Article

ISSN 0006-2979, Biochemistry (Moscow), 2023, Vol. 88, No. 11, pp. 1719-1731 © Pleiades Publishing, Ltd., 2023.

Russian Text © The Author(s), 2023, published in Biokhimiya, 2023, Vol. 88, No. 11, pp. 2084-2100.

1719

REVIEW

DNA Instability in Neurons:

Lifespan Clock and Driver of Evolution

Varvara E. Dyakonova

Koltzov Institute of Developmental Biology, Russian Academy of Sciences, 119334 Moscow, Russia

e-mail: dyakonova.varvara@gmail.com

Received July 17, 2023

Revised July 19, 2023

Accepted September 20, 2023

Abstract— In the last ten years, the discovery of neuronal DNA postmitotic instability has changed the theoretical land-

scape in neuroscience and, more broadly, biology. In 2003, A.M. Olovnikov suggested that neuronal DNA is the “initial

substrate of aging”. Recent experimental data have significantly increased the likelihood of this hypothesis. How does

neuronal DNA accumulate damage and in what genome regions? What factors contribute to this process and how are they

associated with aging and lifespan? These questions will be discussed in the review. In the course of Metazoan evolution,

the instability of neuronal DNA has been accompanied by searching for the pathways to reduce the biological cost of

brain activity. Various processes and activities, such as sleep, evolutionary increase in the number of neurons in the ver-

tebrate brain, adult neurogenesis, distribution of neuronal activity, somatic polyploidy, and RNA editing in cephalopods,

can be reconsidered in the light of the trade-off between neuronal plasticity and DNA instability in neurons. This topic is

of considerable importance for both fundamental neuroscience and translational medicine.

DOI: 10.1134/S0006297923110044

Keywords: nervous system, neuronal DNA, postmitotic mutagenesis, DNA repair, lifespan, epigenetics, evolution of

the nervous system

Abbreviations: DSB,double-strand DNA break; indel,small insertion or deletion; NMDA,N-methyl-D-aspartate; Parp1,poly(ADP-

ribose) polymerase1; SNV,single nucleotide variant; SSB,single-strand DNA break; TopoIIβ,topoisomerase Iiβ.

INTRODUCTION

“A combination of various facts indicates that it is

the brain that is the initial substrate of aging,

and in a brain cell, this substrate is DNA.”

A.M. Olovnikov, 2003

Alexey Matveyevich Olovnikov is widely known for

his outstanding theoretical contribution to the field of

biological chronometry and studies of mechanisms reg-

ulating life expectancy. He predicted the existence of

telomeres, the “counters” of cell divisions, in proliferat-

ing cells even before their experimental discovery [1,2].

After creating the telomere hypothesis of cell aging,

A. M.Olovnikov has retained his interest in the prob-

lem of biological time [3]. In 2003, he publishes a large

theoretical work on the redusome hypothesis of aging,

in which he suggested the following: “A combination of

various facts indicates that it is the brain that is the initial

substrate of aging, and in a brain cell, this substrate is

DNA”. At that time, experimental data were still sparse

and scattered; however, in recent years, the likelihood

of this hypothesis has been increased by the results of

numerous molecular neurobiology studies.

The aim of this article is to analyze experimental

data published mostly during the last decade, that have

shed light on the accumulation of damage by neuronal

DNA and relation between this process and organism’s

aging and lifespan. We will discuss factors contributing

to the accumulation of postmitotic DNA damage in

neurons and specific genome regions, where this accu-

mulation takes place. New data on a high instability of

neuronal DNA have been changing our understanding

of multiple processes in the nervous system, as well as of

the evolution of nervous system itself.

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BIOCHEMISTRY (Moscow) Vol. 88 No. 11 2023

IF NOT UNDERREPLICATION,

THEN WHAT?

One of the problems with the existence of neuronal

“clock” measuring biological time in neurons is terminal

differentiation of these cells, i.e., the absence of mitotic

division in their adult life [4, 5]. Therefore, the “clock”

mechanism proposed by A. M. Olovnikov for dividing

cells monitoring the shortening of specific DNA se-

quences due to the underreplication of chromosomal

ends during mitosis [4] cannot be used for a postmitotic

neuron. Olovnikov was sure that “for the sake of clock

operation (in neurons – author’s note), nature had to

invent a mechanism that could work even in the absence

of DNA replication” [4]. In his latest publication in

2022, Olovnikov proposed the idea of epigenetic label-

ing of specialized temporal DNA [3]. Long before that,

he had suggested that such mechanism could be based

on the formation of DNA breaks (e.g., during active

transcription) and incomplete DNA repair, especially in

the terminal sections of hypothetical “clock” DNA [4].

And only ten years after publication of his work [4], the

first experimental data were obtained that demonstrated

formation of single- and double-strand DNA breaks in

neurons and their subsequent repair even in the course

of normal physiological activity of these cells.

NORMAL PHYSIOLOGICAL ACTIVITY

RESULTS IN THE FRAGMENTATION

OF NEURONAL DNA

Year 2013 has not yet been recognized by the neu-

roscience community as a year of a very important and,

perhaps, one of the most unexpected discoveries in

neuroscience, the full consequences of which would be

realized later. A group studying a mouse model of Alz-

heimer’s disease discovered a higher content of dou-

ble-strand DNA breaks (DSBs) in some brain regions

after two hours of normal activity in an open field, which

was observed not only in the diseased mice, but also

in healthy control animals [6]. After 24 h, most DNA

breaks in the control mice were repaired, while in ani-

mals with the Alzheimer’s disease, both the number of

neurons with fragmented DNA and the extent of DNA

fragmentation (assessed by the length of fluorescent

“tail” formed in the electromagnetic field by the nu-

clei with damaged DNA) remained significantly higher.

Toexclude the effect of stress caused by an open field the

authors used adrenalectomised animals with implanted

corticosterone pellets to hold their corticosterone level

constant. This operation did not decrease the content of

DSBs. Thus, stress seems not to account for the effect

of novelty on DSBs content. Moreover, sensory stim-

ulation in healthy animals led to the DNA fragmenta-

tion in the brain areas associated with sensory input.

Thus, lateral optical stimulation increased the number

of neurons DSBs in corresponding side of the visual

cortex. Optogenetic stimulation of the striatum also in-

creased the number of DSB-containing neurons[6].

Already two years later, it was demonstrated using

next-generation whole-genome DNA sequencing that

the electrical activity of neurons, their excitation, in-

duce formation of DSBs [7]. Moreover, it is the DNA

damage that links the electrical activity of neurons and

transcription of neuronal immediate early genes (IEGs),

such as Fos, Npas4, and Egr1 (it should be noted that

expression of these genes had been used as a marker of

active neurons). Formation of DSBs in the promoter re-

gions of IEGs was sufficient to activate their expression.

It was suggested that the enzyme linking electrical activ-

ity of neurons and DSB formation is DNA topoisomer-

ase IIβ (Topo IIβ). This enzyme introduces temporary

breaks in DNA and then restores two DNA strands,

leading to DNA demethylation in the promoters of ear-

ly response genes and their transcription. The knock-

out of the Topo IIβ gene abolished formation of DSBs

induced by the electrical activity of neurons and tran-

scription of early response genes [7]. In 2022, Delint-

Ramirez et al. [8] published an article that explained

how cell excitation affects the activity of Topo IIβ [8].

Calcium influx in response to the activation of excitato-

ry glutamate NMDA (N-methyl-D-aspartate) receptors

activates the phosphatase calcineurin. Calcineurin de-

phosphorylates Topo IIβ at S1509 and S1511 residues,

which stimulates its DNA cleavage activity and leads to

the formation of DSBs. During the electrical activity

of neurons, calcineurin interacts with Topo IIβ mostly

at the nuclear periphery, where DNA breaks occur [8].

By showing that DNA breaks and their consequences

took place in particular genome regions, the authors pro-

vided another important point in favor of the Olovnikov’s

concept. Beside involvement of calcium signaling in the

formation of DSBs, we should mention the influence

(although less specific) of intense neuronal metabolism,

leading to the generation of free radicals and reactive

oxygen species, which are commonly recognized as fac-

tors contributing to the instability of neuronal DNA [9].

The genome of neurons has been also searched for

the hotspots of single-strand DNA breaks(SSBs), an-

other type of DNA damage [10, 11]. It was found that

postmitotic human neurons derived from pluripotent

cells demonstrate unexpectedly high levels of SSBs in

particular genome regions. Using genome-wide map-

ping, it was suggested that these breaks were located at or

near CpG dinucleotides and sites of DNA demethylation

within gene enhancers [10]. Similar conclusions were

reached in 2021 by Reid et al. [12], who demonstrated

that the processes of DNA repair (and hence the initial

damage) often take place at the well-defined hotspots

adjacent to important genes. These hotspots are en-

riched with histone γH2AX isoforms andRNA-binding

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proteins and associated with evolutionarily conserved

regulatory elements of the human genome. These stun-

ning results required comprehension.

DNA BREAKS: A MECHANISM

OF NEURONAL PLASTICITY

OR A COST OF IT?

Year 2013 was signified by another development due

to the publication of a theoretical work by the Russian

scientist Alexei Krushinskii [13] (later, a revised version of

this article was published in an open access journal [14]).

Based on the Leon Brillouin’s negentropy principle of

information, Krushinskii suggested that to receive a new

information, the brain should pay not only with energy,

but also with a loss of original information. At present,

this hypothesis looks as a remarkably beautiful descrip-

tion of events that molecular neuroscientists encounter

in their studies of neuronal DNA, the “initial substrate”

of intelligence and aging.

Madabhushi et al. [7], who were the first to link

neuronal activity and transcription of early neuronal

genes with the formation of DSBs, made a shocking

conclusion that disruption of brain genome integrity is

anatural physiological phenomenon essential for synap-

tic plasticity, learning, and memory, since these process-

es require participation of early response genes. “If you

don’t break DNA, you can’t memorize” – this inter-

pretation has been accepted by a number of researchers

who have experimentally confirmed the need for tem-

porary DNA damage for the formation of various types

of memory and learning [15] or who have adhered to

this concept in their review [16]. Some researchers were

more wary of this new “tear, repair, remember” para-

digm of memory formation at the DNA level [17-19].

After all, DNA repair is not a 100% efficient process,

and if DNA breaks occur so often, the damage to the

brain genome will inevitably accumulate in a form of

both incomplete repair and errors (i.e., mutations).

Domutations accumulate?

ARE THERE MANY MUTATIONS

IN THE GENOMES OF INDIVIDUAL NEURONS

AND HOW ARE THEY DISTRIBUTED?

Sequencing of genomes of individual mouse neu-

rons [20] revealed an unusually high frequency of post-

mitotic mutations in evolutionarily conserved genome

regions, such as exons and promoters of genes with the

highest expression levels. In other words, mutations ac-

cumulate at the hotspots, which are also the hotspot of

DNA breaks. The most common type of mutation is a

single nucleotide variant (SNV) [20] that might result

from an error in the activity of repair DNA polymerases.

A similar conclusion was reached in the analysis of mu-

tations in human neurons [21-23]. Luquette et al. [23]

used an improved primary template-directed ampli-

fication (PTA) method, which allowed to reduce the

number of artifacts in detection of somatic mutations by

DNA sequencing. Whole-genome sequencing data from

fifty-two PTA-amplified single neurons were analyzed

using SCAN2, a new genotyping method developed by

the authors to identify SNVs and small insertions and

deletions(indels). The results of analysis confirmed that

the number of somatic mutations in individual human

neurons increased with age and that mutations accumu-

lated in functional genome regions, such as enhancers

and promoters [18,22,24-26].

The table shows the data on the types of DNA

damage in neurons occurring during normal physiolog-

ical activity or induced by various physiological factors.

NEURONAL ACTIVITY

REDUCES LIFESPAN

A strong argument in favor of the hypothesis that an

organism pays for the neuronal activity with something

truly significant was obtained in 2019 [27], when it was

found that the level of neuronal excitation directly affects

the lifespan. Zullo et al. [28] searched for the genes ex-

pressed in the human frontal cortex and associated with

healthy longevity. Differential analysis of their expression

was carried out in two groups of cognitively healthy peo-

ple who died at the age of 70-80 and 85-100 years, re-

spectively. Previously, the same group of researchers had

found an increase in the content of protein encoded by

the REST gene in centenarians [28]. The authors revealed

an inverse correlation between the level of REST mRNA

and the content of mRNAs of the genes associated with

neuronal excitation [27]. The expression level of these

genes, whose promoter regions contained the binding site

for the repressor protein REST, was significantly lower

in humans with extended longevity [27]. The authors

also studied the relation between the REST expression

and electrical activity of neurons in mice. Neurons of

REST-deficient mice accumulated fluorodeoxyglucose

(

F-FDG), indicative of increased neural activity. Inter-

mittent epileptiform discharges were significantly more

frequent in REST-deficient animals vs. control mice [27].

These findings suggested a link between the neuronal

excitation and lifespan in mammals, which was proven

in a model very distant from mammals– the nematode

Caenorhabditis elegans. These worms have an ortholog

of mammalian REST gene, spr-4, the protein product

of which protects cells from the damaging effects of re-

active oxygen species and some other adverse factors.

The authors used a broad arsenal of tools, from phar-

macological agents that altered (promoted or inhibited)

excitation to animals with suppressed or hyperactivated

DYAKONOVA172 2

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DNA damage in neurons occurring during normal physiological activity or induced by various physiological factors

Type

of DNA damage

Organism Factor Year Source

DSBs mice (wild-type) exploratory behavior in open field 2013 [6]

DSBs

mice expressing hAPP

(Alzheimer’s disease model)

exploratory behavior in open field 2013 [6]

DSBs mice (wild-type) visual stimulation 2013 [6]

DSBs mice (Adora2a-Cre) optical stimulation of striatum 2013 [6]

DSBs cultured mouse primary neurons

β-amyloid oligomers;

NMDA-dependent effect

2013 [6]

DSBs cultured mouse primary neurons

potassium chloride (KCl),

NMDA, or bicuculline

(promotion of neuronal excitation)

2015,

2023

[7, 19]

DSBs mouse hippocampal slices NMDA receptor agonist

2015,

2022

[7, 8]

DSBs mouse hippocampus memory reconsolidation 2020 [15]

DSBs zebrafish Danio rerio normal daily activity 2018 [32, 33]

DSBs Drosophila larvae and imagoes – 2020 [75]

SSBs

human neurons derived

from pluripotent cells

– 2021 [10-12]

Mutations

(SNVs, indels)

mouse neuron clones – 2016 [20]

Mutations

(SNVs, indels)

human neurons –

2012,

2018,

2022

[21, 23, 24]

spr-4 gene (obtained using the CRISPR-Cas9 system),

as well as with activated or repressed groups of excitatory

or inhibitory neurons. The results clearly indicated that

neuronal excitation decreased the lifespan, while sup-

pression of excitation extended it. Why? It might seem

surprising, but the authors of this discovery, which was

published in the Nature journal, did not discuss the link

between the neuronal excitation, disruption of genome

integrity, and induction of DSBs! They did not even cite

the reports of Suberbielle etal. [6] from 2013 and Mad-

abhushi etal. [7] from 2015 that declared the role of neu-

ronal excitation in the formation of DSBs in their titles.

A deeper insight into the mechanisms underlying

the influence of neuronal excitation on the lifespan pres-

ents several possibilities. The first one suggests a direct

effect of accumulated DNA damage. Thus, it is known

that an incomplete repair of DSBs can trigger apoptosis

in neurons (see review by Boutros etal. [16]). Another

one is direct reduction of the overall viability of an organ-

ism by accumulated mutations [18, 26]. Indeed, there is

an inverse correlation between the somatic mutation

load and lifespan in Drosophila melanogaster [29] and

mammals (however, in mammals, this association was

described for the intestinal epithelium cells, as neurons

were not assessed) [30]. Moreover, the effect of muta-

tions on fitness can be greater than previously thought,

since even synonymous mutations, earlier considered as

mostly neutral, turned out to be strongly non-neutral

because they change the level of gene expression and af-

fect the organism [31].

The second option, which assumes an existence of

a neuronal chronosome [4], would explain the influence

of neuronal excitation on the rate of DNA shortening

in such chronosome. Critical shortening leads to the

neuronal death and, according to Olovnikov’s hypoth-

esis[4], has a hormonal effect on the entire organism.

Given that mitosis and excitation are the main fac-

tors of mutagenesis in dividing cells and neurons, re-

spectively, the author of the current article proposed in

2020 [5] an existence of indirect neural mutation counter.

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Similar to telomeres that “count” cell divisions in pro-

liferating cells and indirectly determine the number of

accumulated mutations, neurons might have a “counter”

of excitations that estimates the duration of their histo-

ry of excitation and determines the lifespan of a neuron.

This counter could be a gene, whose expression gradual-

ly and irreversibly declines upon neuronal excitation and

whose product acts as a repressor ofapoptosis.

The report of Zada et al. [32, 33] indicated that the

DNA repair enzymes themselves can activate various

processes (e.g., sleep) at the organismal level. Thus, it

was found that poly(ADP-ribose) polymerase 1 (Parp1),

which initiates the repair of DSBs accumulated during

the daytime activity, induces sleep in fish hatchlings and

adult mice. The activity of Parp1 increases with sleep

deprivation, while inhibition of this enzyme reduces the

nighttime chromosome movement and suppresses re-

pair of DNA damage accumulated during the daytime.

These data suggest that the DNA repair system may also

signal to the mutation counter.

The mechanisms underlying the influence of neuro-

nal excitation on the lifespan remain to be elucidated, but

the participation of neuronal DNA breaks in these mech-

anisms seems very likely. Interestingly, A. M. Olovnikov

in his 2003 paper [4] referred to an older work showing

that irradiation of the brain, leading to the DNA frag-

mentation in neurons, shortened the life of Drosophila

larvae [34]. Unfortunately, at that time, it was still un-

known that neuronal excitation affects both DNA stabili-

ty and lifespan. However, Olovnikov’s idea that “it is the

brain that is the initial substrate of aging, and in a brain

cell, this substrate is DNA” has been confirmed.

The finding that DNA of differentiated neurons

is extremely unstable could significantly change many

concepts of how the brain works and how it evolves.

The  following sections will provide examples how the

well-known facts and recent discoveries can be reinter-

preted when viewed from this perspective.

THE COST OF INTELLIGENCE:

TRANSITION TO THE MOLECULAR LEVEL

It has been long known that although cognitive

activity provides enormous benefits, it comes at a cer-

tain biological cost. For humans, this was discussed a

century and a half ago in the famous work of Cesare

Lombroso “The man of genius” [35], as well as in more

modern works (for example, Gale et al. [36] and Smith

et al. [37]). A new area of psychology has emerged, cog-

nitive epidemiology, that studies the relationship between

IQ and various physiological, genetic, and social factors

in people [38]. In general, IQ positively correlates with

health and longevity, which could be explained by a sim-

ple inability of a sick body to provide a high cognitive per-

formance [38]. Obviously, social factors (e.g., access to

better food, living conditions, and medicine) play an im-

portant role in ensuring this positive connection. People

with an average and high IQ do not show an increased

predisposition to mental illness, unlike people with either

low or very high IQ that exhibit a higher predisposition

to developing bipolar disorders [36]. In recent years, this

research area has been significantly enriched by the data

of genetic studies [39, 40]. It was shown that genes typi-

cal for people with pronounced creative abilities are also

risk factors for various pathologies [39]. Later, a similar

correlation with a predisposition to neuropathology was

found for genes associated with the ability of individuals

to obtain higher education [40].

Some negative consequences of cognitive activity

have been described in mammals [13, 14, 41-45]. At the

beginning of this century, the first experimental results

were obtained indicating a high biological cost of learn-

ing, as well as selection for cognitive abilities in inver-

tebrates (flies) [46-50]. “Smart” fruit flies had reduced

stress resistance, fertility, and life expectancy. A similar

correlation between the ability to learn and susceptibility

to stress was found in two different populations of pond

snails [51,52]. However, methodological limitations and

lack of understanding of where and at what level to look

for the mechanisms providing the link between these

functions have hindered the development of this inter-

esting and important area of research. Partly, this was

also due to a simple, falsely reassuring explanation: the

brain consumes a lot of energy during cognitive activi-

ty, so that other organs or functional systems may suf-

fer from the energy deficit [49,50]. Now the situation

has changed. Recent methods of genome sequencing

and transcriptomic and epigenetic analysis of neurons

have made it possible to study the molecular basis of the

“cost of intelligence.”

In previous sections, we discussed the influence of

neuronal excitation on the formation of DNA breaks.

However, there exists an epigenetic substrate of cogni-

tive functions that can also increase the susceptibility of

neuronal genome to the accumulated damage.

The statement that cognitive functions are based on

the plasticity of expression of the neuronal genome ap-

peared more than 15 years ago and has been confirmed

experimentally in both vertebrates [53, 54] and inverte-

brates [55, 56]. As a rule, the open chromatin state and

DNA demethylation correspond to higher cognitive lev-

els [57-61]. Recently, additional evidence at the genome

level has emerged indicating that not only learning and

memory, but also a new environment which stimulates

neurogenesis and learning, are associated with chroma-

tin decondensation [62]. The same effects are caused

by the motor activity (which also stimulates neurogen-

esis and memory) even in the first-generation progeny

[63-65]. Finally, according to some data, prolonged ex-

citation predisposes neuronal genome to DNA demeth-

ylation and/or heterochromatin decondensation [66-68].

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Theoretically, both processes reduce the protection of

DNA from possible mutations and increase the likelihood

of transposable element insertion. The above-mentioned

age-related accumulation of indels in the promoters of

neuronal genes can be associated with the activity of

transposable elements (see Dumitrache and McKinnon

[69]) and not only with the repair of DNA breaks.

In other words, it is possible that brain “pays”

for the acquisition of new information, thinking, and

plasticity with the damage of its genetic information.

Post-mitotic DNA damage in neurons is a relatively new

topic (the first data were obtained only 10 years ago).

From the evolutionary point of view, on the contrary,

itis an ancient problem that might have appeared simul-

taneously with the appearance of first neurons (approx-

imately 600 million years ago) and, one way or another,

had been resolved in the course of evolution and, prob-

ably, differently in different taxa. The following sections

of the review will discuss some possible evolutionary

solutions to the problem of minimization of mutational

cost of brain plasticity and its biological consequences.

EVOLUTIONARY SOLUTIONS

TO REDUCE THE BIOLOGICAL COST

OF BRAIN PLASTICITY

DNA repair systems in the brain. It is obvious that

if neuronal activity poses an inherent risk to the genome

stability, one way to adapt to it would be to improve the

DNA repair systems in the brain. Indeed, the knockout

of the DNA repair genes involved in the repair of SSBs

caused multiple errors in the functionally significant

genome regions, mainly in the enhancers [10]. Theau-

thors suggested that disruptions of this system could be

associated with the development of human neurodegen-

erative diseases. Indeed, the risk of brain pathologies

should increase with any DNA repair deficiency in neu-

rons. Analysis of this relationship and studies of neuro-

nal DNA repair systems have recently received consider-

able attention of researchers (for more information, see

the review by Li etal. [25]).

The study published in February 2023 demonstrated

that in the course of evolution, neurons have acquired

specialized mechanisms for genome protection allow-

ing them to withstand for decades the effects of vari-

ous damaging factors during the periods of increased

activity [19]. A DNA repair mechanism dependent on

the neuronal activity has been identified that involved

accumulation in the activated neurons of a novel form

of NuA4-TIP60 chromatin modifier. This accumula-

tion was observed in the regions also expressing the

neuron-specific inducible transcription factor NPAS4.

By examining the pattern of DSBs caused by the brain

activity, the authors showed that NPAS4–NuA4 binds

to the damaged regulatory elements in the genome.

Disturbances in the NPAS4–NuA4 repair system result

in multiple cellular defects, including changes in tran-

scription, loss of the neuronal inhibition control, and

genomic instability, which can shorten the lifespan of

an organism. Therefore, the discovered neuron-specific

complex directly links neuronal activity to the genome

stability maintenance. These results are in good agree-

ment with the hypothesis on the improvement of DNA

repair systems as one of the important directions in the

evolution of nervous system.

Minimization of synchronism between chromatin

open state and electrical excitation? Although neuronal

excitation is often followed by the upregulation of gene

expression [66-68, 70, 71], it would be safer for the neu-

ron to avoid simultaneous occurrence of electrical activ-

ity and epigenetic processes characterized by the open

DNA state. In 1966, the first data showing that electri-

cal stimulation of mollusk neurons causes a transient

inhibition of mRNA synthesis with a strong rebound

effect (activation of mRNA synthesis above the initial

level) after cessation of electrical activity were obtained

[72,73], that were interpreted within the framework of

hypothesis suggesting redistribution of metabolism and

reduction of energy expenditures during cell excitation.

However, these data take on a new meaning with current

understanding of excitation as a threat to DNA stabil-

ity. The transient shutdown of gene expression can be

viewed as a mechanism protecting neuronal DNA.

Other data mentioned above indicate that the re-

modeling of active chromatin and DNA repair in fish

motor neurons occurred during the sleep characterized

by a shutdown of the electrical activity of these neu-

rons[32]. Several recent reviews have summarized a cur-

rent state of affairs in the studies of the gene expression

dependence on the neuronal activity in mammals and

model invertebrates [70, 71, 74], illustrating its complex

and controversial nature.

Polyploidy of neurons. Accumulation of a large num-

ber of DNA mutations in the neurons of healthy people

and animals indicates that DNA repair mechanisms are

not efficient enough to ensure the stability of the neu-

ronal genome. In this situation, an existence of “genetic

safety net” could be another effective approach for re-

ducing the biological cost of the nervous system activity.

Since mutation is a random event, simply increasing the

number of genome copies can be such safety mechanism.

However, the number of genome copies can increase in

different ways. Within a single neuron, it is a well-known

phenomenon of somatic polyploidy. The number of neu-

rons can be increased as well. Different animal taxa have

implemented different solutions. Thus, vertebrates have

an increased the overall number of cells, while some pro-

tostomes, for example, gastropods (but not only them),

use somatic polyploidy [75,76].

Giant neurons (up to 1mm in diameter) of gastro-

pods have been known for a long time. Most of cell body

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in these neurons is occupied by the nucleus [77, 78].

It was found that this giant nucleus contains multiple

genome copies (up to 600,000) [76, 78, 79] that are be-

lieved to be essential for the metabolism of giant cells

and associated with neuronal gigantism. With the emer-

gence of data on the DNA damage in neurons, it was

logical to assume that neuronal polyploidy reflects one

of the evolutionary solutions aimed to reduce the conse-

quences of DNA damage. Experimental data in favor of

this hypothesis have been obtained in the representatives

of another taxon– insects, in which neuronal polyploi-

dy also occurs by to a much lesser extent [75]. Several

species-specific, as well as sex- and age-related differ-

ences in the number of polyploid neurons in the brains

of three different Drosophila species were described in

detail. On average, a fraction of polyploid (mostly tetra-

ploid, 4 C) neurons was estimated as 10-15% of all neu-

rons in the adult brain. The largest number of polyploid

neurons was found in the optic lobes. It was shown that

tetraploidy was not a result of cell fusion. Interesting-

ly, the largest number of DSBs in the neurons has been

identified in the optical lobes as well. Finally, using

etoposide to induce DSBs, it was demonstrated that the

brain response to this drug involved a significant increase

in the proportion of polyploid neurons. And, perhaps,

most importantly, polyploid neurons survived after in-

duced DNA damage better than diploid neurons [75].

Therefore, this work confirmed two assumptions regard-

ing the role of neuronal polyploidy. First, polyploidy,

indeed, protects the cells in the event of DNA damage;

second, neurons have a mechanism for activation of

DNA endoreplication in response to DNA damage.

These data suggest a new look at the neuronal poly-

ploidy in human brain. The association between neu-

rodegenerative diseases and increase in the number of

polyploid neurons had been demonstrated, and for some

time, polyploidy had even been considered as a possi-

ble cause of these pathologies [80, 81]. However, it now

seems more likely that polyploidy arises in response to

DNA damage (which is apparently the main cause of

neurodegeneration) as one of the protective mechanisms

in neurons.

Altruistic neurons of vertebrates and glutamate.

Out of the two approaches to increasing the number or

genome copies (in the same cell or by increasing the

number of cells), somatic polyploidy seems to be a less

efficient solution. First, healthy copies of a damaged

gene in a polyploid cell will play their protective role

only until the “toxic” products encoded by the damaged

gene and detrimental to the cell activity are synthesized.

Second, it does not allow to sacrifice a damaged neuron

without severe consequences for the rest of the brain.

So, it seems that the vertebrate brain has chosen the

second option. Neurons that accumulate mutations and

DNA damage die, but their epigenetic and functional

clones retain the information that remains unchanged.

Such redundancy makes it also possible to distribute the

activity between the neurons, which reduces mutational

load on an individual cell and allows the body to main-

tain neurons longer. This assumption is in good agree-

ment with numerous data showing that the same task

is performed each time by slightly different populations

of neurons [82, 83].

Looking at the evolution of vertebrate brain from

this point of view, we can argue that the rapid increase

in the number of neurons in the evolution of vertebrates

has primarily served the purpose of protecting the brain

and ensuring a sufficient lifespan of an organism. At the

same time, it was also a pre-adaptation to the develop-

ment of cognitive abilities. This assumption is in good

agreement with the fact that the increase in the size of

human brain in the evolution has not been accompanied

by the development of more sophisticated tools over a

period of millions of years [84]. As well as the patho-

genesis of some neurodegenerative diseases (e.g., Par-

kinson’s disease), characterized by the asymptomatic

death of up to 30-70% of neurons in particular brain

areas [85]. These data suggest an existence in human

brain of a developed “safety net” in a form of multiple

epigenetically similar and interchangeable neurons.

Adult neurogenesis might also be a subject of func-

tional revision. Previously, its activation had been consid-

ered as a mechanism for promoting cognitive activity, i.e.,

an ability to learn and memorize information in a new

environment [86]. Now, it can also be viewed as a pro-

tective mechanism triggered in anticipation of increased

cognitive load.

Recent data suggest that the increase in the number

of neurons in the evolution of vertebrate brain has been

achieved mainly due to the increase in the proportion of

glutamate neurons [87] (compare 50% in rodents vs. 80%

in humans). This seems paradoxical, since more complex

systems tend to be more diverse. However, this sugges-

tion makes sense if we assume that these additional neu-

rons had originally served as a “safety net”, a source of

genome “guardians”. Why glutamatergic neurons could

be used for this purpose was explained in several recent

publications by Moroz etal. [87-89]. To put it briefly, the

development of glutamatergic phenotype is energetical-

ly cheap and epigenetically simple (only vesicular gluta-

mate transporter has to be expressed, while glutamic acid

is already present in all cells as their main metabolite).

Another interesting finding is that vertebrates have lost

the diversity of glutamate receptors compared to other

groups and retained only the excitatory receptors [87].

It would be interesting to think about the benefits that

might have been gained from this.

Excitability at the physiological level provides con-

ditions for exit from a stable state and formation of new

ensembles of neurons. Increasing the plasticity of the

epigenome can achieve even greater plasticity and diver-

sity. Indeed, there is an evidence of intracellular cascades

DYAKONOVA172 6

BIOCHEMISTRY (Moscow) Vol. 88 No. 11 2023

linking NMDA and AMPA glutamate receptors to the

heterochromatin decompaction factor Gadd45 [90], i.e.,

excitatory glutamate appears to be associated with both

functions that increase plasticity at the physiological and

epigenetic levels. This can be another advantage of gluta-

matergic phenotype. However, glutamate, through ion-

otropic receptors, stimulates formation of DNA breaks

and their repair [91], while excitation shortens the lifes-

pan [27]. If this is the case, then the emphasis on the

glutamate phenotype of neurons in the evolution of ver-

tebrates could have and should have stimulated natural

selection to promote an increase in the number of neu-

rons in order to reduce the biological cost of DNA dam-

age during glutamatergic signaling [92]. Positive feed-

back loop in the evolution of vertebrate brain?

Transition of plasticity to the RNA level in cepha-

lopods. DNA instability in neurons could be a cause of

another interesting phenomenon recently found in ceph-

alopods, namely, modification of transcripts at the RNA

level. RNA undergoes large-scale editing (or recoding)

especially in the nervous system, where almost all tran-

scripts are edited [93, 94]. Moreover, 65% of editing

sites found in the coding sequences are nonsynonymous.

Only one type of editing, replacement of adenosine

with inosine (A-to-I), occurs at more than 70,000 sites

in cephalopods vs. ~1000 sites in Drosophila and ~3000

in humans [95]. Moreover, unlike mammals, cephalo-

pods demonstrate clear evolutionary selection for the in-

crease in the number of RNA editing sites in functionally

significant genes [93, 95], the editing of which affects the

structure and function of encoded proteins [93]. In one

case (recoding of potassium channel protein transcript in

octopuses), RNA editing was found to provide an adap-

tation to different living temperatures [96].

However, the most important finding was a discov-

ery of the protective role of RNA editing in the main-

tenance of genome stability that was announced in the

title of the article “Trade-Off Between Transcriptome

Plasticity and Genome Evolution in Cephalopods”[93].

The authors assessed the distribution of mutations (de-

letions, synonymous and nonsynonymous single nucleo-

tide substitutions) and found that the DNA regions close

to the RNA editing sites accumulated the least number

of mutations. From 23 to 41% genome regions in dif-

ferent species of cephalopods were stabilized and these

stabilized regions encoded RNAs that underwent edit-

ing. The mechanisms of such plasticity transfer remain

to be explored. It can be assumed that the driver for the

plasticity transition to the RNA level is protection of

unstable DNA in neurons. Perhaps, this non-standard

solution, combined with the increase in the number

ofneurons, ensures an outstanding cognitive evolution

The main risk factors for DNA stability in neurons, identified types of DNA damage, and possible evolutionary adaptations reducing the biological

cost of neuronal DNA instability. The risk factors include high energy metabolism of neurons, formation of reactive oxygen species (ROS), neu-

ronal excitation, and activation of NMDA receptors by glutamate. Activation of NMDA receptors leads to the formation of DSBs in the promoter

regions of early response genes, which induces their expression and subsequent DNA repair. This mechanism involves an increase in the intra-

cellular calcium concentration and activation of calcineurin, which phosphorylates TopoIIβ. Other risk factors include changes in the chromatin

state and chromatin decondensation, which increases DNA susceptibility to various mutagens, e.g., mobile genetic elements (MGEs). Chromatin

decondensation can also be induced by glutamate receptors via activation of Gadd45 heterochromatin. All these factors are necessary for normal

functioning of the central nervous system to provide its plasticity and cognitive functions. Accumulation of neuronal DNA damage such as DSBs,

SSBs, SNVs and indels is actively studied (table). These damages can directly or indirectly lead to the development of neurodegenerative diseases

and might determine the lifespan. Possible evolutionary adaptations that reduce the cost of DNA instability in neurons include DNA repair de-

pendent on the electrical activity of neurons (NPAS4–NuA4), Parp1-dependent repair of DSBs accumulated during the animal’s daytime activity

and activating sleep in vertebrates, neurogenesis and increase in the number of neurons in the central nervous system, somatic polyploidy (which is

especially pronounced in gastropods), mRNA editing, and transfer of plasticity from the DNA to RNA level in cephalopods

DNA INSTABILITY IN NEURONS 1727

BIOCHEMISTRY (Moscow) Vol. 88 No. 11 2023

of cephalopods, which rank first among protostomes in

terms of their mental abilities.

CONCLUSION

The understanding that the post-mitotic DNA in-

stability in neurons is a cost of their electrical activity

and high genome plasticity is changing the theoretical

landscape not only in neuroscience, but in biology as

well. As Olovnikov suggested, neuronal DNA can be

the “clock” determining the lifespan of an organism.

Numerous accumulated data have significantly in-

creased the likelihood of this hypothesis. In addition,

the instability of neuronal DNA seems to have promoted

the search for various ways to reduce the biological cost

of brain functioning in the evolution of Metazoa and

has become a driver of this evolution. Various phenom-

ena, such as sleep, increase in the number of neurons

in vertebrate evolution, adult neurogenesis, distribution

of neuronal activity, somatic polyploidy, and RNA edit-

ing, have received a new meaning and new understand-

ing when considered in the light of the resolution of

the “plasticity vs. neuronal DNA instability” trade-off.

Thetopic is very important not only for basic neurosci-

ence, but also for translational medicine. The main risk

factors, types of neuronal DNA damage, and possible

evolutionary adaptations that reduce the biological cost

of DNA instability are shown in the figure.

There are still many problems that require exper-

imental verification and represent today’s challenges

inmolecular neurobiology, which is especially true for

the neurobiology of invertebrates as organisms that pro-

vide unique experimental opportunities. There are also

other unsolved urgent problems.

(i) Verification of the assumption that neuronal

excitation affects not only DSB formation, but also ac-

cumulation of DNA damage and mutations in neuronal

DNA. This can be done in C. elegance and D. melano-

gaster using optogenetic approaches or by electrophysi-

ological stimulation in gastropods whose giant neurons

can be easily identified and isolated for electrophysio-

logical studies with microelectrodes. For these species,

DNA sequencing can be done for neurons with a sig-

nificant baseline difference in their electrical activity or

for the same neurons with and without electrical stimu-

lation.

(ii) The search for specific genetic and epigene-

tic changes that predispose animals with higher cogni-

tive abilities to shorter lifespans, reduced stress toler-

ance, and reduced fertility. The existing studies [46-52]

should be continued at the level of neuronal DNA and

epigenetic regulation of neuronal genome. In particular,

do “smart” invertebrates express more actively the genes

associated with neuronal excitation or chromatin open

state? Do “smart” animals accumulate more mutations?

Is the biological cost (stress intolerance, low fertility,

and lifespan) in “smart” animals a direct consequence

of accumulation of mutations?

(iii) It is easier to search for the chronomeres or

other intracellular neuronal substrates responsible for

the regulation of lifespan in invertebrates.

(iv) In the light of new data on the influence of neu-

ronal activity on the damage of neuronal DNA, we can-

not provide a convincing explanation or justification of

the benefits of cognitive load on the brain health, which

is now given so much importance. One can only as-

sume a preconditioning for the cognitive load, similar to

hypoxic or ischemic training. The exact mechanisms of

this cognitive preconditioning still have to be discovered.

Funding. This work was supported by the Russian

Science Foundation (project no.22-24-00318).

Acknowledgments. I express my gratitude to

A. I. Kal mykova, I. A. Olovnikov, and I. S. Zakharov

for advice and comments during manuscript editing

and to D.D. Vorontsov for help in preparing the figure.

Ethics declarations. The author declares no con-

flict of interest. The work does not contain description

of studies involving humans or animals performed by

the author.

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