Neurobiology of Disease 132 (2019) 104591

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Neurobiology of Disease
journal homepage: www.elsevier.com/locate/ynbdi

Review

Multigenerational epigenetic inheritance: One step forward, two generations T

back
⁎ ⁎⁎
Jennifer J. Tuscher , Jeremy J. Day
Department of Neurobiology, Evelyn F. McKnight Brain Institute, University of Alabama at Birmingham, Birmingham, AL 35294, USA

A R T I C LE I N FO A B S T R A C T

Keywords: Modifications to DNA and histone proteins serve a critical regulatory role in the developing and adult brain, and
Epigenetics over a decade of research has established the importance of these “epigenetic” modifications in a wide variety of
Gene expression brain functions across the lifespan. Epigenetic patterns orchestrate gene expression programs that establish the
Heritability phenotypic diversity of various cellular classes in the central nervous system, play a key role in experience-
Transgenerational
dependent gene regulation in the adult brain, and are commonly implicated in neurodevelopmental, psychiatric,
DNA methylation
and neurodegenerative disease states. In addition to these established roles, emerging evidence indicates that
Non-coding RNA
Histone modifications epigenetic information can potentially be transmitted to offspring, giving rise to inter- and trans-generational
epigenetic inheritance phenotypes. However, our understanding of the cellular events that participate in this
information transfer is incomplete, and the ability of this transfer to overcome complete epigenetic repro-
gramming during embryonic development is highly controversial. This review explores the existing literature on
multigenerational epigenetic mechanisms in the central nervous system. First, we focus on the cellular me-
chanisms that may perpetuate or counteract this type of information transfer, and consider how epigenetic
modification in germline and somatic cells regulate important aspects of cellular and organismal development.
Next, we review the potential phenotypes resulting from ancestral experiences that impact gene regulatory
modifications, including how these changes may give rise to unique metabolic phenotypes. Finally, we discuss
several caveats and technical limitations that influence multigenerational epigenetic effects. We argue that
studies reporting multigenerational epigenetic changes impacting the central nervous system must be inter-
preted with caution, and provide suggestions for how epigenetic information transfer can be mechanistically
disentangled from genetic and environmental influences on brain function.

1. Introduction
Epigenetic modifications provide an essential mechanism for the
fine tuning of gene expression in response to extracellular signals and
changes in the environment. These alterations can occur in the form of
chromatin remodeling, direct covalent modifications to the DNA itself,
post-translational modifications to histone proteins, and activity of non-
coding RNAs. In the central nervous system, epigenetic mechanisms are
responsible for shaping early developmental programming (Fagiolini
et al., 2009; Feng et al., 2010; Bale, 2015), responses to stressful or
appetitive stimuli (Renthal and Nestler, 2008; McEwen et al., 2012; Day
et al., 2013; Stankiewicz et al., 2013; Klengel and Binder, 2015), ac-
tivity-dependent changes in synaptic plasticity (Cortes-Mendoza et al.,
2013; Karpova et al., 2017; Clayton et al., 2019), and the formation and
long-term storage of memory (Miller and Sweatt, 2007; Lubin et al.,
2008; Gupta et al., 2010; Day and Sweatt, 2011; Day et al., 2013;
Zovkic et al., 2014). Despite the well-established role of epigenetic
regulation of a number of these neural processes, whether experience-
dependent epigenetic marks acquired in an individual can be inherited
by the subsequent generation remains an open topic of debate.
Accumulating evidence supports the notion that external stimuli,
including environmental exposure to chemicals, extreme heat, psy-
chological stressors, traumatic experience, substances of abuse, and
dietary intake, can all shape epigenetic state at the cellular level in the
exposed individual (Waddington, 1953; Francis et al., 1999; Anway
et al., 2005; Baccarelli and Bollati, 2009; Mathers et al., 2010; Gapp

⁎
Correspondence to: J. J. Tuscher, Department of Neurobiology, UAB The University of Alabama at Birmingham, SHEL 930, 1825 University Blvd, Birmingham, AL
35294-2182, USA.
⁎⁎
Correspondence to: J. J. Day, Department of Neurobiology, Evelyn F. McKnight Brain Institute, UAB The University of Alabama at Birmingham, SHEL 910, 1825
University Blvd, Birmingham, AL 35294-2182, USA.
E-mail addresses: jtuscher@uab.edu (J.J. Tuscher), jjday@uab.edu (J.J. Day).

https://doi.org/10.1016/j.nbd.2019.104591
Received 2 April 2019; Received in revised form 22 July 2019; Accepted 26 August 2019
Available online 27 August 2019
0969-9961/ © 2019 Elsevier Inc. All rights reserved.
J.J. Tuscher and J.J. Day Neurobiology of Disease 132 (2019) 104591

Fig. 1. Intergenerational vs. Transgenerational Inheritance. A, Intergenerational inheritance involves exposure of the parent generation (F0, male or non-pregnant
female) to an environmental factor or external stimuli (e.g., drugs of abuse, high fat diet, stress) that leads to an epigenetic alteration in the parent and parental
germline cells (F1; e.g., sperm or oocytes). Epigenetic modifications may be observed in somatic tissues of the F1 adult, but do not persist in the F2 generation. B, For
epigenetic effects to be considered transgenerational, modifications acquired in the F0 generation would need to be observed in the F1 and F2 generations, and
potentially beyond. For pregnant females that experience environmentally-induced epigenetic changes, the pregnant dam (F0), fetus (F1), and fetal germline cells
(F2) would all be exposed to the external stimulus. In this case, intergenerational effects encompass transmission from F0 to F2, and only persistence to the F3
generation would be considered a transgenerational effect.

et al., 2014; Norouzitallab et al., 2014; Vickers, 2014; Klengel and
Binder, 2015; Cadet, 2016; Nilsson et al., 2018; Walker and Nestler,
2018). Alterations in epigenetic profile have also been observed in the
offspring of exposed individuals (i.e., intergenerational inheritance
(Perez and Lehner, 2019)). However, whether the transfer of such
epigenetic marks can occur strictly via germline-dependent transmis-
sion, in the absence of any direct exposure to the driving external sti-
mulus, remains controversial. This review will first define the distinc-
tion between intergenerational and transgenerational epigenetic
inheritance (Skinner, 2008; Perez and Lehner, 2019), as these terms are
frequently conflated in the literature. Next, cellular transmission of
epigenetic modifications will be discussed, followed by putative me-
chanisms of heritable, multigenerational epigenetic transmission. Fi-
nally, preclinical evidence for multigenerational propagation of epige-
netic marks in mammals will be reviewed, as well as the translational
implications of such work. For the purposes of this review, multi-
generational inheritance encompasses both intergenerational in-
heritance (from F0 to F1), and transgenerational inheritance (F0 to F2
or beyond; Fig. 1).
2. Germline reprogramming and intergenerational vs.
transgenerational epigenetic inheritance
Epigenetic inheritance across multiple generations has been most
well documented in plant species, which do not require the passage of
epigenetic marks to occur through the germline (Quadrana and Colot,
2016). The intriguing notion that mammalian gene expression pro-
grams could similarly be influenced by external stimuli to alter phe-
notypic traits has more recently become a topic of great interest and
debate in biomedical sciences. This is particularly true for instances in
which DNA sequence-based inheritance (i.e., Mendelian genetics)
cannot fully explain the pattern heritability of certain phenotypic traits
across multiple generations. Despite the passage of epigenetic mod-
ifications becoming an attractive candidate mechanism for the in-
heritance of such traits, clear evidence that observable phenotypes are a
direct result of germline-dependent transmission of epigenetic altera-
tions has also been severely lacking.
This pursuit is complicated by several remarkable features of re-
productive biology. In mammals, haploid gamete cells (ovum and
sperm) each possess their own unique epigenetic landscapes prior to
fertilization. These patterns are dominated by repressive marks,
yielding transcriptionally inactive cells and (for sperm) a highly com-
pacted genome architecture in which the majority of histones have been
replaced by protamines (Balhorn, 2007). However, upon fertilization
these landscapes are rapidly altered, and established epigenetic marks
are removed. This reprogramming is critical to establishing a totipotent
state in the zygote, but also necessary to ensure the genetic and epi-
genetic blueprint responsible for determining the fate and function of

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J.J. Tuscher and J.J. Day
thousands of distinct cell types cannot simply be undone by parental
experience. Without normal reprogramming of the germline, proper
cellular differentiation cannot be achieved. However, the erasure of
epigenetic marks does not occur with equal efficiency at all sites across
the genome (Guibert et al., 2012; Seisenberger et al., 2012; Tang et al.,
2015), providing a small window of opportunity for some epigenetic
modifications to escape early periods of reprogramming. The matter of
which loci are protected during these phases of epigenomic repro-
gramming, and why some regions escape while others do not, remains
an important open question. Another point that merits consideration is
why might modifications imposed by the environment be stably main-
tained across multiple generations? Such a mechanism that allows en-
vironmental stimuli to influence gene expression programs in response
to external cues without directly altering the DNA has the potential to
be adaptive. As such, understanding the complex interactions between
the genome, epigenome, and environment may have important im-
plications for the heritability of disease states or adaptive fitness of the
species in general.
Although the transfer of environmentally induced epigenetic mod-
ifications through mitotic and meiotic processes is relatively well es-
tablished in non-mammalian species (e.g., plants, nematodes, droso-
phila (Cavalli and Paro, 1998, 1999; Quadrana and Colot, 2016;
Serobyan and Sommer, 2017; Minkina and Hunter, 2018; Moore et al.,
2019; Posner et al., 2019)), mammalian research on multigenerational
epigenetic inheritance has proved considerably more challenging. This
is in part due to a number of caveats that must be carefully considered
when designing and interpreting experiments. First, it is important to
distinguish between intergenerational vs. transgenerational epigenetic
inheritance. The importance of this distinction extends beyond se-
mantics, as the mechanisms that govern epigenetic alterations to germ
cells as a result of direct exposure to an environmental stimulus may
differ from the mechanism through which acquired epigenetic marks
are maintained and propagated during cellular division and across
generations. Intergenerational inheritance involves exposure of the
parent generation (F0, male or non-pregnant female) to an environ-
mental factor (e.g., drugs of abuse, high fat diet, stress, etc.) that leads
to an epigenetic alteration in the parent and likely the parental germ-
line cells (F1; e.g., sperm or oocytes). Observation of epigenetic marks
or phenotypic traits in offspring through this type of F0 to F1 trans-
mission would be considered an intergenerational effect (see Fig. 1A).
Unlike intergenerational inheritance, transgenerational inheritance re-
lies strictly on germline transfer of epigenetic marks in the absence of
any direct environmental exposure. As such, for an effect to be con-
sidered transgenerational, an inherited epigenetic mark and corre-
sponding trait would need to persist in the F2 generation, and poten-
tially beyond (Fig. 1B). For pregnant females that experience
environmentally-induced epigenetic changes, the pregnant dam (F0),
fetus (F1), and fetal germline cells (F2) would all be exposed to the
external stimulus. In this case, intergenerational effects encompass
transmission from F0 to F2 (Fig. 1A), and only persistence to the F3
generation would be considered a transgenerational effect (Fig. 1B). At
present, limited evidence exists to support either mode of transge-
nerational epigenetic inheritance in mammals. Understanding the pu-
tative mechanisms through which epigenetic alterations may be passed
from generation to generation first requires an understanding of how
such marks are maintained at the cellular level during DNA replication
and cellular division. Therefore, the cellular transmission of epigenetic
modifications will be discussed in the following section, followed by a
description of the current evidence in support of transgenerational
epigenetic inheritance and its potential underlying mechanisms.
3. Putative cellular mechanisms for epigenetic inheritance
Over the past several decades, much research has focused on the
transmission of phenotypic traits or disease states via non-genetic
modes of inheritance, and consequently, much debate has ensued
3
Neurobiology of Disease 132 (2019) 104591
regarding the putative mechanisms through which such effects would
be perpetuated. In addition to direct alterations to the DNA itself, a host
of epigenetic factors, including DNA methylation, histone post-trans-
lational modifications (PTMs), and small non-coding RNAs, can be
passed from one generation to the next via mitosis and meiosis. Below
we discuss potential epigenetic routes for cellular inheritance of gene
expression profiles, including DNA methylation, post-translation his-
tone modifications, and small non-coding RNAs. Evidence for the
transmission of associated heritable phenotypic states will be outlined
in later sections of the review.
3.1. Cellular transmission of epigenetic marks via DNA methylation
To discern how epigenetic inheritance across generations may
occur, it is first useful to understand how epigenetic marks are trans-
mitted at the cellular level. DNA methylation involves the covalent
addition of a methyl group to cytosines in the 5th position of pyr-
imidine rings in the DNA backbone, forming 5-methylcytosine (5mC).
This predominantly occurs within cytosine-guanine dinucleotides
(CpGs) in most cells, and is dynamically regulated by DNA methyl-
transferases (DNMTs; (Holliday and Pugh, 1975; Bird, 1992)). The
stable inheritance of DNA methylation patterns requires not only high
fidelity DNA replication, but also faithful maintenance of cytosine
methyl marks. DNA replication is incredibly accurate, with an error
occurring only once per 107–108 bases copied (Kunkel, 2004). During
replication, methylation marks on the parent DNA strand are en-
zymatically copied to the complimentary daughter strand by main-
tenance methyltransferase DNMT1, which coordinates the transfer of
methyl groups to hemi-methylated CpG sites throughout cellular re-
plication (Bird, 1992). This provides a potential route for methylation
marks at hemi-methylated sites to be reestablished and perpetuated
across multiple rounds of cell division. Methylation of non-methylated
sites can also occur throughout the life cycle of the cell, and this process
is coordinated by de novo methyltransferases DNMT3a and DNMT3b
(Okano et al., 1999). A key issue regarding the transfer of epigenetic
marks is the stability of methylation patterns across multiple rounds of
cell division. DNA methylation is considerably less accurate than DNA
replication with a ~96% fidelity rate, which roughly translates to 1
error for every 25 methylation marks copied per cell division (Laird
et al., 2004). This elevated error rate introduces variation in methyla-
tion profiles due to faulty copying during replication and cell division,
which in turn yields cell populations with diverse methylation patterns
(Silva et al., 1993). Thus, even at the cellular level within a single in-
dividual there are multiple opportunities for errors to be introduced
into methylation patterns, preventing highly accurate transmission
during cell division. Taken together, this suggests that although DNMTs
may largely preserve methylation programs across the cell life cycle,
this process is inherently less accurate than DNA replication. This issue
creates one of several substantial hurdles that must be overcome in
order for methylation marks to be stably transmitted not only during
cell division, but also across multiple generations.
The processes of active and passive DNA demethylation are also key
to understanding the cellular maintenance of methylation programs.
Cytosines can be actively demethylated by TET (ten eleven transloca-
tion) enzymes, which are responsible for the addition of a hydroxyl
group to 5-methylcytosines, forming 5hmC (Tahiliani et al., 2009; Ito
et al., 2010). This is followed by a series of oxidation steps resulting in
several intermediates including 5fC (5-formylcytosine) and 5CaC (5-
carboxylcytosine), prior to base excision and repair with an unmodified
cytosine (He et al., 2011; Ito et al., 2011). It remains to be determined
whether these intermediates represent passive transitional stages on the
road to demethylation, or if each modification serves its own distinct
function. Passive demethylation occurs when DNMT1 activity is not
present or inhibited and 5mC marks are not able to be maintained
during the replication process, leading to gradual dilution of the me-
thylation profile over time (Rougier et al., 1998; Lee et al., 2014). Both
J.J. Tuscher and J.J. Day Neurobiology of Disease 132 (2019) 104591

Fig. 2. Methylation levels during epigenetic reprogramming and development. The initial wave of mammalian methylome reprogramming occurs after fertilization
in the pronuclei of the paternal and maternal genomes via distinct demethylation mechanisms. Paternal pronuclei undergo active demethylation mediated via TET
enzymes, whereas maternal pronuclei are passively demethylated. Global methylation levels remain low in the zygote and blastocyst stage, until widespread re-
establishment of methylation by de novo DNMTs 3a and 3b occurs in the embryo after implantation, during the differentiation process. Once methylation programs
are established in differentiated cells of the developing embryo, cell-type specific patterns will be maintained in somatic cells during maturation and throughout the
lifespan by DNMT1. Methylome reprogramming also occurs in the primordial germline cells of the developing embryo. Unlike the distinct mechanisms experienced
by the paternal and maternal pronuclei shortly after fertilization, both active and passive modes of demethylation participate in primordial germ cell reprogramming.

active and passive demethylation mechanisms contribute to widespread
post-fertilization demethylation in the developing zygote, and thus in-
fluence the propagation of methylation patterns inherited from the
maternal and paternal genomes. Each type of demethylation process
will be discussed in greater detail in the following section.
3.2. Multigenerational epigenetic inheritance via DNA methylation
Given its relatively stable perpetuation during cell division, DNA
methylation has long been considered a putative route through which
epigenetic patterns could be transferred from one generation to an-
other. However, the methylome undergoes heavy reprogramming at
least twice during development in mammals (Lee et al., 2014), making
many skeptical about the likelihood of transgenerational epigenetic
inheritance via DNA methylation. The initial wave of mammalian me-
thylome reprogramming occurs in the pronuclei of the paternal and
maternal genomes via distinct demethylation mechanisms (Fig. 2;
Rougier et al., 1998; Mayer et al., 2000; Oswald et al., 2000; Santos
et al., 2002; Iqbal et al., 2011; Smith et al., 2012). Active demethylation
in paternal gametes is mediated via TET enzymes, which are re-
sponsible for converting 5mC to an unmethylated state through a series
of successive oxidation steps (Morgan et al., 2005; Guo et al., 2014a).
Concurrently, the maternally inherited genome undergoes passive de-
methylation prior to implantation, wherein methyl marks fail to be
maintained during replication, and are thus gradually diluted in the
zygote (Mayer et al., 2000; Guo et al., 2014a). Global methylation le-
vels remain low in the blastocyst stage, until widespread re-establish-
ment of methylation by de novo DNMTs 3a and 3b occurs in the embryo
after implantation (Fig. 2; (Santos et al., 2002; Smith et al., 2012). This
re-establishment of methylation patterns by DNMT3a and 3b governs
cell fate and function during the differentiation process. Once methy-
lation programs are established in differentiated cells of the developing
embryo, cell-type specific patterns will be maintained in somatic cells
during maturation and throughout the lifespan by DNMT1 (Fig. 2;
Rougier et al., 1998). These periods of widespread demethylation fol-
lowed by re-methylation in the post-implantation embryo create a
substantial obstacle for the inheritance of any methylation marks that
may have been established in the parental epigenome.
While this sequence of methylation events appears to be the general
rule of thumb, notable examples of persistent DNA methylation after
post-fertilization reprogramming have been documented. For example,
in mice, DNA methylation persists at specific differentially methylated
regions (DMRs) in oocytes during post-fertilization periods that are
typically characterized by widespread hypomethylation (Smith et al.,
2012). Certain DMRs also remain partially methylated during the pre-
implantation stage in human oocytes (Guo et al., 2014b), indicating a
possible route for epigenetic inheritance via maternally transmitted
methylation marks at these sites. Retrotransposable elements known as
intracisternal A particles (IAPs) may also be resistant to DNA de-
methylation during embryonic development, as some of these loci re-
main mostly hypermethylated while primordial germ cells undergo
widespread demethylation (Lane et al., 2003; Guibert et al., 2012;
Seisenberger et al., 2012). Persistent DNA methylation within gene
bodies at intragenic enhancers has also been reported at the pre-im-
plantation stage in humans. Given that intragenic enhancer elements
are also subject to control by DNA methylation state, this represents a
potential platform for methylation-based epigenetic inheritance of
certain cis-regulatory states (Birnbaum et al., 2012; Blattler et al.,
2014).
Methylome reprogramming also occurs in the primordial germline
cells of the developing embryo (Fig. 2; (Smith et al., 2012). Unlike the
distinct mechanisms experienced by the paternal and maternal pronu-
clei shortly after fertilization, both active and passive modes of de-
methylation participate in primordial germ cell reprogramming. More
specifically, primordial germline demethylation is first exerted through
active TET-mediated demethylation, followed by subsequent replica-
tion-dependent passive demethylation (Guibert et al., 2012;
Seisenberger et al., 2012; Hackett et al., 2013; Hill et al., 2018).

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J.J. Tuscher and J.J. Day
However, similar to the post-implantation embryo, not all loci appear to
be subject to methylation reprogramming. For example, some im-
printed genes and retrotransposons are also able escape demethylation
in the primordial germline (Hackett et al., 2013). Although imprint
control regions typically undergo the initial wave of demethylation
during germline development (Brandeis et al., 1993; Hajkova et al.,
2002; Guo et al., 2014a), these sites can escape methyl mark erasure
during the second period of demethylation (Kafri et al., 1993; Oswald
et al., 2000; Sakashita et al., 2014), providing a putative route for
methylation patterns to be transmitted from parent to offspring. Despite
periods of largescale demethylation during embryonic development,
over 115,000 regions remain hypermethylated during methylation re-
programming of the primordial germline in human development, and
many of these same regions similarly escape demethylation in mice
(Tang et al., 2015). Interestingly, a number of genes that escape me-
thylation reprogramming are expressed in the brain, and are linked to
the regulation of neurobiological processes and metabolism (Tang et al.,
2015). These gene targets are prime candidates for future investigation
of how the transmission of their corresponding epigenetic marks may
set the stage for transgenerational inheritance of metabolic disease
states or neurological disorders.
Collectively, these findings suggest at least some fraction of genomic
loci (e.g., particularly imprint control regions, DMRs, IAPs, transposons,
and some enhancer elements), may be more resistant to the global
methylation reprogramming that occurs during early development.
Why and how these particular regions of the genome are able to escape
methylation reprogramming remains largely unknown. Nevertheless,
such sites provide a putative route for DNA methylation patterns to be
conserved and transferred from the parent genome to future genera-
tions.
3.3. Cellular transmission of epigenetic marks via post-translational histone
modifications
Histone modifications also alter availability of DNA to the necessary
transcriptional machinery required for regulating gene expression.
Histones are modified at the N-terminal portion of their tails that ex-
tends beyond the nucleosome to interact with regulatory proteins,
neighboring histones, and the DNA itself (Marmorstein, 2001; Tsankova
et al., 2007). A variety of post-translational modifications (PTMs) to
histone tails have been observed in the literature, including methyla-
tion, phosphorylation, ubiquitination, SUMOylation, and acetylation,
which is perhaps the most well characterized. As with DNA methyla-
tion, relatively stable transmission of any of these epigenetic marks
during DNA replication and cell division would be required for histone
alterations to be heritable. However, the brief lifespan of most histone
modifications, and limited knowledge regarding the fidelity of histone
modifying enzymes, has dampened enthusiasm for histone PTMs as a
plausible route for transgenerational epigenetic inheritance. For ex-
ample, histone lysine methylation, a relatively stable histone mod-
ification, can be maintained on a timescale of hours-to-days (Zee et al.,
2010). Histone acetylation and phosphorylation states can fluctuate
within minutes (Jackson et al., 1975; Chestier and Yaniv, 1979). Thus,
the transient half-life of most histone modifications calls into question
the likelihood that such marks could be easily propagated during re-
plication and cell division. For modifications that do survive long en-
ough to be transmitted during replication, the mechanisms through
which parent histone marks are established on daughter histones re-
main poorly understood. Nonetheless, recent work in yeast has de-
monstrated that certain histone modifications, such as H3K9 methyla-
tion, can be stably propagated during mitosis and meiosis in a manner
independent of DNA methylation (Ragunathan et al., 2015). One pu-
tative mechanism involves the reassembly of two displaced parent
histones into the nucleosome of the newly synthesized DNA strand,
along with two new daughter histones (Annunziato, 2005). This partial
recycling of histones and their corresponding post-translational
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Neurobiology of Disease 132 (2019) 104591
modifications would allow for semi-conservative propagation of histone
modification patterns. However, it is unclear how parent histone marks
are established on daughter histones that have been recently in-
corporated into nucleosomes of the newly synthesized DNA strand. This
may be facilitated in part by the recruitment of histone modifying en-
zymes to the replication fork during DNA synthesis, or histone mod-
ifying enzymes may be able to interact directly with DNA in a sequence-
specific manner, allowing the enzyme to remain associated with the
DNA during replication (Budhavarapu et al., 2013). In support of this
notion, the histone modifying enzyme methyltransferase G9a, which
interacts with DNMT1 to coordinate H3K9 dimethylation, localizes to
the replication fork during DNA synthesis (Esteve et al., 2006). Simi-
larly, PRC2 (Polycomb Repressive Complex 2), which methylates un-
modified H3K27 sites when recruited by previously established tri-
methylated H3K27 marks, is also found near replication foci (Hansen
et al., 2008; Margueron et al., 2009). These examples suggest certain
histone modifying enzymes are physically recruited to replication sites,
and may participate in the reinstatement of post-translational mod-
ifications on daughter histones. Thus, although many details regarding
how histone PTMs are maintained during replication remain to be es-
tablished, this evidence suggests that certain histone modifying en-
zymes can be recruited to the site of DNA replication and may interact
with other epigenetic modifiers (such as DNMTs) to maintain estab-
lished epigenetic patterns during DNA replication and across cell divi-
sion.
3.4. Multigenerational epigenetic inheritance via histone modifications
Similar to global DNA methylation reprogramming, histones and
their associated modifications experience chromatin remodeling events
that prove challenging for maintaining and transferring epigenetic
marks from one generation to the next. For example, during mamma-
lian spermatogenesis, most histones are evacuated from nucleosomes
and replaced by small arginine-rich nuclear proteins known as prota-
mines in sperm to facilitate the compaction of paternal DNA (Balhorn,
2007; Casas and Vavouri, 2014). This process serves to protect and
stabilize the DNA, but also results in the removal of histones and any
corresponding post-translational modification to histone tails. Although
this reduces the likelihood of epigenetic transmission of histone mod-
ifications, recent studies suggest protamines are also post-translation-
ally modified, and therefore may also be capable of transferring epi-
genetic information from one generation to the next (Brunner et al.,
2014). Whether such modifications can be epigenetically inherited
across generations remains to be investigated. Despite the apparent
hurdle of widespread histone removal during sperm maturation, a small
percentage of histone modifications are preserved in humans and mice
(Hammoud et al., 2009; Brykczynska et al., 2010). This has been ob-
served with both active (H3K4me2, H3K4me3) and repressive
(H3K27me3, H3K9me3, H4K20me3) histone marks, however specific
PTMs and the loci at which they occur may differ by species. In humans,
evidence suggests repressive histone marks H3K9me3 and H4K20me3
can be transferred from sperm to zygote, and these alterations are ac-
companied by highly condensed constitutive heterochromatin (van de
Werken et al., 2014). Some retention of histone marks has also been
reported near the promoter region of key developmental and house-
keeping genes (Erkek et al., 2013). These findings suggest epigenetic
transfer of histone modifications may occur via the paternal epigenome,
although the opportunity for such contribution is limited given the
small percentage of histone PTMs preserved in sperm. Alternatively,
paternal chromatin compaction in the developing zygote may be driven
by maternal donation of the polycomb repressive complex (Puschendorf
et al., 2008). This putative mechanism suggests heritable changes in the
paternal epigenome can also be regulated by maternal contribution of
repressive complexes that shape the chromatin landscape during em-
bryogenesis.
In oocytes, histones are largely retained during development (Gu
J.J. Tuscher and J.J. Day
et al., 2010). Thus, stable transmission of histone marks from one
generation to another maybe be more plausible via a maternal route. In
support of this notion, recent examination of the maternal epigenome
has uncovered patterns of H3K4me3 and H3K27me3 marks in mouse
oocytes that appear to impact embryonic development (Dahl et al.,
2016; Zhang et al., 2016; Zheng et al., 2016; Inoue et al., 2017; Xu and
Xie, 2018). For example, a reduction in active histone methylation
mark H3K4me2 at the promoter regions of genes that shape develop-
mental processes and metabolic function have been linked to altered
gene expression in F1 generation embryos. This modification induced
by lysine specific demethylase 1 (LSD1) overexpression was associated
with developmental defects that were present in F1 offspring and could
be transferred across 3 generations (Siklenka et al., 2015). Although
limited at present, these studies suggest that both active and repressive
histone marks may be perpetuated across generations in mammals, and
that these transferable epigenetic marks may yield important implica-
tions for embryogenesis and multigenerational inheritance.
3.5. Cellular transmission of epigenetic marks by non-coding RNAs
Small non-coding RNAs may also act as carriers of heritable epige-
netic information. These small RNAs are comprised of a variety of RNA
species under 200 nucleotides in length, and include micro RNAs
(miRNAs), PIWI-interacting RNAs (piRNAs), and transfer RNA frag-
ments (tRFs). In comparison to DNA methylation and histone mod-
ifications, relatively little is known about the role of small non-coding
RNAs in germline-dependent epigenetic transfer across generations.
Nonetheless, a variety of small non-coding RNA species have been
documented in sperm and oocytes, which may be transferred to the
zygote at fertilization (Rassoulzadegan et al., 2006; Pauli et al., 2011;
Liu et al., 2012; Chen et al., 2016; Cropley et al., 2016; Sharma et al.,
2016; Yang et al., 2016). Although the precise mechanisms involved
remain to be defined, a host of exogenous factors such as exposure to
pathogens, stress, elevated temperature, and dietary composition have
been reported to produce alterations in RNA species found in germline
cells (Rassoulzadegan et al., 2006; Rechavi et al., 2011; Gapp et al.,
2014; Rodgers et al., 2015; Chen et al., 2016; Cropley et al., 2016;
Sharma et al., 2016). These alterations may contribute to inheritance of
certain metabolic phenotypes linked to high fat diet, which will be
discussed further in the following section. Although limited, emerging
evidence suggests tRNA fragments, piRNAs, and miRNAs may also
contribute to epigenetic silencing at certain transcriptional domains.
For example, miRNA inheritance has been linked to altered embryonic
development (Tang et al., 2007; Liu et al., 2012), and transmission of
stress-induced behavioral phenotypes (Gapp et al., 2014; Rodgers et al.,
2015). Finally, research focused on tRNA fragments has begun to
identify a role for these non-coding RNAs in the intergenerational
transfer of metabolic phenotypes (Chen et al., 2016; Cropley et al.,
2016; Sharma et al., 2016). In sum, like DNA methylation and histone
modifications, non-coding RNAs also have the potential to convey
epigenetic information from parental germline to their offspring and
future generations.
4. Effect of epigenetic inheritance on phenotypic traits and brain
function
4.1. Nutritional intake and inheritance of metabolic phenotypes
The prevalence of metabolic disorders is on the rise in the United
States and worldwide (Finucane et al., 2011; Ogden et al., 2014),
making the elucidation of cellular and molecular contributions to these
diseases particularly relevant. Mendelian inheritance does not ade-
quately account for the apparent heritability of observed phenotypic
traits and metabolic disorders. Although environmental exposure to
things like shared diet and other sociological and cultural factors most
certainly contribute to the passage of impaired metabolic function
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Neurobiology of Disease 132 (2019) 104591
across generations in humans, several lines of research suggest that
epigenetic factors may also confer risk for the development of certain
metabolic conditions. Exposure to high fat or low protein diets in the
parent generation, as well as intrauterine exposure to maternal caloric
restriction, have all been linked to disruption of metabolic function in
offspring, in some cases beyond the F2 generation. To date, the vast
majority of multigenerational studies have focused on the inheritance
of metabolic phenotypes in offspring, with little characterization of
heritable epigenetic changes to somatic cells in the brain regions that
regulate energy homeostasis and satiety. Nonetheless, some evidence
does suggest metabolic health of future progeny can be impacted by
poor nutrition in the parent generation. Specific examples of how high
fat or low protein diet in the parent generation can influence metabolic
function of offspring will be discussed in the following sections.
4.2. High fat diet
Parental exposure to a diet high in fat content has been linked to
phenotypic transmission of impaired metabolic function in offspring. A
recent study involving the in vitro transfer of oocytes and sperm from
parents both fed a high fat diet to a healthy foster mother reported
significant weight gain in F1 offspring relative to offspring from mice
fed a normal diet (Huypens et al., 2016). Specifically, female offspring
from obese parents gained the most weight, and this pattern of weight
gain in the offspring persisted regardless of whether the dam, sire, or
both parents received the high fat diet. Although potential epigenetic
modifications accompanying the observed phenotypes were not eval-
uated in this study, the authors did report that obesity in female off-
spring was accompanied by insulin resistance (Huypens et al., 2016),
suggesting future studies should investigate the epigenetic landscape
surrounding genes important for insulin regulation. Pregnant female
mice exposed to a fatty diet resulted in decreased insulin sensitivity and
increased body size in F1 and F2 offspring, an observation authors at-
tributed to both maternal and paternal lineage despite gestational ex-
posure (Dunn and Bale, 2011). Interestingly, in the F3 generation, in-
creased body size was only observed in female offspring, and appeared
to occur by a paternal mode of transmission. Although epigenetic
modifications were not directly measured, the increased body weight
phenotype was accompanied by a disrupted pattern of expression in
paternally imprinted genes involved in growth regulation in liver tissue
collected from F3 female offspring (Dunn and Bale, 2011). Taken to-
gether, the above studies suggest both maternal and paternal modes of
transmission may contribute to inheritance of altered metabolic phe-
notypes. The fact that increased body size and altered gene expression
patterns persisted in the F3 generation suggest transgenerational me-
chanisms of inheritance may be at play, although more definitive
characterization of accompanying epigenetic alterations to somatic and
germline cells for each generation will be important for substantiating
this claim.
Maternal transmission of epigenetic inheritance can be particularly
challenging to study, as diet-induced phenotypic traits in offspring
could arise from nuclear or mitochondrial DNA contributions, effects of
maternal diet and metabolism during gestation (e.g., diabetes, obesity),
or epigenetic modifications to female germ cells. One benefit of fo-
cusing solely on paternal transmission is that sperm carries only genetic
and epigenetic material, eliminating some of the potential confounds
inherent to studies of this nature. Studies investigating exclusively pa-
ternal transmission after exposure to a high fat diet have observed
weight gain, hypertension, and glucose intolerance in both F1 and F2
generations (Masuyama et al., 2016). Further, these metabolic impair-
ments were accompanied by H3K9ac and H4K20me1 in the promoter
regions of adiponectin and leptin genes in adipose tissue from adult F1
offspring, genes involved in insulin resistance (Fasshauer and Paschke,
2003) and satiety and energy homeostasis (Myers Jr., 2004), respec-
tively. Similar work examining paternal transmission of phenotypic
metabolic traits found that sperm from obese male mice exhibited
J.J. Tuscher and J.J. Day
altered DNA methylation patterns and corresponding changes in insulin
signaling genes, effects also observed in the pancreatic islets of F1 and
F2 offspring (Wei et al., 2014). In addition to DNA methylation and
histone PTMs, small non-coding RNAs have also been implicated in the
epigenetic transfer of metabolic phenotypes. For example, injection of
total RNA extracted from the sperm of mice on a high fat diet into
zygotes yielded impaired metabolic function in F1 offspring (Zhang
et al., 2018). This effect was not observed when sperm was donated
from obese mice with genetic deletion of Dnmt2, a methyltransferase
that alters the stability of tRNAs. Such findings suggest that high fat-
diet induced changes in metabolic function may be transferred to the F1
generation via paternal donation of non-coding RNAs in sperm.
Nevertheless, it currently remains unclear whether high fat diet-in-
duced epigenetic alterations also occur in brain tissue, and if such
marks are heritable across multiple generations.
4.3. Caloric restriction and low protein diets
Caloric restriction has also been shown to alter the epigenome in a
heritable way. Male offspring of calorically restricted dams have low
birth weights and suffer from several metabolic defects (Radford et al.,
2012). When dietary restrictions were in place during primordial
germline reprogramming, this led to DNA hypomethylation at 111 re-
gions in F1 sperm (Radford et al., 2012). However, changes in methy-
lation patterns at specific loci in F1 sperm were not observable in brain
or liver cells of F2 embryos, suggesting this particular epigenetic effect
is only intergenerational in nature. As with high fat diet studies, small
non-coding RNA species such as miRNAs and tRNA fragments are likely
also involved in the transmission of phenotypic traits related to meta-
bolism. For example, low protein diets have been linked to altered tRNA
fragment abundance, and this effect can be paternally transmitted via
sperm to alter embryogenesis (Chen et al., 2016; Cropley et al., 2016;
Sharma et al., 2016). Other work examining the role of non-coding
RNAs in epigenetic transmission of metabolic traits reported elevated
cholesterol synthesis in the liver of IVF-derived offspring from sires on a
low protein diet (Sharma et al., 2016). Phenotypic changes in metabolic
function were accompanied by dysregulation of hepatic cholesterol
biosynthesis genes in the F1 generation. These effects appear to be
mediated in part by diet-induced changes in the abundance of tRNA-
Gly-GCC fragments in F0 sperm, which repress genes normally regu-
lated by the endogenous retroelement MERVL (Mouse endogenous
retrovirus with leucine tRNA primer). Collectively, such work suggests
low protein diet-induced alterations in non-coding RNAs can be pa-
ternally transmitted to subsequent generations via germ cells. At pre-
sent, these effects appear to be mostly intergenerational in nature.
Notably, non-coding RNA species may prove an attractive candidate for
future research on transgenerational epigenetic inheritance of meta-
bolic traits, as they may not be subject to the same type of epigenetic
reprogramming events experienced by DNA methylation and histone
modifications during early development.
Taken together, the above studies provide evidence for inter-
generational inheritance of metabolic phenotypes that occur as con-
sequence of diet-induced changes in the F0 generation. Present findings
suggest intergenerational alterations in metabolic function are medi-
ated in part by non-coding RNA species transferred to offspring via
paternal germline cells. However, whether diet-induced epigenetic al-
terations observed in germ cells are also present in somatic cells of the
CNS (i.e., in the brain), and the heritability of such marks across mul-
tiple generations, remain open questions.
5. Stress exposure and inheritance of altered stress responsivity
Activation of the endocrine system by maternal or paternal exposure
to stressful events can alter gene expression and have long-term beha-
vioral consequences in offspring. Male mice that experienced chronic
social defeat or variable stress prior to breeding yielded progeny with
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Neurobiology of Disease 132 (2019) 104591
increased anxiety and depression-like behavior, as well as elevated
plasma levels of the stress hormone corticosterone (Dietz et al., 2011).
However, most of these traits could not be recapitulated with in vitro
fertilization using sperm from defeated sires, suggesting these effects
are likely not due to transmission of epigenetic marks across the
germline. However, other studies involving chronic paternal stress re-
sulted in hypoactivation of the hypothalamic-pituitary-adrenal (HPA)
axis in offspring, and alterations in glucocorticoid-responsive genes in
the bed nucleus of stria terminalis and paraventricular nucleus (Rodgers
et al., 2013). Changes in gene expression were also accompanied by an
upregulation in 9 different miRNAs in the sperm of stressed vs. non-
stressed sires. Given that miRNAs in sperm can impact post-fertilization
gene expression during early development (Rassoulzadegan et al.,
2006), and that DNMT3a (a key regulator of imprinted gene methyla-
tion) was identified as one of the top predicted miRNA targets, these
findings provide a potential mode of action through which paternal
stress can epigenetically alter germ cells. However, whether changes to
stress responsivity and the expression of stress-associated genes extends
beyond the F1 generation remains an open question.
Paternal transmission of traumatic experience has also been in-
vestigated using fear conditioning paired with specific scent cues.
Olfactory receptors are selectively expressed by sensory neurons of the
olfactory epithelium, with each individual sensory neuron expressing
only one of the hundreds (in humans) or thousands (in mice) of ge-
netically encoded olfactory receptors (Godfrey et al., 2004; Monahan
et al., 2017). Given the relatively defined nature of olfactory receptor
activation by distinct odor stimuli, this system presents an opportunity
for elegant investigations into transgenerational epigenetic mechan-
isms. In a landmark study by Brian Dias and Kerry Ressler, mouse sires
were fear conditioned in the presence of a specific odor cue (acet-
ophenone), which is known to activate the M71 odor receptor encoded
by the Olfr151 gene (Dias and Ressler, 2014). Surprisingly, this learned
association was transferred to F1 and F2 progeny, measured as an
elevated “startle” response to the conditioned odor but not to another
control odor. These inherited fear responses were accompanied by al-
tered promoter methylation of Olfr151 in F0 sperm, and neuroanato-
mical differences in the olfactory system of F1 and F2 offspring, in-
cluding more M71 receptor expressing olfactory sensory neurons and
larger M71 glomeruli in the olfactory bulb (Dias and Ressler, 2014;
Fig. 3A). Interestingly, in vitro fertilization (IVF) and cross-fostering
approaches were incorporated in this study to substantiate the idea that
observed phenotypic behavioral responses and molecular changes were
not likely due to social transmission from the parent generation. Per-
sistence of behavioral sensitivity to the conditioned scent, and neu-
roanatomical alterations in the olfactory bulb of IVF-derived F2 pro-
geny, as well as hypomethylation near the odorant receptor gene
Olfr151 in sperm from the F0 generation and F1 odor naïve males,
provides compelling evidence for epigenetic transmission of these
phenotypic traits. Although it is unclear at present how olfactory cues
and corresponding alterations in somatic cells of the olfactory system
led to methylation changes in sperm of the F0 and F1 generation, this
work provides intriguing preliminary evidence that information re-
garding stressful signals encountered in the environment may be in-
corporated into the germline and passed on to offspring.
Maternal stress during pregnancy leads to elevated production of
stress hormones and this heightened activation of the HPA axis has been
linked to increased stress sensitivity, cognitive deficits, and dysregula-
tion of stress-related gene expression in offspring (Mueller and Bale,
2007, 2008). When maternal stress is experienced during early preg-
nancy (i.e., within the first week), male, but not female, offspring dis-
play worse performances in tail suspension and forced swim tests, in-
dicative of depression-like behavior (Mueller and Bale, 2007, 2008).
Early gestation stress also resulted in elevated production of the stress
hormone corticosterone, increased corticotropin-releasing factor in the
amygdala, and decreased glucocorticoid receptor expression in the
hippocampus of male offspring (Mueller and Bale, 2008). Further,
J.J. Tuscher and J.J. Day Neurobiology of Disease 132 (2019) 104591

Fig. 3. Putative mechanisms for transgenerational epigenetic inheritance of neuronal and behavioral phenotypes. A, Multigenerational epigenetic transmission of a
startle response after paternal olfactory fear conditioning. Fear conditioning in the presence of acetophenone odorant leads to reduced methylation of the Olfr151 gene in
the olfactory bulb of the paternal F0 generation (left) and in sperm (F1; right). Hypomethylation of Olfr151 (a gene that encodes M71 odor receptors) is associated
with an increase in the number of olfactory sensory neurons expressing M71 receptors, and larger corresponding glomeruli in the olfactory bulb of the F0 generation
(left). More M71-expressing olfactory sensory neurons and larger M71 glomeruli are also observed in adult F1 and F2 progeny without any direct exposure to the
acetophenone odor cue. Neuroanatomical adaptations observed in the F1/F2 generation are thought to increase sensitivity to acetophenone when offspring are later
exposed to this specific odorant, resulting in elevated startle to the odor cue in the absence of direct pairing with fear conditioning. B, Multigenerational epigenetic
transmission of a cocaine resistant phenotype after paternal cocaine exposure. Cocaine self-administration leads to increased H3K9K14ac2 at Bdnf promoter IV and
elevated BDNF protein and mRNA transcripts in somatic cells of the mPFC in the F0 generation (left), as well as increased H3K9K14ac2 at all Bdnf promoters in F0
sperm (F1 generation; right). During adulthood, parallel changes in BDNF protein, mRNA, and H3 acetylation are observed in the mPFC of F1 offspring. Elevated
BDNF levels in the mPFC have previously been linked to a reduction in the reinforcing effects of cocaine, and subsequently, a decline in drug-seeking behavior. Thus,
elevated Bdnf mRNA and protein levels in the mPFC of F1 progeny prior to drug exposure are thought to confer a protective phenotype by blunting the rewarding
properties of cocaine and reducing the propensity for drug seeking.

8
J.J. Tuscher and J.J. Day
methylation of the glucocorticoid receptor promoter was also elevated
in hippocampal tissue, suggesting epigenetic mechanisms are likely
involved, although most likely as a result of direct exposure to stress
hormones in utero, and not through germline inheritance of such
modifications. In guinea pigs, maternal stress induced by repeated ex-
posure to flashing light similarly increased anxiety-related behaviors
and basal corticosterone levels in offspring while reducing glucocorti-
coid receptor expression in the hippocampus when the stressor oc-
curred during mid, but not late, pregnancy (Kapoor and Matthews,
2005; Kapoor et al., 2009). Taken together, these studies suggest timing
of perturbation during pregnancy may play a key role in the extent to
which developmental processes will be disrupted or induce long-term
epigenetic changes in the neural tissues of their progeny. At any rate,
the vast majority of studies examining the effects of maternal trauma on
stress vulnerability in offspring to date involve direct in utero exposure
to the F1 (developing embryo), and F2 (embryo primordial germline)
generations, and as such are intergenerational in nature. Additional
research will be required to determine whether epigenetic modifica-
tions that occur as a consequence of maternal stress are transgenera-
tional, extending out to the F3 offspring.
Another important consideration for research examining the con-
sequence of parental stress on offspring is that maternal stress can also
alter the level of maternal care dams provide to their pups after birth
(Ivy et al., 2008), in addition to affecting the hormonal milieu of the
placenta. Poor maternal care during this early postnatal period is
traumatic in its own right (Weaver et al., 2004; Rice et al., 2008; Ivy
et al., 2010; Korosi and Baram, 2010; McClelland et al., 2011), and can
thus act as its own direct exposure to stress early in life, making in-
terpretation of transgenerational vs. intergenerational social transmis-
sion of such effects on behavior and gene expression challenging. For
example, stressed mothers provide poor maternal care, and the stress of
poor maternal care acts as its own direct insult to the developing pup.
This elevates stress sensitivity in adult F1 progeny, likely affecting their
own ability to provide adequate maternal care, thus perpetuating the
cycle. An important mechanistic distinction is that this continued pas-
sage of a stress sensitive phenotype and any accompanying epigenetic
alterations likely occurs from direct exposure to a stressful postnatal
experience in each generation, making this a repeating cycle of inter-
generational effects, rather than transgenerational effects, which re-
quire transfer of epigenetic modifications via the germline in the ab-
sence of direct exposure. To avoid these type of experimental
confounds, some investigators have opted to focus on patrilineal in-
heritance, as male rodents are not typically involved in rearing off-
spring in a laboratory setting. Exposure to paternal stress prior to
breeding is thought to minimize potential social or behavioral con-
founds that can be imposed during early postnatal periods. However, it
is worth noting that mate quality can impact maternal investment of
female rodents in caring for their offspring (Drickamer et al., 2000;
Mashoodh et al., 2012). Therefore, even subtle interactions between the
sire and dam during or after mating could theoretically impact maternal
behavior and postnatal care of offspring, and thus it should not be as-
sumed that separation of an exposed sire from the dam after mating will
guarantee avoidance of any potential behavioral confounds.
6. Substance abuse and inheritance of behavioral phenotypes
Exposure to drugs of abuse can modify the epigenome and alter
neural plasticity, and each of these phenomena is thought to establish
and maintain the addicted state (Renthal and Nestler, 2008; Heller
et al., 2014). The evidence for epigenetic modifications and changes in
gene expression in reward circuitry of the drug-exposed individual is
well established, but more recently epigenetic modifications have also
been documented in the sperm and oocytes after drug exposure
(Vassoler and Sadri-Vakili, 2014). Prenatal (i.e., in utero) exposure to
cocaine in rodent addiction models can result in elevated self-admin-
istration of cocaine (Keller Jr. et al., 1996), altered stimulant-induced
9
Neurobiology of Disease 132 (2019) 104591
sensitization (Crozatier et al., 2003), and impaired synapse maturation
during development (Bellone et al., 2011). Although such work pro-
vides valuable translational information regarding the deleterious ef-
fects of cocaine exposure during pregnancy, numerous studies focused
on epigenetic transmission of behavioral traits in the context of ad-
diction have opted to focus on paternal transmission, as this circum-
vents potential interpretational confounds that can arise in transge-
nerational inheritance studies. For example, past drug experience in
female rats can negatively impact maternal care for pups later in life
(Johnson et al., 2011), and a single instance of maternal separation has
been shown to influence drug intake in pups (Martini and Valverde,
2012). Thus, investigation of paternal drug use on neurobiological
processes and behavioral phenotypes in offspring from drug naïve dams
evades these potential confounds. This approach also rules out the
potential for transfer of epigenetic marks by direct drug exposure to the
F1 or primordial germline of F2 generations in utero.
At present, a majority of the evidence for transmission of phenotypic
traits across generations as a consequence of paternal or maternal drug
use is behavioral and correlational in nature. Nonetheless, a variety of
behavioral phenotypes have been observed in offspring from cocaine
experienced sires, including hyperlocomotion, impaired spatial
memory, and elevated anxiety levels (Abel et al., 1989; Fischer et al.,
2017). Some of the earliest work to report intergenerational transmis-
sion of addiction-related phenotypic traits found that both male and
female adolescents (P16) from cocaine-sired offspring displayed hy-
perlocomotive activity (Abel et al., 1989). More recently, sex-specific
effects on locomotion have been reported in male, but not female,
offspring of cocaine-sired mice (Fischer et al., 2017). Anxiety-like be-
haviors have also commonly been observed in the progeny of cocaine-
experienced rodents. For example, male offspring from cocaine-sired
rats reportedly display defensive burying and novelty-induced hypo-
phagia, which are considered measures of anxiety-like behavior (White
et al., 2016). Similarly, male offspring of rats receiving chronic cocaine
injections also demonstrated increased anxiety levels relative to saline
controls, as measured by amount of time in the open arm of the ele-
vated plus maze (Fischer et al., 2017).
Despite these intriguing observations, it should be noted that at least
one study measuring locomotion was not able to recapitulate a similar
hyperactive phenotype in adult mouse progeny (P60) sired by cocaine-
experienced fathers (Killinger et al., 2012). Similarly, this same study
also observed no impact on paternally transmitted anxiety-like traits
after cocaine experience in the elevated plus maze or open field test
(Killinger et al., 2012). One potential difference that could account for
these discrepancies is that sex-specific statistical analyses were used for
some studies (Fischer et al., 2017), while others collapsed sex during
analysis (Killinger et al., 2012), which would alter power for detecting
behavioral effects within each sex. Differences in amount of handling,
behavioral protocols, and total amount of cocaine exposure may have
also contributed to these incongruent observations. Despite these dis-
crepancies, at least some research suggests that paternal exposure to
cocaine can lead to elevated locomotor activity and other anxiety-like
traits in F1 offspring, and that male offspring may be particularly sus-
ceptible to these behavioral abnormalities.
In addition to hyperactivity effects, paternal exposure to cocaine
may also disrupt normal memory function in F1 progeny, as impair-
ments have been observed in the performance of certain memory tasks
in cocaine-sired rodents. For example, adolescent (P35) offspring from
cocaine-sired rats demonstrated perseverative behavior in a T-Maze
task (Abel et al., 1989), and similar impairments were observed in
cocaine-sired CD1 mouse offspring in the 5-Arm Maze, a spatial
working memory task (He et al., 2006). In a self-administration para-
digm, adult male offspring also exhibited impaired spatial memory in
the object location task (Wimmer et al., 2017). Although most of this
evidence suggests spatial memory tasks are particularly susceptible to
disruption by paternal drug use, others have reported no impairment in
the Morris Water Maze, another type of hippocampus-dependent spatial
J.J. Tuscher and J.J. Day
task (Killinger et al., 2012; Fischer et al., 2017). Nonetheless, the bal-
ance of studies to date suggest tasks that recruit the hippocampus may
be more heavily influenced by the consequences of paternal exposure to
cocaine. However, several factors may have contributed to the observed
differences in memory task performance. For example, Killinger and
Fischer used experimenter administered cocaine, whereas Wimmer and
He utilized self-administration models, allowing the subject to lever
press for cocaine reward. Differences in these experimental approaches
should therefore be considered when interpreting the data, as each
approach would likely engage distinct brain regions to a different ex-
tent. Self-administration relies on learning about contingencies between
behavioral responses, environmental cues, and the rewarding proper-
ties of the drug, whereas experimenter administration is more passive.
Further, self-administration studies likely resulted in more overall co-
caine experience vs. the experimenter delivered studies, and this could
also be an important factor in paternal transmission of certain drug-
induced phenotypic behaviors.
Perhaps not surprisingly, cocaine-sired offspring of both sexes dis-
play behavioral differences in response to their own experience with
cocaine. One example of this is stimulant-induced hyperlocomotion,
suggesting increased sensitivity to cocaine experience (Fischer et al.,
2017). Cocaine exposure in the parent generation may shift the dose-
response curve for female mice, which show reduced magnitude in their
conditioned place preferences, and in response to a lower dose of co-
caine than usual (5 mg/kg vs. 10 mg/kg; Fischer et al., 2017). Male
offspring appear to experience the effects of parental exposure to co-
caine slightly differently. For example, cocaine-sired male rats acquire
self-administration more slowly, and took less total drug overall
(Vassoler et al., 2013), suggesting a sex-specific protective effect against
drug seeking in male progeny.
In a model that divided rats into groups based on motivation level to
voluntarily self-administer cocaine, researchers found that the highly
motivated behavioral phenotype (termed “Addict” rats in the study)
could be transmitted to F1 and F2 generations (Le et al., 2017). Inter-
estingly, the heritable addict-like behavioral phenotype was linked to
high motivation to drug seek, rather than higher levels of cocaine intake
alone, as transmission of this behavioral phenotype was not observed in
offspring when the F0 generation received passive experimenter-ad-
ministered cocaine injections (Le et al., 2017). This addict-like pheno-
type was also not observed when the F0 generation sired offspring prior
to drug experience, suggesting the phenotype is acquired as a con-
sequence of contingent drug experience, and is not likely a result of
direct genetic inheritance.
Cocaine use is known to shape changes in gene expression and
plasticity in the reward circuitry that underlies addiction-related be-
haviors. However, currently evidence for which corresponding epige-
netic modifications are also observed in the brains of the F1 generation
after paternal drug experience, and the potential mechanisms through
which this might occur, are quite limited. One of the earliest studies to
link cocaine-induced epigenetic alterations to heritable changes in be-
havior found a significant decrease in Dnmt1 and increased Dnmt3a in
the seminiferous tubules of the testes in male mice trained to self-ad-
minister cocaine (He et al., 2006). Although epigenetic modifications
were not measured in adult offspring, several morphological and be-
havioral abnormalities were noted, including decreased cerebral vo-
lume, reduced head diameter, impaired attention and working memory.
This study was one of the first to show drug experience alters not only
the self-administering rodent, but also their germline cells, hinting at a
potential mechanism through which cocaine-induced changes to the
epigenome could be transferred to the next generation. A more recent
study also observed epigenetic alterations to the sperm of cocaine-ex-
perienced sires, this time in the form of elevated H3 acetylation at Bdnf
promoters I, IV, and VI (Vassoler et al., 2013). These effects in the F0
generation were accompanied by sex-specific increases in H3 acetyla-
tion at Bdnf promoter IV in the medial prefrontal cortex (mPFC) of
cocaine-sired male offspring, as well as increased Bdnf mRNA and
10
Neurobiology of Disease 132 (2019) 104591
protein levels (Vassoler et al., 2013; Collo et al., 2014). Interestingly,
Bdnf levels in the offspring of drug-exposed sires is reported to posi-
tively correlate with amount of cocaine intake in the parent generation
(Le et al., 2017). This aligns with previous research suggesting elevated
mPFC BDNF levels coincide with reduced drug-seeking behavior, and
may play a key role in mediating the protective effects some have ob-
served in the offspring of cocaine-sired rodents (Berglind et al., 2007;
Vassoler et al., 2013;Fig. 3B). Elevated DNA methylation has also been
observed in the sperm of highly motivated “addict” vs. non-addict co-
caine-exposed F0 rats, a pattern which persisted in their F1 descendants
(Le et al., 2017). Consistent with these distinct methylation patterns,
transcriptome sequencing of the Nucleus Accumbens (NAc) in the F1
generation revealed that reduced global methylation in F0 sperm co-
incided with significantly lower expression levels of genes important for
developmental regulation, gap junction formation, and axon guidance
in the addict-like phenotype vs. non-addict F1 progeny (Le et al., 2017).
Overall, these findings suggest highly motivated drug-seeking behavior
may alter DNA methylation in a way that is distinct from methylation
that occurs as a result of drug exposure alone, and that these epigenetic
modifications may be transmitted to F1 offspring. Additional research
will be required to determine if heritability of the epigenetic patterns
observed in the F1 generation extends to F2 progeny.
Collectively, this body of work provides a preliminary glimpse into
the generational effects of cocaine exposure on anxiety, memory, sen-
sitivity to drugs of abuse, and corresponding alterations to the epi-
genome and gene expression. Although converging evidence does sug-
gest transmission of behavioral phenotypes, much work remains to be
done to see if these observations extend beyond the F1 generation,
making them true transgenerational rather than intergenerational ef-
fects. Further, characterization of which epigenetic marks are trans-
ferred from drug experienced parent to offspring, the brain regions and
cell types they occur in, how such modifications survive epigenetic
reprogramming during development, and how such alterations would
manifest as the observed behavioral phenotype remain to be explained.
Thus, future studies will be important to further dissect the multi-
generational transmission of these phenotypic traits, and the epigenetic
remodeling events that may subserve them.
7. Current limitations and challenges, and approaches to move
forward
Many gaps remain in our understanding of how epigenetic mod-
ifications acquired in the parent generation might contribute to the
transmission of phenotypic traits in their offspring. Despite evidence
that certain behaviors and disease states seemingly persist across gen-
erations, the idea that these traits are caused by germline-dependent
inheritance of epigenetic marks remains controversial. This is in part
due to a number of issues that remain unresolved, including un-
ambiguous proof of heritability, demonstration of survival of epigenetic
marks during epigenome reprogramming, their selective maintenance
in specific somatic tissues, and the extent to which these inherited
modifications are causal of an observable phenotype. The following
section highlights current limitations and key issues in transgenera-
tional epigenetic inheritance research, and proposes guidelines for how
future research might address these existing gaps.
7.1. Technical limitations and challenges in experimental design
Epigenetic factors help shape cell fate and function, and accord-
ingly, methylation patterns likely differ between distinct subpopula-
tions of cells both within the brain and throughout the periphery. As
such, the tissue collected to generate bulk homogenate for most EWAS
studies likely differs in composition, with some cell types being over-
represented while others remain underrepresented (Jaffe and Irizarry,
2014). Thus, observations made from methylation patterns consisting
largely of one cell type may not generalize to other cell populations.
J.J. Tuscher and J.J. Day
Single-cell level resolution of methylation and histone profiles in future
studies will be necessary to overcome this technical limitation, and
provide a more accurate reflection of the heterogeneity of epigenetic
patterns across unique cell populations.
In addition to the technical limitations of currently available ap-
proaches, careful attention must be paid to experimental design in
order to reduce potential variability and contribution of non-epigenetic
factors to studies of this nature. For example, preclinical research
should consider using inbred rodent strains with tightly regulated en-
vironments to reduce the potential for genetic and environmental
confounds. Further, if transgenerational effects are to be evaluated,
studies must be extended long enough to allow for evaluation of epi-
genetic modifications and phenotypic traits in the appropriate genera-
tion (i.e., F2 for male or non-pregnant female exposure, F3 for exposure
to pregnant females; see Fig. 1). For some studies, an attractive alter-
native may involve the use of in vitro fertilization and transfer to foster
mothers, which allows for strict observation of transmission of epige-
netic marks via the gametes and reduces the possible influence of al-
tered behavioral effects in the parent generation (e.g., changes in ma-
ternal care) indirectly altering behavior or epigenetic changes in
progeny.
7.2. Crosstalk between different epigenetic modifications
The choreography of various epigenetic factors that act in concert to
initiate transcriptional programs and define cellular fate are complex
and interconnected. Combinatorial approaches will be required to more
accurately define methylation state in conjunction with histone mod-
ifications by using approaches that incorporate bisulfite sequencing
information with ChIP data from the same cellular populations
(Brinkman et al., 2012; Statham et al., 2012). Because epigenetic
modifications are dynamic and reversible, intermediate states of DNA
methylation (e.g., 5fC and 5caC) may also need to be taken into con-
sideration in EWAS studies to capture a deeper level of resolution on
these dynamic states. At present, it is unclear whether these are truly
intermediate states, or if they serve unique functions of their own.
7.3. Contribution of genetic influence on epigenetic landscape
In addition to the complex interplay amongst different epigenetic
factors, there is also the potential for indirect genetic contribution to
the transmission of epigenetic marks. That is, normal variation in DNA
sequence between individuals may underlie some DNA methylation
differences observed between individuals, making it particularly chal-
lenging to disentangle genetic vs. epigenetic transmission of epigenetic
marks and corresponding effects on phenotypic traits (Gibbs et al.,
2010; Bell et al., 2011; Gertz et al., 2011). For this reason, epigenetic
inheritance studies should include DNA sequencing controls whenever
possible to ensure observed effects are not an indirect result of genetic
rather than epigenetic differences between individuals (Lappalainen
and Greally, 2017).
This consideration is also important for monitoring gene variants
that could lead to an indirect epimutation that impacts transcription
patterns in the sense or antisense orientation at the region of interest.
This phenomenon was recently reported to accompany a rare disease
across several generations, wherein genetic mutation in one gene re-
sulted in elimination of its usual transcription termination signal, and
subsequently led to atypical methylation at a gene neighboring the
region of interest (Chan et al., 2006; Ligtenberg et al., 2009; Gueant
et al., 2018). Although ultimately methylation marks were altered at
the disease-linked gene, this change was an indirect consequence of
direct mutation in the DNA sequence, rather than through gamete in-
heritance of the methylation mark, and as such is not truly re-
presentative of epigenetic inheritance.
11
Neurobiology of Disease 132 (2019) 104591
7.4. Unambiguous demonstration of heritability
One challenge transgenerational epigenetic inheritance studies have
yet to overcome is definitively demonstrating that epigenetic marks
present in the germline of the parent generation are truly inherited by
the F1 generation, rather than removed and re-established in offspring.
During early prenatal development, epigenetic marks present in the
parent germline (e.g., DNA methylation and histone PTMs) are largely
removed in the developing zygote after fertilization. At present, it re-
mains unclear how specific epigenetic modifications in the maternal
and paternal gametes would survive this largescale removal of epige-
netic marks. Further, it is not clear how epigenetic modifications that
do survive this period of reprogramming would be transferred from
undifferentiated embryonic cells to specific differentiated somatic tis-
sues (i.e., neurons) in the adult offspring. Although DNMTs assist in
preserving methylation marks during cellular differentiation and in
somatic tissues, the accuracy of DNA methylation during replication is
imperfect (~96% fidelity rate). Although this may sound relatively
high, in reality it allows for errors to be introduced into methylation
patterns across multiple rounds of DNA replication and cell division.
Therefore, accurate sustained transmission of such epigenetic mod-
ifications, which would be necessary for the transgenerational passage
of such marks, would be difficult to achieve. Even less is known about
how post-translational modifications of histones are maintained during
DNA replication and cell division, so this would also be an issue for
studies focused on a histone-mediated mode of epigenetic inheritance.
Given the high probability for the loss of both histone PTMs and DNA
methylation modifications over multiple rounds of replication and cell
division, future studies should focus on providing convincing evidence
that epigenetic marks observed in the parent germline are also observed
at several other key developmental stages (gametes > zygote >
blastocyst > post-implantation embryo > F1 offspring) with parallel
evidence of the heritable mark across these same stages in the F2
generation. Bisulfite sequencing, methylated DNA immunoprecipitation
(MeDIP), or chromatin immunoprecipitation (ChIP)-based approaches
with antibodies against specific histone marks may be best suited to
track histone PTM or DNA methylation profiles across generations.
Sequencing data (e.g., RNA-seq, ChIP-seq) demonstrating gene expres-
sion profiles and phenotypic traits expected to accompany the observed
pattern of epigenetically inherited marks would further strengthen such
work, and provide a plausible proximal mechanism linking inheritance
of these marks to biological functions in the affected cell population.
7.5. Transfer of germline modifications to target somatic tissues
In order for epigenetic modifications laid down in the germline to
influence brain function, it is likely that these modifications would have
to survive through early development in order to alter gene expression
in target neurons that control the phenotype in question. However, it is
presently unclear what determines which somatic tissues will maintain
the epigenetic marks originating from undifferentiated stem cells. It
seems unlikely that such modifications would be transferred from the
germline to all somatic tissues, so how are such marks passed on only to
specific cell populations of cells but not others? For example, if sub-
stance abuse leads to an epigenetic alteration at gene X only in nucleus
accumbens neuron populations, how does this mark 1) get transferred
from parental neural tissue to the germline, and 2) from the germline
containing undifferentiated cells back to just a distinct subpopulation of
neurons in a specific brain region (but not in other somatic tissues)? It is
easier to imagine how this might occur for phenotypes that are encoded
by highly specific expression of specific genes in specific cell popula-
tions (e.g., olfactory receptors), but it is far more challenging to un-
derstand how this selectivity could occur for more complex polygenic
or omnigenic phenotypes.
J.J. Tuscher and J.J. Day
7.6. Evidence demonstrating the causal nature of epigenetic alterations
In addition to the limited proof concerning unambiguous herit-
ability, evidence demonstrating causality is also currently lacking.
Future studies should include rigorous evidence that epigenetic al-
terations passed from one generation to the next are the root cause of
the observed phenotype, rather than a co-occurrence. Recent advances
in epigenetic editing techniques will be particularly important for de-
monstrating both necessity and sufficiency of key epigenetic mod-
ifications in the manifestation of observable phenotypic traits. For ex-
ample, CRISPR/dCas9-based strategies allow for epigenetic modifying
enzymes, including DNMTs and HATs, to be tethered to a catalytically
inactive dCas9 protein (Liu et al., 2016; Vojta et al., 2016; Kwon et al.,
2017; Pflueger et al., 2018; Savell et al., 2019). This dCas9/epigenetic
modifier construct can then be directly targeted to specific loci
throughout the genome. These type of directed epigenetic alterations
will allow for subsequent changes in gene expression, behavior, and
phenotypic traits to be observed in response to specific epigenetic
modifications, which can then be monitored across multiple genera-
tions. Such work will be essential for providing more rigorous and
compelling evidence regarding the causal nature of transgenerational
epigenetic inheritance.
8. Conclusions
Although it has quickly captured the attention of the neuroscience
community, research on multigenerational epigenetic inheritance in
mammalian species remains in its infancy. Despite evidence of inter-
generational and transgenerational phenotypes in many different model
systems, a number of critical questions remain to be answered. Perhaps
most importantly, it is currently unclear what biological problem this
form of inheritance solves. Here, it is worth considering whether gen-
erational passage of epigenetic changes make sense from an evolu-
tionary biology perspective. For example, if an external stimulus (e.g.,
drug use, stress, poor nutrition) results in overwhelming negative
consequences for the individual and their ability to create optimally fit
progeny, why might such epigenetic modifications and consequent
phenotypes be allowed to persist for multiple generations? Why would
the same malleable system that allowed for epigenetic marks to be
deposited in the first place not respond in a manner more adaptive for
the species? Although it is worth noting that some evidence in the ro-
dent literature indicates drug experience may confer a protective phe-
notype in offspring, this is not necessarily reflective of what happens in
humans. If anything, parental drug experiences confer more – not less –
vulnerability for substance use disorders in offspring generations
(Bierut et al., 1998; Goldman et al., 2005; Vink, 2016). This problem is
inherent to all epigenetics studies, as the nature of the system is
somehow both stable (i.e., transmissible across generations) yet plastic
in response to external stimuli.
A second concept that is valuable to consider is the relative weight
of epigenetic changes in comparison to genetic influences. Genetic
contributions to observable phenotypes in mammals are powerful, nu-
merous, and extremely reproducible. Key examples include primary sex
determination by Y-linked genes, severe neurodevelopmental disorders
arising from de novo mutations, and the highly penetrant nature of
familial forms of neuropsychiatric or neurodegenerative disease states.
Are epigenetic modifications simply occurring on top of the large set of
robust genetic factors that contribute to development and adult func-
tions of the nervous system, providing only slight tweaks or temporary
changes? Or are epigenetic modifications able to overturn or usurp
genetically defined programs, establishing new qualitatively different
transcriptional landscapes and behavioral phenotypes? Addressing this
question will be essential to determining the position of epigenetic
mechanisms in the hierarchy of molecular mechanisms that contribute
to brain development, function, and disease.
Although we have gleaned a considerable amount of information
12
Neurobiology of Disease 132 (2019) 104591
about epigenetic mechanisms in somatic and dividing cells over the past
several decades, the mechanisms that drive heritable, germline-de-
pendent transmission of epigenetic marks remain poorly understood. At
present, germline-dependent transmission appears to be the exception
rather than the rule for mammalian species. However, while currently
limited, these exceptions have provided dramatic evidence for the po-
tential implications for transgenerational epigenetic contributions to a
number of global public health issues, including obesity, substance use
disorders, and stress response in the face of traumatic experience.
Future research will be required not only for providing more conclusive
evidence that epigenetic alterations acquired in one generation can be
stably inherited and propagated to future generations, but also for
unravelling the molecular mechanisms that make this type of in-
formation transfer possible.
Acknowledgements
This work was supported by NIH grants DA039650, DA034681, and
MH114990 (J.J.D.). Illustrations were generated using BioRender.
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