Figure 1.
Body weight is not affected by treatment with LM11A-31 in male APPL/S mice.
Body weights of (A) 9–11 month old male APPL/S mice treated with LM11A-31 (50 mg/kg) for 3 months (WT Veh, n = 9 mice; WT-31, n = 10; APP Veh, n = 10; APP-31, n = 9) and (B) 13–14 month old male APPL/S mice treated with LM11A-31 (50 mg/kg) for 1 month (WT Veh, n = 5 mice; APP Veh, n = 4; APP-31, n = 5). APP mice weighed significantly less than WT mice in both of the age groups examined. LM11A-31 did not affect body weight at either age or genotype. Statistical significance was determined using repeated measures ANOVA with Dunnett's post-hoc test.
Figure 2.
Brain concentrations of LM11A-31 delivered ad libitum in drinking water of C57BL/6 mice.
LM11A-31 was given to C57BL/6 mice ad libitum in drinking water for 3 weeks at targeted doses of 10, 50, 75 and 100 mg/kg/day (n = 3–4 mice/dose). Brain concentrations were determined by LC-MS/MS by Absorption Systems (Exton, PA).
Figure 3.
Body weight and water consumption are not affected by LM11A-31 treatment in Tg2576 mice.
(A) Body weight of 17 month old female Tg2576 mice treated with LM11A-31 for 3 months (nTg-Veh, n = 7 mice; Tg2576-Veh, n = 6; Tg2576-31, n = 5; nTg–LM11A-31, n = 4). Starting at treatment week 6, Tg2576 mice weighed less than WT mice; LM11A-31 had no effect on this measure. Statistical significance was determined using repeated measures ANOVA with Dunnett's post-hoc test. (B) Average grams of water consumed by Tg2576 mice over 3 days was significantly greater than nTg mice; LM11A-31 had no effect on this measure. Statistical significance was determined using two-tailed Student's t-test. *p≤0.05 vs. nTg Veh.
Figure 4.
LM11A-31 normalizes increased p75NTR levels in the basal forebrain of late-stage APPL/S mice.
Representative photomicrographs show p75NTR immunostaining in the vertical limb of the diagonal band of the basal forebrain in (A) WT Vehicle (Veh), (B) APPL/S (APP) Veh, and (C) APP LM11A-31 (-31) mice at 13–14 months old. Scale bar = 40 µm. Quantitative analysis showed that, at this age, the (D) area occupied by (**p≤0.01) and (E) the density (*p<0.05) of p75NTR immunostaining is increased in APP Veh compared to WT Veh mice, while the increase in the number of p75NTR-stained cells was not statistically significant. LM11A-31 normalized p75NTR levels (+p≤0.05 vs. APP Veh for both area and density). Statistical significance was determined using a one-way ANOVA with Dunnett's post-hoc test (WT Veh, n = 5 mice; APP Veh, n = 4; APP-31, n = 5).
Figure 5.
LM11A-31 prevents and/or reverses basal forebrain cholinergic neurite atrophy in mid-stage APPL/S mice.
Representative photomicrographs show ChAT-immunostained neurons in VDB of the basal forebrain of (A) WT Veh, (B) WT LM11A-31 (-31), (C) APPL/S (APP) Veh, and (D) APP-31 mice at 9–11 months of age. Arrowheads indicate the distal part of neurites. Below each photomicrograph are reconstructed drawings from Neurolucida tracings of two ChAT-stained neurons per treatment group. The left drawing is the neurite and corresponding soma indicated by the arrowhead in the photomicrograph (orientations were altered). The right drawing is of a cell outside the field displayed in the photomicrograph but within the field analyzed. Scale bar in A = 20 µm and also applies to the line drawings. Quantification indicates that treating APPL/S mice with LM11A-31 for 3 months increases the (E) length, (F) area occupied by, and (G) branching of BFCN neurites compared to those given vehicle. Statistical significance was determined using an ANOVA with Dunnett's post-hoc test and, for branching, a 2×2 contingency table with Fisher's exact test (WT Veh, n = 9 mice; WT-31, n = 10; APP Veh, n = 10; APP-31, n = 9). **p≤0.01 and ***p<0.001 vs. WT Veh; +p≤0.05 and ++p≤0.01 vs. APP Veh.
Figure 6.
LM11A-31's effect on cholinergic dystrophic neurites in cortex of mid-stage APPL/S mice.
Representative photomicrographs show ChAT-immunostained dystrophic neurite clusters in the cortex of (A) APPL/S (APP) Veh and (B) APP-31 mice. Scale bar in photomicrograph A = 50 µm. Quantitative analysis showed that LM11A-31 decreases the total area occupied by the clusters (C) by decreasing their number (D) but not the mean area per cluster (E). Statistical significance was determined using a two-tailed Student's t-test (APP Veh, n = 10; APP-31, n = 9). +p = 0.03 and +++p = 0.001 vs. APP Veh.
Figure 7.
LM11A-31 prevents atrophy of cholinergic neurites in basal forebrain of late-stage APPL/S mice.
Representative photomicrographs show ChAT-immunostained neurons in the basal forebrain of (A) WT Veh, (B) APPL/S (APP) Veh, and (C) APP-31 mice at 13–14 months of age. Arrowheads indicate the distal part of neurites. Scale bar in A = 40 µm. Below each photomicrograph are reconstructed drawings from Neurolucida tracings of two ChAT-stained neurons per treatment group indicated by the arrowhead in the photomicrograph. Scale bar in drawing under A = 40 µm. Quantitative analysis showed that treating late-stage APPL/S mice with LM11A-31 for 1 month prevented the decreases in (D) length, (E) surface area, and (F) branching of cholinergic neurites in basal forebrain, although the latter measure did not reach statistical significance. Statistical significance was determined using an ANOVA with Dunnett's post-hoc test and/or two-tailed Student's t-test for BFCN neurite degeneration and, for branching, a 2×2 contingency table with Fisher's exact test (WT Veh, n = 5 mice; APP Veh, n = 4; APP-31, n = 5). *p<0.05, **p≤0.01 and ***p<0.001 vs. WT Veh; +p<0.05 and ++p≤0.01 vs. APP Veh.
Figure 8.
LM11A-31 prevents cholinergic dystrophic neurites in cortex of late-stage APPL/S mice.
Representative photomicrographs show clusters of cholinergic dystrophic neurites in the cortex of APPL/S (APP) Veh (A) and APP-31 (B) mice. Scale bar in B = 50 µm. Quantitative analysis showed that LM11A-31 did not significantly affect the total area (C) or number (D) of clusters but did decrease the size or mean area per cluster (E). Statistical significance was determined using a two-tailed Student's t-test. ++p≤0.01 vs. APP Veh.
Figure 9.
LM11A-31 reverses dystrophy of cholinergic neurites that occurs with aging in APPL/S mice.
Quantitative analysis of the effect of aging showed that 13–14 month old vehicle-treated APPL/S (APP) mice had ChAT neurites in basal forebrain with decreased (A) length and (B) area and had dystrophic neurite clusters in cortex that occupied more area (C) and were larger (D) compared to the 9–11 month old APP Veh mice. In 13–14 month old APP mice, LM11A-31 increased the length and area of ChAT neurites in basal forebrain while decreasing the area of cortical dystrophic neurites compared to vehicle-treated mice (A–D; absolute numbers shown in Figs. 4 and 5). The effect of LM11A-31 in the basal forebrain was a reversal as neurite length (A) and area (B) were significantly greater in 13–14 month old APP-31 mice than 9–11 month old APP Veh mice. In the cortex, LM11A-31 did not reverse the area occupied by dystrophic neurites as 13–14 month old APP-31 mice still had significantly larger clusters than 9–11 month old APP Veh mice (C, D). Results are expressed as a percentage of the 9–11 month old APP Veh group. Statistical significance was determined using one-tailed Student's t-test (For 9–11 month old APP Veh n = 10; For 13–14 month group: APP Veh, n = 4; APP-31, n = 5). *p≤0.05, **p≤0.005 and ***p≤0.001 vs. 9–11 month old APP Veh; +p≤0.05 and ++p≤0.01 vs. 13–14 month old APP Veh.
Figure 10.
LM11A-31 prevents dystrophy of cholinergic neurites in mid- to late-stage female Tg2576 mice.
Quantitative analysis showed that treating female Tg2576 mice starting at 14 months of age with LM11A-31 for 3 months prevented and/or reversed the decreases in (A) length, (B) surface area, and (C) branching of BFCN neurites. This treatment also decreased the total area (D) occupied by the clusters of cholinergic dystrophic neurites in the cortex and the mean area per cluster (F) but did not affect the number (E). Statistical significance was determined using an ANOVA with Dunnett's post-hoc test for BFCN neurite degeneration, a 2×2 contingency table with a Fisher's exact test for branching, and a two-tailed Student's t-test for clusters (nTg-Veh, n = 7 mice; Tg2576-Veh, n = 6; Tg2576-31, n = 5; nTg–LM11A-31, n = 4). *p≤0.05 vs. nTg veh; +p≤0.05 vs. Tg2576-Veh.