Abundant pyroglutamate-modified ABri and ADan peptides in extracellular and vascular amyloid deposits in familial British and Danish dementias

Abstract

Familial British and familial Danish dementia (FDD) are progressive neurodegenerative disorders characterized by cerebral deposition of the amyloidogenic peptides ABri and ADan, respectively. These amyloid peptides start with an N-terminal glutamate residue, which can be posttranslationally converted into a pyroglutamate (pGlu) modified form, a mechanism which has been extensively described to be relevant for amyloid-beta (Aβ) peptides in Alzheimer's disease. Like pGlu-Aβ peptides, pGlu-ABri peptides have an increased aggregation propensity and show higher toxicity on human neuroblastoma cells as their nonmodified counterparts. We have generated novel N-terminal specific antibodies detecting the pGlu-modified forms of ABri and ADan peptides. With these antibodies we were able to identify abundant extracellular amyloid plaques, vascular, and parenchymal deposits in human familial British dementia and FDD brain tissue, and in a mouse model for FDD. Double-stainings using C-terminal specific antibodies in human samples revealed that highly aggregated pGlu-ABri and pGlu-ADan peptides are mainly present in plaque cores and central vascular deposits, leading to the assumption that these peptides have seeding properties. Furthermore, in an FDD-mouse model ADan peptides were detected in presynaptic terminals of the hippocampus where they might contribute to impaired synaptic transmission. These similarities of ABri and ADan to Aβ in Alzheimer's disease suggest that the posttranslational pGlu-modification of amyloid peptides might represent a general pathological mechanism leading to increased aggregation and toxicity in these forms of degenerative dementias.

Fig. 1

Aggregation profile and cytotoxicity of pGlu-modified ABri/ADan peptides. Compared to ABri peptides, pGlu-ABri peptides showed a much more rapid formation of intermediate oligomeric assemblies and strongly enhanced fibril formation in a Thio-T assay (A). Application of 5 µM non-modified ABri or ADan peptides did not influence cell viability in SH-SY5Y cells as measured by LDH release, however, incubation with both pGlu-modified ABri as well as ADan peptides resulted in a significantly increased toxicity (B). Dotted lines in A indicate s.e.m.

Fig. 2

Dot-blot and Western-blot analyses of ABri/ADan N-terminal antibodies. AB76-2 showed a clear preference for pGlu-modified peptides, whereas AB77 detected ABri and pGlu-ABri peptides in a similar fashion (A). Western-blot analysis corroborated this finding showing only a minor cross-reactivity with non-modified ABri peptides at higher amounts of peptide (250 ng) and a clear preference for pGlu-modified ABri and ADan peptides using AB76-2 (B).

Fig. 3

AB77 and AB76-2 immunoreactivity in FBD and FDD patients. Abundant AB77 (A) and AB76-2 (B) positive extracellular parenchymal deposits are seen in the cerebellum of a FBD patient. In addition, prominent vascular staining was detected in the dentate gyrus (C,D) and the CA4 region of the hippocampus (E,F) in a patient suffering from FDD. AB77: A, C, E; AB76-2: B, D, F. Scale bar: 100 µm

Fig. 4

Double immunofluorescent stainings with a C-terminal ABri antibody combined with the novel N-terminal antibodies in an FBD patient. Abundant ABri (B) positive parenchymal deposits were observed in the hippocampus of an FBD patient, the majority of these deposits were also positive with AB77 (A) apart from the periphery of the plaques (C, arrowhead). AB76-2 (D) stained parenchymal plaques and ABri (antibody 338, E) deposited within the blood vessels, whereas the perivascular deposits remained unstained (F, arrow). Similar staining was observed with AB77 (G) or AB76-2 (J) when combined with Thioflavin-S (H,K) in FBD tissue, however, a complete colocalization was detected using AB76-2 (Fig. 4J). Original magnification: 200×

Fig. 5

Age-dependent accumulation of ADan/pGlu-ADan in ADanPP7 mice. Using AB77 (A–H) and AB76-2 (I–P), ADan/pGlu-ADan immunoreactivity was detected already at 2 months of age in frontal cortex (A,I) and hippocampus (E,M) of ADanPP7 mice showing a strong increase at 4 (B,F,J,N), 13 (C,G,K,O) and 20 months of age (D,H,L,P). Scale bar: 200 µm (A–P)

Fig. 6

Most of the larger blood vessels stained positive with AB77 (A,D), AB76-2 (B,E) and Thio-S (C,F) in ADanPP7 mice. Almost all of the deposits that stained with AB76-2 (H) were also Thio-S positive (I, arrows). However, some deposits could be only labeled with AB77 and showed neither AB76-2 nor Thio-S staining (G, arrowheads). Scale bar: 50 µm (A–I)

Fig. 7

Immunofluorescence stainings using the dendritic marker MAP2 (A) and AB77 (B) revealed only minor co-localization in dendritic processes (C). However, the synaptic marker Synaptophysin (D) showed co-localization with AB77-positive material (E) in the stratum lucidum in the hippocampus (F), indicating the presence of ADan/pGlu-ADan peptides in presynaptic terminals. Scale bar: 33 µm (A–C), 50 µm (D–F).