Table Of ContentEssentials of Noncoding
RNA in Neuroscience
Essentials of Noncoding
RNA in Neuroscience
Ontogenetics, Plasticity
of the Vertebrate Brain
Edited by
Davide De Pietri Tonelli
Neurobiology of miRNA Lab, Neuroscience
and Brain Technologies Department,
Istituto Italiano di Tecnologia, Genoa, Italy
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List of Contributors
Daniel G. Abernathy
WashingtonUniversity inSt. Louis School ofMedicine, St. Louis, MO, United States
BenediktBerninger
University Medical Center oftheJohannesGutenbergUniversity Mainz, Mainz, Germany
Pascal Bielefeld
University of Amsterdam, Amsterdam, The Netherlands
Irene Bozzoni
Sapienza University ofRome,Rome, Italy
Murray J. Cairns
University of Newcastle, Callaghan,NSW, Australia; Schizophrenia Research Institute,Sydney,
NSW,Australia; Hunter Medical ResearchInstitute, Newcastle,NSW, Australia
Nathalie Core´
Aix-Marseille Universite´,CNRS, Marseille, France
Harold Cremer
Aix-Marseille Universite´,CNRS, Marseille, France
Antoine de Chevigny
Aix-Marseille Universite´,CNRS, Marseille, France
DavideDe Pietri Tonelli
IstitutoItaliano diTecnologia, Genoa,Italy
Andrea Erni
University of Basel, Basel, Switzerland
Carlos P. Fitzsimons
University of Amsterdam, Amsterdam, The Netherlands
Michael Geaghan
University of Newcastle, Callaghan,NSW, Australia; Hunter Medical ResearchInstitute,
Newcastle, NSW,Australia
GeorgiosGeorgakilas
University of Thessaly, Volos, Greece; HellenicPasteurInstitute, Athens, Greece
Stefanie Grosswendt
Berlin Institute for Medical Systems Biology(BIMSB), Berlin, Germany
ArtemisG. Hatzigeorgiou
University of Thessaly, Volos, Greece; HellenicPasteurInstitute, Athens, Greece
Wieland B.Huttner
Max Planck Institute ofMolecularCellBiology and Genetics, Dresden, Germany
xiii
xiv List of Contributors
DimitraKaragkouni
University ofThessaly,Volos,Greece; Hellenic Pasteur Institute,Athens, Greece
Ivano Legnini
Sapienza University ofRome, Rome, Italy
YangjianLiu
Washington University inSt. Louis School of Medicine,St. Louis,MO, UnitedStates
Qing Richard Lu
Cincinnati Children’sHospital Medical Center,Cincinnati, OH, UnitedStates; University of
Cincinnati, Cincinnati,OH, United States
FedericaMarinaro
Istituto Italiano diTecnologia, Genoa, Italy
DeboraNapoli
Scuola NormaleSuperiore, Pisa, Italy
Stefania Nicoli
Yale School of Medicine,New Haven,CT,United States
TomaszJan Nowakowski
University ofCalifornia,San Francisco, San Francisco, CA,United States
MariaD.Paraskevopoulou
University ofThessaly,Volos,Greece; Hellenic Pasteur Institute,Athens, Greece
Amy E.Pasquinelli
University ofCaliforniaatSan Diego, LaJolla, CA,UnitedStates
TommasoPizzorusso
InstituteofNeuroscience, Pisa, Italy; University ofFlorence,Florence,Italy
Meritxell Pons-Espinal
Istituto Italiano diTecnologia, Genoa, Italy
Thomas Pratt
University ofEdinburgh, Edinburgh,UnitedKingdom
David Jonathan Price
University ofEdinburgh, Edinburgh,UnitedKingdom
BenPustjens
University ofAmsterdam, Amsterdam, The Netherlands
NikolausRajewsky
Berlin Institute for MedicalSystems Biology (BIMSB),Berlin, Germany
Emma Ristori
Yale School of Medicine,New Haven,CT,United States
Chiara Rolando
University ofBasel, Basel, Switzerland
List of Contributors xv
Marijn Schouten
University of Amsterdam, Amsterdam, The Netherlands
William P. Schreiner
University of California atSan Diego, La Jolla, CA, United States
Spyros Tastsoglou
University of Thessaly, Volos, Greece; HellenicPasteurInstitute, Athens, Greece
Stefania Tavano
Max Planck Institute ofMolecularCellBiology and Genetics, Dresden, Germany
Verdon Taylor
University of Basel, Basel, Switzerland
Neha Tiwari
University Medical Center oftheJohannesGutenbergUniversity Mainz, Mainz, Germany
Ioannis S.Vlachos
University of Thessaly, Volos, Greece; HellenicPasteurInstitute, Athens, Greece
Haibo Wang
Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States
Andrew S. Yoo
WashingtonUniversity inSt. Louis School ofMedicine, St. Louis, MO, United States
XianghuiZhao
Fourth Military Medical University,Xi’an,China; CincinnatiChildren’sHospital Medical Center,
Cincinnati, OH, UnitedStates
Preface
The brain is not just “any organ.” Indeed, (1) it comprises billions of electrogenic and neuromodu-
latory cells, such as neurons and glia, respectively, (2) which are remarkably heterogeneous and
hyperspecialized cells that (3) are interconnected by hundreds of trillions of synapses, forming the
most fascinating and perhaps the most important cellular network in our body: the central nervous
system. (4) As cell turnover in the human brain is almost negligible, brain cells and its networks
arealsohighlyadaptableor“plastic”allowingustolearnandconstantlycope withdiverseenviron-
mental cues, from early postnatal along the whole lifetime.
How are brain development and itsplasticity controlled at the molecular level? these are two of
the relevantquestions, we will try toaddress inthis book.
Neurogenesis can serve as example of both brain development and plasticity. Most of the neu-
rons and glial cells that compose the six layers of mammalian neocortex arise during development.
However, in restricted “niches” of the adult brain, stem cells are preserved and generate neurons
and glia throughout life. Adult neurogenesis may influence learning and memory and also being
regulated by the environment and activity. Thus, it is considered a form of brain’s “structural”
plasticity.
Interestingly, despite both embryonic and adult “neurogenesis” contribute to the generation of
new neurons (and glia), these two processes are very different. In fact, during the intrauterine corti-
cal development, neural stem cells undergo a “program” leading to the orderly generation of hun-
dreds of neurons first, then glial cells. In contrast, adult neural stem cells are “flexible.” They can
give rise to very few neurons or glia simultaneously, though during the entire lifespan of an indi-
vidual. Thus, embryonic and adult neurogenesis require distinct (i.e., in space and time) coordina-
tion of the intrinsic signaling pathways and extrinsic factors, leading to maintenance of stem cell
pools and tothe generation of their differentiated progenies.
Following these considerations, other questions that we will discuss in this book are: Do differ-
ent key players control brain development or “plasticity”? Or Vice versa, are the same players dif-
ferently regulated?Whichofthesemechanisms were leveraged inevolution?
Several years of studies provided important evidence of the transcriptional cascades and signal-
ing pathways that govern neurogenesis, gliogenesis, and “plasticity” in brain. However, after the
“completion” of the human-genome sequencing (though due to the presence of several hundreds of
repetitive elements, the exact sequence of our genome is still not entirely known), it became appar-
ent that less than 2% of the human DNA encodes for proteins. In contrast, in evolution, the com-
plexity of the noncoding transcriptome has increased much more than coding one. These and many
other postgenomic “surprises” instilled the idea that this 98% of the human genome must be “not
sojunkDNA.”Actually,these discoveriesopenthepossibility thatthe regulationofproteinexpres-
sion might be crucial to understand the molecular control of brain development and function.
Moreover, some ofthesemechanisms might have been leveraged inevolution.
The idea of this book originated in the symposia “MicroRNAs in brain development and func-
tion” at the 9th FENS meeting in Milan, in July 2014. The seminar room was full, and delegates
following the lectures from outside screens largely outnumbered those inside. This confirmed that
the topic of noncoding RNAs raises a great interest in the broad field of neuroscience. Having in
mind the above questions, inthis book, we will focus on noncoding RNAsand epigenetics.
xvii
xviii Preface
microRNAs(miRNAs)arethe“founders”ofthelargefamilyofsmallnoncodingRNAs.Thefirst
miRNA gene was isolated in Caenorhabditis elegans in Sidney’s Brenner laboratory in the early
1980s and characterized as the first “heterochronic mutant” by Marty Chalfie, John Sulston, and
RobertHorvitz,whonamedthisgene“lin-4”(lin,celllineageabnormal).Ittookmorethanadecade
before Victor Ambros, Gary Ruvkun, and colleagues discovered that the two-heterochronic genes,
lin-4 and let-7, did not encode for protein products but rather encode small RNAs. As of today, the
field has grown dramatically: miRNAs are known to be present in most plants, insects, animals and
arecruciallyinvolvedinthecontrolof“virtuallyall”biologicalpathwaysandhumandiseases.
For historical reasons, several chapters of this book, authored by leading scientists in noncoding
RNA and neuroscience, focus on miRNAs. We have attempted to highlight key concepts, mechan-
isms, and challenges for the functional investigation of miRNAs in the framework of the neural
system development, plasticity, dysfunction, and evolution. However, we also summarize novel
classes of noncoding RNAs, such as piRNAs and circular RNAs, and discuss epigenetics, as well
as cross talks between different noncoding RNAs andpathways with relevance toneuroscience.
The first section of this book (Introduction and Methods) comprises four chapters on basic biol-
ogy of animal miRNAs and methods to infer their functions. Schreiner and Pasquinelli introduce
miRNA biogenesis and their turnover. Then, Grosswendt and Rajewsky analyze in details miRNA-
mediated mechanisms of target repression, relevance of miRNA-dependent control for silencing of
“gene expression noise” and methods to directly investigate miRNA: target interactions.
Hatzigeorgiou and colleagues highlight the state of the art and challenges in computational
approaches to investigate miRNA functions, targets identification, and novel tools for integrative
biology.Finally,Marinaro,PonsEspinal,andmyselfsummarize current methodological approaches
and unresolved challenges for the functional investigation and therapeutic use of miRNAs and
siRNAs, invivo.
The second section (Neurogenesis) comprises five chapters and is focused on cell biology and
molecular control of embryonic and adult neurogenesis. Tavano and Huttner write on corticogen-
esis in rodents and in human brain evolution. Erni, Rolando, and Taylor present miRNA-dependent
and -independent regulations in neural stem cells. Finally, the functions and use of epigenetics and
noncoding RNAs are discussed by Liu, Abernathy, and Yoo in the context of cell reprograming; by
de Chevigny, Cremer, and Core´ in adult neurogenesis of the olfactory system, and by Bielefeld,
Pustjens, Schouten,and Fitzsimons for the hippocampus.
The third section (Gliogenesis) comprises two chapters focusing on molecular and epigenetic
control of gliogenesis and relevance of these pathways for diseases. Tiwari and Berninger describe
transcriptional, epigenetic, and miRNA-dependent control of astrogliogenesis. Zhao, Wang, and Lu
write about miRNA-dependent control of oligodendrocyte development and implications of these
regulatorymechanismsindemyelinatingdiseases and myelin repair.
The fourth section (Plasticity and Brain dysfunctions) comprises two chapters discussing func-
tions of small noncoding RNAs in the molecular control of brain physiopathology. Napoli and
Pizzorusso present the functions of miRNAs and piRNAs in neuronal networks maturation and
plasticity, whereas Gahagan and Cairns focus on small RNA dysregulation in neurocognitive and
neuropsychiatric disorders.
The fifth and last section of this book (Emerging complexity in small RNA classes and func-
tions) reviews novel types of noncoding RNAs, such as circular RNAs, ceRNA, human accelerated
evolutionary genomic regions (HARs), transposable elements, and ALUs in light of comparative
Preface xix
development and function of the neural system. Legnini and Bozzoni write on circular RNAs in the
context of neural system function and regulation. Ristori and Nicoli discuss on the comparative
functions of miRNAs in development of the nervous system in invertebrates versus vertebrates.
Finally, in the last chapter of the book Pratt, Nowakowski, and Price write on the functions of non-
coding RNAs,in the context of human neocortical evolution.
Through thisbook,several guiding principleswill becomeapparent.
(cid:129) Regulatory capacity and diversity ofnoncodingRNA-dependent control is particularly valuable
inthe brain.Indeed, inbrain, poorcellular turnover andthe constant flow of information
require specializationand adaptability, also at the level ofmolecular control.
(cid:129) Only very few miRNAs (e.g., miR-9, miR-124, miR-134, miR-132,etc.)that here we define as
“hubs” seem individually capable ofexerting relevantbiological functionsin brain.In contrast,
mostmiRNAs-dependent regulations are notstrong: Thedegreeof protein down regulationby a
miRNAis generally lessthan twofolds.
(cid:129) To compensate for such low activity,multiple miRNAs act incombinatorial (also known as
convergent, synergic,or cooperative) manner to buffer genetic noise. Combinatorial miRNA
actions confer robustness tothe expression ofspecificproteins, ortobiological functions.This
explains why single miRNA knockouts often don’t show any phenotype. This also warrants
careful consideration ofresultsobtained upon manipulationof asingle miRNA, especially in
gain of function studies.
(cid:129) It therefore follows that miRNAs seem toevolve more rapidly than protein coding RNAs.
Indeed,de novo miRNAs are well tolerated by evolution and mutationsin precursorand mature
miRNAs arise more frequently in“young” miRNAs comparedto “ancient” miRNAs. Although
this exciting evidence indicates that our miRNA repertoireis evolving rightnow, it also
warrants careful consideration for their use asbiomarkers ofdisease.
Ontheotherhand,severalopenquestionsandchallengesemergingfromthisbookremainunan-
swered. Even just considering miRNAs, perhaps the best characterized class of noncoding RNAs,
we still do notknow much. Forexample, transcriptional control ofmiRNA inbrain is poorly inves-
tigated. Most of the miRNA targets have not been identified or experimentally validated.
Moreover, with respect to the use of miRNAs for brain therapy, we still miss efficient delivery
methods, as well as crucial information about their toxicity and side effects. Finally, we know very
little about the integration of miRNAs with epigenetics and other noncoding RNA classes and
mechanisms, such as circular RNAs andpiRNAs.
Provocative hypotheses are also discussed in this volume. For example, in contrast tolower ver-
tebrates, it seems that sequestering miRNAs is not a general phenomenon for mammalian circular
RNAs. This finding therefore opens the intriguing possibility that mammalian circular RNAs might
betranslated. Ifjust asubsetofthem will befound tobetranslated,a complete new set ofmamma-
lian proteome would bediscovered.
In light of this evidence, it is clear that we still have much work to do. A simple search of
“miRNA” or “microRNA” in PubMed (www.ncbi.nlm.nih.gov/pubmed) retrieves more than 12k
publications in 2015, and certainly new classes of noncoding RNAs are being discovered, while we
are writing thisbook.
With these 16 chapters, we aim toprovidethe “essential” knowledge of“noncoding RNA” biol-
ogy, function(s), and their exploitation in the context of “neuroscience”. Indeed, we made an effort
xx Preface
to use a language that will be readily intelligible to any student or scientist from different fields
and disciplines, thus to reach the widest possible audience. We hope this selection will not reflect
too much our personal opinion and biases and apologize to those researchers whose important work
we were not able to cite because of space limitations. Our ultimate goal is to equip readers withthe
instruments to evaluate the functions of noncoding RNAs in healthy and diseased brain and their
potential exploitations for therapy. We hope that after reading this book many of you will take a
walk on the wild side ofthe transcriptome.
Davide De Pietri Tonelli
IstitutoItalianodiTecnologia,Genoa,Italy
