Distinct deposition of amyloid-β species in brains with Alzheimer’s disease pathology visualized with MALDI imaging mass spectrometry

Human cortical specimens for IMS and IHC were obtained from the Brain Bank at Tokyo Metropolitan Institute of Gerontology. Brains were removed, processed, and stored at −80 °C within 8 h postmortem. Patients were placed in a cold (4 °C) room within 2 h after death. All brains registered at the brain bank came with written informed consents for their use in medical research from the patients or their families. Each brain specimen was taken from the occipital cortex of five AD patients and five age-matched controls. This study was approved by the ethics committee at Doshisha University and Tokyo Metropolitan Geriatric Hospital.

In the present study, the extent of Aβ deposition as shown by an Aβ monoclonal antibody (12B2, 1:50 dilution; IBL, Gunma, Japan), was defined by Braak SP (amyloid) stages [2, 3]. At stage O, there are almost no senile plaques throughout the isocortex. At stage A, low densities of Aβ deposits are detected in the isocortex, particularly in the basal portions of the frontal, temporal, and occipital lobes. Furthermore, some plaques are found in the presubiculum and Pre-β and Pre-γ layers of the entorhinal complex. Stage B shows an increase in Aβ deposits in almost all isocortical association areas, and only the primary sensory areas and primary motor field remain practically devoid of deposits. There are mild amounts of deposits in the hippocampal formation, and Aβ deposits may be found in the entorhinal cortex. At stage C, virtually all the isocortical areas are affected, whereas deposits in the hippocampal formation display the same pattern as that of stage B. AD brains are invariable at stage C.

Fresh frozen sections were post-fixed using 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). After a brief wash in PBS, sections were stained by Sudan Black B for elimination of the autofluorescence due to lipofuscin [12]. Following immersion in 10% goat serum in PBS and 0.1% Tween20 (PBS-T) for 60 min, the sections were incubated with primary antibodies diluted in 1% BSA-PBS-T for 18 h at room temperature. Sections were then rinsed with PBS-T, and bound antibodies were visualized with secondary antibodies conjugated with Alexa dyes (Life Technology). The specimens were visualized by confocal-laser-scanning-microscope (LSM 700; Carl Zeiss Inc.). Whenever necessary, bound antibodies were labeled by biotinylated anti-mouse or anti-rabbit IgG antibodies (Vector Laboratories, Inc., Burlingame, CA), followed by avidin and biotinylated HRP complex (Vectastain Elite ABC kit; Vector Laboratories, Inc.). Bound HRP was developed with 3,3-diaminobenzidine (DAB) in the presence of hydrogen peroxide.

The antigen peptide corresponding to Aβ37–41 amino acid residues, GGVVI, conjugated to thyroglobulin was immunized into rabbits four times at 2-week intervals. Subsequently, the serum was collected and purified with Aβ41 affinity chromatography. To confirm the purified antibodies’ specificity, synthetic Aβ1–40, Aβ1–41, and Aβ1–42 were run on a 12% Tris-tricine gel [7, 8] followed by western blotting. All Aβs were detected with 82E1 (IBL, Japan), while Aβ41 was detected with purified polyclonal antibody.

Aβ was dissolved in hexafluoro isopropanol (HFIP) to 1.8 mg/ml. Each of the 230 μM Aβ samples were completely dried up. Samples were then dissolved in 50 mM NaPO₄ (pH 7.4) and 0.25 mM NaOH. An equal volume of 56 mM thioflavin T was then added to each Aβ sample. Fifty microliters of the mixture was then added into the black wall plate individually and incubated at 37 °C with preventive light cover. The final Aβ concentration was 115 μM in each well. During sample incubation, thioflavin T intensity was measured for 24 h, at an excitation wavelength of 465 nm and emission wavelength of 535 nm.

To characterize the broad range of deposited and accumulated Aβ species in postmortem brain tissues, we adopted MALDI-IMS technology combined with formic acid pretreatment of brain tissues. Samples were obtained from sporadic AD patients with CAA (n = 5; mean age = 83.2 y) and SP free aged subjects (SP O) brains (n = 5; mean age = 77.2 y) as shown in Table 1.

In case No. 4, with the most advanced Braak stage [2, 3], MALDI-IMS analysis identified distinct deposits of Aβ1–40 and Aβ1–42 in the AD brain tissue (Fig. 1A, B). The Aβ1–40 was distributed predominantly in the leptomeningeal vessels of the subarachnoid space and arterioles in the cerebral parenchyma. However, Aβ1–42 formed SP in the cerebral parenchyma, and was mostly deposited in the pyramidal cell layer and subpial molecular layer (Fig. 1A, B). Subpial depositions of Aβ1–42 are seen lining the subarachnoid space, but each MALDI-IMS signal pattern was more dispersed compared with pyramidal layer depositions due to the 100 μm pitch resolution. As shown in detail in Fig. 1C, the mass spectrum from leptomeningeal blood vessels in the subarachnoid space, arterioles, and cerebral parenchyma displayed different mass numbers corresponding to each Aβ ion. Aβ1–40 was detected not only in the blood vessels, but also along with Aβ1–42 in the cerebral parenchyma (Fig. 1C). The MALDI-IMS signal of Aβ1–40 from CAA is stronger than that of SP in the cerebral parenchyma (Fig. 1A, B). In normal brains, MALDI-IMS detected Aβ distribution as weak dot-like patterns (Additional file 1: Figure S1). Different distributions of Aβ40 and Aβ42 were also demonstrated by IHC using serial frozen tissue sections. The anti-Aβ40 antibody preferentially labeled CAA, which is in clear contrast to the distribution of Aβ42 in the cerebral parenchyma (Fig. 1D). It should be noted that these findings are further confirmed when the bound antibodies were visualized by the avidin-biotin-complex-DAB detection method (Additional file 1: Figure S2).

The CAA phenotypes of case No. 3 were the most advanced amongst subjects of this study. Interestingly, Aβ1–42 deposition in the subpial molecular layer was less prominent than in case No. 4 (Fig. 1A, E and Additional file 1: Figure S3). Furthermore, Aβ1–36 to Aβ1–41 were preferentially deposited in the leptomeningeal vascular areas, while both Aβ1–42 and Aβ1–43 were distributed in the cerebral parenchyma of the occipital cortex (Fig. 1E). This is the first study which detected Aβ1–41 in human brain tissue. We have hypothesized that one single amino acid alteration at the C-terminus of Aβ results in drastic changes in Aβs distribution. The spatial resolution used with MALDI-IMS is a key parameter and must be chosen carefully because high-resolution imaging often results in decreased sensitivity. In Fig. 1A and E, MALDI-IMS with 100 μm pitch resolution was used, and we obtained an overall distribution profile in a relatively wide area. To portray fine tissue structures, such as vessels, high-resolution MALDI imaging (20 μm) was performed. The MALDI-IMS clearly demonstrates that Aβ1–36 to Aβ1–41 are distributed in the leptomeningeal vessels and arteriole walls and are quite different from the SP distribution of Aβ1–42 and Aβ1–43 (Additional file 1: Figure S4). These results are in line with recent IHC findings citing that not only Aβ1–40, but also Aβ37 to Aβ39, are deposited in the leptomeningeal blood vessel walls [17].

A variety of different N-truncated Aβ peptides have been identified starting with amino residue Ala-2, N3pE, Phe-4, Arg-5, Asp-7, Ser-8, Gly-9, Tyr-10, and N11pE [15, 24]. In this study, the mass spectrometric profile of SP and CAA gave rise to serial Aβs like N3pE-Aβ and N11pE-Aβ in addition to many N-terminally truncated forms of Aβs (Fig. 1C). As shown here, MALDI-IMS distinguished between N-terminal truncated Aβ40 and Aβ42 with individual localization profiles in the brain tissues (Fig. 2). MALDI-IMS demonstrated that N3pE-Aβ40 is deposited in the leptomeningeal vessels and arterioles similarly to Aβ1–40, but was not present in SP. The m/z 4126.5 for N3pE-Aβ40 and 4132.5 for Aβ1–38 showed close to the mass number respectively (Fig. 1C). However, a detailed analysis with MALDI-IMS demonstrated that the m/z 4126.5 for N3pE-Aβ40 was distributed both in leptomeningeal blood vessels and arterioles, while the m/z 4132.5 for Aβ1–38 was detected only in leptomeningeal blood vessels (Fig. 1C). The mass number of N3pE-Aβ42 was detected not only in SP, but also in leptomeningeal blood vessels with MALDI-IMS (Fig. 2 and Additional file 1: Figures S5 and S6). Furthermore, MALDI-IMS obtained the detailed distributions of both Aβx-40 and Aβx-42 (x = 2, N3pE, 4, 5, 6, 7, 8, 9, 10, and 11pE) in AD accompanied with CAA brains (Additional file 1: Figures S5 and S6). These N-truncated Aβ species were largely similar in their distribution to the full-length Aβ1–40 and Aβ1–42. As shown here, MALDI-IMS can individually track the whole distribution of complex molecules having multiple modifications, an advantage over conventional IHC. Not only does C-terminus truncation cause different distribution patterns than N-terminus truncation, (Fig. 1A, B), it is plausible that the structures of its C-terminus structure predefine the dynamics of Aβs rather than those of the N-terminal end. It must be noted that the N3pE-Aβ42 may particularly behave independently from Aβ1–42. Furthermore, N11pE-Aβ42 was also detected in SP with MALDI-IMS as a relatively major peak (Fig. 1C and Additional file 1: Figures S5 and S6). This pyroglutamate modified Aβ has only been seen in SP [15, 24], while N11pE-Aβ40 was detected in leptomeningeal vessel walls, but not in SP (Additional file 1: Figures S5 and S6).

In Fig. 1C and E, we demonstrated the presence of Aβ1–41 peptide in leptomeningeal vessels similar to the full length Aβ species, Aβ1–36 to Aβ1–40. However, Aβ1–42 was predominantly deposited within the cerebral parenchyma. Aβ1–41 and Aβ1–42 showed clear differences in deposition patterns. To characterize Aβ1–41 in the brain, we have generated antibodies against Aβ41, as shown in Additional file 1: Figure S7. The IHC using anti-Aβ41 IgG labeled vessels with CAA as Aβ40, but was not in present in SP and showed a clear contrast to the antibodies against Aβ42 (Fig. 3A). To further test the hypothesis that such a drastic alteration of Aβ peptides distribution depends on its self-aggregation ability, we examined the in vitro aggregation characteristics of Aβ1–40, Aβ1–41, and Aβ1–42 using thioflavin T. Synthetic Aβ1–42 peptide aggregated immediately, while both synthetic Aβ1–40 and Aβ1–41 peptides aggregated slowly during a 24 h incubation (Fig. 3B). Thus, a C-terminal structure alteration from Aβ41 to Aβ42 is sufficient to alter its self-aggregation ability, and this structural difference may lead to SP formation in the cerebral parenchyma.

We analyzed the accumulation of Aβ41 in contrast to Aβ38, Aβ40, and Aβ42 in the brain at various stages of SP. Surprisingly, Aβ38 and Aβ41 accumulated in the leptomeningeal blood vessels in the aged SP O subject brains as punctate (Fig. 3C). Furthermore, Aβ38 and Aβ41 were also detected in CAA in the brains of all cases having SP. Among the cases which had SP analyzed here, Aβ40 and Aβ42 were deposited in both SP and CAAs (Additional file 1: Table S1). In contrast to the Aβ40- and Aβ42-specific antibodies, the antibodies specific for Aβ38 and Aβ41 labeled only vessels regardless of the pathology. Therefore, it is presumed that Aβ38 and Aβ41 accumulate in vessels before the formation of the SP. Although we cannot confirm whether Aβ38 and Aβ41 positive vessels develop CAA, these Aβ species may be generated and accumulate into cerebral vessel walls in pre-pathological conditions.