Anti-PrPC monoclonal antibody infusion as a novel treatment for cognitive deficits in an Alzheimer's disease model mouse

Abstract

Background: Alzheimer's Disease (AD) is the most common of the conformational neurodegenerative disorders characterized by the conversion of a normal biological protein into a β-sheet-rich pathological isoform. In AD the normal soluble Aβ (sAβ) forms oligomers and fibrils which assemble into neuritic plaques. The most toxic form of Aβ is thought to be oligomeric. A recent study reveals the cellular prion protein, PrPC, to be a receptor for Aβ oligomers. Aβ oligomers suppress LTP signal in murine hippocampal slices but activity remains when pretreated with the PrP monoclonal anti-PrP antibody, 6D11. We hypothesized that targeting of PrPC to prevent Aβ oligomer-related cognitive deficits is a potentially novel therapeutic approach. APP/PS1 transgenic mice aged 8 months were intraperitoneally (i.p.) injected with 1 mg 6D11 for 5 days/week for 2 weeks. Two wild-type control groups were given either the same 6D11 injections or vehicle solution. Additional groups of APP/PS1 transgenic mice were given either i.p. injections of vehicle solution or the same dose of mouse IgG over the same period. The mice were then subjected to cognitive behavioral testing using a radial arm maze, over a period of 10 days. At the conclusion of behavioral testing, animals were sacrificed and brain tissue was analyzed biochemically or immunohistochemically for the levels of amyloid plaques, PrPC, synaptophysin, Aβ40/42 and Aβ oligomers.

Results: Behavioral testing showed a marked decrease in errors in 6D11 treated APP/PS1 Tg mice compared with the non-6D11 treated Tg groups (p < 0.0001). 6D11 treated APP/PS1 Tg mice behaved the same as wild-type controls indicating a recovery in cognitive learning, even after this short term 6D11 treatment. Brain tissue analysis from both treated and vehicle treated APP/PS1 groups indicate no significant differences in amyloid plaque burden, Aβ40/42, PrPC or Aβ oligomer levels. 6D11 treated APP/PS1 Tg mice had significantly greater synaptophysin immunoreactivity in the dentate gyrus molecular layer of the hippocampus compared to vehicle treated APP/PS1 Tg mice (p < 0.05).

Conclusions: Even short term treatment with monoclonal antibodies such as 6D11 or other compounds which block the binding of Aβ oligomers to PrPC can be used to treat cognitive deficits in aged AD transgenic mice.

Figure 1

Radial Arm Maze Cognitive Testing. Figure 1 shows the results of radial arm maze cognitive testing. The number of errors is plotted versus the day of testing. Two-way ANOVA revealed a significant treatment effect in Tg 6D11 treated (n = 10) versus vehicle treated (n = 8) or murine IgG treated (n = 9) APP/PS1 Tg mice (p < 0.0001) with a Bonferroni post-hoc analysis showing no difference between Tg 6D11 treated and wild-type mice which were injected with either PBS alone (n = 8) or 6D11 (n = 9). APP/PS1 Tg non-treated mice and APP/PS1 mice treated with mouse IgG made significantly more errors than wild-type animals and 6D11 treated Tg mice (p < 0.01).

Figure 2

Bar Graphs of Amyloid Quantitation by Stereology. A and B shows a bar graphs of the amyloid quantitation by stereology in the cortex (A) and hippocampus (B) of Tg vehicle injected (n = 8) and 6D11 treated Tg mice (n = 10). There were no significant differences in the amyloid burden (% area occupied by 6E10 immunoreactivity) in both the cortex and hippocampus.

Figure 3

Representative Sections Immunostained with anti-Aβ Antibody. A-D show representative immunostained sections with anti-Aβ antibody 6E10 in the cortex and hippocampus at low power (A and B) and in the cortex (C and D) at higher magnification of 6D11 treated Tg mice (A and C) and vehicle injected Tg mice (B and D). Scale bar = 200 μm for A and B. Scale bar = 50 μm for C and D.

Figure 4

Quantitation of Synaptophysin Immunoreactivity. A shows a bar graph representation of synaptophysin immunoreactive presynaptic terminals in the molecular layer of the dentate gyrus of the hippocampus. The differences between 6D11 treated Tg mice (n = 10) and vehicle treated Tg mice (n = 8) are statistically significant by one-tailed t-test (p = 0.0267). B-E show representative sections immunostained with anti-synaptophysin antibody in the hippocampus at 10x magnification (B and C- Scale bar = 100 μm) and at 100x magnification (D and E- Scale bar = 10 μm) with the green box indicating the area of molecular layer magnified to the higher power. Images are of representative 6D11 treated Tg mice (B, D) and vehicle treated Tg mice (C, E).

Figure 5

Aβ40/42 Quantitation Biochemically. Shows the levels of Aβ40 and Aβ42 in the FA and DEA extracted material from brains of vehicle treated Tg (n = 8) and 6D11 treated Tg mice (n = 10). Levels from the FA extract fraction of Aβ40 and Aβ42 are shown in A and B, respectively. Levels from the DEA extract fraction of Aβ40 and Aβ42 are shown in C and D, respectively. There were no significant differences in the levels of Aβ40 or Aβ42 in either the FA or DEA fractions.

Figure 6

Quantitation of Aβ Oligomer and Aggregated Aβ Levels. A shows a Western blot using Aβ oligomer specific antibody A11 on the left from each of two representative 6D11 treated and vehicle treated Tg mice. On the right, a Western blot using anti-Aβ monoclonal antibody 6E10 is shown. B shows a bar graph of the densitometric analysis of the major A11 immunoreactive band at ~55 kDa, in arbitrary O.D. units. C shows a bar graph of the levels of aggregated Aβ in 6D11 treated (n = 10) and vehicle treated Tg mice (n = 8) as determined by ELISA. There is no significant difference between the 6D11 treated and vehicle treated Tg mice in the levels of Aβ oligomers determined by Western blot or of aggregated Aβ determined by ELISA.

Figure 7

Quantitation of PrP^C Levels. A shows representative Western blots using anti-PrP mAb 6D11 showing immunostainging bands corresponding to the non-, mono-, and diglycosylated isoforms of PrP^C from a Tg 6D11 treated Tg mouse, vehicle treated Tg mouse and a wild-type mouse. B depicts a bar graph of the densitometric analysis (in arbitrary units) of the non-, mono-, and diglycosylated PrP^C bands. There are no significant differences the mouse groups in the levels of PrP^C.