Patients and brain specimens

Two patients from a Swedish and two from an American family were included in the study. The presence of the Arctic mutation was verified by sequencing of exon 17 of the AβPP gene according to the methods previously described [11]. Although not proven, these two families are believed to descend from the same Swedish ancestor, (for respective pedigrees, see Figure 1a-b). The Swedish patients died at 62 (Sw1 = IV:10) and 64 (Sw2 = IV:29) years of age, after six and eight years of disease duration, respectively. The American patients died at 72 (Am1 = III:1) and 59 (Am2 = IV:1) years of age after 16 and 8 years of disease duration, respectively.

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 [3].

Histopathology and immunohistochemistry

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 Aβ_x-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.

Mass spectroscopy

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 Aβ_17–24 and Aβ_arc) and analysed by MALDI-TOF, as described in our previous study [18].

Macroscopic pathology

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 [17].

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 [20]. 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)

Aβ immunohistochemistry

General features

The area fraction of Aβ immunopositivity (Aβ_x-42) in frontal, temporal and parietal cortices was about 25%. Plaques were present in all cortical layers, being most numerous and compact in layers 2 and 3, while in deeper layers they were somewhat larger and less distinct. In addition, in layer 1 there were often small diffuse plaques of variable numbers, shapes and staining intensities, as well as thin patchy variably immunopositive subpial bands (Figure 2a-h). The variation in the cortical pattern appeared e.g. in occipital cortex, where layers 4 and 6 were virtually devoid of plaques and layer 5 displayed only a lesser number of diffuse plaques (Figure 2d and Additional file 2: Figure S7c). Variation between patients was also observed e.g. as a paucity of plaques in layer 4 in frontal cortex of patients Am1 and Am2 compared to more abundant plaques in the same region in patients Sw1 and Sw2 (Additional file 3: Figure S1a-g, Additional file 4: Figure S2a, and Figure 2a).

Staining with general Aβ antibodies

With the C-terminal abAβ_x-42 a majority of neocortical plaques in all four patients were ring-shaped (Figure 2c), as described in the first study on patient Sw1 [17], i.e. the weakly stained centres of larger plaques were surrounded by distinct immunoreactive coronas.

In patient Sw1 the ring pattern visualized with abAβ_x-42 [11] was also clearly noticeable with abAβ_x-40, abAβ_8–17, abAβ_5–10, and abAβ_arc. The staining was progressively weaker and less distinct with the more N-terminal abs (Additional file 4: Figure S2a-g) and nearly negative with abAβ_1–5, 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 patients Sw2 and Am 1, on the contrary, the neocortical plaques displayed a targetoid pattern: In these two brains the plaques were ring-shaped only with the most C-terminal antibody abAβ_x-42 (Figure 2c)_, whereas with abAβ_x-40 the centre was clearly immunopositive (Figure 2b). The mid-domain abAβ_17-24 rendered the plaques most compact of all antibodies (Figure 2e), whereas antibodies with epitopes towards the N-terminus stained the plaque coronas more weakly and centres more intensely (Figure 2f-h, Additional file 3: Figure S1d-f). Confocal analysis of sections from patient Sw2 double immunostained with abAβ_x-42 and abAβ_1-5 clearly distinguished the two Aβ components in plaques: the peripheral corona was positive with abAβ_x-42, while the centre was strongly positive with abAβ_1-5 (Figure 3a). Therefore, the plaques could best be described as targetoid, not ring-shaped.

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).

Allocortical brain regions (phase 2)

Aβ immunohistochemistry

With Aβ immunostaining, the plaques were numerous throughout hippocampus, but their frequency and pattern varied in different hippocampal regions (Figure 4a-f).

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 abAβ_x-42 (Figure 4a) and abAβ_x-40 (Figure 4b), as well as the mid-domain abAβ_17–24 (Figure 4c), the staining was recognizably stronger and the plaques were slightly more frequent than with the N-terminal antibodies abAβ_8–17, abAβ_5–10 and abAβ_1–5 (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 abAβ_arc (Figure 4e), abAβ_3pE (Figure 4f) and abAβ_11pE (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)

Brain stem (midbrain, pons and medulla; phase 4)

Cerebellum (phase 5)

Aβ immunohistochemistry

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 patients Sw1, Sw2 and Am2 with similar Aβ staining pattern, the C-terminal abAβ_x-42 (Figures 5a and 6b) and abAβ_x-40 (Figure 5b) as well as the mid-domain abAβ_17–24 (Figure 5c) displayed diffuse patchy staining in the Purkinje cell layer, from where irregular immunoreactive streaks extended across the molecular layer towards the surface, often following the penetrating blood vessels as wide and irregular cuffs (Figure 5a-c). A similar pattern, although with markedly weaker intensity, was obtained with the pyroglutamate specific abAβ_3pE (Figure 5h) and abAβ_11pE (Figure 5i). The staining was still weaker with the other N-terminal antibodies abAβ_8–17, abAβ_5–10 and abAβ_1–5 (Figure 5d-f), as well as with abAβ_arc (Figure 5g). All Aβ antibodies used gave robust staining of blood vessel walls (Figure 5a-i).

In Am1 patient the staining pattern with abAβ_x-42 and abAβ_17–24 (Additional file 9: Figure S6a and c) was almost similar as in the three brains described above, whereas with the C-terminal abAβ_x-40 (Additional file 9: Figure S6b) and mid-domain abAβ_8-17 (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 abAβ_8–17, abAβ_5–10 and abAβ_1–5 (Additional file 9: Figure S6d-f) stained almost exclusively arterial vessel walls. Among the specific antibodies the staining with Aβ_arc was faint, whereas both abAβ_3pE and abAβ_11pE gave clear staining Additional file 9: Figure S6g-i).

Microscopy of cellular pathology

General alterations

In all Arctic AD patients’ brains neurons could be frequently identified within the cortical plaques. Interestingly, many of these neurons displayed relatively minor degenerative changes with characteristic vesicular nuclei and prominent nucleoli but had often somewhat condensed cytoplasm (Figure 7a). These neurons were similar as those found within the large cotton wool plaques in PS1Δ9 AD patients (Figure 7b). Moreover, confocal microscopy analysis of cortical sections double-labelled for Aβ and neurofilament showed that some NF-positive axons traverse the plaques (Figure 7c-d). Similarly, Purkinje cells surrounded by abundant Aβ did not appear degenerating, not even those with intracellular abAβ_17–24 positive deposits (Figure 6a-b).

Tau immunohistochemistry

Hp-tau immunopositivity was abundant in cerebral cortices and it was mainly present as NTs. The frequency of NTs was accentuated within the Aβ plaques and thus plaques were delineated also by hp-tau staining (Figure 8a-g, Additional file 2: Figure S7a and c). Similar accentuation was also observed in PS1Δ9 AD patients’ cotton wool plaques (Additional file 1: Figure S9i and j). NTs were usually abundant in cortical layers 2 and 3, markedly lesser in layer 4, scarce to absent in layer 5, and slightly accentuated in layer 6. Thus, the frequency of NTs by and large corresponded to that of compact Aβ plaques (Figure 8a-c; Additional file 2: Figure S7a and c). In calcarine cortex, the abundance of NTs corresponded to Braak stage VI, according to the BrainNet Europe recommendation (Additional file 2: Figure S7a). Within Aβ plaques NTs were usually delicate, but occasionally they appeared thicker approaching the appearance of DNs (Figure 8b and g).

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 all four brains, there was slight to moderate reactive astrogliosis in cerebral cortices. GFAP immunopositivity was accentuated within the Aβ-immmunoreactive plaques (Figure 9a-b), mainly as a meshwork of thin processes. Although the number of astrocytic cell bodies in cerebral cortices appeared to be slightly increased, they were relatively diffusely distributed instead of being strictly oriented within or around plaques (Figure 9a-b). Microglial reaction, as determined by Iba1 staining, in cerebral cortices was relatively modest and the distribution of microglial cells did not appear to follow the distribution of plaques (Figure 9b-c). However, their frequency seemed to vaguely correspond to the overall density of the NT meshwork (not shown).

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).

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 [22], 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, [20]). As for the neurofibrillary tau pathology (component B), the presence of NFTs throughout the neocortical regions fulfils the criteria of Braak stage V-VI [26], i.e. NIA-AA component B = 3. Braak stage V-VI is also met, if the Brain Net Europe (BNE) staging is applied [27], 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 [28].

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 [24]. 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 [3]. 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 [3].

Hierarchical order of Aβ deposition

In sporadic AD, Aβ deposition has been suggested to occur hierarchically in five phases [20]. 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 [29] 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 abAβ_x-42 [17], were − with the use of additional Aβ antibodies − shown to be targetoid rather than ring-shaped (as preliminarily demonstrated in our previous article [18] 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 [30]. 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 [31].

The pattern of cerebellar Aβ deposits differed remarkably from the commonly observed restriction of deposits to the molecular layer in sporadic AD [20] 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 [23]) may be explained by a relatively profuse transport of extracellular Aβ through the molecular layer to the subarachnoid space [29].

Differential truncation of Aβ

The composition of Aβ deposits in both sporadic and familial AD brains has been shown to be highly variable, with both N- and C-terminal truncations and modifications [32–37].

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 (ArcAβ_17–24 and wtAβ_17–24), 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 abAβ_17–24 (clone 4G8) appeared to best fulfil such requirements (even though it also recognizes AβPP [21]), whereas the other widely used mid-portion antibody abAβ_8–17 (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 [39]. 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 [18]), 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 [41], 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 [42]. Recent in vitro experiments on the aggregation process of ArcAβ_1–40 [41] demonstrated that at least four types of fibrils can be identified. The intermediate phase of spherical aggregates appeared at earlier time points and ArcAβ_1–40 fibrils polymerized more rapidly and at lower concentrations than wt Aβ_1–40 fibrils. At late stages fragmentation and clustering of ArcAβ_1–40, but not of wtAβ_1–40, fibrils were observed [41]. 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 [41], 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 wtAβ_1-40 led to kinetic stabilization of Arctic protofibrils [47]. 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 [48].

Cellular pathologies

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 [49]. 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 [50], including a transgenic model expressing pyroglutamate Aβ_3pE-42 [51].

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 [3]. 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 [52]. 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 [3]. 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.