Myelin Whole Brain

Figure 1. Thin-layer chromatography of lipids from brain and myelin of normal and ASPA KO mice. A: Neutral and zwitterionic lipids were subjected to TLC on silica gel 60 plates as described. The main observed difference was significant depletion of the upper cerebroside band (Cereb 1) and moderate reduction of the lower band (Cereb 2) in the KO samples (see Table). Chol, cholesterol; Cer, ceramide; EPG, ethanolamine phosphoglycerides; CPG, choline phosphoglycerides; SM, sphingomyelin. B: Standard curves obtained with known amounts of brain cerebrosides; Cereb 1 and Cereb 2 were separately quantified.

In addition to the major alterations of cerebroside content, less pronounced changes were suggested in other lipids, e.g. modest decrease in ethanolamine phosphoglycerides and possible increase in ceramide of KO myelin. The latter might represent unutilized precursor for cerebroside synthesis. However, those and other changes require more detailed study.

3.2. Method for Assay of ANAT

Subcellular fractions were isolated from whole brains of 30-60 day old rats as described (Lu et al., 2004). In addition to microsomes and mitochondria, this included an intermediate fraction obtained by centrifuging the supernatant from crude mitochondria (obtained by

Figure 2. Application of ANAT assay to brain homogenate and subfractions. A: Representative image of TLC plate on storage phosphor screen. B, C: Variation of ANAT activity with protein and time, respectively. Asp = aspartate; STD = standard. Reproduced from Lu et al. (2004) with permission of Elsevier.

Figure 2. Application of ANAT assay to brain homogenate and subfractions. A: Representative image of TLC plate on storage phosphor screen. B, C: Variation of ANAT activity with protein and time, respectively. Asp = aspartate; STD = standard. Reproduced from Lu et al. (2004) with permission of Elsevier.

centrifugation at 10,000g x 10 min) at 35,000g x 20 min. As previously described (Lu et al., 2004), the microsomal fraction had the highest specific activity, ~4-5x that of mitochondria, while the intermediate fraction showed intermediate activity indicative of a mixture of these 2 fractions (Fig. 2). The latter result indicated ANAT was not localized in a subfraction such as small mitochondria that occur in axons and nerve endings. As shown, ANAT activity varied linearly with protein content and time for the 3 fractions.

3.3. Assay of ANAT in Pig Retina and Brain Cells

Applying the above assay to the various sections of pig retina revealed ANAT activity in the ganglion cell-enriched layer to be several times greater than that enriched in photoreceptor cells (Fig. 3A). This was especially true for microsomes, whose specific activity in the ganglion cell-enriched layer was ~8x that of microsomes in the photoreceptor cell-enriched layer. As with whole brain, ANAT activities in mitochondria were significantly lower in all cases. As indicated, the tissue sections analyzed in this manner represented enriched- rather than pure cell populations.

Analysis of isolated brain cells (without subcellular fractionation, in this case) also revealed significant differences in ANAT activity between cells that do not extend myelinated axons (CGN) and cell cultures of mixed types (CN) that include some projection neurons that do ultimately extend myelinated axons (Fig. 3B). That the difference in this case was not as striking as with retinal neurons was likely due to the presence of non-myelinating interneurons in the CN as well as the early stage of development. Even though myelinated axons have not yet formed in the analyzed cultures, upregulation of ANAT has likely begun at that stage.

□ mitochondria

□ mitochondria

Whole Retina Ganglion Photoreceptor

Figure 3. Comparison of ANAT activity in various neuron types. A: Sections of pig retina. B: Cultures of cortical neurons (CN) and cerebellar granule neurons (CGN). Two-tailed student t test: * p<0.05; ** p<0.01; *** p<0.001; compared to value of corresponding fraction in whole retina (panel A) or to value of CN (panel B).

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