To our knowledge, this is the first report describing normal lifespan changes in multiple CSF biomarkers used for Alzheimer’s disease. We examined, in normal subjects, the relationship between age and ApoE genotype for five CSF AD biomarkers. The results showed age by genotype interactions, for some analytes and for others only age effects. No CSF biomarker marker showed effect restricted to genotype. Specifically, both P-tau231 and IP showed age by ApoE genotype interactions in the direction consistent with the known risk that carriers have for AD. Separate age and genotype effects were found for T-tau, no effects were found for the Aβ ratio or the individual Aβ peptide fragments studied. Because other work suggests that P-tau231 and IP are excellent univariate discriminators of AD (Brys et al., 2007
), these biological data highlight the importance of increasing age and the ApoE ε4 genotype in modifying the risk for AD in normal subjects.
T-tau levels increased with age and were higher in ApoE ε4 carriers. The finding of positive association between age and T-tau is in agreement with other previously published data (Blomberg et al., 2001
; Briani et al., 2002
; Itoh et al., 2001
; Shoji et al., 1998
; Sjogren et al., 2001
). However, there are also contrary findings (Andreasen et al., 1998
; Blomberg et al., 1996
; Burkhard et al., 2004
; Jensen et al., 1995
; Lewczuk et al., 2004a
). However, in some of the published negative reports, the samples were much smaller than in our study. T-tau reflects the non-specific neuronal and axonal turnover and damage due to any pathological condition (for review (Blennow and Vanmechelen, 2003
)). The increase in T-tau with age is not unexpected as age is related to multiple sources of neuronal damage and pruning (for review (Uylings and de Brabander, 2002
)). We observed that over the lifespan, the ApoE ε4 positive group had higher overall T-tau levels. This result is in agreement with the report by Golombowski et al. (1997)
, who described this ApoE ε4 effect in group of three healthy ApoE ε4 carriers and nine non-carriers. However, two other studies showed negative results (Blomberg et al., 2001
; Sunderland et al., 2004
). The current study examined risk factors for atherosclerosis in the sample. We hypothesized that the E4 positive carriers might be predisposed to vascular diseases which in turn could contribute to age-related brain damage and therefore to elevated T-tau levels. Our ApoE groups were similar with respect to the distribution of medical (atherosclerosis and medication related) factors. It remains a possibility that our careful screening, which removed from the sample those individuals who had clinical evidence for brain damage due to vascular causes differentially reduced the prevalence associated with the ApoE genotype and diminished the age and interaction effects.
The P-tau231 concentration increased with age in both ApoE groups; and the increase was significantly steeper in the ApoE ε4 positive group. There is not much information about age effects on P-tau231 levels in normal controls, but our results are at variance with three other published reports, which did not show age-dependent changes either in P-tau181 (Lewczuk et al., 2004a
), P-tau199 (Itoh et al., 2001
) or P-tau231 (Buerger et al., 2002
). Greater age-related P-tau231 elevations in the ε4 positive group are consistent with the increased risk of this group for AD and for an earlier accumulation of AD pathology. Several reasons led us to use P-tau231. (1) Neuropathological data shows that P-tau231 is a marker for the earliest stages of the disease. Augustinack et al. (2002)
showed that TG3 antibody (for thr231 epitope) was one of three, which stained predominantly pre-neurofibrillary tangles stage while staining with the antibody specific for thr175/181 was more prominent in the later stage of NFT evolution. This distinction is potentially of great value while assessing first, disease- related signal in normal population (as our study group). (2) Antemortem CSF P-tau231 correlates with post mortem
NFT pathology in neocortical regions (Buerger et al., 2006
) and P-tau181 does not (Buerger et al., 2007
; Engelborghs et al., 2007
). (3) Excellent diagnostic specificity data exist for P-tau231 (Hampel et al., 2004
Interestingly, across the lifespan and in relationship to ApoE genotype, the P-tau231 data parallels the results observed for IP. IP levels also showed steep age-related slope in ε4 carriers. This may reflect the possible link between oxidative stress and tau phosphorylation and is consistent with the hypothesis of an early role for oxidative damage in AD (Zhu et al., 2003
). Examination of the relationship between these two analytes showed they were associated (r
= .48, p
< .01). We are not aware of other studies addressing the effect of ApoE genotype on CSF isoprostanes levels in healthy subjects. There is nonetheless, evidence of their higher levels in plasma in healthy ε4 carriers (Dietrich et al., 2005
). Oxidative damage to peptidyl prolyl-cis, trans
-isomerase (Pin), an enzyme regulating phosphorylation-dephosphorylation of tau has been suggested as an initial event prompting tangle formation (Sultana et al., 2006
). Oxidative stress results in the elevation of stress-activated kinases (SAPK) (Zhu et al., 2003
), which in turn are crucial for the phosphorylation of tau protein. The correlations between IP and P-tau levels in our sample, speaks in favor for the hypothesis linking oxidative stress and tau phosphorylation. Our data, however, does not address the issue of temporal and causative relationships between biomarkers; to fully answer this question longitudinal and animal model studies are needed. Nevertheless, the association between P-tau231 and IP suggest the early diagnostic potential for these biomarkers.
The reports about lifespan changes in the CSF Aβ levels of normal aging subjects are controversial. In a recent study, Peskind et al. (2006)
found an age effect for Aβ42 and Aβ42/Aβ40. The authors showed a decrease in CSF Aβ42 after the age of 60, the decrease in Aβ42/Aβ40 occurred somewhat earlier. Significantly more pronounced age-related Aβ reductions were observed in subjects with an ApoE ε4 allele. However, others have not reported age effects for the Aβ ratio (Shoji et al., 1998
). In our sample only a statistical trend for lower Aβ42/40 ratio with increasing age was found and we did not find an ApoE effect nor an interaction contrary to other reports (Peskind et al., 2006
; Sunderland et al., 2004
). We believe the discrepancy might have come from different assays used. Moreover, the Fagan et al. (2000)
findings support ours, as she found significantly elevated Aβ40/Aβ42 ratios (inverse of Aβ42/Aβ40) between ε4 groups only in the lipoprotein fraction of CSF, but not in the unfractionated CSF (as was ours). Additionally, the possibility exists CSF levels may not be exclusively brain derived, as the platelets are the biggest source of the soluble amyloid in the blood (Chen et al., 1995
). Thus, the interpretation of level changes may be problematic.
As different ApoE isoforms differentially affect lipid metabolism (Xhignesse et al., 1991
), atherosclerosis (Greenow et al., 2005
) and subsequent lipid peroxidation, it is possible that this influence accounts for our observed increase in the IP of ε4 positive subjects. Similarly, drugs may influence the levels of some biomarkers (De Caterina et al., 2002
; Sutherland et al., 2007
). This later issues are not likely to be a major confounding factors in our study, as the ε4 groups did not differ in variables associated with atherosclerosis, lipid metabolism, or the use of lipid modifying, antioxidant or other drugs. Although we did find a difference in BMI values, in neither group the means were in the obesity range. Similarly, there was a trend toward more favorable lipid profile in ε4 positive individuals, but this was limited only to triglycerides. We do not think the slightly higher number of subjects taking statins among the ε4 positive individuals, could be an explanation. Statins are known to have only negligible influence on triglyceride levels. None of our subjects were taking fibrates. However, the ε4 positive group was a little (but not significantly) younger, this could influence the triglyceride levels. Nevertheless, our analysis may be also compromised by the lack of information pertaining the types and dosages of statins and antioxidants.
The presence of an ApoE ε4 allele modifies the age of onset of AD (Khachaturian et al., 2004
). Cognitively intact ApoE ε4 positive individuals display brain glucose metabolic patterns similar to those observed in AD patients (Reiman et al., 1996
) and more recently it was reported that E4 carriers with subjective memory complaints showed elevated IP and P-tau231 levels in association with reduced medial temporal lobe glucose metabolism (Mosconi, 2007
). Our CSF findings are in agreement with PET and other data indicating biological differences between ApoE ε4 positive and ε4 negative subjects that point to an AD predisposition or possibly to already existing preclinical pathology. Overall, this suggests a mechanism for their greater risk for cognitive decline and an early age of AD onset. However the observation of symptom development predicted by lower metabolism or altered biomarkers remains unexamined. The ApoE influence on CSF biomarkers may be exerted on different pathways: Lipid metabolism, neurite outgrowth, tau phosphorylation, mitochondrial alteration (Mahley et al., 2006
) or oxidative stress (Ramassamy et al., 2000
). It remains unclear whether the CSF differences between ε4 carriers and non-carriers are a reflection of increased sub-clinical disease or whether there is a life long trait of biomarker differences found in association with the APOE genotype. This important question can only be answered with longitudinal studies with post-mortem or other validations, assessing the impact of both genotype and biomarkers levels on progression rates from normal to MCI and to AD.
In summary, the present study examines the cross sectional effect of age (through the adult life-span) and ApoE genotype for five CSF analytes. The data show that CSF P-tau231 and IP are particularly sensitive to the interaction of age and genotype and therefore may be useful early predictors of the pathology leading to clinical recognition of AD.