| Alzheimer’s disease (AD) is an irreversible neurodegenerative disease, which occurs in the central nervous system, and characterized by progressive cognitive dysfunction, including loss of learning and memory, and dementia finally. The main pathological features of AD consist of high density of senile plaque (SP) in the brain, neurofibrillary tangles (NFTs) and loss of neurons. However, the etiology and pathogenesis of AD are not clear. The accumulation of amyloid-β (Aβ) in brain is thought to be causative for the progression of AD. Consequently, it is critical to clear the aggregated AP or block the AP toxicity in the brain for the prevention and clinical treatment of AD.Humanin (HN) and its derivatives have been thought to have potential therapeutic application in AD. Hashimoto et al. found that the occipital lobe did not change in the whole process of AD in patients. So they speculated that some genes in occipital lobe neurons must have been activated in the process of AD. HN is discovered as a24-amino acid peptide, and the cDNA was identified from an AD patient’s occipital lobe of brain in2001. HN can effectively protect neuronal cells against almost all AD-related insults, such as various FAD genes, anti-APP antibody, and neurotoxic Aβ in vitro. However, the underlying molecular mechanisms of HN’s neuroprotective roles remain unclear. Furthermore, it was found that HN also existed in rat, mouse, monkey and nematode. Caricasole et al. cloned a homologue gene of HN in the rat named as Rattin, which encodes a peptide of38amino acids (14residues longer than Humanin), with73%identity in the conserved region to HN. The availability of Rattin facilitates the studies in rats aimed at elucidating the mechanism of action of HN-like peptides and the study of their pharmacological properties in vivo. Therefore, on the basis of preparing AD rat model with Aβ injection, the present study investigated the neuroprotecitve effects and mechanisms of Rattin against Aβ-induced impairments in behavioral, electrophysiological and molecular levels.We carried out the research in three parts as follows:(1) considering that MWM test is the classic method to evaluate the ability of spatial learning and memory of rats, we investigated the effects of Rattin on the Aβ31-35-induced impairment of spatial learning and memory of rats by bilateral intrahippocampal injection and using MWM test;(2) in view of the close correlations between spatial cognitive behavior and hippocampal LTP, which is widely accepted as one of the cellular models of learning and memory, the present study investigated the effects of Rattin on the Aβ31-35-induced impairment in in vivo LTP in rat hippocampal CA1region by recording field excitatory postsynaptic potentials (fEPSPs);(3) the protective effects and mechanisms of Rattin on Aβ31-35-induced neurotoxicity in primary cultured rat hippocampal neurons were observed, and three different tyrosine protein kinase signal pathway (MAPK, IP3K and JAK/STAT3) and intracellular calcium concentration ([Ca2+]i were checked by using real-time PCR and laser confocal image techniques. Part ⅠThe Neuroprotection of Rattin against Neurotoxic Amyloid β Protein in Spatial Learning and Memory of RatsObjective:The deposition of amyloid β protein (AP) is thought to be responsible for the loss of memory in Alzheimer’s disease (AD), and Aβ31-35should be a shorter active sequence for the neurotoxicity of Aβ. Rattin, a rat homolog of humanin (HN), shares the ability with HN to protect neurons against amyloid β protein (Aβ)-induced cellular toxicity but with much more effectiveness than HN. Significantly, Rattin facilitates the studies of HN-like peptides in rat model and avoids the interspecific differences that directly using HN in rats. However, it is still unclear whether Rattin can prevent against the Aβ-induced cognitive deficits. In the present study, we investigated the effects of Rattin and Aβ31-35on the spatial learning and memory of rats by using Morris Water Maze (MWM) test.Methods:Sprague-Dawley (SD) rats (200-300g) were divided randomly into six groups:Control, AP31.35, Rattin(2nmol), and Rattin (0.02,0.2,2nmol)+Aβ31-35group (n=10, per group). Rats were anesthetized with urethane and placed in the stereotaxic apparatus for surgery and injection. MWM tests (Hidden platform test, probe trials, visible platform test) were performed2weeks after drugs injection. The escape latency (s), distance traveled (cm) and swimming speed (cm/s) were calculated in acquisition phase (hidden platform tests), and the percentage of the total time and the distances in the different quadrants was recorded in probe trials. To exclude the possibility that the results above such as the change in escape latency were due to the impairment of visual or motor ability of rats, the escapelatencies of rats were tested again in visible platform condition after probe trials, and the average swim speeds of rats in all groups during5days of successive learning were also calculated.Results:(1) Aβ31-35impaired the spatial learning and memory of rats in MWM test. In MWM test, the learning ability of rats to acquire spatial information was first assessed by five consecutive days of hidden platform test. Subsequently, the spatial memory was tested by probe trials on day six. As expected, the average escape latency and distance of rats in searching for the hidden underwater platform decreased with the increase of training days in each group. However, the spatial learning ability of rats in the Aβ31-35group (n=10) was significantly affected, with longer escape latency and distance in searching for the underwater platform. The average escape latencies were55.32±1.68seconds (P<0.01),33.54±1.41seconds (P<0.01),24.17±0.55seconds (P<0.01), and18.38±0.51seconds (P<0.01) on training days2-5, respectively, significantly larger than the values of28.61±0.99seconds,20.61±0.97seconds,16.50±0.45seconds, and14.47±0.52seconds in control group (n=10). Similarly, the average escape distances of the rats in Aβ31-35group increased on training days2-5, being685.19±18.68cm (P<0.01),491.34±13.60cm (P<0.01),393.80±9.33cm (P<0.01) and262.05±6.59cm (P<0.01) respectively, significantly larger than the values of540.11±14.45cm,349.33±16.30cm,281.56±13.15cm and208.92±3.55cm in the control group on the same training days. In the memory test with probe trials, the percentage of total time and distance in the target quadrant significantly decreased in the Aβ31-35group, from47.63±1.43%and45.09±1.41%in control group decreased to29.35±1.09%(P<0.01) and29.26±1.18%(P<0.01), respectively. These results indicate that bilateral intrahippocampal injection of Aβ31-35seriously impaired the spatial learning and memory of rats.(2) Rattin prevented against Aβ31-35-induced deficits in spatial learning and memory of rats. To investigate the neuroprotective roles of Rattin against Aβ31-35, the effects of Rattin alone on the spatial learning and memory of rats were observed first. We found that Rattin alone, even at a high concentration (2nmol), did not affect the escape latency and the escape distance of rats in the hidden platform tests. Similarly, the percentage of swimming distance in target quadrant was not changed by Rattin alone, compared with the control group. In addition, pretreatment with different concentrations (0.02,0.2,2nmol) of Rattin dose-dependently prevented the Aβ31-35induced deficits in spatial learning on almost all training days. Compared with Aβ31-35alone group, the average escape latency and distance in low concentration (0.02nmol) Rattin plus Aβ31-35group (n=10) were not significantly changed. However, in0.2nmol Rattin plus Aβ31-35group (n=10), the average escape latency decreased to42.11±0.72seconds (P<0.01),27.36±0.29seconds (P<0.01),20.18±0.50seconds (P <0.01), and15.48±0.27seconds (P<0.01); the mean escape distances decreased to626.58±6.52cm (P<0.01),427.67±10.77cm (P<0.01),323.52±4.00cm (P<0.01), and226.01±2.94cm (P<0.01) on training days2-5, respectively. In2nmol Rattin plus Aβ31-35group (n=10), the average escape latency further decreased to33.15±0.72seconds (P<0.01),26.10±0.71seconds (P<0.01),18.66±0.55seconds (P<0.01),14.92±0.19seconds (P<0.01); the mean escape distances decreased to585.26±9.20cm (P<0.01),381.80±6.54cm (P<0.01),306.31±4.34cm (P<0.01), and208.52±2.15cm (P<0.01) on training days2-5, respectively. Also, compared with the lower concentration of Rattin (0.02nmol), higher concentrations (0.2nmol and2nmol) of Rattin showed enhanced protective effects, with significant decreases in the average escape latency and distance in coapplication groups. In the probe trials, pretreatment with different concentrations of Rattin dose-dependently prevented the Aβ31-35-induced memory deficit. The percentage of total time and total distance for rats spent in the target quadrant significantly increased in higher concentrations of Rattin plus Aβ31-35groups, in which the time percentages increased to36.60±1.67%and42.71±1.58%in0.2nmol and2nmol Rattin groups respectively, significantly larger than the value of29.35±1.09%in AP31.35alone group (P<0.05); the distance percentages increased to33.99±1.68%(P<0.05) and39.99±1.81%(P<0.01) in the two Rattin groups, respectively, larger than the value of29.26±1.18%in Aβ31-35alone group. The results indicate that Rattin alone do not affect the spatial cognition of rats, but pretreatment with Rattin dose-dependently prevent against AP31-35induced deficits in spatial learning and memory.(3) Neither Rattin nor Aβ31-35did affect the vision and motor ability of rats. To exclude the possibility that the results above such as the change in escape latency were due to the impairment of visual or motor ability of rats, the escape latencies of rats were tested again in visible platform condition after probe trials, and the average swim speeds of rats in all groups during5days of successive learning were also compared. There was no difference in the escape latency among all groups in the visible platform test, and the average time reaching to the visible platform was approximately14s. In addition, there was no significant statistical difference (P>0.05) in swimming speeds among all groups in5days of successive learning acquisition, with an approximately19cm/s of average swim speed. These results indicated that the vision and the motor ability of rats were not affected by Rattin or Aβ31-35in the MWM tests.Conclusion:These findings show that bilateral intrahippocampal injection of AP31.35could impair the spatial learning and memory, while Rattin could dose-dependently prevent the Aβ31-35-induced decline in spatial cognitive behavior of rats. Therefore, the present study strongly suggests that application of exogenous Rattin or up-regulation of endogenous HN in the brain might be beneficial to the prevention and treatment of Aβ-related cognitive deficits such as in AD. Part ⅡRattin Protects against Aβ31-35-Induced Impairment of Hippocampal Long Term Potentiation in Rat Hippocampal CA1Region in vivoObjective:Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease in the elderly leading to progressive loss of memory and cognitive deficits. Amyloid β protein (Aβ) is thought to be responsible for loss of memory in AD, and Aβ31-35should be a shorter active sequence responsible for the neurotoxicity of Aβ. In the behavior study above, we found that Rattin has a protective effect against Aβ31-35-induced behavior impairment. However, its underlying mechanisms are almost unclear. As an electrophysiological neuronal model of synapse plasticity, hippocampal long term potentiation (LTP) has been widely used for the research of cellular basis of learning and memory. Therefore, in the present study, we investigated the effects of Rattin and Aβ31-35on the hippocampal LTP of rats by using in vivo hippocampal field potential recording.Methods:In vivo electrophysiological recording of LTP in hippocampal CA1region of rats was performed after finishing the MWM test. Rats were anesthetized with urethane and placed in the stereotaxic apparatus for surgery and electrophysiological recording. A pair of parallel bound stimulating/recording electrodes was inserted into the hippocampus. The tip of the monopolar recording electrode was positioned at the stratum radiatum in the CA1region and the tip of bipolar stimulating electrode was inserted into the hippocampal Schaffer-collateral region. LTP was induced by using a high frequency stimulus (HFS), and fEPSPs were monitored for a further1h to observe the induction and maintenance of LTP.Results:(1) Aβ31-35suppressed in vivo hippocampal LTP in the CA1region of rats. The change in fEPSP amplitude was used to represent the synaptic efficacy in the CA1region. Immediately after delivering three sets of HFS, the average amplitude of fEPSPs in control group (n=6) increased abruptly to187.67±7.24%from the initial control value set as100%, remaining at150%1hour after HFS, indicating a successful induction of LTP in this in vivo experimental condition. Compared with control, Aβ31-35(10nmol, n=6) injection significantly suppressed the hippocampal LTP. The average standardized fEPSP amplitude in Aβ31-35group decreased to165.96±4.11%(P<0.01),133.83±3.25%(P<0.01),126.11±3.94%(P<0.01), and112.14±1.94%(P<0.01) from187.67±7.24%,167.01±1.66%,161.21±2.31%and150.53±3.01%in control group at0min,15min,30min, and60min after HFS, respectively. However, injection of Rattin alone did not change the fEPSP amplitude at the same four time points post-HFS (10nmol, n=6), compared with the control group.(2) Rattin partly and dose-dependently prevented the Aβ31-35-induced depression of hippocampal LTP. Furthermore, we investigated the effects of Rattin on the Aβ31-35-induced impairment of LTP by co-application of different concentrations of Rattin (0.02nmol,0.2nmol, and2nmol) and Aβ31-35(10nmol).0.2nmol and2nmol, but not0.02nmol, of Rattin significantly prevented10nmol Aβ31-35-induced suppression of LTP (n=6, per group). In0.02nmol Rattin plus Aβ31-35group, the percentage of fEPSP amplitude was165.33±4.08%(P>0.05),138.63±2.43%(P>0.05),125.95±2.33%(P>0.05), and114.01±2.13%(P>0.05) at the four time points, respectively, with an increase but without significant difference compared with Aβ31-35alone group. In0.2nmol Rattin plus AP31-35group, the average fEPSP amplitude increased to166.40±5.95%(P>0.05),143.24±4.03%(P<0.05), 135.25±3.61%(P>0.05), and124.43±1.54%(P<0.01) at the same time points. In particular, in2nmol Rattin plus Aβ31-35group, the average fEPSP amplitudes further increased to172.10±5.21%(P>0.05),145.42±4.45%(P<0.05),136.44±4.23%(P<0.05), and131.48±1.50%(P<0.01) at the same four time points post-HFS, respectively, significantly larger than the values in Aβ31-35alone group. The results indicate that higher concentrations (0.2nmol and2nmol) of Rattin effectively protected hippocampal synaptic plasticity in the CA1region of rats.(3) Neither Rattin nor Aβ31.35did affect the hippocampal PPF. To clarify whether the presynaptic mechanism was involved in the effects of Aβ31.35and Rattin on synaptic plasticity, PPF in the hippocampal CA1region was examined in all groups immediately before HFS. After paired pulses were applied to the Schaffer collaterals, the PPF always appeared, with the second fEPSP larger than the first one. The PPF ratio values were170.05±5.20%,169.53±3.60%,173.18±2.68%,173.89±2.88%,169.68±3.78%, and171.68±3.43%in control, Aβ31-35, Rattin, and different concentrations of Rattin plus Aβ31-35groups, respectively, without any significant statistical difference (P>0.05). These results indicate that Rattin and AP31-35do not affect the presynaptic neurotransmitter release in the hippocampal CA1region of rats.Conclusion:Rattin effectively prevente against Aβ31-35-induced LTP suppression in dose dependent manner. These findings may partly explain the cellular mechanism of Rattin in improving spatial learning and memory, suggesting that Rattin might be one of the promising candidates for the treatment of AD in the future. Part ⅢEffects of Rattin on Aβ31-35-Induced Neurotoxocity and Molecular Mechanisms in Cultured Primary Rat Hippocampal NeuronsObjective:To further investigate whether Rattin can inhibit Aβ31-35toxic effect on cultured primary rat hippocampal neurons, and which tyrosine kinase signaling pathway is involved in the protective role of Rattin, we observed the effects of Rattin on Aβ31-35-induced neuronal death and calcium influx in cultured primary rat hippocampal neurons.Methods:CCK-8assay, real-time PCR, flow cytometry and calcium image techniques were used to observe the effects of Rattin on Aβ31-35-induced neurotoxocity, molecular signaling pathways and [Ca2+]i on cultured primary rat hippocampal neurons.Results:(1) Rattin prevented Aβ31-35-induced decline in cell viability. The growth of cultured hippocampal neurons were observed at different days after plating, and the mature cells were used for further experiments7-10days after plating. CCK-8cell viability assay was used to measure the toxicity of the pre-incubated Aβ31-35(20μM) with or without added Rattin in different concentration (1,10,100μM) on primary cultured hippocampal neurons. As shown in Fig.2, the percentage of cell viability in the Aβ31-35group (n=6) significantly decreased to42.5±3.3%from100%of control (P<0.01). Meanwhile, we found that Rattin alone, even at a high concentration (100μM), had no effect, the value of cell viability being98.3±2.9%(P<0.05) compared with control group. Interestingly, Rattin inhibited cytotoxicity induced by Aβ31-35in a dose-dependent manner. Compared with Aβ31-35alone group, the percentage of cell viability in low concentration (1μM) Rattin plus Aβ31-35group (n=6) were not significantly changed (45.8±2.77%). However, the percentage of cell viability significantly increased in higher concentrations of Rattin plus AP31.35groups, in which the cell viability percentages increased to62.23±3.0%and96.5±4.6%in10μM and100μM Rattin groups, respectively, significantly larger than the Aβ31-35alone group (P<0.05). The values in Genistein plus Rattin and Aβ31-35group (n=6) was44.07±3.65%, similar to that in Aβ31-35alone group.(2) Rattin did not affect Aβ31-35-induced increase in MAPK and IP3K mRNA, but effectively blocked Aβ31-35-induced down-regulation of STAT3mRNA in cultured primary rat hippocampal neurons. To clarify the probable molecular mechanism underlying the neuroprotective roles of Rattin against Aβ in spatial cognition and synaptic plasticity, the expression levels of MAPK, IP3K, and STAT3mRNA in the hippocampus of rats were measured by using real-time PCR technique. As shown in Fig.3, the expression of STAT3mRNA in20nmol Aβ31-35group (n=6) was significantly down-regulated, from1.0090±0.07034in control group (n=6) decreased to0.7226±0.05393(P<0.01). On the contrary, p38MAPK and IP3K mRNA in10nmol Aβ31-35group significantly increased to1.2890±0.04055and1.3223±0.0554from0.9657±0.05092and0.9647±0.07828in the control group (P<0.01). Interestingly, compared with Aβ31-35alone group, the relative mRNA levels for STAT3in the co-application of100μM Rattin and20μM Aβ31-35group increased to1.0188±0.11248(P<0.01), while the expression of p38MAPK and IP3K mRNA were not changed, and the values were1.3647±0.0783and1.2752±0.03528, respectively. The results indicate that the activation of STAT3mRNA expression may be involved in the neuroprotective mechanism of Rattin.(3) Rattin effectively blocked Aβ31-35-induced down-regulation of STAT3mRNA in cultured primary rat hippocampal neurons. To clarify the probable molecular mechanism underlying the neuroprotective roles of Rattin against Aβ31-35in spatial cognition and synaptic plasticity, the expression levels of p-STAT3in the hippocampus of rats were measured by using BD Cytometric Bead Array (CBA) Phospho Stat3(Y705) Flex Set kit and BD CBA Cell Signaling Master Buffer Kit (BD Biosciences) with a FACSCalibur flow cytometer (BD Biosciences). As shown in Fig.4, the expression of p-STAT3in20nmol Aβ31-35group (n=6) was significantly down-regulated, from43.2467±3.3689pg/ml in control group (n=6) decreased to17.5533±2.8854pg/ml (P<0.01). On the contrary, compared with Aβ31-35alone group, the levels for p-STAT3in the co-application of100μM Rattin and20μM Aβ31-35group increased to29.8133±2.3655pg/ml (P<0.01). The results indicate that the activation of p-STAT3expression may be involved in the neuroprotective mechanism of Rattin.(4) Pretreatment with Rattin significantly protected against Aβ31-35-induced elevation of [Ca2+]i, which could be abolished by JAK inhibitor. The mechanism of Aβ-induced neurotoxicity involves in the perturbation of Ca2+homeostasis. Firstly, we investigated the change of [Ca2+]; level in rat primary cultured hippocampal neuron by applying20μM Aβ31-35using laser scanning confocal fluorescent imaging technique. As shown in Fig.4A, the relative fluorescent intensity at resting condition in the control group (n=20) was very stable, nearly being a straight horizontal line. After application of Aβ31.35(n=20), the fluorescent intensity in most of neurons gradually and persistently increased during all of recording time. Fig.4B showed the relative fluorescent intensity values of [Ca2+]i in different experimental groups at20min after application of Aβ31-35. Obviously, Aβ31-35increased the relative fluorescent intensity of [Ca2+]i, being192.1±2.71%, significantly larger than the value in control group (P<0.01). This result indicates that Aβ31-35can increase [Ca2+]i which might be responsible for the neurotoxicity of Aβ seen in cultured hippocampal neurons. Further, we investigated the effects of pretreatment with Rattin on AP31-35induced [Ca2+]i elevation. As shown in the Fig.5, Aβ31-35-induced elevation of [Ca2+]i level was mostly reversed by Rattin (100μM) and the relative fluorescent intensity decreased to120.15±3.6%(n=20, P<0.01). These results indicated that Rattin can protect against Aβ31-35-induced intracellular calcium overloading. To investigate the molecular mechanism of the protection of Rattin against Aβ31-35, we also observed the effects of Rattin on Aβ31-35induced [Ca2+]i elevation in the presence of100μM AG490, a JAK inhibitor. The result showed that pretreatment with AG490for30min, essentially blocked the protective effect of Rattin against Aβ31-35induced [Ca2+]i elevation. Compared with co-application of Rattin and Aβ31-35, the relative fluorescent intensity in co-application of AG490(100μM), Rattin (10μM) plus Aβ31-35group increased to184.07±2.98%(n=20, P<0.01). These results indicated that the protective roles of Rattin against Aβ31-35induced [Ca2+]i elevation are closely associated with the activation of JAK.Conclusion:Rattin has neuroprotection against Aβ31-35-induced neurotoxicity in a dose-dependent manner, and the underlying protective mechanism of Rattin might be mediated via JAK/STAT3of protein tyrosine kinase signal pathways. Meanwhile, the results also indicate that the mechanism of Rattin is probably associated with maintaining intracellular calcium homeostasis. Therefore, application of Rattin or activation of its signaling pathways in the brain might be beneficial to the prevention and treatment of AP-related cognitive deficits. |
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