Ceftriaxone Approves Learning And Memory Deficit In Early Stage Of Alzheimer’s Disease Model In APP/PS1 Transgenic Mice

Alzheimer’s disease(AD), the most prevalent form of dementia, is a typical neurodegenerative disorder characterized clinically by progressive memory and cognitive impairment and pathologically by accumulation of extracellular amyloid plaques and intraneuronal neurofibrillary tangles in the brain. The exact etiology of AD is still unknown. Possible mechanisms include β-amyloid deposition, inflammatory damage, oxidative stress, free radical damage, et al. In all of them, Aβ cascade theory is considered to be the most important process in the pathogenesis of AD.Aβ aggregation is believed to be the initial factor, consequently leading to synaptic damage, activation of glial cells, inflammation, impairment of synaptic transmission and dysfunction of neurons, and then eventually leading to dementia. Recent studies have found that the neurotoxic Aβ is generally presented as soluble and oligomeric form in the brain. Transgenic mice for amyloid precursor protein(APP) usually occur memory impairment and increased soluble Aβ levels before the amyloid plaques formation. Therefore, soluble Aβ in the brain plays an important role in motivating the pathogenesis of early AD. Toxic effects of soluble Aβ oligomers include oxidative damage, inflammatory responses, inducing apoptosis of neurons, and dysfunction of synapses.Glutamate is the major excitatory neurotransmitter in the central nervous system of the mammalian. Glutamate is released to the synaptic cleft by presynaptic terminals and binds with the glutamate receptors on the presynaptic and postsynaptic membrane. The glutamatergic system participates in a variety of physiological and pathophysiological processes such as cognition, learning, and memory, ischemic neuronal damage and brain degenerative diseases. Glutamate released to the synaptic cleft is removed primarily by the excitatory amino acid transporters. Five types of membrane glutamate transporters have been cloned, which include EAAT1, EAAT2, EAAT3, EAAT4 and EAAT5. EAAT2 mainly expressed in hippocampus and cerebral cortex which is closely related to learning and memory. EAAT2 removes more than 90% of the glutamate in the synaptic cleft. The transporter is expressed mainly in astrocytes, so it is also called glial glutamate transporter 1(GLT-1).In recent years, the effect of Aβ on the glutamatergic neuronal system and glutamate transporters has been paid highly attention. It is found that the neurotoxic Aβ could reduce the expression of GLT-1 in early stage of AD mice and patients. According to this phenomenon, it can be hypothezied that the reduced expression of GLT-1 will decrease the uptake of glutamate, the glutamate-glutamine cycle, blocking synaptic tranmission and resulting in the deficit of learning and memory. Thus,up-regulation of GLT-1 expression and function might improve the cognitive dysfunction in early stage of AD. Rothstein et al. reported in Nature that β-lactam antibiotics, such as ceftriaxone(Cef) can greatly and selectively up-regulation GLT-1 expression and function. Therefore, the present study was undertaken to investigate the effect of Cef against learning and memory deficit in early stage of AD model in APP/PS1 AD mice, as well as the expression of GLT-1 and proteins associated with glutamate-glutamine cycle. This study will provide new clues for clinical treatment for AD. Part Ⅰ Cef improves the cognitive impairment of APP/PS1 double-transgenic mice in the early stage of Alzheimer’s disease.The APPswe/PS1 d E9 transgenic mice for Alzheimer’s disease model(APP/PS1 AD mice) can over-express human APP with the Swedish(K594M/N595L) mutation together with presenilin1(PS1) deleted in exon 9 in a C57BL/6J genetic background mice. These mice presented amyloid plaques at 5 months of age and reached peak at 12 months, and showed AD related characters.The effect of Cef on the cognitive ability at different months’ APP/PS1 AD mice were observed in order to provide evidence for the improvement of Cef on the cognitive dysfunction in early stage of AD model mice.Methods:1 The effect of Cef on cognitive function of 7-month APP/PS1 AD mice.Eighty-two 6.5-month APP/PS1 AD mice and wild-type C57 mice were divided into three groups:1) Wild type group(n=18): The cognitive function of the wild C57 mice were evaluated at the age 7 months without any treatment.2) APP/PS1 model group(n=25): The APP/PS1 AD mice received normal saline(NS) injection with the same protocols with those for Cef treatment group.3) Cef treatment group(n=39): The APP/PS1 AD mice were intraperitoneally injected with Cef(25% concentration) for 14 days which started at the age of 6.5 months. The mice were further devided into 100 mg/kg(n=12), 200 mg/kg(n=15) and 300 mg/kg(n=12) subgroups according to the doses of Cef used. After the completion of the injection for 14 days, the animals in all the groups received Novel object recognition and Morris water maze test to evaluate the functions of recognitional memory and spatial learning and memory.2 The effect of Cef on cognitive function of 6-month APP/PS1 AD mice.Another set of 5.5-month APP/PS1 AD mice(n=76) and wild-type C57 mice(n=12) were used and divided into three groups. The animal grouping, treatment method, and behavioral testing were the same as those in the Methods 1.Results:7-month miceNovel object recognition test showed that at 1-hour time point, the recognition index of APP/PS1 AD mice was significantly declined compared with the wild type mice(P<0.05). Cef treatment(200 mg/Kg and 300 mg/Kg) significantly reversed the decline in the recognition index of APP/PS1 group Objective: mice. No significant change was observed in the Cef 100 mg/Kg group.The results at 24-hour point time were similar with those at 1-hour.Morris water maze test showed that compared with wild type mice, the escape latency of APP/PS1 AD mice was significantly prolonged in navigation test, the frequency of crossing target quadrant and the time spent in the target quadrant were significantly reduced(P<0.05). Compared with APP/PS1 model mice, Cef treatment(200 mg/kg and 300 mg/kg) significantly decreased the escape latency(P<0.05), and increased the frequency of crossing target quadrant and the time spent in the target quadrant(only Cef 200 mg/kg group)(P<0.05). There were no effects of Cef 100 mg/Kg treatment on the test performance of the APP/PS1 AD mice. The results showed that Cef treatment could improve the deficits in spatial learning and memory function of APP/PS1 AD mice at 7-month age.6-month miceNovel object recognition test showed that at 1-hour time point, the recognition index of APP/PS1 AD mice was significantly reduced compared with the wild type mice(P<0.05). Cef treatment(200 mg/Kg) significantly reversed the decline in the recognition index of APP/PS1 group mice. No significant change was observed in the Cef 100 mg/Kg group. However, at 24-hour point, Cef 300 mg/Kg group showed an significantly improved recognition index compared with APP/PS1 model group(P<0.05). It was indicated that Cef can approve the recognition ability of APP/PS1 AD mice in an earlier stage.Morris water maze test showed that compared with wild type mice, the escape latency of APP/PS1 AD mice was significantly prolonged in navigation test, the frequency of crossing target quadrant and the time spent in the target quadrant were significantly reduced(P<0.05). Compared with APP/PS1 model mice, Cef treatment(200 mg/kg and 300 mg/kg) significantly decreased the escape latency(P<0.05), and increased the frequency of crossing target quadrant and the time spent in the target quadrant(only Cef 300 mg/kg group)(P<0.05). There were no effects of Cef 100 mg/Kg treatment on the test performance of the APP/PS1 AD mice. These results showed that Cef treatment could improve the deficits in spatial learning and memory function of APP/PS1 AD mice at 6-month age as well.Summary:The above results suggested that Cef treatment could improve the deficits in the ability of recognizing novel objects and spatial learning and memory function of APP/PS1 AD mice in an earlier stage of AD. Part Ⅱ Cef up-regulated GLT-1 expression in the hippocampus of APP/PS1 AD miceObjective:The effect of Cef on GLT-1 expression in the hippocampus of 6-month and 7-month APP/PS1 AD mice were assayed by Immunohistochemical staining and western blot.Methods:Twenty-five 6.5-month and twenty-five 5.5-month APP/PS1 AD mice and twenty companion wild-type C57 mice with the same age were divided into the three groups:1) Wild type group(n=10): Wild type mice at the age 7 months(6 months) were used.2) APP/PS1 model group(n=10): The APP/PS1 AD mice received normal saline(NS) injection with the same protocols with those for Cef treatment group at 6.5 or 5.5 months.3) Cef treatment group(n=15): The APP/PS1 AD mice were intraperitoneally injected with Cef(25% concentration) for 14 days which started at the age of 6.5 or 5.5 months. These mice were further devided into 100 mg/kg(n=5), 200 mg/kg(n=5) and 300 mg/kg(n=5) subgroups according to the doses of Cef used.After the completion of the injection for 14 days, the animals were sacrificed by decapitation in all groups, and the hippocampus of the animals was collected for immunohistochemical staining and western blot assay.127-month miceImmunohistochemical staining showed that brown, fine GLT-1 immunoreactive particles distributed in hippocampus in wild mice. Compared with wild mice, APP/PS1 AD mice showed a significant decrease of GLT-1 immunoreactive particles and integral optic intensity(P<0.05). Compared with APP/PS1 AD mice in APP/PS1 model group, Cef treatment(100 mg/kg, 200 mg/kg and 300 mg/kg) induced a significant increase of GLT-1 immunoreactive particles and integral optic intensity(P<0.05).Western blotting analysis showed that there was basal expression of GLT-1 in wild type mice. Compared with wild type mice, the GLT-1 protein was significantly down-regulated in APP/PS1 AD mice(P<0.05). Compared with APP/PS1 model group, intraperitoneal administration of Cef(200 mg/kg and 300 mg/kg) significant up-regulated GLT-1 expression(P<0.05).6-month miceThe effect of Cef on GLT-1 expression in hippocampus of APP/PS1 AD mice at 6-month age stage was consistent with those of 7-month APP/PS1 AD mice.Summary:The results suggested that Cef up-regulated GLT-1 expression in the hippocampus of 6 and 7 months APP/PS1 AD mice. Part Ⅲ Cef upregulated protein expressions or activity associated withglutamate-glutamine cycle and glutamate release in thehippocampus of APP/PS1 AD mice.Objective:In order to evaluate the effect of Cef on the glutamate-glutamine cycle in the hippocampus of APP/PS1 AD mice, the expression and activity of glutamine synthetase(GS) and system N glutamine transporter 1(SN1) were assayed by western blotting and spectrophotometric method. Similiarly, Group Ⅱ metabotropic glutamate receptor(m Glu R2/3) that modulates presynaptic glutamate release was assayed by western blotting to reflect indirectly the release of glutamate from presynaptic terminals. Results:Methods:Twenty-five 5.5-month APP/PS1 AD mice and wild-type C57 mice were divided into three groups:1) Wild type group(n=5): Wild type mice at the age 6 months were used.2) APP/PS1 model group(n=5): The APP/PS1 AD mice received normal saline(NS) injection with the same protocols with those for Cef treatment group.3) Cef treatment group(n=15): The APP/PS1 AD mice were intraperitoneally injected with Cef(25% concentration) for 14 days. These mice were further devided into 100 mg/kg(n=5), 200 mg/kg(n=5) and 300 mg/kg(n=5) subgroups according to the doses of Cef used.After the completion of the injection for 14 days, the animals in all groups were sacrificed by decapitation, and the hippocampus of the animals was collected for western blot analysis to assay the expression and activity of GS, SN1, and m Glu R2/3.Results:The western blot assay showed that there was basal expression of SN1 and m Glu R2/3 in Wild type group. Compared with Wild type group, the expressions of SN1, and m Glu R2/3 were significantly down-regulated(P<0.05). Compared with APP/PS1 model group, intraperitoneal administration of Cef(200 mg/kg and 300 mg/kg) significant up-regulated the expression of SN1, and m Glu R2/3(P<0.05).There was no change in the expression of GS after the treatment of Cef in any dose used. However, compared with Wild type group, GS activity significantly decreased in APP/PS1 AD mice(P<0.05). Compared with APP/PS1 model group, intraperitoneal administration of Cef in each dose used significant increased the activity of GS(P<0.05).Summary:Cef up-regulateed the expression of SN1 and m Glu R2/3 expression and increases the activity of GS.Conclusion:It could be concluded that Cef could approve APP/PS1 AD mice’s learning and memory deficit. The effect of Cef might be associated with the up-regulation of GLT-1 expression, promotion of glutamate-glutamine cycle and increase of glutamate releasing. These changes might increase the efficacy of glutamate utilization as a neurotransmitter and then facilitate synaptic transmission.