| Alzheimer’s disease (AD) is a primary central nervous system degenerative disease. Themain clinical manifestations of AD are progressive cognitive impairment, learning and memoryintellectual decline and severe dementia. Currently, there are around 36 million AD patients andADI estimated that this figure will be doubled within 20 years and will change into 66 million. in2030. In our country, AD patients are much more than 5 million. With the increase of thenumber of aged population, the incidence and total number of AD patients will continue toincrease. There is no doubt that AD has currently become one of the most serious diseases threatto human health, as well as cardiovascular diseases, cerebrovascular diseases and cancer.One prominent pathological hallmark of Alzheimer’s disease (AD) is the presence of highdensity of senile plaques, which is composed of amyloidβprotein (Aβ) in AD patient’s brain.The neurotoxicity of natural Aβ(Aβ1-40 and Aβ1-42) has been widely reported and thesequence 25-35 in Aβhas been recognized to be the active center of the whole molecule of Aβfor its similar neurotoxicity to natural Aβmolecule. Our recent studies in electrophysiology,membrane patch and cultured neuron also show that Aβ25-35 can impair the function of neurons.Since the mechanism of Aβneurotoxicity is gradually being clarified and“Aβdoctrine”hasbeen established, the blocking of Aβneurotoxicity maybe become an effective treatment of AD.However, the effective medicine to block the Aβneurotoxicity is still seriously absent until now.Humanin（HN）is a 24 amino acid linear peptide, firstly discovered in 2001 by a Japanesescholar, which can specifically inhibit neuronal cell death caused by a variety damage related toAD. Colivelin (CLN), firstly reported in 2005, is a new HN derivatives composed of 26 aminoacids. There are some studies show that Colivelin has a strong neuroprotective effect, which caneffectively reverse the neurotoxcity of Aβin primary cultured cortical neurons and behavioralexperiments. However, the in vivo effect of Colivelin, especially on the electrophysiologicalmodel, long-term potentiation (LTP) of synaptic transmission in hippocampus, and the protectivemechanism of Colivelin is still an open question now.Therefore, our study will use hippocampus injection to deliver Aβ25-35, Colivelin, saline,Colivelin (with different dose) + Aβ25-35, to observe the effects of Colivelin on rat’s behavior and to explore whether it can reverse learning and memory impairment caused by Aβ25-35. Thisresearch includes 2 parts: (1) observing the effect of Colivelin pretreatment on Aβinducedlearning and memory impairment by using Morris water maze (MWM) test; (2) observing theeffect of Colivelin on Aβinduced LTP suppression, and further clarifing its presynaptic andpostsynaptic mechanism. Through those two studies, we will provide sufficient experimentalevidences on the effect of Colivelin on Aβneurotoxicity and its possible mechanism, andprovide new ideas and theories for the prevention and treatment of AD.PartⅠ：Colivelin prevents Aβ-induced impairment of spatial cognition inratsObjective: To investigate whether Colivelin could affect spatial learning and memory inrats, and clarify the effect of Colivelin pretreatment on Aβinduced cognitive functionimpairment by using a Morris water maze test.Method: SD (230-250 g) was randomly divided into 6 groups: control, Aβ25-35, Colivelinalone, Colivelin (0.2 pmol, 2 pmol, 0.2 nmol) + Aβ25-35 groups. All drugs were directlyinjected into the hippocampus of rats. Morris water maze tests (Hidden platform and visibleplatform tests) were performed 2-3 weeks after drugs injection to observe the ability of spatiallearning and memory. The main indicators include the average escape latency and travel distanceof the rats to find the platform, and the swiming time (limited time: 120 seconds) and distance inthe target quadrant after withdrawal of the platform. At the same time, the swimming speed andvision of rats in each group was tested, to exclude the impact of motor ability and visualimpairment on the determination of indicatorsResults: (1) In control group, the average escape latencies and distances of rats forsearching the underwater platform were 79.0±3.3 s, 36.6±3.0 s, 20.2±2.0 s, 19.9±1.3 s and16.5±1.7 s as well as 1427.5±135.1 cm, 689.4±80.8 cm, 369.2±50.7 cm, 322.4±40.6 cmand 265.3±44.0 cm on training days 1-5, respectively. The total time elapsed and distanceswum in target quadrant was 55.4±3.0 s and 1150.4±63.9 cm, respectively. (2) In Aβ25-35injection group, the latencies and distances for searching for the platform under water on 2-5training days were 52.8±4.3 s, 39.5±1.6 s, 29.3±2.6 s, 26.8±1.4 s and 899.1±108.9 cm,551.7±54.9 cm, 448.5±68.7 cm, 369.6±28.6 cm, significantly lager than the control values(P<0.01 or P<0.05). In the probe trails, the total time elapsed and distance swum in targetquadrant were 32.4±2.9 s and 612.0±52.9 cm, respectively, larger than the values in control group (P<0.01). The results indicate that Aβ25-35 obviously impaired spatial learning andmemory of rats. (3) Hippocampal injection of Colivelin alone (0.2 nmol) did not affect thebehavior of learning and memory, the latencies and distances for searching for the under waterplatform on 2-5 training days were 35.3±4.4 s, 22.1±2.9 s, 21.4±2.6 s, 19.4±0.7 s and 670.0±90.6cm, 398.3±80.9 cm, 357.2±51.5 cm, 260.0±37.0 cm (P>0.1), respectively. But three differentdoses of Colivelin (0.2 pmol, 2 pmol, 0.2 nmol) pretreatment can dose-dependently reverse theimpairment of spatial learning and memory induced by Aβ25-35. The average escape latenciesand distances compare with Aβ25-35 group were significantly reduced on training days 2-5(P<0.05). At 0.2 pmol Colivelin+ Aβ25-35 group (n=11) the latencies and distances forsearching for the platform under water on 2-5 training days were 45.4±3.5 s, 33.9±1.7 s,24.6±3.8 s, 22.3±1.9 s and 728.0±78.3 cm, 425.1±43.8 cm, 339.9±84.4 cm, 286.1±41.5 cm,shorter than with the values in Aβ25-35 alone group (P<0.01). In 2 pmol Colivelin+ Aβ25-35group (n=12), the values were 33.8±3.8 s, 29.8±2.1 s, 22.0±1.6 s, 20.0±1.6 s and 582.1±115.2cm, 385.6±59.1 cm, 281.5±33.3 cm, 228.8±27.3 cm. being also reversed compare with Aβ25-35alone group (P<0.01). In 0.2nmol colivelin+Aβ25-35 group (n=12), it was 28.1±2.4 s, 20.8±2.1 s,19.3±1.6 s, 17.9±2.4 s and 509.5±63.6 cm, 302.9±56.7 cm, 247.9±28.8 cm, 203.8±47.9 cm,significantly larger than the values in Aβ25-35 group（P<0.01）. In the probe trials, the totaltime elapsed and distance swum in the target quadrant was increased in Colivelin pretreatmentgroups. In 0.2 pmol Colivelin+ Aβ25-35 group, it was 39.1±2.1 s and 799.0±74.0 cm; in 2 pmolColivelin+ Aβ25-35 group, it was 43.8±3.0 s and 934.9±53.8 cm; in 0.2 nmol Colivelin+ Aβ25-35 group, it was 54.0±3.7 s and 1148.4±63.0 cm, respectively. (4) The visible platform testsshowed that all drugs did not affect the vision and the swimming speeds of rats (P>0.05).Conclusion: Hippocampal injection of Aβ25-35 impaired spatial learning and memory ofrat. Colivelin alone had no affect on learning and memory function of rats, but Colivelinpretreatment could dose-dependently reverse Aβ25-35 induced learning and memoryimpairments.Part II: Colivelin prevents Aβ-induced depression of LTP in the rathippocampal CA1 region in vivoObjective: This experiment use the same batch of rats after Morris water maze test torecord hippocampal field excitatory postsynaptic potentials (fEPSPs) and long-term potentiation (LTP). We will observe the effect of different dose Colivelin pretreatment on Aβ25-35-inducedLTP depression in hippocampal CA1 region in vivo and investigate the possible mechanism.Method: The same rats after Morris water maze test were placed in a stereotaxic deviceafter anesthetized. The bound stimulating/recording electrode was inserted into the hippocampalSchaffer-collateral/CA1 region. The baseline fEPSPs, HFS-induced LTP and paired pulses-induced PPF were recorded in hippocampal CA1 region by delivering test stimuli, high-frequency stimulus and paired test stimuli to Schaffer-collateral/commissural pathway.Results: (1) Injection of 4 nmol Aβ25-35 did not affect baseline fEPSPs but significantlysuppressed HFS-induced LTP. For example, at 0 min, 30 min and 60 min after HFS, theaveraged amplitude of fEPSPs decreased to 166.6±3.0%, 116.2±1.6 and 106.2±2.8% in Aβ25-35group from 176.8±4.1%, 150.6±2.8% and 141.3±2.3% in control group (P<0.01) respectively,significantly lower than that in control group (P<0.01). (2) Colivelin (0.2 nmol) alone did notaffect baseline fEPSPs and HFS-induced LTP, it was 178.5±2.9%，139.9±2.9%，138.5±2.1%.But pretreated with different dose Colivelin (0.2 pmol, 2 pmol, 0.2 nmol) could reverse Aβ25-35induced LTP suppression in hippocampal CA1 region. In 0.2 pmol Colivelin+ Aβ25-35 group, itwas: 164.5±1.8%, 122.4±1.9%, 110.3±1.1% at three time points; In 2 pmol Colivelin+ Aβ25-35group, it was: 166.0±2.3%, 132.2±2.1%, 119.2±1.2%; in 0.2 nmol Colivelin+ Aβ25-35group, itwas 178.7±2.8%, 139.8±2.4%, and 126.0±2.6%. The results indicated a dose-dependent increasein the LTP (P<0.01) compared with Aβ25-35 alone group; (3) All drugs used in the study did notaffect PPF (P>0.05).Conclusion: Hippocampal injection of Aβ25-35 significantly suppressed HFS-induced LTP,while delivered Colivelin alone did not affect baseline fEPSPs and HFS-induced LTP, butpretreatment with Colivelin can dose-dependently reverse the Aβ25-35 induced LTP suppression.The results suggest that the Colivelin application could be a new treatment of AD by blockingthe neurotoxcity of Aβ. Therefore, the study provides a new clue and idea for the prevention andtreatment of AD. |
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