The Arctic AβPP mutation leads to Alzheimer’s disease pathology with highly variable topographic deposition of differentially truncated Aβ
© Kalimo et al.; licensee BioMed Central Ltd. 2013
Received: 5 August 2013
Accepted: 5 August 2013
Published: 10 September 2013
The Arctic mutation (p.E693G/p.E22G)fs within the β-amyloid (Aβ) region of the β-amyloid precursor protein gene causes an autosomal dominant disease with clinical picture of typical Alzheimer’s disease. Here we report the special character of Arctic AD neuropathology in four deceased patients.
Aβ deposition in the brains was wide-spread (Thal phase 5) and profuse. Virtually all parenchymal deposits were composed of non-fibrillar, Congo red negative Aβ aggregates. Congo red only stained angiopathic vessels. Mass spectrometric analyses showed that Aβ deposits contained variably truncated and modified wild type and mutated Aβ species. In three of four Arctic AD brains, most cerebral cortical plaques appeared targetoid with centres containing C-terminally (beyond aa 40) and variably N-terminally truncated Aβ surrounded by coronas immunopositive for Aβx-42. In the fourth patient plaque centres contained almost no Aβ making the plaques ring-shaped. The architectural pattern of plaques also varied between different anatomic regions. Tau pathology corresponded to Braak stage VI, and appeared mainly as delicate neuropil threads (NT) enriched within Aβ plaques. Dystrophic neurites were scarce, while neurofibrillary tangles were relatively common. Neuronal perikarya within the Aβ plaques appeared relatively intact.
In Arctic AD brain differentially truncated abundant Aβ is deposited in plaques of variable numbers and shapes in different regions of the brain (including exceptional targetoid plaques in neocortex). The extracellular non-fibrillar Aβ does not seem to cause overt damage to adjacent neurons or to induce formation of neurofibrillary tangles, supporting the view that intracellular Aβ oligomers are more neurotoxic than extracellular Aβ deposits. However, the enrichment of NTs within plaques suggests some degree of intra-plaque axonal damage including accumulation of hp-tau, which may impair axoplasmic transport, and thereby contribute to synaptic loss. Finally, similarly as the cotton wool plaques in AD resulting from exon 9 deletion in the presenilin-1 gene, the Arctic plaques induced only modest glial and inflammatory tissue reaction.
KeywordsFamilial Alzheimer’s disease Arctic AβPP mutation β-amyloid peptide Mass spectrometry Truncation of Aβ Topography of Aβ Hyperphosphorylated tau Neuronal damage
Amyloid-β precursor protein
Antibody to indicated amyloid-β
Brain Net Europe
The Consortium to Establish a Registry for Alzheimer’s disease
Cerebral amyloid angiopathy
Enzyme linked immunosorbent assay
Familial Alzheimer’s disease
Glial fibrillary acidic protein
Matrix-assisted laser desorption/ionization-time of flight mass spectrometry
National Institute on Aging and Alzheimer’s Association
Periodic acid schiff
Pittsburgh compound B-positron emission tomography
Presenilin 1 with deletion of exon 9-mutation
Deposition of amyloid-β (Aβ) peptides and hyperphosphorylated tau (hp-tau) as neurofibrillary tangles (NFT), dystrophic neurites (DN) and neuropil threads (NT) are invariably found in the brains of patients with both sporadic and familial forms of Alzheimer’s disease (AD). The majority of the pathogenic mutations in the amyloid-β precursor protein (AβPP) gene (http://www.alzforum.org/res/com/mut/app/default.asp) results in either an increase in total Aβ or Aβ42/Aβ40-ratio and often in aggressive plaque pathology. The recently reported protective effect against AD of the p.A673T substitution in AβPP (p.A2T in Aβ peptide) further suggests that Aβ is pivotal for the disease development .
Distinct pathological features are seen in AβPP mutation carriers as well as in other early-onset familial forms of AD. For example, brains from carriers of mutations in exons 8 and 9 of the presenilin 1 gene (e.g. PS1 Δ9) harbour cotton wool plaques; large ball-like plaques without an amyloid core [2–4].
Similarly, there is a marked phenotypic variation also among patients with various AβPP mutations , which is exemplified by different substitutions in AβPP codon 693. For example, both the Dutch AβPP mutation (p.E693Q; reviewed in ) and the Italian mutation (p.E693K; [7, 8]) within the Aβ sequence cause amyloid angiopathy with intracerebral hemorrhages, whereas parenchymal AD pathology and dementia are subsidiary. Moreover, the Osaka AβPP deletion mutation (p.E693Δ) identified in a Japanese pedigree also causes a primarily dementing disease . The Arctic AβPP mutation (p.E693G; in Aβ-peptide p.E22G) was initially reported as a polymorphism of unclear pathogenic significance . Subsequently, the same mutation was found to segregate with AD in a Swedish family . Interestingly, the Arctic AβPP mutation increased the formation of large soluble Aβ oligomers/protofibrils [11, 12], while symptomatic carriers showed low CSF-Aβ42 levels but remained PIB-PET negative . The Osaka AβPP deletion mutation (p.E693Δ) also accelerated Aβ oligomerization  but it did not cause deposition of fibrillar Aβ in vivo, neither in transgenic mice nor in AD patients [9, 14].
Elevated intra- or extracellular levels of Aβ oligomers/protofibrils are believed to be of pathogenic significance and neurotoxic effects have been demonstrated both on cultured cells and in vivo. For example, intrathecal administration of Aβ oligomers in rats caused impaired learning  and extracellular accumulation of soluble dodecameric Aβ in the brains of AβPP transgenic mice impaired cognition independently of plaques or neuronal loss .
We have previously reported epidemiological and clinical as well as a limited description (based on two Arctic AD brains) of some neuropathological features resulting from the p.E693G mutation in AβPP . Apart from concluding that the clinical features of this mutation are compatible with AD, we identified ring-formed Aβ plaques but did not further study the overall neuropathology. In another, more recent, biochemically oriented study  we analysed the composition of Arctic Aβ plaques in the frontal and temporal cortex of two Arctic AD patients (patients Sw1 and Sw2 of this study) using biochemistry (including Aβ ELISA and MALDI-TOF and MALDI-imaging mass spectrometry) and complementary immunohistochemistry and electron microscopy. Aβ ELISA and mass spectrometry analyses on brain cortex samples from one of our patients (patient Sw2 of this study) with the Arctic AβPP mutation revealed deposition of a heterogeneous mixture of Aβ peptides with a significant contribution of N-truncated and N-terminally modified Aβ. Moreover, by applying mid-domain, N- and C-terminal specific Aβ antibodies we demonstrated in the temporal cortex (patient Sw2 of this study), the presence of targetoid plaques composed of both N- and C-terminally truncated Aβ .
Here, we have extended and made a comprehensive analyses of the neuropathology in Arctic AβPP mutation carriers, based on four autopsied brains. We have applied nine well-characterized antibodies to different epitopes spanning the Aβ molecule, including an antibody specific to the Arctic mutation p.E22G and two antibodies recognizing posttranslational modifications of glutamates 3 and 11 of Aβ (cyclization into pyroglutamate). Thus, we could define the pattern of differentially truncated and N-terminally modified Aβ deposition in different regions of the Arctic AD brains, and also correlate these to mass spectroscopic findings in cerebral cortex. In addition, we describe local effects of Aβ on neurons, the association of Aβ with other pathological features, such as accumulation of hp-tau, macro- and microglial reactions, and basic vascular alterations.
Patients and brain specimens
This study has been carried out in compliance with the Helsinki Declaration and it has been approved by the Regional ethical committee in Uppsala, Sweden (2009)/089; 2009-04-22 and 2005-103; 2005-2006-29.
The brains were routinely fixed in buffered 4% formaldehyde, within twelve hours post mortem and widely sampled for embedding in paraffin. For comparison we used brain samples from five AD patients with the cotton wool plaque-associated PS1Δ9 mutation .
Histopathology and immunohistochemistry
The list of antibodies used in immunohistochemistry and immunoprecipitation experiments
IBL, Hamburg, Germany
Covance, Berkeley, CA, USA
Novocastra, Newcastle, U.K.
Biosource/Invitrogen, Camarillo CA, USA
Aβ20-24 with Arc-mutation p.E22G
Lord et al. 2009 
Synaptic Systems, Göttingen, Germany
Antibodies to cellular alterations
Innogenetics, Zwijndrecht, Belgium
Glial fibrillary acidic protein
Dako, Glostrup, Denmark
mAb HLA-DP, DQ, DR
Wako, Osaka, Japan
rpAb cathepsin D
Dako (production discontinued)
After pre-treatment relevant for each antigen, the sections were incubated with the primary antibodies overnight at +4°C, followed by incubation with relevant secondary antibodies and visualization using the avidin-biotin-peroxidase method with diaminobenzidine as chromogen (Vectastain, Vector Laboratories, Burlingame, CA, USA). Neurons and their axons were double-labelled with a polyclonal Aβx-40 antibody and a monoclonal antibody to neurofilament, followed by Alexa 633 labeled anti-rabbit and Alexa 488 labeled anti-mouse secondary antibodies (Molecular Probes, Eugene, OR, USA). The details of antibody sources and specifications are listed in the Table 1.
For comparative immunohistochemical and mass spectroscopic analyses of Aβ in cortical plaques, samples of fresh frozen temporal cortex from patient Sw2 were immunoprecipitated (with antibodies to Aβ17–24 and Aβarc) and analysed by MALDI-TOF, as described in our previous study .
Patient Sw1 (IV:10): ApoE genotype 3/3. The brain weighed 1385 g. Gross examination revealed focal moderate atrophy of parietal superior lobules. There were no signs of infarcts or hemorrhages.
Patient Sw2 (IV:29): ApoE genotype 3/3. The brain weighed 1490 g. A mild degree of atrophy with dilatation of the ventricular system was seen in frontal, parietal and occipital lobes as well as in different parts of the temporal lobe, including gyrus parahippocampalis, hippocampus, and amygdala (Figure 1c). Brainstem and cerebellum had normal macroscopic appearance, apart from mild atrophy of the anterior part of vermis.
Patient Am1 (III:1): ApoE genotype 2/3. The weight of the brain was 822 g. There was a severe degree of atrophy in frontal, temporal and parietal cortices .
Patient Am2 (IV:1): ApoE genotype 2/3. After fixation, the brain weighed 1220 g and showed moderate cortical atrophy.
Microscopic pathology of Aβ deposition
The deposition of Aβ in human brain has been suggested to follow a distinct hierarchical sequence, classified as phases 1 to 5 . In the following, we present the structural and immunohistochemical findings in the anatomical regions corresponding to these phases. The pattern of Aβ deposits varied both with respect to the antibodies applied and to the different brain regions analysed. Furthermore, there were some noticeable differences between the four Arctic AD brains studied.
Cerebral neocortices (phase 1)
In H&E stained sections, senile plaques appeared as compact rounded structures with remarkably homogenous texture (Figure 1d). The plaques were devoid of an amyloid core, as shown by the absence of Congo red (Figure 1d, inset) and thioflavin S (not shown) positivity and thus resembled cotton wool plaques (Additional file 1: Figure S9) [2, 3]. With Bielschowsky silver impregnation (without gold enhancement) the plaques were moderately brownish with accentuation of peripheral parts and negative or weakly stained centres, giving the plaques a vaguely ring-like pattern (Figure 1e).
Staining with general Aβ antibodies
With the C-terminal abAβx-42 a majority of neocortical plaques in all four patients were ring-shaped (Figure 2c), as described in the first study on patient Sw1 , i.e. the weakly stained centres of larger plaques were surrounded by distinct immunoreactive coronas.
In patient Sw1 the ring pattern visualized with abAβx-42  was also clearly noticeable with abAβx-40, abAβ8–17, abAβ5–10, and abAβarc. The staining was progressively weaker and less distinct with the more N-terminal abs (Additional file 4: Figure S2a-g) and nearly negative with abAβ1–5, although with this antibody the small subpial plaques were still positive (Additional file 4: Figure S2f). No central accentuation with the N-terminal antibodies was observed (Additional file 4: Figure S2e-f).
In patient Am2 the staining pattern was more variable. In this patient’s frontal cortex it was similar as in patient Sw1, i.e. the more N-terminal abs also rendered the plaques ring shaped without intensely stained centres, although the staining was less distinct and much weaker (cf. Additional file 4: Figure S2). On the other hand, in Am2 patient’s temporal and occipital cortex many plaques displayed intensely stained centres, similarly to those found in patients Sw2 and Am1 (cf. Figure 2 and Additional file 3: Figure S1).
Staining with specific Aβ antibodies
In patient Sw1 almost all plaques were ring-shaped with the Arctic specific antibody abAβarc (Additional file 4: Figure S2g). In patients Sw2, Am1 and Am2 the staining pattern with abAβarc resembled that with the mid-domain abAβ17-24, although it was somewhat weaker (Figures 3b vs. 2e and Additional file 3: Figure S1g vs. S1c).
In patients Sw2 and Am1 the antibodies abAβ3pE and abAβ11pE showed that N-terminally truncated Aβ starting with pyroglutamate (Aβ3pE and Aβ11pE) colocalized in frontal cortical plaques, although the staining with abAβ11pE was weaker than with abAβ3pE (for Sw2 Figure 3c-d and for Am 1 Additional file 3: Figure S1h-i). Aβ3pE stained most plaques relatively homogeneously and much more extensively than the other N-terminal abAβ1–5 (cf. Figure 2h). Some strongly abAβ1–5 positive centres were also abAβ3pE positive (Additional file 3: Figure S1h). Moreover, we observed vague predominant deposition of Aβ11pE to the plaque centres (Figure 3d).
Correspondence between Aβ immunohistochemistry and mass spectrometry
Analysis by MALDI-TOF of Aβ peptides immunoprecipitated from Sw2 patient’s temporal cortex resulted in various peaks within the m/z range of 2000–5000 Da (Figure 3e and Additional file 5: Table S1). Noteworthy, several of the Aβ species (see labels) corresponded well with the predicted masses of AβpE species, as detected by specific Aβ antibodies (abAβ3pE and abAβ11pE; Figure 3c-d). The observed and predicted m/z values with their relative intensities, corresponding to the peaks shown in Figure 3e are presented in Additional file 5: Table S1.
Allocortical brain regions (phase 2)
Plaques in hippocampus were not as easily discernible with H&E staining as in neocortex–except for those located in dentate gyrus, where they were found to displace granular cells (Additional file 6: Figure S3a). In adjoining occipito-temporal cortex the pattern was similar as elsewhere in cerebral cortex. Although with Bielschowsky silver impregnation, both DNs and NFTs were strongly positive (Additional file 6: Figure S3b and inset), hippocampal plaques were virtually silver negative. However, in the same sections plaques in the nearby occipito-temporal cortex were clearly silver positive and the Aβ immunostainings of hippocampal plaques were intensely positive (see below). Congo red staining was negative (not shown).
In the CA1-CA4 sectors, the general Aβ antibodies disclosed abundant Aβ deposits of variable size and irregular shape. Remarkably, in hippocampus the plaques did not show such a distinct targetoid pattern as in cerebral cortex. Instead, they were small with a diffuse pattern when stained with different Aβ antibodies (insets of Figure 4a-f). In CA3 and CA4 sectors, in addition to the better defined plaques there were also background-like diffuse Aβ deposits, whereas in CA1 and CA2 such deposits were uncommon (Figure 4a-f).
Using the C-terminal abAβx-42 (Figure 4a) and abAβx-40 (Figure 4b), as well as the mid-domain abAβ17–24 (Figure 4c), the staining was recognizably stronger and the plaques were slightly more frequent than with the N-terminal antibodies abAβ8–17, abAβ5–10 and abAβ1–5 (Figure 4b). Diffuse plaques were also abundant in stratum radiatum, subiculum (Figure 4a-f) and transentorhinal cortex, whereas in entorhinal cortex they were relatively sparse. In the adjoining occipito-temporal cortex the plaques appeared similar as elsewhere in neocortex (cf. Figure 2a-h and Additional file 3: Figure S1 and Additional file 4: Figure S2).
Staining of allocortical sections with the specific Aβ antibodies abAβarc (Figure 4e), abAβ3pE (Figure 4f) and abAβ11pE (not shown) gave similar patterns as the general Aβ antibodies. The staining intensities were comparable to that of N-terminal abAβ1−5 (Figure 4d).
Subcortical grey matter nuclei (phase 3)
In basal nuclei, plaques were most often not discernible with H&E or silver staining, but they were selectively positive for Aβ with immunohistochemistry (see below). In this region, the Congo red staining was negative in the parenchyma.
Among the basal nuclei, claustrum was remarkably deviant: Aβ was deposited as large compact plaques, which had similar targetoid staining pattern as plaques in neocortex (Additional file 7: Figure S4a-g). On the contrary, in the neighbouring putamen the plaques were small and diffusely stained (Additional file 7: Figure S4h-k). These plaques were positive with the C-terminal abAβx-42 (Additional file 7: Figure S4h) and abAβx-40, as well as with mid-domain abAβ17-24 (not shown) and were weakly positive with abAβarc (Additional file 7: Figure S4j) and abAβ11pE (not shown). With the N-terminal abAβ1-5 plaques were almost negative (Additional file 7: Figure S4i), whereas with the pyroglutamate specific N-terminal abAβ3pE the plaques were clearly discernible (Additional file 7: Figure S4k). In amygdala the Aβ deposition was similar to that in putamen, though with C-terminal antibodies the number of small diffuse plaques was greater (Additional file 7: Figure S4l). In thalamus (Additional file 7: Figure S4m) and caudate nucleus (not shown) the plaques were ragged and weakly stained with all antibodies. Globus pallidus was completely negative for Aβ-immunoreactivity (not shown).
Brain stem (midbrain, pons and medulla; phase 4)
In midbrain, pons and medulla, Aβ deposits were not discernible with H&E and only weakly positive with silver staining. None of the parenchymal Aβ deposits were positive for Congo red (not shown).
In midbrain, the deposition of Aβ was scarce. Diffuse weakly stained Aβ deposits were discernible almost exclusively in nucleus ruber. Among the different Aβ antibodies, only abAβx-42 and abAβ17–24 stained these plaques. However, amyloid angiopathy could be clearly visualized with all Aβ antibodies (not shown). In pons, virtually no parenchymal deposits were observed despite brisk staining of blood vessels (not shown). In medulla, a few distinct plaques, strongly positive with all Aβ antibodies used were present in inferior olivary (Additional file 8: Figure S5a-i) and dorsal vagal nuclei (not shown). The pattern, number and size of plaques in medulla were approximately similar with both general and specific Aβ antibodies, although some variation in the intensity was observed (Additional file 8: Figure S5d-i). Remarkably, abAβx-42 rendered the neuropil in inferior olivary nucleus distinctly positive (Additional file 8: Figure S5a and d), whereas with all other Aβ antibodies it was negative (e.g. Additional file 8: Figure S5b-c and e-i). Olivary neurons within the plaques appeared fairly well preserved (Additional file 8: Figure S5d-k), but both abAβx-42 and abAβ17–24 stained cytoplasmic inclusions within inferior olivary neurons (Additional file 8: Figure S5d-e), as did also PAS and an antibody to lysosomal cathepsin D (Additional file 8: Figure S5j-k; see also paragraph Intracellular Aβ immunoreactivity).
Cerebellum (phase 5)
In H&E stained sections the Aβ deposits were not detectable (not shown). Bielschowsky silver showed no impregnation in the Purkinje cell layer (see below) and only a small number of weakly positive perivascular streaks or smaller deposits in the molecular layer perpendicular to the surface (not shown). As elsewhere, Congo red did not reveal any parenchymal staining.
The amount of Aβ deposited in cerebellum was much more abundant than that normally seen in AD. Furthermore, the pattern of the deposits was remarkably different from elsewhere in the Arctic AD patients’ brains, especially compared to the cerebral cortices. The immunopositive Aβ deposits were highly variable in size and had very irregular configurations, while distinct rounded Aβ plaques were completely absent. Furthermore, there were marked inter-individual differences, e.g. Aβ deposits in patient Am1 were distinctly different from those in patients Sw1, Sw2 and Am2 (see below).
In Am1 patient the staining pattern with abAβx-42 and abAβ17–24 (Additional file 9: Figure S6a and c) was almost similar as in the three brains described above, whereas with the C-terminal abAβx-40 (Additional file 9: Figure S6b) and mid-domain abAβ8-17 (Additional file 9: Figure S6d) both the deposits in the granular/Purkinje cell border zone and the streaks in the molecular layer were scarce and weak, although blood vessels were clearly positive. In this brain the more N-terminal abAβ8–17, abAβ5–10 and abAβ1–5 (Additional file 9: Figure S6d-f) stained almost exclusively arterial vessel walls. Among the specific antibodies the staining with Aβarc was faint, whereas both abAβ3pE and abAβ11pE gave clear staining Additional file 9: Figure S6g-i).
Intracellular Aβ immunoreactivity
In all brains definite cytoplasmic immunoreactivity was observed in inferior olivary neurons with abAβx-42 and abAβ17–24 (Additional file 8: Figure S5d-e). The cytoplasmic Aβ(or AβPP ) immuno-positivity persisted, even if the formic acid pretreatment was omitted, whereas the extracellular Aβ deposits in the medulla were immunonegative (not shown). These granular cytoplasmic inclusions in inferior olivary neurons were also positive with PAS (Additional file 8: Figure S5j), cathepsin D (Additional file 8: Figure S5k), and α1-antitrypsin (not shown).
Cytoplasmic immunoreactivity with abAβ17–24 was observed also in some other locations, most prominently in cerebellum, both in Purkinje cells (Figure 6a) and in neurons of the dentate nucleus (Figure 6c). Markedly less intense staining was occasionally seen in cerebral cortical and hippocampal pyramidal neurons (not shown).
Microscopy of cellular pathology
Variable numbers of neurons with hp-tau positive NFTs were detected in all cerebral cortical areas examined (Figure 8a, d-e). Neurofibrillary deposits were found in the cytoplasm of small or atrophic neurons (Figure 8a, d-e), whereas larger (non-degenerated) neurons in cortical layers 3, 5 and 6, including those located within Aβ plaques, were usually devoid of NFTs (Figure 8b-c). Most commonly neurons with NFTs were not distributed within though nearby plaques (Figure 8d).
Many hippocampal pyramidal neurons from CA4 to subiculum as well as in adjoining entorhinal, transentorhinal and occipito-temporal cortices harboured prominent NFTs (Figure 8f). Furthermore, many of the granule cells in the dentate gyrus contained distinct hp-tau positive inclusions (Figure 8g). In general, NTs were abundant in the hippocampal neuropil and DNs were more prominent within hippocampal than cortical Aβ plaques (Figure 8g).
In claustrum, the pattern of NTs, DNs and neurons with NFTs was − like that of Aβ plaques − similar as in neocortex (cf. Figure 8a-b and d). In thalamus, where the Aβ plaques were small and diffuse, only few NFTs and very delicate NTs were discernible. In putamen, where plaques were even less conspicuous, almost no NTs were present (not shown). In globus pallidus, where no Aβ deposits were detected, neither were hp-tau immunoreactive structures seen (not shown).
Despite the abundance of Aβ deposits in cerebellar cortex and around Purkinje cells, no neurons (Purkinje cells, granule cells or other neurons) harboured NFTs. In addition, the number of NTs or DNs associated with Aβ deposits was insignificant (not shown).
Macro- and microglial changes
In cerebellum there was pronounced GFAP staining around Purkinje cells (Bergmann astrocytes), although not as prominent as for Aβ. In the molecular layer GFAP positivity did not follow the perivascular streaks of Aβ deposits. Instead, loose GFAP-immunopositive astrocytic network of varying intensity was found throughout the molecular layer (Additional file 10: Figure S8a-b). In all patients, the Iba-1 immunoreactivity in cerebellum was weak and diffuse compared to the Aβ deposition and astroglial reaction and it did not specifically associate with Aβ deposits (Additional file 10: Figure S8c).
No major atherosclerosis in the circle of Willis was present, neither were there infarcts, haemorrhages nor microbleeds.
In all four patients’ brains leptomeningeal and cortical penetrating arteries of widely different calibres were variably immunopositive with the different Aβ antibodies used (e.g. Figures 2d and g, 5a-i and Additional file 9: Figure S6a-i). In addition, capillaries in many particular anatomic locations were positive for Aβ, most commonly with abAβx-40 (Figure 2d and Additional file 2: Figure S7c). Presence of fibrillar Aβ within the vessel walls, i.e. evidence of true cerebral amyloid angiopathy (CAA), was verified by Congo positivity with green birefringence in arteries, but not in capillaries (Figure 1d, inset). Details of the composition, distribution and severity as well as illustrations of CAA in these Arctic AD patients’ brains will be presented in a separate article (in preparation).
Fulfilling the neuropathological criteria of AD
We performed neuropathological examinations of the brains from four patients who carried the Arctic AβPP mutation and whose clinical picture complies with AD. We could demonstrate that the neuropathological hallmarks of AD, Aβ plaques and deposition of hp-tau, were prominent features in all four patients’ brains. However, the Arctic AD pathology has certain features that deviate from the common AD pathology , as it is referred to in both of the latest consensus criteria [23–25].
When the ABC system of the new 2012 NIA-AA criteria [23, 25] is applied, the distribution of Aβ plaques, i.e. NIA-AA component A = 3, since the accumulation of Aβ is florid even in cerebellum (= Thal phase 5, ). As for the neurofibrillary tau pathology (component B), the presence of NFTs throughout the neocortical regions fulfils the criteria of Braak stage V-VI , i.e. NIA-AA component B = 3. Braak stage V-VI is also met, if the Brain Net Europe (BNE) staging is applied , as the neocortical NTs are abundant in the striate area (occipital cortex; Additional file 2: Figure S7a). The grading of component C in the Arctic brains according to CERAD as recommended in NIA-AA criteria is somewhat problematic, because of the exceptional plaque structure. Although Aβ plaques are compact, they are devoid of fibrillar amyloid cores and only rarely harbour robust DNs. Nevertheless, we propose that they do fulfil the CERAD criteria for neuritic plaques, since they display delicate hp-tau positive NTs and occasional DNs. The frequency of plaques should be considered frequent, i.e. the component C = 3 .
Taken together, our neuropathological findings in the Arctic AD patients’ brains meet both the 2012 NIA-AA criteria for high level of AD- related changes (A3, B3, C3) [23, 25] and the previous NIA-RI criteria for a high likelihood of AD . The very rare α-synuclein positive neurons encountered (not shown) most likely did not contribute to the patients’ clinical picture. Neither did we find any significant ischemic pathology, although CAA was relatively prominent. Thus, the dementia caused by the Arctic AβPP mutation is due to AD, albeit with unique features of AD neuropathology.
Interestingly, the large, round cortical Arctic plaques bear resemblance to the cotton wool plaques, such as the Aβ deposits found in AD patients with PS1 mutations [2–4]. Similarly, the cotton wool plaques and the Arctic plaques are devoid of amyloid cores and harbour very few hp-tau-positive DNs . However, cotton wool plaques have a homogeneous composition (Additional file 1: Figure S9), whereas the Arctic plaques are composed of variably truncated and spatially differentially distributed Aβ and therefore when immunostained with different Aβ antibodies they appear targetoid.
Amount of Aβ deposition
The extent of Aβ deposits in the Arctic AD patients’ brains was massive, which may explain why the weights of Sw1, Sw2 and Am2 brains were considerably higher than what is commonly recorded in sporadic AD. Alzheimer brains are usually atrophic, but no studies have systematically compared the brain weight with the Aβ burden. Interestingly, the brain weights in three of the five PS1Δ9 AD patients with large cotton wool plaques of similar abundance as Aβ plaques in our Arctic AD patients’ brains were within normal limits .
Hierarchical order of Aβ deposition
In sporadic AD, Aβ deposition has been suggested to occur hierarchically in five phases . The distribution of Aβ deposits in the Arctic AD patients’ brains corresponds to Thal’s most advanced phase 5, for which the requirement is the presence of Aβ also in cerebellum. Aβ deposits were highly abundant in all Arctic AD patients’ cerebellum, but they were few in brain stem nuclei–the criterion for phase 4. These features suggest that, in these brains, cerebellum may have become involved at an earlier phase or at a faster pace than the brain stem. Thus, the Arctic AβPP mutation may alter the hierarchy according to which the Aβ deposits are generally seen to emerge in AD patients’ brains. Perhaps abundant extracellular perivascular Aβ aggregates in Arctic AD brain overwhelm the perivascular drainage pathways  in cerebellum leading to an early pathology. Moreover, the pattern of Aβ deposition and distribution in our Arctic patients’ cerebellum was exceptional (see next section).
Variation in the distribution of Aβ deposition
The pattern and composition of Aβ deposits in the Arctic AD brain revealed additional topographic variability. The ring plaques, originally discovered by Bielschowsky silver and immunostaining with abAβx-42 , were − with the use of additional Aβ antibodies − shown to be targetoid rather than ring-shaped (as preliminarily demonstrated in our previous article  and now in greater detail in this article). These plaques were almost only observed in cerebral cortex, whereas in other anatomical locations the plaques were usually of more irregular and diffuse types.
Interestingly, in claustrum the pattern of Aβ deposits was similar to that in cerebral cortex. This is consistent with a previous observation of cotton wool plaques in PS1Δ9 AD patients’ brains . In addition, the tau pathology in claustrum was similar as in cerebral cortex. Claustrum is generally considered to be a part of the basal ganglia and the presence of plaques in this region would thus correspond to Thal’s phase 3. However, this pattern was strikingly different from that in the other subcortical grey matter nuclei. For example, globus pallidus was virtually negative for Aβ plaques, while only small diffuse plaques could be found in putamen, thalamus and caudate nucleus. This discrepancy may be related to the fact that, of these regions, only claustrum has bidirectional connections with almost all cortical regions .
The pattern of cerebellar Aβ deposits differed remarkably from the commonly observed restriction of deposits to the molecular layer in sporadic AD  and from the abundance of cored plaques in PS1Δ9 AD patients’ cerebellum. In the Arctic AD patients’ brains, the abundant Aβ deposition next to the Purkinje cells may indicate active Aβ production in these cells. Moreover, the apparent pattern of Aβ deposition along the perivascular drainage pathways (also depicted in NIA-AA article by Hyman et al. 2012 ) may be explained by a relatively profuse transport of extracellular Aβ through the molecular layer to the subarachnoid space .
Differential truncation of Aβ
Our mass spectrometric analyses, in which we immunoprecipitated both wt and mutated Aβ from the temporal cortex of the patient Sw2 with antibodies against two distinct Aβ epitopes (ArcAβ17–24 and wtAβ17–24), demonstrated that the deposits contained variably truncated and modified Aβ species, both wt and Arctic Aβ. Furthermore, our immunohistochemical stainings revealed that although all four patients carried the same AβPP mutation, both the distribution and the composition (truncations or modifications) of the Aβ deposits showed considerable inter- and intra-individual variability. This should not actually be surprising, since it is most likely that the “machinery and milieu” in the AβPP production, processing and/or Aβ aggregation are not identical in the four individual Arctic AD patients, nor in different anatomic regions within each patient’s brain. Whether a similar variability in the composition of Aβ deposits exists in other forms of FAD has been the subject of several studies [5, 34–37].
The variability in the composition of deposited Aβ also sets new requirements for the antibodies used in diagnostic work. Since truncations can occur at many different sites of the Aβ peptide, some antibodies give virtually negative parenchymal staining. Thus, either an antibody against the mid-portion of Aβ preferably against an epitope located C-terminally to aa 11 (to avoid problems with 11pE truncated Aβ species) and N-terminally to aa 40 (to avoid problems with the different C-terminal truncations), or a mixture of different antibodies should be recommended for routine analyses. In our hands, the antibody abAβ17–24 (clone 4G8) appeared to best fulfil such requirements (even though it also recognizes AβPP ), whereas the other widely used mid-portion antibody abAβ8–17 (clone 6F/3D) yielded inconsistent results. For instance, it gave only vague staining of the cerebellar parenchymal deposits, although it stained blood vessels in this region clearly positively.
It is known that the Arctic mutation interferes with the processing of AβPP by making it less prone for α-secretase cleavage, while elevating β-secretase cleaved fragments [12, 38]. Natural processing of AβPP at the β′-site (at Aβ aa 10/11) is also favoured over the β-site (at Aβ aa −1/1) in situations when accession to it by BACE1 enzyme is affected, e.g. through structural twists in AβPP . Moreover, N-terminally truncated and modified Aβ peptides (e.g. AβpE3-x and AβpE11-x) have been shown to be significantly increased in the brains of AD patients with various PS1 mutations [36, 40]. Our data show that the Arctic mutation like the PS1 mutations referred to above may increase cleavages at aa 3 and 11 since we observed 3pE40arc, 11pE40arc and 11pE42arc in our MS measurements (Figure 3e, Additional file 5: Table S1 and ), and considerable immunopositivities with ab3pE and ab11pE antibodies. However, since we also observed a heterogeneous population of N-terminally truncated Aβ peptides, additional (primary/secondary) cleavages are also likely to occur in the Arctic AD brain. Aβ-peptides might also aggregate in a way that exposes cleavage sites and facilitates peptide truncation and their modifications as a secondary process.
Lack of fibrillar Aβ and amyloid cores is a characteristic feature of both Arctic plaques and PS1Δ9 cotton wool plaques. The reason for this feature in these two genetic variants of AD is unknown. Certain properties of the mutated forms of Aβ such as posttranslational modifications and altered propensity to oligomerize/aggregate, could offer an explanation for the limited formation of structurally ordered Aβ fibrils in Arctic AD patients , whereas the underlying reason in the PS1Δ9 AD patients is even less clear, because the cotton wool plaques in such brains contain wild-type Aβ only. It might be that in these two types of FAD Aβ aggregation is too rapid and does not lead to Congo-red positive, fibrillar Aβ deposits.
It has been shown that the Arctic mutation leads to an accelerated oligomerization and disordered fibrillogenesis of Aβ, measured both in vitro [11, 41–44] and in vivo [11, 45, 46]. The diameter of Arctic Aβ fibrils correlated with decreased neuronal viability . Recent in vitro experiments on the aggregation process of ArcAβ1–40  demonstrated that at least four types of fibrils can be identified. The intermediate phase of spherical aggregates appeared at earlier time points and ArcAβ1–40 fibrils polymerized more rapidly and at lower concentrations than wt Aβ1–40 fibrils. At late stages fragmentation and clustering of ArcAβ1–40, but not of wtAβ1–40, fibrils were observed . The results of these experiments are in agreement with the suggestion that spherical aggregates (containing abundant β-hairpin and/or β-sheet structures), and/or Aβ oligomers have a pathogenic role in the AD brain. Especially the larger soluble oligomers, i.e. protofibrils, are known to have neurotoxic properties. However, an alternative Aβ aggregation pathway, different from simple assembly of spherical aggregates and protofilaments into fibrils, has also been proposed , which may contribute to the distinct morphology of Aβ plaques in Arctic AD patients (i.e. Congo-red negativity of the Arctic AD deposits). Moreover, co-incubation of ArcAβ with wtAβ1-40 led to kinetic stabilization of Arctic protofibrils . An increase in the ratio of ArcAβ to wtAβ in Arctic AD may result in the rapid accumulation of neurotoxic protofibrils and acceleration of the disease process .
The large plaques in both the Arctic and PS1Δ9 AD patients’ brains were found to embrace neurons. More specifically, in all four Arctic brains seemingly viable neocortical pyramidal neurons and cerebellar Purkinje cells could be identified within several plaques of variable Aβ composition. Interestingly, the perikarya of these neurons seemed intact and did not appear to be under way to develop neurofibrillary pathology. This sparing of neuronal perikarya is in accordance with the notion that intraneuronal Aβ triggers neuron loss in AD . In several animal models it has been demonstrated that extracellular Aβ plaques do not seem to instigate neuronal death, whereas accumulation of intraneuronal Aβ correlated well with the loss of neurons , including a transgenic model expressing pyroglutamate Aβ3pE-42 .
We showed that even apparently intact axons traversed the plaques. These observations are phenomena, similar to those in PS1Δ9 AD patients, i.e. axons are not pushed aside to wind around the accumulated Aβ. However, the relatively low number of neurofilament positive intraplaque axons may indicate that axons traversing Arc plaques suffer from some degree of degeneration, as it was interpreted to occur within PS1Δ9 AD patients’ cotton wool plaques . The intraplaque accentuation of NTs (i.e. axons containing hp-tau) in both Arc and PS1Δ9 plaques supports the suspicion of axonal degeneration. Accumulation of pathogenic species of microtubule associated tau protein can impair axoplasmic transport and consequently contribute to synaptic loss, which may be pivotal in the pathogenesis of AD . If the synaptic contacts are lost, sparing of the perikarya or axons en route cannot prevent functional loss (and further degeneration). The fate of axons within Aβ plaques obviously merits further analysis.
Furthermore, the non-fibrillar type of Aβ deposits in the Arctic brain induced only a limited reactive response. Although the density of astrocytic processes was increased within the Arctic Aβ deposits (clearly visible around Purkinje cells), the astrocytic cell bodies appeared not to cluster around the non-fibrillar Aβ deposits. Likewise, in PS1Δ9 AD patients’ brains astrocytes did not cluster around the non-fibrillar cotton wool plaques, although in these brains the scarce cored plaques were surrounded by an increased number of astrocytes . Similarly, the Arctic non-fibrillar plaques did not appear to attract microglial cells. The presence of preserved neuronal perikarya and axons as well as the lack of activated glial cells strengthen the perception of extracellular non-fibrillar Aβ deposits as being relatively non-toxic. Thus, it is conceivable that Aβ oligomers, which are considered pathogenic, exert their effects already within the neurons or by being diffusely distributed (not in plaques) in the parenchyma.
We have demonstrated special neuropathologic characteristics in the brains of four deceased AD patients with the Arctic AβPP mutation (p.E693G/p.E22G). The amount of Aβ deposited in the brains was profuse, but virtually all parenchymal deposits were composed of non-fibrillar, Congo negative Aβ aggregates Aβ Congo red only stained the walls of moderately to severely angiopathic vessels. Mass spectrometric analyses on temporal cortex samples showed that the Aβ deposits contained variably truncated and modified wt and mutated ArcAβ species. The structure and composition of the Aβ deposits varied considerably between the patients. In three of the four analysed Arctic AD brains, the plaque centres containing C-terminally (beyond aa 40) and variably N-terminally truncated Aβ were surrounded by Aβx-42 immunopositive coronas giving the plaques a targetoid appearance. Furthermore, in each individual the architectural pattern of plaques was found to vary between different anatomic regions–from diffuse deposits to targetoid or ring-shaped plaques. Tau pathology appeared mainly as delicate neuropil threads with accentuation within Aβ plaques. Thicker dystrophic neurites were only occasionally observed. Neurons within the large Aβ plaques appeared relatively intact. Thus, the extracellular deposits of non-fibrillar Aβ did not seem to directly damage neuronal perikarya or to induce formation of neurofibrillary tangles, supporting the present view of intracellular Aβ oligomers being neurotoxic. The enrichment of NTs within plaques may indicate axonal damage, even though neurofilament positive axons traversing plaques were detected. Finally, similarly as the cotton wool plaques in PS1 Δ9 AD, the Arctic plaques induced only a modest glial and inflammatory tissue reaction.
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Hannu Kalimo and Maciej Lalowski: These authors share the first authorship.
This study was funded by grants from Helsinki University Central Hospital EVO research funds (HK); Magnus Ehrnrooth Foundation (ML, MB); Uppsala University, Landstinget i Uppsala län, the Swedish Research Council (#2009-4567; #2009-4389; #2006-6326; # 2006–3464), Alzheimerfonden, Gamla Tjänarinnor, Gun och Bertil Stohnes Stiftelse, Åhlén-stiftelsen, Frimurarstiftelsen and Trolle-Wachtmeisters stiftelse (MI, LNGN, OP, RMB, HB, LL); NIA # AG05136 to the ADRC and NIA # AG06781-06 to the ADRP, University of Washington (DN); Veteran Affairs Research Funds (TB); NIA/NIH (P01-AG-017586-11) (GS). The skilled technical assistance by Ms. Liisa Lempiäinen, Ms. Randy Small and Ms. Christiane Ulness in histopathology is gratefully acknowledged.
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