Neuroprotective Effect Of A New Chiral Nitroxide In Alzheimer’s Disease

Objectives:Alzheimer’s disease （AD）, an age-related neurodegenerative disorder, is the most commonform of dementia. AD is characterized by the deposition of β-amyloid （Aβ） plaques,intracellular neurofibrillary tangles, loss of neurons in the brain, progressive decline ofmemory and cognitive functions, and behavioral and personality changes. In thepathogenesis and progression of AD, aging is the most critical risk factor. Moreover,oxidative stress has an important function in the early stages of AD. Reactive oxygenspecies （ROS）-mediated pathways are involved in AD development. Numerous studieshave reported the presence of elevated DNA, RNA, lipid, and protein oxidation in brainsof subjects with AD and mild cognitive impairment （MCI）, suggesting that oxidative stressis an early event in AD pathogenesis. It illustrate that oxidative stress occurs at early stages, before the appearance of amyloid plaques and neurofibrillary tangles. For mostantioxidant drugs, beneficial effects have been reported in cell cultures, and partially, inanimal models. However, success in human clinical trials is much less frequent.Nitroxide radicals （NRs） are stable free radicals. NRs are utilized as biophysical tools inelectronic spin resonance spectroscopic studies and spin-label oximetry in early days.Recently studies found that NRs has some special biological activities, include radiationprotection, anticancer, against ischemia-reperfusion injury and so on. Unlike otherantioxidants that act in a sacrificial mode, NRs can provide protection in a catalyticmanner. Through the continuous exchange between these forms NRs act asself-replenishing antioxidants that degrade superoxide and peroxide. For example, NRshave Superoxide dismutase （SOD） mimetic action. Similar to endogenous SOD, thenitroxide acts as a catalyst and is not consumed in the process of dismutation of O₂^-toH₂O₂and oxygen. The catalytic rate is higher than SOD. Because its function of spintracer, we can observe its distribution and understand the changes in the body. Therefore,NRs have a better view in anti-oxidative damage as new type antioxidant drugs.Methods:In our study, Lucigenin chemiluminescence models derived from xanthine–xanthineoxidase reaction were used to evaluate the free radical-scavenging activity CHP-inducedlipid peroxidation system to evaluate inhibition of lipid peroxidation of Curcumin, Tempoland L-NNNBP. In primary cortical neuronal cultures, the neurons were rinsed briefly withPBS at the10th day and then pretreated with10μM Curcumin, Tempol and L-NNNBP for24h, respectively, followed by exposure to25μM of Aβ1-42for12h in the same medium.Afterward, cells were washed3times and returned to the original culture medium for24h.Cell viability was determined by cell counting kit-8（CCK-8）. Measurement of apoptoticcells by the TUNEL. Male APP/PS1double-transgenic mice were used in this study toevaluate the protective effect of L-NNNBP. Mice were divided into five groups: Curcumintreatment, Tempol treatment, L-NNNBP treatment, Wild-type and Transgenetic group.Male mice were treated with curcumin, tempol or L-NNNBP （1mM in drinking water）. Treatment was started when the mice were6months old and was continued for1month.After treatment for1month, spatial learning and memory were evaluated by the Morriswater maze test. The mice brain sections were stained with Congo red solution to identifythe Aβ plaques. The culture neurons and hippocampal tissues were normalized viabicinchoninic acid protein assay to generate homogenates.3-Nitrotyrosine （3-NT）measurements were performed in the supernatants.Mitochondrial membrane potential（Δψm） in the culture neurons was detected by the cationic fluorescent probetetramethylrhodamine methyl ester （TMRM）. Western blot analysis was performed to theexpression of phosphorylated Tau and GFAP. Using immunohistochemistry method, wetest the activation of astrocytes.Results:We first examined the ability of L-NNNBP to scavenge the superoxide anion radical.Lucigenin chemiluminescence models derived from xanthine–xanthine oxidase reactionwere also used to evaluate the free radical-scavenging activity of L-NNNBP.L-NNNBPshowed more potent free-radical scavenging activities, which increased thechemiluminescence compared with curcumin and tempol at the same concentrations（#P<0.05，##P<0.01）. In the CHP-induced lipid peroxidation system, L-NNNBP alsoshowed a higher inhibiting rate on lipid peroxidation compared with curcumin and tempol（#P<0.05，##P<0.01）. In cultures, L-NNNBP attenuated Aβ1-42-induced cell death in aconcentration-dependent manner. However, tempol （0.1–10μM） did not exhibit anyneuroprotective activity against Aβ1-42-induced toxicity. Curcumin exhibitedneuroprotection only at a high concentration （10μM）. Treatment with L-NNNBP alonedid not affect the cell viability. TUNEL staining was performed to identify the apoptoticneurons. Pretreatment of L-NNNBP （10μM） significantly reduced apoptotic neurons to22.7%±2.6%, and this anti-apoptotic activity was more significant than that mediated bythe same concentrations of curcumin （42.9%±3.1%） and tempol （51.1%±1.1%）. Cellimmunofluorescence results show that the L-NNNBP have anti-apoptotic effect throughdecreasing the activated Caspase-3. Aβ can cause oxidative stress injury lead tomitochondrial dysfunction, affect the normal mitochondrial membrane potential and make its depolarization. L-NNNBP （0.1–10μM） prevented the depolarization of Δψm caused byAβ1-42treatment, and this action was stronger than those of curcumin and tempol at thesame concentration （#P<0.05）. In addition to oxidative stress, nitrification stress is anothermain source of free radicals generation. LNNNBP attenuated Aβ1-42-induced3-NTincrease. The inhibition of3-NT by curcumin and tempol was weaker compared with thatmediated by L-NNNBP at the same concentration （#P<0.05）.Aβ plaque deposition is one of the main pathological symptom of AD. Treatment withL-NNNBP and tempol markedly reduced Aβ plaque accumulation in the hippocampus andsomatosensory cortex, whereas curcumin reduced Aβ plaque acumulation in thehippocampus only. Besides Aβ plaque deposition, another feature AD pathologicalprogress is neurofibrillary tangles caused by abnormal phosphorylation of Tau. A markeddecrease in Tau phosphorylation at Ser235and Thr205was observed in theL-NNNBP-treated APP/PS1mice compared with the vehicle-treated mice. LNNNBPmarkedly decreased tau phosphorylation compared with curcumin or tempol （**P<0.01，#P<0.05，##P<0.05）.In the brains of AD patients and transgenic AD mouse models, theinfiltration of activated astrocytes are seen in the area of Aβ plaques, which arecharacteristic components of an inflammatory process that develops around an injury inthe brain. Quantitative analysis showed a58.5%±3.2%decrease in GFAP expression inthe L-NNNBP-treated APP/PS1mice compared with that in the control APP/PS1mice（**P<0.01）. In Morris water maze text, the L-NNNBP-treated APP/PS1mice reachedthe platform, which resulted in significantly reduced escape latency across the trialscompared with the control APP/PS1mice, and curcumin-and tempol-treated mice（**P<0.01,##P<0.01）. L-NNNBP markedly improved the learning capability and memoryof APP/PS1mice compared with curcumin or tempol.Conclusions:This study demonstrates that both in oxidative damage vitro model, Aβ1-42-inducedtoxicity model or APP/PS1double transgenic mice model, treatment with the new chiralNRs （L-NNNBP）, shows the exciting results. It can scavenger superoxide anion, inhibitlipid peroxidation, Reduce the cytotoxicity induced by Aβ1-42, anti-apoptosis, reduce oxidation and nitration stress, decrease Aβ plaque deposition, decrease level ofphosphorylated Tau protein, inhibit astrocyte activation and Improve spatial learning andmemory of AD transgenetic mice. In a word, L-NNNBP is likely to become the candidatedrugs for AD clinical therapy.