Background
The progressive accumulation of senile plaques composed of β-amyloid (Aβ) is a main pathological hallmark of Alzheimer’s disease (AD), the most common dementing disorder in the elderly. The Aβ accumulation promotes synaptic loss and neuronal degeneration apparently by activating microglia, the resident macrophages of the brain [
1‐
4]. In the healthy brain, microglia cells are long-lived cells using highly motile processes to survey parenchymal territory for the presence of pathogens and cell debris. In addition, microglia secrete factors that support neuronal survival and synaptogenesis [
5]. In the early stages of AD, microglia migrate towards amyloid deposits and express certain cell-surface receptors to promote the clearance and phagocytosis of Aβ [
6‐
8]. Furthermore, deficits in microglia activation favor accelerated amyloid deposition [
9]. However, it has been hypothesized that microglial reactions are overwhelmed by the massive Aβ deposition in later AD stages [
10,
11]. This suggestion is supported by the finding that plaque-associated microglia ultimately show decreased expression of Aβ-binding receptors, which leads to a significant reduction in Aβ degradation by microglia in the aging brain [
11]. Moreover, plaque-associated microglial cells show a threefold higher mortality rate compared to non-plaque-associated microglia in vivo [
12].
The use of small animal positron emission tomography (μPET) with Aβ tracers enables longitudinal investigations of cerebral amyloidosis in rodents in vivo [
13,
14]. Confirmation of the hypothesis of a ceiling effect in microglial reactions has been hampered by the technical difficulty in following the fate of aging microglial cells in living mice. The past decade has seen the introduction of Aβ-μPET in rodents [
15,
16], using the same radioligands employed in the clinical routine for the differential diagnosis of AD [
17,
18]. A series of PET radiotracers targeting the microglial marker 18-kDa translocator protein (TSPO), formerly known as the peripheral benzodiazepine receptor (PBR) [
19‐
23], has been developed in recent years.
The basal availability of TSPO binding sites is low in the healthy living brain (21), such that local upregulation presents a sensitive marker for the detection of microglial activation in afflicted brain regions [
24‐
26]. This is supported by findings of elevated TSPO expression in the hippocampus and the frontal, temporal, and parietal cortices of postmortem AD brain [
25,
27,
28]. Our group has recently established cross-sectional dual tracer μPET imaging of Aβ and TSPO in transgenic AD mouse models [
29]. Given this background, we aimed in the present longitudinal Aβ/TSPO double tracer μPET study to explore the longitudinal association between amyloidosis and microglial response during aging of an amyloid mouse model in vivo. By using mice with a range of baseline age, we were able to perform correlation analysis with the longitudinal biomarker progression over 7 months. Final immunohistochemistry supported the interpretation of μPET results by mapping of individual plaques and microglial cells.
Discussion
We present the first longitudinal in vivo dual tracer μPET study aiming to directly compare the time courses of microglial activation and fibrillar amyloidosis with age in a transgenic amyloid mouse model. Our results clearly indicate that both biomarkers increase with age, but that microglial activation is disproportionately elevated at an early age and seems to saturate relative to amyloidosis, which continues to progress. Detailed immunohistochemical analyses revealed a significant decrease of microglial brain fraction around amyloid plaques with increasing plaque radius to be the cellular correlate of our in vivo μPET findings. Moreover, we found that the microglia brain fraction in the plaque-free brain parenchyma of APP-SL70 mice was lower than in wt mice. This depletion of microglial cells distal to plaques is likely related to the massive microglial migration towards zones of fibrillar Aβ deposition [
6,
39].
With this serial in vivo study, we aimed to investigate longitudinal relationships between microglial activation and amyloidosis during the life course of the APP-SL70 AD mouse model. We performed dual-tracer small animal μPET examinations with the novel tracer [
18F]-GE180 for TSPO and [
18F]-florbetaben for fibrillar amyloidosis, in conjunction with immunohistochemical analyses after the final imaging studies. To enable a reliable comparison of the relationship between the two μPET readouts, we took pains to develop a standard procedure for quantification, entailing a biphasic calculation method: First, we calculated standardized tracer specific
Z-scores of individual mice at different time points by considering mean and standard deviation values of age-matched wt mice. We next calculated differences between TSPO- and Aβ-μPET Z-scores as a measure of microglial activation relative to fibrillar amyloidosis. We deemed this calculation of a difference score to be more reliable than a ratio method, as values close to zero would potentially have distorted the results at the onset of fibrillar amyloidosis in young mice. The two radioligands have different sensitivities for their specific targets, resulting in distinct detection thresholds unequal magnitudes of signal alterations during the progression of the AD model. To address these issues, we used the standardized
Z-score calculation as our main endpoint. In fact, our analysis showed positive
Z-score differences at early ages of APP-SL70 mice, which suggest that microglial activation precedes fibrillar amyloidosis at the onset of amyloid pathology. However, even with standardized
Z-scores, there remains some possibility that this effect may be related to a higher sensitivity of the TSPO ligand to its target compared to the applied Aβ tracer. In contrast, due to its baseline dependency, the longitudinal decrease of the
Z-score difference towards late ages is a rather compelling readout unlikely to be biased by possible tracer sensitivity differences. Furthermore, ceiling effects are unlikely as far higher magnitudes of TSPO activation and amyloidosis can be detected with these tracers in other circumstances [
31,
40]. Thus, our serial dual tracer μPET imaging proves that microglial activation saturates during an ongoing fibrillar amyloid deposition in this mouse model (Figs.
2 and
3). Our PET findings are absolutely in line with a recently observed plateau during TSPO PET imaging in aged APP23 mice by the same radioligand [
41]. The results are also in line with findings for other biomarkers of microglial function, i.e., the peak in sTrem2 levels in cerebrospinal fluid of patients with mild cognitive impairment [
42], followed by a drop in patients who have converted to dementia [
43]. Even more importantly, computed longitudinal courses of sTrem2 in individuals with dominantly inherited AD decrease after symptom onset whereas amyloid deposition continues to progress [
44], thus concurring with the presently observed relations between TSPO expression and fibrillar amyloidosis in aging APP-SL70 mice.
Although an important strength of μPET lies in its fitness for longitudinal monitoring and target quantification, molecular imaging has limitations in spatial resolution and in its applicability for resolving mechanistic processes. For this reason, we supplemented μPET with a detailed immunohistochemical study of activated microglia and histological staining of fibrillar Aβ, which together supported automatized volumetric computations. Our data clearly indicate that microglia fraction adjacent to plaques declines with increasing plaque size (Fig.
4d). Given that plaque size but not density increases with advanced age (Fig.
4b,
c), it seems obvious that the microglial activity must decrease relative to fibrillar amyloidosis over time. We validated these findings by comparing mid- and late aged APP-SL70 groups, concluding that the decreasing microglial brain fraction with increasing plaque size is consistent with our μPET results in vivo. Since a recent study showed that brain location of microglia is a relevant factor for its morphological classification [
45], our specific analysis of frontal cortical microglia cells in wt and APP-SL70 mice seems appropriate as it matched to the regional PET analysis.
So far, it remains unclear when and why microglial activity decreases adjacent to plaques. It is known that microglia migrate within 1–2 days towards newly formed amyloid deposits, where they promote the clearance and phagocytosis of Aβ by expressing certain cell-surface receptors [
6‐
8]. However, during disease progression, plaque-associated microglial cells show a decrease in fiber mobility [
46], lower expression of Aβ-binding receptors [
11], and moreover also show a threefold higher mortality rate compared to non-plaque associated microglial cells [
12]. Nonetheless, the same in vivo study reported a threefold higher proliferation rate of microglia distal to plaques in AD compared to wt mice, suggesting that new microglial cells migrate from the periphery to the plaque border [
12]. However, we observed even lower microglial brain fraction distal to plaques of aged APP-SL70 mice compared to wt animals (Fig.
6). We conclude that the rate of microglia cell migration towards Aβ depositions in APP-SL70 mice eventually exceeds the rate of proliferation of microglia cells staying in the peripheral zone. With aging, this potentially leads to an exhaustion of microglial cells for migration towards Aβ depositions and, together with the increased rate of microglial cell loss around plaques [
12], this may explain the observed decrease in microglia brain fraction with increasing plaque radius.
As a limitation of this study, we note that the amyloid tracer does not distinguish soluble and oligomeric Aβ. Thus, we cannot disentangle if there is a stronger association between microglia and soluble or oligomeric proportions of Aβ, which could show different growth rates with aging. Furthermore, given the nature of our longitudinal PET design, we were not able to acquire immunohistochemistry from mice at younger ages but instead focused on the late stage of the disease. Proliferation rates, mortality, and spatial distribution of microglia during their whole life cycle should therefore receive attention in future cross-sectional designs.