The effects of melatonin in the glymphatic system in an Alzheimer’s disease mouse model

Student: Eleni Miliotou
Major: Neuroscience
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.

Slide 1:

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.

Slide 3:

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)

67 thoughts on “The effects of melatonin in the glymphatic system in an Alzheimer’s disease mouse model”

  1. Thank you Eleni for showing us your IS work. Your ideas about how melatonin can positively impact the brain were interesting and your data suggestive of a true impact on behavior. An IS student will extend this work next year, thank you for bringing this idea to the lab!

  2. Hi Eleni! This is such significant work. Will you continue to pursue Alzheimer’s research beyond Wooster?

    1. Hi Melissa and thank you for taking the time to look at my IS. At the moment I will switch my research attention to developmental neuroscience, but I am planning to return to Alzheimer’s research in the future.

  3. How did you measure anxiety?
    How did you measure the Aβ42 levels in the brain?

    1. Hi Frank. Anxiety was measured in the open field comparing the time spend in the perimeter vs the center. ELISA was used to quantify Aβ42 levels.

      1. Eleni, I had the same question about measuring anxiety in mice. Can you expand on your answer, is it that increased perimeter time spent is considered more anxious or the opposite?

        1. Your research is interesting and I hope you have a chance to further explore this topic or related ones. Best wishes for wherever your research takes you.

        2. Hi Lori. Increased anxiety will result in less locomotion and a preference to move and stay close to the walls of the field, which includes perimeter and corners. Where decreased levels of anxiety lead to increased exploratory behavior and more entries at the center. It is similar to human, for example think about a time that you attend an event and you didn’t know anyone beside the host, that would have increased anxiety levels and subconsciously you would prefer to stand in the perimeter of the room. I hope this answer your question.

          1. It does thanks and congratulations on your graduation.

  4. Hi Eleni – This is such incredible research – thank you and congratulations! I’ve lost two family members to this terrible disease and it is so meaningful to see Wooster students like you doing this important work. What inspired you to study this topic? And of course, do you have plans to continue your research in the future? Again, congratulations on your accomplishments, thank you for sharing your work today, and we look forward to welcoming you to the Wooster alumni community!

    Carolyn ’08

    1. Hi Carily. Thank you very much! I wanted to study Alzheimer’s because the most common symptom of the disease is memory lost and I think memories play such an important role in defying our personalities. My biggest inspiration for examining this project was the fact that we underestimate sleep and its therapeutic effects, that is why I decided to use melatonin which is an already endogenous hormone released by our bodies. The idea is to try and look at more natural ways of treating diseases, like sleep. My current plans involve working in a lab that conducts research on developmental neuroscience.

      1. I couldn’t agree more! Thank you for responding and good luck on your post-Wooster path.

  5. This was great! I had no idea about melatonin and what impact it may have on Alzheimer’s. I am happy to say I look forward to learning more. Thanks for sharing and I hope you continue your work in the future.

    1. I am glad you like my IS. If there are any questions I can answer for you please let me know.

  6. Really nice work Eleni! I appreciate you sharing the results and look forward to seeing how this project evolves. Do you think that there are any potential concerns for use of melatonin as a preventative treatment?

    1. Hi Dr. Sobeck. In my knowledge I haven’t read about any negative effects of melatonin, independent of dosage. However, the only concern I have is that the longer and the more exogenous melatonin supplementation is used the less endogenous melatonin the brain will release, which means the individual will eventually have to rely on supplementation to fall asleep.

      1. Thank you and congratulations! I hope our paths cross in the future so I can hear about the wonderful things you are doing in person.

  7. Congratulations, Eleni, on your fascinating project and clear presentation. Can you tell us what your favorite and most challenging part of the research process were? Thanks!

    1. The most challenging part of this project was definitely conducting the ELISAs. It was the first time I had to do an ELISA and the protocol was not very clear. Beside when I turned in my IS, my favorite part was at the begging when I was writing my intro and my attitude switched from “I have to been done with this” to “I love this project” and the more I was writing the more I was getting into it.

      1. Love this. That switch to “I love this project” is the absolute best part of advising ISs 🙂

      2. I’m so glad this happened while you were still working on it–it’s such a great feeling to be at that stage of the writing! Is it possible to briefly explain ELISAs for non-scientists? I’m familiar with some of the other lab protocols but not this one.

        1. Absolutely, ELISA is a technique used to detect and measure atnibodies in the body. Antibodies are proteins that your body produces and in this project the protein Aβ42 was measure in the brain’s hippocampus.

  8. Hi Eleni,
    Thanks for sharing your project. Do you think sampling CSF or ISF (or blood, should the AB42 waste make it that far in that form. I’m not super familiar with the glymphatic system!) would provide a more sensitive or real time measure of the efficacy of your melatonin treatment? I would be curious to see if you could get in vivo biomarkers.

    1. You are absolutely right! A measure of Aβ42 in CSF would have been a much better indicator of melatonin’s effect in the glymphatic system. However, not much literature has looked at Aβ42 CSF, since the glymphatic system is relatively new, and I wouldn’t be able to base my results on previous literature. By looking at Aβ42 in the hippocampus it confirms the results of the Morris water maze and supports the hypothesis that melatonin decreases memory deterioration, a major symptom in Alzheimer’s disease.

      1. I’m definitely “eavesdropping” on this exchange. But, I wanted to comment that this is a very thoughtful answer and demonstrates your research prowess. I appreciate the use of ELISAs for the reasons you state here, although they can be tricky. 🙂 Experimental design and the consideration of different techniques are one of the most challenging but rewarding aspects of scientific research. Your work is incredibly interesting and novel, and kudos on developing a whole new project (based on Dr. Stavnezer’s comment). I hope you’re going to graduate school; you’ll do amazing things there!

  9. Hi Eleni,
    First congratulations! My father died from AD. One of the constraints, as I understand it, of using melatonin is the negative effect with those on blood pressure medication. Was an exploration of contra-indicators for use of melatonin explored in your study? Or is that a future direction? Again, thank you for this important work.

    1. These are some very interesting questions, but they were not explore in this study. Further research needs to be conduct to examine implications of melatonin in combination with other medications.

  10. Thank you, Eleni, for this work. The role of sleep in Alzheimer’s disease is fascinating, and your work on this issue is encouraging. I hope that you continue to find this line of work rewarding.

  11. Hi Eleni! GOOD WORK! I wish I could have read your whole IS! I’m glad you were able to see the fruits of your long and hard work. Here is my question: If AB42 levels were not significantly reduced, then what do you think accounts for the improvement in Morris Water Maze performance in the Melatonin rats?
    Here is another question: How is your family? When can you go home? Or are you home now? I hope all good things for you. It was a privilege to learn with you in my classes.

    1. Hi Dr. Thompson! Very interesting question. A better indicator of Aβ plaque accumulation is the ration Aβ40/42, individually (Aβ42 or Aβ40) they are weaker indicators. However due to time and funding I wasn’t able to looked at both and even though Aβ42 was not significant it was leaning toward that direction, maybe a longer treatment would have revealed significant results. Worth to mentioned that the memory improvement might be due to still unknown factors, there might be biomarkers that we are still haven’t discovered that will help treat Alzheimer’s.
      I had no idea Herpes 1 causes Aβ formation, thank you for sharing.
      Thank you for asking, I did make it home yesterday and family is good.
      I hope you are safe and healthy and thank you for taking the time to look at my IS.
      Best,
      Eleni

  12. Oh– I forgot — I just read an article today that the Herpes 1 virus can trigger AD because the virus also induces amyloid plaque formation. I just thought I’d mention it because you seem to want to do further AD research!

  13. Eleni, thanks for sharing your research! Over the few years, it’s been awesome to see you connect your Wooster coursework, your internships and APEX Fellowship, and your research to culminate in this significant original project. I wish you all the best!

    1. Thank you Krista! I wish things have ended differently and I could have at least say goodbye.

  14. Hi Eleni,
    I am curious if these effects also are relevant for people with early onset Alzheimer’s or not? Any thoughts on that? Also, your I.S. is very well presented—clear, concise and understandable for someone like me who is not a scientist! Congratulations on a terrific accomplishment and best of luck as you move forward with your research and career aspirations!

    1. Hi Elizabeth and thank you for being here. A limited number of studies has found the beneficial effects of melatonin in patients with early onset of the disease. I think the inconsistency in the results might be due to the fact that in order for melatonin to work and have beneficial results has to be taken at the same time of day and right before going to bed, in order to balance circadian rhythm disturbances. Patients with Alzheimer’s will probably forget to take it at the same time every day and will need someone else to make sure the medication is taken consistently. This is the a problem that I didn’t face with mice because I was able to keep their treatment consistent at all times.

      1. Super interesting and yes, a good point! Thanks for replying! And my son just told me that you were his TA—very cool! Thanks for sharing your research and good luck!!

  15. This is such important work, Eleni — thank you! Like many who have posted comments, Alzheimer’s disease runs in my family and I have seen way too many people suffer from it. It’s exciting to see research like this that suggest we’ll have better treatments in the future!

    1. Thank you Dean Craven. I hope a cure is found soon and we stop losing our love ones.

  16. Congratulations, Eleni!
    This looks like an ambitious project with really interesting results.
    Best wishes for your next steps as you continue to grow as a researcher.

  17. Eleni, first of all congratulations. Your research is so interesting and well explained, thank you for sharing it with us.🎓👍📚✨💫

    1. I am glad you enjoyed it! I would love to answer questions if there are any.

  18. Hi Eleni: Congratulations and great job! This research area has the ability to touch so many lives. Do you have any recommendations for others who would like to learn more about Alzheimers research? Do you have any advice for students that might like to pursue this type of research in the future?

    1. Thank you! There are many approached to examine Alzheimer’s but personally I prefer looking at more natural ways of treating diseases and illnesses and using what the brain and body already offer us. I would love to answer any questions and share ideas about Alzheimer’s and sleep to other students.

  19. Congratulations, Eleni! Your research gives me a lot of hope and I’m so grateful you shared your IS today!

  20. Eleni, your IS is fascinating! Thank you for sharing it with us. How would you like to see future Wooster seniors expand on your research?

    1. Thank you! I do see why not, if a student finds this project interesting and has some good ideas why not explore it further and maybe find some important results.

  21. Eleni, great job on your IS project! It was very interesting to learn about how melatonin can act as a preventative measure in the early stages of AD. As we know, AD has no cure so it is critical that we find ways to prevent its progression in the early stages–and that we diagnose it early enough. Also, thanks for being such a helpful TA this semester. All the best!

    1. Thank you Matt! I wish you all the best in your journey at Wooster and I am looking forward to reading your IS next year.

  22. Congratulations to you, Eleni! This is a fabulous project and I am so very proud of you!! All the Best to you as you move on from Wooster. Until we meet again…

    With much respect,
    Sandi

    1. Thank you Sandi! I hope our paths will cross again soon. Best wishes to all the family.

  23. Eleni,
    I really enjoyed learning about your research project. It is such an interesting subject and your presentation was very well written . I was intrigued by your interest in how memories might play a role in defining personality and how natural processes, like sleep, can have therapeutic effects. I hope you are able to continue exploring these topics in future work in developmental neuroscience. Best wishes to you!

    1. Thank you Ms. Mandych for taking the time to read my IS. I wish you and your family all the best and hopefully our paths will cross soon.

  24. Thank you for sharing your study. Was there any indication that this was clearly linked to the melatonin or would other ways of improving sleep be similarly beneficial?

  25. Hi, Eleni, (from the mom of a COW alum), your research is very significant for my family. Although I am familiar with melatonin as a sleep aid, I was not aware of its use for people with Alzheimers.

    My husband’s younger sister (age 60) has Down’s Syndrome, and my mother-in-law (her caregiver) noticed the beginning of AZ symptoms about 18 months ago. I am going to share your research with my mom-in-law.

    Thank you for this very important research. Congratulations on a job well done!

  26. Eleni,

    You have convinced me to look into the use of melatonin for sleep. I am 61 years old and finding sleep increasingly difficult. I will redouble my efforts to find a way to sleep better. I hope this includes the use of melatonin. Do you have any suggestions as to what product to use? I have avoided melatonin because of its dietary supplement category. As you know, it is not FDA approved. I’m curious to know how you found a reliable melatonin product. My mother had early-onset Alzheimers, so I am deeply concerned about my sleep deprivation.

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