Intraneuronal N-acetylaspartate supplies acetyl groups for myelin lipid synthesis: evidence for...

Journal of Neurochemistry, 2001, 78, 736±745

Intraneuronal N-acetylaspartate supplies acetyl groups for myelin

lipid synthesis: evidence for myelin-associated aspartoacylase

Goutam Chakraborty, Praveen Mekala, Daniel Yahya, Gusheng Wu and Robert W. Ledeen

Department of Neurosciences, New Jersey Medical School, Newark, New Jersey, USA

Abstract

Despite its growing use as a radiological indicator of neuronal

viability, the biological function of N-acetylaspartate (NAA) has

remained elusive. This is due in part to its unusual metabolic

compartmentalization wherein the synthetic enzyme occurs in

neuronal mitochondria whereas the principal metabolizing

enzyme, N-acetyl-L-aspartate amidohydrolase (aspartoacylase),

is located primarily in white matter elements. This study demon-

strates that within white matter, aspartoacylase is an integral

component of the myelin sheath where it is ideally situated to

produce acetyl groups for synthesis of myelin lipids. That it

functions in this manner is suggested by the fact that myelin

lipids of the rat optic system are well labeled following

intraocular injection of [14C-acetyl]NAA. This is attributed to

uptake of radiolabeled NAA by retinal ganglion cells followed

by axonal transport and transaxonal transfer of NAA into

myelin, a membrane previously shown to contain many lipid

synthesizing enzymes. This study identi®es a group of myelin

lipids that are so labeled by neuronal [14C]NAA, and demon-

strates a different labeling pattern from that produced by

neuronal [14C]acetate. High performance liquid chromato-

graphic analysis of the deproteinated soluble materials from

the optic system following intraocular injection of [14C]NAA

revealed only the latter substance and no radiolabeled acetate,

suggesting little or no hydrolysis of NAA within mature neurons

of the optic system. These results suggest a rationale for the

unusual compartmentalization of NAA metabolism and point

to NAA as a neuronal constituent that is essential for the

formation and/or maintenance of myelin. The relevance of

these ®ndings to Canavan disease is discussed.

Keywords: aspartoacylase, axon to myelin transfer, Canavan

disease, myelin lipids, myelin, N-acetylaspartate.

J. Neurochem. (2001) 78, 736±745.

N-Acetylaspartate (NAA) was identi®ed as a major amino

acid derivative in mammalian brain by Tallan et al. (1956)

and shown to be present in all regions of the CNS with

highest concentrations in cerebral gray matter (Tallan 1957).

This was estimated as 5±9.5 mm (Marcucci et al. 1966;

Burri et al. 1990), while NAA in other tissues of various

species was 1% or less of this level (Miyake et al. 1982).

During development NAA is found in both neurons and

oligodendrocytes (OLs) but becomes localized in the former

at maturity (Simmons et al. 1991; Urenjak et al. 1992;

1993), where its intraneuronal concentration reaches an

estimated 10±14 mm. It is thus slightly less than glutamate,

the most abundant amino acid of brain. The interstitial space

concentration is only 80±100 mm, indicating a large

outward-directed transport gradient (Sager et al. 1997).

NAA has become widely utilized as a neuronal marker and,

by virtue of its distinctive chemical shift in magnetic

resonance spectroscopy, an in vivo indicator of neuronal

viability in multiple sclerosis (Gonen et al. 2000) and other

neurological disorders (Tsai and Coyle 1995).

Despite its growing usefulness in clinical studies, the

biological function of NAA has remained elusive. Con-

tributing to the enigma is its unusual metabolic compart-

mentalization. It is synthesized in neuronal mitochondria by

the enzyme N-acetyl-l-aspartate transferase and transported

through the mitochondrial membrane to neuronal cytoplasm

(Patel and Clark 1979; Truckenmiller et al. 1985). On the

other hand the principal metabolizing enzyme, N-acetyl-l-

aspartate amidohydrolase II (aspartoacylase) was reported to

736 q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 736±745

Received February 5, 2001; revised manuscript received May 14, 2001;

accepted May 14, 2001.

Address correspondence and reprint requests to Dr Robert Ledeen,

New Jersey Medical School, UMDNJ, Department of Neurosciences,

MSB-H506, Newark, New Jersey 07103, USA.

E-mail: [email protected]

Abbreviations used: AU, absorbance units; aspartoacylase, N-acetyl-

l-aspartate amidohydrolase; DTT, dithiothreitol; LGN, lateral genicu-

late nucleus; NAA, N-acetylaspartate; OL, oligodendrocyte; ON, optic

nerve; OT, optic tract; PBS, phosphate-buffered saline; RSA, relative

speci®c activity; SC, superior colliculus.

predominate in white matter (D'Adamo et al. 1973; Kaul

et al. 1991), with highest activity in OLs among cultured

rat macroglial cells (Baslow et al. 1999). The metabolic

importance of this enzyme (and of NAA itself ) was indi-

cated in the discovery that Canavan disease, involving

an autosomal recessive defect in aspartoacylase, gives rise

to NAA accumulation and spongy degeneration in brain

associated with edema and progressive loss of OLs and

myelin (Matalon et al. 1988; Baslow and Resnick 1997).

These aberrations have been suggested to result from loss of

osmoregulation with resultant water imbalance and edema

(Baslow and Resnik 1997), in keeping with proposed

intercompartmental cycling of NAA between neuron and

OL that may function as a molecular water pump (Baslow

1999). Alternatively, the leukodystrophy of Canavan disease

was suggested as due to impaired acetylcholine/lipid

synthesis based on the proposed function of NAA as a

storage form of acetate for acetyl-CoA formation (Mehta

and Namboodiri 1995).

The latter proposal follows earlier observations that the

acetyl group of NAA is ef®ciently incorporated into rat

brain lipids (D'Adamo and Yatsu 1966; D'Adamo et al.

1968; Burri et al. 1991). These studies employed intra-

cerebral injection of radiolabeled NAA but did not identify

the locus of NAA hydrolysis. They also left unanswered the

question of whether NAA within the neuron can contribute

acetyl groups for myelin lipid synthesis. The present study

attempts to address these issues by showing that aspartoa-

cylase occurs at a high level of activity in puri®ed myelin

and that NAA of neuronal origin contributes acetyl groups

for biosynthesis of myelin lipids, evidently through a trans-

axonal process. For this purpose we have used the rat optic

system to show that radiolabeled NAA taken up by retinal

ganglion cells undergoes axonal transport and eventual

incorporation into myelin lipids. The role of myelin aspar-

toacylase is thus seen as the agent for releasing acetyl

groups within myelin for direct incorporation into myelin

lipids. In that respect NAA would constitute another

example of a lipid precursor undergoing axon-to-myelin

transfer for utilization by myelin-associated enzymes.

Experimental procedures

Assay of aspartoacylase in puri®ed myelin and other fractions

Myelin was isolated from brainstems of rats approximately 30±50

days of age by a modi®cation of the Norton and Poduslo method

(1973) that utilizes a third `¯oating up' sucrose gradient to reduce

contaminants to a very low level (Haley et al. 1981). A cocktail of

protease inhibitors was present throughout isolation (Chakraborty

et al. 1997). Following buffer washes, the myelin was dispersed

in medium A containing 20 mm Tris-HCl (pH 8.0), 0.1 mm

dithiothreitol (DTT) and protease inhibitors (without EDTA);

aliquots were taken for protein determination (Lees and Paxman

1972) and aspartoacylase assay. Our assay procedure was similar to

that described (Matalon et al. 1988; Kaul et al. 1991), with

modi®cations. The reaction medium was 50 mm Tris-HCl (pH 8.0),

50 mm NaCl, 1.0 mm CaCl2, 0.1 mm DTT, 0.05% (w/v) NP-40,

3.0 mm NAA (Medium B) to which was added the appropriate

amount of myelin (or other subfraction) in 500 mL. After addition

of enzyme source the reaction mixtures were shaken at 378 for 4 h

and the reaction was then stopped by placing in a boiling water bath

for 3 min. Blanks (0 h) were obtained by assaying equivalent

samples after boiling for 3 min. The amount of aspartate released

was quanti®ed in a coupled reaction system containing

2-ketoglutarate, NADH, and an excess of malate dehydrogenase

and aspartate aminotransferase (Fleming and Lowry 1966).

Decrease in absorbance at 340 nm indicated conversion of

NADH to NAD1 in the coupled reaction.

To test for intrinsic vs. loosely associated enzyme, myelin

samples were dispersed in medium A with added NaCl (0.5 m) or

Na-taurocholate (0.1%) and stirred for 30 min at 08C. Resulting

myelin was pelleted and assayed in medium B (see above). To

determine distribution, other subcellular fractions were subjected to

aspartoacylase assay by the same procedure used for myelin. These

were prepared as previously described (Chakraborty et al. 1997),

utilizing cerebral hemispheres and brain stems. The initial

homogenate in 0.3 m sucrose was centrifuged at 1500 g for

10 min to give a P1 pellet, followed by centrifugation of the

resulting supernatant at 18 000 g for 30 min to give a P2 pellet.

Finally, the resulting supernatant was centrifuged at 105 000 g for

60 min to give a P3 (microsomal) pellet and a supernatant (cytosol).

In assaying homogenate and cytosol, correction for endogenous

aspartate was made by subtracting the boiled (0 h) blank.

Aspartoacylase activity in whole rat eye was assayed following

homogenization in 0.3 m sucrose with DTT (0.1 mm) and protease

inhibitors.

Labeling of myelin by neuronal NAA

To determine whether intraneuronal NAA is able to contribute

acetyl groups for the synthesis of myelin lipids, groups of 5±7 rats,

30±50 days of age, were given an intraocular injection (rear

chamber vitreus) of 20 mCi of [14C]acetylNAA (55 mCi/mmol;

ARC, St. Louis, MO) in 5 mL of sterile phosphate-buffered saline

(PBS); this was preceded by anesthetization with a combination of

ketamine and xylazine and local application of 1% lidocaine 1 1%

tropicamide ophthalmic solution. Following injection, an ophthal-

mic ointment (mixture of tetramycin and dexamethasone) was

applied to prevent postoperative infection. Similar injections were

carried out with 20 mCi of [1±14C]acetate (56.7 mCi/mmol, ARC,

St Louis, MO, USA) in 5 mL of PBS. After varying periods of time

(2, 5, 8 or 33 days) each group of animals was killed with ether/

decapitation and the components of the optic system obtained by

dissection; in the case of optic nerve (ON), only the distal half was

employed, the proximal half being discarded to avoid error due to

periaxonal diffusion from the injection site (Haley et al. 1979).

Since injection was into the right eye, tissues labeled through

axonal transport included right ON, left optic tract (OT), left lateral

geniculate nucleus (LGN) and left superior colliculus (SC). The

control (uninjected) pathway included the contralateral tissues: left

ON, right OT, right LGN, and right SC. A small percentage of

®bers make ipsilateral connections. Each individual tissue was

thoroughly homogenized in 5 mL of buffered 0.3 m sucrose and

aliquots removed for counting. The counts in each tissue from the

injected pathway were corrected for background labeling by

N-Acetylaspartate for synthesis of myelin lipids 737

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 736±745

subtracting the counts in the corresponding control (uninjected)

tissue. Depending on the experiment, tissues were either processed

individually or pooled. To enhance myelin yield, 0.3 m sucrose

homogenates of white matter of other (unlabeled) animals were

added to the above. These combined homogenates were processed

for myelin isolation as above. The ®nal preparations were

homogenized in medium A (above) and aliquots taken for counting.

The myelin pellets were extracted with ether/ethanol (3 : 2) to

solubilize the neutral and zwitterionic lipids, followed by chloro-

form/methanol/2N HCl (1 : 1 : 0.1) to dissolve acidic lipids and

some of the proteins. The former fraction, comprising . 80% of

myelin lipids, was counted prior to separation into individual

components (see below). The acidic lipid fraction was evaporated

to dryness and redispersed in chloroform/methanol (1 : 1), which

resulted in precipitation of the proteins (proteolipids) that had

initially dissolved in this solvent. The latter were combined

with the proteins that had not dissolved and were solubilized in

5% SDS/0.5N NaOH for counting.

Identi®cation of myelin lipids labeled by neuronal NAA

The above neutral/zwitterionic lipids solubilized with ether/ethanol

were resolved into individual components by two previously

described thin layer chromatography (TLC) systems (Chakraborty

et al. 1997) employing Merck silica gel 60-coated plates (Fisher

Scienti®c, Spring®eld, NJ, USA). Prior to TLC the myelin-derived

lipids were mixed with standard lipids. The separated components

were revealed by iodine vapor and the scraped zones of silica gel

counted.

Identi®cation of intraneuronal precursor

To determine whether [14C-acetyl]NAA injected into the optic

system remained intact or was hydrolyzed to [14C]acetate, ®ve rats

of approximately 55 days of age (220±250 g) were injected as

above in the right eye with 30 mCi of [14C]NAA in 5 mL of PBS.

Five days later the animals were killed and optic system

components removed by dissection. Due to the low level of counts

the four tissues of the injected pathway (right ON, left OT, left

LGN, left SC) were combined, as were the four contralateral tissues

of the control (uninjected) pathway. Each set of tissues was

homogenized in 5 mL of methanol±water (90 : 10), followed by

addition of 5 mL of methanol to give a tissue dispersion in 10 mL

of methanol±water (95 : 5). This was rehomogenized thoroughly

and centrifuged at 10 000 g for 15 min. Each supernatant was

treated with 50 mL of 0.01 m NaOH (aq) and evaporated to 2 mL,

to which was added 4 mL chloroform 1 1.2 mL water with

thorough mixing. After brief centrifugation the upper phase was

removed and the lower phase treated twice with 2 mL of

methanol±water (1 : 1) followed by mixing and phase separation.

Small aliquots were taken at various stages to monitor recovery.

The combined upper phases were carefully evaporated with

nitrogen to near-dryness, then treated with 100 mL of 50 mm

sodium acetate, 20 mL of 30 mm NAA (Na salt), and 155 mL of

HPLC running buffer consisting of 25 mm KH2PO4, 2.8 mm

tetrabutylammonium hydroxide, 1.25% methanol, pH 7. From the

total volume of 275 mL, a 25-mL aliquot was counted and the

remainder applied to HPLC. The latter was performed with 2

Sephasil C18 columns in series (40 cm combined length � 4.6 mm;

5 mm particle size), employing the above running buffer with

elution at 0.75 mL/min (Tavazzi et al. 1999). Absorbance units

measured at 210 nm revealed peaks at approximately 6 and 10 min,

corresponding to acetate and NAA, respectively. Fractions were

collected every half-minute and counted.

All animal procedures were in accordance with the National

Institutes of Health Guidelines for the Care and Use of Animals and

approved by the Local Animal Care Committee.

Results

Detection of aspartoacylase in myelin

Highly puri®ed myelin was assayed as described with vary-

ing concentrations of NAA (Fig. 1). The optimal concentra-

tion of the latter was approximately 3.0 mm, the concentration

used in subsequent experiments. A Lineweaver±Burk plot

(inset) yielded a Km of 0.710 mm in reasonable agreement

with previously reported values (Kaul et al. 1991; Namboodiri

et al. 2000). Enhancement by NP-40 was optimal at 0.05%

(w/v) and activity was linear up to 150 mg of myelin protein.

Linearity with respect to time was also observed (Fig. 2);

Fig. 1 Variation of myelin aspartoacylase

activity with NAA concentration. Puri®ed

myelin was subjected to aspartoacylase

assay as described, but with varying NAA

concentration. Lineweaver±Burk plot (inset)

yielded the Km and Vmax values shown.

738 G. Chakraborty et al.


however, a difference was noted between aspartoacylase in

myelin vs. whole brain in that the former showed a delay of

1 h before reaction began. This difference was possibly due

to different isoforms of aspartoacylase or latent (seques-

tered) status of the enzyme within myelin. To determine

whether the enzyme is truly intrinsic to myelin, samples of

the latter were treated with 0.5 m NaCl or 0.1% taurocholate

prior to aspartoacylase assay, involving stirring for 30 min

at 08C. Virtually full activity was observed in the resulting

pelleted myelin following both treatments, and myelin

recovery was close to quantitative (data not shown). Since

these low temperature treatments are standard methods for

releasing loosely bound proteins from membranes, retention

of activity indicated that aspartoacylase is an integral part of

the myelin sheath and not adventitiously adsorbed during

isolation. A brief study compared myelin aspartoacylase in

comparatively young (40±50 days old) vs. older (. 6

months) rats: values of 137 ^ 12.7 and 124 ^ 5.2 nmol/

mg/h, respectively, indicated no signi®cant difference.

Comparison of myelin aspartoacylase to that in other

subcellular fractions

Cerebral hemispheres and brain stem were separately

fractionated as described into three membranous fractions

and cytosol; enzyme activities of these fractions were

compared to that of puri®ed myelin and tissue homogenate

from the same source (Table 1). Myelin showed high

relative activity that was signi®cantly greater than any

other subfraction from cerebral hemispheres. The appreci-

able activities in P1, P2 and P3 from both tissues indicated

widespread membrane-associated activity in brain beyond

that present in myelin and myelin-producing cells. Compar-

ing whole brain to brain stem, higher activity was seen in

homogenate and most subfractions of the latter. The higher

activity in brain stem, a white matter-rich structure, com-

pared to cerebral hemispheres accords with reports showing

highest activity in white matter (see below). Cytosol

contained the lowest RSA of all fractions. Analysis of

whole rat eye revealed very low activity at the borderline of

detection (data not shown).

Radiolabeling of myelin by neuronal NAA

Measurement of 14C-radioactivity in homogenized tissues

following intraocular injection of [14C-acetyl]NAA revealed

axonal transport of radiolabeled materials to all components

of the optic system (Fig. 3a). The Y-axis values represent

axonally transported counts, obtained by subtracting counts

of the uninjected pathway from those of the corresponding

injected pathway (right ON minus left ON; left OT minus

right OT, etc.). This subtraction corrected for nonspeci®c

labeling due to leakage of radiolabeled NAA from the eye

into brain and/or general circulation, which (following brain

Fig. 2 Variation of aspartoacylase activity with time. Puri®ed myelin

and cerebral hemisphere homogenate were each subjected to

aspartoacylase assay with varying time of incubation. A difference

was noted in that myelin remained inactive during the ®rst h. After

4 h, the usual assay period, the two assay curves converged.

Table 1 Comparison of aspartoacylase activity in subcellular fractions

Cerebral hemispheres Brainstem

Speci®c activity

(nmol/mg/h) RSA

Total protein

(mg)

Total activity

(mmol/h)

Speci®c activity

(nmol/mg/h) RSA

Total protein

(mg)

Total activity

(mmol/h)

Homogenate 76.6 �^ 4.62 1�.00 62.9 �^ 3.52 4.81 �̂ 0.35 154 �^ 6.39 1�.00 34.7 �̂ 2.43 5.34 �̂ 0.15

P1 (1500 g, 10 min) 36.9 �̂ 1.63 0�.48 16.1 �^ 0.43 0.57 �̂ 0.046 160 �^ 5.00 1�.03 3.19 �̂ 0.23 0.51 �̂ 0.29

P2 (18 000 g, 30 min) 40.3 �̂ 3.04 0�.53 24.6 �^ 1.60 1.02 �̂ 0.14 189 �^ 8.88 1�.22 7.27 �̂ 0.36 1.36 �̂ 0.048

P3 (105 000 g, 60 min) 60.0 �̂ 3.78 0�.78 2.70 �^ 0.21 0.16 �̂ 0.025 94.6 �^ 8.42 0�.61 0.92 �̂ 0.078 0.09 �̂ 0.014

Cytosol 85.7 �^ 5.68 1�.12 25.3 �^ 1.95 2.15 �̂ 0.17 144 �^ 12.5 0�.94 22.9 �̂ 1.54 3.28 �̂ 0.29

Myelin 131 �^ 4.84 1�.71 ± ± 121 �^ 5.64 0�.79 13.9* 1.68*

Subcellular fractions were prepared and assayed as described. P1, P2 and P3 are membranous particulate fractions of heterogeneous composition.

Relative speci®c activities (RSA) are expressed for each fraction relative to homogenate. While highly puri®ed myelin had appreciable activity in

both areas of brain, the data also indicate distribution of aspartoacylase in other brain components. Mean ^ SEM, n � 4.*These values were

calculated assuming myelin contains < 40% of brain stem protein. Similar calculation was not made for myelin from cortical hemispheres which

comprise a small percentage of total protein.



uptake) would label virtually all parts of the brain. Numbers

in parentheses indicate the ratio of counts in injected- to

uninjected tissue; values . 2 are generally considered

evidence for axonal transport. Higher ratios were found in

most cases at longer times (. 2 days), while the lower

values at 2 days could be due to limited arrival of axonally

transported material at this early time, especially in the more

distal components of the optic system. This ratio also

re¯ects the fact that a small but measurable number of

ganglion cell axons form ipsilateral (nondecussating)

connections to the OT and LGN.

Myelin isolated from the above tissues contained 5±20%

of the radiolabel present in the original pooled homogenates

(Fig. 3b); this represents losses incurred in the multistep

procedure employed to obtain myelin of high purity, and to

the fact that a portion of homogenate counts likely repre-

sented free [14C]NAA remaining in the axon. Extraction of

neutral and zwitterionic lipids of myelin by ether/ethanol

(3 : 2) solubilized the large majority of myelin counts

(Table 2; compare with Fig. 3b). Relatively little radio-

activity was recovered in the acidic myelin lipids or myelin

proteins (data not shown). As above, values in parentheses

indicate ratio of counts in injected to corresponding

uninjected (control) samples, all but one of which were

2.5 or higher. In general there was an increase in radiolabel

in each sample during the 33 days, suggesting that the

process of transport and transcellular supply of NAA from

axon to myelin occurs over an extended period. The 5 day

study was repeated once with similar results (data not

shown). A relatively small proportion of counts (,4±8% of

myelin) remained in the protein residues after removal of

lipids, possibly representing incorporated acyl moieties.

Such counts were signi®cantly higher for acetate than NAA

in proteins from unfractioned tissues of the optic system

harvested one day after intraocular injection of precursors (not

shown). A similar experiment carried out with [14C]acetate

also produced labeling of myelin lipids comparable to that

produced by NAA (Table 2). Despite this similarity we

believe the mechanisms involving these two precursors

differ fundamentally (see below).

Identi®cation of myelin lipids labeled by neuronal NAA

and acetate

At speci®c times following intraocular injection of

[14C]NAA or [14C]acetate, the neutral and zwitterionic

lipids were extracted from isolated myelin with ether/

ethanol and subjected to preparative TLC as described.

Results for the 8- and 33-day postinjection periods are

shown in Table 3. Data indicate the percentage of recovered

counts from the TLC plate for each lipid. Labeling of

individual lipids showed differences as well as similarities

with respect to the 2 precursors. Cholesterol was well

labeled by both precursors at both times, albeit more by

acetate. Choline phosphoglycerides were also well labeled

by both precursors, but the heavier labeling by acetate at 8

Fig. 3 Axonal transport and myelin incorporation of radiolabel from

[14C]NAA in rat optic system. [14C]NAA was administered intra-

ocularly to groups of six rats, and the four components of the optic

system harvested at the times indicated. Radioactivities were deter-

mined in tissue homogenates (a) of six animals (mean ^SEM) and

in myelin isolated from the latter (b) after pooling the respective

tissues (hence a single value for each). y-Axes indicate transported

radioactivity (injected minus control) and numbers in parentheses

are the ratios injected : control. Results indicate axonal transport of

radioactivity and incorporation of liberated acetyl groups into myelin

lipids. Radioactivity of all samples rose after the initial 2-day period,

and that of myelin peaked roughly in parallel with that of tissue

homogenate.

Fig. 4 Separation of NAA and acetate standards by HPLC. This

procedure employed 2 Sephasil C18 columns in series as described.

Absorbance units were measured at 210 nm. The indicated peaks

were obtained with standards of 1 nmol and 7.5 nmol of NAA and

acetate, respectively.



Table 2 Incorporation of NAA and acetate into myelin lipids of optic system

Day post-injection

2 5 8 33

[14C] NAA

Optic nerve 504� (4.3) 1470� (9.8) 2060� (16) 1680� (7.6)

Optic tract 330� (2.5) 720� (5.5) 2230� (6.3) 2470� (6.8)

Lateral geniculate nucleus 298� (4.1) 1010� (5.8) 1990� (9.2) 1430� (6.7)

Superior colliculus 208� (4.3) 608� (5.2) 503� (18) 864� (14)

[14C] acetate

Optic nerve ± 1890� (7.2) 1620� (41) 1660� (31)

Optic tract ± 1530� (2.7) 1150� (9.2) 2960� (12)

Lateral geniculate nucleus ± 540� (7.0) 660� (4.1) 1540� (5.1)

Superior colliculus ± 197� (4.1) 400� (14) 1970� (14)

Dried myelin samples� (from Fig. 3b) were extracted with ether : ethanol� (3 : 2) to solubilize the neutral/zwitterionic lipids� (, 80% of total lipids) and

aliquots were counted. Values shown were obtained by subtracting counts for uninjected� (control) tissues from those for injected tissues, thus

indicating the amount of intraneuronal precursor that was incorporated into myelin lipids. These values tended to increase over time, suggesting

transcellular supply from axon to myelin occurs over an extended period. Values in brackets indicate ratio of counts in the injected to corresponding

uninjected sample. Data represent DPM in total myelin obtained from each set of pooled tissues.

Table 3 Labeling of myelin lipids by NAA and acetate

Right optic nerve Left optic tract

Left lateral geniculate nucleus

and superior colliculus

Lipid NAA Acetate NAA Acetate NAA Acetate

8 days

Choline phosphoglycerides 20�.1 33�.8 27�.6 40�.2 24�.7 24�.6

Ethanolamine phosphoglycerides 7�.6 9�.6 10�.5 11�.8 8�.6 8�.6

Sphingomyelin 4�.6 0�.8 6�.0 0�.4 5�.7 1�.7

Cholesterol 24�.5 36�.4 17�.2 33�.8 19�.6 30�.1

Ceramide 2�.6 0 2�.2 0 3�.3 0

Cerebrosides 10�.2 11�.4 11�.9 7�.8 8�.7 9�.1

Diacylglycerol 3�.8 6�.1 6�.6 1�.8

Cholesterol esters 4�.3 {7�.9 1�.6 {6�.0 2�.2 0

LIPID X 19�.9 15�.4 18�.4 24�.0

33 days

Choline phosphoglycerides 26�.3 5�.3 28�.9 15�.5 24�.1 19�.3

Ethanolamine phosphoglycerides 24�.7 0 31�.5 2�.1 36�.4 3�.9

Sphingomyelin 2�.0 0 2�.0 2�.9 1�.3 1�.8

Cholesterol 28�.5 47�.2 20�.6 35�.7 20�.6 34�.5

Ceramide 0 0 0 0 0 1�.8

Cerebrosides 18�.5 8�.1 17�.0 13�.1 17�.5 12�.6

Diacylglycerol 0 3�.2 0 1�.1 0 3�.5

Cholesterol esters 0 0 0 0�.8 0 1�.1

LIPID X 0 36�.2 0 28�.8 0 20�.8

Neutral/zwitterionic lipids from myelin samples (Table 2) obtained at the indicated times after intraocular injection of [14C]NAA or [14C]acetate were

subjected to preparative TLC. Data indicate the percentage of recovered DPM (from TLC) in each lipid after applying most of the counts shown for

the corresponding samples in Table 3. Data are expressed as percentage of recovered DPM from TLC. Acetate-labeled diacylglycerol, cholesterol

esters, and lipid X at 8 days had too few counts for clear resolution, and were pooled.



days was reversed with signi®cantly more labeling by NAA

at 33 days. Ethanolamine phosphoglycerides were about

equally labeled by the 2 precursors at 8 days while at 33

days labeling by NAA was much more pronounced.

Cerebrosides showed a similar pattern with signi®cantly

more labeling by NAA at 33 days. Lipid X, of unknown

structure, showed the opposite pattern in being highly

labeled by NAA at 8 days but not at all by this precursor at

33 days; it was, however, well labeled at the latter time by

acetate. On TLC this lipid migrated slightly behind

cholesterol oleate (standard), suggesting it might be one

such compound with a more unsaturated fatty acid than the

cholesterol esters in myelin that migrate with the standard

on TLC. Sphingomyelin, a less abundant phospholipid, was

labeled proportionately by NAA, especially at 8 days, but

scarcely at all by acetate. Ceramide showed a similar pattern

while diacylglycerol and cholesterol esters appeared to be

labeled by NAA only at 8 days.

Detection of NAA in the optic system

To determine whether NAA taken up by retinal ganglion

cells remained as such or was hydrolyzed to acetate within

the neuron, ®ve rats were each given an intraocular injection

of [14C-acetyl]NAA followed by harvesting of tissues 5 days

later, as described above. The tissues of the injected

pathway were pooled: right ON, left OT, left LGN, and

left SC. Similarly, the tissues of the uninjected (contra-

lateral) pathway were pooled. After homogenizing in

methanol-water and centrifuging, aliquots of the supernants

were counted to give 10 120 DPM and 480 DPM for the

injected and control samples, respectively. To each super-

nantant was added chloroform and water to give two phases,

the upper phases of which were counted to give 2290 DPM

and 80 DPM for the injected and control samples, respec-

tively. Separation of soluble radiolabeled constituents was

carried out by HPLC, with clear resolution of NAA and

acetate (Fig. 4). Collected fractions corresponding to NAA

contained 1420 DPM, or 68% of the applied counts whereas

fractions corresponding to acetate contained nine DPM. This

experiment was repeated twice with similar results. We

found no absorption of acetate by the column matrix in trial

runs.

Discussion

A principal ®nding of this study is that puri®ed myelin

contains a high level of aspartoacylase (amidohydrolase II),

the enzyme required to release acetyl groups from NAA.

This enzyme was observed only in OLs among cultured

macroglial cells (Baslow et al. 1999), but has not yet been

examined in mature astroglia which were shown to have an

active uptake mechanism (Sager et al. 1999). Biochemical

studies in brain have shown aspartoacylase to predominate

in white matter (McIntosh and Cooper 1965; D'Adamo et al.

1973; Goldstein 1976; Kaul et al. 1991), suggesting a

possible role in myelin formation and/or maintenance. These

®ndings are consonant with developmental data in the rat

showing negligible activity at birth and maximal activity at

3 weeks, the peak of myelination (Goldstein 1976). Initially

considered a supernatant enzyme (D'Adamo et al. 1973,

1977), NAA amidohydrolase activity was also claimed to be

membrane-bound as well (Goldstein 1976), a result supported

by our ®ndings. The fact that signi®cant activity was found

in all subcellular fractions (Table 1) suggests that membrane

aminoacylase (amidohydrolase II) in brain is not con®ned to

myelin or myelin-producing glia. In view of its apparent

absence from neurons (Baslow 2000), the widespread

activity found in cerebral hemispheres suggests a glial

locus ± possibly satellite OLs or mature astrocytes. It is not

clear how much of the observed activity might be attributed

to amidohydrolase I, a different aminoacylase of broad

speci®city that accounts for the high activities observed in

extraneural tissues (D'Adamo et al. 1977; Miller and Kao

1989; Kaul et al. 1991; Mehta and Namboodiri 1995) and at

least some of the activity in brain (Miller and Kao 1989).

Amidohydrolase I from brain was shown to have approxi-

mately 7% the activity of aspartoacylase (amidohydrolase

II) toward NAA (Goldstein 1976). The delayed reactivity of

aspartoacylase in myelin, compared to that in cerebral

hemispheres (Fig. 2), also suggests enzyme heterogeneity.

Our results further suggest that acetyl groups liberated in

this manner within myelin, from NAA originating in the

neuron, are incorporated into myelin lipids. Such incorpora-

tion would logically be catalyzed by lipid synthesizing

enzymes in the myelin sheath, a large number of which are

known to be integral components of this membrane (for

review: Norton and Cammer 1984; Ledeen 1992). Occur-

rence of several lipid-synthesizing enzymes in myelin is

consistent with the myelin labeling pattern elicited by

neuronal [14C]NAA in the present study, while the latter also

suggests the presence of several more such enzymes yet to

be demonstrated.

These ®ndings imply inclusion of NAA among the pre-

cursors shown to undergo axon to myelin transfer with

subsequent incorporation into myelin lipids, a list that includes

choline (Droz et al. 1978, 1981), phosphate (Ledeen and

Haley 1983), acyl chains (Toews and Morell 1981;

Alberghina et al. 1982), and serine (Haley and Ledeen

1979). For NAA to be utilized in this manner, liberation of

acetyl groups is required and the present ®ndings show

myelin to have this capability. Radiolabeling of myelin

lipids by transaxonal ¯ow, following uptake of radiolabeled

precursor by the neuronal perikaryon, has been shown to

involve two simultaneous processes: (i) axon to myelin

transfer of intact lipid that was synthesized in the cell body

and axonally transported, and (ii) synthesis of new lipid

within myelin from axonally derived substrates. These

conclusions were based on studies in both the CNS (Haley



and Ledeen 1979; Alberghina et al. 1982, 1985; Ledeen and

Haley 1983) and PNS (Droz et al. 1978, 1981; Gould et al.

1982; Toews et al. 1988) which employed autoradiographic

localization and direct measurement of speci®c lipids from

whole tissue or isolated myelin. In the present study the fact

that myelin from all components of the optic system

acquired radiolabel following intraocular injection suggests

that NAA taken up by retinal ganglion cell perikarya

undergoes axonal transport and transaxonal movement.

Although the amount taken up by the retinal neurons may

be limited (Sager et al. 1999), it was suf®cient to indicate

these processes via the observed labeling. This interpretation

is supported by the observation that low molecular weight

substances, including those that are not metabolized or

incorporated into macromolecules, undergo rapid axonal

transport (Margolis and Grillo 1977; Gross and Kreutzberg

1978; Weiss 1982). While such transport is required in

the present model, it could have little or no physiological

signi®cance since mitochondria, the organelles producing

NAA, are themselves axonally transported (Forman 1987).

An alternative mechanism to explain labeling of myelin

lipids by neuronal NAA might be prior hydrolysis of

the latter with subsequent acetate utilization. Such hydro-

lysis might occur intraneuronally, as proposed (Mehta and

Namboodiri 1995), or in the eye. The former would entail

incorporation of liberated acetate into lipids within neuronal

perikarya followed by axonal ¯ow and axon-myelin transfer

of intact lipid as outlined above, and/or axonal transport of

free acetate followed by axon to myelin transfer and

utilization by myelin associated enzymes. One or both of

the latter processes likely explain the observed myelin

labeling by injected acetate, but appear unable to account for

labeling of myelin by NAA in view of the many substantial

differences in labeling pattern of individual lipids produced

by the two precursors over time (Table 3). An additional

result arguing against intraneuronal hydrolysis of NAA to

acetate is our observation that free NAA, but not acetate,

was detected within the optic system 5 days after intraocular

injection of [14C]NAA, supporting the concept of aspartoa-

cylase absence from neurons (Baslow 2000). Hydrolysis of

NAA in the eye seems unlikely to contribute signi®cantly in

view of the very low aspartoacylase activity we found in

whole eye, although a more careful look at individual

components of the eye seems warranted in view of the low

but detectable level of aspartoacylase reported in the ocular

¯uid of rainbow trout (Yamada et al. 1993). The extent of

incorporation of NAA acetyl into myelin lipids seems even

more signi®cant in light of the isotope dilution effect from

the in vivo pool of NAA, which is roughly 10 times that for

acetate (Knowles et al. 1974). Thus, even assuming that

uptake of some liberated acetate may occur in parallel with

NAA into retinal ganglion cells, its contribution to myelin

labeling should be relatively minor compared to that of

NAA. The results en toto suggest different processing

mechanisms and/or kinetics of incorporation for the two

precursors, contrary to what would be expected if NAA

labeling depended on prior hydrolysis to acetate.

These studies were undertaken to address the long

standing question of NAA function in the nervous system,

which has yet to be clari®ed despite growing use of this

substance as a diagnostic marker for neuronal loss or

dysfunction in a variety of neurodegenerative disorders

(Tsai and Coyle 1995; Gonen et al. 2000). The latter use is

facilitated by primary localization of NAA in neurons

(Simmons et al. 1991; Urenjak et al. 1993; Moffett and

Namboodiri 1995), consistent with localization of NAA

synthesis in neuronal mitochondria (Patel and Clark 1979).

It was shown that the acetyl group of NAA is ef®ciently

incorporated into brain fatty acids (D'Adamo and Yatsu

1966; Burri et al. 1991), and comparative labeling of

proteins and individual lipids led to the conclusion that

NAA and acetate enter by separate metabolic pathways

(Burri et al. 1991). The present study supports and extends

that idea by showing different kinetics of incorporation into

myelin lipids by these two precursors when originating in

the neuron. The above mentioned in vivo studies employing

intracerebral injections (D'Adamo and Yatsu 1966; Burri

et al. 1991), and another utilizing tissue slices (Mehta and

Namboodiri 1995), involved precursor presentation in an

extracellular mode, in contrast to the present experiments in

which the anatomical features of the optic system were

utilized to localize NAA in the neuron prior to lipid

incorporation. We believe this model is more physiologi-

cally relevant in view of NAA synthesis being con®ned to

neurons and also because intracerebrally injected NAA was

reported to be metabolized more rapidly and in a different

manner than endogenous NAA (Nadler and Cooper

1972).

A number of roles have been proposed for NAA (Tsai and

Coyle 1995) including that of neuronal osmoregulation

(Taylor et al. 1995; Baslow 1999, 2000). Conceivably NAA

could have more than one function, as suggested by its

presence in the lens of some species (Baslow and Yamada

1997). Its proposed role as an acetyl source for myelin lipid

synthesis may be considered in light of the ®nding that

Canavan disease, a condition of spongy degeneration associ-

ated with progressive loss of OLs and myelin, involves an

autosomal recessive defect in aspartoacylase (Matalon et al.

1988; Matalon and Michals-Matalon 1999). The observed

dysmyelination-demyelination could result from loss of

acetyl groups required for myelin formation and/or main-

tenance. This does not imply a selective, or even major role

for NAA during myelinogenesis, but a signi®cant contri-

bution is suggested in the recent report of a child lacking

NAA who showed aberrant myelination (Martin et al. 2001).

Creation of suitable animal models, such as the mouse

aspartoacylase knock-out (Matalon et al. 2000), would

likely help to elucidate this question.



Acknowledgements

This study was supported by Research Grant 2874-A3 and Pilot

Project PP0673 from the National Multiple Sclerosis Society.

We are happy to acknowledge the assistance of Mr Vamsi

Gullapali with intraocular injections.

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