Student: Eleni Miliotou
Advisors: Dr. Amy Jo Stavnezer, Dr. Ashley Abraham
Melatonin is an endogenous antioxidant and free radical scavenger that regulates sleep/wake cycle and circadian rhythm. Previous studies indicated that melatonin is decreased during the aging process and that patients with AD have a significant reduction of this hormone. However, melatonin supplementation has been found to reduce Αβ neurotoxicity and formation while improving cognitive performance. In this study, the long-term influence of melatonin on behavior and neuropathologic changes were evaluated, after 24 weeks of 10 mg/kg melatonin treatment in the 5xFAD mice model. The 5xFAD is a transgenic mouse model for Alzheimer’s disease that mimics the accumulation of senile plaques, neuronal apoptosis and memory impairment. Overall, transgenic mice showed decreased anxiety, impaired learning, and significant increased in Aβ42 plaque load, replicating previous results with this strain. Melatonin improved learning in transgenic mice by decreasing distance travelled in the Morris water maze and slightly decreasing Aβ42 concentration in the hippocampus of 5xFAD mice compared with untreated 5xFAD. These results supported the hypothesis that long-term melatonin supplementation may be beneficial at early stages of the disease process. Early melatonin interventions may be one of the most promising strategies in the development of approaches to retard or prevent Aβ and memory deficits during this stage of the disease. In sharp contrast to conventional antioxidants, melatonin crosses the blood brain barrier, is relatively devoid of toxicity, and constitutes a potential therapeutic candidate in AD treatment, perhaps due to improving sleep quality and clearance of Aβ42 plaque by increasing glymphatic system efficiency.
Welcome and thank you for being here.
In my independent study I looked at the effects of melatonin in the glymphatic system in an Alzheimer’s disease mouse model.
Alzheimer’s Disease (slide 2):
Alzheimer’s disease (AD) is a progressive and chronic neurodegenerative disease characterized by deterioration in cognition, function, and behavior. AD is the main case of dementia and the fifth leading cause of death among people above the age of 65. Statistics of 2019 show that 5.8 million Americans are living with AD and those numbers are projected to rise to 14 million by 2050. As the numbers of AD rise so does the need for a treatment.
As the most common aging disease, AD is characterized by accumulation of amyloid beta plaques and neurofibrillary tangles. In a healthy brain plaques and tangles are being clear out and thus preventing their devastating effects by a system that gets activated during sleep.
Specifically, deep sleep is important in this clearance process by promoting glymphatic system activity. The glymphatic system is known as the waste clearance pathway of the brain by the continuous interchange of fluids. With AD and aging, glymphatic function is reduced due to the loss of the water channels, AQP4 which facilitate the fluid movement, with impaired clearance of interstitial solutes and increased aggregation.
Alzheimer’s Disease (slide 4):
Interestingly a significant amount of AD patients report increasing sleep disturbances along with the severity of the disease. AD and sleep disturbances denote a bidirectional relationship observed before clinical onset of AD, where sleep disturbances are present with the occurrence of amyloid beta, but also cause the augmentation of soluble amyloid beta. Sleep disturbance do not allow the active process of the glymphatic system to perform its function leading to build up of toxins and the effects are becoming more apparent in cognitive abilities, behavior, and judgement.
While sleep disturbances have many negative consequences, a sleep hormone might be a solution to the problem. During dark periods the pineal gland releases the endogenous hormone melatonin, once released it acts both as an endocrine product and as an antioxidant. However, with aging and AD there is a decrease of melatonin levels which can explain the decrease in sleep quality and disturbances, therefore leading to a decremental efficiency of glymphatic clearance. Melatonin’s antioxidant effect and free radical ability supports all the classical hypotheses in AD suggesting that with early detection, melatonin treatment may be qualified to be an anti-AD therapy.
Research Question (slide 5):
Although there are many approaches to AD therapy, melatonin has advantages that can benefit and possibly reverse AD degeneration. With the increased number of studies implicating melatonin as a potential therapeutic agent against AD, the aim of this study was to assess whether melatonin can slow down the progression of AD by improving glymphatic system efficiency and thus decreasing amyloid beta accumulation.
Research design and methods (slide 6):
Here the 5xFAD mouse model was used which co-overexpresses human APP and PS1 with 5 familial AD mutation, representing one of the most early-onset and aggressive amyloid mouse models, with Αβ deposits being visible as early as 2 months of. A total of 38 mice (n = 19 wild type and n = 19 transgenic) were used in this study and PCR was used to divide animals to their groups based on genotype: control WT, control Tg, melatonin WT, and melatonin Tg. At one month of age mice in the treatment group received 10mg/kg/day melatonin supplementation for 5 consecutive months.
At 6 months of age, the open field test was conducted to evaluate exploratory behaviors followed by the Morris water maze for spatial learning and memory function evaluation. The Morris water maze task was performed over 6 consecutive days with six trials per day.
Following the completion of the behavioral tasks hippocampal tissue was collected to assess the effect of melatonin in Αβ clearance, levels of Αβ42 in mouse hippocampus were measured by ELISA.
Hypotheses (slide 7):
In this experiment, melatonin treatment was expected to improve memory performances and increase activity through all conditions, but significant changes are expected to be observed in the treated transgenic mice. In addition, melatonin supplementation will decrease Αβ plaque load in the brain.
Results (slide 8):
Open field task (slide 9): no significant effects of melatonin on general activity of transgenic or wild type mice were found. However, a significant effect of genotype was observed in the total distance travelled and time spent away from the perimeter and corners. As expected, transgenic mice showed decreased anxiety levels, which agrees with the results of other studies also performed in transgenic mice.
Morris water maze (slide 10): Supporting the literature, this study found that the 5xFAD mouse line had significantly impaired cognitive performance at 6 months of age in the Morris water maze, but melatonin revealed its beneficial effects in learning and memory deficits in transgenic mice. In contrast to the impaired performance of untreated transgenic mice in cognitive tasks, here it was found that performance of melatonin-treated transgenic mice was equivalent to control wild type mice, indicating that melatonin enhanced spatial memory and learning of AD to be at normal levels of non-AD.
ELISA (slide 11):
Although results from this study did not indicate a statistically significant difference between melatonin and control transgenic mice, means show a decrease in Aβ42 levels in melatonin treated transgenic mice compared to matched controls. Similar results were obtained from previous studies that explored melatonin effects in Αβ42 of transgenic mice at different ages and reported that melatonin partially inhibited the expected time-dependent elevation of Aβ42, but the difference was not statistically significant until 8 to 9.5 months old
Conclusion (slide 12):
Overall, results from this study support the hypothesis that due to melatonin’s ability to promote sleep and serve as an antioxidant, it can decrease AD progression. Therefore, it improves cognitive performance and reduces neuropathology in the 5xFAD mouse line, possibly through activation of the glymphatic system. Altogether, at the foundation of behavioral tests and ELISA, the mechanism by which melatonin ameliorates cognitive impairment is thought to be through increased sleep quality. The sleep dependent role of the glymphatic system suggests that increased sleep facilitates Αβ clearance and NREM sleep activates the glymphatic clearance and clears Αβ. In summary, the results of the current investigation promote melatonin as a main mechanism of neuroprotective and memory rescue action against AD pathology, by slowing down the progression of the disease. AD pathogenesis has been found to be degraded by an already endogenous hormone, which raises the interesting point that maybe we should turn our attentions to our biological and innate functions. A decent amount of evidence suggest that melatonin plays a significant neuromodulator and neuroprotective role, which makes it a potent endogenous antioxidant, possessing several advantages over other antioxidants. Early melatonin interventions forestalled the development of AD suggesting that this devastating brain disorder may be preventable.
Acknoldgement (slide 13):
I would like to thank my advisor Dr. Stavnezer for her support and guideence. Our animal technisian Julie Pringle. Also, Copeland Fund and the Neuroscience program for their funding and support. Lastly, my friends and family for theyr love and encouragment.
Eleni will be online to field comments on May 8:
8am-10am EDT (Asia: evening, Africa/Europe afternoon)