Antroquinonol administration in animal preclinical studies for Alzheimer's disease (AD): A new avenue for modifying progression of AD pathophysiology

Abstract

Despite the rise of Alzheimer's disease (AD) in an ageing population, no cure is currently available for this disorder. This study assessed the role of a natural compound, Antroquinonol, in modifying the progression of AD when administered at the start and/or before appearance of symptoms and when the disease was well established, in a transgenic animal model. Antroquinonol was administered daily for 8 weeks, in 11 week (early stage) and 9 month (late stage) male transgenic mice (3 times Transgenic mice PS1_M146V, APP_Swe, and tau_P301L, 3 ​Tg XAD) and their respective aged controls. Behavioural testing (including Elevated Plus Maze Watermaze, Recognition object testing and Y maze) was performed at the end of the drug administration. In addition AD biomarkers (Amyloid beta 42 (Aβ42), tau and phospho-tau levels), oxidative stress and inflammatory markers, were assessed in tested mice brains after their sacrifice at the end of the treatment. When administered before the start of symptoms at 11 weeks, Antroquinonol treatment at 34 ​mg/kg (D2) and more consistently at 75 ​mg/kg (D3), had a significant effect on reducing systemic inflammatory markers (Interleukin 1, IL-1β and TNF-α) and AD biomarker (Amyloid Beta 42, Aβ42 and tau) levels in the brain. The reduction of behavioural impairment reported for 3TgXAD mice was observed significantly for the D3 drug dose only and for all behavioural tests, when administered at 11 weeks. Similarly, beneficial effects of Antroquinonol (at higher dose D3) were noted in the transgenic mice in terms of AD biomarkers (tau and phosphorylated-tau), systemic inflammatory (IL-1β), brain anti-inflammatory (Nrf2) and oxidative (3-Nitrotyrosine, 3NT) markers. Improvement of memory impairment was also reported when Antroquinonol (D3) was administered at late stage (9 months). Since Antroquinonol has been used without adverse effects in previous successful clinical trials, this drug may offer a new avenue of treatment to modify AD development and progression.

Keywords: AD biomarkers; AICD, APP Intracellular Cytoplasmic/C-terminal Domain; AMPK, 5′ adenosine monophosphate-activated protein kinase; APP, Amyloid precursor protein; APPβ, secreted amino-terminals APPβ fragment; Alzheimer's disease (AD); Animal model of AD; Antroquinonol; Aβ, amyloid-β peptides; Behavioural testing; CTFα, carboxyterminal fragment-α; CTFβ, carboxyterminal fragment-β; GSK3β, Glycogen synthase kinase 3 beta; IFNγ, Interferon gamma; IL-1, Interleukin 1; IL-6, Interleukin 6; Inflammatory markers; MAPK, Mitogen activated protein kinase; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells; NMDA Rc, methyl-D-aspartate receptors receptor; ROS, reactive oxygen species; TNF-α, Tumor Necrosis factor alpha; Transgenic mice; cdck5, cyclin dependant kinase 5; sAPPα, secreted amino-terminals APPα fragment.

Fig. 1

Schematisation of Antroquinonol administration at early and late stage in two mice cohorts (3 and 9 month-old mice 3TgXAD and their related control).

Fig. 2

Behavioural testing for 3TgXAD and control mice treated or not with Antroquinonol (D1 at 3 month old (n ​= ​7 to 14, after removal of outliers). Graphs were plotted as mean values ​± ​SEM.∗ ​= ​p ​< ​0.05, ∗∗ ​= ​p ​< ​0.01, ∗∗∗ ​= ​p ​< ​0.001(A). EPM results with time spent in open arms, time spent in closed arms and total distance travelled during the test.(B). Object recognition test. The analysis of Objection Recognition (OR) test revealed a significant difference between the tested groups in the 3TgXAD mice model (p ​< ​0.001).(C). Y maze results. A significant difference in the mean time required to perform ten alternations between vehicle treated mice and D3 treated mice was found in 3TgXAD group (p ​< ​0.05), but no significant difference was reported in the control group (p ​> ​0.05).(D). Time spent in platform quadrant in Watermaze Morris test. In the Morris test, spatial memory was improved significantly within 3TgXAD treated mice, with D2 (p ​< ​0.01) and with D3 (p ​< ​0.001) spending around 40% more time in quadrant platform than the vehicle treated mice.

Fig. 3

Assessment of AD biomarkers, oxidative stress anti-inflammatory markers in brain tissue (half-hemisphere randomly assigned in groups) and plasmatic inflammatory markers from 3TgXAD and control mice administered or not with Antroquinonol or vehicle (n ​= ​4 to 8 after removal of outliers) at 3-month-old. (A) Aβ42, (B) tau, (C) p-tau, (D) IL-1 β, (E) TNF-α, (F) Nrf2 and (G) 3NT levels. Graphs were plotted as mean values ​± ​SEM.∗ ​= ​p ​< ​0.05, ∗∗ ​= ​p ​< ​0.01, ∗∗∗ ​= ​p ​< ​0.001.

Fig. 4

Behavioural testing for 3TgXAD and control mice treated or not with Antroquinonol (D1 at 9 month old (n ​= ​10 to 18, after removal of outliers). Graphs were plotted as mean values ​± ​SEM.∗ ​= ​p ​< ​0.05, ∗∗ ​= ​p ​< ​0.01, ∗∗∗ ​= ​p ​< ​0.001 (A). EPM results with time spent in open arms, time spent in closed arms and total distance travelled during the test. The 3TgXAD mice spent significantly less time in open and closed arms compared to their respective controls p ​< ​=0.001, Fig. 4A). Transgenic mice showed significant difference of time spent in open arms for the mice treated with D3 compared to vehicle treated mice (p ​< ​0.05).(B) Object recognition test. The 3TgXAD mice spent significantly less time with new object compared to the control mice (p ​< ​0.001) while the transgenic D3 treated mice spent significantly more time with the new object (p ​< ​0.001) compared to 3TgXAD vehicle mice.(C) Y maze. Transgenic treated with D3 and ALLO treated mice were significant faster to complete the task in Y maze compared to 3TgXAD vehicle treated mice (p ​< ​0.001).(D). Time spent in platform quadrant in Watermaze Morris test. Spatial memory tested in Watermaze was significantly lower in transgenic mice compared to control mice (p ​< ​0.001). Transgenic treated mice with D2, D3 and ALLO spent more time in the platform quadrant of the Watermaze compare to the 3TgXAD vehicle treated mice.

Fig. 5

Assessment of AD biomarkers, oxidative stress anti-inflammatory markers in brain tissue (half-hemisphere randomly assigned in groups) and plasmatic inflammatory markers from 3TgXAD and control mice administered or not with Antroquinonol or vehicle (n ​= ​4 to 8 after removal of outliers) at 9-month-old. A) Aβ42, (B) tau, (C) p-tau, (D) IL-1β, (E) TNF-α, (F) Nrf2 and 3NT levels. Graphs were plotted as mean values ​± ​SEM∗ ​= ​p ​< ​0.05, ∗∗ ​= ​p ​< ​0.01, ∗∗∗ ​= ​p ​< ​0.001.

Fig. 6

Summary of the role of Antroquinonol in the context of AD pathophysiology. Antroquinonol administration leads to a decrease of Aβ levels in the brain, which is forms through the cleavage of APP. Briefly, APP can be processed through non-amyloidogenic or amyloidogenic pathways, with the latter being more prominent in AD brains. Cleavage by β-secretase of APP produces sAPPβ and CTFβ (after APP endocytosis). CTFβ is then cut by γ-secretase into Aβ and AICD fragments. Exocytosis of Aβ and sAPPβ back into the extracellular space then occurs. Aβ accumulates, and its oligomerisation, leads to the formation of amyloid plaques in the brain. The non-amyloidogenic pathway consists of APP cleavage by α-secretase; which produces sAPPα, and CTFα, with CTFα being further cleaved by γ-secretase, leading to generation of p3 and AICD fragments. All products part of non-amyloidogenic pathway are non-pathogenic. Antroquinonol can also reduce inflammation in the neurons notably by its action on Nrf2 expression and reduction of NF-kB, TNF-α and IL-1β levels produced by reactive neuroglial cells. Actions of Antroquinonol can therefore lead to reduction of learning and memory impairment, neuronal loss and inflammatory and oxidative responses.