Intranasal Administration Of Exendin-4 Ameliorates Aβ31-35-induced Circadian Rhythm Disruption In Mice And Possible Mechanisms

Circadian rhythm is a ubiquitous phenomenon in all living organisms, which synchronizes behavior, biochemical and physiological processes with environmental factors. Circadian rhythm disorders are common in many elderly persons with neurodegenerative disease. Alzheimer’s disease(AD) is a common neurodegenerative disease. Amyloid β-protein(Aβ), one of major pathological features in the human AD brain, is closely related to progression of disease. Triple transgenic model of AD(3x Tg-AD) mice with Aβ plaques showed circadian rhythm disruption. However, it is not clear whether Aβ will affect the disruption of circadian rhythm directly. At present, there are still no effective methods to improve disrupted circadian rhythms in AD in clinic.Type 2 diabetes mellitus(T2DM) is similar to AD in some respects, and Exendin-4, a glucagon-like peptide-1(GLP-1) receptor agonist, which is widely used as the T2 DM therapeutic drug has been proved to significantly enhance learning and memory deficits in AD model mice. However, it is unknown whether Exendin-4 can reverse the circadian rhythm disruption caused by Aβ. At present, the lateral ventricle and hippocampus injection which are usually used to explore neuroprotective effect of Exendin-4 are limited by being invasive. The intranasal administration which is a new administration of nerve therapy dugs has acquired increasing attention in recent years.Therefore, the study innovatively treated Exendin-4 with intranasal administration,tested the concentration of Exendin-4 in the hippocampus tissue through ELISA experiment, monitored that Exendin-4 ameliorated obviously circadian rhythm disruption induced by intra-hippocampal injection of Aβ31-35 via wheel-running experiment,observed that Exendin-4 reversed partly alteration of per1 and per2 gene expression in the mouse suprachiasmatic nucleus caused by Aβ31-35 through Real-time PCR at the molecular level, provided theoretical basis for improving circadian rhythm disruption in the early stages of AD.Part Ⅰ Intra-hippocampal injection of Aβ31-35 caused circadian rhythm disruption in C57BL/6 miceObjective:To monitor the effect of intra-hippocampal injected Aβ31-35 on circadian rhythm of mice.Methods:We injected tri-distilled water and Aβ31-35 into hippocampus via stereotaxic instrument respectively, and then observed circadian rhythm of wheel-running activity of Vehicle and Aβ31-35-treated mice which were entrained to 12:12 Light-Dark(LD) cycles for one week and then transferred to constant darkness(DD) for two weeks. The data was collected by Vital View system and analysed by Acti View system.Results:Two group mice showed a derivable rhythm which expressed obvious division between moving phase and resting phase with an approximately 24-h period under LD conditions. After transferred to DD conditions, the locomotor activity of Vehicle mice has two distinct phases, moving phase and resting phase. Aβ31-35 which was injected into hippocampus caused abnormal circadian rhythm with dispersive locomotor activity and made free-running period lengthened(23.69 ± 0.06 h) compared with Vehicle group(23.54 ± 0.11 h)(P(27) 0.05). Meanwhile, Aβ31-35 induced the activity declined obviously(719.3 ± 107.0 counts/h) compared with Vehicle group(1113.2 ± 133.8 counts/h)(P(27)0.05).Conclusion:Intra-hippocampal injection of Aβ31-35 caused circadian rhythm disruption and decreased activity in C57BL/6 mice.Part Ⅱ Intranasally administered Exendin-4 entered hippocampus in C57BL/6 miceObjective:To measure the concentration of Exendin-4 in the hippocampus tissue after intranal administration.Methods:(1) Intranal administration: keeping mice awake, fixing its head, exposing nasal cavity, administrating by nasal drops.(2) Vehicle: Tri-distilled water was intranasally administered; The concentration of Exendin-4 in hippocampus was measured at 10 min, 30 min and 3 h post-administration with 25 nmol/kg bw dose of Exendin-4 via ELISA experiment respectively.(3) Using ELISA experiment, concentrations of Exendin-4 in the hippocampus and in the cerebral cortex were measured at 30 min post-administration with 5 nmol/kg bw,25 nmol/kg bw of Exendin-4 and tri-distilled water respectively.Results:(1) There was no significant increase of Exendin-4 at 10 min post administration in the hippocampus of mice compared with Vehicle group. However, significant levels of Exendin-4 were found at both 30 min and 3 h post administration(P(27) 0.05), and the peptide level 3 h post administration was lower than 30 min post administration(P(27)0.05).(2) Compared with Vehicle group, 5 nmol/kg bw and 25 nmol/kg bw of Exendin-4were also sufficient to raise peptide levels in the hippocampus 30 min post administration(P(27) 0.05), and the level of 25 nmol/kg bw group was higher than 5 nmol/kg bw group(P(27) 0.05). However, there were no increased levels of Exendin-4 found in the cerebral cortex of mice administered with a 5 nmol/kg bw or 25 nmol/kg bw dose of peptide, and the level was significantly lower than hippocampus(P(27) 0.05).Conclusion:Intranasally administered Exendin-4 could enter hippocampus of C57BL/6 mice.Furthermore, the concentration of Exendin-4 in the hippocampus was higher than in the cerebral cortex.Part Ⅲ Intranasal administration of Exendin-4 meliorated ciracadian rhythm disruption induced by Aβ31-35 in C57BL/6 mice and possible mechanismsObjective:To observe effects of intranasally administrated Exendin-4 on circadian rhythm induced by Aβ31-35 and possible mechanisms.Methods:Pretreatment with Exendin-4: Exendin-4 was intranasally administrated with a dose of 25 nmol/kg body weight and repeated this step in 2 hours. Then Aβ31-35 was injected into hippocampus. Observed circadian rhythm of locomotor activity of Vehicle-treated mice, Aβ31-35-treated mice, Exendin-4-pretreated mice and Exendin-4-treated mice which were entrained to 12:12 LD cycles for one week and then transferred to DD for two weeks. Furthermore, according to circadian time(CT), we extracted total RNA from SCN of mice at CT2, CT8, CT14 and CT20, and then tested the expression of per1 and per2 via Real-time PCR.Results:Under DD conditions, mice pretreated with Exendin-4 showed an obvious moving phase- resting phase, and free running period(23.56 ± 0.07 h) was significantly shorter than Aβ31-35 group(23.69 ± 0.06 h)(P(27) 0.05). Exendin-4 didn’t affect the rhythm of C57BL/6 mice and free running period was 23.50 ± 0.05 h. Rhythmic expressions of per1 and per2 were disturbed by Aβ31-35. per1 m RNA level caused by Aβ31-35(1.09 ±0.18) was lower obviously than Vehicle group(1.53 ± 0.35) at CT14(P(27) 0.05), but pretreatment with Exendin-4 could ameliorate the alteration(1.43 ± 0.34). Aβ31-35 caused per2 m RNA expression decreased(0.99 ± 0.10), which was obviously different comparing with Vehicle group(1.47 ± 0.35)(P(27) 0.05), but pretreatment with Exendin-4could reverse the change(1.40 ± 0.38). Exendin-4 didn’t affect the expression of per1 and per2.Conclusion:Intranasal administration of Exendin-4 ameliorated circadian rhythm disruption caused by Aβ31-35, which might be related to the regulation of per1 and per2.