A novel process driving Alzheimer's disease validated in a mouse model: Therapeutic potential

Abstract

Introduction: The neuronal mechanism driving Alzheimer's disease (AD) is incompletely understood.

Methods: Immunohistochemistry, pharmacology, biochemistry, and behavioral testing are employed in two pathological contexts-AD and a transgenic mouse model-to investigate T14, a 14mer peptide, as a key signaling molecule in the neuropathology.

Results: T14 increases in AD brains as the disease progresses and is conspicuous in 5XFAD mice, where its immunoreactivity corresponds to that seen in AD: neurons immunoreactive for T14 in proximity to T14-immunoreactive plaques. NBP14 is a cyclized version of T14, which dose-dependently displaces binding of its linear counterpart to alpha-7 nicotinic receptors in AD brains. In 5XFAD mice, intranasal NBP14 for 14 weeks decreases brain amyloid and restores novel object recognition to that in wild-types.

Discussion: These findings indicate that the T14 system, for which the signaling pathway is described here, contributes to the neuropathological process and that NBP14 warrants consideration for its therapeutic potential.

Keywords: 5XFAD; Alzheimer's disease; Braak stage; NBP14; T14; acetylcholinesterase; alphaLISA; amyloid beta; basal forebrain; cortex; hippocampus; novel object recognition.

FIGURE 1

(A) Quantification of hippocampal T14 (mean ± SEM) normalized to the data for Braak 0‐II (n = 14), showing a significant increase at Braak VI (n = 6, Mann‐Whitney test). (B) T14‐alpha‐7 receptor binding using AlphaLISA in early stage (Braak I‐II, n = 6) and late stage (Braak V‐VI, n = 12, Mann‐Whitney test) hippocampal tissue. (C) Examples of western blots for hippocampal T14 at Braak I, II, and VI, showing an increase at the later stage. (D, E) T14‐ and p‐tau‐immunoreactivity in Alzheimer's disease; representative photomicrographs of the CA1 region of the hippocampus in an 85‐year‐old man, Braak VI. (D) T14‐immunoreactive neurons in proximity to T14‐immunoreactive plaques. (E) T14‐ and p‐tau‐immunoreactivity in adjacent sections. (F, H, J) Representative photomicrographs of hippocampal T14‐immunoreactivity in the 5XFAD mouse; (G, I, K) adjacent coronal sections showing loss of T14‐immunoreactivity following immunoneutralization of the primary antibody. (L) The Cornu ammonis 1 (CA1) region of the hippocampus in an FAD‐negative mouse, showing light T14‐immunoreactivity in pyramidal neurons and no T14‐immunoreactive plaques. (M) Dose‐dependent effects of NBP14 in displacing T14‐alpha‐7 binding in advanced AD (Braak III‐VI, n = 3‐4, two‐way ANOVA) hippocampal tissue (NBP‐14 IC50 = 61.4 μM); data are mean ± SEM. *P < .05, ** P < .01. Scale bars: 70 μm (D), 210 μm (E), 140 μm (F, G, L), 40 μm (H‐K), sub, subiculum (F). Arrows (H, J), examples of a T14‐immunoreactive neuron adjacent to a T14‐immunoreactive plaque

FIGURE 2

(A‐C) Blood and brain concentrations of NBP14 in 5XFAD mice following intranasal treatments with NBP14 (10 mg/kg) and brain‐to‐blood ratio. The samples (n = 3 for each time point) were collected 30 minutes after a single initial treatment in T0W and after twice weekly treatments for 6 weeks or 14 weeks; additional samples were collected 2 days after the final treatment in T14W, to test for any accumulation of the drug. Friedman multiple comparison tests showed differences in NBP14 concentrations in blood between T0W and T6W (P = .0286) but not T0W and T14W, and in the brain between T0W and T14W (P = .0495) but not T0W and T6W; the blood‐to‐brain ratio increased significantly by T14W (P = .0306) but not by T6W. (D) Body weight (mean of biweekly measurements) of 5XFAD mice (5XFAD‐VEH and 5XFAD‐NBP14) over the 14‐week treatment period. Weights taken immediately before (Day 0) and following vehicle or NBP14 treatment twice weekly for 14 weeks (n = 14–28). Analysis of covariance revealed no overall effect of treatment per se (F(1, 39) = 2.17, P = .1484), but an effect of time (F(13, 520) = 90.22, P < .0001) and a difference for the main effect of treatment x time (F(13, 520) = 1.90, P = .0281). Pairwise comparison showed a significant difference between the 5XFAD‐VEH and 5XFAD‐NBP14 mice at 12 to 14 weeks of treatment. (E‐H) Novel Object Recognition Test performance in wild‐type (WT) and 5XFAD mice. (E) Time (s = seconds) spent in general activity by untreated WT and 5XFAD mice prior to and after 6 and 14 weeks of vehicle or NBP14 treatment (n = 13‐28; one‐way ANOVA showed no significant differences> P > .05). (F‐H) Total object exploration time (s = seconds) for familiar and novel objects in 5XFAD mice: (F) before treatment (T0W) and (G) after 6 weeks (T6W) or (H) 14 weeks (T14W) of intranasal vehicle or NBP14 (10 mg/kg); untreated age‐matched WT were tested concurrently (n = 13 for vehicle, 28 for NBP14 one‐way ANOVA). (I) Recognition Index showing percentage of recognition above chance (50%) levels, calculated as time spent exploring the novel object/time exploring novel + familiar object in untreated WT and 5XFAD mice prior to and at 6 and 14 weeks of vehicle or NBP14 treatment (n = 13–28); data are mean ± SEM. * P < .05, ** P < .01 versus 5XFAD‐VEH; ## P < .01 versus baseline value (T0W) of the group in question, two‐way ANOVA followed by Bonferroni`s post hoc). T0W, Week 0; T6W, Week 6; T14W, Week 14.

FIGURE 3

Immunohistochemistry for Aβ (gold) in sagittal sections from 5XFAD mice following intranasal treatment with vehicle (saline) or NBP14 (10 mg/kg) for (A, B, C) 6 weeks or (D, E, F) 14 weeks; scale bars: hippocampus 6/14 weeks (w) = 200 μm; frontal cortex 6w = 100 μm, 14w = 200 μm; basal forebrain 6/14w = 200 μm. Aβ deposits were detected in the hippocampus and frontal cortex in both groups at both time points; in contrast, Aβ was detected in the basal forebrain only in the groups that had been treated for 14 weeks, at which time the animals were 21‐ to 24‐weeks‐old. Levels of intracellular Aβ were calculated in the groups receiving treatment for 6 weeks as the mean intensity in the perinuclear region (AU, arbitrary units), and in the groups receiving treatment for 14 weeks extracellular Aβ was calculated as the mean area of immunoreactivity. (G) Higher magnification examples showing intracellular amyloid and extracellular plaques in frontal cortex after, respectively, 6 and 14 weeks of vehicle or NBP14. Arrows show examples of intraneuronal Aβ; asterisks show examples of extracellular Aβ plaques; scale bars = 50 μm. At 6 weeks of treatment (A, B, C) n = 10 for vehicle, n = 8 for NBP14; at 14 weeks of treatment (D, E, F) (n = 4 for vehicle, n = 8 for NBP14. Data are mean ± SEM. * P < .05; **P < .01, unpaired t‐tests)

FIGURE 4

The T14 system participates in at least four interactive cascades. Cascade 1 (brown): (A) T14 binds at its allosteric site on the alpha‐7 receptor unless (B) this is blocked by NBP14., , (C) T14 binding increases calcium influx (dotted line), causing (D) depolarization and activation of voltage sensitive calcium channels (L‐VOCC). (E) Elevated calcium triggers AChE release (G4 tetramer) from intracellular storage, for example, smooth endoplasmic reticulum (SER) into extracellular space. Subsequently (F) proteases, for example, Insulin Degrading Enzyme, cleave T14 from the parent molecule, leaving the G1 monomer without the T14‐containing disulfide bonds and therefore unable to oligomerize. (G) T14 diffuses to act (H) on alpha‐7 receptors on neighboring cells, thereby perpetuating the cycle. Cascade 2 (blue): (I) The T14‐induced increase in intracellular calcium activates the enzyme GSK‐3 resulting in (J) tau phosphorylation and (K) cleavage of amyloid beta from membrane bound APP by secretases, resulting (L) in enhanced calcium toxicity via alpha‐7 receptors. (c) Cascade 3 (pink): (M) The T14‐induced increase in intracellular calcium causes upregulation of its target alpha‐7 receptors and their trafficking to the plasma membrane, further facilitating T14 effects. (d) Cascade 4 (black): The T14‐induced increase in intracellular calcium leads to (N) calcium uptake into mitochondria, decreasing ATP synthesis accompanied by electron leakage, thereby (O) increasing free radical generation (ROS), (P) triggering cytochrome C release, followed by (Q) caspase‐3 activation and (R) cell death. (S) Consequent membrane disintegration makes previously membrane‐bound AChE (G4) vulnerable to protease degradation, leading (T) to increased T14 for diffusion to neighboring neurons (U) perpetuating the T14 process