PMC5797595_01
PMC5797595_02
MR imaging of cortical atrophy and myelin degeneration. (A) Mid-sagittal T2W image of the brain demonstrating atrophy of the intra-thalamic adhesion (*) height 4.76 mm. (B) Transverse T2W at the level of temporal lobes demonstrating cortical atrophy with widening of the subarachnoid space (green arrow) and enlargement of the lateral ventricle (orange arrow). (C) Transverse FLAIR at level of temporal lobes and (D) transverse FLAIR at level of occipital lobes and midbrain demonstrating white matter hyperintensity (blue arrows).
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The brain MRI scan revealed a diffuse cortical atrophy and a hyperintensity in the white matter on T2W, particularly in the corona radiata (Figure 1); changes consistent with age-associated cognitive decline (Hasegawa et al.,).
Cortico-neuropathological microscopic lesions. (A) Cortical degeneration (vacuolation) and neuronal death observed on routine H&E stained sections of prefrontal cortex and diffuse cerebral periventricular white matter degeneration (vacuolation and pallor); (B) Neuronal degeneration and loss (arrows) confirmed with neuron-specific marker, NeuN. (C) Specific labeling of diffuse Abeta plaques with anti-Abeta specific antibody in the prefrontal cortex. (D) Extensive gliosis in prefrontal cortex revealed by GFAP stain and associated with (E) microglia activation demonstrated with Iba1 staining. (F) Specific labeling of CAA with anti-Abeta specific antibody in the cortical blood vessels in the prefrontal cortex.
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Neuronal loss and degeneration was marked in the cortical region of the subject (Figures 2A,B) while intense binding of large extra-cellular diffuse Abeta plaques, recruitment, and activation of astrocytes and microglial cells (Figures 2C-E) were observed in the neocortex and hippocampus. Pronounced cerebral amyloid angiopathy (CAA) was observed in several cortical blood vessels (Figure 2F), but was less intense in the tunica media of leptomeningeal arteries. Furthermore, white matter degeneration took the form of myelin vacuolation and isomorphic gliosis (Figure 2A) with macrophages containing pale yellow cytoplasmic material evident as small clusters of cells and as perivascular aggregates (Figure 2A).
Immuno-detection of Abeta soluble oligomers. (A) Western blot showing the ability of PRIOC10 anti-oligomer antibody to strongly immunocapture precipitates from the CSF (sub-CSF) but not the serum (sub-serum) derived from the dog. Positive control derived from CSF from Alzheimer's disease patients (patient case number 51486:age 79 years [pos1-CSF] and 60649-65 years [pos2-CSF]) displayed PRIOC10-positivity for soluble oligomers. In contrast, CSF (neg-CSF) and serum (neg-serum) derived from a Rottweiler failed to display PRIOC10 labeling on Western blotting. (B) A customized Sandwich ELISA was used to detect specific Abeta oligomers. Biotinylated anti-Abeta 4G8 (Abeta) (Thakker et al.,), PrioAD12 (Abeta1-40) or PrioAD13 (Abeta1-42) (David et al.,) were used to immunocapture Abeta species contained in the CSF (sub-CSF) and the serum (sub-serum) precipitates derived from the dog. Purified PRIOC10 anti-oligomer antibody was added to immunodetect specifically Abeta soluble oligomers. The sandwich format of the assay has established the specificity of PRIOC10 antibody for Abeta oligomers. Values shown are the mean Abeta +- SD from 12 observations.
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Western blotting and Sandwich ELISA. Western blotting results indicate that PRIOC10 mAb bound to soluble oligomers in sub-CSF but not in sub-serum or negative control CSF (neg-CSF) derived from the Rottweiler (Figure 3A). Further, PRIOC10 mAb pattern of recognition revealed several bands ranging between 50 and 160 kDa (Figure 3A); and positive control CSF (pos-CSF) isolated from two patients with AD also led to detection of soluble oligomers (Figure 3A). PRIOC10-specific bands detected in the sub-CSF were mainly composed of Abeta using our customized sELISA as described previously (Tayebi et al.,). PRIOC10, 4G8 (Abeta) (Thakker et al.,), PrioAD12 (Abeta1-40), or PrioAD13 (Abeta1-42) (David et al.,) were used as immunocapture antibodies to detect soluble oligomers in the sub-CSF and sub-serum, followed by immunodetection with biotinylated PRIOC10 (Figure 3B). Of note, sELISA lead to detection of Abeta in the sub-serum (Figure 3B) in contrast with western blotting, albeit with significantly lower OD intensity as compared to levels detected in the sub-CSF.
CSF but not serum derived from the aged dog leads to neurotoxicity of neuron-like SH-SY5Y cell line. The effect of CSF and serum on the survival of SH-SY5Y cell line was compared with monoAbeta1-40, monoAbeta1-42, scramAbeta25-35, oligoAbeta1-40, oligoAbeta1-42, fibAbeta1-40, fibAbeta1-42 as well as CSF (neg-CSF) and serum (neg-serum) derived from a Rottweiler. Values shown are the mean cell survival +- SD from 12 observations.
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MonoAbeta1-40, monoAbeta1-42, scramAbeta25-35, and sub-serum displayed no toxicity toward RA-SH-SY5Y neuroblastoma cells as compared to untreated cells (p <= 0.05) (Figure 4). In contrast, treatment with oligoAbeta1-40, oligoAbeta1-42, fibAbeta1-40, fibAbeta1-42, and sub-CSF lead to significant cell death as compared with untreated cells, resulting in <=61% cell viability for treatment with both oligoAbeta1-40 and fibAbeta1-40 (p <= 0.05) and <=44% cell viability for treatment with oligoAbeta1-42, fibAbeta1-42, and sub-CSF (p <= 0.05) (Figure 4).
Aggregation kinetics of "seed-free" synthetic monomeric Abeta peptide. Synthetic seed-free Abeta monomers were used to produce (A) PRIOC10-labeled Abeta soluble oligomers and (B) Abeta ThT-labeled fibrils in a kinetic reaction (0-72 h). t0 is the initial time just before starting the kinetic reaction (t0 = seed-free monomers only); t12 represents maximal OD value of PRIOC10-specific Abeta oligomers (t12 = maximal oligomer yield); and t72 represents maximal value of ThT-specific Abeta fibrils (t72 = maximal fibrils yield). (C) seed-free Abeta1-40 (t0) aggregation kinetic was assessed after addition of known quantities of preformed Abeta1-40 amyloid seeds (t12 and t72) and compared with the aggregation kinetic after addition of conversion buffer or scrambled Abeta25-35 (t12 and t72). (D) seed-free Abeta1-40 (t0) aggregation kinetic was assessed after addition of known quantities of preformed Abeta1-42 amyloid seeds (t12 and t72) and compared with the aggregation kinetic after addition of conversion buffer or scrambled Abeta25-35 (t12 and t72). (E) seed-free Abeta1-40 (t0) aggregation kinetic was assessed after addition of known quantities of sub-CSF and sub-serum and compared with the aggregation kinetic after addition of conversion buffer, negative control CSF (neg-CSF) or serum (neg-serum). (F) seed-free Abeta1-42 (t0) aggregation kinetic was assessed after addition of known quantities of sub-CSF and sub-serum and compared with the aggregation kinetic after addition of conversion buffer, negative control CSF (neg-CSF) or serum (neg-serum). Error bars represent the mean level derived from n = 4 wells.
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We first demonstrated that PRIOC10 immunodetected Abeta soluble oligomer species derived from monoAbeta1-40 and monoAbeta1-42 peptides (Figure 5A). ThT fluorescence intensity of the fibrilar species was measured following conversion of monoAbeta1-40, monoAbeta1-42, and scramAbeta25-35 peptides was assessed and was shown to be inversely proportional to levels of PRIOC10-specific oligomer species (Figure 5B). ThT did not bind to scramAbeta25-35 peptide before and after being incubated in conversion buffer and to monoAbeta1-40 and monoAbeta1-42 peptides before conversion. Abeta peptide following addition of Abeta oligomers or fibrils (Figures 5C,D). A known concentration of Abeta prepared by conversion during 12 h (t12) and 72 h (t72), as described above, was used in the seeding reaction; as t12 represents maximal optical density (OD) expression of Abeta soluble oligomers immunodetected with PRIOC10 (Figure 5A) and t72 represents maximal fluorescence expression of Abeta fibrils bound to ThT (Figure 5B). We report that t12 oligoAbeta1-40, oligoAbeta1-42 but not post-conversion scramAbeta25-35 led to substantial reduction of the "lag-phase" (p <= 0.05) compared to t0 Abeta (Figures 5C,D). Both t72 fibAbeta1-40 and fibAbeta1-42 but not post-conversion scramAbeta25-35 affected the Abeta aggregation kinetic by shortening the reaction's "lag-phase," albeit the effect was limited when compared to the addition of the oligomers (Figures 5C,D), reflecting a weaker seeding ability of the fibrils. Surprisingly and for the first time, we show that the dog's CSF led to a substantial shortening of the reaction's "lag-phase" (p <= 0.05) and acceleration of human synthetic monomeric Abeta1-40 and Abeta1-42 aggregation as compared to a negative control CSF derived from the Rottweiler or scramAbeta25-35 (Figures 5E,F). Sub-CSF was more efficient in speeding up Abeta1-42 then Abeta1-40 aggregation. In contrast, sub-serum and negative control serum led to complete inhibition of the Abeta kinetics and the "lag-phase" was not observed (Figures 5E,F).