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Keywords:

  • humanin;
  • Morris water maze;
  • long-term potentiation;
  • STAT3;
  • caspases-3

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES

Rattin, a specific derivative of humanin in rats, shares the ability with HN to protect neurons against amyloid β (Aβ) peptide-induced cellular toxicity. However, it is still unclear whether Rattin can protect against Aβ-induced deficits in cognition and synaptic plasticity in rats. In the present study, we observed the effects of Rattin and Aβ31–35 on the spatial reference memory and in vivo hippocampal Long-term potentiation of rats by using Morris water maze test and hippocampal field potential recording. Furthermore, the probable molecular mechanism underlying the neuroprotective roles of Rattin was investigated. We showed that intra-hippocampal injection of Rattin effectively prevented the Aβ31–35-induced spatial memory deficits and hippocampal LTP suppression in rats; the Aβ31–35-induced activation of Caspase-3 and inhibition of STAT3 in the hippocampus were also prevented by Rattin treatment. These findings indicate that Rattin treatment can protect spatial memory and synaptic plasticity of rats against Aβ31–35-induced impairments, and the underlying protective mechanism of Rattin may be involved in STAT3 and Caspases-3 pathways. Therefore, application of Rattin or activation of its signaling pathways in the brain might be beneficial to the prevention of Aβ-related cognitive deficits. © 2013 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES

Alzheimer's disease is the most prevalent neurological disease with cognitive dysfunction. The loss of neurons in the association cortex and limbic system is the major pathological hallmark of AD, which results in impairment of cognitive ability in clinic and leads to dementia finally (Albert, 2011; Dadic-Hero et al., 2011; Bateman et al., 2012). It is believed that increased levels of toxic amyloid β (Aβ) are correlated to the neuronal dysfunction and cell death. Amyloid imaging studies support a model in which amyloid deposition is an early event on the path to dementia, beginning insidiously in cognitively normal individuals, and accompanied by subtle cognitive decline and functional and structural brain changes suggestive of incipient AD (Rabinovici and Jagust, 2009). Yet there are other reports showing that Aβ may be the products rather than the cause of neurodegeneration in AD (Fjell and Walhovd, 2012). A modification to the amyloid cascade hypothesis is proposed, which may better explain the pathogenesis of AD, especially of late-onset cases of the disease (Armstrong, 2011). Even so, the neurotoxicity of Aβ including different Aβ fragments such as Aβ1–42, Aβ1–40, and Aβ25–35 have been widely reported (Manzoni et al., 2011; He et al., 2012; Izuo et al., 2012; Luo et al., 2012). Aβ31–35, a shorter synthetic Aβ fragment, was also identified to be neurotoxic in cell culture, animal behavior and electrophysiological recording in vitro and in vivo (Yan et al., 1999; Kanski et al., 2002b; Misiti et al., 2005; Li et al., 2009, 2011; Han et al., 2012). Consequently, it is critical to clear the aggregated Aβ or block the Aβ toxicity in the brain for the prevention and clinical treatment of AD.

Humanin is a newly discovered 24-amino acid peptide, and the cDNA of HN was isolated from an AD patient's occipital lobe of brain (Hashimoto et al., 2001b). A series of studies indicated that HN could decrease neuronal cell death induced by enforced expression of familial AD-related genes (Hashimoto et al., 2001a,b; Kariya et al., 2002). Meanwhile, HN can protect neurons from being killed by toxic Aβ in vitro (Onoue et al., 2002). In addition, it is reported that the neuronal dysfunction-associated dementia of mice caused by antagonists of muscarinic receptor and toxic Aβ was improved by HN (Matsuoka, 2009). Therefore, HN and its derivatives have been thought to have potential therapeutic application in AD. Further, it was found that HN also existed in the brain of rats. Rattin, a HN homologue identified and cloned in rats, encodes a peptide of 38 residues (14 residues longer than HN), with 73% identity in the conserved region to HN. The full-length Rattin peptide was equally effective as HN in protecting rat- and mouse-cultured cortical neurons against Aβ-induced toxicity. Moreover, Rattin was much more effective than HN against excitotoxic neuronal death induced by a toxic pulse with NMDA (Caricasole et al., 2002). Importantly, as a specific derivative of HN in rats, Rattin facilitated the studies of HN-like peptides in rat model and avoided the interspecific differences that perhaps happened in the previous experiments by directly using HN in rats (Zou et al., 2003; Yang et al., 2008; Guo et al., 2010). Therefore, by using Morris water maze (MWM) test, in vivo electrophysiological techniques, real-time PCR and flow cytometry techniques, the present study investigated for the first time the neuroprotective effects and possible signaling mechanism of Rattin against the Aβ31–35-induced impairments in spatial cognition and hippocampal synaptic plasticity of rats.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES

Animal and Surgical Procedure

Adult male Sprague–Dawley (SD) rats (200–300 g), supplied by the Research Animal Center of Shanxi Medical University, were divided randomly into four groups: saline + vehicle, saline + Aβ31–35; Rattin + vehicle and Rattin + Aβ31–35. Aβ31–35 was purchased from Sigma (St. Louis, MO) and Rattin was provided by Prof. Tomohiro Chiba from KEIO University in Japan. All drugs dissolved in normal saline, and Aβ31–35 was “aged” before surgery by incubation at 37°C for 4 days (Maurice et al., 1996; Yenkoyan et al., 2009; Li et al., 2010). Three days after adaptation, rats were anesthetized with Chloral hydrate (0.3 g kg−1 i.p.) and placed in a stereotaxic apparatus (Narishige, Japan) for bilateral intra-hippocampal injection. Saline (0.9% NaCl, 2 µl) or Rattin (2 nmol µl−1, 2 µl) was slowly administered to the CA1 region in 5 min using a Hamilton microsyringe, with a retaining for another 5 min to make sure the drug fully dispersed. Then Aβ31–35 (10 nmol µl−1, 2 μl) or vehicle (0.9% NaCl, 2 µl) was given 15 min after the first injection in the same way. Two weeks later, Morris water maze (MWM) test and in vivo hippocampal LTP recording were performed.

Morris Water Maze Test

The classical MWM test was performed as described by Terry AV Jr (Morris, 1984; Prediger et al., 2007; Terry, 2009). A circular pool (diameter 150 cm, height 50 cm, painted black) was filled with 30 cm tap water (maintained at 23°C ± 2°C), in which a circle platform in black was hidden 1 cm beneath the water level. In hidden platform tests, the rats were allowed to swim in the water to search for the under-water platform. The tests were performed four times per day for consecutive 5 days. In every trial, the rats were placed into the water facing the pool wall at one of the four equal quadrants (Zone 1, 2, 3, and 4) designated by computer software. The order for rats to entry into individual quadrant is randomized by random number table so that all four quadrants are used once every day. In probe trials on the sixth day, the platform was removed and the rats were permitted to swim in the water for 120 s. A software system (Ethovision 3.0, Noldus Information Technology, Wageningen, Netherlands) was used to analyze the escape latency and distance of rats in hidden platform tests. The percentages of time and distance for rats to swim in target quadrant in the probe trials were also calculated.

In Vivo Hippocampal LTP Recording

A parallel bound stimulating/recording electrode was inserted into the hippocampal Schaffer-collateral/CA1 region (4.2 mm posterior to bregma and 3.8 mm lateral to the midline for the tip of recording electrode). Test stimuli were delivered to the Schaffer-collateral/commissural pathway every 30 s and a maximal field excitatory postsynaptic potential (fEPSP) in the CA1 region was evoked by increasing the intensity of single stimulus gradually. For further observing the LTP of fEPSPs, the amplitude of baseline fEPSPs was chosen as 50% of the maximal fEPSP amplitude by adjusting the pulse intensity. The baseline fEPSPs were recorded for at least 30 min to ensure steady synaptic transmission. LTP was induced by using a high frequency stimulus (HFS) protocol with three trains of 20 pulses at 200 Hz at an interval of 30 s and a stimulus intensity that evoked a fEPSP of 80% of the maximum response. After HFS, fEPSPs were monitored for a further 1 h to observe the induction and maintenance of LTP. An electronic stimulator (SEN-3301, Japan) and a coupled isolator (ss-102J, Japan) were used to give pulse stimulation. The signals from the recording electrode were filtered at 1 kHz, amplified and displayed by a multichannel biological signal acquisition/processing system (Chengdu Instruments, China).

Real-Time PCR

After finishing MWM test and LTP recording, the rats were rapidly decapitated and the bilateral hippocampuses were removed. Total RNA from the hippocampus was isolated with the RNA iso Plus (RAKARA, China) and the concentration of RNA was determined by NanoDrop 2000 Spectrophotometer (THERMO, USA). Nearly 2 μg of total RNA was reversely transcribed with the RevertAid First Strand cDNA Synthesis Kit (THERMO, USA). Quantitative real-time PCR amplification was performed using the MaximaTM SYBR Green qPCR Master Mix (THERMO, USA). PCR reactions and data analysis were performed by Mx3005P QPCR Systems (STRATAGENE, USA). The relative quantity of mRNA was calculated from a GAPDH standard curve. The sequences of the primers were as follows: GAPDH, sense primer 5′-GGCACAGTCAAGGCTGAGAAT-3′, antisense primer 5′-ATGGTGGTGAAGACGCCAGTA-3′; STAT3, sense primer 5′-AGAGCCAGGAGCACCCTGAA-3′, antisense primer 5′-GGTCAATGGTATTGCTGCAGGTC-3′; Caspase-3, sense primer 5′-GAGACAGACAGTGGAACTGACGATG-3′, antisense primer 5′-GGCGCAAAGTGACTGGATGA-3′.

Primary Cell Culture and Flow Cytometry

Primary hippocampal cultures were prepared from 24 h postnatal SD rats. Briefly, pups were anesthetized with ether, and sterilized in 75% ethanol. Rat brains were quickly removed into ice-cold dissection solution. The hippocampus was dissected and cut into small pieces (each cube <1 mm3). The tissue pieces were incubated with 0.125% trypsin at 37°C for 15–20 min, and then complete culture medium was added to stop enzymatic reaction. Single-cell suspensions were obtained by mechanical dissociation using a Pasteur pipette with a fire-polished tip in complete culture medium. After filtration (200 meshes) and centrifugation (5 min, 1,000 rpm), the cells were plated on poly-d-lysine coated culture dishes at a density of 5 × 105 cells ml−1. Cultures were maintained in 5% CO2 at 37°C in complete culture medium for 16 h. The culture media were then changed to serum-free B27/neurobasal medium. Afterward, half of the culture medium was replaced with fresh serum-free B27/neurobasal medium every 3 days. Mature cells were exposed to Aβ31–35 (20 μM) for 24 h. In coapplication group, Rattin (10 μM) were added to medium before Aβ31–35 treatment. And then phospho Stat3 of neurons was measured 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). Data analysis was performed using FCAP Array Software.

Statistics

All values were expressed as means ± standard errors (SEM). In MWM, the escape latency was analyzed using three-way repeated measures analysis of variance (ANOVA) and other data were analyzed using two-way ANOVA. The data of LTP, real time-PCR and Flow cytometry were analyzed by a two-way ANOVA, all statistical analysis were performed by using the statistical software package SPSS13.0. The statistical significance level was defined as P < 0.05.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES

Rattin Treatment Prevented Aβ31–35-Induced Impairments of Spatial Learning and Memory of Rats

In MWM test, the learning ability of rats to acquire spatial information was first assessed by five consecutive days of hidden platform test. The time (escape latency) to find the hidden platform was decreased following the 5-day training sessions (F(1,39) = 25.901; P < 0.01). There was a significant main effect of intra-hippocampal injection (Aβ31–35 or vehicle) (F(1,39) = 20.874; P < 0.001) and drug treatment (saline vs. Rattin) (F(1,39) = 11.660; P < 0.01) on the escape latency and a significant interaction between Aβ31–35 injection and drug treatment (F(1,39) = 9.186; P < 0.01).The escape latency was significantly different between rats that received intra-hippocampal injection of vehicle and Aβ31–35 (P < 0.05), suggesting that animals that received intra-hippocampal injection of Aβ31–35 learned more slowly to find the hidden platform than animals that received vehicle (Fig. 1A). Rattin treatments had no significant effect on the escape latency in vehicle-injected animals (P > 0.05), but significantly decreased the escape latency of Aβ31–35-injected animals. Thus, Rattin treatments reversed Aβ31–35-induced impairment of spatial learning.

image

Figure 1. Rattin treatments reversed Aβ31–35-induced impairment in spatial learning and memory. (A) Plots showing the average escape latencies of rats in searching for the hidden platform over five consecutive training days. The escape latency was significantly decreased in Aβ31–35-treated rats at the 2, 3, 4, and 5 training sessions (P < 0.001), which was reversed by Rattin treatments (P < 0.001) (n = 10 rats per group). (B) During probe trial, Aβ31–35 decreased the time in the target quadrant (P < 0.001), which was reversed by Rattin treatments (P < 0.001). ***P < 0.001. (C) All groups had no significant difference in the visible platform test (P > 0.05). (D) All groups had no significant difference in the swimming speed (P > 0.05). (E) Representative swimming tracings of all the four group animals in 4th training day of hidden platform test. (F) Representative swimming tracings of all the four group animals during the probe trial. The large circle represents the water maze pool and the small circle represents the platform. Error bars indicate SEM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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To further assess spatial memory ability of rats in Morris water maze, probe trials without platform were performed on day 6. Two-way ANOVA showed that Aβ31–35 and Rattin treatments had significant main effects on the time in the targeted quadrant in which the platform was previously located (Aβ31–35: F(1,39) = 68.149, P < 0.001; Rattin treatment: F(1,39) = 21.697, P < 0.001; Aβ31–35 by Rattin interaction: F(1,39) = 25.901, P < 0.001; Fig. 1B). Tukey's post hoc test showed that Aβ31–35 injection produced a significant decrease in the time in the targeted quadrant (P < 0.001), and this decrease was reversed by Rattin treatment (P < 0.001). The results indicated that pretreatment with Rattin protected against Aβ31–35 induced deficits in spatial learning and memory.

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 with visible platform after probe trials. As shown in Figures 1C,D, the average time for rats to reach the visible platform was ∼14 s, and the average swimming speed was 20 cm s−1, without any significant difference (P > 0.05) between all groups. These results indicated that the vision and the motor ability of rats were not affected by Aβ31–35 or Rattin.

Rattin Treatment Protected Against Aβ31–35-Induced Impairment of Hippocampal LTP

To clarify whether the presynaptic mechanism was involved in the effects of Aβ31–35 and Rattin on synaptic plasticity, PPF in the hippocampal CA1 region was examined in all groups before HFS. After paired pulses were applied to the Schaffer collaterals, the PPF always appeared, with the second fEPSP larger than the first one (Fig. 2A). Two-way ANOVA showed that Aβ31–35 and Rattin treatments had no significant main effects on the PPF (Fig. 2A), suggesting that Rattin and Aβ31–35 did not affect the presynaptic neurotransmitter release in the hippocampal CA1 region of rats.

image

Figure 2. Rattin administration reversed Aβ31–35-induced impairment of hippocampal LTP. (A) Aβ31–35 and Rattin treatments did not affect the PPF (fEPSP2/fEPSP1) in the hippocampal CA1 region (n = 6, P > 0.05). Inset, representative paired fEPSPs. (B) Typical fEPSP traces recorded from the four groups of rats. Scale bars, 1 mV and 20 ms. (C) All groups had no significant difference in the magnitude of LTP induction (P > 0.05). (D) The normalized fEPSP amplitude of the last 5 min of recordings was used to calculate the magnitude of LTP. Aβ31–35 produced a significant decrease in the magnitude of LTP in saline-treated rats (n = 6, P < 0.001), but this decrease was reversed by Rattin treatments (n = 6, P < 0.001). Error bars indicate SEM. ***P < 0.001.

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We next examined the effects of Aβ31–35 and Rattin on in vivo hippocampal LTP. The change in fEPSP amplitude was used to represent the synaptic efficacy in the CA1 region. After stable baseline recordings of fEPSPs for 30 min, HFS was applied to induce LTP (Fig. 2B). Two-way ANOVA showed that Aβ31–35 and Rattin treatments had no significant effects on the induction of LTP (Fig. 2C). However, Two-way ANOVA showed that Aβ31–35 and Rattin treatments had significant main effects on the fEPSP amplitude of the last 5 min (Aβ31–35: F(1,23) = 164.364, P < 0.001; Rattin treatment: F(1,23) = 17.931, P < 0.001; Aβ31–35 by Rattin interaction: F(1,23) = 20.056, P < 0.001; Fig. 2D). Tukey's post hoc test showed that Aβ31–35 injection induced a significant decrease of the magnitude of LTP in saline-treated rats (P < 0.001), this decrease was reversed by Rattin treatment (P < 0.001).

Rattin Effectively Prevented Aβ31–35-Induced Change in the Levels of STAT3 and Caspase-3 in the Hippocampus of Rats

To clarify the probable molecular mechanism underlying the neuroprotective roles of Rattin against Aβ in spatial cognition and synaptic plasticity, the expression levels of STAT3 and Caspase-3 mRNA in the hippocampus of rats were measured after LTP recording by using real-time PCR technique. As shown in Figure 3A, Aβ31–35 and Rattin treatments had significant main effects on the expression of STAT3 mRNA (Aβ31–35: F(1,23) = 5.499, P < 0.05; Rattin treatment: F(1,23) = 8.783, P < 0.01; Aβ31–35 by Rattin interaction: F(1,23) = 4.404, P < 0.05). Tukey's post hoc test showed that Aβ31–35 injection produced a significant decrease of the expression of STAT3 mRNA in saline-treated rats (P < 0.01), and this decrease was reversed by Rattin treatments (P < 0.01). There was also a significant main effect of intra-hippocampal injection (Aβ31–35 or vehicle) (F(1,23) = 5.927; P < 0.05) and drug treatment (saline vs. Rattin) (F(1,23) = 6.014; P < 0.05) on the expression of Caspase-3 mRNA and a significant interaction between Aβ31–35 injection and drug treatment (F(1,23) = 11.139; P < 0.01, Fig. 3B). Tukey's post hoc test showed that Aβ31–35 injection produced a significant upregulation of the expression of Caspase-3 mRNA in saline-treated rats (P < 0.001), and this up-regulation was reversed by Rattin treatments (P < 0.001). These results indicate that the activation of STAT3 mRNA expression and the suppression of Caspase-3 may be involved in the neuroprotective mechanism of Rattin.

image

Figure 3. Rattin effectively prevented Aβ31–35-induced decrease of STAT3 mRNA and increase of Caspase-3 mRNA in the hippocampus of Rats. (A) Aβ31–35 produced a significant decrease in the STAT3 mRNA levels in saline-treated rats (n = 6, P < 0.01), but this decrease was reversed by Rattin treatments (n = 6, P < 0.01). (B) Aβ31–35 produced a significant increase in the Caspase-3 mRNA levels in saline-treated rats (n = 6, P < 0.001), but this increase was reversed by Rattin treatments (n = 6, P < 0.001). Error bars indicate SEM. **P < 0.01, ***P < 0.001.

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We further detected the expression levels of p-STAT3 protein in primary cultured hippocampal neurons by using flow cytometry. Two-way ANOVA revealed that Aβ31–35 and Rattin treatments had significant main effects on the expression levels of p-STAT3 (Aβ31–35: F(1,23) = 97.372, P < 0.001; Rattin treatment: F(1,23) = 6.733, P < 0.05; Aβ31–35 by Rattin interaction: F(1,23) = 16.344, P < 0.001; Fig. 4D). Tukey's post hoc test showed that Aβ31–35 injection produced a significant decrease in p-STAT3 protein in saline-treated rats (P < 0.001), and this decrease could be blocked by Rattin treatments (P < 0.001). These results indicate that JAK-STAT3 signal pathway may be involved in the neuroprotective mechanism of Rattin.

image

Figure 4. Rattin reversed Aβ31–35-induced change in the levels of p-STAT3 protein in the primary cultured hippocampus neuron of rats. (A) Original figure of sample acquisition on the flow cytometer. Bead population of sample is located in gate R1. (B) FL2 histogram of capture p-STAT3 beads. (C) Standard curve of BD CBA Phospho Stat3 (Y705) Flex Set, R2 = 99.94%. (D) Aβ31–35 treatment produced a significant decrease in p-STAT3 protein in saline-treated neurons (n = 6, P < 0.001), but this decrease was reversed by Rattin treatments (n = 6, P < 0.01). Error bars indicate SEM. ***P < 0.001. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES

As the primary constituent of senile plaques in AD, the neurotoxicity of Aβ has been widely studied in in vivo and in vitro experiments (Mattson, 2004; Alkam et al., 2007; Ferreira et al., 2007). For example, soluble Aβ oligomers have been demonstrated to be neurotoxic in synaptic plasticity and memory formation (Selkoe, 2008); Aβ1–40 induced deficits in learning and memory have been reported in various animal behavioral tests including Y-maze (Alkam et al., 2007), T-maze (Kunesova et al., 2008), and MWM (Wang et al., 2010); the sequence 25–35 of Aβ is further recognized as the active center of the full length of Aβ molecule for its similarity in neurotoxicity to natural Aβ (Chen et al., 2000; Freir et al., 2003; Pan et al., 2010). Interestingly, Aβ31–35, a shorter fragment than Aβ25–35, could also induce apoptosis in cultured cortical neurons (Yan et al., 1999) and form new ion channels in the membrane patches excised from hippocampal neurons (Qi and Qiao, 2001). Further, some investigators, by using amino acids-substitution, justified that the neurotoxicity of Aβ1–42 was associated with three critical amino acids, i.e., isoleucene-31, glycine-33, and methionine-35 (Kanski et al., 2002a; Varadarajan et al., 1999), which are all included in the Aβ31–35 we used. In the present study, we reported for the first time that bilateral intra-hippocampal injections of synthetic Aβ31–35 resulted in marked deficits in spatial learning and memory of rats, as well as a depression of in vivo hippocampal LTP. These results support the hypothesis we previously suggested that the sequence 31–35, as a critical active center of Aβ, may play an important neurotoxic role in the endogenous Aβ (Qi et al., 2004).

It is reported that HN could suppress the neurotoxicity induced by the AD relevant insults, such as Aβ and mutants of APP, PS1, and PS2 in primary cortical neurons and PC12 cell (Hashimoto et al., 2001a,b; Terashita et al., 2003; Matsuoka, 2009). Additionally, HN could also antagonize some non-AD-related such as hypoxia, tetramethylpyrazine, prion protein (PrP) 118–135 fragment, and dentatorubral-pallidoluysian atrophy (DRPLA) protein induced neurotoxicity in cortical neurons, PC12 cells and retinal ganglion cells (Zou et al., 2003; Sponne et al., 2004; Kariya et al., 2005; Yang et al., 2008). However, HN could not antagonize etoposide, poly-Q (Q79), and glutamate induced neurotoxicity (Hashimoto et al., 2001a; Onoue et al., 2002; Jung and Van Nostrand, 2003; Cui et al., 2006). Notably, Rattin, a rat homolog of HN, elicits neuroprotective action against glutamate toxicity as well as the HN-like anti-AD activity (Caricasole et al., 2002). Rattin is composed of the N-terminal HN-homologous domain and an additional 14 amino acid domain in the C-terminus. Earlier reports found that the full-length Rattin peptide and its 1–25 fragments were equally effective as HN in protecting rat- and mouse-cultured cortical neurons against Aβ-induced toxicity. However, Rattin and its short C-terminal fragment were much more effective than HN against excitotoxic neuronal death induced by toxic NMDA (Caricasole et al., 2002). These results suggest that Rattin has a broader spectrum of neuroprotective activity than HN. The C-ternimal region of Rattin is closely related to the protection against excitatory toxicity, while the N-terminal HN-homologous domain is relevant to the biological activity of Rattin against Aβ31–35. In the present study, the neuropretective effect of Rattin was investigated for the first time in the cognitive behavior experiment. We found that the escape latencies and distances of rats in the Rattin plus Aβ group were significantly decreased in searching for the hidden platform of MWM, while the swimming time and distance elapsed in the target quadrant after removing the platform significantly increased in the probe trials. These results indicate that Rattin can effectively prevent the Aβ31–35 application-induced impairment of spatial learning and memory in rats. Also, we noticed that the control and Aβ31–35-injected animals did not show significant difference on the last training day. A similar phenomenon was also reported by other group (Yau et al., 2002; Limback-Stokin et al., 2004). One possible explanation is that the neurons in different hippocampal subregions were compensating the cognitive function. It is reported that the dorsal hippocampus has a preferential role in certain forms of learning and memory, notably spatial learning (Moser et al., 1993; Hock and Bunsey, 1998; Bannerman et al., 2002), while ventral hippocampus may associated with anxiety-related behaviors (Talalaenko et al., 1992; Bannerman et al., 2003; McHugh et al., 2011); rats with excitotoxic dorsal hippocampal lesions can eventually acquire a spatial reference memory in the Morris water maze if given sufficient training. These reports suggest that the ventral hippocampus may contribute to the spatial learning after the dorsal hippocampal lesion (de Hoz et al., 2003).

Learning and memory are thought to be encoded by modification of synaptic strength (Bliss and Collingridge, 1993). Hippocampal LTP, a prominent form of synaptic plasticity, has been widely considered one of the major cellular mechanisms underlying learning and memory (Bliss and Collingridge, 1993; Cooke and Bliss, 2006). The persistent increase in synaptic efficacy induced by HFS results from the modification of pre-existing proteins, and can last for at least 1 h (Barria et al., 1997). Previous studies showed that Aβ could inhibit LTP in the hippocampus both in vitro (Wang et al., 2002) and in vivo (Freir et al., 2001; Walsh et al., 2002), while Aβ25–35-induced LTP inhibition in mouse hippocampal slices could be reversed by [Gly14]-Humanin (HNG), a derivative of HN (Zhang et al., 2009). In the present study, the protective effect of Rattin on in vivo hippocampal LTP strongly supports the behavioral improvement of rats in the MWM test, and partly explains why HN and its derivatives could reverse the Aβ-induced cognitive impairments. Of course, we also noticed that Rattin nearly completely suppressed Aβ toxicity in Morris water maze (Fig. 1), but only partially suppressed Aβ toxicity in hippocampal LTP (Fig. 2). The inconsistence between behavioral test and electrophysiological recording suggests that the alteration of hippocampal LTP could not always proportionally reflect the spatial learning and memory of rats, and both of them could be inconsistent in some conditions (Burwell et al., 2004; Quan et al., 2011). In addition, the hippocampal LTP recording in the present study was performed after 5 days of Morris water maze test. At that time, the protective effects of Rattin on LTP might be gradually decaying because of its limited half-life.

The molecular mechanisms by which HN and its derivatives ameliorate Aβ-induced cognitive impairments have not been well understood yet. There is no report showing that Rattin can directly bind with Aβ peptide. However, the neuroprotective function of HN could be completely inhibited by Genistein, a tyrosine kinase inhibitor (Hashimoto et al., 2001a; Tajima et al., 2005). Our recent research also showed Genistein nearly completely abolished the protective action of HNG in Aβ25–35-induced depression of LTP in hippocampal CA1 region (Guo et al., 2010). These findings suggest that certain tyrosine kinases are involved in the signal transduction mechanism of HN and its derivatives. Indeed, as a kind of nonreceptor tyrosine kinase, the activation of JAK2 and the phosphorylation of STAT3 has been linked to the neuroprotective function of HN and its derivatives (Hashimoto et al., 2001a; Hashimoto et al., 2005; Yamada et al., 2008); an age-dependent decrease in p-STAT3 level was reported in both AD model mice and AD patients, whereas passive immunization with anti-Aβ antibody conversely restored hippocampal p-STAT3 levels in Tg2576 mice (Chiba et al., 2009). Meanwhile, the loss of neuron in AD has been closely associated with the over activation of Caspases (Selznick et al., 2000; D'Amelio et al., 2011; Zhang et al., 2011). In the present study, we observed the gene expression of STAT3 and Caspase-3 using real-time PCR and measured the protein expression level of STAT3 with flow cytometry. Our results indicated that Aβ31–35 decreased the levels of STAT3 mRNA and p-STAT3 protein, increased the level of Caspase-3 mRNA in the hippocampus of rats, while Rattin pretreatment effectively prevented the decrease of STAT3 and the increase of Caspase-3. The evidence suggests that STAT3/Caspase-3 could be common targets of Aβ and Rattin; the activation of STAT3 and the suppression of Caspase-3 in the brain may be involved in the neuroprotective mechanism of Rattin in improving spatial memory and synaptic plasticity of rats. In addition, it is reported that HN is secreted out of cells, dimerizes, and specifically binds to the cell surface of F11 neuronal cells (Terashita et al., 2003; Yamagishi et al., 2003). Although the exact membrane receptors of Rattin are still unknown, a G protein-coupled formylpeptide receptor-like-1 (FPRL-1) molecule, originally identified as a receptor for Aβ1–42 (Le et al., 2001) was thought to be a functional receptor of HN (Ying et al., 2004). Probably, HN or Rattin binds to G protein-coupled FPRL-1 and blocks Aβ-induced neurotoxicity by competing with Aβ molecules for binding FPRL-1 (Ying et al., 2004). This putative HN receptor exerts cytoprotection by regulating certain intracellular signaling, such as tyrosine kinases and STAT3.

In conclusion, the present study provided for the first time behavioral and electrophysiological evidence that intra-hippocampal injection of Aβ31–35 could impair the spatial memory and hippocampal LTP of rats, and Rattin pretreatment could prevent the Aβ31–35-induced decline in cognitive behavior and synaptic plasticity. The neuroprotective function of Rattin against Aβ31–35 may be, at least partly, mediated by up-regulating STAT3 signaling and down-regulating Caspase-3 pathways. Therefore, application of exogenous Rattin or HN derivatives would probably contribute to the prevention of Aβ-related cognitive deficits seen in AD.

Acknowledgments

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES

The authors are very grateful to Prof. Tomohiro Chiba for offering Rattin peptide.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES
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