Behavioral and Gene Expression Study on the Neuroprotective Function of [Gly14]-humanin
|Keywords||[Gly14]-humanin Amyloid-β protein Genistein Alzheimer’s disease Morris watermaze Spatial learning and memory STAT3 Caspase-3 Real-time PCR|
Alzheimer’s disease (AD) is one of the commonest causes of dementia in the elderly, which, as a serious public health problem, is afflicting tens of millions of people worldwide. AD, the insidious and progressive neurodegenerative disorder, is characterized by global cognitive decline. One of the hallmarks of AD is the presence of high density of senile plaques in the brain. Extracellular amyloid plaques are found most commonly in the hippocampus and neocortex, two memory-related brain regions. The major component of AD senile plaques is amyloid-β protein (Aβ), a39-43residue peptide. The neurotoxicity of natural Aβ (Aβ1-40and Aβ1-42) has been widely reported, but the mechanism by which Aβ impairs cognitive function and the active center of Aβ is still not well clarified up to now. Also, it is seriously short of effective neuroprotective drugs or measures against AD or Aβ.It is reported that the sequence25-35in Aβ has been recognized as an active center of the whole molecule of Aβ for its similarity in neurotoxicity to natural Aβ molecule. Our recent studies showed that AP31-35, similar to Aβ25-35or Aβ1-40, also impaired the function of neurons in cell culture, in vitro membrane patch and in vivo electrophysiological experiments, suggesting that the sequence31-35may be a shorter active center than25-35in Ap molecules. However, it is still an open question up to now whether Aβ31-35can impair the cognitive function of rats in learning and memory. Humanin (HN) is a novel24-amino acid peptide cloned from a cDNA library of the occipital lobe of an AD brain by Hashimoto et al. An important clue in the development of AD therapy is the finding of HN and its potent derivative [Glyl4]-humanin (HNG). HN can prevent neuronal cell death caused by various AD-relevant insults such as neurotoxic Aβ protein, and is the only factor that is effective in suppressing various types of AD-related neuronal death so far compared with many other neurotrophic factors. Our previous studies also indicated that HNG could protect against Ap-induced impairments of hippocampal LTP. However, the underlying molecular mechanisms by which HNG plays neuroprotective roles remain largely unknown.Therefore, the present study, by using behavioral and real time RT-PCR molecular biology methods, investigated the effects of bilaterally intrahippocampal injection of AP31-35and HNG on the spatial learning and memory of rats and its possible signaling mechanism so as to provide a new idea for the prevention and treatment of AD. The experiments include the following two parts:Part Ⅰ:[Glyl4]-humanin Prevents Against Aβ31-35-Induced Impairment of Spatial Learning and Memory in RatsObjective:To investigate the neuroprotective effects of [Glyl4]-humanin (HNG) against Aβ31-35induced spatial learning and memory impairment in rats by using Morris water maze test.Methods:Sprague-Dawley (SD) rats (220-250g) were divided randomly into ten groups: control, Aβ31-35(2.0nmol,10.0nmol,20.0nmol), Aβ35-31, HNG、HNG (0.02nmol,0.2nmol,2.0nmol)+Aβ31-35, and40.0nmol Genistein+HNG+Aβ31-35group. All drugs/vehicle were delivered by intrahippocampal injection under the guidance of a brain stereotaxic apparatus. Morris water maze tests (Hidden platform test, probe trials. Visible platform test) were performed2weeks after drugs injection to obtain the ability of rat spatial learning and memory.Results:(1) In control group, the average escape latencies for searching for the under platform were96.9±5.2s,40.3±3.8s,24.5±2.1s.21.2±1.5s and18.4±1.2s on the training day1-5, respectively; the traveled distances of rats were1855.5±133.5cm,645.4±95.1cm,413.1±60.7cm,296.13±35.2cm and218.6±36.0cm at training day1-5, respectively. After withdrawing the platform, the percentages of the swimming time elapsed and distance swam in target quadrant were49.4±1.2%and47.8±1.6%, respectively.(2) Bilateral intrahippocampal injections of AP31-35resulted in a significant decline of spatial learning and memory in a dose-dependent manner, with longer latencies and distances for searching for the under water platform (P<0.05or P<0.01). In the probe trials after removing platform, the percentage of total time elapsed and distance swam in target quadrant were decreased to38.9±1.2%,35.9±2.6%,30.3±1.5%and36.0±1.8%,32.6±1.4%,28.0±1.4%in2.0nmol,10.0nmol and20.0nmol Aβ1-35group, respectively (P<0.01). While Aβ35-31, a reversal sequence of Aβ31-35, had no discernible effect on spatial learning and memory (P>0.05).(3) In the bilateral intrahippocampal injections of2.0nmol HNG alone group. no significant change in learning and memory function was found compared with control group at any training day (P>0.05).(4) In co-application of HNG and Aβ31-35group, HNG pretreatment dose-dependently protected against the Aβ31-35induced impairments in spatial learning and memory.0.02nmol HNG did not induce any significant protective effect (P>0.05) as compared with Aβ31-35alone group. However, co-application0.2nmol or2.0nmol HNG and Aβ31-35reversed the spatial learning and memory impairment induced by Aβ31-35. The escape latencies and distances were significantly decreased (P<0.05or P<0.01) when compared to Aβ31-35only group. At the same time, the percentage of total time and distance elapsed swam in the target quadrant were increased in these higher concentrations of HNG groups. The percentage of total time elapsed and the distance swam in right quadrant were increased to45.2±1.0%(P<0.05),48.6±1.2%(P<0.01) and40.9±1.4%(P<0.05),46.8±1.6%(P<0.01) for0.2nmol and2.0nmol HNG plus Aβ31-35group respectively.(4) Genistein nearly completely abolished the protective action of HNG against Aβ31-35-induced neurotoxicity by co-application of Genistein (40nmol), HNG (2nmol) and Aβ31-35(2nmol). The average escape latencies and distances were significantly larger than the corresponding values in HNG+Aβ31-35group (P<0.01and P<0.05). In the probe trials, the percentage of elapsed time and distance in the target quadrant in the presence of Genistein (Genistein+HNG+Aβ31-35) were decreased to34.3±1.8%and32.1±1.3%respectively, significantly less than the values in co-injection of HNG plus Aβ31-35group (P<0.01).(5) The visible platform tests showed that all drugs did not affect the swimming speeds and vision of the rats (P>0.05).Conclusion:Aβ31-35, but not Aβ35-31, can result in a significant decline of spatial learning and memory in a dose-dependent manner, which supports the hypothesis we previously suggested that Aβ31-35might be a shorter active sequence responsible for the neurotoxicity of Aβ. HNG can dose-dependently prevent against Aβ31-35-induced impairment in spatial learning and memory of rats, while Genistein can attenuate the protective function of HNG against the impairments in spatial learning and memory, suggesting that up-regulation of the tyrosine kinase signaling by using HNG might be of great significance for the improvement of cognitive function in AD.Part Ⅱ:The Effects of HNG on the Gene Expression of STAT3and Caspase-3in RatsObjective:In order to clarify the possible molecular mechanism underlying the neuroprotective effect of HNG in Aβ-induced spatial learning and memory impairment, the present study, by using Real-time RT-PCR technique, explored the influence of HNG on the gene expression of STAT3and caspase-3in the AD related injury animal.Methods:SD rats were randomly divided into five groups:control, Aβ31-35(2.0nmol), HNG (2.0nmol), HNG (2.0nmol)+Aβ31-35, and Genistein (40.0nmol)+HNG+Aβ31-35. The rats were anesthetized with urethane and decapitated after finishing Morris water maze behavioral test. The bilateral hippocampuses were rapidly removed; the total RNA in hippocampus was isolated with the Trizol; and the expressions of STAT3and caspase-3mRNA in the hippocampus were analyzed via real-time PCR.Results:(I) The expression levels of STAT3and caspase-3mRNA in control group were0.99±0.02and0.99±0.03, respectively.(2) Aβ31-35treatment significantly decreased the level of STAT3mRNA (0.74±0.05, P<0.01) of hippocampus, but increased the expression of caspase-3mRNA (1.25±0.06, P<0.01) compared with control group.(3) Bilateral intrahippocampal injections of2nmol HNG up-regulated STAT3but down-regulated caspase-3gene expression, being1.23±0.07and0.69±0.05(P<0.01) respectively.(4) In the co-injection of HNG plus A(331-35group, the expression of Caspase-3mRNA (0.64±0.05) was markedly reduced and the level of STAT3mRNA (1.11±0.05) was increased compared with AP31-35alone group (P<0.01). However, after pretreatment with Genistein, the neuroprotective effect of HNG was significantly reduced compared with HNG plus Aβ31-35group (P<0.01). The level of caspase-3and STAT3mRNA was1.28±0.05and0.65±0.06, respectively in Genistein+HNG+Aβ31-35group.Conclusion:The activation of caspase3-dependent intrinsic apoptotic pathways, as well as the suppression of STAT3mRNA expression may be involved in the Aβ-induced impairments of hippocmpal synaptic plasticity and animal cognitive behavior. The neuroprotective effects of HNG against Aβ neurotoxicity might be mediated by activating tyrosine kinase and STAT3transcription factor, and blocking caspase3-dependent intrinsic apoptotic pathways.