| Background and Objective: Alzheimer's disease (AD) is the most common cause of dementia in the elderly population, characterized by progressive cognitive dysfunction. Extracellular amyloid plaques, intraneuronal neurofibrillary tangles, and neuron loss are considered as the major neuropathological hallmarks of the disease. Since no specific and causal therapy for this disease has been developed currently, AD is unfortunately still incurable disease, which severely affects the physical and mental health as well as life quality of the elderly population. Although the pathogenesis of AD still remains unclear, increasing evidence has shown that aggregation, fibrillization, and deposition of amyloid-beta protein (Aβ) as well as formation of senile plaques and its neurotoxicity play a crucial role in the development of AD. Therefore, the important treatment targets and strategies for AD are how to effectively prevent or reduce these factors respectively. Humanin (HN) and its derivative, S14G-Humanin (HNG), is a novel 24 amino acid peptide best known for its ability to markedly suppress neuronal cell death caused by AD-related toxitic insults in vitro, however the exact mechanism that how HNG can improve the cognitive dysfunction still remains to be elucidated. By using in vitro and in vivo experiments, the aim of this study is to investigate the effect and its mechanism of HNG for preventing and treating AD. Methods and Results: (1) By using thioflavine-T fluorometric assay and transmission electron microscopy, we observed whether HNG could block/reduce the Aβ1-42 aggregation and fibrillization in vitro. The effects of HNG on the neurotoxicity of Aβ1-42 were further examined in cultured PC12 cells by MTT method. The data from thioflavine-T fluorometric assay and transmission electron microscopy disclosed that HNG could markedly inhibit fibrillar Aβformation in dose-dependent manner; by using MTT method to assay the cell activity, HNG dose-dependently reduced Aβ1-42 on the cultured PC12 cells. These findings suggest that HNG could prevent Aβ-induced neurotoxicity via inhibiting Aβaggregation and fibrillization. (2) By using a mouse AD model with intracerebroventricular injection (icv) of aggregated Aβ25-35, we sought to determine the effects of HNG on neuroinflammatory responses and apoptosis associated with behavioral deficits induced by Aβ25-35 in vivo. The results from this study indicate that icv injection of aggregated Aβ25-35 induced impairment of learning and memory, markedly elevated numbers of reactive astrocytes, activated microglia, and apoptotic cells, as well as remarkable increased levels of IL-6 and TNFα. Moreover, intraperitoneal HNG treatment ameliorated behavioral deficits, and reduced neuroinflammatory responses and apoptotic cells in the brain. Cumulatively, these findings demonstrate for the first time that HNG may have the potential for attenuating Aβ-induced cognitive deficits by reducing inflammatory responses and apoptosis in vivo. (3) By using young transgenic AD model (APP/PS-1 mice; 3-month-old), we investigated the effects of HNG on behavioral deficits, and Aβexpression and formation of senile plaques. The data from behavioral tests (i.e. Morris water maze, step-through test, and shuttle-box test) revealed that the behavioral deficits in APP/PS-1+HNG group were significantly improved than in APP/PS-1+saline group, suggesting that intraperitoneal HNG treatment may ameliorate behavioral deficits associated with hippocampus, amygdale, and cerebrocortex. Furthermore, neuromorphological investigation disclosed that no significant influence of HNG on diffuse Aβplaques in cerebrocortex and hippocampal region was observed in APP/PS-1 mice, while HNG might significantly reduce the fibrillar Aβformation and plaques in those regions, indicating that HNG may slow the development of AD by inhibiting Aβaggregation and fibrillization as well as reducing Aβ-induced pathological impairments. Conclusions: (1) It has been shown for the first time that HNG may effectively inhibit Aβaggregation and fibrillization and its neurotoxicity, suggesting that HNG probally plays its neuroprotective role by reducing Aβaggregation and fibrillization. (2) The study is the first to show beneficial effects of HNG on neuroinflammatory responses and apoptosis in an animal model of AD. As a result of its ability to cross the blood-brain barrier into the brain, HNG may have therapeutic potential as a protective agent against Aβ-induced cognitive dysfunction. (3) It is the first time to demonstrate beneficial effects of HNG on preventing behavioral deficits in transgenic AD models (APP/PS-1 mice), and its underlying mechanism for slowing the development of AD may be through its ability of inhibiting fibrillar Aβformation.
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