Correlation of Specific Amyloid-β Oligomers With Tau in Cerebrospinal Fluid From Cognitively Normal Older Adults

Abstract

Objective

To investigate two specific amyloid-β (Aβ) oligomers, Aβ trimers and Aβ*56, in human cerebrospinal fluid (CSF), evaluate the effects of aging and Alzheimer's disease (AD), and obtain support for the hypothesis that they may be pathogenic by determining their relationships to CSF tau.

Design

A CSF sampling study.

Setting

The University of Minnesota Medical School in Minneapolis, Minnesota, and the Salhgrenska University Hospital, Sweden.

Participants

Older adults with mild cognitive impairment or Alzheimer's disease (Impaired), age-matched cognitively intact controls (Unimpaired), and younger, normal controls.

Main outcome measures

Measurements of CSF Aβ trimers, Aβ*56, Aβ1-42, total tau (T-tau), and phospho-tau (ptau-181).

Results

We observed that Aβ trimers and Aβ*56 levels increased with age, and within the Unimpaired group were elevated in subjects with T-tau/Aβ1-42 ratios above a cutoff that distinguished the Unimpaired group from AD subjects. In the Unimpaired group, T-tau and ptau-181 were found to correlate strongly with Aβ trimers and Aβ*56 (r > 0.63), but not with Aβ(1-42) (-0.10 < r < -0.01). The strong correlations were found to be attenuated in the Impaired group.

Conclusions

In cognitively intact older adults CSF Aβ trimers and Aβ*56 are elevated in individuals at risk for AD, and show stronger relationships with tau than does Aβ1-42, a surrogate for amyloid deposition. These data support the hypothesis that Aβ trimers or Aβ*56 are pathogenic in preclinical AD. However, the attenuation of these associations in symptomatic subjects suggests an uncoupling between the Aβ oligomers and tau in later stages of AD.

Introduction

Alzheimer's disease (AD), the most common form of dementia in the elderly, threatens to become a major public health hazard as more people live beyond the eighth decade of life. Treatments that are administered after the symptoms of dementia appear have not effectively altered the course of illness, possibly because the pathophysiological processes causing neuronal loss in demented patients have become self-sustaining in ways that are difficult to curb. To improve our ability to detect incipient AD and develop treatments that prevent it from progressing to overt AD, it is important to identify and understand the molecular basis of the earliest pathophysiological abnormalities.

Although the exact cause of AD is unknown, it is widely believed to be triggered by abnormal aggregates of amyloid-β (Aβ), which collaborate with the microtubule binding protein tau to produce widespread neuronal degeneration and dysfunction (reviewed in ¹). The disease process begins one or two decades prior to the onset of neuron loss or overt symptoms.^2-4 There have been many investigations of various Aβ species present in end-stage AD (reviewed in ⁵), but our understanding of Aβ species in the initial stages of AD remains limited. One species of Aβ with the potential to be pathogenic in the initial, preclinical stages of AD is Aβ*56, a soluble 56 kilodalton (kDa) oligomer that correlates with memory dysfunction independently of neuron loss or plaque deposition in several lines of mice over-expressing Aβ,^6-8 and disrupts cognition when injected into the cerebral ventricles of young, healthy rats.^7,9

It has been suggested that Aβ*56 consists of four Aβ trimers, a conjecture that is based on its pattern of dissociation in a polar solvent.⁷ Aβ trimers were the only oligomeric species present in mice prior to the appearance of Aβ*56 in Tg2576 mice modeling preclinical AD,⁷ providing additional support for the idea that they are basic compositional units for higher order oligomers. Interestingly, low levels of Aβ trimers were the only Aβ oligomers found in the brains of children and adolescent humans (S. Lesne and K. Ashe, unpublished data). Aβ trimers were not shown to disrupt cognition when applied to rats,⁹ and do not correspond well to memory deterioration in mice.⁷ There is at least one report of Aβ trimers disrupting neural function in vitro,¹⁰ but their presence in children and adolescent humans who, presumably, are free from AD suggests that they are benign at low concentrations.

Longitudinal studies in humans indicate that subtle memory deficits as well as functional and metabolic brain abnormalities, presumably the result of synaptic dysfunction, precede neuron loss at least a decade before overt cognitive symptoms emerge.^2-4 The 42-amino acid Aβ isoform Aβ1-42, T-tau and tau phosphorylated at threonine 181 (p-tau181), have emerged as CSF biomarkers for the preclinical stages of AD. Not only are CSF T-tau and p-tau181 elevated in cognitively normal elderly individuals who later go on to develop AD,¹¹ their levels also robustly correlate with glucose hypometabolism,¹² making them presumptive biomarkers for synaptic dysfunction during the preclinical stages of AD.

In this study, we measured Aβ trimers and Aβ*56 in lumbar CSF using a highly sensitive and specific immunoblot assay. Our aims were to evaluate the effects of aging and AD, and to examine relationships to CSF tau. Our goal was to obtain support for the hypothesis that specific Aβ oligomers induce changes in tau in the preclinical stages of AD, thereby advancing our understanding of how Aβ may be involved very early in its pathogenesis.

Subjects and Methods

We obtained CSF samples from 107 subjects, including 10 young, normal controls (YNC) and older subjects consisting of 26 with AD, 22 with mild cognitive impairment (MCI), and 49 age-matched healthy controls.¹³ We made clinical diagnoses of AD according to the National Institute of Neurological and Communicative Disorders and Stroke (NINCDSADRDA) criteria,¹⁴ and MCI according to the Petersen criteria.¹⁵ We combined the AD and MCI subjects to form the Impaired group. The Unimpaired control group consisted of 49 age-matched individuals without signs of psychiatric or neurologic disease, malignant disease, or systemic disorders (e.g., rheumatoid arthritis or infectious disease). Cognition in the Unimpaired and Impaired groups was assessed using the Mini-Mental State Examination (MMSE).

We obtained CSF samples by lumbar puncture (LP) in the L3/L4 or L4/L5 inter-space. The CSF samples were collected in polypropylene tubes, gently mixed to avoid possible gradient effects, centrifuged, aliquoted and stored at -80°C pending biochemical analyses, without being thawed or re-frozen. All procedures were approved by the institutional review board (IRB) of the University of Göteborg.

Levels of CSF T-tau, p-tau181, and Aβ1-42 for all but two specimens were obtained using ELISA's, as described in the Supplementary text.

To detect Aβ trimers and Aβ*56 in the CSF, we optimized the sensitivity of our immunoprecipitation and immunoblot protocols to measure and quantify Aβ trimers and Aβ*56 in 240 μL of CSF (detailed methods in Supplementary text). We received 750 μL of CSF from each subject, permitting us to perform each measurement in triplicate. The percent coefficient of variance of triplicate measurements was 13% for Aβ trimers and 15% for Aβ*56. All measurements were performed blind to the clinical and demographic characteristics of the subjects.

All statistical analyses were performed and graphs plotted using SPSS version 19. Non-normal data were log-transformed. Chi-square and Student's t-tests were used to compare demographic and clinical data and CSF measurements. Best fitting exponential curves were generated and provided for illustration purposes only.

Results

DEMOGRAPHICS OF STUDY PARTICIPANTS

The demographic and clinical characteristics of the subjects are presented in Table 1.

Table 1.

Subject demographic characteristics

Characteristics	YNC	Unimpaired	MCI	AD	p *
n	10	49	22	26
Mean age at LP (SD), yr	41.7 (4.9)	64.8 (7.9)a	65.3 (8.7)	68.9 (6.5)	0.094
Sex, F/M (%F)	7/3 (70)	32/16 (65)b	14/8 (64)	17/9 (65)	0.902
Mean MMSE score (range, 0-30) (SD)	NA	29.3 (0.97)c	28.2 (1.66)	21.6 (4.59)d	<0.001

^*comparison of Unimpaired, MCI, and AD groups

^an=46

^bn=48

^cn=47

^dn=25

AD = Alzheimer's disease; LP = lumbar puncture; MCI = Mild cognitive impairment; MMSE = Mini Mental State Examination; SD = standard deviation; YNC= Young normal controls.

INCREASED CSF Aβ TRIMERS AND Aβ*56 WITH AGING

To determine the effects of aging on the levels of CSF Aβ trimers and Aβ*56, we calculated Pearson correlation coefficients between these oligomers and age in the combined YNC and Unimpaired groups. The correlation coefficients were 0.31 between age and Aβ trimers (P = 0.02; n = 59) and 0.25 between age and Aβ*56 (P = 0.07) (Figure 1A). We also compared the levels of the oligomers in Unimpaired subjects ≥65 years of age and in younger subjects between 35 and 50 years of age, and found 9.2% higher levels of Aβ trimers (P < 0.01) and 6.9% higher levels of Aβ*56 (P = 0.04) in the older subjects (Figure 1B). These results suggest that CSF Aβ trimers and Aβ*56 increase with aging, but do not distinguish between an age-dependent phenomenon that is related to AD from one that is unrelated to AD.

Figure 1.

Alzheimer's-related, age-dependent increase in CSF Aβ*56 and Aβ trimers. In cognitively normal subjects CSF Aβ*56 and Aβ trimers increased with aging (A, B), and were higher in subjects with T-tau/Aβ1-42 ratios above 1.005 (C), which provided a sensitivity of 92.3% and a specificity of 89.6% for distinguishing cognitively normal older adults from AD subjects.

CSF Aβ TRIMERS AND Aβ*56 HIGHER IN SUBJECTS AT RISK FOR AD

To determine whether elevated levels of Aβ trimers or Aβ*56 in cognitively intact subjects were related to AD, we identified subjects at high risk for AD based on CSF T-tau/Aβ1-42 ratios, which have been shown to be elevated in AD and in cognitively intact individuals that go on to develop AD ^16-18. Using ROC (receiver operating characteristic) analysis, we demonstrated that a T-tau/Aβ1-42 cutoff of 1.005 provided a sensitivity of 92.3% and a specificity of 89.6% for distinguishing the Unimpaired group from AD subjects. We found that subjects in the Unimpaired group with T-tau/Aβ1-42 ≥ 1.005 had 10.7% higher levels of Aβ trimers (P < 0.01) and 10.9% higher levels of Aβ*56 (P < 0.01) (Figure 1C). These results support the hypothesis that age-dependent elevations in Aβ trimers and Aβ*56 are related to AD.

CSF Aβ AND TAU IN COGNITIVELY NORMAL OLDER ADULTS

To examine relationships between CSF Aβ oligomers and tau in cognitively normal older adults, we calculated age-adjusted partial correlation coefficients and p-values in the Unimpaired group. Correlation coefficients were 0.64 between Aβ*56 and T-tau (P <0.01) (Figure 2A), and 0.70 between Aβ*56 and p-tau181 (P < 0.01) (Figure 2B). The correlation coefficients were 0.65 between Aβ trimers and T-tau (P < 0.01) (Figure 2C), and 0.71 between Aβ trimers and p-tau181 (P < 0.01) (Figure 2D).

Figure 2.

Strong relationships between CSF Aβ*56, Aβ trimers and tau in cognitively normal older adults. In cognitively normal older adults, strong relationships were observed between CSF Aβ*56, Aβ trimers and T-tau (A, C) and p-tau181 (B, D), but not between Aβ1-42 and T-tau (E) or p-tau181 (F).

The relationships between CSF Aβ1-42 and tau were also examined by calculating age-adjusted partial correlation coefficients and p-values. Correlation coefficients were -0.10 between Aβ1-42 and T-tau (P = 0.50) (Figure 2E), and -0.01 between Aβ1-42 and p-tau181 (P = 0.95) (Figure 2F).

These results suggest that in cognitively normal older adults there are strong relationships between CSF tau and both Aβ trimers and Aβ*56, which contrasts with the absence of relationships between Aβ1-42 and tau.

CSF Aβ AND TAU IN COGNITIVELY IMPAIRED OLDER ADULTS

The relationships between CSF Aβ oligomers and tau in subjects with MCI/AD were assessed by calculating age-adjusted partial correlation coefficients and p-values in the Impaired group. Correlation coefficients were 0.14 between Aβ*56 and T-tau (P = 0.35) (Figure 3A), 0.19 between Aβ*56 and p-tau181 (P = 0.210) (Figure 3B), 0.37 between Aβ trimers and T-tau (P =0.01) (Figure 3C), and 0.44 between Aβ trimers and p-tau181 (P < 0.01) (Figure 3D). The relationships between CSF Aβ1-42 and tau were similarly assessed. Correlation coefficients were -0.40 between Aβ1-42 and T-tau (P < 0.01) (Figure 3E), and -0.33 between Aβ1-42 and ptau181 (P = 0.025) (Figure 3F).

Figure 3.

Attenuated relationships between CSF Aβ*56, Aβ trimers and tau in impaired adults. In subjects with MCI or AD, relationships between the CSF Aβ*56, Aβ trimers and T-tau (A, C) or p-tau181 (B, D) were attenuated relative to age-matched controls (see Figure 2A-D), but were stronger between Aβ1-42 and tau (E, F).

These results suggest an attenuation of the relationships between CSF tau and both Aβ oligomers in symptomatic individuals with MCI/AD compared to their respective relationships in cognitively normal individuals, while a moderate relationship appeared between CSF tau and Aβ1-42 in the symptomatic group.

Comment

We measured two Aβ oligomers, Aβ*56 and Aβ trimers in CSF, and found age-dependent increases in Aβ oligomers in cognitively normal adults, and elevated levels of both oligomers in subjects that were at greater risk for AD. We found strong positive relationships between the Aβ oligomers, but not Aβ1-42, and tau in cognitively normal older adults, and attenuations of these relationships in MCI/AD. Since Aβ1-42 is a surrogate for Aβ fibril deposition,¹⁹ these findings suggest that in the years prior to the onset of overt symptoms, one or both of the Aβ oligomers, but not fibrillar Aβ, is coupled to tau, but that this coupling is weakened or broken when AD advances to symptomatic stages. The uncoupling in MCI/AD is interesting in light of the consistent failure of experimental Aβ therapies to alter the clinical course of patients with MCI or AD, which has prompted a shift in the timing of Aβ therapies to asymptomatic subjects.²⁰ Knowing which Aβ species to target in asymptomatic subjects may enhance the success of future drug development.

To our knowledge, this is the first report in which the levels of specific Aβ oligomers were measured in CSF in cognitively normal older adults and shown to correlate with tau. It is interesting to speculate that the strengths of the correlations between tau and the Aβ oligomers (r > 0.63) or Aβ1-42 (-0.10 < r < -0.01) reflect the relative participation of the respective Aβ species in the molecular events causing abnormalities in tau or synaptic dysfunction in preclinical AD. If the speculation is true, it suggests that targeting Aβ fibrils alone will not prevent preclinical AD from progressing to symptomatic AD. In addition, it indicates that tracking amyloid deposition or CSF Aβ1-42 may not be useful as measures of how well Aβ therapies are blocking the ability of Aβ to engage tau or disrupt synaptic function.

Although animal studies show more evidence supporting a pathogenic role for Aβ*56 than for Aβ trimers,^1,7,9 we do not know whether Aβ trimers or Aβ*56 are more likely to play a pathogenic role in humans. The data in this study do not resolve this question, since the relationships between tau and each oligomer were equivalently robust. The strong relationships between Aβ trimers and tau may indicate a pathogenic role for trimers or reflect a molecular equilibrium between Aβ*56 and Aβ trimers, which would be expected to exist if Aβ trimers cluster to form Aβ*56 as is suspected.⁷ Additional molecular studies in animals and cells and longitudinal clinical studies in humans may better define the pathogenic roles of these oligomers and elucidate their molecular interactions with tau.