Role Of Melatonin And PARP-1 In The Memory Impairment Of Rats And Its Underlying Mechanisms

Alzheimer's disease is characterized by intracellular neurofibrillary tangles and extracellular senile plaques. Neurofibrillary tangles are composed of aberrantly hyperphosphorylated tau protein, andβ-amyloid (Aβ), a peptide derived from cleavage ofβ-amyloid precursor protein (APP), is the major component of senile plaques. Numerous studies have demonstrated that an imbalanced regulation of specific phosphatases and kinases in neurons represents a critical step for the cytoskeleton hyperphosphorylation. But till now the upstream factors leading to cytoskeleton hyperphosphorylation are not defined. Lots of evidences suggest that melatonin deficit may serve as an upstream effecter in AD-like pathology and melatonin may be a potent drug for the therapy of AD. Recent studies show that the level of melatonin in serum is decreased in aged people, especially in AD patients and the cognitive function of the patients is improved after melatonin supplement. We have found recently that melatonin protects SH-SY5Y neuroblastoma cells from calyculin A-induced neurofilament impairment and neurotoxicity. Growing evidences suggest that melatonin deficit may serve as an upstream effector in AD-like pathology. The aim of the present study is to investigate the in vivo effect of inhibiting melatonin biosynthesis on spatial memory retention of rats and tau phosphorylation, and the possible underlying mechanisms. The results as follows: (1) Effect of haloperidol on serum melatonin level: To confirm the suppression of melatonin synthesis by haloperidol, the serum level of melatonin was measured by HPLC. The melatonin level decreased dramatically compared with vehicle control after administration of haloperidol. We also observed that exogenous supplementation of melatonin did not significantly restore the serum level of melatonin. (2) Effect of inhibited biosynthesis of melatonin on spatial memory of rats: To detect the effect of inhibited biosynthesis of melatonin on spatial memory of rats, Morris water maze test was used. Compared with vehicle-control rats, injection of haloperidol significantly prolonged the latency of rats to find the hidden platform, and these rats took a random searching strategy for the platform in the maze. Supplementation of melatonin with both low and high doses before and during haloperidol injection partially restored the haloperidol-induced impairment in spatial memory. (3) Effect of inhibition melatonin synthesis on tau phosphorylation: To detect the effect of inhibited melatonin synthesis on tau phosphorylation, we carried out western blot by using phosphorylation-dependent mAb tau-1 and PHF-1, as well as nonphosphorylation dependent pAb R134d. We found that the immunoreactivity of tau-1 was remarkably weaker, and of PHF-1 was obviously stronger in haloperidoltreated rats than in vehicle control rats. The immunoreactivity of tau was partially restored to the vehicle control level by supplement of melatonin both with low and high doses. The treatments used in the study did not alter the immunoreactivity of total tau stained by R134d. These results suggested that tau was hyperphosphorylated at Ser199/202 (tau-1 epitope) and Ser396/404 (PHF-1 epitope) when synthesis of melatonin was inhibited, and the supplement of melatonin partially arrested the haloperidol-induced hyperphosphorylation of tau at the above epitopes. (4) Effect of haloperidol and melatonin on the activity of PP2A: To determine the connection of PP2A with haloperidol-induced melatonin deficits and tau hyperphosphorylation, we measured the activity of PP2A by 32P-labeling assay using specific substrates. We found that the infusion of haloperidol significantly inhibited PP2A activity. The supplementation with melatonin at the high dose not only restored but also significantly elevated PP2A activity. An obvious increased activity of PP2A was also observed in low dose melatonin supplemented group, but it was not statistically significant. (5) Effect of inhibition of melatonin biosynthesis on the oxidative stress: Melatonin is a well-known antioxidant. It was shown that treatment of the rats with haloperidol significantly depressed the activity of SOD with a concomitant elevation of MDA. Supplementation of high and low doses of melatonin significantly restored SOD activity and arrested MDA overproduction. Conclusion: In summary, we have found in the present study (i) that inhibition of melatonin biosynthesis by haloperidol induces hyperphosphorylation of tau at both PHF-1 and tau-1 epitopes and compromises spatial memory retention in rats; (ii) that inhibition of melatonin biosynthesis inactivates PP2A, which may play a major role in haloperidol-induced tau hyperphosphorylation; (iii) that administration of haloperidol inhibits SOD and elevates MDA which might be the upstream alterations of decreased PP2A activity, and as well as tau hyperphosphorylation and spatial memory impairment; (iv) that melatonin supplementation can at least partially reverse elevated haloperidol-induced oxidative stress, tau hyperphosphorylation/PP2A inhibition, and as well as behavioral changes. Alzheimer's disease (AD), the most common cause of dementia in the elderly, is characterized by the presence of two pathological hallmarks: extracellular b-amyloid deposits in senile plaques and intracellular neurofibrillary tangles (NFTs). It is reported that the abundance of NFTs correlates well with the clinical degree of dementia and numerous studies have demonstrated that tau hyperphosphorylation plays a crucial role in the formation of NFTs. An imbalanced regulation of specific phosphatases and kinases in neurons represents a critical step for the initiation of tau hyperphosphorylation. The aim of this study is to investigate the effect of inhibiting melatonin biosynthesis on activities of protein kinase A (PKA), glycogen synthase kinase-3 (GSK-3) and tau phosphorylation at PS214 and M4 epitopes using haloperidol, a specific inhibitor of 5-hydroxyindole-O-methyltransferase.By 32P-labeling assays of protein kinase activities and western blot, we found that: Haloperidol injection through the lateral ventricle and intraperitoneal reinforcement significantly stimulated PKA activity with a concurrent hyperphosphorylation of tau at M4 (Thr231/Ser235) and pS214 (Ser214) sites. Prior treatment of the rats using melatonin supplement for one week and reinforcement during the haloperidol administration arrested PKA activity and attenuated tau hyperphosphorylation. GSK-3 activity showed no obvious change after haloperidol injection, however, melatonin supplements and reinforcements during haloperidol infusion inactivated basal activity of GSK-3. We concluded that: Decreased melatonin may be involved in Alzheimer-like tau hyperphosphorylation, and overactivation of PKA may play a crucial role in this process. Decreased level of melatonin and hyperphosphorylation of neurofilament proteins have been reported in Alzheimer's disease (AD). However, the direct evidence linking melatonin and neurofilament phosphorylation is still lacking. Here, we investigated the effect of inhibiting melatonin biosynthesis on phosphorylation of neurofilament proteins and the involvement of cyclin-dependent kinase 5 (cdk-5) in rats. We observed that injection of haloperidol, a specific inhibitor of 5-hydroxyindole-O-methyltransferase, resulted in significantly decreased level of serum melatonin with a concomitantly increased phosphorylation of neurofilament proteins and activation of cdk-5 in rats. Exogenous supplementation of melatonin partially arrested the hyperphosphorylation of neurofilament and the activation of cdk-5. These results suggest that inhibition of melatonin biosynthesis may activate cdk-5 and thus induces Alzheimer-like hyperphosphorylation of neurofilament proteins. Synaptic activity-dependent de novo gene transcription is crucial for long-lasting neuronal plasticity and long-term memory (LTM), while short term memory (STM) involves gene expression-dependent synaptic plasticity in the hippocampus. PolyADP-ribose-polymerase-1 (PARP-1) is a multifunctional enzyme that can modulate gene expression and LTM. The cAMP-PKA pathway is involved in CREB activation, which is recognized as a molecular switch for LTM formation in hippocampus. Little is known whether PARP-1 is involved in the formation of hippocampus-dependent memory. Here, we studied the effect of PARP-1 on hippocampus-dependent memory in rats and the underlying mechanisms. The PARP-1 inhibitor (3-Aminobenzamide) and activator (NGF) were adiministered respectively to rats through lateral ventricle injection. The results were as follows: (1) Inhibition of PARP-1 by injection of 3AB (50 mM, 20μl) impaired the hippocampus-dependent contextual memory but not the cue memory measured by fear conditioning tasks (2) Injection of 3AB at 50 mM and 100 mM inhibited LTM but not STM measured by inhibitory avoidance tasks, and 3AB at 1mM did not affect the memory of the rats. (3) 3AB (50 mM, 20μl) impaired the spatial memory measured by Morris water maze, and the impairment was partially improved by simultaneous administration of NGF. Adminitration of NGF alone stimulated the memory functions. (4) The relative amount of polyADP-ribosylated PARP-1, representing activated PARP-1, in 3AB (50 mM, 20μl) treated hippocampal neurons was significantly decreased, and a significant shift to acid PI was found in samples treated with NGF. (5) Western blotting results showed that polyADP-ribosylation protein decreased when the activity of PARP-1 was inhibited by 3AB. Activition of PARP-1 by NGF increased the level of polyADP-ribosylation proteins. Immunocytochemical results also demonstrated that after treated 3AB, staining with PAR was much weaker in hippocampus and cortex, while staining of 3AB+NGF group was stronger than group treated with 3AB alone. (6) Inhibition of PARP-1 impaired the LTP. When the high-dose 3AB was given (3AB50, 50mM; 3AB100, 100mM), the ampitude and slope of LTP were decreased compared with the controls. No statistical significance between 3AB1 and control group was observed. (7) Western blotting and quantitative analysis of PKAcαshowed that there was no significance between 3AB group and control. The level of PKAcαwas increased after NGF was administered. The activity of PKA was significantly decreased after inhibition of PARP-1 by 3AB, whereas NGF could provent 3AB-induced inhibition of the kinase. Additionally, NGF alone could increase significantly the activity of PKA. The results of immunohistochemistry in hippoampus showed that the staining of PKA was much stronger in region CA3, while weaker in region CA1 compared with control when 3AB was adiministered. When the animals were then treated with NGF, the staining became weaker in CA3 but stronger in CA1 region than that of 3AB group; In animals treated with NGF alone, the staining of PKA was the weakest in CA3 and the strongest in CA1 region. (8) The cAMP level was significantly decreased in the group treated with 3AB while NGF could increase the level of cAMP. cAMP level was significantly increased when NGF was administrated alone. (9) Western blots of homogenized tissue from rat hippocampal neurons were probed with anti-CREB and anti-phospho-CREB (p-CREB) antibodies. The total amount of CREB did not change in each group. The phosphorylated CREB level was decreased in 3AB-treated animals, and NGF supplementation could provent the inhibition of CREB phosphorylation. But when NGF was given alone, the phosphorylation level of CREB did not increase compared with control. (10) The mRNA levels of memory-associated genes, including PKA cαand NF-κB, were measured by RT-PCR and no change was found. Conclusion: these results disclose a novel hierarchical transcriptional network involving PARP-1, PKA, and CREB that leads to concerted nuclear transduction of synaptic signals in neurons, accounting for the critical function of PARP-1 in learning and memory.