Novel Botanical Therapeutic NB-02 Effectively Treats Alzheimer's Neuropathophysiology in an APP/PS1 Mouse Model

Abstract

Alzheimer's disease (AD) is an incurable neurodegenerative disorder and a major cause of dementia. Some of the hallmarks of AD include presence of amyloid plaques in brain parenchyma, calcium dysregulation within individual neurons, and neuroinflammation. A promising therapeutic would reverse or stymie these pathophysiologies in an animal model of AD. We tested the effect of NB-02, previously known as DA-9803, a novel multimodal therapeutic, on amyloid deposition, neuronal calcium regulation and neuroinflammation in 8- to 10-month-old APP/PS1 mice, an animal model of AD. In vivo multiphoton microscopy revealed that two-month-long administration of NB-02 halted amyloid plaque deposition and cleared amyloid in the cortex. Postmortem analysis verified NB-02-dependent decrease in plaque deposition in the cortex as well as hippocampus. Furthermore, drug treatment reversed neuronal calcium elevations, thus restoring neuronal function. Finally, NB-02 restored spine density and transformed the morphology of astrocytes as well as microglia to a more phagocytic state, affecting neuroinflammation. NB-02 was effective at reversing AD neuropathophysiology in an animal model. Therefore, in addition to serving as a promising preventative agent, NB-02 holds potential as a treatment for AD in the clinic.

Keywords: Alzheimer’s disease; astrocyte; neuron; therapy.

Figure 1.

Chronic treatment with NB-02 halts deposition of cortical amyloid plaques in vivo. A, Experimental schematic showing the time-points of viral injections, craniotomies, multiphoton imaging sessions, and gavage treatments with NB-02 or vehicle of APP/PS1 mice. B, E, Multiphoton microscopy images of red dextran angiograms in APP/PS1 mice before treatments with vehicle (B) or NB-02 (E). C, F, Multiphoton microscopy images of methoxy-X04-positive amyloid plaques in cortices of APP/PS1 mice before treatments. Note B, C were taken during the same imaging session and constitute the same field of view. Also, E, F were taken during the same imaging session and constitute the same field of view. D, G, Images of amyloid plaques taken after treatments with vehicle or NB-02. Note C, D were taken during different imaging sessions and constitute the same field of view. Similarly, F, G were taken during different imaging sessions and constitute the same field of view. H, Plaque numbers per cubic millimeter of cortex across the two conditions over time. Statistical comparisons are made to baseline (0 months). I, Size of amyloid plaques in the course of treatment across conditions. J, Amyloid plaque burden, which takes into account plaque number and size, over time across conditions K, Methoxy-X04 intensity change at the end of treatment. Scale bar: 100 μm. Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; n.s., not significant.

Figure 2.

NB-02 treatment results in lower amyloid load compared with vehicle when assessed postmortem. A–H, Cortical images and analysis. I–P, Hippocampal images and analysis. A, D, 82E1 immunoreactivity against amyloid in cortical sections obtained postmortem after treatment with vehicle (A) or NB-02 (D). B, E, Methoxy-X04-positive amyloid plaques in cortical sections. Images in A, B were acquired from the same field of view. Similarly, images in D, E were acquired from the same field of view. C, F, Colocalization of 82E1 and methoxy-X04. G, H, Cortical amyloid plaque burden as assessed by 82E1 immunoreactivity (G) and methoxy-X04 signal (H). I, L, 82E1 immunoreactivity against amyloid in hippocampal sections obtained postmortem after treatment with vehicle (I) or NB-02 (L). J, M, Methoxy-X04-positive amyloid plaques in hippocampal sections. Images in I, J were taken from the same field of view. Similarly, images in L, M were taken from the same field of view. K, N, Colocalization of 82E1 and methoxy-X04. O, P, Hippocampal amyloid plaque burden as assessed by 82E1 immunoreactivity (O) and methoxy-X04 signal (P). Each dot represents amyloid burden in a cortical or hippocampal section. Open circles or squares represent females and filled circles or squares represent males. Scale bars: 50 μm. Mean ± SEM; *p < 0.05, **p < 0.01.

Figure 3.

NB-02 does not alter soluble amyloid levels. A, B, Human amyloid β (A, 40; B, 42) levels in cerebrospinal fluid (CSF) of APP/PS1 mice treated with vehicle or NB-02. C, D, Human amyloid β (C, 40; D, 42) levels in TBS-soluble brain fractions of APP/PS1 mice treated with vehicle or NB-02. Each dot represents amyloid measurement in CSF or brain fraction obtained from individual mice; n.s., not significant.

Figure 4.

NB-02 restores neuronal calcium overload in vivo. A, B, Multiphoton microscopy images of calcium reporter YC3.6 expressed in neurons pseudocolored according to intracellular calcium concentrations in APP/PS1 brains treated with vehicle (A) or NB-02 (B). Yellow arrows point to neuronal cell bodies exhibiting calcium overload, while the yellow arrowhead points to a neuronal process exhibiting calcium overload. C, D, Histograms showing percentages of neurites binned into categories according to the YFP/CFP ratios over the course of treatment with vehicle (C) or NB-02 (D). Percentage of neurites with calcium overload, YFP/CFP ratio > 1.79, are shaded in red. E, Bar graph showing percentages of neurites exhibiting calcium overload at the end of treatment. Each dot represents percent calcium overload in an individual mouse. Open circles or squares represent females while filled circles or squares represent males. Scale bar: 50 μm. Mean ± SEM; **p < 0.01.

Figure 5.

NB-02 increases spine density. A, B, Multiphoton microscopy images of calcium reporter YC3.6 expressed in neurons of APP/PS1 mice treated with vehicle (A) or NB-02 (B). Individual dendrites are outlined in red and spines are labeled. C, Spine density assessed posttreatment. Each dot represents an individual mouse. D, E, Bar graphs displaying the comparative levels of synaptic proteins PSD-95 (D) and synaptophysin (E) assessed by Western blot analyses of vehicle-treated and NB-02-treated brains. Representative bands of PSD-95, synaptophysin, and β-tubulin as control are shown for each condition. Scale bar: 50 μm; *p < 0.05; n.s., not significant.

Figure 6.

NB-02 transforms morphology of reactive astrocytes and microglia. A, E, Iba-1 immunoreactivity in cortical sections of vehicle-treated (A) and NB-02-treated (E) APP/PS1 mice. B, F, Iba-1 immunoreactivity in hippocampal sections of vehicle (B) and NB-02-treated mice (F). C, G, GFAP immunoreactivity in cortical sections of vehicle (C) and NB-02-treated mice (G). D, H, GFAP immunoreactivity in hippocampal sections of vehicle (D) and NB-02-treated mice (H). I, Cortical Iba-1-positive cell counts across conditions. J, Hippocampal Iba-1-positive cell counts across conditions. K, GFAP-positive cell counts in cortex across conditions. L, GFAP-positive cell counts in hippocampus. M, S, Higher magnification images of a single microglia in the brain of an APP/PS1 mouse treated with vehicle (M) or NB-02 (S). N, T, Higher magnification images of a single astrocyte in the brain of an APP/PS1 mouse treated with vehicle (N) or NB-02 (T). O, Microglia process length across conditions. P, Microglia cell body diameter across conditions. Q, Microglia soma area across conditions. R, Microglia cell roundness across conditions. U, Astrocyte process length across conditions. V, Astrocyte cell body diameter across conditions. Scale bars: 100 μm (A–D) and 10 μm (M, N). Each dot represents an individual mouse; n.s., not significant; **p < 0.01, ****p < 0.0001.

Figure 7.

Inflammatory cytokine levels trend down in NB-02-treated brains. A–F, Levels of proinflammatory cytokines IL-10 (A), TNF-α (B), IL-6 (C), IL-1β (D), chemokines KC/GRO (E), and MCP-1 (F) are represented as pg per mg of total protein as assessed by electrochemiluminescent assay; n.s., not significant; *p < 0.05.