This project was reviewed by the University of Surrey Ethics Committee and verified that the aspects of the study pertaining to the Samoyed dog including use of body fluids excess and post mortem material did not come under the auspices of Animals (Scientific Procedures) Act 1986 (ASPA). The subject's owner gave informed consent to participation in the study. Full clinical and neurological examination and presentation, including MR imaging assessment are found in Supplementary Materials. Negative control CSF was obtained from a 10-year-old male Rottweiler suffering from nodular granulomatous episclerokeratitis following submission for routine teaching post-mortem and not subject to animal ethics guidelines. CSF derived from a 79-year-old patient with advanced sporadic AD (pos1-CSF) and from a 65 year old patient with advanced sporadic AD (pos2-CSF) were provided by The UK Multiple Sclerosis Tissue Bank.

A 12-year-old neutered male Samoyed dog was presented for pain management and evaluation of difficulty in rising. Neurological examination revealed tetraparesis and reduced spinal reflexes and muscle tone consistent with a polyneuropathy. The difficulty rising was attributed to this, complicated by the sedative polypharmacy. The historical and consulting room behavior suggested a cognitive function deficit possibly complicated by a urinary tract infection. 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., 2005). Signs were controlled for the next 6 months after which the dog deteriorated and described as being extremely agitated and distressed during the night which was unresponsive to changing dose of medication and resulting in significant sleep deprivation for the owners. A full post mortem examination was undertaken at the pathology facility at the University of Surrey and the brain and other organs were removed for further analysis. A more extensive description of the clinical and neurological examination and presentation is included as supplementary results.

Neuronal loss and degeneration was marked in the cortical region of the subject (Figures 2A,B) while intense binding of large extra-cellular diffuse Aβ 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. Of note, PHF and the signaling adapter p62 were not detected (data not shown) (Babu et al., 2005). 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). Iba1 stain demonstrated more widespread microglial/macrophage activation while GFAP confirmed the brisk gliosis (data not shown).

The ability of PRIOC10 mAb to bind to Aβ soluble oligomers in sub-serum and sub-CSF was assessed by 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).

We then set out to investigate the specificity of the soluble oligomers and confirm that the PRIOC10-specific bands detected in the sub-CSF were mainly composed of Aβ using our customized sELISA as described previously (Tayebi et al., 2011). PRIOC10, 4G8 (Aβ) (Thakker et al., 2009), PrioAD12 (Aβ_1–40), or PrioAD13 (Aβ_1–42) (David et al., 2014) were used as immunocapture antibodies to detect soluble oligomers in the sub-CSF and sub-serum, followed by immunodetection with biotinylated PRIOC10 (Figure 3B). We show that the 4G8 vs. biot-PRIOC10 combination detected highest levels of Aβ in sub-CSF as expressed in OD-values (p = 0.0001) and almost matched CSF levels detected with the PRIOC10 vs. biot-PRIOC10 combination. This was followed by the PRIOAD13 vs. biot-PRIOC10 combination that displayed higher CSF levels of detection compared with the PRIOAD12 vs. biot-PRIOC10 combination (p = 0.0279). Of note, sELISA lead to detection of Aβ 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.

Genome assembly CanFam3.1 and transcripts ENSCAFT00000013599.4 for APP, ENSCAFT00000026626.1 for PSEN1, and ENSCAFT00000025451.3 for PSEN2 were used for primer design (See Supplementary Results: Table S2) and as the reference for sequence analysis. The subject's DNA was used to sequence the genes that when mutated are known to cause AD in humans. No variants expected to be pathogenic were identified. Synonymous variants were found in APP (p.G120G; p.K178K; p.A242A; p.T266T), and PSEN2 (p.P436P).

The toxic effects of monoAβ_1–40, monoAβ_1–42, scramAβ_25–35, oligoAβ_1–40, oligoAβ_1–42, fibAβ_1–40, fibAβ_1–42, sub-serum, and sub-CSF on differentiated human neuroblastoma cell line RA-SH-SY5Y viability was investigated using the MTT assay. To achieve similar concentrations of synthetic Aβ and CSF/serum-borne Aβ, standard curves of all synthetic Aβ was generated and the subject's CSF and serum Aβ oligomers values were determined by comparison to the appropriate standard curve. MonoAβ_1–40, monoAβ_1–42, scramAβ_25–35, 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 oligoAβ_1–40, oligoAβ_1–42, fibAβ_1–40, fibAβ_1–42, and sub-CSF lead to significant cell death as compared with untreated cells, resulting in ≤61% cell viability for treatment with both oligoAβ_1–40 and fibAβ_1–40 (p ≤ 0.05) and ≤44% cell viability for treatment with oligoAβ_1–42, fibAβ_1–42, and sub-CSF (p ≤ 0.05) (Figure 4).

Cell viability was significantly affected following treatment with oligoAβ_1–42 compared with treatment with the fibrillary species of Aβ_1–42 (17 vs. 27%; p ≤ 0.05), while treatment with sub-CSF lead to 44% cell death. These results show that the subject's CSF induced RA-SH-SY5Y cell death and confirmed the potent toxic effects of Aβ soluble oligomers previously shown to affect neurons (Bate et al., 2008).

We first demonstrated that PRIOC10 immunodetected Aβ soluble oligomer species derived from monoAβ_1–40 and monoAβ_1–42 peptides (Figure 5A). Secondly, ThT fluorescence intensity of the fibrilar species was measured following conversion of monoAβ_1–40, monoAβ_1–42, and scramAβ_25–35 peptides was assessed and was shown to be inversely proportional to levels of PRIOC10-specific oligomer species (Figure 5B). ThT did not bind to scramAβ_25–35 peptide before and after being incubated in conversion buffer and to monoAβ_1–40 and monoAβ_1–42 peptides before conversion. Of note and as shown previously, PRIOC10 failed to bind the fibrilar species (Tayebi et al., 2011).

We then assessed aggregation kinetics of “seed-free” synthetic monomeric Aβ peptide following addition of Aβ oligomers or fibrils (Figures 5C,D). A known concentration of Aβ 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 Aβ soluble oligomers immunodetected with PRIOC10 (Figure 5A) and t₇₂ represents maximal fluorescence expression of Aβ fibrils bound to ThT (Figure 5B).

Here, we added 10 pmol oligoAβ_1–40/oligoAβ_1–42 (t₁₂), fibAβ_1–40/fibAβ_1–42 (t₇₂), or scramAβ_25–35 (t₁₂ and t₇₂) prepared by conversion during t₁₂ and t₇₂ to 2 mM monoAβ_1–40, or monoAβ_1–42 in order to assess their effects on the “lag-phase” kinetic as measured by ThT fluorescence. We report that t₁₂ oligoAβ_1–40, oligoAβ_1–42 but not post-conversion scramAβ_25–35 led to substantial reduction of the “lag-phase” (p ≤ 0.05) compared to t₀ Aβ (Figures 5C,D). Importantly, we show that oligoAβ_1–42 was more efficient in shortening the “lag-phase” compared to oligoAβ_1–40 (p ≤ 0.05). Both t₇₂ fibAβ_1–40 and fibAβ_1–42 but not post-conversion scramAβ_25–35 affected the Aβ 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.

Finally, the subject's serum and CSF was added to “seed-free” synthetic monomeric Aβ to investigate whether pre-existing oligomer seeds contained in the serum and CSF of the aged dog can affect the Aβ aggregation kinetics through reduction of the “lag-phase” and to explore if cross-species interaction of dog Aβ with human Aβ synthetic peptide overcomes the so-called “species barrier” as applies for prion disorders (Hill and Collinge, 2002). First, we tested whether the precipitation protocol altered the conformation of Aβ soluble oligomer content of sub-CSF and sub-serum. We show that PRIOC10-specific Aβ oligomers in TCA/acetone treated sub-CSF and sub-serum were preserved (Figure S1). Total protein (20 μl of 10 mg/ml) derived from sub-serum and sub-CSF samples were incubated with solutions of monoAβ_1–40, and monoAβ_1–42 peptides. 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 Aβ_1–40 and Aβ_1–42 aggregation as compared to a negative control CSF derived from the Rottweiler or scramAβ_25–35 (Figures 5E,F). Sub-CSF was more efficient in speeding up Aβ_1–42 then Aβ_1–40 aggregation. In contrast, sub-serum and negative control serum led to complete inhibition of the Aβ kinetics and the “lag-phase” was not observed (Figures 5E,F). Of note, similar concentrations of synthetic Aβ and CSF/serum-borne Aβ were used.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.