Alzheimer therapy with an antibody against N-terminal Abeta 4-X and pyroglutamate Abeta 3-X

Abstract

Full-length Aβ1-42 and Aβ1-40, N-truncated pyroglutamate Aβ3-42 and Aβ4-42 are major variants in the Alzheimer brain. Aβ4-42 has not been considered as a therapeutic target yet. We demonstrate that the antibody NT4X and its Fab fragment reacting with both the free N-terminus of Aβ4-x and pyroglutamate Aβ3-X mitigated neuron loss in Tg4-42 mice expressing Aβ4-42 and completely rescued spatial reference memory deficits after passive immunization. NT4X and its Fab fragment also rescued working memory deficits in wild type mice induced by intraventricular injection of Aβ4-42. NT4X reduced pyroglutamate Aβ3-x, Aβx-40 and Thioflavin-S positive plaque load after passive immunization of 5XFAD mice. Aβ1-x and Aβx-42 plaque deposits were unchanged. Importantly, for the first time, we demonstrate that passive immunization using the antibody NT4X is therapeutically beneficial in Alzheimer mouse models showing that N-truncated Aβ starting with position four in addition to pyroglutamate Aβ3-x is a relevant target to fight Alzheimer's disease.

Figure 1. NT4X antibody binds specifically to Aβ_pE3-42 and Aβ_4-42 peptides.

Sensorgram profiles generated by binding studies using the Biacore T200 showing the association and dissociation binding kinetics of the NT4X antibody to freshly prepared Aβ_pE3-42 (a), Aβ_4-42 (b) and Aβ_1-42 (c) peptides. NT4X binds to Aβ_pE3-42 and Aβ_4-42 but does not bind to Aβ_1-42. The heterogeneous nature of the Aβ peptides limits this application in obtaining accurate kinetic data. However, the theoretical fit of binding of Aβ_pE3-42 (d) and Aβ_4-42 (e) peptides to the NT4X antibody and their kinetics of binding (f) is also shown. Abbreviations: RU: response units; K_on: association rate, K_off: dissociation rate; KD: equilibrium constant.

Figure 2. NT4X protection of Aβ_pE3-42 and Aβ_4-42 induced toxicity in primary rat neuronal cultures.

NT4X IgG2b and an isotype control antibody were assayed to assess potential protective properties in an amyloid peptide induced cellular toxicity assay using primary rat cortical cultures. Cellular toxicity was measured using an LDH release assay and results converted to percentage inhibition of cell death compared to control wells (n = 3 repeats on cell preparations from 3 separate rats). Values plotted are means +/− SEM. The NT4X IgG2b antibody shows inhibition of Aβ_pE3-42 and Aβ_4-42 induced toxicity but has no effect on the toxicity induced by full length Aβ_1-42.

Figure 3. Age-dependent spatial reference memory deficits in homozygous Tg4-42 (Tg4-42_hom) mice.

Spatial reference memory in Tg4-42_hom mice was assessed at 4, 5, 6 and 7 months of age using the Morris water maze. (a,b) Cued training of the water maze revealed that Tg4-42_hom exhibited intact eyesight and the motor abilities to perform the test. (b) Tg4-42_hom mice showed comparable swimming speeds. (c) Spatial learning was assessed using the acquisition training of the Morris water maze. All Tg4-42_hom mice showed reduced escape latency over 5 days of acquisition training irrespectively of age. (d) A probe trial was given at the end of the learning phase to assess spatial reference memory. Quadrant preference was analyzed for 60 seconds. Tg4-42_hom showed no impairment in spatial reference memory at 4 months of age as mice spent a significant greater percentage of the time in the target quadrant. At 5 months of age, spatial reference learning is already slightly impaired. Furthermore, the probe trial revealed a significantly reduced learning behavior for 6- and 7-month-old Tg4-42_hom mice as they showed no preference for the target quadrant. Four months old Tg4-42_hom mice performed significantly superior to 6-months-old mice on day 3 to 5 (one-way-ANOVA, day 3: p < 0.001; day 4 and 5: p < 0.01). Furthermore, 4-month-old mice showed significantly shorter escape latencies compared to 5- and 7-month-old mice on day 3 (one-way-ANOVA, p < 0.05). ***p < 0.001; **p < 0.01; *p < 0.05; n = 8–17 per group (4 m: n = 8, 5 m: n = 16, 6 m: n = 17, 7 m: n = 9). Swimming Speed and escape latency: repeated measures ANOVA followed by Bonferroni multiple comparisons. Quadrant preference: one-way analysis of variance (ANOVA) followed by Bonferroni multiple comparisons. T target quadrant, L left quadrant, R right quadrant, O opposite quadrant. Data presented as mean ± S.E.M; m = months.

Figure 4. Age-dependent neuron loss in hippocampus of Tg4-42_hom mice.

Quantification of neurons in the CA1 using unbiased stereology. Tg4-42_hom show an age dependent reduction in number of neurons in the CA1 region, which is significant at 5 m, 6 m, 7 m, and 8 m. One-way analysis of variance (ANOVA) followed by Bonferroni multiple comparisons; n = 4–8; ***p < 0.001; data presented as mean ± S.E.M; m = months.

Figure 5. Passive immunization with NT4X rescues learning deficits in Tg4-42_hom mice.

Tg4-42_hom mice that received weekly injections with the full-length NT4X-antibody, the Fab fragment of NT4X, an IgG control antibody and PBS for a period of 12 weeks as well as a group of non-treated (NT) mice were tested at 6 months of age in the Morris Water Maze. (a,b) Cued training revealed that all mice exhibited intact eyesight and the motor abilities to perform the test. (a) Escape latency decreased progressively over 3 days of cued training for all mice. (b) Mice showed comparable swimming speeds. (c) Spatial learning was assessed in the acquisition training. All Tg4-42_hom mice showed reduced escape latency over the 5 days of acquisition training. (d) Spatial reference memory was impaired in non-treated Tg4-42_hom as well as in PBS- and IgG-treated Tg4-42_hom mice as they showed no preference for the target quadrant in the probe trial. In contrast, Tg4-42_hom mice immunized with the full-length NT4X and the Fab fragment of NT4X antibody displayed no learning deficits at this age. ***p < 0.001; **p < 0.01; *p < 0.05. n = 10–17 per group (Tg4-42_hom NT: n = 17, Tg4-42_hom NT4X: n = 15, Tg4-42_hom Fab: n = 10, Tg4-42_hom IgG: n = 14, Tg4-42_hom PBS n = 15). Swimming Speed and escape latency: repeated measures ANOVA followed by Bonferroni multiple comparisons. Quadrant preference: One-way analysis of variance (ANOVA) followed by Bonferroni multiple comparisons. T target quadrant, L left quadrant, R right quadrant, O opposite quadrant. Data presented as mean ± S.E.M; m = months.

Figure 6. NT4X mitigates neuron loss in Tg4-42_hom mice.

Quantification of neurons in the CA1 of 6-months-old Tg4-42_hom using unbiased stereology. Tg4-42_hom mice that received weekly injections with the NT4X antibody or its respective Fab fragment, both an IgG control and a PBS control group for a period of 12 weeks as well as a group of non-treated mice were analyzed at 6 months of age. Tg4-42_hom mice immunized with NT4X full length or Fab fragment both displayed significantly more neurons than same-aged untreated, IgG and PBS injected mice. In contrast, the number of neurons did not differ significantly between untreated, IgG and PBS injected Tg4-42_hom mice. One-way analysis of variance (ANOVA) followed by Bonferroni multiple comparisons; n = 9–15 (Tg4-42_hom NT: n = 9, Tg4-42_hom NT4X: n = 15, Tg4-42_hom IgG: n = 9, Tg4-42_hom PBS n = 15). *p < 0.05; **p < 0.01; ***p < 0.001; data presented as mean ± S.E.M; NT = non-treated.

Figure 7. Full-length and Fab fragment of NT4X rescue Aβ_4-42 induced working memory deficits in wildtype mice.

(A,B) Working memory deficits were induced by intraventricular injection of Aβ_4-42 as the mice performing at chance level (dashed line). Both treatment with (a) full-length antibody and (b) Fab fragment rescued memory deficits in a dose-dependent manner. Mice treated with the vehicle control and the vehicle in combination with full-length or Fab fragment of NT4X respectively demonstrated normal working memory performance. One-way analysis of variance (ANOVA) followed by Bonferroni multiple comparisons; n = 6–8 per group; **p < 0.01; *p < 0.05; data presented as mean ± S.E.M.

Figure 8. Reduced plaque loads in immunized 5XFAD mice.

Plaque load analysis of NT4X and 1–57 immunized 5XFAD mice compared to PBS injected 5XFAD mice. (a) NT4X treatment significantly reduced the percentage of brain area occupied by fibrillar Aβ deposits in the cortex in 5XFAD mice demonstrated by Thioflavin S staining. (b) Immunostaining with the antibody 1–57 against pyroglutamate Aβ_3−x revealed a reduced plaque burden in the cortex of NT4X immunized 5XFAD. D Moreover, immunostaining with an antibody against Aβ_x-40 showed a reduced plaque burden in the cortex of NT4X immunized 5XFAD mice. (c,e) Immunostaining with antibodies againt Aβ_1−X and Aβ_X−42 revealed no significant differences in the plaque load of NT4X or 1–57 immunized 5XFAD and the PBS control group. (f) Representative images from Thioflavin S and pyroglutamate Aβ_3-X staining for respective treatment groups of PBS, NT4X and 1–57, both showing a decreased staining for NT4X treatment group. One-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test against the PBS control group; n = 7–9; **p < 0.01, *p < 0.05 data presented as mean ± S.E.M.