Use of bomb pulse carbon-14 to age senile plaques and neurofibrillary tangles in Alzheimer’s disease

Abstract

The time course of formation of neurofibrillary tangles (NFT) and senile plaques (SP) in Alzheimer’s disease (AD) brain is unknown. Above ground nuclear weapons testing in the late 1950s and early 1960s led to significantly increased levels of ¹⁴C in the atmosphere and carbon cycle. Because the amyloid beta peptide of SP and paired helical filaments of NFT, once formed, are relatively resistant to degradation, ¹⁴C levels observed in SP and NFT should reflect their year of formation. The purpose of this study was to develop a method to determine whether ¹⁴C levels could be used to define NFT and SP ages. Using accelerator mass spectrometry to measure bomb-pulse ¹⁴C levels, we determined the average age of formation of isolated SP and NFT fractions in bulk brain samples of 6 AD subjects. Although preliminary, the results demonstrate that it is possible to use bomb pulse ¹⁴C to determine the average year of formation of NFT and SP in the brain in AD. In addition, the data show that these structures, once formed, have a much slower carbon turnover rate than normal brain and are not in a formation/enzymatic degradation equilibrium.

Introduction

Alzheimer’s disease (AD), an age-associated dementing disorder, is the most common form of adult onset dementia and the fourth leading cause of death in the United States [23]. The mean duration from diagnosis to death is 8.4 years with a range of 1 to 21 years [22]. The major histopathologic features of AD are senile plaques (SP), composed primarily of amyloid beta peptide (Aβ) and neurofibrillary tangles (NFT), composed of paired helical filaments containing hyperphosphorylated tau. These pathologic markers remain the key criteria for the neuropathologic diagnosis of AD [34]. Both of these filamentous proteins are essentially insoluble [40].

There is a paucity of knowledge about the time course involved in the formation of NFT and SP. Because no current diagnostic procedure can detect the presence of these pathologic features in living subjects, it is unclear whether Aβ deposition occurs initially and influences neuron degeneration and NFT formation, or if NFT occur first followed by Aβ deposition, or they occur concomitantly. Although recent studies suggest that NFT formation is a primary event [12], [38], these autopsy studies only represent an endpoint measure and do not provide definite temporal information. The temporal relationship between these pathologic features and cognitive impairment is also unclear. Current evidence indicates that NFT and SP may exist in aged prospectively evaluated nondemented subjects and their formation may begin even before age 30 [7], [8], [37]. Studies of the relationship between NFT and SP densities and clinical parameters have been contradictory with nearly equal numbers of reports supporting a positive correlation between severity of cognitive impairment and degree of SP [4], [10], [13], [16], [39] or NFT [1], [2], [3], [14], [27], [31], [33], [51] accumulation as those that do not [11], [26], [30], [31], [35], [41], [46], [47]. Hippocampal NFT formation may be a continuous process throughout the course of AD [5]. The duration of NFT survival in the hippocampus has been estimated to be from 3.4 years [6] to 20 years [32] in AD and 4.7 years in the amygdala in patients with Down syndrome [50]. The average duration of NFT in the hippocampus of patients with Parkinsonian dementia complex of Guam is 2.5 years [42].

Carbon-14 is naturally produced in the atmosphere through cosmic ray interaction with nitrogen [44]; however, atmospheric testing of nuclear weapons in the early 1960s doubled the concentration of this radioisotope as shown in Fig. 1 (adapted from [20], [21], [24], [25], [36]). After the peak in 1963, ¹⁴C levels began decreasing exponentially to the contemporary level of 110% of that of 1950 (Fig. 1) and may reach prebomb levels within 20 years. The observed decrease is due to exchange of CO₂ in environmental reservoirs and the introduction of ¹⁴C depleted CO₂ from burning fossil fuels. As previously demonstrated in analyses of gallstones [15], the elevation in ¹⁴C above the contemporary level can be used to date biologic samples in which carbon does not turn over. As time progresses, lower levels of incorporated ¹⁴C will make the technique less useful as a method of dating NFT and SP in AD brain. However, because NFT and SP may be formed years before the onset of symptoms, it is possible that subjects currently coming to autopsy would have NFT and SP formed during a time earlier on the bomb pulse curve and thus demonstrate ¹⁴C levels that are more amenable to measurement. The ¹⁴C content of any tissue is a function of dietary ¹⁴C levels and the residence time of carbon in that tissue. Several studies of ¹⁴C in human tissues during the late 1960s and early 1970s demonstrated incorporation of bomb-pulse ¹⁴C into a variety of organs with variable turnover rates [9], [19]. The slowest turnover rates are observed in collagen and bone, whereas brain exhibits the fastest [9], [19]. The aim of this study is to develop a method using ¹⁴C incorporation to determine the average age of NFT and SP in the brain in AD. Because NFT and SP are essentially insoluble [40], we hypothesized that they turn over carbon at a slow rate and that the ¹⁴C levels in these structures would be indicative of their average age of formation. Inherent in this approach is the assumption that rates of degradation of NFT and SP are similar and considerably slower than non-involved tissue. It is possible that if turnover rates of NFT and SP are significantly different, leading to changes in ¹⁴C, the apparent ages may be skewed relative to one another. However, the results obtained using this approach allow an assessment of when, on average, the pathologic features of the AD brain are being formed and provide more information regarding the ages of NFT and SP than can be obtained using currently available techniques. Although current evidence suggests that soluble amyloid in the brain of mice expressing mutant amyloid precursor protein is degraded, there is no evidence to suggest that neuritic SP in the AD brain are significantly degraded. Because of the severity of the isolation procedure, it appears from electron microscopy that the ¹⁴C measured in this study results from neuritic SP and not from diffuse SP, which would likely be degraded and lost during processing.

We present here the average year of formation of isolated NFT and SP fractions from bulk brain samples of cerebral hemispheres of 6 AD subjects. Ages of carbon in the cerebellum (CER) were also measured as an internal control representative of brain actively turning over carbon and as a method to demonstrate that the observed results are not due to a decrease in cerebral metabolic rate.