a u g u s t 2 0 2 1 v o l u m e 5 - i s s u e 2

an undergraduate biomedical researchjournal by and for university college

students in The Netherlands

Volume 5 - Issue 2August 2021

COLOPHON

Science of the Human Body publishes research papers and literature reviews written by biomedicaland premedical students of the university colleges of Amsterdam, Utrecht, Groningen, Maastricht,Rotterdam, and Middelburg. The aim is to create a platform where students can read about each

other’s research, learn about and experience the process of publication and peer reviewing, but mostimportantly the students will get the credit they deserve and inspire others with their research.

Editor-In-Chief

Samantha Scharringa - University College Roosevelt

Senior Editors

Amsterdam University College

Jasmin Ronach

Editors


Preksha Bapna

Terezia Klasova (Junior Editor)

University College Roosevelt

Emma Verheijen

Aroha Oyugi (Junior Editor)

Isabel Vaz (Junior Editor)

University College Utrecht

Vela Kaludjerovic (Junior Editor)

Peer Reviewers


Tessa Zonneveld


Daan Pijs

Nick Goldin

IT Team


Jonathan Seib (alumnus)

HUMAN BODY | Volume 5 - Issue 2 | August 2021

Advisory Staff Members

Prof. dr. Ger Rijkers - University College Roosevelt

Dr. Frans van Overveld - University College Roosevelt

Prof. dr. Edward Nieuwenhuis - University College Roosevelt

Isabel Hageman - University College Roosevelt (alumna)


TABLE OF CONTENTS

VOLUME 5 - ISSUE 2: August 2021 pages 226-262

Letter from the Editors

SAMANTHA SCHARRINGA - UNIVERSITY COLLEGE ROOSEVELT 226

Performance and Experiences across a Traditional, Hybrid, and Online University College Cur-riculum

VERONIQUE DIERKX - UNIVERSITY COLLEGE ROOSEVELT 227-234

COVID-19 risk perception in Germany and its implications for policy making

EMMA WEßEL - UNIVERSITY COLLEGE ROOSEVELT 235-239

The Physiology of Erythropoietin, Medical Application, and Abuse in Sport

OLIVIER BRAAS - UNIVERSITY COLLEGE ROOSEVELT 240-245

A literature review on Guillain-Barre syndrome

DAAN PIJS - UNIVERSITY COLLEGE ROOSEVELT 246-250

Harnessing the power of exosomes: Using exosome-delivered miRNA to modify dysregulatedα-synuclein expression in PD patients

ELEANOR DARE SWANSON - AMSTERDAM UNIVERSITY COLLEGE 251-256

The Role of DNA Methylation in Regulation of BDNF and Depression Neuropathophysiology

MAXENCE LIESENBORGS - AMSTERDAM UNIVERSITY COLLEGE 257-262


LETTER FROM THE EDITORS

Dear Reader,

Welcome to the special second issue of our fifth volume!

If this is your first time hearing about us, allow me to introduce our journal to you. Science of theHuman Body (SOTHB) is an undergraduate (bio)medical research journal run by and for studentsfrom University Colleges (UC) across the Netherlands. This journal was established as a platform forUC students to share their academic work and discuss their interests with peers, as well as a space toimprove their academic writing skills; which is a valuable asset to have in academia.

From surviving a national lockdown, partying at a nightclub, standing in line to receive one of the mostimportant vaccines in our decade, to graduating (online), this semester has seen it all. Incredibly, ourauthors have achieved to write six incredible papers; including two special edition COVID-19 relatedpapers. Similar to our previous issue, this issue will also include COVID-19 related topics from otherdisciplines such as psychology, to give you a better understanding on how this pandemic affects us fromdifferent perspectives, as well as the value of a multidisciplinary approach.

Veronique Dierkx will start off this issue with an original study on student performance and experienceacross different curriculums during the pandemic. Emma Weßel continues the psychology theme byanalysing the characteristics of COVID-19 risk perception in the German public, highlighting theimportance of communication between policy makers and the general public. Olivier Braas’ paper onthe physiology of EPO, its application, and abuse in sport leads our transition towards more biomedicalpapers, where Daan Pijs follows with his literature review on GBS. As Eleanor Dare Swanson exploresa new hypothesised novel approach to Parkinson’s disease treatment, Maxence Liesenborgs probesinto the significance of DNA methylation on brain-derived neurotrophic factor and its involvement indepression neuropathophysiology; sealing off this issue with two detailed papers from our two AUCauthors.

This issue wraps up my board year as Editor-in-Chief and I would really like to thank the senioreditor, the editors, and the amazing peer reviewers for their time, effort, and enthusiasm; as well asthe continuous support from the IT team and teachers to make this year possible in these special times.Second of all, I would like to thank you – our readers and authors – for your interest and contributiontowards our journal. For without you, we are nothing but a blank page :)

Without further ado, I hope you will enjoy reading this issue and keep an eye out for our next issuearranged by the new board!

Sincerely yours,

Samantha ScharringaEditor-In-Chief Science of the Human Body 2020-2021

226| HUMAN BODY | Volume 5 - Issue 2 | June 2021

Performance and Experiences across a Traditional, Hybrid,

and Online University College Curriculum

Veronique Dierkx, University College Roosevelt

Abstract

The switch from traditional to online education amidst the COVID-19 pandemic has left many wonder-ing what the impact will be on the learning experience of students. The current study compared examgrades obtained by university college students in a psychology course with a traditional, hybrid andonline class format (N=142) and distributed a questionnaire among other university college students(N=89) including items about levels of satisfaction, interaction and motivation in an online curriculumas compared to a traditional one. The main findings were that there was a statistically significant dif-ference in performance on one of three exams, in which the online grades were significantly higher thanthose obtained in the traditional and hybrid format. Moreover, levels of satisfaction, interaction andmotivation all dropped when students switched to an online curriculum. These findings are consistentwith previous research and could be explained by the inexperience of both students and professorswith an online curriculum before the pandemic, causing the learning experience to be less optimalbecause of the suddenness of the transition. As the frequency of interaction in class decreased, so didthe motivation and satisfaction of students, as these three have been shown to be highly correlatedby previous studies. The higher online grades in the last exam could be explained by the gaining ofexperience with online teaching towards the end of the semester, the lack of social activities that werepossible during this time or the increased cheating efforts in the online format.

Introduction

The performance of students in online or hybrid class for-mats compared to the traditional in-person class formatis dependent on various factors including student satisfac-tion, interaction, and motivation. [1, 2] Previous stud-ies that examined the relationship between these variableswere either dated, since improvements in technology aremade at a very rapid pace, or conducted before the pan-demic, meaning they failed to capture the suddenness ofthe switch and the effect that this had on students andtheir learning. Many of these studies found no differencesin the performance of students, but did report a drop instudent satisfaction, motivation and interaction in an on-line class format as compared to a traditional class format.[3, 4, 5, 6].

Present Study

The present study consists of two parts, the first exam-ining student performance across different class formatsand the second examining a self-report by students aboutthe differences between traditional and online education.

For Study 1 it was hypothesized that there would be nodifference in performance based on exam grades betweentraditional, online, and hybrid education in an introduc-tory psychology class. Study 2 examines the difference be-tween online and traditional education in terms of studentsatisfaction, interaction, and motivation. It was hypothe-sized that student satisfaction, motivation and interactionwould be higher in the traditional class format than in theonline class format.

Study 1 - Analysis of grades

Methods

Participants. The participants in the first section of thecurrent study were four groups of university college stu-dents (N =142) enrolled in the Introduction to Psychologycourse in the Fall 2019 semester, the Spring 2020 semester,the Fall 2020 semester and the Spring 2021 semester atUniversity College Roosevelt.

227| HUMAN BODY | Volume 5 - Issue 2 | August 2021

Materials

Instruments. The grades that were used in the com-parison were obtained by in-person exams conducted inthe Fall 2019 semester, the first half of the Spring 2020semester and the Fall 2020 semester, as well as onlineexams conducted in the second half of the Spring 2020semester and in the Spring 2021 semester. The in-personexams as well as the online exams included 50 multiplechoice questions with one correct answer and 3 alterna-tives sorted per topic. Three different setups were usedin the four semesters as a result of the teaching methodschanging according to COVID-19 restrictions (see Table1).

Analysis. To analyse the variations in grades be-tween the exams in the four semesters a one-way repeatedmeasures analysis of variance ANOVA was conducted inSPSS. Only two out of the three exams were used forthis analysis, as the results for the third exam of thespring 2021 students were not available at the time ofthe analysis. A second analysis was conducted compar-ing the results of the third exam of the fall semester 2019,spring semester 2020 and fall semester 2020 using a one-way ANOVA. The grades were analysed using a one-wayANOVA. To analyse the data SPSS version 26 was used,with an alpha level of .05 for all statistical tests.

Results

Two separate analyses were conducted to analyze the ob-tained grades in the three different class formats. Firstly,an ANOVA was conducted to evaluate the null hypothesisthat there is no change in student’s exam grades in Exam1 and 2 based on the class format (N = 116, see Table 2).The results of the ANOVA did not indicate a statisticallysignificant effect, Wilks Lambda = .98, F (2, 113) = .95,p = .39, η p2 = .02. There was no statistically significantmain effect for the exams, F (1, 71.26) = 1.16, p = .29.There was no statistically significant interaction effect be-tween the exams and class format, F (2, 58.26) = .95, p =.39. Thus, the null hypothesis was not rejected.

A one-way between subjects ANOVA was conductedto evaluate the null hypothesis that there is no difference instudent performance in Exam 3 in terms of grades, basedon the class format (N =119). There was a statisticallysignificant interaction between class format and studentperformance in Exam 3, F (2, 71.78) = 5.65, p =.005,therefore the null hypothesis was rejected. Levene’s testshowed that the variances for the grades of Exam 3 werenot equal, F (2,116) = 4.37, p = .015, η p2 = .07. Post-hoccomparisons to evaluate pairwise differences among groupmeans were conducted with the use of the Games-Howelltest since equal variances were not assumed. The test re-vealed statistically significant pairwise differences betweenthe mean scores of students in the online class format andtraditional class format, as well as between those in theonline class format and hybrid class format, p < .05. Themean scores of students in the traditional class format andthe hybrid class format did not significantly differ, p > .05(see Table 3).

Study 2 - Questionnaire

Methods

Participants. Participants in the second section of thecurrent study were university college students, from threedifferent university colleges, N =89: University CollegeRoosevelt (UCR), University College Utrecht (UCU) andUniversity College Maastricht (UCM), N =70, N =10, N=9, respectively. The ages of the participants ranged from18-26, with M=20.43. The participants were in differentyears of their bachelor’s degree, with N =14 being in their


first year, N =27 in their second year and N =48 in theirthird year. Out of the participants, N =75 identified asfemale, N =10 as male, and N =4 as neither male nor fe-male. The majority had the Dutch nationality (N=47),the other participants reported 22 other nationalities. 65Of the participants reported that they were currently re-siding in the city where they study, and 24 reported thatthey did not. All participants reported using a laptop orcomputer most frequently when following online classes.

Materials

Instruments. To analyse the students’ motivation, sat-isfaction, time spent on studies and participation in onlineclasses compared to traditional classes, students filled ina questionnaire (see Appendix) using Google Forms. Thequestionnaire was developed by Meeter et al. (2020) [6]and adapted to fit the educational format used at Univer-sity College Roosevelt.

Procedure. The questionnaire items were presentedin a Google Forms document, the link for the question-naire was distributed in the student Facebook groups ofUniversity College Roosevelt, University College Utrecht,and University College Maastricht, as well as in a generalFacebook group for university colleges in the Netherlands.Prior to the data collection participants were asked to signan informed consent item.

Analysis. Bivariate correlation analyses and fre-quency analyses were conducted to both explore the re-lationship between variables in the current online edu-cational format as well as to analyze how variables havechanged since switching to the online format.

Results

Frequency analyses show that the frequency of reported in-teraction and active participation in class (7-point Likert-scale items 15 to 18) are lower in the online class format(M=3.92, SD=1.37; M=4.24, SD=1.73) as compared tothe traditional class format (M=6.22, SD=.77; M=5.60,SD=1.57). Bivariate correlation analyses showed that theinteraction and active participation in class were both sig-nificantly correlated with student satisfaction in the onlineclass format in both lectures and meetings (r(87) = .260,p <.05; r(87) = .456, p<.01 for lectures and r(87) = .427,p < .001; r(87) = .336, p<.001 for meetings) and moti-vation in the online class format (r(87) = .308, p <.01;r(87) = .541, p<.01). Moreover, the level of student satis-faction in online lectures and meetings (M=4.13, SD=1.44;M=4.54, SD=1.34) was also lower than that in tradi-tional lectures and meetings (M=5.68, SD=.93; M=5.69,SD=1.03). The student satisfaction in online lectures wasalso significantly correlated to motivation (r(87) = .513, p<.01). Student motivation for their studies declined, from

M=5.82, SD=1.11 before COVID to M=3.79, SD=1.72after COVID. The motivation in the online format wassignificantly correlated with interaction and active partic-ipation and satisfaction which were all mentioned above,as well as hours per week spent on studies (r(82) = .264, p<.05). The reported attendance rate went down for bothlectures and meetings after the classes switched to the on-line format during COVID-19. The participants reportedattending 94% of all lectures and 93% of all meetings onaverage before the pandemic and attending 90% of bothafter the switch to online education occurred. In bothscenarios, attendance was required.

For the two qualitative items in the questionnaire (40and 41) the replies were coded and then sorted into varioustopics based on their similar content (see Appendix).

Discussion

The results of Study 1 indicate that out of the three ex-ams that are conducted in each semester, the performanceof students based on their grades did not show a statisti-cally significant difference in different class formats on thefirst two exams, but it did differ significantly on the thirdexam which means the hypothesis that students’ perfor-mance would not differ between class formats was onlypartly rejected. On the third exam, students obtaineda significantly higher score in the online format than inthe traditional and hybrid format. It was hypothesizedthat there would be no difference in performance basedon exam grades between traditional, online, and hybrideducation, and this hypothesis was therefore rejected forthe third exam but corroborated for the first two exams.The results concerning the first two exams are in line withprevious research [5] which found no differences in perfor-mance of the students but rather a decreased satisfactionrate, which in Study 2 of the present study is also the case.

The results of the second study show that the hy-potheses about motivation, interaction and satisfactionwere all corroborated, and this suggest that the overall at-tendance has gone down since switching to the online for-mat and that students’ expectations of their performancethis semester have also gone down. Students reported thatthe most demotivating elements of online education wererelated to the lack of interaction in class, the high work-load, and the increased amount of screen time. Not onlywas the interaction in class rated lower in the online classformat, but participants also rated their own level of ac-tive participation as being lower than it used to be in thetraditional class format. This was reflected in the openquestions, in which participants reported feeling discon-nected from their peers as well as their instructors andfinding the material more difficult to follow online. Thesereplies are consisted with the theory of the three types of


interaction as defines by Moore (1989), [7] the interactionof students with each other, with their instructor and withthe course content. Lowering these interactions could re-sult in a decrease in motivation and student satisfaction[2, 8, 9] and this exact effect could be observed in theresults of the second study. A lower amount of studentsatisfaction and motivation has been linked to a decreasein performance and this effect is even greater in online ed-ucation. [1, 10] The results of both Study 1 and Study 2are in line with previous research that reported that stu-dents performed equally well in a hybrid format and fullyonline format, but that they preferred the hybrid class for-mat over a fully only class format which led to a higherlevel of reported satisfaction. [11, 12]

An important element that plays a significant role inthe interpretation of the results in the current study is thecontext in which the switch to online education was made.As Graham, Woodfield and Harrison (2013) [13] pointedout, the proper infrastructure for online education is anessential component in providing high quality classes. Asuniversities were not fully prepared for the change to afully online class format, this could have led to the qualityof the education not being as optimal as it could have beenwith adequate preparation and training for both studentsand professors. [14] However, as classes get organized withadequate online interaction in mind and students learn toadapt their studying habits to an online setting, satisfac-tion could increase which in turn has a positive impact onthe grades, which in Study 1 is demonstrated by the sig-nificantly higher grades on Exam 3 in the online format.[15]

Unfortunately, a more negative interpretation of theimproved online results also exists. As students familiar-ized themselves with the online format, cheating efforts ononline exams have also increased. [16] Even though pre-cautions were taken by the instructors of the psychologycourse in Study 1 to minimize the possibility of cheating(by for example shortening the time limit on the exam),it still remains easier to cheat on an online exam thanon an exam in the traditional format. Measures that haveshown to be effective against cheating during online examsinclude proctoring [17] but as this was not implemented inthe psychology course the possibility that the grades weresignificantly higher because students cheated on the examexists.

Limitations

A limitation of the present study is that it analyses a hy-brid and online class format in the midst of a pandemic, itis thus more difficult to generalize these results to a situa-tion where the decision to switch to an online educationalformat was a deliberate and well-prepared one. The re-

sults of this study should therefore mostly be interpretedwith the context in mind and not outside of the COVID-scenario [14].

Directions for future research

A follow up study could be conducted at a later time asstudents will most likely have had much more experiencewith online and hybrid teaching by then and might havedeveloped new levels of efficiency, and institutions andtheir instructors might have had more time to set up amore permanent distance education system. In a differentscenario, students and instructors might get more drainedthe longer online teaching goes on. Moreover, future stud-ies should focus on improving the balance between thevarious sexes used in the study.

Conclusion

To conclude, when comparing a traditional, hybrid, andonline university college curriculum, there was only a sta-tistically significant difference in students’ performance inthe third exam, showing higher grades were obtained in theonline format. When looking at student satisfaction, mo-tivation, and interaction it became evident that the levelsof all three dropped when students received online classesas opposed to traditional classes. These findings are im-portant when studying the effects of the sudden transitionfrom traditional to online education in university collegesduring the COVID-19 pandemic, which continues to im-pact education worldwide.

References

1. Cho, M. H. & Kim, B. J. (2013). Students’ self-regulation for interaction with others in online learn-ing environments. The Internet and Higher Educa-tion, 17, 69-75.

2. Alqurashi, E. (2019). Predicting student satisfactionand perceived learning within online learning envi-ronments. Distance Education, 40(1), 133-148.

3. He, L., Yang, N., Xu, L., Ping, F., Li, W., Sun, Q.... & Zhang, H. (2020). Synchronous distance ed-ucation vs traditional education for health sciencestudents: A systematic review and meta-analysis.Medical Education.

4. Hortos K, Sefcik D, Wilson SG, McDaniel JT, Zem-per E. Synchronous videoconferencing: impact onachievement of medical students. Teach Learn Med.2013; 25(3): 211- 215.


5. Lyke, J. & Frank, M. (2012). Comparison of studentlearning outcomes in online and traditional class-room environments in a psychology course. Journalof Instructional Psychology, 39(3/4), 245.

6. Meeter, M., Bele, T., den Hartogh, C., Bakker, T.,de Vries, R. E. & Plak, S. (2020). College students’motivation and study results after COVID-19 stay-at-home orders..

7. Moore, M. G. (1989). Three types of interaction.

8. Hartnett, M. (2016). Motivation in online education.Singapore: Springer.

9. Tinto, V. (1998). Colleges as communities: Takingresearch on student persistence seriously. The reviewof higher education, 21(2), 167-177.

10. Woods Jr, R. H., (2002). How much communica-tion is enough in online courses?–exploring the re-lationship between frequency of instructor-initiatedpersonal email and learners’ perceptions of and par-ticipation in online learning. International Journalof Instructional Media, 29(4), 377.

11. Protopsaltis, S. & Baum, S. (2019). Does onlineeducation live up to its promise? A look at the evi-dence and implications for federal policy. Center forEducational Policy Evaluation.

12. Tallent-Runnels, M. K., Thomas, J. A., Lan, W.Y., Cooper, S., Ahern, T. C. & Shaw, S. M.(2006). Teaching courses online: A review of theresearch. Review of Educational Research, 76,93–135.10.3102/00346543076001093

13. Graham, C. R., Woodfield, W. & Harrison, J. B.(2013). A framework for institutional adoption andimplementation of blended learning in higher educa-tion. The internet and higher education, 18, 4-14.

14. Gacs, A., Goertler, S. & Spasova, S. (2020). Plannedonline language education versus crisis-prompted on-line language teaching: Lessons for the future. For-eign Language Annals, 53(2), 380-392.

15. Hodges, C., Moore, S., Lockee, B., Trust, T. & Bond,A. (2020). The difference between emergency remoteteaching and online learning. Educause review, 27,1-12.

16. Bilen, E. & Matros, A. (2021). Online cheating amidCOVID-19. Journal of Economic Behavior & Orga-nization, 182, 196-211.

17. Dendir, S. & Maxwell, R. S. (2020). Cheating in on-line courses: Evidence from online proctoring. Com-puters in Human Behavior Reports, 2, 100033.


Appendix


COVID-19 risk perception in Germany and its implications

for policy making

Emma Weßel, University College Roosevelt

Abstract

This world is in the midst of a COVID-19 pandemic. For successful virus containment, the adherenceof the public to behavioural measures is of great importance. The following paper is analysing the char-acteristics of risk perception in the German public, its intertwining with the adherence to governmentmeasures and subsequent implications for policy makers. Models for risk perception range from therational “Simplified Conjoint expected risk model” to the emotion based “Psychometric Paradigm”to combined, holistic models. The risk perception in Germany during the first months of COVID-19was high and in line with expert’s risk assessments. Still, overconfidence and an underestimation ofexponential growth were seen. The risk perception in Germany was driven by irrational dread risk.However, there is a substantial influence of rational factors on risk perception, driven by knowledgeand perceived control towards the virus. It is concluded that policy makers can rely on the rationalityof the German public towards both the implementation of and adherence to behavioural measures.This implies that communication towards the public of both the risks of the virus and the benefits ofthe measures are of great importance for successful policies against COVID-19 in Germany.

Introduction

Since it first emerged in China at the end of 2019, COVID-19 has substantially changed life all across the globe.As of 11 November 2020, the John’s Hopkins Universitycounted a total number of 51.817.846 COVID-19 infec-tions and 1.278.086 deaths related to the new virus world-wide. COVID-19 is posing a new and unknown healthrisk onto every individual and the general public has toreact to this new thread. People consciously and uncon-sciously perceive and process the risk of COVID-19 ona daily basis, leading to an individualised perceived risktowards COVID-19. During the pandemic, those percep-tions are additional variables to be taken into account inall decisions in daily life, both in personal and professionalcircumstances. It is known that people’s behaviour canfundamentally influence and alter the spread of a pan-demic (1). Governments are therefore acting to containthe spread of the virus by implementing widespread be-havioural measures. The behavioural guidelines are simi-lar in countries all around the globe and range from advis-ing 1.5m distance at all times to wearing face masks andclosings schools, shops and other public institutions.

However, measures are only successful if the publicis willing to cooperate and adopt this new, health protec-tive behaviour (2). There are three important relation-ships within the concepts discussed, as seen in figure 1.

The overall goal of containing virus spread can only bemet when the public is willing to cooperate in the healthprotective behaviour. Health behavioural change in a pop-ulation however heavily depends on risk perception. Themore accurate the perception of personal and societal risksin the public, the more successful public policies will be(2). Lastly, risk perception itself depends on several so-cial, emotional, cognitive and cultural variables that arespecific to both population and hazard (3,4).

Figure 1. : Summary of the main relationships be-tween the concepts of the paper. Individual risk perceptionis determined by hazard and population specific variablesas summarized in the risk perception models. Changein risk perception drives behavioural change that in turnleads to the intended change in public health.

In the following, those relationships are investigatedfor the German public in a time frame ranging from thebeginning of the pandemic in early March 2020 to the be-ginning of the second wave in the beginning of October


2020. Firstly, a good understanding of ’Risk perception’as a psychological concept is needed. The paper there-fore starts by introducing the most important models ofrisk perception. Then, the characteristics of risk percep-tion towards COVID-19 in Germany are analysed, includ-ing the factors that influence individual risk perception.This is combined with information on the adherence togovernment measures in the country. The models of riskperception will be applied to the COVID-19 situation inGermany to draw a conclusion on the implications for suc-cessful governmental measures in Germany. The objectiveof this paper is to give implications on how policies for theGerman public will be most successful when consideringthe variable risk perception.

Risk Perception Models

In the natural sciences, risk assessment can be a measur-able and objective unit such as probability or hazard whilein the social sciences risk is seen as subjective (5). Thepsychological construct of risk perception is influenced bycognitive, emotional, social and cultural factors as well asinterpersonal differences (2,4,6). Risk perception of indi-viduals is therefore not necessarily equal to the objective,scientific risk assessments of a hazard. Risk perceptioncan be both deliberative and affective, referring to judge-ments being both reason-based and based on uncertainties(3). To measure and conceptualise subjective risk percep-tion and the factors that influence it, several models havebeen proposed. The Simplified Conjoint Expected RiskModel has been designed by Luce and Weber in 1986. Itis a rational risk perception model derived from gamblingbehaviour. Perceived risk is seen as a weighted functionof the probability of harm, the probability of benefit, theprobability of the status quo, the expected harm and theexpected benefit of an activity (7,8). This model, be-longing to the axiomatic measurement paradigm of riskperception, describes single dimensional risk assessmentand is applicable in a situation where subjective or objec-tive information about a situation is present (5,7). Sec-ondly, using the Psychometric Paradigm, Paul Slovic hasincluded the irrational side of risk perception, evaluatingpeoples emotional response to a risk (5,6). It classifiesrisk as determined by two categories of factors, dread riskand unknown risk (9). Dread risk increases if the risk isseen as uncontrollable, fatal, having global catastrophicconsequences and posing a high risk to future generations.Additionally, dread risk rises if the risk cannot be easilydecreased and is involuntary. Unknown risk rises if a riskis not observable, unknown to those exposed, if the ef-fect is delayed or if a new risk is unknown to science (5).Covello has added to risk science by proposing three lin-ear relationships of risk perception. Firstly, the Mental

Noise Model highlights that the more stress an individualhas, the smaller is their ability to evaluate risk. The Neg-ative Dominance Model rules that fear, dread, and anx-iety create an environment where people focus more onthe negative messages of a risk. The Trust DeterminationModel shows that people trusting the actors and institu-tions dealing with a risk have a decreased perception ofrisk (9). Lastly, the holistic risk perception approach byvan der Linden aimed at building a comprehensive modelcombining the several determinants of risk perception seenbefore (4). The model includes socio-demographic, cog-nitive, empirical and socio-cultural factors forming onecombined theory (4). Each category has several variables.Gender, socio-economic status, age and education definethe socio-demographic category while affect and personalexperience are empirical variables. Knowledge is the cog-nitive variable and culture, values and worldviews relateto socio-cultural experiences (4).

Influence of Risk Perception on be-haviour

Risk perception is an important factor in this analysis as itdirectly relates to behaviour. This can for example be seenin the Health Belief Model where risk perception is onemajor determinant of change in behaviour (9). Althoughrisk perception is not the only variable for behaviouralchange, interventions that successfully change risk percep-tions have been shown to have great potential to changehealth behaviour (9).

Risk perception to COVID-19 inGermany

Health behaviour is hazard and population specific (10).Risk perception in Germany towards COVID-19 will bereviewed, dividing the current COVID-19 crisis into threestages; the first wave in the beginning of March 2020, theexponential growth phase during the end of March 2020and a phase of decreasing infections in May 2020.

Within the time of March 3 and October 27, between57% and 67% of Germans rated their own chance of get-ting infected with the virus as high or very high. Duringthe weeks, 56,3% to 74% rate the risk of their COVID-19infection as ‘severe’ or ‘very severe’ and 70% to 91,3% ofthe people have a medium or high perception of affectiverisk towards the illness. Still, the first wave is dominatedby an underestimation of exponential growth and a feelingof overconfidence in the public (11). Adults who have notyet tested positive for COVID-19 perceive a higher risk of


infection for members of their family or friends than forthemselves (12). Elderly people think they are less likelyto get infected while being more afraid of the consequencesof an infection than younger people (12). When estimat-ing the probability of being infected by the end of the year,the public’s perceptions were in line with experts’ estima-tions (11,13). However, the probability of the rare event ofbecoming severely ill and being hospitalized was substan-tially overestimated (11)..The public had a high knowledgeof COVID-19 and widespread trust in the acting author-ities (13,14). 76.2% said to trust hospitals, emergencyservices and other aid organizations to be able to protectthem; 71.0% trust authorities and medical experts while54.5% trust the political landscape (12). As compared tothe first wave, perceived risk was rising in the exponentialgrowth phase. Especially the perceived risk for a severeclinical course were higher (11,15). Risk perception washigher when COVID-19 was perceived as close, if someonewas chronically ill or when people had a negative feelingtowards the situation (16). When the curve flattened, theperceived risk was dropping. The probability of getting in-fected was seen as much lower which is in line with expertsestimations, however, especially people older than 50 yearsof age showed an optimistic bias (15), while the probabil-ity of getting severely ill was continuously overestimated(11). Variables that have been found to significantly in-crease peoples risk perception towards COVID-19 were be-ing female, being older, working in health care, knowinginfected people, having less knowledge about the diseaseand having an overall negative feeling towards COVID-19 (15). When people were asked to rate the dread risksof COVID-19, most people rated it as a global catastro-phe with a high risk for future generations that has fatalconsequences and is not voluntary but that can be eas-ily reduced and is controllable. Unknown risks variablesnamed were ’new’, ’observable’ and ’having delayed effects’(10,12). From the beginning of the pandemic onward, theGerman government took severe measures to prevent thespread of the virus. Those included, among others, a lock-down where stores, schools and other institutions had toclose, a travel ban to other countries as well as withinGermany and wearing face masks in public spaces. Asseen in the COSMO waves 1, 4, and 10, the measures hadwidespread public support in the beginning of the crisis.The support peaked in March and decreased since (15–17).In general, it can be seen that females, inhabitants ofcities, and people who personally know infected peopleadhered to the measures better (15–17). Approval also in-creased with increasing perception of ones own infectionrisk, severity of infection consequences, assumed positivelong-term consequences and the assumption that negativeshort-term consequences will decrease (11). What shouldbe noted is that regardless the high adherence to the mea-sures, protective behaviour was not voluntarily initiated

but only followed after government implementation (13).

Discussion

In Germany, risk perception towards COVID-19 is multi-dimensional and influenced by both rational and irra-tional factors. Variables from the axiomatic measure-ment paradigm, socio-cultural paradigm and psychometricparadigm can all be detected. An analysis of the psycho-metric paradigm as seen in figure 2 shows that the COVID-19 crisis fulfills variables for both dread and unknownrisk. While there are some variables, such as ’controllabil-ity’ and ’observability’, that decrease dread and unknownrisk, both variables play a significant role in the high riskperception for COVID-19 (11,12). The multi-dimensionalperformance of risk perception can be shown best whenapplying the holistic risk perception model of van der Lin-den (see figure 2). Socio-demographics are seen to play abig role in the perception of COVID-19. Being of femalegender, of older age or belonging to a risk group for thedisease increases the risk perception for COVID-19. Edu-cation has not been found to be an explanatory variableswhile the socio-economic status has not been thoroughlystudied in the reviewed papers. Of the empirical variables,personal experience can be seen to play a very importantrole. Personal experiences with COVID-19 patients, bothduring work and in ones personal life significantly increasesrisk perception. The second empirical variable ’affect’ wasalso significantly correlated with higher risk assessment.That a negative attitude towards the situation leads tohigher risk perception additionally supports the NegativeDominance Model by Covello. The socio-cultural variableshave not been reviewed sufficiently in this paper to makea statement about their influence. However, the cognitivevariable ’knowledge’ is noteworthy. Risk perception is seento increase when knowledge of the topic increases. This isa reversed effect to what has been proposed by risk percep-tion theories (11). The same can be seen for the variable’perceived control’. Opposite to what is proposed in theliterature, risk perception rises with increasing values ofperceived control towards the virus (11). A possible ex-planation of the reversed effects is the rational componentof risk perception in Germany. While the previous exam-ples and the highly perceived dread risks for COVID-19show the irrational part of risk perception, rationality ishigh in the German response. In theory, in cases of overes-timated, rare risks, the effects of knowledge and perceivedcontrol lead to a decrease in risk perception. However,in situations where risks are potentially underestimated,knowledge and perceived control stimulate higher risk per-ceptions. As this applies especially in the exponential riskphase of COVID-19, the reversed effects highlight the ra-tional side of the risk response (11).


Figure 2. : Variables increasing (arrow up) and de-creasing (arrow down) COVID-19 risk perception in Ger-many, as seen in the holistic risk perception approach (A)and psychometric paradigm (B). .

Further rationality in German risk perception canbe seen when looking at the second part of analysis,the reaction to the implemented government measures.There is widespread support for government measures withproblem-focused strategies seen to be favored in big partsof the German population. There is a will to follow ex-perts advise and to not act in the heat of the moment(12). The relationship between risk perception and be-haviour is undermined as approval increases with increas-ing perception of ones own infection risk, severity of infec-tion consequences and assumed positive long-term conse-quences (11). Applying the Simplified Conjoined ExpectedRisk Model shows that the same variables that have beennamed to influence the adherence to the government mea-sures can be seen to be part of risk perception. Infectionrisk is a probability of harm and severity of infection isan expected harm, while assumed positive long-term con-sequences are seen as expected benefits. All three vari-ables are part of the function of perceived risk by Luceand Weber. Adherence to the government measures istherefore connected to rational risk perception. However,next to the rational criteria, adherence to the measures isalso driven by dread and decreases slightly when negativeshort-term consequences are experienced (11).

Conclusion

The reaction to the first months of the COVID-19 pan-demic can already be studied and analysed but the pan-demic is an ongoing development. To further decreasethe detrimental spread of the virus, government measureswill remain important and so does the adherence to the

measures by the public. A decrease in acceptance of themeasures in the public has already been seen since March.In newer COSMO summaries it is visible that with thebeginning of the second wave in October the acceptanceincreases again, however, it has not reached the high levelsof March. It is therefore important that knowledge fromthe first months of the pandemic will be used to improvethe measures in place, the communication between policymakers and the public, and predict public responses. Forall of those goals, risk perception remains an importantvariable to consider (6).

What can be concluded from the previous risk per-ception analysis is that the German public has a rationalview on COVID-19. While being faced with a very un-known and unexpected situation the irrational factors ofrisk perception are present but not overwhelming. Instead,the German public was well informed and trusted its au-thorities. This implies that the communication of both thecurrent COVID-19 situation and current policies will playan important role. The willingness of people to informthemselves and follow experts advise should be used bypolicy makers. Measures should be taken to decrease thenumber of people underestimating the exponential growthphases and to tackle overconfidence (11). Potential fa-tal consequences for the individuals themselves and short-term negative consequences of the measures should besufficiently communicated as the German public did notpresent as short sided (11). Instead, they looked at thelong term effects and were willing to handle the short termnegative consequences (11). Additionally, future researchcould look into socio-cultural variables of risk perceptionand their influence on adherence. The sample in this pa-per can be seen to be rather homogeneous as all studiesfocused on the German public indicating similar culture,values and worldviews. However, both the cultural influ-ence on risk perception between countries and within theGerman public should be further researched to be able touse and apply other countries knowledge of public adher-ence and successful measures. The COVID-19 pandemicis far from over but using new knowledge gained duringthe first wave, both medical and psychological, can makechallenges in the coming months easier to tackle.

References

1. Epstein JM, Parker J, Cummings D, HammondRA. Coupled contagion dynamics of fear and dis-ease: Mathematical and computational explorations.PLoS One. 2008;3(12).

2. Dryhurst S, Schneider CR, Kerr J, Freeman ALJ,Recchia G, van der Bles AM, et al. Risk percep-tions of COVID-19 around the world. J Risk Res[Internet]. 2020;23(7–8):994–1006. Available from:


https://doi.org/10.1080/13669877.2020.1758193

3. Ferrer R, Klein WM. Risk perceptions and healthbehavior. Curr Opin Psychol. 2016;5:85–89.

4. van der Linden S. The social-psychological deter-minants of climate change risk perceptions: To-wards a comprehensive model. J Environ Psy-chol [Internet]. 2015;41:112–24. Available from:http://dx.doi.org/10.1016/j.jenvp.2014.11.012

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6. Slovic P. Risk Perception. Science (80- ).1987;236(4799):280–5.

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8. Luce RD, Weber EU. An axiomatic theoryof conjoint, expected risk. J Math Psychol.1986;30(2):188–205.

9. Paek H, Hove T. Risk perceptions and risk char-acteristics risk and risk perception: Definitionsand dimensions. Oxford Res Encycl Commun.2017;(March):1–15.

10. Aven T, Bouder F. The COVID-19 pandemic:how can risk science help? J Risk Res [In-ternet]. 2020;23(7–8):849–54. Available from:https://doi.org/10.1080/13669877.2020.1756383

11. Glockner A, Dorrough A, Wingen T, Dohle S. ThePerception of Infection Risks during the Early and

Later Outbreak of COVID-19 in Germany: Con-sequences and Recommendations. 2020; Availablefrom: https://doi.org/10.31234/osf.io/wdbgc

12. Gerhold L. COVID-19: Risk perception and Copingstrategies. Results from a survey in Germany. 2020;Available from: https://psyarxiv.com/xmpk4

13. Fuhrer A, Frese T, Karch A, Mau W, MeyerG, Richter M, et al. COVID-19: Wissensstand,Risikowahrnehmung und Umgang mit der Pandemie.Z Evid Fortbild Qual Gesundh Wes. 2020;(Jan-uary):153–4.

14. Betsch C, Korn L, Felgendreff L, Eitze S, Schmid P,Sprengholz P, et al. COVID-19 Snapshot Monitoring(COSMO Germany) - Thema Risikowahrnehmung(30.10.2020). 2020; Available from:https://projekte.unierfurt.de/cosmo2020/web/topic/risiko-emotionen-sorgen/10- risikowahrnehmung/

15. Betsch C, Korn L, Felgendreff L, Eitze S, SchmidP, Sprengholz P, et al. German COVID-19 Snap-shot Monitoring (COSMO) - Welle 10 (05.05.2020).PsychArchives [Internet]. 2020; Available from:https://doi.org/10.23668/PSYCHARCHIVES.2900




The Physiology of Erythropoietin, Medical Application, and

Abuse in Sport

Olivier Braas, University College Roosevelt

Abstract

Erythropoietin, a 34-kDa glycoprotein, is an essential hormone for the induction of erythropoiesis. Pri-marily occurring in the renal cortex, hepatocytes also contribute to erythropoietin synthesis. The 165amino acid chain consists of four α-helices, two β-pleated sheets and two disulphide bridges. Unableto be stored in the body, erythropoietin is under constant regulation by partial pressure of oxygen,haemoglobin -O2 affinity and -concentration, in a negative feedback loop. In hypoxic conditions,hypoxia-inducible transcription factors promote the expression of the erythropoietin gene, allowingrenal erythropoietin producing cells to transition from OFF-REP to ON-REP. When activated, ON-REPs produce erythropoietin, which binds to erythropoietin-specific receptors in the bone marrow,where it encourages differentiation and proliferation of haematopoietic stem cells. A variety of medicalconditions result in anaemia, including iron deficiency, chronic kidney disease and anaemia of chronicdisease. Renal fibrosis and chemotherapy for cancer patients also cause anaemia because renal cellsare consequently destroyed. Anaemic patients can be treated with erythropoietin stimulating agentssuch as human recombinant erythropoietin; however, this treatment cannot completely replace bloodtransfusions. In professional sports, erythropoietin has been abused as a performance enhancer, in-creasing haemoglobin count, and thus stamina and overall performance. In recent years, erythropoietinhas demonstrated potential for tumour cell marking, possibly opening a door to a new type of cancertreatment.

List of Abbreviations

• ACD – Anaemia of chronic disease

• Asn – Asparagine

• BFU-E – Erythroid burst forming unit

• CFU-E – Colony forming unit erythroid

• Cys – Cystine

• CKD – Chronic kidney disease

• Epo – Erythropoietin

• EPO – Erythropoietin gene

• Epo-R– Erythropoietin receptor

• ESA – Erythropoiesis-stimulating agent

• Hb – Haemoglobin

• HIF – Hypoxia-inducing transcription factors

• HSC – Haematopoietic stem cell

• PHD – Prolyl hydroxylase domain proteins

• pO2 – Partial pressure of oxygen

• RBC – Red blood cell

• REP – Renal erythropoietin producing cells

• rhEpo – Recombinant human erythropoietin

• Ser – Serine

Introduction

Erythropoietin (Epo) is a 34-kDa glycoprotein essentialfor the induction of erythrocyte production in mammals:erythropoiesis. The first mention of Epo occurred in the19th century, where it was observed that blood viscosityof people living in, or arriving from, high altitudes wasgreater than those living at sea level. Causal factors forincreased viscosity were unknown until an erythropoietichumoral factor was discovered in the first half of the 20thcentury by Miyake et al. in 1977 (1).

In human adults, the hormone is mainly producedin the renal cortex of the kidneys; however, during the


foetal stage of development, Epo is manufactured by hep-atocytes in the liver (2). Encoded by a gene on chro-mosome 7, located centrally (q11-q22) on the long arm(3), this relatively small protein consists of a 165 aminoacid chain, forming four α-helices, two β-pleated sheetsand two disulphide bridges (4). These bridges are locatedbetween the Cystine7 (Cys7)- Cys161 and Cys29-Cys33amino acids, and aid in solidifying the structure of theprotein. In addition to the disulphide bonds, the proteinhas four attached glycans (carbohydrate-based polymers),three of which are Nitrogen (N) linked, and one of whichis Oxygen (O) linked. These are attached to Asparagine24(Asn24), Asn38, Asn83, and to Serine126 (Ser126), respec-tively (4,5). Glycosylation, although marginally obstruct-ing the biological activity of Epo, is critical for guarantee-ing extended circulation of Epo in the plasma (2,6).

Because Epo plays a central role in erythropoiesis, itis in high demand in conditions where the red blood cellcount (RBC) is low. Additionally, it cannot be stored inthe body and must thus be produced based on demand.As a result, the concentration of Epo increases tremen-dously when the concentration of haemoglobin (Hb) in thebloodstream decreases (4). An Hb concentration below thenormal range (130-180 g/L and 115-165 g/L in males andfemales respectively), is labelled as anaemia (7). Anaemiacan be instigated by a list of physiological dysfunctions,the most prevalent being iron deficiency. In 2010, it wasestimated that around 32.9% of the world’s populationsuffered from anaemia resulting from iron deficiency (8).Anaemia, attributed to iron deficiency or another cause,poses serious health issues. As individuals have less avail-able RBCs, and therefore Hb, their bodies become poorlyoxygenated. Depending on the severity, this can result inserious tissue damage.

Tissue partial pressure of oxygen (pO2) is the variableregulating factor when it comes to Epo production andsecretion, and is influenced by Hb concentration, Hb-O2affinity, and arterial pO2 (4). When these values fluctu-ate due to hypoxia, so does the amount of circulating Epoand, ultimately, the number of erythrocytes produced inthe body by the bone marrow (1). By focusing on thetransport, synthesis, and effects of Epo, this paper aimsto discuss the process of erythrocyte regulation in the hu-man body. In addition, both the role of Epo in medicineand its highly debated use in sports will be discussed.

Synthesis, Secretion, and Effect ofEpo

As previously mentioned, in adults Epo is primarily pro-duced in the renal cortex which accounts for roughly 90%of total Epo, while the remaining 10% is (mostly) derivedfrom the liver. Specifically, Epo synthesis occurs in the re-

nal interstitial fibroblasts, located in the interstitial spacebetween the renal tubules (9,10). The cells that produceEpo are called renal erythropoietin-producing (REP) cells,and come in two states: OFF-REP and ON-REP. The for-mer being most prevalent, acting as a reserve for the lattertype, which is in shorter supply and the producer of Epo.The total number of REP cells remains constant in thebody, however, REP cell activation to ON fluctuates de-pending on the stress induced by hypoxia (9). This changeis depicted in Figure 1, for a normal, acute and chronicanaemic state.

Figure 1. : Normal, acute and chronic anaemia andthe corresponding levels of OFF-REP and ON-REP cells.OFF-REPs are depicted by red dots and are the mostprevalent in normal conditions. As the anaemia progressesfrom acute to chronic, the number of ON-REPs increases.The increase in ON-REPs is area-specific, as illustrated bythe acute anaemic condition.

The differentiation between OFF and ON-REPs isregulated by the erythropoietin gene (EPO). This isswitched on or off by the EPO promoter, which in nor-mal conditions, is suppressed by GATA-binding factor 2(GATA-2). However, in hypoxia, GATA-2 levels decrease,and an EPO enhancer is activated by hypoxia-inducibletranscription factors (HIF) (11). HIFs are complexes madeup of one α-subunit (HIF1α, HIF2α or HIF3α) and oneβ-subunit (HIF1β). HIF1α is usually hydroxylated byHIF prolyl hydroxylases (referred to as prolyl hydroxy-lase domain proteins, PHD) and degraded via von HippelLindau protein-mediated ubiquitination (1,12). However,during hypoxic conditions, PHD activity is blocked, al-lowing HIF2α proteins to dimerise with HIF1β subunits.The newly formed complex is then able to bind to EPO ’sregulatory region, hypoxia response element and ‘turn on’the gene. This binding has been suggested to occur up-stream from the start of the transcription site in the kid-ney, whereas in the liver it is known to be in the proximaldownstream region (13,14).

Once Epo has been synthesised and secreted into cir-


culation, it travels through plasma to the bone marrowof flat bones where it is then able to bind to an erythro-poietin receptor (Epo-R). This occurs at the colony form-ing unit erythroid (CFU-E) stage in erythrocytic progen-itors (Epo acts as an anti-apoptotic agent for erythrocyteprogenitors) (11). Once bound, Janus kinase 2 is acti-vated, resulting in the phosphorylation of a signal trans-ducer and activator of transcription 5 (STAT5), which hasbeen found to activate certain genes that are critical forthe survival, proliferation and differentiation of erythroidprogenitors. Moreover, during stress caused by hypoxia,the phosphorylation of STAT5 has been shown to speedup erythropoiesis (15). A haematopoietic stem cell (HSC)differentiates into a CFU-E (stage at which Epo bindsto Epo-R), from there the cell continues to differentiateto an orthochromatic erythroblast removing the nucleusby exocytosis. The result is a reticulocyte, which is thenreleased into circulation. Once released, the reticulocytecommences the maturation stage, in which the cell expelsits mitochondria and ribosomes, to finally become a ma-ture erythrocyte capable of carrying oxygen throughoutthe body (12,14,15).

Production of erythrocytes is regulated by an induc-ing signal and a negative feedback loop. When tissue is ina hypoxic state, HIF acts as an inducing signal to triggererythropoiesis. Once the tissue returns to normal condi-tions, a negative feedback loop causes the production ofEpo to subside, and erythropoiesis to stop. The action ofthis feedback loop is visible on Figure 2, where the hy-poxia as determined by O2 carrying capacity, saturationand affinity of Hb, regulate HIF to express EPO (6,11).

Figure 2. : Negative feedback loop of Epo. Low O2carrying capacity, saturation and affinity triggers HIF toexpress EPO (primarily in the kidneys), and thus result-ing in erythropoiesis. Increased circulating erythrocytesinhibit HIF and EPO expression.

Applications of Epo in Medicine

Evidenced by the description of Epo’s structure and rolein the body, it is clear that it is an essential protein tosustain life, as it induces erythrocyte production. Withouterythrocytes, the body would shut down as there would beno oxygen supplied to tissues throughout the body. Regu-lar functioning of Epo production and action is thus criti-cal, however, there are several conditions whereby erythro-poiesis is limited, and external intervention is required.

As mentioned above, iron deficiency-derived anaemiais the most common cause of anaemia world-wide. Thisrelatively simple form of anaemia is followed by anaemiaof chronic disease (ACD), or chronic anaemia (16). ACDis primarily caused by above-normal levels of interleukin6 circulating in the blood stream. This cytokine inhibitsthe production of tumour necrosis factor-α and stimulatesferritin production. Despite an increase in ferritin, the ab-sorbed iron is sequestered in intestinal endothelium cells,ultimately leading to anaemia (16).

Chronic kidney disease (CKD) is the most upcomingdisease contributing to constricted erythropoiesis. Dia-betes is becoming more prevalent every year, being themain inducer of CKD and speculated to be the cause of30-50% of cases, in high-, middle- and low-income coun-tries. CKD is classified by the kidney’s inability to fil-ter, where values of glomerular filtration rate below 60mL/min per 1·73 m² are indicative of the disease. A com-mon consequence of CKD is anaemia, the severity of whichincreases with decreasing filtration rate (17). More causesof anaemia include, but are not limited to, physical traumaand heavy metal poisoning (6).

Developments in technology and understanding ofEpo have allowed the production of erythropoiesis-stimulating agents (ESAs) such as human recombinanterythropoietin (rhEpo), which as the name suggests, stim-ulate the production of erythrocytes. Treatment foranaemia include the administration of ESAs. Syntheticdrugs like rhEpo function through the increased prolifera-tion of erythrocyte progenitors like BFU-E by encouragingthe differentiation to reticulocytes and finally erythrocytes(16). Although rhEpo has demonstrated to be quite ef-fective, with haematocrit levels returning within normalrange after a few days of treatment, it is also associatedwith a few complications. Most notably, an increased mor-tality rate and risk of thrombosis. Furthermore, 5-10% ofpatients treated with rhEpo have developed resistance tothe drug due to inflammation and infection, preventingthem from functioning in the body (19). Hypertensionis also a common comorbidity in patients treated withrhEpo, as the sudden surge of erythrocyte production in-creases blood viscosity and vascular tension (18). As risksof rhEpo treatment are not negligible, the use of this treat-ment should be carefully considered and monitored.


Administration of rhEpo has reduced the requirementfor blood transfusions in patients (decreasing chances ofblood transfusion related complications), however, it isimportant to note that it cannot be used to substitute atransfusion when blood loss is severe, as erythrocyte pro-duction is a relatively slow process (11,17). Effectivenessof ESA treatment can be hindered by iron deficiency; apatient might be able to produce more erythrocytes dueto the treatment, but without Fe2+ they fail to carry anyoxygen, and thus their condition would show no improve-ment (17). Various anaemic complications discussed aboveare summarised in Table 1, including the current treat-ment methods.

Patients may also receive rhEpo treatment as a resultof other underlying conditions, as anaemia derived fromdisease is not the only form in which anaemia presents it-self. For example, cancer patients who have been treatedwith chemotherapy often experience permanent damageto their renal cells, leading to anaemia derived from treat-ment. This is commonly treated with rhEpo. However,the mortality, and thrombosis risks are accentuated by thistreatment (6,20). In addition, rhEpo treatment may alsobe used as a precautious measure to reduce risk of anaemiaresulting from surgery. For example, patients scheduledfor hip (one of the primary erythrocyte-producing bones)replacements, are given rhEpo injections before and aftersurgery to reduce the need of blood transfusions. Note,however, that patients are only eligible for rhEpo admin-istration when their Hb levels are≥120 g/L, and treatmentshould be stopped when Hb values exceed this limit (18).Momentarily the cost-effectiveness and efficacy of this typeof treatment is still a topic of discussion within the clinicalcommunity (21).

Although the literature is relatively limited, it hasbeen suggested that Epo has a significant function outsideof erythropoiesis, where the focus lies around the centralnervous system. Research being conducted alludes to theneurotrophic, neuroprotective, antioxidative, angiogeneticand antiapoptotic potential of Epo (22). In cancer re-search, Epo receptors have been positively identified ontumour cells of the breasts. This discovery is notewor-thy because healthy non-erythroid precursor cells do notcontain Epo receptors, implying that this could allow spe-

cific targeting of a drug to tumour cells in breast cancerpatients (23).

Epo Abuse in Sport

As with any competition, participants of sport tourna-ments sometimes attempt to gain an edge on their oppo-nents, with disregard of ethics or health risks. This holdstrue for the use of Epo as a doping agent in sports. Oneof the first and most well-known cases occurred during the1998 Tour de France, when the physiotherapist of the Fes-tina cycling team was arrested for possession of more than400 Epo bottles (24). Since this incident, sports adminis-trations such as the World Anti-Doping Agency have beenerected, and development of procedures that detect sub-stance abuse in athletes has begun, thus officially startingthe fight for fair play in professional sport (25).

As drug detection methods became more efficient andreliable, so did the methods of doping. The desire to out-compete an opponent has grown to become significantlymore important than the health risks posed by use ofEpo. To such an extent that the increased risk of pul-monary embolisms, coronary thrombosis, arrythmias andstroke are being ignored (24). Even in the 2020 Tour deFrance, there have been accusations of Epo use to enhanceathletes’ performance by increasing their ability to carryoxygen to their muscles (26). The most effective, safe andlegal way to increase an athletes’ oxygen carrying capacityremains to be high-altitude training (27).

Discussion and Conclusion

This paper has outlined the physical, chemical, and struc-tural attributes of Epo and demonstrated the necessity ofnaturally occurring Epo in the body to function and sur-vive. Given the role of Epo in the physiological functioningof a body, it also plays a crucial role in the treatment of pa-tients suffering from anaemia derived from iron deficiency,ACD, CKD and other treatments including those for can-cer. Current treatment consists of the use of ESA’s suchas rhEpo. However, the increased risk of thrombosis andmortality still place this type of treatment up for debate,despite the concluded positive effect it has on the promo-tion of effective tissue oxygenation through erythropoiesisregulation. Besides medical applications, Epo has alsoreached the world of competitive sports, where it has beenabused as a performance enhancer by increasing oxygena-tion levels, boosting stamina, and overall performance.

The wide-spread use of performance enhancing drugsin sports creates an ethical problem among athletes as well.Namely, that the competition becomes unfair, and thoseother athletes are, as a result, encouraged to also startdoping to level the playing field, and have a chance at


competing. If every athlete in a competition is competingwith external performance enhancers, sport might becomemore about the technological application of medicine in-stead of human performance. The health risks that comewith such a development, would also mean that the prob-lems that may arise from such practice, are not only anissue for the rules of the game and the ethics they arebased on, but potentially also pose a threat to healthcaresystems.

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A literature review on Guillain-Barre syndrome

Daan Pijs, University College Roosevelt

Abstract

Guillain-Barre syndrome is an autoimmune inflammatory demyelinating polyneuropathy disorder inwhich the patient’s immune system damages the peripheral nerves. An infection often initiates the onsetof the condition. Guillain-Barre syndrome is hard to study, since it is a rare disease with mechanismsthat are not yet fully understood. Because of this, the medical community has yet to come withefficient drugs focused on Guillain-Barre syndrome. Once a drug that delays the progression of thedisorder is found, the life quality of patients with Guillain-Barre syndrome can be improved, and thescientific community will gain new insights into autoimmune diseases. With this knowledge, futurepatients could be saved and experience a reduced amount of trauma. Since the disease can be fatal, itis important to focus future research on Guillain-Barre syndrome.

Introduction

Guillain-Barre syndrome (GBS) is a rare neurological au-toimmune disorder that triggers inflammatory demyelinat-ing polyneuropathy. GBS rapidly affects the peripheralnervous system with a maximum disability peak on av-erage on day 14 [1]. The condition starts with sensorycomplications and reduced strength in the distal extremi-ties, and ascends rapidly to more proximal extremities ofthe body. The clinical presentation shows a heterogenouspath with various subtypes [2]. It is estimated that 1.70in 100.000 people will suffer from the condition [1]. Eventhough GBS is a rare condition, there are some meth-ods that can be used to diagnose the disorder, such asneurological examinations and a lumbar puncture whichanalyses elevated proteins [2,3]. The patient’s bodily func-tion and respiratory system are closely monitored to aidwhenever the disorder progresses. Patients are given ei-ther immunoglobulin infusions or plasma exchange pro-cedures, which otherwise could result in 3-7% mortalitychance [3,4,5]. Six months after reaching the maximumdisability peak, 1 out of 5 patients is still unable to walkindependently. A minority of patients will have residualpain and fatigue after the recovery phase [2,3].

Some patients may experience an atypical GBS vari-ant, with various symptoms deviating from the more com-mon GBS pathway. There are at least four different sub-types of GBS, which include acute inflammatory demyeli-nating polyneuropathy (AIDP), Miller Fisher syndrome;acute motor and sensory axonal neuropathy (AMSAN);and acute motor axonal neuropathy (AMAN) [20]. Virtu-ally all variants have overlapping symptoms, which makesit difficult to distinguish between the variants (fig.1).

Figure 1: Different GBS variants [2].

The most common variant of GBS is AIDP, AIDPpathogenesis gives an elevated cerebrospinal fluid (CSF)protein level, which supports the hypothesis of proteinleakage from the blood through the blood-nerve barrier[6]. The elevated CSF protein level in AIDP activates pro-tein degradation that causes autoantibodies to target celladhesion proteins, localized at Ranvier’s nodes [6]. Datasuggests that after analysing the AIDP CSF the levels ofcell adhesion proteins are degraded, but any dependenceon the autoantibodies levels cannot be found [7]. Thedamage to the nodes of Ranvier in the peripheral nervoussystem, caused by the autoantibodies, start an innate im-munity inflammation [6]. Figure 2 shows how the neuroncan get damaged in the most prominent forms of GBS.The damage to the peripheral nervous system is believedto cause the weakness and sensory signs in the legs andeventually escalate to other complications, although theexact mechanism is not understood yet [7].


Figure 2: The most prominent forms of GBS andtheir antibody-mediated effector pathway, resulting in dif-ferent kinds of neuron injury [3].

T-cells activate macrophages in the peripheral ner-vous system, which release free oxygen radicals and stimu-late cytokine production in the body. This results in dam-age to the blood-nerve-barrier and myelin sheaths, whichcauses demyelination and a reduced T-regulatory (Treg)cell concentration. The reduction of CD4+ and CD25+Treg levels are reversible with intravenous immunoglobu-lin treatment since it restores the old concentration levelsof Treg cells. However, the decline in Treg cells is notlinked to the different forms of subtypes in GBS. IFN-γproduction is stimulated in the peripheral blood of GBSpatients due to the interaction with GM-1 specific helper Tcells. During the acute phase of GBS, the cytokines inter-leukin 17 (IL-17), interleukin 22 (IL-22), and interleukin4 (IL-4) are increased. This results in the maturation ofT-cells into Th1 cells.

IgG, IgA, and IgM are associated with GBS. However,in contrast to oligoclonal antibodies, IgM antibodies arepolyclonal and on the same level at the T-cell formationagainst components in GBS. Note that GM-11 concentra-tions are fo1und more frequently in neurons with a ventralroot than in neurons with a dorsal root. This justifies, inpatients with anti-GM1, a more frequent incidence rate ofmotor neuropathy.

Although the exact cause of the disease is still beingstudied, there is a correlation between the disease and in-fections. Infections cause an autoimmune response, whichinitiates a reaction on the myelin sheaths and Schwanncells of sensory and motor nerves. Generally, it is possi-ble for peripheral nerve remyelination to naturally occur,while axonal regeneration is rare and slow. Damage to the

axons is, in most cases, irreversible if the damage occursalong the whole length of the nerve fiber [3]. After aninfection, GBS can develop in a matter of days or weeksafter the initial infection. Studies have shown that thereis also a link between the frequency of GBS cases andthe Zika virus, certain vaccinations, surgery, and possiblyHepatitis A, B, C and E [8]. Other risk factors include sexand age, such as patients who are male and patients whoare in their adolescent phase are more at risk [9]. Addi-tionally, elderly patients are also more susceptible towardsdeveloping GBS [7,10].

Certain infectious organisms have also been linked toGBS like C. jejuni, which creates antiganglioside antibod-ies that infiltrate the nerves [9]. The different antibodiescreated, and the different target areas in the nerve cell,support the idea of different variants in GBS. However,there is not enough evidence of patients with GBS directlyresulting from a C. jejuni infection. In essence, the patho-logical process is significantly influenced by other factorslike genotype. More research is required to find the exactgenes involved in GBS.

GBS can often be experienced as an impactful event,so it is important to offer psychological help to aid patientsin their physical recovery. While the psychosocial healthstatus in GBS patients was not altered significantly after a6-12 month period, the health status of the patient groupwas consistently worse than the control group throughoutthe duration of the study [11]. As the psyche aids in thephysical recovery, most patients with a typical form of thedisease will be admitted to a rehabilitation program, witha multidisciplinary team involved in their recovery. De-pending on the severity of the patient’s episode, a rangeof supportive care is required to help the patient with theireveryday activities. Rehabilitation ranges from weeks tomonths, which helps to recover daily activity in the patient[2].

How to Diagnose GBS

If there is a lack of adequate GBS biomarkers, the di-agnosis can be made based on clinical history, physicalexaminations, a CSF examination, neurological examina-tion, and electrodiagnostic studies [2]. Differential diag-nosis must be considered when GBS is suspected. Toexclude differential diagnosis, clinicians may subject sus-pected GBS patients to a complete blood count and ablood test for liver enzymes, kidneys, electrolytes, and glu-cose [2]. It is important to note that, while a positive testof anti-ganglioside antibodies can confirm GBS in a pa-tient, a negative test does not necessarily mean that thereis no active GBS [2,12]. Anti-GQ1b antibodies are mostprofound in the Miller Fisher Syndrome (MFS), which isused to confirm MFS [2].


CSF examination is used to rule out other causes ofmuscle weakness than GBS. The procedure should be per-formed during the initial evaluation of the patient [2,3]. InGBS the physician would observe a normal cell count butin combination with an elevated protein level [3].

Once a patient is admitted with bilateral weakness ofthe extremities, without any indicators for complicationsregarding the central nervous system, GBS should be con-sidered [2]. Patients often describe that the weakness isascending and had an origin in the distal extremities [3].Reflex responses are generally decreased or not present atall and dysautonomia is common followed by pain [13].Patients with GBS can exhibit symptoms in an atypicalmanner, children under the age of 6 years old are especiallyprone to this. Localized pain or the bearing of weight, canbe symptoms of GBS in babies. Signs of weakness andsensory problems are always bilateral, and predominantlyproximal or distal. It often starts in the legs, but can alsostart in the arms or all limbs simultaneously [2]. Clini-cians should be aware of the atypical forms of GBS to notfurther delay diagnosis. Even though in the typical form,the reflex responses are expected to be decreased, normalor exaggerated reflexes can be observed throughout thecourse of the disease in an atypical variant of GBS [2,14].

Treatment

When patients are having evolving respiratory distress,swallowing dysfunction, or rapid progression of weakness,they are admitted to the intensive care unit (ICU) [2].Around 22% of all the GBS patients need mechanical ven-tilation within the first week of hospitalization [2]. In ad-dition to regular check-ups of the body functions, treat-ment should be started as soon as possible. Treatmentexists out of either Intravenous Immune Globulin (IVIg)or plasma exchange. Both treatments have been shown tobe equally effective when you use 0.4 g/kg body weightdaily for 5 days of IVIg or when you use the plasma ex-change treatment, 200-250 ml of plasma/kg body weightin five sessions [2].

The combination of both treatments or giving onetreatment after the other does not show any benefit in thepatient’s health [3]. Data suggests that on the seven-gradedisability scale, following either treatment after a monthdoes not make a difference in grade [15]. Both these treat-ments are used for pregnant women & Children althoughIVIg is preferred as a treatment, as it seems to create lessdiscomfort in the patient [16].

Since GBS is an immune-mediated inflammatory pe-ripheral neuropathy disease, it might result in nerve dam-age that brings a reduced quality of life to the patient foran indefinite period [17]. Pain medication should, there-fore, start as soon as symptoms are noticed to relieve the

suffering of the patient, enabling faster rehabilitation [18].GBS causes the patient often to become immobilized

due to muscle weakness, which forms a risk factor for deepvein thrombosis. Patients, who are unable to walk inde-pendently, are recommended to use unfractionated hep-arin and support stockings to avoid the risk of deep veinthrombosis formation and the linked complications [2].

Recurrences are reported in 2-5% of patients, eventhough GBS is considered to be a monophasic disease [19].Recurrences are defined as two or more episodes of AIDPwith a disability peak difference of two months. It is diffi-cult to conclude why some patients have recurrences, andif this occurs more frequently in certain variants of thedisease. In the group of recurrences, patients are often un-der 30 years old. Studies have shown that in comparisonwith the non-recurrent group, the recurrent group oftenhas mild episodes [19].

Most studies regarding GBS were conducted in west-ern Europe or in the USA, in which certain types of GBSare more prevalent than in other parts of the globe [3]. Inwestern countries, the disease is on average observed in 1out of 100.000 people. Researchers conducted an interna-tional study in 2019, in which the results showed that theincidence chance of GBS increases by 20% every 10 yearsin a person’s life [2,5]. The age rate of GBS is among 0- to9-year-olds per 100.000 people, which result in 0.62 casesper year, while in 80- to-89-year-olds per 100.00 peoplethe GBS rate is 2.66 cases per year [5]. The disease occursmore in men than in women due to reasons scientists haveyet to discover [2,20]. Epidemiological studies show thatGBS has a slight peak in late adolescence, which may bedue to an increased risk of infections by cytomegalovirus(CMV) and C. jejuni [2,20]. The elderly are, also, moresusceptible to GBS, and possibly more susceptible to au-toimmune disorders due to decreased immune suppressormechanisms [20]. The different variants have different in-cident percentages around the globe due to geographicaldifferences. The typical GBS form, AIDP, is more preva-lent in the western world. It is believed that 90% of theGBS cases are AIDP’s [6]. GBS formation due to an in-fection by the C. jejuni is predominant in Asian countries,where 25-50% of adult GBS patients had a prior C. jejuniinfection [3].

There is still an insufficient amount of research con-ducted to show the possibility of a connection betweenimmunization and GBS. Even though the benefits of im-munization, such as the influenza vaccination, outweighthe potential risks for vaccines regarding GBS, the effectsof immunization should be researched more extensively.Recurrences seem to be rare after immunization, althoughthis is based on limited data regarding the relationship be-tween GBS and immunization [6]. During the acute phaseof the disease, immunizations are not recommended. Ifa specific immunization causes GBS within 6 weeks, the


specific immunization should be avoided in the future [4].More research should be conducted on the mechanisms ofthe pathology and the genes involved in the disease, toform an efficient drug against GBS.

Conclusion

There is still a long way to go to fully understand themechanisms behind GBS. With the possibility of futureoutbreaks similar to the Zika virus placing large popula-tions at risk, it becomes a necessity to study the effectsof GBS further. There are different ways to treat GBS,but a lot of patients will often be long term hospitalizedand have a difficult recovery process ahead. Therefore,more efficient medicine must be developed to reduce theeffects of GBS. Although medication is widely available forpatients in most medical centres, it should be noted thatpeople who are affected by GBS have a reduced qualityof life. Future research should focus on the mechanismsof the disease and the genes involved, contributing to abetter understanding of autoimmune diseases and savingmany lives in the process.

References

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2. Leonhard, S. E. (2019, September 20). Di-agnosis and management of GuillainBarre syn-drome in ten steps. Nature Reviews Neurol-ogy. https://www.nature.com/articles/s41582-019-0250-9

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5. Sejvar, J. J. (2011). Population inci-dence of Guillain-Barre syndrome: a sys-tematic review and meta-analysis. PubMed.https://pubmed.ncbi.nlm.nih.gov/21422765/

6. Ziganshin, R. H. (2016). The Pathogenesis ofthe Demyelinating Form of Guillain-Barre Syn-drome (GBS): Proteo-peptidomic and Immunolog-ical Profiling of Physiological Fluids. PubMed.https://pubmed.ncbi.nlm.nih.gov/27143409/

7. The Pathogenesis of the Demyelinating Formof Guillain-Barre Syndrome (GBS): Proteo-peptidomic and Immunological Profiling of Phys-iological Fluids. (2016, July 1). ScienceDirect.https://www.sciencedirect.com/science/article/pii/S1535947620335222

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10. Rath, C. (2017). Neuroinfectious Dis-eases: Guillain Barre Syndrome Clinical Fea-tures Suggestive of Early Diagnosis. Neuro-science and Neurological Surgery, 1(1), 01–03.https://doi.org/10.31579/2578-8868/020

11. Bernsen, R. A. (2010, April 1). Psychoso-cial dysfunction in the first year after Guil-lainanBarrA© syndrome. Wiley Online Library.https://onlinelibrary.wiley.com/doi/abs/10.1002/mus.21536

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Harnessing the power of exosomes: Using

exosome-delivered miRNA to modify dysregulated

α-synuclein expression in PD patients

Eleanor Dare Swanson, Amsterdam University College

Abstract

Parkinson’s disease (PD), a common neurodegenerative disease, is largely caused by the aggregationα-synuclein protein in neurons, which results in severe impairment of motor function among othersymptoms. α-synuclein protein levels are modulated by specific microRNAs in vivo. This study syn-thesizes the growing body of literature surrounding microRNAs and PD, identifying miR-34b and 34cas inhibitory towards α-synuclein -aggregates, among many other microRNAs. Exosomes, a small typeof extracellular vesicle, have been recently used in therapeutic approaches to cancer treatment. Thismeta-synthesis links exosome therapy cancer research to the role exosomes play in PD pathogenesis.Previous research shows that microRNAs may be shuttled by exosomes to the regions in the brainthat are implicated in PD. Using this knowledge about microRNA, this study proposes a novel genetherapy for PD using exosomes. It is hypothesized here that principles from exosome therapy can beapplied to PD treatment. The research focuses on reviewing existing literature on exosome shuttlemicroRNA with microRNA in PD research. The study describes how since microRNAs can downreg-ulate α-synuclein protein levels to prevent aggregation formation in PD, shuttling microRNA-loadedexosomes to crucial brain regions may be used to treat Parkinson’s disease. This hypothesized novelapproach to PD treatment is target-specific, low in side effects and capitalizes on grant money beingspent on the now-trendy exosome therapy research.

Introduction

“There is no cure” is the devastating news that is deliv-ered to new Parkinson’s disease (PD) patients, 60,000 peryear in the USA. (22) Like many other neurodegenerativediseases, PD is largely caused by abnormal protein forma-tion in the brain. (20) Its pathogenesis is linked to theaggregation of the protein α-synuclein in the brain, whichleads to impaired motor function. (25)

Unfortunately, all treatment options for PD aresymptomatic (not disease-modifying) and many of thepharmacological approaches have unpleasant side effects.(1) However, exosome therapy, a novel therapeutic ap-proach to cancer treatment, exosome therapy, may proveto be a new method for treating PD. In exosome ther-apy, scientists use extracellular vesicles called exosomesas prognostic biomarkers, anticancer drug-carriers, andtherapeutic targets. In many cell types, exosomes par-ticipate in intercellular communication by carrying bioac-tive metabolites or nucleotides, such as microRNAs.(5) Among exosome contents, microRNAs (miRNAs)are crucial since they maintain brain function by post-

transcriptional modification via gene expression. Recentresearch has shown that miRNAs play a critical role inPD pathogenesis. While the exact mechanisms of actionremain largely unknown, (18) several studies have notedthe influence of miRNA expression on α-synuclein levels.(4, 26) It is thought that abnormal expression of miRNAcauses dysregulation of α-synuclein formation, causing itsmonomers to cluster into oligomers and eventually largefibrils, which prove toxic to the brain.

While miRNA in exosomes have been studied for can-cer treatment, there has been little research into appli-cations of exosome-based therapy for the diagnosis andtreatment of neurodegenerative diseases. This paper ar-gues that exosome-delivered miRNA may be used to mod-ify dysregulated α-synuclein expression in PD patients,thereby exploring a potential disease-modifying treatmentfor PD. This meta-synthesis will find links between recentexosome therapy research and research on the role of miR-NAs in PD pathogenesis.


Understanding the basics of exo-some therapy

MicroRNA in exosomes

Exosomes, small, single-membrane vesicles, have recentlygained popularity in biomedical research as potentialbiomarkers and cellular messengers. They are generatedfrom multivesicular bodies and carry contents such as pro-tein complexes and nucleic acids from their origin cell toother cells via the circulatory system. (14) Before leavingthe origin cell, exosomal contents are marked by ubiquiti-nation whereby the cell decides to encapsulate them in anexosome. (5) At their destination, exosomes interact withthe recipient cell via specific receptor-ligand interaction.(25) The vesicles carry a “message in a bottle”, (13 p1)delivering cellular signals in the form of proteins, lipids,and nucleic acids.

One type of exosome content is miRNA. MiRNAsgenerally take part in regulating cell development, death,and differentiation, among other cell processes. (14) MiR-NAs can be selectively loaded into exosomes (19) andtransported between mammalian cells. In target cells,they modify protein production. (19) Over 2000 miRNAshave been mapped in the human genome, each of whichis thought to act on the expression of thousands of genes,making exosome shuttle miRNA a critical pathway of con-trolling gene expression. (19)

Protein level modification by exosome miRNA

Exosome shuttle miRNAs are a speedy and specificmethod to transmit cellular signals across great distances.Before being loaded into an exosome, miRNAs are sortedby the hnRNP protein family that recognizes 3’-end se-quences on the nucleic acid chains. The packaging of ex-osome cargo is influenced by environmental stimuli andthe physiological health of the organism. (20) After trans-portation, miRNAs arrive at the target cell and are re-leased from the exosome. There are currently three hy-potheses for mechanisms of release into the exosome, themost common of which is receptor/raft-mediated endocy-tosis. (19)

Once endocytosed into the target cell, the miRNAcan alter gene expression. The functioning miRNA com-plex, RISC, consists of minimally an AGO protein and amiRNA molecule. RISC is an RNA-induced silencing com-plex which uses miRNA or siRNA as a template for rec-ognizing a complementary mRNA strand. AGO protein isan Argonaute protein involved in RNA silencing, as well asdevelopment and differentiation. MiRNAs recognize theirtarget mRNA via the complementary base-pairing princi-ple, while the AGO proteins act as binding platform fortranscription factors and other accessory molecules. To-

gether, the miRNA and AGO attract a host of moleculeswhich ultimately alter gene expression and thus proteinproduction. (3)

Exosomes shuttle microRNA as a form of genetherapy

Exosome therapy capitalizes upon the ability of the vesi-cles to carry contents to a specific target cell via the circu-latory system. While exosomes have been studied since thelate 20th century, (22) up until recent years, exosomes weremerely regarded as vesicles to hold and transport cellularwaste products within the cell. (12) Today, major researchacknowledges the important role exosomes play in intercel-lular communication. This breakthrough led scientists toexplore the applications of exosomes and to treat and diag-nose cancers. Tumour-derived exosomes carry importantbiological information about the cancerous growth. ThemiRNA delivered by exosomes may be involved in can-cers in three ways: tumour suppression, initiation, and cellpreparation. In this way, certain miRNAs are up or down-regulated in different types of cancers. For example, thereis an increase in the miR-17-92 cluster in many cancertypes. While many RNAs get destroyed by the increasedlevels of RNases in cancer, miRNAs in exosomes stay in-tact, protected by the lipid membrane, making them viablecandidates for targeted treatment or for use as diagnosticbiomarkers. (19) Exosome shuttle drugs have several ad-vantages over free drugs: (1) they have transmembraneand membrane anchoring proteins, that enable a fast en-docytosis, (2) their surface proteins add specificity, whichenables highly targeted treatment. These proteins mayalso be modified to change the exosome’s delivery speedor specificity. An added benefit is the reduced side ef-fects of exosome-delivered drugs, compared to free drugs.(5) This targeted gene therapy approach has already beenproven in several studies. For example, the use of miR-21 sponge constructs to downregulate the expression ofmiR-21 in glioma cell lines U87-MG and C6 (17) or thetargeted delivery of miR-497 to treat non-small cell lungcancer. (10)

Exosome therapy and Parkinson’sdisease

MicroRNAs and Parkinson’s pathogenesis

PD symptoms have a dual cause: loss of dopaminergic neu-rons in the substantia nigra pars compacta and the forma-tion of Lewy bodies in neuronal cell bodies and processesfrom α-synuclein protein clusters. This study will focuson treating the protein aggregation in the brain. Pro-tein aggregation is controlled by non-coding nucleic acidsequences, among which are miRNAs. MiRNAs regulate


posttranscriptional gene expression via epigenetic mecha-nisms, and are thus implicated in many genetic disordersincluding PD. Changes in miRNA levels have been shownin various PD studies: many different miRNAs are cor-related with PD pathogenesis (Table 1). Goh et al. ex-amined the role of miRNAs in PD in a 2019 study bytesting 15 different miRNAs for implication in PD patho-genesis. They concluded that several miRNAs influencedisease progression by contributing to the accumulationof α-synuclein, among which are miR-30, miR-29, let-7,miR-485, and miR-26. Researchers of another study mea-sured miRNA levels in PD-affected vs normal brains andmeasured dysregulated levels of hsa-miR-671-5p, hsa-miR-19b-3p, and hsa-miR-24-3p. (24) Hu et al. saw decreasedlevels of miR-34b and miR-34c, which was accompaniedby a decrease in cell viability of dopaminergic neurons.(9) A further study identified involved miRNAs, profilingmiR-55, miR-26a, miR-146a, and mi132. (3) The studyconcluded that miR-155-5p was upregulated in most PDpatients compared to controls, while miRNA-146a-5p weredownregulated, while the other miRNAs did not showa significant change. (3) Replicating the findings of thestudy by Hu et al. (2012), Minones-Moyano et al. (2011)found miR-34b and miR-34c to be downregulated in PDpatients during clinical motor stages, affecting brain re-gions in the amygdala, frontal cortex, substantia nigraand cerebellum. These miRNA studies indicate a causallink between miRNA expression and the development ofParkinson’s via α-synuclein cluster formation.

Figure 1

MicroRNAs and α-synuclein

A crucial aspect of PD pathology, α-synuclein aggregation,is directly impacted by miRNA strands. Before becominga fully functioning control complex, miRNAs are createdand modified in several processes. First, miRNAs are tran-scribed in the cell nucleus, producing long pri-miRNAs.Later these pri-miRNAs are converted to pre-miRNAs byRNase III enzyme. The pre-miRNAs are transported intothe cytoplasm, where they undergo further modification toform RISC, a complex which can suppress and/or promotethe degradation of target mRNAs. (26)

According to Zhao and Wang, α-syn aggregation maybe initially caused by a mutation in the SNCA gene (theprocess is later modified by interaction with miRNA).When α-syn synthesis, aggregation, and clearance is outof balance, oligomers start to form. During disease de-velopment, aggregates slowly form bigger and bigger con-glomerates, ultimately shaping into large, insoluble fibrils.Smaller aggregates are found in axons and presynaptic ter-minals; the largest fibrils tend to form in the neuronal cellbody. (26)

There are several different miRNAs that act on α-synuclein expression. The 2019 study by Zhao and Wangsummarize miRNAs that directly and indirectly affect PDprogression. MiRNA-7 was shown to directly bind to α-synuclein mRNA, causing a decreased expression of theprotein by inhibiting translation of the mRNA. (11) WhenmiR-7 is knocked out in mice, it causes overexpression ofα-synuclein protein. (15) MiR-153 impacts mRNA degra-dation in such a way that it also downregulates α-synucleinexpression. (15) MiR-34b and 34c target α-synuclein di-rectly, inhibiting protein expression and aggregation for-mation. (15) In PD, levels of these last two miRNAs


are decreased in the amygdala. Finally, miR-214 plays asimilar neuroprotective role: upregulation of α-synucleinis linked to downregulation of miR-214. (15) AdditionalmiRNAs act indirectly on α-synuclein translation and ex-pression. These findings clearly show that expression ofα-synuclein can be manipulated by several different miR-NAs.

Treating Parkinson’s with exosome shuttle miRNA

α-synuclein levels have been shown to be influenced bymiRNA levels, making this protein a prime candidate forexosome shuttle miRNA therapy. Conveniently, most celltypes in the brain release exosomes. Oligodendroglia,where the α-synuclein fibrils are found in PD, are part ofthe exosome messaging network. (6) α-synuclein protein istransmitted via exosomes between synaptically connectedneurons. Applying techniques used in successful exosometherapy strategies for other diseases, this paper suggeststhe use of exosome shuttle miRNA to treat α-synucleinaggregations in the brain of PD patients. Loading exo-somes with specific cargo has been shown to be a success-ful strategy to artificially engineer transport of the vesic-ular contents. (20) The suggested strategy is thus to loadexosomes with miRNAs that downregulate α-synuclein inPD and inject them into the patient. The chosen miRNAswould be miR-7, miR-34b and 34c, and miR-214. Once in-jected, the miRNAs would be transported to the oligoden-drocytes. At their destination, the miRNAs would be en-docytosed and would enter the neuronal cell bodies, wherethey would act on the α-synuclein mRNA. This wouldinhibit translation/promote mRNA degradation, therebypreventing protein production, and ultimately, decreasingα-synuclein aggregation formation.

Conclusion

In summary, this research described the significant rolemiRNAs play in PD progression. Based on previous ex-osome therapy research, a hypothesis for a new form ofPD treatment is laid out. In short, a major pathway ofPD progression is caused by α-synuclein aggregation inoligodendrocytes, which is initially caused by a gene mu-tation but regulated by miRNAs. By injecting PD patientswith miRNA-loaded exosomes, the dysregulated produc-tion of α-synuclein protein in the brain can be modifiedand restored to healthy levels. Several different miRNAshave been shown to act directly on α-synuclein produc-tion. These would be the first to test for efficacy in thisnew treatment.

Given the published research about exosome therapyand the connection between miRNA and PD, it is plausibleto hypothesize that exosome therapy can be applied to PDby acting on miRNAs. Further testing is needed to decide

which specific miRNAs are most crucial for aggregation ofα-synuclein in the brain. Once these miRNAs are known,there are two strains of research to focus on: (1) identify-ing the best miRNA in exosomes for early diagnosis (usingexosome-delivered miRNA as a biomarker), and (2) engi-neering an exosome to contain the miRNA that will targetα-synuclein expression in crucial brain regions. Further-more, it should be investigated at which point in diseaseprogression α-synuclein levels are critical for pathogene-sis. In this way, administration of the exosome-deliveredmiRNA can be given at the correct time to decrease aggre-gation as much as possible, and therefore alleviate mostsymptoms. Additionally, the trajectory of the exosomefrom the point of injection needs to be mapped, to ensurethat the exosome will reach its target. It is possible thatthe exosome surface proteins would need to be engineeredto guide the exosome to the correct location.

This research is socially relevant, as it investigates asafe treatment option for the approximately 6 million peo-ple suffering from PD worldwide. (1) Exosome-deliveredtreatment has fewer side effects and capitalizes on the ex-osome’s natural function. Exosome-therapy research forPD would capitalize on grant money already being spenton exosome research and may clarify the still elusive mech-anisms of PD

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The Role of DNA Methylation in Regulation of BDNF and

Depression Neuropathophysiology

Maxence Liesenborgs, Amsterdam University College

Abstract

In December 2019, COVID-19 swept across the world, which resulted in a worldwide mandated lock-down and a surge of depression. Depression symptoms may include fatigue, appetite changes, insomnia,poor concentration, anhedonia, and suicide contemplation. At first, depression was thought to arise dueto a lack of monoamines, such as dopamine and serotonin, and thus the ‘monoamine hypothesis’ wascreated. Unfortunately, scientists quickly discovered this was outdated and created the ‘neurotrophinhypothesis’. This hypothesis explained that when facing stressors, neurotrophin levels decreased anda person’s vulnerability to depression increased. Neurotrophins are vital for neurogenesis and theirdecrease can lead to dysfunctional neurogenesis in brain regions responsible for emotion and cognition,such as the hippocampus. While neurogenesis is extremely crucial throughout embryonic and foetaldevelopment, the mechanism actually never stops. A neurotrophin that stood out was brain-derivedneurotrophic factor (BDNF). Through several studies, it was understood that stressors could increaseDNA methylation of BDNF, which reduced BDNF expression. Reduced expression meant that therewas a lower concentration of peripheral BDNF and thus, the likelihood of developing depression ishigher. In addition, the val66met BDNF genotype could also increase susceptibility to depression. Thecurrent research sheds light on how intricately DNA methylation of BDNF is involved in depressionneuropathophysiology and that its status may be a suitable biomarker for subtypes of depression.

Introduction

Mental illnesses, especially depression, spiked throughoutthe COVID-19 pandemic. In the United States alone, thepercentage of adults experiencing symptoms of anxietyand/or depression went from 11% in January-June 2019 to41.1% in January 2021 (1). In addition, before COVID-19,the price of depression for the U.S. economy was aroundUS$210 billion yearly. This cost is likely to continue torise, signifying that depression is very much relevant to to-day’s society’s mental well-being and the economy. Whilethere is a genetic explanation, environmental factors suchas social isolation, increased uncertainty, and decrease inexercise can also be triggers for depression (2,3). Depres-sion symptoms vary from neurovegetative, examples in-clude fatigue, changes in appetite or weight, and insomnia,to neurocognitive, including poor concentration, worsenedmemory, and taking longer to complete tasks (4). Addi-tionally, anhedonia, depressed mood and suicidal contem-plation can be counted as symptoms of depression (4).

Over the years, research on depression advanced fromthe ‘monoamine hypothesis’ to the ‘neurotrophin hypoth-esis’ (5,6). Neurotrophins are crucial for neurogenesis.Though neurogenesis is known to mainly occur during em-

bryonic development, it can continue in adulthood (7).More importantly, Nakagawa and Hishimoto (5) show thata decrease in neurotrophins can lead to dysfunctional neu-rogenesis in brain regions responsible for emotion and cog-nition. Demonstrating that depression is not a receptorbased illness, but rather a neural network malfunction.Along with other complications, this dilemma was why re-search on depression shifted to the neurotrophin hypoth-esis (5,6). One neurotrophin that stood out was brain-derived neurotrophic factor (BDNF). BDNF is crucial forneuronal survival and differentiation, neurogenesis, andsynaptic plasticity in the central nervous system and sincethe late 90s its function and assistance in regards to de-pression neuropathophysiology has been examined closely(8-10). However, Poon et al. (11) have clarified that therole of BDNF expression on behaviour during depressionlacks concrete research. This is partially because BDNFexpression needs to be further understood before the be-haviour during depression can be studied. As depressioncan also be induced by means of environmental factors,one way BDNF expression could be researched is throughan epigenetic lens. One way this can be done is throughstudying the relationship between DNA methylation ofBDNF and the neuropathophysiology of depression. Poon


et al. (11) already suggest that BDNF expression maypartially be modified through DNA methylation of BDNFexon IV.

This deductive synthesis based on existing literaturefirst further explores why BDNF was linked to depres-sion. Secondly, the role of neurotrophins in the humanbody is examined because this will elucidate the relation-ship between depression and neurotrophins. Next, thematuration process and signalling pathways of BDNF iselaborated upon in order to provide a background un-derstanding of the methylation status of BDNF. Further-more, existing clinical trials examining the association ofthe methylation status of BDNF and depression aetiologyis discussed. Lastly, the previously mentioned informationis reflected upon to determine how it can contribute tonew insight surrounding the neurtrophin hypothesis of de-pression. Through this it might be possible to determineto what extent the methylation status of BDNF could ren-der someone more vulnerable to depression. Therefore ex-ploring the possibility that the status may be a suitablebiomarker of depression.

The Monoamine Hypothesis inComparison to the NeurotrophinHypothesis

For several decades, research about depression wasreceptor-based and revolved around the ‘monoamine hy-pothesis’ (5). In brief, it was observed that during de-pressive states the concentrations of monoamines such asserotonin, dopamine and noradrenaline in synaptic gapswere decreased (5). As a result, most antidepressants weredeveloped to target this phenomenon. Unfortunately, twoproblems arise from this hypothesis. Foremost, these an-tidepressants take 2-4 weeks to exert their therapeutic ef-fects even though they are expected to work rapidly. Itmust also be noted, up to 30% of patients with majordepressive disorder may find the antidepressants to beineffective (5). The combined problems show that themonoamine hypothesis is outdated. In response to this,Duman et al. (6) proposed the neurotrophin hypothesisof depression in 1997. The hypothesis explored the beliefthat when facing stressors neurotrophin levels decreasedand led to depression. Neurotrophins are vital for neuro-genesis and their decrease can lead to dysfunctional neu-rogenesis in brain regions responsible for emotion and cog-nition (5). For example, Sanacora and Banasr (12) notedthat neural atrophy and a loss of glial cells was observedafter a decrease in hippocampal neurogenesis, which wasthe result of facing stressors in patients affected by mooddisorders, such as depression.

Why BDNF Specifically?

From 1995 to 2002, there were several research groupsthat explored hypotheses associating low BDNF serumlevels with depression (9,10,13,14). Some researchers es-tablished that depressive symptoms were able to be re-versed through central administration of BDNF (15). Thiswas only demonstrated in animal studies, though, which isonly an indicator that it might be the same in humans andnot a guarantee. However, in 2002, Karege et al. (16) suc-cessfully established the aforementioned in human trials.Their method included assaying BDNF serum levels us-ing ELISA and measuring the severity of depression withthe Montgomery–Asberg-Depression Rating Scale (16). In2005, Schumacher et al. (15) examined the BDNF genelocus to determine a genetic relationship. Now, it has be-come a well-known hypothesis that patients who are de-pressed show low serum levels of BDNF as a result ofreduced gene expression, which has been seen in post-mortem brain tissues and via peripheral blood samples(17,18). Likewise, throughout gestation, BDNF plays acritical role for neuronal development of the foetus, es-pecially regarding future mood disorders (19,20). Fur-thermore, BDNF has become one of the most extensivelystudied neurotrophins, especially in research about depres-sion aetiology. Nonetheless, whether depression is corre-lated or caused by low BDNF serum levels lacks evidence.Therefore, more research is needed in order to determineif BDNF could be a biomarker for depression.

The Role of Neurotrophins in theHuman Body

At first, neurotrophins were thought of as growth factors.However, they have recently been identified as a family ofproteins and their functions are integral for the central andperipheral nervous systems (21). The neurotrophin family,at least in mammals, consists of the nerve growth factor(NGF), brain-derived neurotrophic factor (BDNF), neu-rotrophin 3 (NT3), neurotrophin 4 (NT4/5), which mayall exist as a homodimer and/or heterodimer (22-24). Neu-rotrophins interact with two different classes of receptors,the Trk family of receptor tyrosine kinases and a specifictumour necrosis factor receptor p75NTR (22). Firstly,endogenous Trk receptors that mediate tyrosine kinasesignalling stimulate neuronal survival and differentiation(22). Trk receptors favour binding mature neurotrophinsand are selective when doing so. TrkA is activated throughthe binding of NGF, TrkB is activated through binding ofBDNF or NT4/5, and TrkC is activated through bindingof NT3 (24). Secondly, p75NTR is especially importantfor neuronal survival throughout development (22). Addi-tionally, p75NTR is able to be activated through binding


of all the aforementioned neurotrophins, but usually bindspro-neurotrophins (24). Moreover, the rough endoplasmicreticulum is where neurotrophin synthesis takes place andlater in the process are packaged in secretory vesicles (24).Based on certain signals, the pro-neurotrophins may bereleased to either bind to the p75NTR receptor or be pro-teolytically cleaved to produce mature proteins.

BDNF Precursor and MatureBDNF

For BDNF precisely, synthesis of the precursor, pro-BDNF, takes place mainly in the hippocampus and hy-pothalamus (25). After, pro-BDNF is proteolyticallycleaved to form mature BDNF that exists as a homod-imer. The maturation process of BDNF is supported byother mature neurons and glial cells (25). Moving on, bothpro-BDNF and mature BDNF have different functions asthey bind to different receptors and in turn initiate dif-ferent cascades of intracellular signalling. Pro-BDNF willbind to p75NTR, and launch pathways that lead to apop-tosis and suppressed neuronal growth (26). In contrast,mature BDNF binds to TrkB, which initiates two path-ways. Upon binding to the receptor, phospholipase Cγ1 isactivated and drives the activation of calcium/calmodulin-dependent protein kinases and protein kinase C. Simulta-neously the mitogen-activated protein kinase (MAPK) ex-tracellular signal–regulated kinase (ERK) pathway is stim-ulated. As both pathways function in parallel, they con-currently trigger cAMP response element binding (CREB)dependent transcription (11).

BDNF Signaling Pathways

BDNF gene expression is widely present in the adult ner-vous system, yet the hippocampus, amygdala, and cere-bral cortex are some of the locations where the expressionis at its highest (24,27). The BDNF gene has several non-coding exons and a coding exon. However, the IV exonhas been extensively studied and will be used as an exam-ple to explain one of BDNF’s pathways. The transcrip-tion of exon IV is regulated by the actions of methyl-CpGbinding protein 2 (MeCP2) and CREB protein (11). Onone hand, MeCP2 is involved in DNA methylation andrepresses transcription by recruiting histone deacetylasesand corepressor proteins, and favours binding to BDNFexon IV promoter to reduce its activity (28). On theother hand, an influx of Ca2+ can stimulate the disso-ciation of MeCP2 from the BDNF exon IV promoter inorder to resume BDNF gene transcription (11). Contrast-ingly to MeCP2, CREB is first phosphorylated by differentreceptor-activated protein kinases, calcium/calmodulin-

dependent kinases specifically for BDNF, after an influxof Ca2+ due to neuronal stimulation (11,29). Afterwards,CREB binds to the cAMP response element (CRE) ofthe promoters of the BDNF gene. Once CREB is boundto CRE, it becomes activated and CREB-binding protein(CBP) is recruited, which promotes transcription (29). Itis crucial to understand that BDNF gene transcription ismainly controlled through these two mechanisms. Cor-respondingly, it is interesting to see that through chang-ing BDNF gene transcription, DNA methylation of BDNFmay be influenced and as a result, serum levels of BDNFare altered, which could possibly determine whether or notsomeone is more vulnerable to depression.

Translational Perspective and Dis-cussion

After providing background information that has ex-plained the role of neurotrophins, why BDNF specificallyhas been focused on, and BDNF’s maturation process andsignalling pathways, the translational perspective will bediscussed. In 2015, Januar et al. (30) published a longi-tudinal study over 8 years, where they observed the asso-ciation of BDNF methylation, specifically at promoters Iand IV, and depression using buccal-derived DNA. The re-searchers recruited 1024 participants and determined theirclinical level of depression using an interview based on theDiagnostic and Statistical Manual of Mental Disorder IVcriteria. Their findings revealed diminished BDNF levelsin the plasma and post-mortem hippocampus of depressedindividuals (30). In this manner, the results presented acorrelation between elevated BDNF promoter methylationand depression, which could possibly be explained by theidea that hypermethylation leads to decreased gene expres-sion and eventually disturbed neurogenesis. As a result,decreased BDNF may lead to a decline in function of theBDNF gene in supporting neural growth and repair in de-pression. This was also noted in the paper by Sanacoraand Banasr (12). To recapitulate, Sanacora and Banasr(12) noted a loss of glial cells after a patient affected bya mood disorder, such as depression, faced a stressor andglial cells support BDNF maturation (25). Combined withJanuar et al. (30), this shows that depression is not sim-ply the outcome from an increase of BDNF methylation,but rather also further promotes it. This creates a loopbecause it continues to facilitate dysfunctional neurogen-esis in the hippocampus, therefore worsening depression.This insinuates that low BDNF levels and depression aredefinitely correlated. Moreover, Januar et al. (30) addi-tionally indicate that other studies also found a correlationbetween higher promoter IV methylation and depressionprevalence. Although they came to the conclusion thatBDNF methylation in buccal tissue could potentially be a


biomarker of depression, it is due to the scarcity of longi-tudinal studies in this area that they cannot fully confirmthis.

Limitations

Throughout the research, several limitations arose(16,30,31). To begin with, in the studies discussed, theway they diagnosed the participants with depression var-ied. For example, one study determined depression withMontgomery–Asberg-Depression Rating Scale, but an-other used the Diagnostic and Statistical Manual of MentalDisorder IV criteria (16,30). This variation could possiblyinfluence the perception of the measurement of BDNF lev-els as the difference between control and depressed groupcould become skewed. In a similar manner, depressionis a big umbrella term that represents various subtypes.For more accurate oriented research, terms should be nar-rowed down to specific subtypes, such as major depres-sive disorder and persistent depressive disorder. More-over, some studies measured serum BDNF levels and oth-ers measured plasma BDNF levels. It also became appar-ent that the variation of the BDNF gene in combinationwith the Serotonin Transporter Gene-Linked PolymorphicRegion (5-HTTLPR) genotype is also critical. A studyby Kaufman et al. (31) in 2006 explored how childrenwith the BDNF val66met variation combined with thes/s 5-HTTLPR genotype were the most likely to developdepression after maltreatment compared to children withthe same treatment and BDNF variation but the l/l 5-HTTLPR genotype. In addition, children with the BDNFval66val variation with the s/s 5-HTTLPR genotype aftermaltreatment were also more likely to develop depressioncompared to the l/l 5-HTTLPR genotype for the sameBDNF variation, but not nearly as likely as the val66metvariation (31). This reveals that the val66met BDNF vari-ation renders children more vulnerable to depression andthat when studies observe BDNF levels, they should alsotest for the BDNF polymorphism. Moving on, methyla-tion levels are usually tissue-specific and the levels mea-sured in blood cannot fully reflect the true readings in thebrain. However, the study done by Januar et al. (30)was done on buccal tissue, which has ectodermal origin,the same as neural tissue; compared to blood, which is ofmesodermal origin. Likewise, other advantages to buccalbiomarkers instead of blood-based markers is that they areless invasive and reduce cell heterogeneity (30). Lastly, un-fortunately there is also a lack of longitudinal studies thatmeasure BDNF methylation and depression.

Conclusion

Stressors, which can come in many forms, such as child-hood maltreatment, can possibly increase the level of DNA

methylation of BDNF. Increased levels of BDNF methy-lation normally leads to a diminished BDNF gene expres-sion and eventually to lower levels of BDNF concentration.As previously discussed, a lower BDNF concentration canmake a person more vulnerable to develop depression.BDNF is especially important because it influences neu-ronal survival and development, neuronal plasticity, andneurogenesis in areas of the brain that regulate emotionand cognition, for example the hippocampus. It was alsoseen that if depression is left untreated, it continues to di-minish the brain’s function, worsening the mental state ofthe patient. To conclude, current available research provesthat the DNA methylation of BDNF is highly involved inthe disease progression of depression and that its statusmay be a suitable biomarker for subtypes of depression.

Further Investigations

Further investigations in this area could explore the re-lationship between behaviour of a depressed patient andDNA methylation of BDNF. Similarly, how manipulat-ing the methylation status could alter this depressive be-haviour could also be investigated. Likewise, exon-specificBDNF transcription regulates memory processes and isconcerned in several neuropsychiatric disorders, whichmay include depression (11). Delving deeper in exon-specific BDNF transcription could be another approach tofurthering understanding of the role of BDNF and depres-sion. As a result, depression could potentially be treatedthrough epigenetically focussed approaches. Regardless,further longitudinal studies regarding depression and DNAmethylation are needed.

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