Carnosine Protects Against Aβ42-induced Neurotoxicity In Differentiated Rat PC12 Cells

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by global cognitive decline involving memory, orientation, judgment, and reasoning, and occurs with pathological hallmarks including widespread neuronal degeneration and neuritic plaques containing amyloid-β peptide (Aβ). Evidence indicates that Aβ42 is essential for amyloid deposition in the parenchyma and in vessels. In addition to Aβ, enhancement of excitotoxicity by glutamate receptors especially NMDA receptors has also been implicated in the pathogenesis of AD. Among several hypotheses, the "amyloid hypothesis" has firmly taken root, but it currently lacks detail, and certain observations do not fit easily with the simplest version of the hypothesis. Recently, Snyder and colleagues found that Aβ reduces glutamatergic transmission and NMDA receptor-dependent LTP through intemalization of NMDA receptors, which suggests a potential therapeutic direction in AD. Now that therapies based on this idea serve as a basis for the treatment of AD, it is important to clarify these mechanisms.As an important neurotransmitter and neuromodulator, histamine has many actions in the central nervous system, affecting emotion, locomotor activity, stress behavior, learning and memory. Histamine also participates in pathological process of brain diseases. In AD, neurofibrillary tangles occur extensively in the tuberomammillary nucleus, where the cell bodies of histaminergic neurons are located, and histamine levels and/or histidine decarboxylase activity decrease in affected areas of the brain. But the exact role of histamine in AD remains unclear. In addition, several evidences show that histamine causes direct or indirect facilitation of the NMDA receptor, which is one of the most important receptors in AD, through its polyamine modulatory site or as a result of H₁ receptor activation. So regulation of NMDA receptor may be one action of histamine in AD.Carnosine (β-alanyl-histidine) is a major constituent of excitable tissues, brain and skeletal muscles, but more than a century after its isolation, the biological functions of this dipeptide remain enigmatic. Meanwhile, as a putative neurotransmitter in the brain, carnosine has a protective effect in many diseases such as AD, because of its abundant functions such as an anti-oxidant, in anti-glycation, anti-aging. In addition, as a reservoir for histidine, which is a precursor of histamine, there is an association between carnosine and histamine. Our previous work, demonstrating the protective effect of carnosine on kindled seizures in rats and neurotoxicity induced by NMDA, has revealed that the carnosine-histidine-histamine pathway may be one of the most important potential mechanisms in its protective effect. So it is of interest to examine whether the carnosine-histidine-histamine pathway is involved in the protective effect of carnosine in the case of neurotoxicity induced by Aβ42.The present study was designed to investigate whether histamine is involved in the protective effect of carnosine on Aβ42-induced impairment in differentiated PC12 cells. Various concentrations histamine receptor antagonists or α-fluoromethylhistidine (α-FMH) were added 10 min before carnosine or histamine; carnosine, histamine or the H₃ receptor antagonists were added 18 h before Aβ42. Cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 24 h after application of Aβ42. The amount of glutamate was quantified by HPLC 24 h after application of Aβ42. Clobenpropit was added 10 min before carnosine. Cell surface expression of NMDA receptors was quantified by cell ELISA assays 18 h after application of carnosine or clobenpropit.We found that Aβ42 caused a concentration-dependent reduction of viability in PC12 cells and pretreatment with histamine ameliorated Aβ42-induced impairment. This amelioration was significantly reversed by the H₂ receptor antagonist zolantidine, but not reversed by the H₁ receptor antagonist diphenhydramine. These results indicated that histamine attenuates Aβ42- induced neurotoxicity through H₂ receptor.Pretreatment with carnosine also ameliorated Aβ42-induced impairment. This amelioration was reversed by the H3 receptor antagonists thioperamide and clobenpropit, but neither the H₁ receptor antagonist diphenhydramine nor the H₂ receptor antagonist zolantidine. Further, α-FMH, an irreversible inhibitor of histidine decarboxylase, also had no effect. These results indicated that the carnosine-histidine-histamine pathway may not be involved in the protection of carnosine on Aβ42- induced neurotoxicity, although histamine alone attenuated Aβ42-induced neurotoxicity through H₂ receptor.Since the H₃ receptor, which is a heteroreceptor, not only provides negative feedback to restrict histamine synthesis and release, but also regulates the release of other transmitters such as glutamate and GABA, and also because of the importance of glutamate in AD, we examine the glutamate release, hoping its regulation could go with the cell survival. We found Aβ42 did not affect the glutamate release. In the presence of Aβ42, carnosine significantly decreased glutamate release, which was further reduced by clobenpropit and thioperamide. Then we examine whether NMDA receptor is involved in the protective effect of carnosine on Aβ42-induced neurotoxicity because of the important role of NMDA receptor in AD. And we found that carnosine increased the cell surface expression of NMDA receptor, which was abolished by clobenpropit. These results implicated that regulation of NMDA receptor trafficking may be involved in the protective effect of carnosine on Aβ42-inducedneurotoxicity.In addition, we also found that the H₃ receptor antagonists clobenpropit and thioperamide significantly ameliorated Aβ42-induced impairment. Both of them in the present of Aβ42 increased glutamate release. And clobenpropit increased the cell surface expression of NMDA receptor. It seems that regulation of both glutamate release and NMDA receptor trafficking are worked in the protective effect of carnosine on Aβ42-induced neurotoxicity.Finally, we found that MK801, a complete antagonist of NMDA receptor, abolished increasing cell surface expression of NMDA receptor afforded by carnosine or clobenpropit. These data indicated that NMDA receptor activity is necessary for surface expression of NMDA receptors under these conditions.In conclusion, the results indicate that the mechanism by which carnosine attenuates Aβ42- induced neurotoxicity is independent of the carnosine-histidine-histamine pathway, but may act through regulation of NMDA receptor trafficking, which could be abolished by the H₃ receptor antagonist. Although the mechanism by which histamine attenuates Aβ42- induced neurotoxicity is through H₂ receptors.