IngeZo ¨ller,1MarionMeixner,1DieterHartmann,2HeinrichBu ¨ssow,2RainerMeyer,3VolkmarGieselmann,1and
are thought to play an important role in formation and function of myelin. To prove this hypothesis, we generated mice lacking a
in peripheral nerves. In contrast, nonhydroxylated galactosylceramide was increased in FA2H?/?mice. However, oligodendrocyte
sciatic nerves revealed no significant differences between FA2H?/?and wild-type mice. Moreover, myelin of FA2H?/?mice up to 5
and functionally normal myelin can be formed in the absence of 2-hydroxylated sphingolipids but that its long-term maintenance is
The myelin sheath is a highly specialized membrane, synthe-
sized by oligodendrocytes in the central and Schwann cells in
the peripheral nervous system (Baumann and Pham-Dinh,
2001). Synthesis of compacted myelin is essential for saltatory
nerve conduction. To fulfill its function, myelin has a unique
composition, in terms of both quality and quantity. Several
abundant myelin-specific proteins, such as myelin basic pro-
tein (MBP) and proteolipid protein (PLP), and a high lipid
content (?70%) are characteristic features of the myelin
sheath. Galactosylceramide (GalC) and its sulfated derivative
sulfatide belong to the most abundant lipids in myelin, mak-
ing up ?25% of myelin lipids (Norton and Cammer, 1984).
More than 50% of GalC and sulfatide are hydroxylated at the
1973). Thus, one can estimate that ?25% of the lipids in the
outer leaflet of the myelin membrane carry 2-hydroxylated
fatty acids (HFA). In neurons HFA–sphingolipids are not de-
tectable (Raghavan and Kanfer, 1972). The high HFA-content
in myelin suggests, but does not prove, an important role of
fatty acid 2-hydroxylation in the formation and/or function of
the myelin sheath or the metabolism of oligodendrocytes. So
far, however, no direct evidence for any specific function of
2-hydroxylated sphingolipids in the nervous system has been
Indications for an important role of 2-hydroxylated sphin-
golipids in the myelin membrane came from studies of UDP-
(Bosio et al., 1996; Coetzee et al., 1996) and overexpressing
mice (Fewou et al., 2005). CGT-deficient mice lack GalC and
sulfatide (Bosio et al., 1996, 1998; Coetzee et al., 1996), which
in part is compensated by the synthesis of HFA–glucosylcer-
amide. The total amount of HFA–sphingolipids, however, is
significantly reduced (Bosio et al., 1996; Coetzee et al., 1996).
Maturation of myelin is delayed and myelin structure is ab-
normal in CGT-deficient mice (for review, see Marcus and
Popko, 2002). Although cerebroside sulfotransferase (CST)-
and almost normal HFA–sphingolipid levels, displayed a sim-
ilar pathology, their phenotype is less severe when compared
with CGT-deficient mice (Honke et al., 2002). The more se-
University of Bonn. We thank Ivonne Becker, Birgit Rau, and M. Lindemann for expert technical assistance and
TheJournalofNeuroscience,September24,2008 • 28(39):9741–9754 • 9741
vere phenotype in CGT-deficient mice
might be attributable to the absence of
GalC, but we hypothesized that it could
also be caused by the substantially re-
duced HFA–sphingolipid levels.
Fatty acid 2-hydroxylase (FA2H) was
recently identified as an enzyme in-
sphingolipids (Alderson et al., 2004;
Eckhardt et al., 2005). To prove the hy-
pothesis that FA2H synthesizes HFA–
sphingolipids in myelinating glial cells
and to examine a possible role of HFA–
sphingolipids in myelin formation and
function, we generated mice deficient in
FA2H (FA2H?/?). FA2H?/?mice lack
HFA–GalC and HFA–sulfatide in brain
and peripheral nerves. Unexpectedly,
these mice developed structural and
functional normal myelin up to early
adulthood. Aged FA2H?/?mice, how-
ever, showed scattered axon and myelin
sheath degeneration in the spinal cord
and morphologically similar, but signif-
icantly more pronounced, lesions in pe-
ripheral nerves, indicating that FA2H is
sheath and axon maintenance.
Generation of FA2H-deficient mice. A target-
ing vector was designed to replace part of the
FA2H gene (149 bp of exon 3, intron 3, and
exon 4, and 402 bp of intron 4) by a lacZ/neo
cassette. The lacZ gene was inserted in frame
into exon 3, leading to a potential transcript
encoding a fusion protein of the cytochrome
b5 domain of FA2H and ?-galactosidase.
FA2H genomic fragments were amplified by
PCR from 129/Ola mouse HM-1 embryonic
stem (ES) cell (Magin et al., 1992) genomic
DNA. The 5? homology arm (4.3 kb of intron
2 and 30 bp of exon 3) was amplified by PCR
GG-3?), FH2 (5?-CAGCAGAGGTTTCTGCCAGTCC-3?), and Phu-
sion DNA polymerase (Finnzymes). The PCR product was cloned
into the SmaI site of pLRlacZpA/MCINeopA. The 3? homologous
region was a 1.8 kb PvuII fragment of intron 4, which was subcloned
into the blunted SpeI site of the targeting vector. A HSV–TK cassette
was isolated from the plasmid pHSV–TK by digestion with XhoI and
XbaI and 5? overhangs were filled in using Klenow enzyme. This
fragment was subcloned into the SalI site downstream of the 3? ho-
into HM-1 ES cells cultured on mitomycin C-treated feeder cells. After
48 h, the medium was supplemented with 350 ?g/ml G418 and 2 ?M
tion were expanded, and genomic DNA was screened for homologous
analysis. Therefore, genomic DNA was digested with BglII and analyzed
by Southern blot hybridization with a [32P]-labeled 943 bp 3? external
probe. The probe was amplified by PCR using primers 5?-CCCGGA-
GCACCTGGAGACCTACTT-3? and 5?-CTGGCTGGCTCATGCAT-
was injected into C57BL/6N (Charles River) blastocysts to obtain chi-
meric animals. Chimeric male mice with a high contribution of agouti
coat color were bred to C57BL/6N females, and germ-line transmission
of the disrupted FA2H allele was confirmed by Southern blotting of
genomic tail DNA of agouti F1mice. Subsequent genotyping was per-
formed byPCR using the
TCGCCTTCTATC-3?, 5?-GTGCTGTACCTCAGCTGGTC-3?, and 5?-
GCTCTTCTTCAAGAGCCATCC-3?, which amplified a 1045 bp frag-
ment from the wild-type and a 685 bp fragment from the targeted allele.
Northern blot analysis. Total RNA of murine brain was isolated using
Trizol (Invitrogen) as described by the manufacturer. Total RNA (20
?g/lane) was separated in 1 M formaldehyde/1% agarose gels and trans-
ferred onto Hybond-N?nylon membranes (GE Healthcare). A 568 bp
fragment of the FA2H cDNA (corresponding to exons 3 and 4) was used
to synthesize a [32P]-labeled probe by random priming using
[?-32P]dCTP and Megaprime DNA labeling kit (GE Healthcare), fol-
lowing the instructions of the manufacturer. A ?-actin probe (Strat-
targeting vector, and targeted allele. Restriction sites for BglII (B) are indicated. B, Southern blot analysis of FA2H?/?ES cell
Generation of FA2H-deficient mice. A, Schematic representation of the FA2H wild-type allele, structure of the
9742 • J.Neurosci.,September24,2008 • 28(39):9741–9754Zo ¨lleretal.•FA2H-DeficientMice
dendrocytes on neuronal function (Kaga et al., 2006). Simi-
larly, impaired bidirectional signaling between neurons and
oligodendrocytes could affect neuronal activity, leading to re-
duced activity of FA2H?/?mice in the open-field test, despite
structural and functional normal myelin. Recent studies with
membrane monolayers of a composition similar to the myelin
outer leaflet showed that the size and number of membrane
domains formed by GalC were significantly affected by
2-hydroxylation (Windschiegl and Steinem, 2006). This could
potentially influence signaling between myelinating cells and
neurons that depend on “lipid rafts.” The fibrillar material,
which appears to accumulate in the myelin sheaths in the
spinal cord of 18-month-old FA2H?/?mice, suggests that the
transport and/or degradation of myelin components might be
affected. This could, for example, affect the generation and
release of myelin exosomes (Kra ¨mer-Albers et al., 2007; Tra-
jkovic et al., 2008) and thereby the communication between
myelinating cells and neurons. Because of the slow turnover of
myelin components in adult mice, axonal degeneration and
appearance of the fibrillar material might be the end point of a
lengthy process that might already affect axonal function be-
fore morphological changes are apparent, leading to the be-
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