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Elevation of 1215 lipoxygenase products in AD and mild cognitive impairment.

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BRIEF COMMUNICATIONS
Elevation of 12/15
Lipoxygenase Products
in AD and Mild
Cognitive Impairment
Yuemang Yao, BSc,1 Christopher M. Clark, MD,2,3
John Q. Trojanowski, MD, PhD,4 – 6
Virginia M.-Y. Lee, PhD,4,5 and Domenico Praticò, MD1
The 12/15 lipoxygenase (12/15LOX) enzyme is increased
in pathologically affected frontal and temporal regions of
Alzheimer’s disease (AD) brains compared with controls.
Herein, we measured 12(S)-HETE and 15(S)-HETE levels, products of 12/15LOX, in cerebrospinal fluid (CSF)
of normal individuals, subjects with mild cognitive impairment (MCI) and AD. Compared with controls, there
was a significant increase of both metabolites in CSF
from AD and MCI, which correlated with lipid peroxidation and tau protein levels. These results suggest that
the activation of this enzyme occurs early in the course of
AD, before the onset of overt dementia, thereby implicating 12/15LOX-mediated lipid peroxidation in the pathogenesis of AD.
Ann Neurol 2005;58:623– 626
Alzheimer’s disease (AD) is the most common form of
dementia in the elderly. Recent pathogenetic and biochemical studies have consistently suggested a role for
oxidative stress and inflammation in AD pathogenesis.1,2 Aging is one of the strongest risk factors for developing AD, and oxygen-mediated events are considered possible mechanisms responsible for the increased
neuronal vulnerability in aging. Lipoxygenases (LOXs)
are a family of lipid-peroxidizing enzymes that insert
molecular oxygen into free as well as esterified polyunsaturated fatty acids.3 Among them, the 12/15LOX has
two main functions: formation of biologically mediators/
signal molecules and modification of membrane structure (peroxidation reaction). When the substrate of this
enzyme is arachidonic acid, the stereoselective metabolic
products formed are 12(S)-hydroxyeicosatetraenoic
(HETE) acid and 15(S)-HETE in various ratios.4 This
enzyme has been described in neurons and also in some
glial cells throughout the cerebrum, basal ganglia, and
hippocampus.5 However, despite the fact that 12/
15LOX mRNA and protein and enzymatic activity have
been documented in the central nervous system (CNS),
a precise biological role for this enzyme in the brain has
yet to be established. Recently, we quantified 12/15LOX
protein and activity levels in affected regions of postmortem brains of AD patients as well as normal controls.6
We showed that the amount of its two metabolic products, that is, 12(S)-HETE and 15(S)-HETE, were significantly increased in the brains of AD patients compared with controls. Moreover, semiquantitative analysis
of immunoreactive 12/15LOX protein suggested that at
the early and moderate stages of AD there was more
intense 12/15LOX immunoreactivity than at the more
advanced stages.6 Because these findings suggest that 12/
15LOX may be involved early in the pathogenesis of
AD, we conducted the studies reported here to determine if 12(S)-HETE and 15(S)-HETE levels are increased in CSF of living patients with mild or moderate
probable AD and individuals with mild cognitive impairment (MCI) relative to age and sex-matched normal
controls.
Subjects and Methods
Patients
Received Apr 6, 2005, and in revised form May 28. Accepted for
publication May 28, 2005.
This study was reviewed and approved by the Institutional
Review Board of the University of Pennsylvania. Subjects
were recruited from the University’s AD Center Memory
Disorder Clinic (MDC). Informed consent was obtained
from all participants and their caregivers. The clinical diagnosis of probable AD was based on the National Institute of
Neurological and Communicative Disorders and Stroke–
Alzheimer’s Disease and Related Disorders Association criteria.7 Standard clinical criteria for MCI were followed.8 As
part of their routine cognitive assessment, all patients received the Consortium to Establish a Registry for AD psychometric battery.9 The Dementia Severity Rating Scale
(DSRS) and Mini-Mental Sate Examination (MMSE) assessments were performed to evaluate the clinical severity of the
disease.9,10
Inclusion and exclusion criteria were the same as previously described.11,12 The 30 CSF samples came from a prospective clinical cohort with dementia or memory complaints, in which AD or MCI were diagnosed according to
the criteria described above. Twenty CSF samples from ageand sex-matched control subjects were from a cohort of cognitively normal individuals followed by the AD Center and
from cognitively normal spouses of AD patients attending
the MDC.
Published online Jul 21, 2005, in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.20558
Biochemical Analyses
Address correspondence to Dr Praticò, Department of Pharmacology, 3620 Hamilton Walk, John Morgan Building, Room 124,
Philadelphia, PA 19104. E-mail: domenico@spirit.gcrc.upenn.edu
All the assays were performed without knowledge of the clinical diagnosis of the patient.
Tau protein levels were measured by sandwich enzyme-
From the Departments of 1Pharmacology and 2Neurology, 3Alzheimer’s Disease Center, 4Department of Pathology and Laboratory
Medicine, and the 5Center for Neurodegenerative Disease Research,
University of Pennsylvania, School of Medicine, Philadelphia, PA.
© 2005 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
623
linked immunosorbent assay (ELISA) using the Innotest
hTAU-Antigen kit (Innogenetics, Gent Belgium). A␤1-40
and A␤1-42 levels were assayed by a previously wellcharacterized sandwich ELISA.11 Total 12(S)-HETE and
15(S)-HETE levels were measured as previously described.6
In brief, samples were subjected to solid phase extraction,
dried under nitrogen, resuspended in Enzyme Immune Assay
(EIA) buffer, and assayed by a standardized ELISA sandwich
kit (Assay Design, Ann Arbor, MI). The interassay and intraassay variability of the test is 5% and 4.5%, respectively. Total isoprostane iPF2␣-VI levels were assayed by gas chromatography/mass spectrometry as previously described.11,12
Apolipoprotein E Genotype
DNA was extracted from peripheral leukocytes and apolipoprotein E (ApoE) genotyping was performed as previously
described,11,12 without knowledge of the patient’s clinical diagnosis.
Statistical Analysis
Data are presented as mean ⫾ standard error of the mean
(SEM) and range. Comparison between groups was performed by nonparametric one-way analysis of variance
(Kruskall–Wallis test) with the Dunn’s post-test. Only p values less than 0.05 were regarded as statistically significant.
Correlations between 12(S)-HETE, 15(S)-HETE, other biochemical parameters, and demographic data were examined
by using linear regression.
Results
Twenty AD patients, 10 MCI, and 20 control subjects
were investigated in this study. The mean ( ⫾ SEM)
age for AD was 74.5 (1.8), for MCI was 72.8 (2.5),
and for controls was 71(2) years. No significant difference in gender ratio and years of education was observed among the three groups. Probable AD patients
had mild or moderate dementia and their mean
(⫾ SEM) MMSE score was 23 ⫾ 0.7. On the other
hand, MCI subjects had a score of 26.3 ⫾ 1.3, whereas
control subjects had a score of 28.3 ⫾ 0.7. ApoE ε2,
ε3, and ε4 allele distribution in the AD patients was as
follows: ε2/ε3, n ⫽ 3; ε3/ε3, n ⫽ 5; ε3/ε4, n ⫽ 10;
ε4/ε4, n ⫽ 2. ApoE alleles for MCI subjects were ε2/
ε3 ⫽ 1, ε3/ε3 ⫽ 4, and ε3/ε4 ⫽ 5. The ApoE allele
distribution was not known for the control subjects.
A scatterplot of CSF 12(S)-HETE and 15(S)-HETE
levels for the each patient in the three different groups
is shown in Figure 1. AD patients had CSF mean
(⫾ SEM) concentrations of 20 ⫾ 1ng/ml for 12(S)HETE and 48 ⫾ 5.2ng/ml for 15(S)-HETE. MCI
subjects had CSF levels of 17 ⫾ 2.6ng/ml for 12(S)HETE and 44 ⫾ 5.9ng/ml for 15(S)-HETE, whereas
the control group had, respectively, 9.5 ⫾ 0.8pg/ml
and 19.7 ⫾ 1.1ng/ml ( p ⬍ 0.001 for both metabolites
between AD and controls; and p ⬍ 0.001 between
MCI and controls). A direct correlation was observed
between these two metabolites (r2 ⫽ 0.43, p ⫽ 0.001).
Confirming previous work,11,12 we found that both
624
Annals of Neurology
Vol 58
No 4
October 2005
AD patients (75 ⫾ 8.2pg/ml) and MCI subjects (44 ⫾
8pg/ml) had higher levels of CSF isoprostane iPF2␣-VI
when compared with control subjects (24 ⫾ 2.1pg/ml,
p ⬍ 0.02 for both). A direct correlation was observed
between iPF2␣-VI and 12(S)-HETE (r2 ⫽ 0.35, p ⬍
0.0001; Fig 2A) and 15(S)-HETE (r2 ⫽ 0.28, p ⫽
0.001).
As previously shown by our group11,12 and others,13
we found that CSF tau protein levels were elevated,
whereas the percentage ratios between CSF A␤1-40
and A␤1-42 were lower in AD patients than in
matched controls (Table). Interestingly, we found that
CSF tau protein levels but not A␤1-42 percentage ratios were directly correlated with levels of 12(S)-HETE
(r2 ⫽ 0.35, p ⬍ 0.0001; see Fig 2B) and 15(S)-HETE
(r2 ⫽ 0.35, p ⫽ 0.0003). No correlation was observed
between CSF levels of 12(S)-HETE, 15(S)-HETE, and
the MMSE or DSRS scores of the subjects investigated
(not shown).
Discussion
In this study, we show for the first time that compared
with matched controls, patients with a clinical diagnosis of AD have increased CSF levels of 12(S)-HETE
and 15(S)-HETE, two metabolic products derived
from the activation of the 12/15LOX enzyme. These
data support the hypothesis that this metabolic pathway is increased during the initiation and early progression of AD. Interestingly, we found that the levels of
these metabolites directly correlated with the isoprostane iPF2␣-VI, a specific marker of lipid peroxidation,14 which is known be elevated in AD.1
Fig 1. Cerebrospinal fluid (CSF) 12(S)-hydroxyeicosatetraenoic
(HETE) and 15(S)-HETE levels are increased in Alzheimer’s
disease (AD) and mild cognitive impairment (MCI) patients.
(A) CSF 12(S)-HETE and (B) 15(S)-HETE levels in patients
with probable AD, subjects with MCI, and control subjects.
Fig 2. Correlation between cerebrospinal fluid (CSF) levels of
12(S)-hydroxyeicosatetraenoic (HETE) and iPF2␣-VI (A) and
between 12(S)-HETE and tau protein levels (B).
LOXs are enzymes widely represented in mammals
that oxidize fatty acids thereby generating several different biologically active eicosanoids. Two major functions have been ascribed to these enzymes: formation
of bioactive mediators and modification of membrane
structure (peroxidation reaction).3 One of the most
abundant LOX isoforms in the CNS is 12/15LOX.
Previous work proposed that 12(S)-HETE and 15(S)HETE are involved in learning and memory processes.15 Some circumstantial evidence also suggested
that 12/15LOX may be involved in neurodegeneration.16 Recently, we showed that both 12(S)-HETE
and 15(S)-HETE levels are significantly elevated in
pathologically affected regions of AD patients compared with controls.6 We also found that 12/15LOX
protein levels as well as immunoreactivity for this enzyme was increased in the same AD brain regions.
In this study, we confirm and extend this previous
observation not only to living patients with AD, but
also to subjects who meet the clinical criteria of MCI.
The finding that this metabolic pathway is increased in
individuals with MCI provides additional support for
the novel concept that the activation of 12/15LOX enzyme is an early event in AD pathogenesis. Moreover,
we found that, among the three groups studied, AD
patients had the highest values for CSF tau protein and
the lowest percentage ratio between A␤1-40 and A␤142, but no significant difference was observed between
MCI and control subjects. In contrast, MCI patients
had CSF 12(S)-HETE and 15(S)-HETE levels significantly higher than those of elderly control subjects.
These observations, as well as the correlation between
levels of CSF 12/15LOX metabolites and CSF tau also
are consistent with the notion that the activation of
this enzyme is an early event in the onset of AD and
that elevated levels of 12(S)-HETE and 15(S)-HETE
also may reflect disease severity.
Oxidative stress mechanisms and inflammatory reactions have been implicated in the regulation of
12/15LOX activity and expression levels.17 Because these
events have also been implicated in AD pathogenesis, it
is possible that this metabolic pathway participates in
mechanisms leading to the development of AD. However, this enzyme could also facilitate disease progression
and contribute to neuronal loss by being part of a selfsustaining cycle of autocrine stimulation in the CNS associated with the induction of proinflammatory mediators produced by microglia or astrocytes and in response
to the deposition of amyloid ␤. These stressors could
then stimulate neuronal 12/15LOX, which by producing high levels of HETEs would further reinforce this
vicious cycle of inflammatory and oxidative reactions.
In summary, this study shows for the first time to our
knowledge that metabolic products of the 12/15LOX
enzyme are increased in AD patients and MCI subjects
compared with controls, and that this increase is directly
correlated with brain oxidative stress and disease severity.
We conclude that drugs that specifically reduce or block
Table. CSF tau Protein and CSF A␤1-42 Percentage Ratio Levels in AD, MCI Patients, and Control Subjects
Tau (pg/ml), mean (SE)
Range
A␤ 1–42 (% ratio), mean (SE)
Range
a
AD (n ⫽ 20)
MCI (n ⫽ 10)
Controls (n ⫽ 20)
532 (76)a
146–1513
4.2 (0.29)b
1.8–7.2
353 (91)
177–834
5.6 (0.5)
3.4–7.4
264 (32)
119–437
5.9 (0.7)
1.7–10.9
p ⫽ 0.0005 versus controls;
p ⫽ 0.01 versus controls and p ⫽ 0.02 versus MCI subjects.
b
CSF ⫽ cerebrospinal fluid; AD ⫽ Alzheimer’s disease; MCI ⫽ mild cognitive impairment.
Yao et al: Cerebrospinal Fluid HETEs Levels in AD
625
this enzyme activation could be of potential interest for
future therapeutic interventions in AD.
This work was supported by grants from the NIH (National Institute on Aging, AG-10124, AG14449, J.Q.T., V.M.Y.L.; AG22203, AG-22512, D.P.).
We thank the patients and all their families who made this research
possible.
Mutations in Phenotypically
Mild D-2-Hydroxyglutaric
Aciduria
Eduard A. Struys, MSc,1 Stanley H. Korman, MBBS,2
Gajja S. Salomons, PhD,1 Patricia S. Darmin, BSc,1
Younes Achouri, PhD,3 Emile van Schaftingen, PhD,3
Nanda M. Verhoeven, PhD,1 and Cornelis Jakobs, PhD1
References
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functional events in Alzheimer’s disease. J Alz Dis 2004;6:
171–175.
2. Praticò D, Trojanowski JQ. Inflammatory hypotheses: novel
mechanisms of Alzheimer’s neurodegeneration and new therapeutic targets? Neurobiol Aging 2000;21:441– 445.
3. Brash AR. Lipoxygenases: occurrence, functions, catalysis and
acquisition of substrate. J Biol Chem 1999;274:23679 –23682.
4. Kuhn H, Thiele B. The diversity of lipoxygenase family. Many
sequence data but little information on biological significance.
FEBS Lett 1999;449:7–11.
5. Nishiyama M, Watanabe T, Ueda N, et al. Arachidonate 12lipoxygenase is localized in neurons, glia cells and endothelial
cells of the canine brain. J Histochem Cytochem 1993;41:
111–117.
6. Praticò D, Zhukareva V, Yao Y, et al. 12/15 Lipoxygenase is
increased in Alzheimer’s disease. Am J Pathol 2004;164:
1655–1662.
7. Radebaugh TS, Buckholz NS, Khachaturian ZS. Fisher
symposium: strategies for the prevention of Alzheimer’s disease—an overview of research planning meeting III. Alzheim
Dis Assoc Disord 1996;10(suppl 1):15.
8. Welsh KA, Butters N, Mohs RC, et al. The Consortium to
Establish a Registry for Alzheimer’s Disease (CERAD). Part V.
A normative study of the neuropsychological battery. Neurology 1994;44:609 – 614.
9. Clark CM, Ewbank DC. Performance of the dementia severity
rating scale: a caregiver questionnaire for rating severity of Alzheimer’s disease. Alzheimer’s Dis Assoc Disord 1996;10:31–39
10. Folstein MF, Folstein SE, McHugh PR. “Mini-mental state”: a
practical method for grading the cognitive state of patients for
the clinician. J Psychiatr Res 1975;12:189 –198.
11. Praticò D, Clark CM, Liun F, et al. Increase of brain oxidative
stress in mild cognitive impairment. A possible predictor of Alzheimer’s disease. Arch Neurol 2002;59:972–976.
12. Praticò D, Clark CM, Lee VM-Y, et al. Increased 8,12-isoiPF2␣-VI in Alzheimer’s disease: correlation of a noninvasive index of lipid peroxidation with disease severity. Ann Neurol
2000;48:809 – 812.
13. Arai H, Terajima M, Miura M, et al. Tau cerebrospinal fluid: a
potential diagnostic marker in Alzheimer’s disease. Ann Neurol
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2001;12:243–247.
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of arachidonic acid as second messengers for pre-synaptic inhibition of Aplysia sensory cells. Nature 1987;328:38 – 43.
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cell death caused by gluthatione depletion. Neuron 1997;19:
453– 463.
17. Huang JT, Welch JS, Ricote M, et al. Interleukin-4-dependent
production of PPAR-g ligands in macrophages by 12/15lipoxygenase. Nature 1999;400:378 –382.
626
D-2-hydroxyglutaric
aciduria is a neurometabolic disorder
with mild and severe phenotypes. Recently, we reported
pathogenic mutations in the D-2-hydroxyglutarate dehydrogenase gene as the cause of the severe phenotype of
D-2-hydroxyglutaric aciduria in two patients. Here, we report two novel pathogenic mutations in this gene in one
patient with a mild presentation and two asymptomatic
siblings with D-2-hydroxyglutaric aciduria from two unrelated consanguineous Palestinian families: a splice error
(IVS4-2A3 G) and a missense mutation (c.1315A3 G;
p.Asn439Asp). Overexpression of this mutant protein
showed marked reduction of the enzyme activity.
Ann Neurol 2005;58:626 – 630
Recently, we described the discovery of disease-causing
mutations in the D-2-hydroxyglutarate dehydrogenase gene
in two unrelated patients with a severe presentation of
the neurometabolic disorder D-2-hydroxyglutaric aciduria (D-2-HGA).1 This was the first report revealing, in
two unrelated patients, the underlying molecular defect
in D-2-HGA, since the description of this disorder in
1980.2 In an international survey of clinical data of patients with D-2-HGA, a mild and a severe phenotype
could be distinguished.3,4 The severe phenotype is homogeneous and characterized by an early-infantile-onset
epileptic encephalopathy, facial dysmorphic features, and
often a cardiomyopathy. Magnetic resonance images
(MRIs) show signs of disturbed cerebral maturation and
white matter abnormalities. The mild phenotype has a
more variable clinical presentation and less consistent
MRI findings.
From 1Metabolic Unit, Department of Clinical Chemistry, VU
University Medical Center, Amsterdam, The Netherlands; 2Department of Clinical Biochemistry, Hadassah-Hebrew University Medical Center, Jerusalem, Israel; and 3Laboratory of Physiological
Chemistry, Universite Catholique de Louvain and Christian de
Duve Institute of Cellular Pathology, Brussels, Belgium.
Received Mar 7, 2005 Accepted for publication May 28, 2005.
Published online Jul 21, 2005, in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.20559
Address correspondence to Dr Jakobs, Metabolic Unit, Department
of Clinical Chemistry, VU University Medical Center, Amsterdam,
The Netherlands. E-mail: c.jakobs@vumc.nl
© 2005 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
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