Oligomannurarate, A Novel Anti-Alzheimer’s Disease Drug Candidate, Protects Mitochondria Function Via Inhibiting The Formation Of Aβ Oligomers

Alzheimer’s disease(AD) is in pre-senile or senile a chronic progressive centralnervous system degenerative diseases. With the aging population, AD morbidity anddisability, mortality is rising year by year. Cognitive disorders, memory impairment andpersonality changes are the main clinical features. Senile plaques (SP), neurofibrillarytangles (NFT) and a large loss of neurons constitute the pathological hallmarks of AD.The main constituent of SP is theβ-amyloid protein(Aβ), which is formed aftersequential cleavage of the amyloid precursor protein byβsecretase andγsecretase. Inpathological conditions, Aβform a high state of aggregation of amyloid fibrils, whichproduce toxic effects on nerve cells, causing nerve cell death. Aβmainly exists in twoforms: Aβ1-40 and Aβ1-42 , while latter is more toxic than former. As an importantmolecular target, Aβhas become a hot point of the anti-Alzheimer’s drug research anddevelopment.With further research on AD, the mitochondrial is the most important sub-cellularorgans that Aβdamages nerve cells and induces the development of AD, but also one ofthe main venues that Aβoligomerization at the earliest. Mitochondrial dysfunctionappears at the early phase of dementia is one of proof. As the mitochondria in theneurotoxicity of Aβproduction process not only acts as a target for Aβ, but also servedas an intermediary that Aβdevelops the neurotoxicity, therefore, suppression of Aβ-induced mitochondrial dysfunction become an important strategy against Alzheimer’s. 971, an acidic oligosaccharides developed by Ocean University of China, as aclass of anti-Alzheimer’s drugs is in a state of clinical Phase II. Our previous works havedemonstrated that 971 could specifically bind to Aβin vitro, improving thedepolymerization and inhibiting the fibril formation of Aβand thus antagonize itsneurotoxicity. These results suggest that 971 decreased the injury of nerve by targettingAβ. The fact that a large part of the binding protein of 971 are mitochondrial-associatedproteins by use of affinity chromatography and mass spectrometry suggests that 971 maytarget to mitochondria. Flag-C99, C99-Flag in vitro and APP/PS1 transgenic cell modelwere used, many methods, such as immunoprecipitation, immunofluorescence staining,ELISA and molecular biology methods were applicated to explore the mitochondriaprotective effect and mechanism of 971.1. Protection of mitochondrial function by 971Immunofluorescence methods were used in transgenic cell model APP/PS1 todetermine that 971 can enter the mitochondria. Further, mitochondria content of Aβoligomers isolated from APP/PS1 cells incubated with 971 in 0, 25, 50, 100μg / mlconcentration for 24 hours was detected. The results showed that 971 can significantlyreduced the content of Aβoligomers in mitochondria in APP/PS1 cells, which validatesour early test results. By addition, 971 can significantly increased COX activity ofmitochondria and the overall cellular ATP level after Aβoligomers treatment. Theseresults show that the 971 has significantly protective effect to mitochondrial function.2. 971 suppresses the generation of Aβ(1) 971 plays a protective effect of mitochondrial function by inhibiting Aβgeneration through influencing the maturation process of Aβprecursor proteinAPP, further reduce Aβoligomerization in mitochondriaThe Flag-C99 transgenic cell model was used to evaluated the clearance effect ofintracellular Aβof 971. The content change of intracellular Aβwas detected by usingWestern blot (WB), immunoprecipitation (co-IP) and enzyme-linked immunosorbent(ELISA) after cells incubated with 100μg/ml 971 for 24h. The results showed that 971 had no effect on Aβclearance process in the detection of concentrations. Next weevaluated the impact of 971 on Aβgenerated. Using double transgenic CHO cell modelAPP/PS1 cells, we examined the levels of intracellular Aβ. The results showed that theimmature form of APP intracellular content increased, mature form of APP levelsdecreased after cells co-incubation with 971 for 24 h, with Aβmonomers and oligomerslevels reduced. These results show that 971 inhibits Aβgeneration by affectingmaturation process of the Aβprecursor protein APP. Similar results are obtained fromboth APP transgenic CHO-K1 cell model and APP transgenic model of N2a cells. Theseresults suggest that 971 plays a role in protecting mitochondrial function by influencingthe maturation process of APP, inhibiting of Aβgeneration, reducing Aβoligomerizationin mitochondria.(2) 971 specifically inhibits the formation of Aβ1-42, reduce the proportion ofAβ42/Aβ40, reduces Aβoligomerization in mitochondria by targetting to thetransmembrane region of APP and changing the conformation of C99The C99-Flag transgenic cell model was used to evaluated the effect of 971 ondifferent fragments of Aβ. The results showed that 971 significantly reduced thecontent of Aβ42 and the ratio of Aβ42/Aβ40 with no effect on Aβ40 after co-incubatedwith cells in 100μg/ml for 24h. The results showed that 971 protected mitochondrialfunction by significantly decreasing the generation of high toxicity Aβ42 and theproportion Aβ42/Aβ40. To further reveal the mechanism of 971, we use computersimulation of molecular docking to simulate the combination of 971 with Aβ. Thecomputer simulation results suggest that 971 may bind to the APP transmembraneGXXXG domain. Summing up the above, 971 may target to APP transmembranedomain, and specifically bind to C99, change the C99 conformation, thereby reduce thegeneration of Aβ1-42 and the ratio of Aβ42/Aβ40.Taken together, 971 can protect mitochondrial function in two aspects: one is that971 can inhibit Aβgeneration by influencing the maturation process of Aβprecursorprotein APP; on the other hand, 971 can specifically inhibit of the formation of Aβ42 and reduce Aβ42/Aβ40 by targetting to the APP transmembrane region and changing theC99 conformation. It is reported for the first time that 971 plays anti-AD role byprotection of mitochondrial function, which provides important theoretical basis forclarification of mechanism of action and further clinical application. Inhibiting Aβgeneration by inhibiting the maturation process of APP is a novel strategy for anti-Alzheimer’s development.