Multiple Lines Of Evidence Indicating A Strong Relationship Between Naa And Myelin Lipid Synthesis

3.1. Incorporation Studies

The first report showing that NAA provides acetate groups for lipid synthesis during myelination was published by D'Adamo and Yatsu in 1966. In a subsequent study in 1968, D'Adamo et al. showed that injection of acetyl labeled NAA into rats of various ages resulted in maximum incorporation into fatty acids just before and during myelination.37 Since that time, these observations have been confirmed and extended by at least three other research groups. In 1991, Burri et al. performed a detailed study on incorporation of acetyl labeled NAA into various lipids in the rat brain, and showed that NAA is a major source of acetyl groups for lipid synthesis during rat brain development.33 In 1995, we performed similar incorporation studies using rat brain slices and showed that free acetate and acetyl CoA are formed from the radiolabeled NAA under this condition, indicating acetyl CoA route for this NAA-lipid pathway.35 Finally, in 2001, Chakraborty et al. have shown by intra ocular injection studies that neuronally derived NAA supplies acetyl groups for myelin lipid synthesis.34 Taken together, these results provided strong evidence for the involvement of NAA-derived acetyl groups for lipid synthesis during myelination. However, the quantitative significance of this acetate source for myelin synthesis remained unclear until recently.

3.2. Cellular Localization Studies of ASPA

Earlier enzyme activity studies had indicated that ASPA is enriched in the white matter in the CNS.38 Two recent studies have reported cellular localization of ASPA using polyclonal antibodies against recombinant ASPA.39,40 Both studies showed predominant localization of ASPA in white matter. The study by Madhavarao et al.40

Figure 1. ASPA immunoreactivity in the rat forebrain (A) and hindbrain (B). In forebrain, ASPA was present in oligodendrocytes throughout the corpus callosum (CC), and in fiber bundles in the striatum. Fewer ASPA-positive oligodendrocytes were observed in the superficial layers of cortex. In the hindbrain, ASPA-immunoreactive oligodendrocytes were present in great numbers throughout the cerebellar deep nuclei, white matter, and medulla, but were very sparse in the cerebellar cortex.

Figure 1. ASPA immunoreactivity in the rat forebrain (A) and hindbrain (B). In forebrain, ASPA was present in oligodendrocytes throughout the corpus callosum (CC), and in fiber bundles in the striatum. Fewer ASPA-positive oligodendrocytes were observed in the superficial layers of cortex. In the hindbrain, ASPA-immunoreactive oligodendrocytes were present in great numbers throughout the cerebellar deep nuclei, white matter, and medulla, but were very sparse in the cerebellar cortex.

is detailed here. The polyclonal antibody preparation against ASPA showed a single band at 37kD by Western blot analysis. Immunohistochemical studies with the antibodies showed that ASPA is localized in the CNS predominantly in oligodendrocytes (Figure 1). ASPA was found to be co-localized throughout the brain with CC1, a marker for oligodendrocytes. Many ASPA-positive cells were observed in white matter, including cells in the corpus callosum and cerebellar white matter. Relatively, fewer cells were labeled in gray matter, particularly in the superficial layers of cerebral cortex. In all the stained cells, ASPA staining was restricted to the cell body and proximal processes. Interestingly, the ASPA antibodies labeled not only the cell bodies and proximal processes of oligodendrocytes, but also labeled their cell nuclei, indicating that ASPA is not restricted to the cytoplasm of these cells (Figure 2). No astrocytes were labeled for ASPA, and neurons were unstained in the forebrain, although a small number of reticular and motor neurons were faintly to moderately stained in the brain stem and spinal cord. Finally, it should be mentioned that microglial cells were faintly stained throughout the brain.

The extensive localization of ASPA in oligodendrocytes strengthens the case for the NAA/ASPA system being required for myelination during development. However, it also raised additional questions because of the prominent localization of the substrate NAA in neurons.

Figure 2. ASPA immunoreactivity in the rat corpus callosum. Strongly stained oligodendrocytes are arranged in rows between myelinated axons, with immunoreactive processes visible on many of the stained cells. An unusual finding was that ASPA was present not only in the cytoplasm of oligodendrocytes, but was also strongly expressed in their nuclei.

Figure 2. ASPA immunoreactivity in the rat corpus callosum. Strongly stained oligodendrocytes are arranged in rows between myelinated axons, with immunoreactive processes visible on many of the stained cells. An unusual finding was that ASPA was present not only in the cytoplasm of oligodendrocytes, but was also strongly expressed in their nuclei.

3.3. Developmental Increases in ASPA

Another line of evidence indicating a connection between ASPA and myelination is the parallelism between the developmental increases in ASPA activity and myelination, which was first studied by D'Adamo's group in 1973.22 They showed that the increase of ASPA activity in the rat brain with development almost paralleled the pattern of myelination. In 2001, Bhakoo et al. performed a more detailed developmental study covering different rat brain regions, and have confirmed the similarity between the developmental increases in ASPA enzyme activity and myelination.38 More recently, we used in situ hybridization to study ASPA gene expression in the rat brain during development using a riboprobe based on a 400-bp cDNA fragment of murine ASPA.30 These studies also confirmed a temporal correlation between the developmental increase in ASPA and myelination.

3.4. Myelin Lipid Synthesis Study in CD Mice

Despite the evidence that the acetyl moiety of NAA is used for acetyl CoA/lipid synthesis during myelination, and other evidence that ASPA is associated with myelination, the quantitative significance of this pathway for myelination remained unclear. In most cell types, such as hepatocytes, the enzyme citrate lyase provides the acetyl groups for fatty acid synthesis.41 It remained to be demonstrated that the NAA-ASPA system contributes significantly to lipid synthesis, most likely myelin lipid synthesis, in the CNS. This is a central issue in the context of the pathogenesis of CD, so we addressed this question by examining the rate of myelin lipid synthesis during the period of maximal postnatal myelination in ASPA knockout (ASPA -/-) mice.42 These mice exhibit pathology similar to that of human CD patients (See chapter by Matalon et al., this volume).

In the ASPA knockout mouse lipid synthesis study, the control group consisted of homozygous wild type (ASPA+/+) mice and the experimental group consisted of homozygous knockout (ASPA-/-) mice.42 Comparison of a number of general parameters between the control and knockout mice showed that the two groups differed in only two measures; brain weight and ASPA activity. Other parameters, including animal weight, liver weight and kidney weight were not significantly different between wild type and ASPA knockout mice. The brain weight of the mutant mice was significantly greater than the normal group (461mg ± 17.8 vs. 416mg ± 22.3 respectively; p<0.05). In addition, ASPA activity was undetectable in ASPA-/- mice, as compared with ASPA activity of 18.3 ± 5.6 nmol/h/mg protein in control mice (p<0.001).

The level of lipid synthesis was determined in wild type and ASPA-/- mice using tritiated water incorporation, which has previously been demonstrated to be a reliable method for determining the rate of lipid synthesis,43 cholesterol synthesis44 and myelin synthesis.45 Table 1 shows the incorporation of tritium into lipids from liver, kidney and myelin in control and ASPA-/- mice 5 hours after intraperitoneal administration of 20 mCi [3H]2O per animal. The specific activity of [3H]2O in the serum samples from the control and ASPA-/- mice did not differ significantly (p<0.05).46 There was no difference between the two groups with respect to lipids synthesized in liver, however total myelin lipids were decreased in the brains of ASPA-/- mice by approximately 30% (p<0.005). Total lipid incorporation in kidney was increased by approximately 18% in the ASPA-/-group.

Table 1. Specific activity of 3H-H2O in serum, and radioactivity in lipids from various tissues 5 hours after administration of 20mCi 3H-H2O per mouse.

Lipid Category

Control

ASPA -/-

Specific activity of [3H]2O (dpm/nmol water)

90298±6816

97290 ± 8930 NS

Total lipids-liver (cpm/mg protein)

60056 ± 6850

60499 ± 4402 NS

Total lipids-kidney (cpm/mg protein)

95611±13007

113233 ± 8438 **

Total myelin lipids (cpm/mg myelin protein)

26946 ± 3864

18991±2099 ***

The differences between the mean values (± standard deviations) were assessed for significance by two-tailed paired and unpaired t-test. (** p<0.05; *** p<0.001; NS = not significant).

The differences between the mean values (± standard deviations) were assessed for significance by two-tailed paired and unpaired t-test. (** p<0.05; *** p<0.001; NS = not significant).

Myelin lipids were further analyzed by two-dimensional thin layer chromatography (2D-TLC). Table 2 shows the changes in the various lipid classes from the myelin samples of control and ASPA-/- mice. Among nonpolar lipids separated on the first dimension, four spots showed significant decreases in the mutant mice, corresponding to glycerol 1-fatty acids (decreased by ~35%), cholesterol, (decreased by ~22%), cholesteryl fatty acids, (decreased by ~35%), and glycerol tri fatty acids (trimyristin, tripalmitin, trilaurin, tristearin: decreased by ~21%). Glycerol 1,2 fatty acids (dimyristin, dipalmitin, dilaurin and distearin) did not show statistically significant change.

In the second TLC dimension for the separation of polar lipids, two out of three lipid spots showed significant reductions (p<0.05) in the ASPA-/- mice. The Rf of 0.75 corresponded to phospholipids and sulfatides (phosphatidylinositol, phosphatidyl choline, phosphatidyl glycerol, phosphatidic acid and cerebroside sulfate), which were decreased by approximately 38% in ASPA-/- mice. The Rf of 0.88 corresponded to phosphatidyl ethanolamine, galactocerebroside and hydroxy fatty acid ceramide, which were decreased approximately 35% in the experimental group.

These data show that CNS myelin lipid synthesis is decreased in the murine model of CD, whereas lipid synthesis in other organs such as kidney and liver was either increased, or not affected. This indicates that lipid synthesis in the brain, in part, requires an intact ASPA enzyme, providing the first direct evidence for deficiency of NAA-derived acetate as an etiological mechanism of CD. As mentioned above, earlier studies have demonstrated that the acetate moiety from neuronally-derived NAA is incorporated into myelin in the CNS34, and the present study establishes that the acetate contribution to myelin synthesis from NAA is quantitatively sufficient to decrease myelin synthesis during the period of elevated postnatal myelination in the murine model of CD.

Table 2. Comparison of the myelin lipids in the control and mutant mice1.

(a) Nonpolar lipids2

Table 2. Comparison of the myelin lipids in the control and mutant mice1.

(a) Nonpolar lipids2

Rf

Control

ASPA -/-

Comigrating lipid standards

0.16 ±0.01

342±48 **

224 ± 62

Glycerol 1-fatty acids3

0.45 ± 0.02

3837±564 **

2989±351

Cholesterol

0.53 ± 0.03

548 ± 208 NS

556 ± 98

Glycerol 1,2-fatty acids

0.61 ± 0.02

1699±112 *

1346±330

Glycerol tri fatty acids

0.66 ± 0.02

500±62 **

379 ± 85

Cholesteryl fatty acids (myristate, palmitate)

(b) Polar lipids

0.60 ± 0.06

821 ± 400 NS

788 ± 807

Unknown

0.75 ± 0.02

9995±2525 **

6153± 1320

PC, PI, PG, Sulfatides, PA4

0.88 ± 0.02

7513± 1114 **

4904± 1157

PE, GC, Ceramide

1 All lipid spots are given as cpm/mg myelin protein.

2 Myelin lipids were separated by TLC.

3 Fatty acids include laurate, myristate, palmitate and stearate.

4 PC, phosphatidyl choline; PI, phosphatidyl inositol; PG, phosphatidyl glycerol; Sulfatides, 3-sulfate ester of galactosyl cerebroside; PA, phosphatidic acid; PE, phosphatidyl ethanolamine; GC, galactocerebroside; Ceramide, hydroxy fatty acid ceramides.

1 All lipid spots are given as cpm/mg myelin protein.

2 Myelin lipids were separated by TLC.

3 Fatty acids include laurate, myristate, palmitate and stearate.

4 PC, phosphatidyl choline; PI, phosphatidyl inositol; PG, phosphatidyl glycerol; Sulfatides, 3-sulfate ester of galactosyl cerebroside; PA, phosphatidic acid; PE, phosphatidyl ethanolamine; GC, galactocerebroside; Ceramide, hydroxy fatty acid ceramides.

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