Effects And Mechanisms Of T-006 On Learning And Memory Impairment In Two Dementia Animal Models

Background Alzheimer's disease(AD) is a progressive neurodegenerative disorder characterized by cognition and memory impairment. AD brains are characterized by the presence of extracellular senile plaques composed primarily of ?-amyloid peptide(A?), intracellular neurobrillary tangles(NFTs) and degenerating neurons. The mechanism of AD is still not explicit, while the identification of A? as the major component of the senile plaque is one of the primary causes of AD. It has been proposed in amyloid cascade hypothesis that deposition of amyloid ?-peptide in plaques in brain tissue triggers the pathogenic cascade of AD,such as excitatory neural toxicity, imbalance of the calcium homeostasis, productionof free radicals, hyperphosphorylation of tau protein. These lesions would cause dysfunction of the neurons and neurogliocytes and psyhcological symptoms eventually.Currently, the approved drugs for the treatment of AD are mostly cholinesterase inhibitors, like donepezil, the inhibition effect of acetylcholine degradation was forced by inhibiting acetylcholinesterase activity,increasing local concentration of acetylcholine, thereby improving neurotransmission function. But donepezil only treat for mild AD, when the AD progress develops,the number of neurons reduces,and the role of donepezil will also diminish. Therefore, the exploration of new drugs for AD treatment is extremely important. TMP derives from the Chinese herb Ligusticum wallichii, it has multi-functional pharmacological effects, like anti-inflammatory, antioxidant and inhibition of apoptosis, but with low bioavailability. J147 is a compound synthesized by the Salk Institute from America and proved to have neuroprotective effect in synapse.We combined the TMP molecule with J147, and synthesized T-006, a novel compound obtained through structural modification. Previous experiments showed that T-006 has significant anti-oxidative effect in stressed cells model and it can protect nerve cells from damage, suggesting that the compound has a potential of becoming a new drug for AD treatment.In this study, we explore the effects and mechanisms of T-006 on learning and memory impairment in Kunming mice and SD rats induced by scopolamine and A? 1-42, respectively.Objective To explore the effect and mechanisms of T-006 on learning and memory impairment in two AD models.Methods lateral ventricle injectionA?1-42 was dissolved in sterile saline at a concentration of 2?g/?l and placed in an incubator at 37°C for 72 h to activate the neurotoxic of A?1-42. Rats were anaesthetised with sodium pentobarbital(40 mg /kg i.p.), holes were drilled in the skull over the lateral ventricle according to Paxinos & Watson Rat Brain Stereotaxic Coordinates: 0.96 mm posterior to bregma;2.0 mm lateral,dorsal-ventral 4.0mm.Then, 5?l of peptide was gradually delivered into the hole by syringe within 5 minutes, and the syringe was remained for an additional 10 minutes before withdrew.Control group rats were injected with 5?l distilled saline using the same procedure.The rats were taken to single cages after the surgery and were permitted 11 days for recovery before behavioral assays begin.Morris water maze All rats were trained on Morris Wtaer Maze(MWM) test on Day11 after the injection of A?1-42. The maze is a circular pool with 1.5 meters(1.2 meters for mice)in diameter,containing water at 22±1°C in temperature.The hidden platform was positioned in the middle of north-west quadrant of the maze.The MWM procedure lasted for 5 consecutive days:on Day1 rats were required to find the platform within 90 s. If the rats failed to find the platform after 90 s, they would be placed on it for an additional 20 s by experimenter.Then they were placed in a heated incubator between trials for recover.Day2 to Day4 have the same procedure as Day1.On Day 5, a probe test was done while the platform was removed,each trial lasted for 90 s. The time it took each rat to find the hidden platform area was measured as escape latency.The swimming speed,times crossing the platform area,time and distance spent in target quadrant(north-west quadrant) of each animal were monitored and recorded with a video tracking system.For scopolamine mice experiment, during Day1 to Day4, all mice except for control group were given an i.p. injection of 2 mg/kg scopolamine 20 minutes before the trial, control mice were given distilled saline.Rest of the procedure for mice assay is the same as rats assay.Passive avoidance test The BA-200 passive avoidance testing apparatusconsists of six shuttle boxes,each shuttle box is composed of a dark room and a light room linking by a sliding door.All dark rooms have a grid floor with a shock generator.The two-day passive avoidance test including a training trial on the Day1 and a memory retention test on Day2.On training day, mice was placed in the light box facing opposite to the sliding door, and the door was opened when trial begin and time limit is 5 minutes.Once entering the dark room, mice would suffer a foot shock(39V) and would return to light room. The memory retention test was carried out 48 h after the training in a same procedure but without shock generator.The step through lantency(latency of entering the dark room) and the error times(total times entering the dark room) were recorded.Elevated plus maze The apparatus was made of two open arms and two enclosed arms, each arm has size of 50×15 cm(enclosed arms were surrounded by 20 cm walls),and was set 50 cm above the floor. All arms were connected in the central area(15×15 cm) of the maze. Mice were habituated to the maze for 2 minutes before testing and then tested in the maze for 5 minutes.A video-tracking system is used to automatically detect and record the track for each mice and the time spent in each area.All data measured including distance ratio in open arms, total number of all arms entries and time spent in the open arms. An entry was counted only when the mouse entered the arm with all four paws.The percentage of distance in open arm and the time spent there could indicate the anxiety level of mice.Enzyme Assays Ach E activity, SOD activity, GSH activity and MDA content were analyzed using the corresponding kits respectively.Samples were prepared the same as tissue preparation part. Protein concentrations in the tissue extracts were determined by Bradford protein assay.Ach E, SOD,GSH and MDA activities were then detected according to their manufacturer's guidelines.Tissue preparation and immunoblotting All animals were sacrificed after the last behavioral test for tissue harvesting.Hippocampal and cortex tissue samples were homogenized in 10 volumes of RIPA lysis buffer(20 m M Tris, p H 7.4, 150 m M Na Cl, 1m M EDTA, 1Mm EGTA, 1% Triton, 2.5 m M Na-pyrophosphate, 1 m M Na3Vo4, 1m M?-Glycerophosphate, 1ug/ml Leupeptin, 1 m M AEBSF).Samples were sonicated(4×3 s) and centrifuged at 10000×g for 30 minutes at 4°C.Protein concentrations in the cell extracts were determined by BCA protein assay(Thermo Fisher Scientific,Rockford, IL, USA).Equal amounts of protein were solubilized in 4×SDS-sample buffer, separated on SDS-polyacrylamide gels, transferred to PVDF membrane and immunoblotted with the antibodies indicated in the materials section. Protein levels were normalized to ?-tubulin levels in western blots. ANOVA analysis was used to determine differences between means.Result T-006 can improve spatial memory ability of animals in Morris water maze;improve memory retention ability in passive avoidance assay and reduce AD related anxiety in elevated plus maze.The mechanisms underlying thses may related to its abilities of antioxidative effect;decreasing Acetylcholinesterase activity;decreasing the expression of Pho-tau and HSPs;increasing the BDNF level and the expression of synasins.Conclusion T-006 can significantly improve learning and memory ability of animals in two AD models.T-006 is a multifunctional drug with a short-term treatment efficiency.The ability of T-006 to reduce phosphorylated tau levels, increase neurotrophic factors, maintain synaptic proteins and antioxidative effect is further proved in this study. When compared to currently accepted AD drug donepezil in our studies, T-006 performed equally well or superior.