Background
Alzheimer's disease is characterized not only by the presence of parenchymal amyloid deposits and intracellular tangles but also by the presence of amyloid deposits in the vasculature, a condition referred to as cerebral amyloid angiopathy (CAA). The CAA observed in both Alzheimer's disease patients [
1] and some of the transgenic mouse models [
2] is primarily composed of the shorter form of amyloid beta (Aβ), Aβ
1–40, while the majority of amyloid deposits in the parenchyma are composed of Aβ
1–42, although the compact amyloid deposits also contain Aβ
1–40.
Anti-Aβ immunotherapy has been considered as a potential treatment for Alzheimer's disease for some time [
3,
4]. Active immunization with a vaccine including Aβ
1–42 fibrils progressed to human clinical trials where its administration was suspended due to meningoencephalitits in a subset of patients [
5]. To date there have been pathology reports on two patients who participated in the trial and subsequently died [
6,
7]. Both reports note that while the numbers of parenchymal amyloid deposits appeared lower than expected in these cases, the CAA in these patients did not appear outside the normal range for Alzheimer's disease. In addition, one report mentioned multiple cortical hemorrhages and the presence of hemosiderin around the CAA vessels [
7].
Given the adverse reactions to the active immunization, the irreversibility of such procedures and the variable antibody response to vaccines in older individuals [
8], passive immunization against the Aβ peptide emerged as an alternative immunotherapeutic strategy. Studies in young and middle aged APP-transgenic mice have reported significant amyloid reductions with passive immunization [
9‐
11]. Such treatments also demonstrate rapid improvements of memory function in APP-transgenic mice, sometimes without detectable reductions in amyloid [
12‐
14]. Most recently, intracranial administration of anti-Aβ antibodies has been shown to not only remove Aβ but also clear, early-stage, hyperphosphorylated-tau aggregates [
15]. Importantly, in the only prior study evaluating adoptive antibody transfer in older APP-transgenic mice, Pfeifer
et al. [
16] reported a doubling of cerebral microhemorrhages associated with significant reductions in amyloid burden after administration of an N-terminal specific anti-Aβ antibody.
Discussion
Earlier studies with vaccines against the Aβ peptide demonstrated protection from the learning and memory deficits associated with amyloid accumulation in APP-transgenic mice [
14,
19]. Passive immunization protocols with anti-Aβ antibodies also produced cognitive benefits, in some cases even in the absence of significant reduction in amyloid burden [
12,
13]. Our recent work found that 3 months of anti-Aβ treatment of 18-month-old APP-transgenic mice improved spontaneous alternation performance on the Y-maze [
14]. In the present work we confirmed that passive anti-amyloid immunotherapy can reverse spatial learning deficits in APP-transgenic mice and that this benefit of immunotherapy is retained, even in aged mice (26 and 28 months old at testing) with long-established amyloid pathology.
Additionally, we describe a more rapid means of testing spatial reference memory to reveal learning and memory deficits in APP-transgenic mice. This two-day version of the radial arm water maze included greater spacing of individual trials (mice spent time in their home cage after every trial), combined with less spacing of aggregate trials (fifteen trials per day rather than four or five) to facilitate learning of platform location in the nontransgenic mice, with a clear absence of learning in the age-matched transgenic mice.
A substantial reduction in total Congophilic amyloid deposits was observed in old APP-transgenic mice treated with anti-Aβ antibodies for 2 or more months. This measurement of total Congo-red staining included both parenchymal and vascular amyloid staining. When we analyzed the sections for only vascular amyloid (CAA) we found that this measure was significantly increased following 2, 3 and 5 months of anti-Aβ antibody treatment. The remaining parenchymal amyloid load was almost completely eliminated with this antibody approach. Clearly, because total amyloid load was significantly reduced not all amyloid was shifted into the vessels; but, it appears that at least some of the Congophilic material was redistributed to the vasculature. At the present time the mechanism for this redistribution is unclear. However, one possibility is that the microglia associated with the antibody-opsonized amyloid, either by phagocytosis or surface binding, and transported the material to the vasculature, possibly in an attempt to expel it. We and others have shown evidence for microglial involvement in the removal of amyloid using both intracranial anti-Aβ antibody injections [
11,
21] and systemically administered anti-Aβ antibody treatment [
14], as well as
ex vivo studies [
10,
22]. Here we also report our impression that microglia surrounding CAA vessels in immunized mice expressed more CD45 than control transgenic mice. This increased expression could be due to either increased expression in the same number of microglial cells or an increased number of microglial cells in these animals. It is feasible that this microglial activation was simply in reaction to the presence of increased amyloid in the blood vessels. However, it is equally likely that microglia activated by the opsonized material migrated to the vessels for disposal of the amyloid.
Cerebral amyloid angiopathy (CAA) is defined as the deposition of congophilic material in meningeal and cerebral arteries and arterioles (capillaries and veins can also show CAA but less frequently), and it occurs to some extent in nearly all Alzheimer's disease patients [
23]. Severe CAA, affecting about 15% of cases, can be associated with both infarction and hemorrhagic injury [
24,
25]. It has also been shown that the severity of CAA can be directly linked to the severity of dementia in Alzheimer's disease patients [
26].
In the current study we found a significantly increased number of microhemorrhages in the brain as detected by Prussian-blue staining, associated with the increase in CAA following passive immunization. Another transgenic mouse model of amyloid deposition, the APP23 mice, have been shown to deposit amyloid in both brain parenchyma and blood vessels and show a CAA associated increase in spontaneous cerebral hemorrhages [
27]. Moreover, Pfeifer
et al. [
16] showed that these spontaneous hemorrhages were significantly increased following 5 months of passive immunization of 21-month-old APP23 mice using an anti-Aβ antibody with an N-terminal epitope, similar to those typically developed in active immunization with vaccines [
4,
28,
29]. When young mice (6 months of age) were immunized following the same protocol, no hemorrhages were observed. More recently, DeMattos
et al. [
30] showed that passive immunization with an N-terminal antibody (3D6: directed against amino acids 1–5 of Aβ) of PDAPP transgenic mice also resulted in significantly increased microhemorrhage. They were unable to detect increased microhemorrhage with a mid-domain antibody (266: directed against amino acids 13–28 of Aβ). Notably, antibody 266 fails to bind Aβ deposited in CAA vessels or amyloid plaques [
31]. Importantly, Ferrer
et al. [
7] noted the presence of CAA and microhemorrhage in the brain of one patient that participated in the Aβ-vaccine trial, even though the parenchymal amyloid appeared lower than expected. Also, Nicoll
et al. [
6] noted that CAA appeared unaffected in the brain of another patient that participated in the Aβ-vaccine trial.
It remains to be determined whether these observations regarding increased CAA and microhemorrhage in transgenic mice are relevant to trials of passive immunotherapy in humans. It should be noted that, in spite of extending the period of immunotherapy to 5 months, there was no discernable loss of the cognitive benefits of immunotherapy in the transgenic mice, all of whom showed increased microhemorrhage. While the observation that antibody 266 does not result in vascular leakage encourages testing of this idiotype, data from the Zurich cohort of the Aβ vaccine trial argue that brain-reactive antibodies may be important for cognitive benefits [
32].
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
DMW treated the mice, performed the behavioral analysis, processed the tissue and performed pathological analyses, and drafted the manuscript. ARojiani evaluated slides and provided expert opinion regarding CAA and microhemorrhage. ARosenthal and SS developed, produced and purified the antibodies used in the studies. MJF performed DNA extraction and PCR for genotyping of the mice. MNG oversees the breeding colony generating mice for the studies, collected samples from the mice and assisted in editing the manuscript. DM conceived the design of the study, guided data interpretation and assisted in editing the manuscript.