Conceived and designed the experiments: FS AP PS. Performed the experiments: FS KM. Analyzed the data: FS KM. Contributed reagents/materials/analysis tools: FS JC NK WW BC OL. Wrote the paper: FS AP JC NK BC HB GS PS.
The authors have declared that no competing interests exist.
Apolipoproteins have recently been implicated in the etiology of Alzheimer’s disease (AD). In particular, Apolipoprotein J (ApoJ or clusterin) has been proposed as a biomarker of the disease at the pre-dementia stage. We examined a group of apolipoproteins, including ApoA1, ApoA2, ApoB, ApoC3, ApoE, ApoH and ApoJ, in the plasma of a longitudinal community based cohort.
664 subjects (257 with Mild Cognitive Impairment [MCI] and 407 with normal cognition), mean age 78 years, from the Sydney Memory and Aging Study (MAS) were followed up over two years. Plasma apolipoprotein levels at baseline (Wave 1) were measured using a multiplex bead fluorescence immunoassay technique.
At Wave 1, MCI subjects had lower levels of ApoA1, ApoA2 and ApoH, and higher levels of ApoE and ApoJ, and a higher ApoB/ApoA1 ratio. Carriers of the apolipoprotein E ε4 allele had significantly lower levels of plasma ApoE, ApoC3 and ApoH and a significantly higher level of ApoB. Global cognitive scores were correlated positively with ApoH and negatively with ApoJ levels. ApoJ and ApoE levels were correlated negatively with grey matter volume and positively with cerebrospinal fluid (CSF) volume on MRI. Lower ApoA1, ApoA2 and ApoH levels, and higher ApoB/ApoA1 ratio, increased the risk of cognitive decline over two years in cognitively normal individuals. ApoA1 was the most significant predictor of decline. These associations remained after statistically controlling for lipid profile. Higher ApoJ levels predicted white matter atrophy over two years.
Elderly individuals with MCI have abnormal apolipoprotein levels, which are related to cognitive function and volumetric MRI measures cross-sectionally and are predictive of cognitive impairment in cognitively normal subjects. ApoA1, ApoH and ApoJ are potential plasma biomarkers of cognitive decline in non-demented elderly individuals.
It is estimated that 35.6 million people worldwide currently suffer from dementia
The etiology of sporadic AD is not well understood, and vascular risk factors appear to play an important role
Apolipoproteins (Apo) are a group of proteins related to cholesterol and lipid metabolism
Literature evidence: *, Katzav, Faust-Socher et al. 2011; George and Erkan 2009. **, Lewis, Cao et al. 2010; Roher, Maarouf et al. 2009; Martins, Berger et al. 2009; Bereczki, Bernat et al. 2008.
The
ApoH is involved in diverse physiological processes, including lipid metabolism
Therefore, these seven apolipoproteins, ApoA1, ApoA2, ApoB, ApoC3, ApoE, ApoH and ApoJ, were chosen for the analysis of plasma in MCI and cognitively normal subjects in a community based study. The aims of the present study were, to determine if plasma apolipoproteins are abnormal in MCI subjects at an early stage of cognitive decline, and to establish if baseline apolipoprotein levels are predictive of cognitive impairment in cognitively normal subjects over a two year period. Finally, we wished to examine the effect of
The procedures of this study were approved by Human Research Ethics Committee of the University of New South Wales on human experimentation, and written informed consent was obtained from all participants involved in this study.
Plasma samples were from Wave 1 (baseline) of the Sydney Memory and Ageing study (MAS), described in detail in a previously published paper
Participants were excluded if they had a previous diagnosis of dementia, psychotic symptoms or a diagnosis of schizophrenia or bipolar disorder, multiple sclerosis, motor neuron disease, developmental disability, progressive malignancy (active cancer or receiving treatment for cancer, other than prostate non-metastasized, and skin cancer), or if they had medical or psychological conditions that may have prevented them from completing assessments. Participants were excluded if they had a Mini-Mental State Examination (MMSE)
Participants received a detailed neuropsychological evaluation, and a subset had MRI brain scans at Waves 1 and 2, two years apart. A subgroup of 664 subjects was included in this study, of which the Wave 1 characteristics are shown in
Normal n = 407 | MCI n = 257 | p | P(FDR corrected) | |
|
77.9±4.5 | 78.8±4.7 |
|
––– |
|
175(43%) | 126 (49%) | 0.13 | ––– |
|
11.6±3.5 | 11.3±3.4 | 0.30 | ––– |
|
84 (20.6%) | 72 (28.0%) |
|
––– |
|
205 (49.9%) | 124(47.5%) |
|
|
|
2.97±1.28 | 2.70±1.12 |
|
|
|
158.63±45.78 | 148.50±47.63 |
|
|
|
1.86±0.53 | 1.98±0.68 | 0.07 | 0.08 |
|
64.04±23.59 | 60.12±24.13 | 0.08 | 0.08 |
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36.30±20.40 | 39.86±22.00 |
|
|
|
171.40±43.07 | 156.44±45.10 |
|
|
|
108.97±27.43 | 119.98±33.03 |
|
|
|
0.73±0.38 | 0.86±0.47 |
|
|
Data are presented as mean±SD.
Covariates in ANCOVA for effects of apolipoproteins: age, sex, years of education,
The diagnosis of MCI was based on international consensus criteria
Clinical Dementia Rating (CDR)
Genomic DNA was extracted from peripheral blood leukocytes or saliva samples using standard methods. Genotyping of two apolipoprotein E gene (
Plasma apolipoprotein concentrations were assayed using multiplex fluorescent immunoassay kits (WideScreen™ Human CVD Panel 1; Novagen, EMD Chemicals Inc, WI). The xMAP platform used here was based on the Rules Based Medicine (RBM) fluorescent beads and antibody pairs. These are sensitive, specific and widely used reagents, sourced by numerous manufacturers and data collected using xMAP multiplex beads are widely reported in the literature in studies where simultaneous assay of multiple plasma proteins, including apolipoproteins, are performed. Some recent examples of published work which has made use of human xMAP multiplex technology include;
Total cholesterol, HDL-cholesterol and triglycerides were measured in heparin plasma aliquots using a Beckman LX20 Analyzer using a timed-endpoint method (Fullerton, CA). This direct HDL-cholesterol method requires no off-line pre-treatment steps. LDL-cholesterol was estimated using the Friedewald equation (LDL-cholesterol = total cholesterol - HDL-cholesterol - triglycerides/2.2). These assays were conducted in an independent laboratory of South Eastern Area Laboratory Services.
All participants were invited to undergo an MRI scan, and those who agreed were screened for contra-indications (pacemaker, metallic implant or foreign bodies, cochlear implants, ferromagnetic homeostatic clips, claustrophobia). A subgroup (n = 376) was included in this study at Wave 1, and 282 subjects at Wave 2. Subjects were scanned using a Philips 3 T Intera Quasar scanner (Philips Medical Systems, Best, Netherlands). The main parameters for T1-weighted 3D structural MRI were: TR = 6.39 ms, TE = 2.9 ms, flip angle = 8°, matrix size = 256×256, FOV = 256×256×190, and slice thickness = 1 mm with no gap between; yielding 1×1×1 mm3 isotropic voxels. The T2-weighted fluid attenuated inversion recovery (FLAIR) sequence was acquired with TR = 10000 ms, TE = 110 ms, TI = 2800; matrix size = 512×512; slice thickness = 3.5 mm with no gap between slices, yielding spatial resolution of 0.488×0.488×3.5 mm3/voxel.
All statistical analyses were performed using SPSS Version 18.0 (SPSS Inc., Chicago, IL). Differences between normal and MCI groups on categorical variables (sex and
To assess if plasma apolipoproteins levels predicted cognitive decline in cognitively normal participants, logistic regression analysis was performed, using only those participants with a CDR = 0 at Wave 1, and a CDR of >0 (versus CDR = 0) at Wave 2 as the dependent variable. We used the z-scores of apolipoproteins as independent variables, so that the odds ratios (ORs) reflect the ratio of odds when values of apolipoproteins increase by one standard deviation (SD). Age, sex, years of education and
Linear regression models were used to examine whether Wave 1 apolipoprotein levels were associated with brain volumetric changes from Wave 1 to Wave 2, including changes in CSF volume, grey matter, white matter and hippocampal volume, and brain atrophy between the two waves. The brain atrophy measure during follow-up was calculated as the changes of brain volume (sum of grey matter and white matter volumes) divided by ICV. Transformed apolipoprotein levels were used as independent variables and age and sex were covariates. ICV was also used as a control variable for brain volume variables, except for the atrophy measure.
P-values, corrected for multiple testing using the false discovery rate (FDR) method, were used when evaluating the statistical significance of results
The Wave 1 demographic information on cognitively normal and MCI participants is shown in
Non |
F | p | |||
N | 508 | 144 | 12 | ||
|
2.86±1.22 | 2.91±1.26 | 2.64±1.21 | 0.62 | 0.54 |
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155.13±47.29 | 151.95±43.64 | 170.27±58.37 | 0.70 | 0.50 |
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1.86±0.59 | 2.04±0.62 |
2.10±0.31 | 5.11 | 0.006 |
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64.04±24.44 | 57.80±21.11 | 54.88±22.76 | 4.31 | 0.01 |
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41.16±21.44 | 26.94±14.83** | 19.06±17.54** |
56.44 | <0.0005 |
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167.66±44.78 | 159.95±42.87 | 147.00±41.50 | 2.56 | 0.08 |
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113.64±30.62 | 111.76±28.33 | 113.66±34.88 | 0.07 | 0.94 |
Statistics details: ANCOVA, Post-hoc: Bon Ferroni Covariates: age, sex.
Compared to non
Compared to
When heterozygous and homozygous carriers are pooled and compared with non
After adjusting the p values using the FDR method, MCI subjects had lower levels of ApoA1 (F = 6.34, p = 0.013), ApoA2 (F = 9.20, p = 0.008) and ApoH (F = 18.78, p = 0.00008), and higher levels of ApoE (F = 6.06, p = 0.013), ApoJ (F = 20.76, p = 0.00008) and ApoB/ApoA1 (F = 8.10, p = 0.01).
Most correlations between apolipoproteins were weak to moderate (
ApoA1 | ApoA2 | ApoB | ApoC3 | ApoE | ApoH | ApoJ | ApoB/ApoA1 | |
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– | 0.60*** | 0.01 | 0.39*** | −0.05 | 0.35*** | 0.16*** | – |
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0.60*** | – | –0.04 | 0.57*** | 0.09 | 0.50*** | 0.23*** | –0.50*** |
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0.01 | −0.04 | – | –0.08 | 0.05 | −0.10 | 0.23*** | – |
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0.39*** | 0.57*** | –0.08 | – | 0.43*** | 0.47*** | 0.32*** | –0.36*** |
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−0.05 | 0.09 | 0.05 | 0.43*** | – | 0.25*** | 0.36*** | 0.05 |
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0.35*** | 0.50*** | −0.10 | 0.47*** | 0.25*** | – | 0.30*** | −0.34*** |
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0.16*** | 0.23*** | 0.23*** | 0.32*** | 0.36*** | 0.30*** | – | −0.01 |
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– | −0.50*** | – | −0.36*** | 0.05 | −0.34*** | −0.01 | – |
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0.60*** | 0.22*** | −0.001 | 0.13** | −0.24*** | 0.03 | 0.01 | −0.48*** |
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0.01 | 0.07 | 0.29*** | 0.08 | 0.06 | 0.06 | 0.03 | 0.16*** |
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0.24*** | 0.18*** | 0.25*** | 0.28*** | 0.10 |
0.11** | 0.05 | −0.04 |
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−0.22*** | 0.06 | −0.02 | 0.49*** | 0.53*** | 0.19*** | 0.08 | 0.16*** |
p<0.05, **p<0.01, *** p<0.001(FDR corrected p values) Covariates: age, sex.
ApoA1 | ApoA2 | ApoB | ApoC3 | ApoE | ApoH | ApoJ | ApoB/ApoA1 | ||
|
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0.04 | 0.07 | −0.06 | 0.02 | −0.07 | 0.15** | −0.13 |
−0.05 |
|
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0.07 | 0.12 |
−0.05 | 0.04 | −0.05 | 0.11 |
−0.11 |
−0.05 |
|
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0.01 | 0.03 | −0.03 | −0.04 | −0.11 |
0.09 | −0.09 | −0.03 |
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0.01 | 0.01 | −0.02 | −0.05 | −0.11 |
0.08 | −0.07 | −0.03 |
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−0.01 | 0.02 | −0.07 | 0.003 | −0.009 | 0.09 | −0.06 | −0.03 |
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0.01 | 0.03 | −0.01 | −0.007 | −0.05 | 0.07 | −0.07 | −0.01 |
|
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0.06 | 0.05 | −0.03 | 0.02 | −0.07 | 0.15** | −0.04 | −0.04 |
Subject numbers, n = 657.
p<0.05; **p<0.01 (FDR corrected p values, see text).
Covariates: age, sex, years of education,
Wave 1 apolipoprotein levels were correlated with grey matter and CSF volume (
ApoA1 | ApoA2 | ApoB | ApoC3 | ApoE | ApoH | ApoJ | ApoB/ApoA1 | ||
|
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−0.01 | −0.08 | −0.03 | –0.14 |
−0.14 |
−0.06 | −0.10 |
−0.07 |
|
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0.08 | 0.03 | 0.003 | –0.02 | −0.05 | 0.01 | −0.01 | −0.05 |
|
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−0.03 | 0.05 | 0.02 | 0.11 | 0.16 |
0.05 | 0.14 |
0.08 |
|
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0.08 | 0.05 | −0.06 | –0.03 | –0.02 | 0.01 | −0.03 | −0.13 |
Subject numbers, n = 377.
p<0.05 (FDR corrected p values, see text).
Covariates: age, sex, intracranial volume.
We investigated if apolipoprotein levels at Wave 1 could predict conversion from cognitively normal (CDR = 0) at Wave 1 to cognitively impaired (CDR >0) at Wave 2. For this analysis, 517 participants had a CDR = 0 at Wave 1 and of these 149 participants had a CDR greater than 0 at Wave 2. Individuals who went from CDR 0 to CDR >0 were classified as
Nonconverters (Wave 2 CDR = 0) | Converters (Wave 2 CDR >0) | p | |
N | 368 | 149 | |
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77.3±4.1 | 79.2±4.7 |
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140, 38% | 85, 43% |
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11.8±3.4 | 10.7±3.4 |
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65, 17.9% | 42, 28.6% |
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28.5±1.2 | 27.8±1.4 |
|
Models without CVD risk index includedas a control variable | Models with CVD risk index included as a control variable | Models with CH, TG, HDL,LDL includedas control varaible | |||||||||||||
OR | 95%CI | Wald | P | p (FDR ) | OR | 95%CI | Wald | p | P (FDR) | OR | 95%CI | Wald | p | P (FDR) | |
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0.61 | 0.46–0.80 | 12.82 |
|
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0.65 | 0.49–0.86 | 9.18 |
|
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0.56 | 0.41–0.78 | 11.98 |
|
|
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0.73 | 0.58–0.91 | 7.61 |
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0.76 | 0.61–0.96 | 5.27 |
|
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0.72 | 0.57–0.91 | 7.45 |
|
|
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1.17 | 0.93–1.47 | 1.74 | 0.19 | 0.27 | 1.15 | 0.91–1.45 | 1.33 | 0.25 | 0.33 | 1.15 | 0.91–1.47 | 1.36 | 0.24 | 0.38 |
|
0.87 | 0.69–1.08 | 1.64 | 0.20 | 0.27 | 0.87 | 0.69–1.09 | 1.51 | 0.22 | 0.33 | 0.76 | 0.57–1.00 | 3.76 | 0.52 | 0.66 |
|
1.16 | 0.89–1.50 | 1.23 | 0.27 | 0.31 | 1.13 | 0.86–1.49 | 0.77 | 0.38 | 0.43 | 1.06 | 0.76–1.46 | 0.10 | 0.75 | 0.75 |
|
0.76 | 0.61–0.94 | 6.26 |
|
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0.77 | 0.62–0.96 | 5.50 |
|
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0.74 | 0.59–0.93 | 6.90 |
|
|
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1.06 | 0.85–1.32 | 0.29 | 0.59 | 0.59 | 1.08 | 0.86–1.35 | 0.44 | 0.51 | 0.51 | 1.07 | 0.85–1.33 | 0.31 | 0.58 | 0.66 |
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1.60 | 1.–2.11 | 10.92 |
|
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1.46 | 1.09–1.95 | 6.53 |
|
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1.61 | 1.18–2.21 | 8.90 |
|
|
Covariates: age, sex, years of education and
CH, cholesterol; TG, triglycerides; HDL, high density lipoprotein; LDL, low density lipoprotein.
To study which apolipoproteins had the highest independent predictive value, the z-scores of all apolipoprotein measures were entered together into a stepwise logistic regression (probability of F for entry = 0.05, and for removal = 0.10), while adjusting for age, sex, years of education,
With regard to the relationship between apolipoproteins and brain neuroimaging changes, a slightly different pattern emerged. For this analysis, 232 participants had neuroimaging data for both waves. Linear regression models showed that increased ApoC3 levels were associated with a decrease in grey matter volume (β = 0.19, p = 0.04), and increased ApoJ levels were associated with a decrease in white matter volume (β = 0.16, p = 0.03) after two years follow-up. Lower ApoA1 levels showed a weak but not significant association with an increase in brain atrophy (β = −0.15, p = 0.09). There were no associations between apolipoprotein levels and increase in CSF volume or decrease in hippocampus volume.
The lipid profiles at Wave 1 were analysed to investigate if they could predict conversion from cognitively normal (CDR = 0) at Wave 1 to cognitively impaired (CDR>0) at Wave 2. However, none of the measures examined was associated with cognitive impairment during follow-up; total cholesterol (OR = 1.06, p = 0.94), HDL-cholesterol (OR = 0.87, p = 0.71), LDL-cholesterol (OR = 0.95, p = 0.95) and triglycerides (OR = 1.02, p = 0.95). Furthermore, including lipid profile as covariates did not affect the ability of ApoA1, ApoA2 and ApoH levels and ApoB/ApoA1 ratio to predict cognitive impairment (
In this study, we report for the first time detailed measurements of multiple apolipoproteins in the plasma of elderly non-demented subjects, and relate them to cognition. Our salient findings are that MCI subjects at Wave 1 had lower levels of ApoA1, ApoA2, ApoH and higher levels of ApoE, ApoJ and the ApoB/ApoA1 ratio. Furthermore ApoE, ApoC3 and ApoH show significant downward trends in
Cognitiveimpairment | Cognitive decline over2 years | MRI lower volumeat Wave 1 | MRI volume decreaseover 2 years | |
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+ | + | ||
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+ | + | ||
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+ | + | ||
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+ | + | ||
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+ | + | + | |
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+ | + | + |
+ Significantly indicates relationship in direction of pathology.
Our results therefore indicate that apolipoproteins are associated with cognitive decline independently of the effect of lipid profile. The pathomechanisms underlying this relationship still remain unclear. A few clinical studies have revealed that some apolipoproteins are deregulated in AD patients
In this study, ApoA1 concentration was lower in MCI, and a low level of ApoA1 was the strongest risk factor of cognitive decline in comparison with other apolipoproteins. These observations are consistent with published studies, which have shown a marked decrease of ApoA1 levels in AD plasma and serum
ApoA1 and ApoA2 are the principal apolipoproteins in HDL, and are responsible for the reverse transport of cholesterol, a process that removes excess cholesterol from peripheral tissues to the liver for excretion
ApoB has been found to be increased in AD plasma
The results in the literature on ApoE levels in AD are inconsistent. Darreh-Shori et al reported an
The apparent inconsistencies in the published literature on ApoE levels in AD or MCI may be due to multiple differences between studies, including; (1) the sample, which can be CSF, serum, plasma, urine or tissue (2) experimental design (
Our results showed that MCI subjects had a lower level of ApoH in the cross-sectional comparison, and low levels of ApoH also increased the risk of cognitive decline during follow-up. ApoH, also known as beta2-glycoprotein I (
Our results support current knowledge on the functions of plasma ApoH. Even though a mechanistic link between plasma ApoH and cognitive decline has not been established, it may indicate that subclinical hypercoagulation caused by low ApoH might increase the risk of cognitive decline.
Our results indicate that ApoJ plasma concentration is raised early, before AD and dementia symptoms become clinically evident. We observed that ApoJ levels were negatively correlated with global cognition and associated with white matter volume atrophy during follow-up. Apo J is a disulfide linked glycoprotein composed of two 40 kDa subunits and expressed at higher levels in the brain than in many other tissues
We did not find that plasma ApoJ levels were associated with cognitive decline during follow-up. However, it has been reported that high plasma ApoJ was associated with clinical progression of AD
We have followed up subjects for only two years, and further follow-up will be needed to develop a more complete view of the observed relationship, especially for incident MCI and AD subjects. Serial measurements may also help determine if apolipoproteins play a major casual role in MCI and AD. The findings, moreover, need to be replicated in independent populations. The examination of transgenic animal models of AD may also be informative in this regard. The final objective would be to determine if modification of plasma apolipoprotein levels can reduce the risk of cognitive disorders in the elderly.
In conclusion, this study indicates that apolipoprotein levels are dysregulated in the plasma of MCI subjects at an early stage of cognitive decline, before a clinical diagnosis of AD is possible. ApoA1, ApoH and ApoJ may be potential clinical biomarkers for cognitive impairment. Our results provide further support for a pathophysiological role for these apolipoproteins in cognitive decline in the elderly, and possibly AD. As early indicators of cognitive decline, these apolipoproteins might also become targets of treatment or preventative healthcare measures.
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The authors would like to thank Peter Day and Prof D Naidoo for assistance with sample collection and storage and lipid profile analysis at the South Eastern Area Laboratory Services, and Ms Angie Russell for facilitating purchases of multiplex kits and her excellent administrative help. The DNA was extracted by Genetic Repositories Australia, an Enabling Facility. The genotyping was performed by Arezoo Assareh and Karen Mather in the laboratory of Prof P Schofield and John Kwok at Neuroscience Research Australia. We thank the Research Team and all participants in the Sydney Memory and Ageing Study (MAS).