Regenerating the CNS Using Ctenophoran Epigenetic Mechanisms ----------------Angela

Luo
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Regenerating the CNS Using Ctenophoran Epigenetic
Mechanisms

Abstract
Neuronal degeneration has long been thought of as irreversibleand it is true that adult nerve
cells of the CNS cannot spontaneously repair like many other cells. How is it, then, that
organisms such as ctenophores are able to regenerate their brains in only a few days? The
phenotypic differences between humans and ctenophores are innumerable, yet genotypic
differences only lie in sequencing and expression mechanisms. Epigenetic research seeks to
manipulate the latter, by studying DNA methylation, histone modification, chromatin
remodeling and non-coding RNAs. I am proposing to study the epigenetic mechanisms within
ctenophores for the purpose of applying them to support the continued development of neural
stem cells, and plasticity of existing neural and assisting glial cells in mature adult mice facing
acute CNS injury. I aim to focus on the ctenophoran sophisticated usage of L-glutamate and
iGluRs, RNA-editing, and DNA demethylation in neural gene expression during regeneration. I
would then replicate the ctenophoran neural-regenerative mechanism to test on the brains of
mice suffering from acute CNS, followed by adult human postmortem neural tissue. The
success in these experiments would lead to breakthroughs in not only neural regeneration
procedures for human sufferers of acute CNS injuries, but also for those afflicted with
neurodegenerative conditions such as Alzheimers. Further study in the identification of
specific genes responsible for such diseases, coupled with neural-regenerative epigenetics
research as I have proposed, would lead to significant progress in treatingand curingthe
afflictions embodying one of humanitys greatest fears: losing our minds.
Regenerating the CNS Using Ctenophoran Epigenetic Mechanisms ----------------Angela
Luo
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Background Information
The regenerative abilities of injured neurons in the central nervous system (CNS) are restricted
by the absence of a robust injury-induced gene response supporting axon regeneration [1,5].
CNS regeneration is a complex process that requires the system to: (1) overcome inhibitors to
axonal regeneration and limit scar formation; (2) enabling spontaneous mechanisms of axonal
regeneration and neurite outgrowth; and (3) reestablish long-distance connections [2].

In
regards to the first requirement, In vivo reprogramming has made transformation of neuroglial
tissue into normal neuron tissue possible [3]. Concerning the second requirement, however,
the CNS is unable to spontaneously induce a large cohort of regeneration associated genes
(RAGs) as the peripheral nervous system (PNS) is able to accomplish [4]. Even forced
expression of isolated RAGs in injured neurons of the CNS has only small beneficial effects on
axon regeneration [5], and some even stimulate further degeneration [6].

Transcription factors
(TFs) have been used to express a multitude of beneficial RAGs at once, but they are limited
by their targeting of RAG network hubsonly highly connected genes within hubs would be
expressed [7].

To promote the regeneration of the CNS, more RAGs would need to be targeted
jointly [8]. The final requirement dictates that the CNS must maintain long-distance
connectivity; thus, efficient alternative routes between neural regions must be promoted.
As the study of epigenetics in gene expression rises in prominence, research suggests that
neurodegenerative disorders are partly caused by abnormal epigenetic modifications [9].
Epigenetic processes such as the methylation of DNA, a common enzyme-induced mechanism
that locks genes in the off position [10], also play a large role in inhibiting the plasticity of
nervous injurymaking CNS regeneration nearly impossible [11]. Therefore, manipulation of
various epigenetic factors could significantly impact the CNSs regenerative abilities [12].
Regenerating the CNS Using Ctenophoran Epigenetic Mechanisms ----------------Angela
Luo
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Current Knowledge
Ctenophores, better known as comb jellyfish, are perhaps the best model of epigenetically-
induced regeneration. "Some ctenophores can regenerate an elementary brain in [three and a
half] days [13]." Physiological data supports that
ctenophore neural systems evolved independently from
those in other animals; but despite their unique chemical
language they exhibit the same epigenetic processes as
all other living organisms [14].
Pleurobrachias genome encodes the highest number of
RNA-editing enzymes and RNA-binding proteins
reported among metazoans (Fig. 1), a generalized
mechanism generating post-transcriptional diversity and ctenophore-specific integrative
structures [15]. In human brains, non-protein-coding RNAs (ncRNAs) are highly concentrated
in the CNS and PNS [16], and transfer genes to different parts of the nucleus, where they can
be more freely expressed [17]. It is evidenced that epigenetic mechanisms such as RNA
editing are fundamental for neural development and maintaining mature function [16].
Ctenophores also demonstrate strong demethylation potential; even in adulthood, TET levels
remain high (Fig. 2); these enzymes catalyze active DNA demethylation via formation of 5-
hmC [15, 18], enabling locked methylated genes to be expressed. This increases plasticity in
cells and allows for flexible responses to the environment, such as regeneration [18].The TET
proteins have been shown to function in human cerebellum development [19], tumor
suppression, DNA methylation reprogramming processes, and transcriptional activation [20].
This transcriptional activation, coupled with the aforementioned ncRNA binding/transfer of
Figure 1: Diversity and differential expression of RNA-editing
genes in Pleurobrachia developing and adult tissues
Regenerating the CNS Using Ctenophoran Epigenetic Mechanisms ----------------Angela
Luo
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RAGs to more expressive regions of the nucleus, allows ctenophores to meet the second
requirement of CNS regeneration:
enabling spontaneous mechanisms of
axonal regeneration and neurite
outgrowth [2].
Finally, ctenophores may also hold the
key to overcoming the third obstacle of
CNS regeneration: reestablishing long-
distance connections [2]. Classical
intercellular messengers such as dopamine
are replaced by a prevalence of diverse ionotropic glutamate receptors (iGluRs; Fig. 3) and L-
glutamate [15]; glutamate signaling aids in neural protection against disease and injury, and in
DNA reparation [21]. Glutamate is the primary excitatory neurotransmitter in the mammalian
CNS [21], and has been observed in the regulation of long-distance neurite outgrowth and
survival [21, 22]. iGluRs are ligand-gated ion channels that are densely expressed in
mammalian brains [23]. These channels permit the
flow of increased concentrations of Ca
2+
(caused by
L-glutamate) to rapidly induce immediate-early
gene expression [24], using a diverse array of
signaling pathways [24, 25].


Figure 3: TET family of enzymes catalyzes active DNA demethylation via
formation of 5-hydroxymethyl cytosine (5-hmC). TET-like genes are
predominantly expressed during cleavage and also highly expressed in adult
combs.
Figure 2: L-glutamate as a transmitter candidate in Pleurobrachia
bachei. The ionotropic glutamate receptors (iGluRs) are diverse and
underwent substantial adaptation in the Ctenophora lineage.
Regenerating the CNS Using Ctenophoran Epigenetic Mechanisms ----------------Angela
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Objectives
By studying how comb jellyfish utilize DNA demethylation to regenerate, use ncRNA-binding to
speed up gene targeting in methylation processes, and use glutamate/iGluRs to quickly
reestablish long-distance connections in the CNS, I wish to apply ctenophoran DNA epigenetic
mechanisms into mammals (especially humans), as an addition to neural-regenerative
developments currently being made to regenerate the CNS after acute injury.
Scientific Details
The hypothesis of this project is that acutely injured CNS can be repaired and regenerated with
a combination of epigenetic mechanisms expressed within ctenophores: DNA demethylation,
ncRNA binding, and iGluR activity.
Methodology Design
Trials would be carried out on mice afflicted with acute CNS injury; however, animal models
cannot fully mimic human neurology. Therefore, following confirmed success, trials would then
be carried out on human post-mortem brains from willing donors. Human brain tissue slices
obtained via autopsy within 8 h after death can be maintained in vitro for up to 78 days for
experimental manipulation [26].
(1) Targeted DNA demethylation would be enabled using SINEUP, utilizing ncRNA to
guide experimentally-introduced TET enzymes to respective genes [27].
(2) L-glutamate within iGluR to produce multiple pathways to establish long distance
connections.
Regenerating the CNS Using Ctenophoran Epigenetic Mechanisms ----------------Angela
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Evaluation and Analysis
Depth of analysis can be separated into three levels:
(1) Physical Expression: Successful regeneration of CNS in mice could be evaluated by
comparing post-regeneration activity/ability with pre-regeneration activity/ability levels.
(2) Cognitive Expression: fMRI scans could show whether previously
dysfunctional/disabled areas of the brain were functioning normally after regeneration
trials.
(3) Genotypic expression: The success of these trials would be analyzed with high-
throughput gene expression
techniques such as with microarray
technology (Fig.4) that can detect gene
expression in thousands of coded DNA
sequences in parallel [28].

Relevance
Ctenophores diverged from the standard
evolutionary design of neural circuits; equipped with epigenetic mechanisms enabling rapid
regeneration of their rudimentary brains. Although ctenophores may be considered too
phenotypically alien from our species for us to utilize the same strategies, research has proved
that lifes unity enables the subsystems of living organisms to be highly interlocked [29]. DNA
demethylation, combined with ncRNA-binding and delivery of glutamate/Ca
2+
in iGluRs
Figure 4: microarray assay for gene expression
Regenerating the CNS Using Ctenophoran Epigenetic Mechanisms ----------------Angela
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demonstrated by ctenophore regeneration can be carefully utilized for the same process within
humans. Coupled with recent breakthroughs with chromatin-modifying drugs [30], neural stem
cells [31], and conversion of neuroglial cell scar tissue into functional neural tissue [3], effective
CNS regeneration techniques may well be within reach. Not only would this research enable
regeneration of neurons in acute CNS injury, the findings could lead to new ways to
investigate neurodegenerative diseases, such as
Alzheimers or Parkinsons [13]. Dementia affects
around 5% of the population over 65 years, and
prevalence increases with age (Fig.5) [32]. In an
aging population, dementia becomes an increasing
concernby understanding epigenetic mechanisms
and neural regenerative processes, neurodegenerative
diseases can finally be managed.

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