A Small Molecule p75NTR Ligand, LM11A-31, Reverses Cholinergic Neurite Dystrophy in Alzheimer's Disease Mouse Models with Mid- to Late-Stage Disease Progression

Degeneration of basal forebrain cholinergic neurons contributes significantly to the cognitive deficits associated with Alzheimer's disease (AD) and has been attributed to aberrant signaling through the neurotrophin receptor p75 (p75NTR). Thus, modulating p75NTR signaling is considered a promising therapeutic strategy for AD. Accordingly, our laboratory has developed small molecule p75NTR ligands that increase survival signaling and inhibit amyloid-β-induced degenerative signaling in in vitro studies. Previous work found that a lead p75NTR ligand, LM11A-31, prevents degeneration of cholinergic neurites when given to an AD mouse model in the early stages of disease pathology. To extend its potential clinical applications, we sought to determine whether LM11A-31 could reverse cholinergic neurite atrophy when treatment begins in AD mouse models having mid- to late stages of pathology. Reversing pathology may have particular clinical relevance as most AD studies involve patients that are at an advanced pathological stage. In this study, LM11A-31 (50 or 75 mg/kg) was administered orally to two AD mouse models, Thy-1 hAPPLond/Swe (APPL/S) and Tg2576, at age ranges during which marked AD-like pathology manifests. In mid-stage male APPL/S mice, LM11A-31 administered for 3 months starting at 6–8 months of age prevented and/or reversed atrophy of basal forebrain cholinergic neurites and cortical dystrophic neurites. Importantly, a 1 month LM11A-31 treatment given to male APPL/S mice (12–13 months old) with late-stage pathology reversed the degeneration of cholinergic neurites in basal forebrain, ameliorated cortical dystrophic neurites, and normalized increased basal forebrain levels of p75NTR. Similar results were seen in female Tg2576 mice. These findings suggest that LM11A-31 can reduce and/or reverse fundamental AD pathologies in late-stage AD mice. Thus, targeting p75NTR is a promising approach to reducing AD-related degenerative processes that have progressed beyond early stages

A small molecule p75NTR ligand, LM11A-31, reverses cholinergic neurite dystrophy in Alzheimer's disease mouse models with mid- to late-stage disease progression.

Degeneration of basal forebrain cholinergic neurons contributes significantly to the cognitive deficits associated with Alzheimer's disease (AD) and has been attributed to aberrant signaling through the neurotrophin receptor p75 (p75NTR). Thus, modulating p75NTR signaling is considered a promising therapeutic strategy for AD. Accordingly, our laboratory has developed small molecule p75NTR ligands that increase survival signaling and inhibit amyloid-β-induced degenerative signaling in in vitro studies. Previous work found that a lead p75NTR ligand, LM11A-31, prevents degeneration of cholinergic neurites when given to an AD mouse model in the early stages of disease pathology. To extend its potential clinical applications, we sought to determine whether LM11A-31 could reverse cholinergic neurite atrophy when treatment begins in AD mouse models having mid- to late stages of pathology. Reversing pathology may have particular clinical relevance as most AD studies involve patients that are at an advanced pathological stage. In this study, LM11A-31 (50 or 75 mg/kg) was administered orally to two AD mouse models, Thy-1 hAPPLond/Swe (APPL/S) and Tg2576, at age ranges during which marked AD-like pathology manifests. In mid-stage male APPL/S mice, LM11A-31 administered for 3 months starting at 6-8 months of age prevented and/or reversed atrophy of basal forebrain cholinergic neurites and cortical dystrophic neurites. Importantly, a 1 month LM11A-31 treatment given to male APPL/S mice (12-13 months old) with late-stage pathology reversed the degeneration of cholinergic neurites in basal forebrain, ameliorated cortical dystrophic neurites, and normalized increased basal forebrain levels of p75NTR. Similar results were seen in female Tg2576 mice. These findings suggest that LM11A-31 can reduce and/or reverse fundamental AD pathologies in late-stage AD mice. Thus, targeting p75NTR is a promising approach to reducing AD-related degenerative processes that have progressed beyond early stages

LM11A-31 prevents atrophy of cholinergic neurites in basal forebrain of late-stage APP^L/S mice.

<p>Representative photomicrographs show ChAT-immunostained neurons in the basal forebrain of (<b><i>A</i></b>) WT Veh, (<b><i>B</i></b>) APP^L/S (APP) Veh, and (<b><i>C</i></b>) 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 APP^L/S mice with LM11A-31 for 1 month prevented the decreases in (<b><i>D</i></b>) length, (<b><i>E</i></b>) surface area, and (<b><i>F</i></b>) 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.</p

LM11A-31 prevents cholinergic dystrophic neurites in cortex of late-stage APP^L/S mice.

<p>Representative photomicrographs show clusters of cholinergic dystrophic neurites in the cortex of APP^L/S (APP) Veh (<b><i>A</i></b>) and APP-31 (<b><i>B</i></b>) mice. Scale bar in B = 50 µm. Quantitative analysis showed that LM11A-31 did not significantly affect the total area (<b><i>C</i></b>) or number (<b><i>D</i></b>) of clusters but did decrease the size or mean area per cluster (<b><i>E</i></b>). Statistical significance was determined using a two-tailed Student's t-test. ⁺⁺p≤0.01 vs. APP Veh.</p

Brain concentrations of LM11A-31 delivered <i>ad libitum</i> in drinking water of C57BL/6 mice.

<p>LM11A-31 was given to C57BL/6 mice <i>ad libitum</i> 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).</p

LM11A-31 normalizes increased p75^NTR levels in the basal forebrain of late-stage APP^L/S mice.

<p>Representative photomicrographs show p75^NTR immunostaining in the vertical limb of the diagonal band of the basal forebrain in (<b><i>A</i></b>) WT Vehicle (Veh), (<b><i>B</i></b>) APP^L/S (APP) Veh, and (<b><i>C</i></b>) APP LM11A-31 (-31) mice at 13–14 months old. Scale bar = 40 µm. Quantitative analysis showed that, at this age, the (<b><i>D</i></b>) area occupied by (^**p≤0.01) and (<b><i>E</i></b>) the density (^*p<0.05) of p75^NTR immunostaining is increased in APP Veh compared to WT Veh mice, while the increase in the number of p75^NTR-stained cells was not statistically significant. LM11A-31 normalized p75^NTR 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).</p

LM11A-31 prevents dystrophy of cholinergic neurites in mid- to late-stage female Tg2576 mice.

<p>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 (<b><i>A</i></b>) length, (<b><i>B</i></b>) surface area, and (<b><i>C</i></b>) branching of BFCN neurites. This treatment also decreased the total area (<b><i>D</i></b>) occupied by the clusters of cholinergic dystrophic neurites in the cortex and the mean area per cluster (<b><i>F</i></b>) but did not affect the number (<b><i>E</i></b>). 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.</p

LM11A-31 prevents and/or reverses basal forebrain cholinergic neurite atrophy in mid-stage APP^L/S mice.

<p>Representative photomicrographs show ChAT-immunostained neurons in VDB of the basal forebrain of (<b><i>A</i></b>) WT Veh, (<b><i>B</i></b>) WT LM11A-31 (-31), (<b><i>C</i></b>) APP^L/S (APP) Veh, and (<b><i>D</i></b>) 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 APP^L/S mice with LM11A-31 for 3 months increases the (<b><i>E</i></b>) length, (<b><i>F</i></b>) area occupied by, and (<b><i>G</i></b>) 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.</p

Body weight and water consumption are not affected by LM11A-31 treatment in Tg2576 mice.

<p>(<b><i>A</i></b>) 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><i>B</i></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.</p

LM11A-31's effect on cholinergic dystrophic neurites in cortex of mid-stage APP^L/S mice.

<p>Representative photomicrographs show ChAT-immunostained dystrophic neurite clusters in the cortex of (<b><i>A</i></b>) APP^L/S (APP) Veh and (<b><i>B</i></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 (<b><i>C</i></b>) by decreasing their number (<b><i>D</i></b>) but not the mean area per cluster (<b><i>E</i></b>). 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.</p