MiR-124-3p as a regulator for post-TBI recovery process

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NIINA VUOKILA

MiR-124-3p as a Regulator for Post-TBI

Recovery Process

Dissertations in Health Sciences

PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND

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MIR-124-3P AS A REGULATOR FOR POST-TBI

RECOVERY PROCESS

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Niina Vuokila

MIR-124-3P AS A REGULATOR FOR POST-TBI RECOVERY PROCESS

Publications of the University of Eastern Finland Dissertations in Health Sciences

No 597

University of Eastern Finland Kuopio

2020

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Series Editors

Professor Tomi Laitinen, M.D., Ph.D.

Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences

Associate professor (Tenure Track) Tarja Kvist, Ph.D.

Department of Nursing Science Faculty of Health Sciences Professor Kai Kaarniranta, M.D., Ph.D.

Institute of Clinical Medicine, Ophthalmology Faculty of Health Sciences

Professor Tarja Malm, Ph.D.

A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences

Lecturer Veli-Pekka Ranta, Ph.D.

School of Pharmacy Faculty of Health Sciences

Distributor:

University of Eastern Finland Kuopio Campus Library

P.O.Box 1627 FI-70211 Kuopio, Finland

www.uef.fi/kirjasto

Grano Oy, 2020

ISBN: 978-952-61-3630-1 (print/nid.) ISBN: 978-952-61-3631-8 (PDF)

ISSNL: 1798-5706 ISSN: 1798-5706 ISSN: 1798-5714 (PDF)

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Author’s address: A.I. Virtanen Institute for Molecular Sciences University of Eastern Finland

KUOPIO FINLAND

Doctoral programme: Doctoral Program in Molecular Medicine Supervisors: Noora Puhakka, Ph.D.

A.I. Virtanen Institute for Molecular Sciences University of Eastern Finland

KUOPIO FINLAND

Professor Asla Pitkänen, Ph.D, M.D, D.Sc.

A.I. Virtanen Institute for Molecular Sciences University of Eastern Finland

KUOPIO FINLAND

Professor Katarzyna Lukasiuk, Ph.D.

Nencki Institute of Experimental Biology WARSAW

POLAND

Reviewers: Professor Eleonora Palma, Ph.D.

Department of Physiology and Pharmacology University of Rome, Sapienza

ROME ITALY

Docent Mikko Airavaara, Ph.D.

Neuroscience Center, HiLIFE University of Helsinki HELSINKI

FINLAND

Opponent: Adjunct Professor Tiina Laitala, Ph.D.

Institute of biomedicine University of Turku TURKU

FINLAND

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As you know, in most areas of science, there are long periods of beginning before we really make progress.

– Eric Kandel

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Vuokila, Niina

MiR-124-3p as a regulator for post-TBI recovery process Kuopio: University of Eastern Finland

Publications of the University of Eastern Finland Dissertations in Health Sciences 597. 2020, 88 p.

ISBN: 978-952-61-3630-1 (print) ISSNL: 1798-5706

ISSN: 1798-5706

ISBN: 978-952-61-3631-8 (PDF) ISSN: 1798-5714 (PDF)

ABSTRACT

Traumatic brain injury (TBI) occurs when the brain is subjected to an external force, most commonly during traffic accidents and falling. The primary injury initiates cellular and molecular changes that can lead to the development of secondary injury and post-traumatic comorbidies.

Among the candidate targets for intervention are microRNAs (miRNAs), small non-coding RNAs that act as master regulators of gene expression. The aims of this study were to study post-TBI changes in neuronal miR-124-3p, to investigate the miR-124-3p controlled molecular networks and their connection to post-TBI pathology and recovery process, as well as to evaluate possible targets for therapeutic intervention.

Lateral fluid-percussion injury (FPI) was used to induce TBI in adult, male Sprague-Dawley rats. The post-TBI expression of the miR-124-3p targets was detected from the dentate gyrus with microarray, and from the perilesional cortex with RNA-Seq at 3 months post-TBI. Expression of miR-124-3p was detected from the dentate gyrus with droplet digital PCR (ddPCR) at 3 months post-TBI, and from the perilesional cortex with quantitative reverse transcription PCR (qRT-PCR) at 7 days and 3 months post-TBI. Two of the targets, Plp2 and Stat3, were detected in the dentate gyrus with qRT-PCR, and Stat3 in the perilesional cortex. In situ hybridization was used to detect intracellular level of miR-124-3p in individual cells in the dentate gyrus and perilesional cortex. In situ hybridization combined with immunohistochemical staining with cellular markers and hematoxylin eosin staining was used to investigate the localization of miR-124-3p. To investigate if the observed changes are TBI specific, the level of miR-124-3p, Plp2 and Stat3 in the dentate gyrus was measured with microarray at 7 days after status epilepticus. Samples of the human dentate gyrus and perilesional cortex were used to compare post-TBI miR- 124-3p expression in rats and humans. Bioinformatic methods (STRING, Reactome;

Ingenuity Pathway analysis, L1000CDS2) were used to study the role of miR-124-3p controlled networks, and existing pharmaceutical compounds that could be used to modify them. To further study the connection between miR-124-3p and cortical

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pathologies, tail-vein blood samples were collected at 2 days post-TBI, and magnetic resonance imaging (MRI) was conducted at 2 months post-TBI.

Microarray on the dentate gyrus and RNA-Seq on the perilesional cortex showed upregulation of a portion of predicted targets of miR-124-3p, suggesting that TBI causes downregulation of miR-124-3p. The upregulation of Plp2 and Stat3 in the dentate gyrus and Stat3 in the cortex were confirmed with qRT-PCR. PCR analyses indicated that TBI causes downregulation of miR-124-3p in the dentate gyrus and perilesional cortex next to the necrotic core of the lesion. In situ hybridization confirmed the intracellular downregulation of miR-124-3p in both areas. Microarray on the dentate gyrus indicated that the level of the precursors of miR-124 were not affected by TBI, suggesting impairment in the maturation of miR-124. The analysis in the dentate gyrus pinpointed the dysregulation of miR-124-3p in the granule cell layer. Further on, our in situ hybridization with samples of TBI patients show that similarly to rats, TBI causes intracellular downregulation of miR-124-3p in humans.

qRT-PCR and in situ hybridization suggest that similar changes occur in rat dentate gyrus after status epilepticus. Moreover, the cortices of sham operated controls had lower level of miR-124-3p in comparison to naïve animals, indicating that the loss of miR-124-3p is not TBI specific but rather a more general consequence of brain injuries. Bioinformatics analysis and text mining connected the targets of miR-124-3p to neuroinflammation and apoptosis. MRI analysis on the cortical athropy and perilesional inflammation revealed varied cortical pathologies. Linear regression model revealed that higher plasma level at acute time point after the induction of TBI explains the cortical lesion area at chronic time point, indicating that the miR-124-3p might relate to the cortical lesion development.

Together these findings indicate that brain injuries cause lasting impairment at the level of mature miR-124-3p in both rats and humans, that possibly contributes to post-injury hippocampal and cortical pathologies.

Keywords: microRNA, miR-124-3p, traumatic brain injury

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Vuokila, Niina

MiR-124-3p aivovamman jälkeisen paranemisprosessin säätelijänä Kuopio: Itä-Suomen yliopisto

Publications of the University of Eastern Finland Dissertations in Health Sciences 597. 2020, 88 p.

ISBN: 978-952-61-3630-1 (print) ISSNL: 1798-5706

ISSN: 1798-5706

ISBN: 978-952-61-3631-8 (PDF) ISSN: 1798-5714 (PDF)

TIIVISTELMÄ

Aivovamma aiheutuu aivoihin kohdistuneesta ulkoisesta voimasta, usein kaatumisen tai liikenneonnettomuuden yhteydessä. Alkuvaiheen aivovamma käynnistää molekyyli- ja solutason muutoksia, jotka voivat johtaa vaurion etenemiseen ja liitännäissairauksien kehittymiseen.

Yksi mahdollinen kohde aivovamman hoitoon ovat mikroRNA:t (miRNA:t), pienet ei-koodaavat ribonukleiinihappoketjut, jotka säätelevät geenien ilmentymistä.

Tämän projektin päämääränä oli tutkia hermosoluissa ilmentyvää miR-124-3p:tä, sen kontrolloimia molekyyliverkostoja ja niiden yhteyttä aivovamman liitännäissairauksiin, sekä etsiä mahdollisia molekulaarisia kohteita terapeuttiselle interventiolle.

Aivovamma aiheutettiin aikuisille, urospuolisille Sprague-Dawley rotille nestepulssimallilla. Geenien ilmentymistä aivovamman kolme kuukautta jälkeen tutkittiin dentate gyruksella mikrosirutekniikka käyttäen, sekä aivokuorella RNA- sekvensoinnin avulla. MiR-124-3p:n ilmentymistä mitattiin dentate gyruksessa kolme kuukautta aivovamman jälkeen digitaalisella pisara-PCR:llä (ddPCR), sekä aivokuorella seitsemän päivää ja kolme kuukautta aivovamman jälkeen kvantitatiivisella käänteistranskriptaasipolymeraasiketjureaktio -menetelmällä (qRT-PCR). Kahden miR-124-3p:n kohdemolekyylin, Plp2 ja Stat3:n, taso dentate gyruksessa mitattiin qRT-PCR menetelmällä. Stat3:n määrä mitattiin myös aivokuorelta läheltä vaurioaluetta. In situ hybridisaatio menetelmää käytettiin solunsisäisen miR-124-3p määrän tutkimiseen. Samaa menetelmää käytettiin myös yhdessä immunohistokemiallisten ja hematoksyliini-eosiini värjäysten kanssa miR- 124-3p:n paikallistumisen tutkimiseen dentate gyruksella ja aivokuorella. MiR-124- 3p, Plp2 ja Stat3:n ilmentymistä status epilepticuksen jälkeen mitattiin dentate gyruksesta qRT-PCR-menetelmällä, jotta voitiin päätellä, onko miR-124-3p:n muutos ominaista vain aivovammalle. MiR-124-3p määrä mitattiin myös aivovammapotilaiden dentate gyruksessa ja aivokuorella. Bioinformatiikan välineitä (STRING, Reactome; Ingenuity Pathway Analysis, L1000CDS2) käytettiin miR-124- 3p kontrolloimien molekyyliverkostojen sekä niihin vaikuttavien lääkeaineiden

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tutkimiseen. Saadaksemme lisää tietoa aivokuoren vaurion patologiasta, sekä sen yhteydestä miR-124-3p:hen, mittasimme miR-124-3p määrän rotan plasmassa kaksi päivää aivovamman jälkeen ja suoritimme magneettikuvantamisen samoille rotille kaksi kuukautta aivovamman jälkeen.

Geenien ilmentymisen analysointi osoitti, että kolme kuukautta aivovamman jälkeen dentate gyruksella sekä aivokuorella ilmentyy joukko miR-124-3p:n kohdemolekyylejä, mikä viittaa miR-124-3p määrän laskuun aivovamman jälkeen.

Plp2 ja Stat3 ilmentyminen dentate gyruksella, sekä Stat3:n määrä aivokuorella varmennettiin PCR-menetelmillä. Analyysimme osoitti, että aivovamma aiheuttaa miR-124-3p:n määrän vähenemisen dentate gyruksella sekä aivokuorella lähellä nekroottista vaurioaluetta. Mikrosiruanalyysi myös osoitti, että miR-124-3p:n esimuodot eivät muutu dentate gyruksella, viitaten siihen, että valmiin molekyylin valmistuminen häiriintyy aivovamman seurauksena. In situ hybridisaatio varmensi solunsisäisen miR-124-3p:n määrän laskun. Dentate gyruksessa vaurio nähtiin ennen kaikkea jyväissolukerroksessa. In situ hybridisaatio myös osoitti, että miR-124-3p:n määrä laskee myös aivovammapotilailla. Samankaltaisia muutoksia nähtiin dentate gyruksessa myös status epilepticuksen jälkeen. Tuloksemme osoittavat myös, että aivokuorella miR-124-3p:n määrä vähenee myös sham-operoiduilla kontrollielämillä naiveihin eläimiin verrattuna, mikä osoittaa, että miR-124-3p:n määrän lasku ei ole spesifistä aivovammalle, vaan yleinen seuraus aivon vauriosta. Bioinformatiikka- analyysi liitti miR-124-3p:n kontrolloimat molekyyliverkot erityisesti neuroinflammaatioon sekä ohjelmoituun solukuolemaan. Lineaariregressiomalli osoittaa, että akuutti plasma miR-124-3p:n määrän nousu selittää kroonisen leesion kokoa. Tämä viittaa siihen, että miR-124-3p liittyy aivokuoren vaurion kehittymiseen.

Yhdessä nämä tulokset viittaavat siihen, että aivovamma aiheuttaa pitkäkestoisen miR-124-3p:n määrän vähenemisen sekä rotilla, että ihmisillä, mikä saattaa edesauttaa aivovaurion aiheuttaman hippokampuksen ja aivokuoren patologian etenemistä.

Avainsanat: microRNA, miR-124-3p, aivovamma

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ACKNOWLEDGEMENTS

This study was conducted in the Epilepsy Research Laboratory at the A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, during the years 2015-2019.

I would like to express my sincere gratitude to my principal supervisor Noora Puhakka, PhD. Her genuine passion towards molecular science and research work has been inspirational during these years. I am grateful to Professor Asla Pitkänen, MD, PhD, for offering this opportunity to join the epilepsy research laboratory. She has impressive knowledge in epilepsy and trauma field, and I feel honored for being one of her students. I would like to thank my third supervisor, professor Katarzyna Lukasiuk, PhD, for her help and encouragement during my project.

I thank the reviewers, professor Eleonora Palma, PhD, and docent Mikko Airavaara, PhD, for offering valuable criticism. I am thankful for adjunct professor Tiina Laitala, PhD, for accepting the invitation to oppose this thesis.

My gratitude goes to professor Eleonora Aronica, MD, PhD, and her lab for helping me with setting up the in situ hybridization method, and for providing human samples for my study. Special thanks for Jasper Anink with his help with lab work.

I would like to thank all the co-writers of my articles, Katarzyna Lukasiuk, Anna Bot, Eleonora Aronica, Erwin van Vliet, Anatoly Korotkov, Salma Nuzhat, Jussi Tohka, Riina Huusko, Shalini Das Gupta, Noora Puhakka and Asla Pitkänen, for their valuable help during writing.

I thank all the past and present members of “Epiclub”: Merja Lukkari, Shalini Das Gupta, Tamuna Bolkvadze, Anu Lipsanen, Anssi Lipponen, Xavier Ekolle Ndode- Ekane, Sofya Ziyatdinova, Diana Miszczuk, Francesco Noé, Pedro Andrade, Tomi Paananen, Elina Hämäläinen, Mehwish Anwer, Niina Lapinlampi, Jenni Kyyriäinen, Johanna Hiltunen, Leonardo Lara Valderrabano, Ivette Bañuelos Cabrera, Mette Heiskanen, Natallie Kajevu, Robert Ciszek, and Jarmo Hartikainen. Special thanks go to the soul of the laboratory, Merja Lukkari. My deepest gratitude goes to my dear friends Mehwish Anwer and Niina Lapinlampi. Their support during this journey has been invaluable.

I want to thank the technical and administrative staff of A. I. Virtanen institute for helping me during these years. Special thanks to Joanna Huttunen for helping with the thesis, and Jari Nissinen for solving many technical issues.

I thank all my friends from the UEF Doctoral Student Association, especially Victor Carrasco Navarro, Miia Hurskainen, Juha Halme and Juha-Matti Huusko. I want to also thank Ekaterina Zhurakovskaya, and Ahmad Mahfuz Gazali for the fruitful conversations we had.

Sincere thanks to the wonderful and terrifying people of Xiong Mak Kung Fu Kuopio, especially Loris Borro, Ari Karvinen, Mirva Karhula, Juho Koponen ja

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Ellinoora Sirviö. Our quest for investicating ways to cause TBI without causing it to each other has been – and will continue to be- the highlight of my week.

I want to also thank Henri Karvinen, Jonne Rundgren and Atte Kuosmonen for bringing me back to earth. Jonne Rundgren proofread my thesis, and for that I am in dept. I want to thank all my friends from #Merivirta. Special thanks to Krista Juurikka, Meira Mankinen and Henna Salo.

I want to thank my family from both camps. Thanks for Brita and Seppo Vuokila for their advice in life. I want to thank Sanna, Jukka, Veera, Pekka, Jyrki, Eija, Ville and Eetu Pyykönen as well as Jaana Eskola, Janne Muikkula and Riikka Brofeldt for being on my side. My sincere gratitude goes to my father, Matti Pyykönen. His genuine interest in the nature around us inspired me to study science. In addition, it seems that stubbornness is genetical, and I have to thank him for providing the skills to finish this work.

Finally, I want to thank my husband, Simo Vuokila, who truly knows and understands me.

This project was supported by funding from the Academy of Finland, the European Union’s Seventh Framework Program (FP7/2007-2013) under grant agreement n°602102 (EPITARGET), The Finnish Epilepsy Research Foundation (Epilepsiatutkimussäätiö), Emil Aaltonen Foundation, Oskar Öflunds Stiftelse, and the University of Eastern Finland.

Kuopio, November 4th 2020 Niina Vuokila

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LIST OF ORIGINAL PUBLICATIONS

This dissertation is based on the following original publications:

I Vuokila N, Lukasiuk K, Bot A M, van Vliet E A, Aronica E, Pitkänen A, Puhakka N. miR‑124‑3p is a chronic regulator of gene expression after brain injury. Cellular and Molecular Life Sciences. 75:4557-4581, 2018.

II Vuokila N, Aronica E, Korotkov A, van Vliet E A, Nuzhat S, Puhakka N*, Pitkänen A*. Chronic Regulation of miR-124-3p in the Perilesional Cortex after Experimental and Human TBI. International Journal of Molecular Sciences.

21:7, 2020

III Vuokila N*, Das Gupta S*, Huusko R, Tohka J, Puhakka N, Pitkänen A.

Elevated acute plasma miR-124-3p level relates to evolution of larger cortical lesion area after traumatic brain injury. Neuroscience. 433:21-35, 2020

*These authors contributed equally to this work.

The publications were adapted with the permission of the copyright owners.

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ABBREVIATIONS

Ago Argonaute

aPeCx perilesional cortex adjacent to necrotic lesion core

APP amyloid precursor protein atm standard atmosphere, unit of

pressure

ATP Adenosine triphosphate AUF1 AU-rich-binding protein 1 AV aivovamma, brain injury BA Brodmann area

BACE1 β-site APP cleaving enzyme BBB blood-brain barrier

BCL-2 B-cell lymphoma 2

Bmp6 Bone Morphogenetic Protein 6

C2orf88 Chromosome 2 Open Reading Frame 88

CA3 cornu ammonis 3 CBZ carbamazepine

CCI controlled cortical impact Ccl2 C-C Motif Chemokine Ligand

2

CCR4 C-C Motif Chemokine Receptor 4

Cdk6 Cyclin-dependent kinase 6 circRNA circular RNA

GLT-1 glutamate transporter 1 CT computed tomography Ctdsp1 CTD Small Phosphatase 1

CTE chronic traumatic

encephalopathy

d days

ddPCR droplet digital polymerase chain reaction

DGCR8 DiGeorge syndrome chromosomal region 8 dPeCx perilesional cortex distal to

necrotic lesion core

Eci2 Enoyl-CoA Delta Isomerase 2

f female

FOV field of view Foxq1 Forkhead Box Q1 FPI fluid-percussion injury Frmd8 FERM Domain Containing 8 GAS5 Growth Arrest Specific 5 GCS Glasgow Coma Scale GFAP glial fibrillary acidic protein GSEA Gene Set Enrichment

Analysis Gsn Gelsolin

H&E Hematoxylin-eosin HC hippocampus

HSC70 heat shock cognate 71 kDa protein

HSP90 heat shock protein 90 HuR human antigen R

Iba1 ionized calcium binding adaptor molecule 1

IL-1B interleukin 1 beta

ILAE Internationa League Against Epilepsy

Lamc1 Laminin Subunit Gamma 1 LEV levetiracetam

Limch1 LIM And Calponin Homology Domains 1 Lrrc57 Leucine Rich Repeat

Containing 57 LTG lamotrigine

m male

MFE minimum free energy mGluR1 metabotropic glutamate

receptor 1 miRNA microRNA mm millimeter

mo month

MRI magnetic resonance imaging Mrlp44 Mitochondrial Ribosomal

Protein L44 mRNA messenger RNA Myo10 Myosin X

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NeuN neuronal nuclei

NLS nuclear localization signal NOT Negative Regulator Of

Transcription 1 Nrep neuronal-regeneration-

related protein

PABPC Poly(A) Binding Protein Cytoplasmic

PABPC Polyadenylate-binding protein 1

PAN2 Poly(A) Specific

Ribonuclease Subunit PAN2

PAN3 Poly(A) Specific

Ribonuclease Subunit PAN3 Plp2 Proteolipid Protein 2 Ppfibp2 PPFIA Binding Protein 2 Prkd1 Protein Kinase D1

Prrx1 Paired Related Homeobox 1 Ptbp1 Polypyrimidine Tract

Binding Protein 1 PTE post-traumatic epilepsy Rab27a RAB27A, Member RAS

Oncogene Family

Rbms1 RNA Binding Motif Single Stranded Interacting Protein 1

REST RE1-Silencing Transcription factor

Rhoj Ras Homolog Family Member J

RISC RNA-induced silencing complex

RNA ribonucleic acid RT reverse transcription

qRT-PCR quantitative reverse transcription polymerase chain reaction

SE status epilepticus

Slc7a2 Solute Carrier Family 7 Member 2

Stat3 Signal Transducer And Activator Of Transcription 3 Swap70 Switching B Cell Complex

Subunit SWAP70 TBI traumatic brain injury TC temporal cortex

TDMD target RNA-directed miRNA degradation

TE echo time

Tjp2 Tight Junction Protein 2 Tln1 Talin 1

Tmbim1 Transmembrane BAX Inhibitor Motif Containing 1 TNF tumor necrosis factor alpha TNRC6 trinucleotide repeat

containing 6

TNRC6 Trinucleotide repeat- containing gene 6A protein Tom1L1 Target Of Myb1 Like 1

Membrane Trafficking Protein

TR repetition time

TRBP transactivation response element RNA-binding protein

tSAH traumatic subarachnoid haemorrhage

Vat1 Vesicle Amine Transport 1 WHO World Health Organization Vim Vimentin

X-ALD X-linked

adrenoleukodystrophy Zfp36l2 ZFP36 Ring Finger Protein

Like 2

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1 INTRODUCTION

Traumatic brain injury (TBI) refers to a brain injury caused by mechanical external force to the head (Menon et al. 2010), most commonly caused by falls and traffic accidents (Maas et al. 2017). TBI is the main cause of injury related deaths and disabilities (Majdan et al. 2016; Maas et al. 2017; WHO, 2013). The primary injury triggers cascade of molecular and cellular changes, leading for example to altered gene expression (Huang et al. 2019; Lipponen et al. 2016), and chronic neuroinflammation (Johnson et al. 2013; Thelin et al. 2017). These changes contribute to the development of secondary injury, that might trigger post-traumatic comorbidities such as cognitive impairment (Fleminger et al. 2003; Li et al. 2016) Parkinson’s disease (Gardner et al. 2015; Kenborg et al. 2015; Crane et al. 2016) and epilepsy (Walsh et al. 2017). While effort should be put to reduce the number of new TBI cases, the studying of TBI recovery process is essential to find biomarkers and possible molecular targets for therapeutic intervention.

One of the epigenetic factors possibly contributing to post-TBI response in brain are microRNAs (miRNAs). These small non-coding RNA molecules suppress gene expression by binding to messenger RNA and suppressing its use for translation of genetic code to amino acid sequence (Lee et al. 1993a; Lee & Ambros 2001; Lau et al.

2001). One miRNA can have even hundreds of possible targets (Lewis et al. 2003;

Giraldez et al. 2006; Baek et al. 2008). Hence, a change in miRNA expression can lead to drastic changes in cell function.

The most prevalent miRNA in brain is microRNA-124-3p (miR-124-3p), a neuronal and evolutionally well conserved miRNA that is connected to neuronal identity (Neo et al. 2014; Kutsche et al. 2018; Lagos-Quintana et al. 2002; Smirnova et al. 2005; Papagiannakopoulos & Kosik 2009; Cheng et al. 2009; Makeyev et al. 2007).

The aim of this thesis was to study the post-TBI changes in of miR-124-3p expression, to investigate the role of the targets in post-TBI recovery, and to estimate if modifying effects in this miRNA could serve as a target for treatment.

The level of miR-124-3p was measured with PCR methods from the dentate gyrus at chronic time point and perilesional cortex at subacute and chronic time points. In situ hybridization and intensity analysis of miR-124-3p positive cells was conducted in both experimental TBI model on rat and samples from TBI patients. Microarray and RNA-Seq data were used as a basis for the bioinformatics analysis that aimed to investigate the role of differentially expressed targets of miR-124-3p. Finally, in order to investigate if miR-124-3p has a connection to chronic lesion development, plasma samples were collected from acute timepoint and MRI analysis was conducted on the perilesional cortex at chronic timepoint.

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2 REVIEW OF THE LITERATURE

2.1 TRAUMATIC BRAIN INJURY

2.1.1 Definition and classification

The term traumatic brain injury (TBI) refers to situation, where head is subjected to external mechanical force, to separate it from internal damages caused by - for example- drugs and alcohol (Maas et al. 2017; Carroll et al. 2004; WHO, 2013). TBI can also be defined as “alteration in brain function, or other evidence of brain pathology, caused by external force” (Menon et al. 2010), encompassing better more subtle TBI cases. The external force causes brain to move inside skull and get further damaged by collision with dura, meningeal membrane and neurocranium. The force can be either penetrating or not, meaning that TBI might occur also due to acceleration and deacceleration, and have no obvious superficial signs of head injury.

“Alteration” in brain function might manifest as loss of memory, neurologic deficits, such as loss of balance and aphasia, changes in mental state, meaning for example disorientation and confusion (Menon et al. 2010). The injury might be detected with brain imaging techniques, that can reveal bruising and swelling of the brain. Based on the injury mechanism, the injury can be a focal or diffuse. Focal injury encompasses subdural, epidural and intracranial hemorrhage (Abu Hamdeh et al.

2018b). Diffuse injury instead includes shearing of axons (Smith et al. 2000; Adams et al. 1989). Interestingly, the injury types contribute differently to development of post-TBI comorbidities (Abu Hamdeh et al. 2018a, WHO, 2013). However, patients can suffer from a mixture of the two types. For example, a study conducted on 159 patients with moderate or severe TBI found that 28% had “pure” focal injury, 22%

“pure” diffuse injury and remaining 50% had a mixture of them (Skandsen et al.

2010).

To assess the severity of TBI, patients are classified based on Glasgow Coma Scale (GSC) that assesses patients ocular, verbal and motor response (Teasdale and Jennett 1974). Each of the three categories are scored and total score divides patients to mild (GCS 13-15), moderate (GSC 9-12), and severe (GSC 3-8) TBI. Mild TBI – including concussion – account for 90% of the all TBI cases (Maas et al. 2010).

Severe TBI has high mortality rate, approximately 40% (Rosenfeld et al. 2012), and the patients that survive the acute phase often suffer from variety of disabilities, ranging from cognitive impairment to diseases such as Parkinson’s. However, disabilities are not restricted to severe cases, but they often manifest in patients with mild or moderate TBI, indicating that GCS does not reflect the pathoanatomical changes caused by TBI (Maas et al. 2017). Thus it is recommended that patients undergo computed tomography (CT) or skull X-ray if there is suspicion of TBI (Maas et al. 2017; WHO, 2013).

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2.1.2 Cellular and molecular changes caused by traumatic brain injury The initial physical hit to the brain causes – for example – blood-brain barrier (BBB) damage, axonal injuries and cell loss. However, studies indicate that after first necrotic cell loss occurs a second wave of cell death by programmed cell death, apoptosis (Wagner et al. 2011; Lenzlinger et al. 2002; Clark et al. 2000; Ng et al. 2000;

Zhang et al. 2005; Yakovlev et al. 1997). Studying of the translation of physical phenomenon into molecular level has revealed a complex network of reaction that often intertwine and affect each other (Figure 1).

The mechanical stretching of neuronal membrane is thought to create micropores leading to sodium influx that opens voltage-gated calcium channels and releases neurotransmitters (Greve and Zink 2009; Krishnamurthy and Laskowitz 2016). One of the neurotransmitters, glutamate, is thought to be the primary culprit in triggering excitotoxicity as it increases the level of intracellular calcium by binding to ionotropic receptors, mainly NMDAR (Faden et al. 1989; Palmer et al. 1994). Studies on experimental models of TBI and TBI patients have shown that brain injury causes increase in extracellular glutamate (Baker et al. 1993; Zhang et al. 2001; Faden et al.

1989; Palmer et al. 1994). The elevated level of extracellular glutamate increases with the severity of trauma (Faden et al. 1989; Palmer et al. 1994). When the abnormal level of glutamate persists or continues to increase, it elevates the mortality (Chamoun et al. 2010). The intracellular calcium elevates the amount of free fatty acids and free radicals by activation of peroxidases, proteases and phospholipases, which contribute to membrane degradation and finally to cell death (Werner and Engelhard 2007).

Parallel with this neuronal event, TBI causes downregulation of astrocytic glutamate transporter 1 (GLT-1) , that is needed for clearing of extracellular glutamate (Rao et al. 2001; Yi & Hazell 2006; Van Landeghem et al. 2001), contributing to the excitotoxicity. Further on, TBI is known to increase the number of gap junctions, that are critical during development of central nervous system but are downregulated at by 4^th postnatal week (Wang et al. 2012; Todd et al. 2010; Hartfield et al. 2011). This allows flow of calcium and other pro-apoptotic messengers to neighboring cells creating so called “bystander effect” where otherwise uninjured cells undergo apoptosis (Ripps 2002; Decrock et al. 2009).

Closely related to the excitotoxity is activation of calpains, cysteine proteases, by calcium influx. In non-pathological situation calpains take part in synaptic remodeling (Lynch and Baudry 1987) and Wnt-signaling (Abe & Takeichi 2007; Liu et al. 2002). In pathological situation activation of calpains can lead to degradation of microtubule-associated protein 2, which causes destabilization of microtubules (J.

Liu, Liu, and Wang 2008), damaging the neuronal structure. In addition, calpains are known to interact with metabotropic glutamate receptor 1 (mGluR1), which leads to release of calcium from endoplasmic reticulum. Further on, calcium overload leads to mitochondrial damage causing a release of cytochrome C. This in turn triggers the formation of apoptosome, a protein complex that initiates apoptosis, via caspase-3

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protein (Tait and Green 2010). However, a study on ischemic injury indicated that neuronal injury can lead to direct activation of caspase-3 via calpain (Cao et al. 2007).

Post-TBI mitochondrial damage also promotes formation of free radicals, a process that is further aggravated by acidic conditions following the lactic acidosis after failure of mitochondrial energy production (Gahm et al. 2000; Wada et al. 1998;

Cornelius et al. 2013) highlighting the devastating and complex molecular changes triggered by TBI.

Brain is normally protected by BBB. The primary injury and following formation of free radicals leads to damage to BBB and infiltration of peripheral immune cells to the brain (Chodobski et al. 2011; Morganti-Kossmann et al. 2010; Ndode-Ekane et al.

2018). These cells activate the brains own immune system, astrocytes and microglia (Kumar & Loane, 2012). This has dual consequences. Microglia encloses the damaged area (Dardiotis et al. 2008; Krishnamurthy & Laskowitz 2016) and preventing astrogliosis is known to extent the cortical damage (Benner et al. 2013). On the other hand, overactive microglia can release free radicals (Block & Hong 2005). Studies on post-TBI inflammation dwell around two inflammatory cytokines, IL-1B (interleukin 1 beta) and TNFa (tumor necrosis factor alpha). Studies suggest that IL-1B promotes excitotoxity (Pearson et al. 1999; Viviani et al. 2003; Hu et al. 2000; Relton & Rothwell 1992), tying together neuroinflammation and post-traumatic apoptosis. TNF, that is produced by immune cells and neurons (Gahring et al. 1996), also promotes excitotoxity (Gelbard et al. 1993; Chao & Hu 1994). However, at the same time mice with TNFa or TNF receptor knockout had more extensive BBB damage after TBI (Sullivan et al. 1999; Sullivan et al. 1999).

Together post-injury apoptosis and neuroinflammation form a vicious cycle that promotes the development of comorbidities.

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Figure 1. Physical injury initiates molecular changes that in turn promote cellular damage. Traumatic brain injury (TBI) increases the level of intracellular glutamate by creating micropores to neurons as well as preventing astrocytes from clearing the extracellular space.

The elevated level of glutamate leads to increase in intracellular calcium that in turn triggers excitotoxicity by promoting the formation of free radicals and activation of peroxidases, proteases and phospholipases. Increase level of these compounds also contribute to blood- brain barrier damage, which in turn evokes the immune response. TBI activates calpains, that trigger the release of calcium from endoplasmic reticulum leading to mitochondrial damage and apoptosis. Activation of calpains also destabilizes microtubules leading to disruption of neuronal structure. TBI increases the number of gap junctions leading to the spread of pro- apoptotic messages to the neighboring cells creating so called “bystander effect”.

2.1.3 Post-traumatic co-morbidities

The cascade of molecular changes leads to development of secondary injury, that in turn can initiate development of post-TBI comorbidities. These include many neurodegenerative disorders.

TBI has been shown to increase the risk dementia (Wilson et al. 2017), even after one time loss of consciousness (Fleminger et al. 2003). Mild repetitive TBI has been associated with chronic traumatic encephalopathy (CTE), and so called punch-drunk syndrome (Martland 1928) commonly found in boxers (Hay et al. 2016)

Similarly, the risk of Parkinson’s disease increases after TBI (Wilson et al. 2017) (Gardner et al. 2015; Crane et al. 2016), even in the mild TBI cases (Gardner et al.

2018). At the same time, a study in Finnish population found association between moderate-to-severe TBI and increase risk of dementia but not Parkinson’s (Raj et al.

2017), while another multicohort study on patients from USA found the opposite (Crane et al. 2016), indicating that there is some variation between populations.

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Studies suggest an increased risk of Alzheimer’s disease, as post-mortem studies on patients with history of TBI report higher level of tau and amyloid plaques (Johnson, Stewart, and Smith 2012). However, a study on post-mortem brain samples suggest that patients with diffuse injury have more Tau than the ones with focal injury (Abu Hamdeh et al. 2018a). However, it has been suggested that the post-TBI dementia differs from the Alzheimer’s disease as the TBI patients are more likely depressed, show more agitation and irritability, and have worse motor skills (Sayed et al. 2013). Nevertheless, TBI induces accumulation of toxic Aβ oligomers already at four hours after injury (Abu Hamdeh et al. 2018b)

In addition, TBI seems to also be a risk factor for stroke (Burke et al. 2013; Liao et al. 2014) and epilepsy (Walsh et al. 2017; Christensen 2012; Diaz-Arrastia et al. 2009), both of which cause further damage to brain.

2.2 ANIMAL MODELS OF BRAIN INJURY

2.2.1 Animal models of head injury

Several animal models of TBI have been developed over the years to aid the studies on TBI pathologies. Over the years different animals, including non-human primates, have been used to model TBI. However, in past decades rodents have dominated the field. Most of the TBI models reflect either diffuse or focal injury, which makes these models less comparable to human TBI but allows to study these injury types separately. Focal injury models, such as controlled cortical impact (CCI, Lighthall 1988), produce localized damage and some axonal damage near the injury site. Diffuse injury models use either acceleration or blast wave to produce a diffuse injury without the focal injury, and they have been developed for non-human primates (Gennarelli et al. 1982), rats (Marmarou et al. 1994), and pigs (Smith et al.

2000; Duhaime 2006). However, the studies included in this thesis concentrate on one of the models that cause a mixed injury, fluid percussion injury.

2.2.2 Lateral fluid-percussion injury

In fluid-percussion injuries (FPI) the injury is caused with pendulum that hits a tube filled with fluid. The impact causes a fluid impulse that hits animals exposed dura on the other end of the tube. The pressure of the impulse is measured from the fluid impulse outlet. Based on the craniotomy site FPI injuries are divided to midline FPI (center of the sagittal suture (Ziebell et al. 2016)), parasagittal FPI (less than 3.5 mm lateral to midline, (Floyd et al. 2002)) and lateral FPI (3.5 mm lateral to midline, (McIntosh et al. 1989).

Lateral FPI models human TBI without the skull fracture, as the injury is done on exposed dura. Similar to human TBI, lateral FPI can cause edema, haemorrhage and gray matter damage (Thompson et al. 2005), as well as neurodegeneration in the cortex, hippocampus, thalamus, striatum and amygdala (Liu et al. 2010). The consequences of the injury depend on the severity class. While there is no consensus

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on the definite classifiers of the severity of TBI in animals, animals are often divided to mild, moderate and severe class. The severity can be determined based on mortality, righting reflexes, pressure range, intracranial pressure, blood pressure, post-injury apnea, or combination of some or all of them (Ma et al. 2019). In addition, pressure range corresponding to the three injuries classes varies slightly depending on the instruments used. The pressure ranges across the studies can be roughly divided as 0.9-1.5 atm for mild, 1.6-2.5 atm for moderate, and more than 2.5 atm for severe TBI (Ma et al. 2019).

2.2.3 Status epilepticus

In addition to traumatic brain injuries, other phenomena are known to cause neuronal injury, one of them being epileptic seizures. According to the International League Against Epilepsy (ILAE) seizure is “a transient occurrence of signs and/or symptoms due to abnormal excessive or synchronous neuronal activity in the brain”(Trinka et al. 2015). Further on, if a patient has “at least two unprovoked (or reflex) seizures occurring>24 h apart”, “one unprovoked (or reflex) seizure and aprobability of further seizures similar to the general recurrence risk (at least 60%) after two unprovoked seizures, occurring over the next 10 years”, or “a diagnosis of an epilepsy syndrome”, he or she is considered to have epilepsy (Fisher et al. 2014).

Interestingly, epilepsy is also one of the post-traumatic comorbidities. The seizures might occur either at acute or chronic time point. For example, study on patients with moderate-to-severe brain injuries found that 74% had seizures within the first 14 days after injury (Vespa et al. 1999). Over time, approximately 25% of TBI patients will develop post-traumatic epilepsy (PTE) (Asikainen et al., 1999; Garga &

Lowenstein 2006), which consequently worsens the neuronal damage with each seizure.

An extreme form of seizures is status epilepticus, that ILAE defines as “a condition resulting either from the failure of the mechanisms responsible for seizure termination or from the initiation of mechanisms, which lead to abnormally, prolonged seizures” (Trinka et al. 2015). Status epilepticus often stems from acute brain insults, such as stroke (Knake et al. 2001). A recent study showed that lateral FPI causes rats to have acute non-convulsive status epilepticus (Andrade et al. 2019).

In the projects concerning this thesis, experimental status epilepticus was used as an alternative injury model to investigate the expression miR-124-3p.

2.3 MICRORNAS

2.3.1 Biogenesis and function

MicroRNAs (miRNAs) are short, non-coding RNAs, that count for 1% of predicted genes in genomes of human, C. elegans and drosophila (D.P. Bartel 2004).

First reported miRNA was lin-4, found in Caernohabditis elegans (R. C. Lee, Feinbaum, and Ambros 1993), although the name “microRNA” was first used over decade later

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(Lau et al. 2001; Lee & Ambros 2001). From there the number of known miRNAs sequences has grown rabidly, and 2019 the miRBase database contained already 48 860 mature miRNA sequences from 271 organisms (Kozomara et al. 2019). MiRNA act as suppressors of gene expression by causing either cleavage of targets (complementary binding) or translation inhibition (Tuschl et al. 1999; Hamilton &

Baulcombe 1999). It has been estimated that at least 10% of human protein production is under control of miRNAs (John et al. 2004)

MiRNAs are encoded by either their own genes or from introns of protein coding genes (Baskerville and Bartel 2005). The pri-miRNA transcript forms a hairpin structure with 5’ cap and 3’ poly-A tail (Figure 2)(Lee et al. 2003; Cai et al. 2004), which are clipped out by Microprocessor complex to form pre-miRNA (Lee et al.

2002; Zeng & Cullen 2003). This protein complex is formed minimally from two proteins, RNase III enzyme Drosha together with the double-stranded RNA binding protein DGCR8 (in mammals) also known as Pasha (in flies) (Lee et al., 2002; Han et al. 2006; 2004; Landthaler et al. 2004; Gregory et al. 2004). The pre-miRNA is transported from nucleus to cytoplasm via exportin 5 (Yi et al. 2003). There pre - miRNA is converted to 21-22 nucleotides long RNA duplex by protein complex formed by an endoribonuclease called Dicer, and the transactivation response element RNA-binding protein (TRBP) (Elbashir et al. 2001; Landthaler et al. 2004, Fareh et al., 2016). The half of the duplex, that is presented in 5’-3’ position in the hairpin, is named -5p side. The opposing site is respectively -3p side. Both sides can be functional, although the -3p half is is often found in greater amounts (Bartel 2004).

One or both halves of the duplex are loaded to one of the Argonaute (Ago) f amily proteins to form RNA-induced silencing complex (RISC) (Rehwinkel et al. 2006;

Kobayashi & Tomari 2016). The half that is used for silencing complex, is determined based on the 5’ region, as the 5’terminal U and A are preferred (Frank et al. 2010;

Suzuki et al. 2015) The loading to Ago proteins is assisted by chaperone proteins (HSC70/HSP90), which use ATP (adenosine triphosphate) to push Ago into higher energy state in order to make it accessible for miRNA (Iwasaki et al. 2010).

When loaded to Ago, and protein relaxes to its normal energy state (Kawamata and Tomari 2010), mature miRNA can bind to RNA molecules with Watson-Crick complementarity (Lewis et al. 2003). The 5’ end of miRNA contains so called seed region, more precisely the nucleotides 2-7, that binds to the complementary sequence in 3’ region of the targets mRNA (Lewis et al. 2003; Bartel 2009). While the canonical pairing via seed sequence is used as the gold standard for target recognition, studies suggest that miRNA-target binding can include phenomena such as G-bulge (nucleotides 5-6)(Chi, Hannon, and Darnell 2012) and G-U wobble pairs. In addition, some miRNA bind via centered pairing; meaning they lack the seed pairing and 3’- compensatory pairing, but they bind from the miRNA nucleotides 4-15 (Shin et al.

2010). Moreover, some miRNAs bind to the targets also outside the seed sequence with “3’-supplementary” and “3’-compensatory” sites (Grimson et al. 2007;

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Friedman et al. 2009; Bartel 2009). These additional binding sites might compensate for a seed mismatch, thus enhancing target recognition (Grimson et al. 2007)

The binding of target and seed sequence leads to suppression of mRNA as its poly(A) tail is shortened by TNRC6 (Trinucleotide repeat-containing gene 6A protein), PABPC (Polyadenylate-binding protein 1), and either PAN2-PAN3 (Poly(A) Specific Ribonuclease Subunit) deadenylase complex or the CCR4-NOT (C- C Motif Chemokine Receptor 4, Negative Regulator Of Transcription 1) deanelysase complex (Jonas and Izaurralde 2015). Shortening of poly(A) tail prevents translation initiation and expedites the degradation of the mRNA (Jonas & Izaurralde 2015; Chen

& Shyu 2011).

Studies indicate that more extensive pairing at 3’ end of a miRNA might lead to miRNA being pulled from the Ago, thus exposing it to degrading enzymes and leading to miRNA decay (Ameres et al. 2010; de la Mata et al. 2015). This phenomena, called target RNA-directed miRNA degradation (TDMD) is especially robust in neuronal cells (Bartel 2018).

While the canonical miRNA function occurs in cytoplasm, some miRNAs localize to nucleus (Bartel 2009; Meister et al. 2004). There is three known mechanism that lead to nuclear localization of miRNAs. One option is nuclear biogenesis of miRNAs.

There is evidence of nuclear localization (Gagnon et al. 2014; Doyle et al. 2013; Burger et al. 2017) and function of Dicer (Doyle et al. 2013). Some RISC component have also been detected in the nucleus (Gagnon et al. 2014; Ohrt et al. 2008; Sarshad et al. 2018;

Roya et al. 2016; Rüdel et al. 2008; Robb et al. 2005; Nishi et al. 2013). On the other hand, some proteins critical for loading the miRNA to RISC, are only found in cytosol (Gagnon et al. 2014). Nevertheless, there is some evidence of alternative proteins that can be found in both nucleus and cytosol (He & Schneider 2006). AUF1 (AU-rich- binding protein 1) can load Let-7b to AGO2 (Yoon et al. 2015). Similarly, HuR (human antigen R) has been shown to load Let-7b and Let-7i to AGO2 (Yoon et al. 2013).

However, more studies are needed to confirm the nuclear biogenesis of miRNAs.

Antoher option is translocation of miRNAs. Many studies on HeLA cells and murine epithelial yolk sac cells suggest that the sequence of the miRNA seems to play a part in translocation of miRNAs from cytosol to nucleus (Turunen et al. 2019; Bartel 2009;

Hwang et al. 2007; Kriegel et al. 2018; Kollinerová et al. 2017; Kriegel et al. 2012). A study on neural stem cells show that ASUS-sequence (where S equal to either cytosine or guanine) was found in one-third of nuclear miRNAs (Jeffries et al. 2011).

However, the exact mechanism how the miRNAs are translocated to nucleus, is not known. Finally, some RISC components, such as Dicer and TNRC6A contain nuclear localization signal (NLS), that can mediate transport to nucleus (Doyle et al. 2013;

Nishi et al. 2013; Wente & Rout 2010; Ando et al. 2011; Schraivogel et al. 2015). This, however, needs to be studied more extensively.

The target pool for one miRNA can be surprisingly large, even hundreds of mRNAs (Lewis et al. 2003; John et al. 2004; Giraldez et al. 2006; Baek et al. 2008), prompting a title of “master regulators of gene expression”. As the master regulators of reaction pathways, miRNAs are tempting subject for epigenetic studies.

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Figure 2. The microRNA synthesis and formation of the RNA-induced silencing complex. The pri-miRNA transcript forms a hairpin, that is cut into pre-miRNA by Dicer and DGCR8. The pre-miRNA is transferred to cytoplasm via exportin 5. There pre-miRNA is cut into RNA duplex by Dicer and TRBP. One half of the duplex is loaded into Argonaute protein and the RNA-induced silencing complex is formed. This complex can then silence gene expression by binding to messenger-RNA. Abbreviations: Ago, Argonaute; DGCR8, DiGeorge syndrome chromosomal region 8; miRNA, microRNA; mRNA, messenger-RNA; RISC, RNA- induced silencing complex; TRBP, transactivation response element RNA-binding protein.

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2.3.2 microRNA-124

Brain miRNome is dominated by miR-124 as it accounts for 25% to 48% of all miRNAs, and it is conserved between invertebrates and vertebrates (Lagos-Quintana et al. 2002). Several studies indicate that miR-124-3p is brain specific (Lagos-Quintana et al. 2002; Sempere et al. 2004; Yekta et al. 2004; Lim et al. 2005; Schratt et al. 2006).

However, some studies suggest that miR-124-3p is downregulated in hepatocellular carcinoma (He et al. 2018; Lang & Ling 2012) and gastric cancer (Xia et al. 2012), despite other studies indicate that miR-124-3p is not present in liver (Babak et al.

2004; Lagos-Quintana et al. 2002; Sempere et al. 2004) or stomach (Babak et al. 2004;

Lagos-Quintana et al. 2002) in non-pathological state. Nevertheless, as the most abundant miRNA in brain, miR-124 has a crucial role in normal brain function.

As many miRNAs, miR-124 has multiple predicted targets. At the time of writing, a search in TargetScan (Agarwal et al. 2015) reported 1547 mRNA transcripts with conserved binding sites for miR-124-3p. Based on the expression levels and number of possible targets, it is no wonder that in vitro and in vivo studies have connected miR-124 to functions that are fundamental to function of neurons. MiR-124-3p promotes neuronal splicing via PTBP1/PTBP2 interaction (Boutz et al. 2007; Makeyev et al. 2007; Licatalosi et al. 2012). Transfection with miR-124 causes HeLa cell to shift towards brain expression profile (Lim et al. 2005). It is thought that in non-neuronal cells miR-124-3p is downregulated by REST (RE1-Silencing Transcription factor)(Conaco et al. 2006). Studies also indicate that miR-124 has a key role in hippocampal axogenesis (Sanuki et al. 2011).

miR-124 has been connected to many neurodegenerative diseases some of which are also post-traumatic comorbidities. Gitaí et al. showed that status epilepticus causes upregulation of miR-124-5p in extracellular vesicles derived from rat forebrain (Gitaí et al. 2020). A study in two glioma cell lines suggest downregulation of miR-124-5p in glioma (Chen et al., 2014). Nevertheless, most of the studies are focusing on miR-124-3p. MiR-124-3p is known to target the mRNA encoding β-site APP cleaving enzyme (BACE1), that is most known for cleaving amyloid precursor protein (APP) to plaque forming β-amyloid in Alzheimer’s disease (Fang et al. 2012;

An et al. 2017). Downregulation of miR-124-3p has been observed cellular models of Alzheimer’s (Fang et al. 2012) and Parkinson’s diseases (Dong et al. 2018), in the cortices of mice and patients with frontotemporal dementia (Gascon et al. 2014), as well as in patients with sporadic Alzheimer’s disease (Fang et al. 2012).

Together these findings highlight the crucial role miR-124-3p in brain function and neurodegenerative disorders. Besides the articles discussed in this book, there is only five published articles that deal with the role of miR-124-3p in TBI. Two of them suggest that cultured microglia produces exosomes with miR-124-3p in them after being incubated with brain extract from mouse with TBI (Li et al. 2019; Huang et al.

2018). One shows that miR-124-3p containing extracellular vesicles can enhance hippocampal neurogenesis after TBI (Yang et al. 2019). However, this article does not otherwise investigate the role of miR-124-3p in post-TBI pathology. One article studies oxidative stress in optic nerve injury, and shows that knockdown of long non-

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coding RNA GAS5 alleviates the injury cause to retinal ganglion cells by upregulating miR-124-3p (X. Miao and Liang 2019). Finally, one article measured the effect that voluntary exercise prior to induction of TBI with pneumatic impact on level of miR-124-3p in the rat cerebral cortex (Miao et al. 2015). MiR-124-3p was among the six miRNAs that were found to be expressed at higher level in rats that were exercising in comparison to controls (Miao et al. 2015). Thus, not much is known about chronic post-TBI expression of miR-124-3p, its connection to post-TBI cortical pathologies or its expression in patients with TBI.

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3 AIMS OF THE STUDY

This broad objective of this study was to gain more insight on the role of miRNAs in the progression of the secondary injury and in the development of post-traumatic comorbidities. Earlier studies show that neuronal loss is one of the key features of TBI. Thus our study focused on a neuronal miRNA, miR-124-3p.

The specific aims of this thesis study were:

• to study post-injury alterations of miR-124-3p expression in the dentate gyrus and perilesional cortex in rat and human

• to investigate the connection between miR-124-3p and molecular pathways related to post-TBI recovery process

• to evaluate if miR-124-3p or its targets are suitable canditates for therapeutic intervention

• to study chronic cortical lesion evolution and if circulating miR-124-3p is associated with the lesion outcome

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4 SUBJECTS AND METHODS

The resources, materials and methods used during this thesis project are summarized in following tables and paragraphs. More detailed description can be found from the original publications (I-III) in the end of this book.

All animal procedures using TBI model, were approved by the Animal Ethics Committee of the Provincial Government of Southern Finland. All animal procedures using status epilepticus model were approved by the Ethical Committee on Animal Research of the Nencki Institute. All animal work was carried out in accordance with the guidelines of the European Community Council Directives 2010/63/EU.

4.1 ANIMALS

4.1.1 Animal models

Two animal models of brain injury, lateral FPI (McIntosh et al. 1989; Kharatishvili et al. 2006) and amygdala stimulation-induced status epilepticus (Nissinen et al. 2000;

Bot et al. 2013) were used in the studies included to this thesis.

Briefly, for induction of TBI, rats were first anesthetized and placed to stereotactic frame. The skull was exposed and a circular 5-mm diameter craniectomy was performed on the left parietal lobe, midway between lambda and bregma. TBI was induced with fluid-percussion device after 90 min of anesthesia. Sham-operated controls underwent all procedures except the induction of injury.

In the status epilepticus (SE) study, rats were anesthetized, and wire electrodes were implanted into the left lateral nucleus of amygdala. A steel screw electrode was implanted to the skull over the right frontal cortex. Two steel screws were put bilaterally over the cerebellum. After two week recovery period, SE was induced by electrical stimulation to the intra-amygdala electrode. The SE was stoped 1.5 to 2 hours post-stimulation with diazepam. The control animals had electrode implanted, but they were not subjected to electrical stimulation.

4.1.2 Study design

All together 159 adult male Sprague-Dawley rats, in 6 cohorts, were used during this project to study TBI. The origin and bodyweight of the animals at the time of injury are described in Table 1. In addition, samples from 25 animals (350-400 g, Medical Research Centre, Warsaw, Poland) that were used in status epilepticus experiments, were received from Poland as courtesy of professor Lukasiuk’s lab.

Rats were housed in controlled environment (temperature 22 ± 1°C; humidity 50- 60%; lights on from 07:00 to 19:00 h). Water and pellet food were provided ad libitum.

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Table 1. The number of animals used in this thesis project, their origin, weight at the time of operation, and division to treatment groups.

Cohort Publication Total number of

animals Origin

Weight at the time of

operation (g)

Groups

1 I, II 30

Harlan Netherlands B.V.,

Horst, The Netherlands

330-371 (average 350)

Naive: 0 Sham: 12 TBI 3 mo: 18

2 I, II 23 Envigo, Udine,

Italy 356-391

(average 376)

Naive: 3 Sham 7 d: 4 Sham 3 mo: 4

TBI 7 d: 6 TBI 3 mo: 6

3 II, III 25 Envigo, Udine,

Italy

342-425 (average 392)

TBI 7 d: 5 TBI 3 mo: 10

4 III 30 Envigo, Udine,

Italy 346-384

(average 365)

TBI 2 d: 8 TBI 2 mo: 9

5 III 51 Envigo, Udine,

Italy

353-425 (average 387)

Naive: 7 Sham: 14 TBI 3 mo: 30

Abbreviations: TBI, traumatic brain injury; d, day; mo, month.

4.2 HUMAN SAMPLES

Autopsy samples were obtained from the hippocampus ( I) and the cortex (II) at the Department of Neuropathology at the Academic Medical Center in Amsterdam, The Netherlands. Samples were derived from 5 TBI patients (Table 2., 1 female, 4 males, median age 65, range 20 – 82) and 5 subjects with no history of neurological diseases (3 males, 2 females, median age 48, range 35-71). All samples were collected within 24 hours after death. Informed consent was obtained for the use of brain tissue and for access to the medical records. Tissue was obtained and used in accordance with the Declaration of Helsinki and the AMC Research Code provided by the Medical Ethics Committee. Samples were fixed in 10% buffered formalin and embedded in paraffin.

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Table 2. TBI patient information.

Sex Post-TBI

Age at the time of death

Injury mechanism

Injury

severity Epilepsy CT/

(GCS) yes/no MRI/

_brain

Hippocampus samples in publication I

f 1 mo 57 accident Moderate yes

CT:haemo.c contusions temp. L; tSAH

m 25 y 65 accident Mild yes NA

Temporal lobe samples in publication II

m 5 d 20 accident severe no

Cerebral edema, small hemorrhages, diffuse axonal

injury

m 8 d 79 accident moderate no Cerebral

edema

m 2 mo 82 accident mild no NA

Mild head injury (GCS score of 13 to 15); Moderate head injury (GCS score of 9 to 12), Severe head injury (GSC score 3-8); Abbreviations: CT, computed tomography; f, female; GCS, Glasgow Coma Scale; m, male; MRI, magnetic resonance imaging; TC, tSAH, traumatic subarachnoid hemorrhage.

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4.3 SAMPLING

Table 3. Summary of the sampling and preservation of tissue.

Publication Sample type Preservation method Used for Purpose

I Dentate

gyrus

immersed to ice-cold RNAlater RNA Stabilization

Reagent, snap-frozen

microarray qRT-PCR

ddPCR

Transcriptomics profiling Detections of miR-124-3p, Plp2 and Stat3 after TBI or SE II Perilesional

cortex snap frozen in liquid nitrogen RNA-Seq Transcriptomics profiling after TBI

I, II Human brain Immersion fixing in 10%

formalin

in situ hybridization

Detection of miR-124-3p after TBI

II Rat brain Fresh frozen in -70°C

isopentane qRT-PCR Detection of miR-124-3p and Stat3 after TBI

I Rat brain Fresh frozen in -70°C isopentane

in situ hybridization

Detections of miR-124-3p, Plp2 and Stat3 after SE

I; II Rat brain perfusion, paraffin

embedding in situ

hybridization

Detection of miR-124-3p after TBI

III Plasma snap frozen in dry ice qRT-PCR Detection of miR-124-3p after TBI

III Rat brain Immersion fixing in 10%

formalin Nissl

Characterization of cytoarchitectonic changes in

perilesional cortex

Abbreviations: ddPCR, droplet digital polymerase chain reaction; qRT-PCR, quantitative reverse transcription poilymerase chain reaction; Plp2, Proteolipid Protein 2; SE, status epilepticus; Stat3, Signal transducer and activator of transcription 3;TBI, traumatic brain injury.

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4.4 MOLECULAR ANALYSIS

Table 4. Summary of molecular biology methods used in this thesis.

Publication Method Purpose

I, II, III RNA extraction Detection of the level of miR-124-3p, Plp2 and Stat3

I, II, III Reverse transcription Detection of the level of miR-124-3p, Plp2 and Stat3

I ddPRC Detection of the level of miR-124-3p

I, II, III qRT-PCR Detection of the level of miR-124-3p, Plp2 and Stat3

I, II In situ hybridization Detection of intracellular level of miR-124-3p

I, III Nissl Detection of cytoarchitectonic structures

I, III

Immunohistochemistry (I: STAT3, parvalbumin I, II: NeuN, GFAP, Iba1)

Detection of STAT3 expression In situ hybridization together with immunohistochemistry against parvalbumin, and

cellular markers

I Hematoxylin & eosin Staining of nucleus and cytoplasm in order to detect the cellular localization of miR-124-3p II Crezyl violet Visualization of cellular structures before laser

capture microdissection

Abbreviations: ddPCR, droplet digital polymerase chain reaction; GFAP, glial fibrillary acidic protein; qRT-PCR, quantitative reverse transcription poilymerase chain reaction; Iba1, ionized calcium binding adaptor molecule 1; NeuN, neuronal nuclei; Plp2, Proteolipid Protein 2; RNA, ribonucleic acid; Stat3, Signal transducer and activator of transcription 3.

4.5 STATISTICS AND BIOINFORMATICS

To study the miR-124-3p controlled networks, previously published microarray data from post-TBI dentate gyrus (Puhakka et al. 2017), post-SE dentate gyrus (Bot, Dębski, and Lukasiuk 2013), and RNA-Seq data from post-TBI the perilesional cortex (Lipponen et al. 2016) were obtained.

MiR-124-3p as a regulator for post-TBI recovery process

NIINA VUOKILA

MiR-124-3p as a Regulator for Post-TBI

Recovery Process

Dissertations in Health Sciences

MIR-124-3P AS A REGULATOR FOR POST-TBI

RECOVERY PROCESS

Niina Vuokila

MIR-124-3P AS A REGULATOR FOR POST-TBI RECOVERY PROCESS

ABSTRACT

TIIVISTELMÄ

ACKNOWLEDGEMENTS

LIST OF ORIGINAL PUBLICATIONS

CONTENTS

ABBREVIATIONS

1 INTRODUCTION

2 REVIEW OF THE LITERATURE

2.1 TRAUMATIC BRAIN INJURY

2.2 ANIMAL MODELS OF BRAIN INJURY

2.3 MICRORNAS

3 AIMS OF THE STUDY

4 SUBJECTS AND METHODS

4.1 ANIMALS

4.2 HUMAN SAMPLES

4.3 SAMPLING

4.4 MOLECULAR ANALYSIS

4.5 STATISTICS AND BIOINFORMATICS