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, 10 (1), 179-200
eCollection

Coniferaldehyde Attenuates Alzheimer's Pathology via Activation of Nrf2 and Its Targets

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Coniferaldehyde Attenuates Alzheimer's Pathology via Activation of Nrf2 and Its Targets

Yaqiong Dong et al. Theranostics.

Abstract

Background: Alzheimer's disease (AD) currently lacks a cure. Because substantial neuronal damage usually occurs before AD is advanced enough for diagnosis, the best hope for disease-modifying AD therapies likely relies on early intervention or even prevention, and targeting multiple pathways implicated in early AD pathogenesis rather than focusing exclusively on excessive production of β-amyloid (Aβ) species. Methods: Coniferaldehyde (CFA), a food flavoring and agonist of NF-E2-related factor 2 (Nrf2), was selected by multimodal in vitro screening, followed by investigation of several downstream effects potentially involved. Furthermore, in the APP/PS1 AD mouse model, the therapeutic effects of CFA (0.2 mmol kg-1d-1) were tested beginning at 3 months of age. Behavioral phenotypes related to learning and memory capacity, brain pathology and biochemistry, including Aβ transport, were assessed at different time intervals. Results: CFA promoted neuron viability and showed potent neuroprotective effects, especially on mitochondrial structure and functions. In addition, CFA greatly enhanced the brain clearance of Aβ in both free and extracellular vesicle (EV)-contained Aβ forms. In the APP/PS1 mouse model, CFA effectively abolished brain Aβ deposits and reduced the level of toxic soluble Aβ peptides, thus eliminating AD-like pathological changes in the hippocampus and cerebral cortex and preserving learning and memory capacity of the mice. Conclusion: The experimental evidence overall indicated that Nrf2 activation may contribute to the potent anti-AD effects of CFA. With an excellent safety profile, further clinical investigation of coniferaldehyde might bring hope for AD prevention/therapy.

Keywords: Alzheimer's disease; Aβ clearance; Nrf2; coniferaldehyde; neuroprotection.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
CFA improved neuronal cell viability and protected cells against Aβ stress and mitochondrial toxins. (A) Chemical structure of CFA. (B) The effects of coniferaldehyde (CFA) on the viability of SH-SY5Y cells (neo, APPwt and APPswe) with/without Aβ burden, 300 μM MPP+ (1‐methyl‐4‐phenylpyridinium) (C) or 1 μM rotenone (Rot) (D) 24 h pre-induced SH-SY5Y cells. All kinds of cells were treated with CFA for 36 h before quantification of cell viability by MTS assay. (E) Representative immunofluorescence images of CFA-treated and control primary cultured neurons. Mouse primary neurons were treated with 100 μM CFA for 36 h with or without Aβ42 (5 μM) stress. The neurons were visualized with FITC-labeled Microtubule Associated Protein 2 (MAP2) antibodies (green) and Hoechst (blue). Scale bars, 75 μm. (F) Improvement of cell viability upon CFA treatment with/without Aβ42 (5 μM) stress in primary cultured neurons. Results are mean±SD (n=3). *P<0.05, ***P<0.001 versus control or specific indication.
Figure 2
Figure 2
CFA treatment activated Nrf2 in SH-SY5Y cells with & without Aβ burden responsible for most neuronal protective effects. (A-C) Representative immunofluorescence image of Nrf2 (green) translocation into nucleus (blue) upon treatment of Aβ-expressing SH-SY5Y (neo, APPwt and APPswe) cells with 100 μM CFA. Scale bars, 10 μm. (D) Quantification of nuclear vs cytoplasmic Nrf2 expression based on the imaging (A-C). (E-H) Nrf2 expression (E,F) and HO-1 expression (G,H) were elevated upon CFA treatment (100 μM) but inhibited by pretreatment with all-trans-retinoic acid (ATRA, 5 μM, 24 h); (I) Nrf2 knockdown with siRNA significantly decreased improvement of cell viability by CFA (100 μM). (J) ATRA treatment significantly decreased improvement of cell viability by CFA (100 μM). Results are mean±SE (n=3). *P<0.05, **P<0.01, ***P<0.001 versus CFA group.
Figure 3
Figure 3
CFA treatment improved the mitochondrial morphology and energy production in Aβ-expressing SH-SY5Y cells. (A-B) Morphological changes of mitochondria in SH-SY5Y cells with/without Aβ burden upon CFA treatment. SH-SY5Y cells (neo, APPwt and APPswe) were treated with 100 μM CFA and mitochondria were stained with MitoTracker Red CMXRos. Scale bars, 2.5 μm. (C) Substantial increase in ATP production by CFA treatment. (D-K) Enhancement of mitochondrial aerobic respiration/oxidative phosphorylation by CFA. D,H: illustration of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurement on a Seahorse XF Extracellular Flux Analyzer, in which oligomycin (OM) inhibits ATP synthase to block coupled respiration, FCCP serves as a mitochondrial uncoupler to resume oxygen consumption, AA (a complex III inhibitor) and Rot (complex I inhibitor) halt oxidative phosphorylation, and 2-deoxyglucose (2-DG) inhibits hexokinase to stop glycolysis. As Figure 3H-K showed, CFA stimulated a slight decrease in ECAR over basal glycolysis, but result in an obvious decrease in maximum glycolytic capacity; E-G: The OCR of Aβ-expressing SH-SY5Y cells upon 100 μM CFA treatment; I-K: The ECAR of Aβ-expressing SH-SY5Y cells upon 100 μM CFA treatment. Each experimental group was analyzed using three replicates in each analysis. Results are mean±SD (n=3).
Figure 4
Figure 4
The effect of CFA treatment on Drp1 expression and PKM2 expression on Aβ-expressing SH-SY5Y cells and primary cultured neurons upon Aβ42 treatment. SH-SY5Y cells (neo, APPwt and APPswe) were treated with 100 μM CFA and the levels of Drp1 (A,B) and PKM2 (C,D) were analyzed by western blot. *P<0.05, **P<0.01 versus control. Primary cultured neurons were treated with100 μM CFA in presence/absence of 5 μM of Aβ42 for 36 h, Drp1 (E,F) and PKM2 (G,H) levels were analyzed by western blot. Results are mean±SE (n=3). ***P<0.001 versus Aβ group.
Figure 5
Figure 5
The effect of CFA on Tau phosphorylation in SH-SY5Y cells. Western blot analysis of phosphorylation of Tau (S262) (A,B), Tau (S422) (C,D), Akt (S437) (E,F), GSK-3β (S9) (G,H). Results are mean±SE (n=3). *P<0.05, **P<0.01, ***P<0.001.
Figure 6
Figure 6
CFA treatment strongly activated Nrf2 signaling in vivo. The brain samples were immunostained with the Nrf2 antibody. (A-B) Representative images of Nrf2 staining of brain sections of Normal Elderly controls (A) and Alzheimer's Disease patients (B). (C) Quantification of amounts of nuclear Nrf2 in (A-B). The mean age of the AD patients was 84.7±2.1 years, and the controls 85.2±3.3. Scale bars, 1500 μm. Results are mean±SE (n=7). *P<0.05 versus Normal Elderly. (D-F) Representative images of Nrf2 staining of brain sections of WT mice (D), untreated APP/PS1 control (E) and CFA(H)-treated (F) mice. (G) Quantification of amounts of nucleus Nrf2 in (D-F). Scale bars, 1000 μm. (H, I) Western blot of Nrf2 levels in brain of WT and APP/PS1 mice with/without CFA treatment. The mice were 10 months old and had been treated with CFA in pellet food for 7 months. Results are mean±SE (n=6, with random selection). *P<0.05. Results are mean±SE (n=6, with random selection). *P<0.05, ***P<0.001 versus untreated APP/PS1 or specific indication.
Figure 7
Figure 7
CFA enhanced brain Aβ excretion in both EVs-bound and unbound forms in APP/PS1 AD mice. Eu-labeled amyloid β (Eu-Aβ, 2µl) was injected into the mouse striatum and interstitial soluble amyloid Aβ clearing from the brain parenchyma was evaluated as described in Materials and Methods. (A) Schematic of Aβ intrastriatal injection and fluorescence measurement setup. (B) The time course of brain Aβ content (*P < 0.05, n = 4~6). (C) Time course of Aβ level in peripheral blood (**P < 0.01, n = 4~6). The fluorescence intensity of samples was presented as folds of plasma fluorescence blank. (D-H) In vivo imaging of Aβ flux in mouse cortex surface layer (0~240 μm) after intracisternal injection. D: cerebral vasculature visualized with intra-arterial Cy5-Dextran 70KD. Scale bars, 250 μm. < 0.5 h after intracisternal injection. E: capillaries surrounded by FITC-labeled Aβ highlighted by rectangle marks; F: FITC-labeled Aβ (Green) moving along the outside of cerebral surface blood vessels in a representative area (green circle) in F, Scale bars, 250 μm. G: 3D image of FITC-Aβ spots (Green) moving outside blood vessels. The fluorescence background of free FITC-Aβ was reduced manually to highlight the Aβ spots. (H) Co-localization of EVs and Aβ spots outside cerebral blood vessels. EVs was visualized with an Alexa 647-labeled CD63 antibody. Scale bars, 50 μm. (I) The relative amounts of free plasma Aβ and EVs-bound forms.
Figure 8
Figure 8
Learning and memory protective effects of CFA in male APP/PS1 AD mice. (A) Illustration of dosing and experimental design. CFA treatments on male APP/PS1 mice began in the 3rd month by feeding the animals with pellet food containing desired amounts of CFA at a low dose (0.02 mmol kg-1day-1) and a high dose (0.2 mmol kg-1day-1) denoted CFA(L) and CFA(H), respectively. Step-down passive avoidance tests were conducted from 6 months of age to monitor the cognitive changes and Morris water maze tests were conducted at 9-10 months to evaluate spatial learning and memory. The littermate C57BL/6 mice (WT) were the negative control; (B) The step-down latency and basal number of errors (C) in the step-down passive avoidance tests. (D) Circular images display the representative swimming paths for the mice to locate the escape platform in the water maze during the 60 s test period. The small circle indicates the position of escape platform; (E) The percentage of time animals spent in the target quadrant (IV) compared to the other three quadrants (III, II, I); (F) Time to the first arrival in the escape platform position in the final water maze tests; (G) Times across the escape platform in the final water maze tests; Data represented as mean±SE (n=8~10). *P<0.05, **P<0.01 versus untreated APP/PS1 mice.
Figure 9
Figure 9
Histopathological demonstration of protection effects of CFA in APP/PS1 AD mice. (A-H) Representative images of hematoxylin-eosin (HE) staining of brain sections (hippocampus and cerebral cortex) of APP/PS1 AD model mice (male) upon CFA(H) treatment. Scale bars, 534.5 μm for 6.3× (A,E), 169.5 μm for 20× (B-D, F-H). Magnification: 6.3× (A, E), 20× (B-D, F-H). (I-K) Quantification of the total number of neurons in the DG (i), and the neurons (J) and basophilic cells (K) in CA1, CA2, CA3 and CA4. (L-O) Nissl staining of neurons in the brains APP/PS1 mice upon CFA treatment. Scale bars, 185 μm for (L, N); 46.3 μm for (M, O). The mice were 10 months old and had been treated with CFA in pellet food for 7 months. Results are mean±SE (n=6, with random selection). *P<0.05, **P<0.01, ***P<0.001versus untreated APP/PS1 or specific indication.
Figure 10
Figure 10
CFA treatment eliminated brain Aβ deposition and toxic Aβ accumulation. (A-B) Representative images of brain Aβ deposition. The brain samples (hippocampus, cerebral cortex and cerebellum) were immunostained with the Aβ antibody (6E10) in untreated APP/PS1 control (A) and CFA(H)-treated mice (B). Scale bars, 534.5 μm for 6.3×, 169.5 μm for 20×. (C-F) Aβ oligomerization in APP/PS1 mice upon CFA treatment. Western blot analysis and quantification of soluble Aβ peptides in hippocampal lysates (C, D) and frontal lobe lysates (E, F). The mice were 10 months old and had been treated with CFA in pellet food for 7 months. Results are mean±SE (n=6, with random selection). *P<0.05, **P<0.01, ***P<0.001versus untreated APP/PS1 or specific indication.

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References

    1. Galvin JE, Howard DH, Denny SS, Dickinson S, Tatton N. The social and economic burden of frontotemporal degeneration. Neurology. 2017;89:2049–56. - PMC - PubMed
    1. Morrison C. Hope for anti-amyloid antibodies surges, yet again. Nat Biotechnol. 2016;34:1082–3. - PubMed
    1. Gold M. Phase II clinical trials of anti-amyloid beta antibodies: When is enough, enough? Alzheimers Dement (N Y) 2017;3:402–9. - PMC - PubMed
    1. Koss DJ, Jones G, Cranston A, Gardner H, Kanaan NM, Platt B. Soluble pre-fibrillar tau and beta-amyloid species emerge in early human Alzheimer's disease and track disease progression and cognitive decline. Acta Neuropathol. 2016;132:875–95. - PMC - PubMed
    1. Luo J, Warmlander SK, Graslund A, Abrahams JP. Cross-interactions between the Alzheimer Disease Amyloid-beta Peptide and Other Amyloid Proteins: A Further Aspect of the Amyloid Cascade Hypothesis. J Biol Chem. 2016;291:16485–93. - PMC - PubMed
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