The contribution of mutations in amyloid precursor protein (APP) and presenilin (PSEN) to familial Alzheimer’s disease (AD) is well
may be posttranscriptionally regulated. Therefore, we investigated the role of miRNAs in the regulation of SPT and amyloid ? (A?)
generation. We show that SPT is upregulated in a subgroup of sporadic AD patient brains. This is further confirmed in mouse model
studies of risk factors associated with AD. We identified that the loss of miR-137, -181c, -9, and 29a/b-1 increases SPT and in turn A?
It is well established that amyloid precursor protein (A?) accu-
mechanisms contributing to A? accumulation in sporadic AD
are less well understood. Research thus far consistently demon-
strates that ceramide, a sphingolipid, is increased in AD patients
(Cutler et al., 2004; He et al., 2010) and may contribute to the
disease pathogenesis. Membrane ceramides not only are the ma-
jor component of lipid rafts, but they also contribute to AD pa-
thology by facilitating the mislocation of BACE1 and ?-secretase
to lipid rafts, and thereby promoting amyloid ? (A?) formation
exogenous addition of ceramide increased A? production (Pug-
lielli et al., 2003; Patil et al., 2007). Numerous studies suggest a
connection between ceramides and A? and indicate that in-
creased ceramide levels may be an important risk factor for spo-
radic AD (Puglielli et al., 2003; Mattson et al., 2005).
SPT is the first rate-limiting enzyme in the de novo cer-
amide synthesis pathway (Hannun and Obeid, 2008). Activa-
tion of SPT elevates ceramide levels (Perry et al., 2000) and
inhibition of SPT decreases ceramide levels (Hojjati et al., 2005;
Patil et al., 2007) and neuronal cell death by A? (Cutler et al.,
2004), supporting SPT as an important regulator of ceramide.
SPT is a heterodimer composed of serine palmitoyltransferase
long chain 1 (SPTLC1) and either serine palmitoyltransferase
long chain 2 (SPTLC2) or serine palmitoyltransferase long
chain 3 (SPTLC3) (Rotthier et al., 2010). In the brain, SPTLC3
is lowly expressed, while SPTLC1 and SPTLC2 are the major
subunits (Hornemann et al., 2006). However, the regulation
of these subunits and in turn SPT is not well understood. Cell
culture studies demonstrate that SPT activity increases in re-
sponse to various stimuli (i.e., etoposide or resveratrol), but
without concomitant changes in SPTLC1 and SPTLC2 mRNA
levels (Perry et al., 2000; Scarlatti et al., 2003), which have led
researchers to hypothesize that SPT may be posttranscription-
Gene expression may be posttranscriptionally regulated
degradation of the mRNAs (He and Hannon, 2004). miRNAs
ticity, and memory formation (Sempere et al., 2004; Mehler and
files, several miRNAs are differentially expressed in AD patients
(Lukiw, 2007; Cogswell et al., 2008; He ´bert et al., 2008; Wang et
al., 2008), and several have been reported to be specific or en-
riched in the brain (Sempere et al., 2004). Indeed, a recent study
This work was supported in part by the National Institute of Health (R01GM079688, R01GM089866, and
Correspondence should be addressed to Christina Chan, 2527 EB, Department of Chemical Engineering and
14820 • TheJournalofNeuroscience,October12,2011 • 31(41):14820–14830
(Schonrock et al., 2010), suggesting the involvement of miRNA
diated the posttranscriptional regulation of SPT with respect to
Patient information. The AD (n ? 7) and control (n ? 7) neocortical
brain samples were from the University of Kentucky (UK) Alzheimer’s
GroupRef#AgeSex Braakstage PMIMMSEscore FrontNPcount FrontNFTcountApoEallele
GeekiyanageandChan•miR-137/181cRegulatesSPTandInTurnA?inSporadicAD J.Neurosci.,October12,2011 • 31(41):14820–14830 • 14821
SPTLC1andSPTLC2aremiRNAtargetedgenes.A,HumanSPTLC13?UTRluciferaseand Renilla luciferase constructs were transfected into wild-type (Figure legend continues.)
14822 • J.Neurosci.,October12,2011 • 31(41):14820–14830 GeekiyanageandChan•miR-137/181cRegulatesSPTandInTurnA?inSporadicAD
clinically diagnosed by neurologists, neuropa-
thologists, neuropsychologists, and other staff
been obtained in ?4 h postmortem interval.
All individuals were between the ages of 88–99
years. The reference number, gender, Braak
stage, Mini Mental State Examination scores,
frontal neuritic plaque numbers, neurofibril-
individuals are listed in Table 1. The cause of
death of these individuals is multifactorial or
unclear with pneumonia being the classical
cause of death. The above information was
provided by the UK ADC.
Animals. Wild-type male C57BL/6 mice pur-
chased from The Jackson Laboratory were used
hybrid background, C3H/He (Charles River) ?
C57BL/6, were used in the diet (all male)- and
gender-specific (7 males, 7 females) studies. All
procedures conducted were approved by the In-
stitutional Animal Care and Use Committee at
Primary cell culture. Primary astrocytes were
isolated and cultured from ?24-h-old wild-
type Sprague Dawley rat pups and 3-week-old
TgCRND8 (Centre for Research in Neurode-
generative Diseases) transgenic mouse pups,
containing the APP 695-cDNA with both the
Indiana and the Swedish mutations, in a hy-
brid C3H/He ? C57BL/6 background (Ch-
ishti et al., 2001) as described previously
(Patil et al., 2007). The TgCRND8 mice ex-
press the APP transgene at levels fivefold
higher than the endogenous APP under the
control of the Syrian hamster prion pro-
moter (Chishti et al., 2001).
Protein extraction and Western blot analy-
sis. Cells, mouse brain cortices, and human
brain neocortices (homogenized) were lysed
in buffer: 1% (v/v) Triton, 0.1% (w/v) SDS,
0.5% (w/v) deoxycholate, 20 mM Tris, pH
7.4, 150 mM NaCl, 100 mM NaF, 1 mM
Na3VO4, 1 mM EDTA, 1 mM EGTA, 1 mM
phenylmethylsulfonyl fluoride, and protease
inhibitor cocktail (all chemicals from Sigma).
The lysis was spun at 10,000 rpm for 10 min
and then the total protein concentration of the
supernatant was measured by Bradford assays
and was mixed with reducing loading buffer
and heated at 94°C for 5 min. Immunoblot
conducted by normalizing to GAPDH or
?-actin. Western blots were quantified using
Quantity One (Bio-Rad) version 4.5.
(Figure legend continued.) rat primary astrocytes with the indicated miRNA oligonucleotides at a final concentration of 100 nM. Normalized (to Renilla) sensor luciferase activity is shown as a
transfected into wild-type rat primary astrocytes with the indicated miRNA oligonucleotides at a final concentration of 100 nM. Normalized (to Renilla) sensor luciferase activity is shown as a
with LCB1) and SPTLC2 in wild-type primary astrocytes treated with 100 nM (final concentration) of miRNA (oligonucleotides) or anti-miRs (antisense), with scrambled siRNA as controls. E, F,
GeekiyanageandChan•miR-137/181cRegulatesSPTandInTurnA?inSporadicADJ.Neurosci.,October12,2011 • 31(41):14820–14830 • 14823
ELISA. Protein was extracted from human autopsy brain samples and
ing to the manufacturer’s instruction. The A?42levels were calculated by
Transfection, plasmid, and luciferase assays. Primary wild-type and
transgenic astrocytes were plated in 12-well plates and transfected for
24–72 h with 100–150 nM Syn-miRNA miScript miRNA mimic or
anti-miR-RNA miScript miRNA inhibitor (Qiagen), 500 ng to 2 ?g of
human SPTLC1 cDNA, or 1.5–2 ?g of luciferasevectorconstructusing
Lipofectamine RNAi/MAX or Lipofectamine 2000 following the manu-
facturer’s instructions. The SPTLC1 cDNA plasmid and SPTLC1 and
SPTLC2 luciferase 3?UTR expression clones, containing the luciferase
reporter gene and Renilla tracking gene and driven by the SV40 pro-
moter, were purchased from Genecopoeia. The luciferase assay was con-
ducted with a dual luciferase assay kit (Luc-Pair miR Luciferase Assay
Kit) (Genecopoeia). The luciferase expression levels were normalized to
Renilla expression levels.
Quantitative RT-PCR. Total mRNA was extracted using RNeasy Plus
spectrophotometer. RNA quality control was performed by assessing OD
260/280 ratio. RNA quality control of the control and AD human brain
PCR products were run on agarose gels. Quantitative RT-PCR (qRT-PCR)
was conducted using iQSYBR Green Supermix (Bio-Rad) and MyiQ real-
time PCR detection system following reverse transcription using iScript
cDNA Synthesis Kit according to the manufacturer’s instructions. Primers
include human SPTLC1: 5?-TGGAAGAGAGCACTGGGTCT-3? and 5?-
GCTACCTCCTTGATGGTGGA-3?; human SPTLC2: 5?-GAGACGCCT-
GAAAGAGATGG-3? and 5?-TGGTATGAGCTGCTGACAGG-3?; human
GAPDH: 5?-GAGTCAACGGATTTGGTCGT-3? and 5?-TTGATTTTG-
GAGGGATCTCG-3?; mouse Sptlc1: 5?-AGTGGTGGGAGAGTC-
CCTTT-3? and 5?-CAGTGACCACAACCCTGATG-3?; mouse Sptlc2: 5?-
CCTGTCAGCAGCTCATACCA-3? and 5?-CACACTGTCCTGGGA-
GGAAT-3?; mouse Gapdh: 5?-AACTTTGGCATTGTGGAAGG-3? and 5?-
ACACATTGGGGGTAGGAACA-3?; rat Sptlc1: 5?-ACCTGGAGCGACT-
GCTAAAA-3? and 5?-ATCCCATAGTGCTCGGTGAC-3?; rat Sptlc2: 5?-
TTGAGACTCACTGGCCCTCT-3? and 5?-GGCCAGGAGGAGTC-
ACATAA-3?; rat Gapdh: 5?-AGACAGCCGCATCTTCTTGT-3? and 5?-
CTTGCCGTGGGTAGAGTCAT-3?. Relative human, mouse, and rat
Total miRNAs were extracted using miRNeasy Mini Kit (Qiagen) and
RNeasy MinElute Cleanup Kit (Qiagen) total RNA was quantified using
ND-1000 NanoDrop spectrophotometer. RNA quality control was per-
formed by assessing OD 260/280 ratio. In addition the PCR products were
run on agarose gels. qRT-PCR was conducted using miScript SYBR Green
PCR Kit (Qiagen) and MyiQ real time PCR detection system following re-
verse transcription using miScript Reverse Transcription Kit (Qiagen) ac-
cording to the manufacturer’s instructions. All miRNA primers were
purchased from Qiagen, and the relative expressions were calculated using
man brain neocortices and mouse brain cortices according to Bligh and
Dyer (1959). Tandem mass spectrometry (MS/MS) was performed using
Quattro Premier XE (Waters), Acquity ultra performance liquid chroma-
amide standards were purchased from Matreya and Avanti, Polar Lipids.
Antibodies. The following antibodies were used: LCB1 (BD Transduc-
tion Laboratories), SPTLC1 (Proteintech), SPTLC2 (Abcam), GAPDH
Technology), and ?-amyloid-4G8 clone (Covance).
Statistical analysis. Statistical significances were determined by using
two-tailed t tests andSpearmancorrelation(two-tailedtdistributiontest).
The levels of ceramide and SPT protein expression were mea-
sured in the frontal brain cortices of seven sporadic AD patients
and seven controls (see Table 1 for information on the patients).
3 for each age group). Gapdh was used as the loading control. B, Quantification of SPTLC1 expression from Western blots normalized to GAPDH and the average expressions represented as
14824 • J.Neurosci.,October12,2011 • 31(41):14820–14830GeekiyanageandChan•miR-137/181cRegulatesSPTandInTurnA?inSporadicAD
Of the vast number of distinct ceramide species (?50 species),
d18:1;18:0 and d18:1;16:0 are reported to be the major sphingo-
lipid species in rat neurons (Valsecchi et al., 2007) and human
brain (Ladisch et al., 1994). Consistent with previous reports
(Cutler et al., 2004; He et al., 2010), ceramide levels, d18:1;16:0
(p ? 0.037, Student’s t test) and d18:1;18:0 (p ? 0.033), were
significantly increased in this subgroup of AD patients (Fig. 1A).
Several reports have shown that the sphingomyelin levels either
increased (Pettegrew et al., 2001; Bandaru et al., 2009) or re-
mained unchanged (Han et al., 2002) in AD brains. In contrast,
other researchers have shown that the sphingomyelin levels de-
creased (Cutler et al., 2004; He et al., 2010) in AD brain. We
found that the sphingomyelin d18:1;16:0 levels increased (p ?
0.045), while the d18:1;18:0 levels remained unchanged, in the
subgroup of AD patient brain cortices studied (Fig. 1B). This
suggests that the increased ceramide levels in these patients are
from the de novo synthesis pathway. Accordingly, SPTLC1 (p ?
0.004) and SPTLC2 (p ? 0.007) protein expression were signifi-
cantly elevated in the autopsy AD brain samples (Fig. 1C,D).
inantly unchanged in the AD samples as measured by qRT-PCR
Previously, our group found that palmitate (a saturated fatty
acid) increased de novo ceramide synthesis in astrocytes through
SPT (Patil et al., 2007). Thus, we treated
wild-type primary rat astrocytes with
palmitate for 24 h and found that the
SPTLC1 (p ? 0.032) and SPTLC2 (p ?
0.015) protein levels (Fig. 1F) increased
without a concomitant change in their
mRNA levels (Fig. 1G), which is consis-
tent with previous reports (Perry et al.,
2000; Scarlatti et al., 2003). Overall, these
results support that increased SPTLC1
and SPTLC2 expressions may be post-
transcriptionally regulated in a subgroup
of sporadic AD patients and in primary
astrocytes cultured with palmitate. Thus,
we proceeded to further elucidate the po-
tential regulation of SPT by miRNAs.
2005), Pictar (Krek et al., 2005), and mi-
Randa (Betel et al., 2008) were used to se-
lect potential miRNAs that bind the
human 3?UTR of SPTLC1 or SPTLC2
with strongly conserved (in mammals)
target sites. Likely miRNA candidates
were filtered according to the following
criteria: they must be (1) predicted by at
least two algorithms and (2) downregu-
lated in AD patients or enriched in the
more algorithms to bind the 3?UTR of
SPTLC1, miR-15a and miR-181c (He ´bert
et al., 2008) are reported to be downregu-
lated in sporadic AD patients, while miR-
137 and miR-124 (Sempere et al., 2004)
are reported to be enriched in the brain.
Of the miRNAs predicted by two or more
algorithms to bind the 3?UTR of SPTLC2, miR-29a, miR-29b-1,
and miR-9 are reported to be downregulated in sporadic AD
Two luciferase reporter constructs were generated containing
the 3?UTR of human SPTLC1 or SPTLC2. The miRNAs (sense)
were cotransfected with the constructs and the luciferase expres-
137 (p ? 0.000016, Student’s t test) and miR-181c (p ? 0.0003)
significantly decreased the luciferase expression of the construct
(Fig. 2A). The luciferase expression of the construct containing
the 3?UTR of SPTLC2 decreased significantly upon cotransfec-
or miR-29b-1 (p ? 0.007) (Fig. 2B). These results were con-
firmed by transfecting primary rat astrocytes with either the
sense-miRs or anti-miRs (antisense) of their respective miRNAs
following analysis of the endogenous miRNA expression lev-
els in primary rat astrocytes (Fig. 2I). miR-137 and miR-181c
cellular ceramide, levels while anti-miR-137 and anti-miR-181c
significantly enhanced the endogenous SPTLC1 (Fig. 2C,E) and
cellular ceramide (Fig. 2G) levels upon transient transfection.
Similarly, miR-9, miR-29a, and miR-29b-1 significantly sup-
pressed the endogenous SPTLC2 and cellular ceramide levels,
tions are shown as a percentage of the average control chow diet-fed mice (n ? 3). The samples were normalized to internal
expressions. F, G, The expression levels of miR-137, -181c, -9 (*p ? 6.0 ? 10?5), -29a, and -29b-1 in palmitate-treated
GeekiyanageandChan•miR-137/181cRegulatesSPTandInTurnA?inSporadicADJ.Neurosci.,October12,2011 • 31(41):14820–14830 • 14825
while anti-miR-9 and anti-miRs-29a/b-1
significantly enhanced the endogenous
SPTLC2 (Fig. 2D,F) and cellular cer-
amide (Fig. 2H) levels upon transient
(p ? 0.045), miR-29a (p ? 0.03), and miR-
29b-1 (p ? 0.03) (Fig. 3A–C) and miR-15
(p ? 0.048) and miR-124 (p ? 0.002) (data
Statistically significant negative corre-
lations were observed between SPTLC1
and miR-137 (r ? ?0.807, p ? 0.0005,
Spearman’s correlation) (Fig. 3D), miR-
181c (r ? ?0.569, p ? 0.034) (Fig. 3E),
and miR-15a (r ? ?0.59, p ? 0.026) and
AD patients. Significant negative correla-
and miR-9 (r ? ?0.675, p ? 0.008) (Fig.
3F), miR-29a (r ? ?0.603, p ? 0.023)
(Fig. 3G), and miR-29b-1 (r ? ?0.714, p ? 0.004) (Fig. 3H) in
the subgroup of AD patients. This negative correlation be-
tween the subunits of SPT and their corresponding miRNA
expressions, coupled with the transient transfection results,
suggests the possibility that changes in miR-137 or miR-181c,
to the overall protein expressions of SPTLC1 and SPTLC2,
respectively, in AD.
Given that AD is an age-related disorder (Bachman et al., 1992),
4A,C), and their corresponding miRNAs (Fig. 4D–F) with de-
velopment. The protein, mRNA, and miRNA expressions were
(P0) up to 18 months. This provided an independent confir-
respective miRNAs under nonpathological settings. During de-
velopment, the expression levels of miR-137 and miR-181c (Fig.
4D) and miR-124 (data not shown) increased, while SPTLC1
previous reports (He ´bert et al., 2008), expression levels of miR-
29a and miR-29b-1 were found to increase (Fig. 4E) with devel-
opment, while the expression levels of SPTLC2 decreased with
age (Fig. 4A,C). The Sptlc1 and Sptlc2 mRNA expression levels
remained unchanged (stable) over the period analyzed (Fig. 4F),
137, miR-181c, miR-29a, and miR-29b-1 are developmentally
regulated, with the highest expressions in adult mice. Concomi-
tantly, protein analyses indicate that SPTLC1 and SPTLC2 have
lower expression levels in adult mice, thereby further supporting
Increasing evidence in animal models suggest that a high-fat diet
aggravates the A? burden and thereby the AD pathology (Julien
et al., 2010). Indeed, high-fat/high-cholesterol diets have been
found to increase plasma ceramide levels in rodents (Shah et al.,
2008). Moreover, prior research in our laboratory demonstrated
that palmitate, a saturated fatty acid, increases ceramide levels
and induces AD-like pathology in primary neuronal cell culture
mediated by astrocytes (Patil et al., 2007). Therefore, the expres-
sion levels of ceramide, SPTLC1, and SPTLC2 and their corre-
sponding miRNAs were measured in brain cortices of wild-type
male mice fed a 60% kcal high-fat diet for a period of 5 months
(starting at 4 months of age). While ceramide (Fig. 5A) and
SPTLC1 and SPTLC2 (Fig. 5B) expression levels increased in
mice fed a high-fat diet, Sptlc1 and Sptlc2 mRNA levels remained
unchanged (Fig. 5C), supporting our hypothesis that SPTLC1/2
137 (p ? 0.005, Student’s t test) (Fig. 5D), miR-181c (p ? 0.026)
0.0027) (Fig. 5E) expression levels were downregulated in mice
and miR-9 (p ? 1.9 ? 10?5) (Fig. 5G) expression levels were
downregulated in wild-type primary rat astrocytes treated with
palmitate, whereas SPTLC1/2 protein expression levels were up-
regulated (Fig. 1F). However, miR-29a and miR-29b-1 expres-
sions did not change with either a high-fat diet (in vivo) or
palmitate treatment (in vitro).
Evidence suggests that AD pathology may be more prevalent in
uated the SPTLC1, SPTLC2, and miRNA expression levels in the
brain cortices of female and male wild-type mice (9 months of
expressions. E, F, The expression levels of miR-137 and -181c (*p ? 0.04) (E) and miR-29a/b-1 (*p ? 0.04) and -9 (F) were
Gender-specific differential regulation of miR-137, -181c, -29a, 29b-1, SPTLC1, and SPTLC2. A, Ceramide species
14826 • J.Neurosci.,October12,2011 • 31(41):14820–14830GeekiyanageandChan•miR-137/181cRegulatesSPTandInTurnA?inSporadicAD
GeekiyanageandChan•miR-137/181cRegulatesSPTandInTurnA?inSporadicADJ.Neurosci.,October12,2011 • 31(41):14820–14830 • 14827
age). Ceramide species, d18:1;18:0 (p ? 0.0042, Student’s t test)
and d18:1;16:0 (p ? 0.0045, Student’s t test) (Fig. 6A), and
expression levels were higher in females as compared to males,
while the Sptlc1 and Sptlc2 mRNA levels remained unchanged
(Fig. 6D), further indicating that SPTLC1/2 may be posttran-
scriptionally regulated by miRNAs. Concomitantly, miR-137
(p ? 0.011) and miR-181c (p ? 0.038) (Fig. 6E), miR-124 (data
not shown), and miR-29a (p ? 0.031) and miR-29b-1 (p ?
0.004) (Fig. 6F), but not miR-9, expression levels are downregu-
lated in female mice, while SPTLC1/2 protein expression levels
SPTLC1/2 and their target miRNAs.
A casual relationship between miR-29a/b-1, BACE1 activity, and
A? has been established by He ´bert et al. (2008). Therefore, we
A?, mediated by SPTLC1. Statistically significant positive corre-
lations were observed between SPTLC1 (Western blot) and
A?(42)protein levels (from ELISA) (r ? 0.76, p ? 0.002, Spear-
man’s correlation) (Fig. 7A), and between SPTLC2 (Western
blot) and A?(42)protein levels (r ? 0.67, p ? 0.007) (Fig. 7B) in
the control and the subgroup of AD patients. Additionally, sta-
tistically significant negative correlations were observed between
A?(42)and miR-137 (r ? ?0.75, p ? 0.003), miR-181c (r ?
?0.57, p ? 0.037), miR-9 (r ? ?0.7, p ? 0.007), miR-29a (r ?
?0.64, p ? 0.01), and miR-29b-1 (r ? ?0.569, p ? 0.037) in the
control and the subgroup of AD patients. Furthermore, we per-
cytes derived from transgenic mice expressing the human APP
Swedish mutation. In these cells, overexpressing miR-137 or
miR-181c downregulated the endogenous expression levels of
SPTLC1 (p ? 0.001) and A? (p ? 0.01) (Fig. 7D,F). The func-
tional affects were reversed upon transfection with the comple-
mentary anti-miRs-137 and -181c (Fig. 7C,E). Thus, the loss of
the suppressing activity of miR-137 and miR-181c led to in-
creased A? production in cell culture. Additionally, transient
overexpression of SPTLC1 (p ? 0.033) restored/increased A?
expression levels in cells cotransfected with miR-137/-181c (p ?
0.005) (Fig. 7D,F). To assess the direct role of miR-137, miR-
181c, and thus SPTLC1 on A? expression, “target protectors”
were designed against the targeted site on SPTLC1 for miR-137
and miR-181c. Primary astrocytes expressing the human APP
Swedish mutation were transiently transfected with miR-137 or
miR-181c along with their respective “target protectors” (Fig.
7G–J). Both SPTLC1 and A? expression levels decreased signifi-
cantly upon transfection with miR-137 (Fig. 7G,I) or miR-181c
(Fig. 7H,J) along with a negative target protector. SPTLC1 and
A? expression levels remained unchanged upon transfection
with miR-137 (Fig. 7G,I) or miR-181c (Fig. 7H,J) along with
their respective target protectors. Additionally, the transfection
of anti-miR-137 (Fig. 7G,I) or anti-miR-181c (Fig. 7H,J) signif-
icantly increased A? and SPTLC1 expression levels.
We found that a subgroup of sporadic AD patients exhibited
for the treatment of AD. Increased ceramide levels have been
associated with increased neutral SMase (N-SMase) levels in
AD where A? induced N-SMase production (Jana and Pahan,
2010). In this study, we observed that the A? levels increased
with overexpression of SPTLC1. Therefore, ceramide rise
through the de novo synthesis pathway upregulates A? levels,
and the A? in turn may induce N-SMase activity to reinforce
the production of ceramide, and thereby propagate a contin-
ual cycle of ceramide-A? generation.
increased levels of ceramides with concomitant increase in
tices. This coupled with our animal and cell culture studies sug-
gests that SPT may be a novel target for the treatment of AD.
data suggests that SPTLC1/2 may be posttranscriptionally reg-
ulated through miRNAs. Along these lines, we found negative
correlations/relationships between the expression levels of
miR-137/-181c and SPTLC1, and between miR-9/-29a/b-1 and
SPTLC2 protein expressions, in sporadic AD brains, and devel-
oping diet- and gender-specific mouse brains.
transcription factor NF?B have been identified in the promoter re-
tissue and stimuli specific as the tested experimental conditions did
not impact brain SPT activity (Memon et al., 2001). Further, a sig-
nificant increase in SPTLC2 protein levels was observed in human
2009). Interestingly, miR-29b is downregulated in glioblastomas
(Cortez et al., 2010), suggesting that miR-29b could be involved in
elevating the SPTLC2 protein levels. In this present study, we ob-
was also observed with development and in both genders of mice.
cells suggest that changes in the miRNA levels, miR-137, -181c, -9,
and 29a/b-1, could contribute to altered SPTLC1 and SPTLC2 ex-
Of the miRNAs identified to regulate SPT expression, in-
creased expression levels of miR-137 has been shown to induce
neurogenesis in hippocampus (Szulwach et al., 2010) while
miR-9 is involved in neurogenesis and differentiation (Coolen
and Cogswell et al. (2008) observed downregulated miR-9 levels
in AD patient brains, whereas Lukiw (2007) detected an upregu-
lation in AD. In contrast, miR-29a/b-1 was observed to be con-
14828 • J.Neurosci.,October12,2011 • 31(41):14820–14830 GeekiyanageandChan•miR-137/181cRegulatesSPTandInTurnA?inSporadicAD
sistently downregulated by He ´bert et al. (2008), Wang et al.
al. (2008) also detected downregulated miR-181c expression lev-
cortex of the subgroup of AD patients in this study. In addition,
we observed that miR-137 was also downregulated in the frontal
cortex of these seven AD patients. In support of this, chromo-
somes 1p13.3–q31.1 region, which includes the map location of
miR-137, chromosome 1p21, has been linked to late-onset AD
19p13.13, has also been linked to late-onset AD (Butler et al.,
2009). We observed that the suppression of SPTLC1 by miR-137
and miR-181c reduced A? expression levels in a target-specific
137 and miR-181c increased A? expression levels. This, coupled
with the fact that overexpression of BACE1 did not increase A?
leaves open a possible role of ceramide, mediated by SPT, in
transporting BACE1 and ?-secretase to the lipid rafts for amy-
loidogenic processing of APP. Inactive BACE1 and ?-secretase
resides outside of the lipid rafts under nonpathological settings,
allowing nonamyloidogenic processing of APP, while under dis-
ease state the ceramides facilitate the trafficking of these patho-
genic secretases to lipid rafts, where they become active to
produce A? (Cordy et al., 2003; Vetrivel et al., 2005; Ebina et al.,
2009). Ceramide also increases A? production by stabilizing
BACE1 (Puglielli et al., 2003; Costantini et al., 2007; Patil et al.,
2007) through increased acetylation (Ko and Puglielli, 2009).
miR-9 and miR-29a/b previously have been identified as poten-
tial suppressors of BACE1 and thus associated with sporadic AD
(He ´bert et al., 2008). Given that SPT is also regulated by miR-9
and miR-29a/b-1, it further strengthens the contribution of SPT
to the etiology of sporadic AD.
Of the miRNAs identified to regulate SPTLC1 expression, miR-
epigenetically and transcriptionally by MeCP2 and Sox2 through
direct binding to the 5? regulatory region (Szulwach et al., 2010).
explanation for the reduced miR-137 levels in mice fed a high-fat
positively regulated by Akt1 at the transcriptional level (Androuli-
(Tremblay et al., 2001), providing a possible mechanism for our
sion, miR-9 is negatively regulated by RE1-silencing transcription
factor (REST) but positively regulated by cAMP-response element
binding protein (CREB) (Laneve et al., 2010). High fat has been
al., 2005), providing a possible explanation for the reduced miR-9
is aging-associated and contributes to AD (Niwa et al., 2008).
lated expressions of miR-137 (Langevin et al., 2010) and miR-29
family (Koturbash et al., 2011) in females, indicative of differen-
supporting our observations in the mice study. Furthermore,
expression of mouse hepatic miRNAs in offspring, including
downregulation of miR-29a (Zhang et al., 2009). We observed
a reduction of miR-137, -181, and -29a/b-1 expressions in
females compared to males and a downregulation of miR-137,
-181c, and -9 expression levels with high dietary fat intake.
This raises an intriguing possibility that women consuming
high-fat diets may be at higher risk for SPT dysregulation and
thus AD. Therefore, our results lend support to epidemiolog-
ical factors such as age, gender, and diet epigenetically regulating
and 29a/b-1, resulting in reduced suppression of SPT expression,
and thereby increasing the ceramide levels and A? generation
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