genetics proposal

5/22/2018 Genetics Proposal

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Regenerating the CNS Using Ctenophoran Epigenetic Mechanisms ----------------AngelaLuo 1

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.


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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 vivoreprogramming 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 processessuch asthe methylation of DNA, a common enzyme-induced mechanism

that locks genes in the offposition [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].


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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].

Pleurobrachiasgenome 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-

genes in Pleurobrachiadeveloping and adult tissues


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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 Ca2+

(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 ad

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.


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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 vitrofor 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.


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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/Ca2+ in iGluRs

Figure 4: microarray assay for gene expression


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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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Figure 5: Age-specific incidence of dementia (per 1 000

person years) across continents and countries.


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