Human Endogenous Retroviruses in Autism Spectrum Disorders: Recent Advances and New Perspectives at the Gene-Environment Interface

*Emanuela Balestrieri, Chiara Cipriani, Enrico Garaci, Claudia Matteucci and Paola Sinibaldi-Vallebona*

## **Abstract**

Human endogenous retroviruses (HERVs) are genetic elements, derived from their exogenous retroviral counterpart by a process of germline infection and proliferation within the human genome, and their integration as proviruses led to the fixation and the vertical transmission, following Mendelian laws. HERVs currently make up ~8% of the genetic material, and some of them have been cooped for physiological functions. Otherwise, their activation in response to environmental factors has been associated with human pathological conditions. In the setting of neurodevelopmental disorders, HERVs have been proposed as contributing factors involved in Autism Spectrum Disorders (ASD), spanning the bridge between genetic susceptibility, environmental risk factors and immune response. We described a distinct expression profile of some HERV families and cytokines in lymphocytes from autistic children and in their mothers suggesting a close mother-child association in ASD. Moreover, *in vitro* treatment with an antiretroviral drug was able to restore the expression level of HERVs and cytokines providing new insights into the potential role of HERVs as biomarkers of ASD and raising the possibility of using HERVs expression as a therapeutic target for a tailored approach to patient care.

**Keywords:** human endogenous retroviruses; HERVs, biomarker, mother-child association, gene expression, aetiology, antiretrovirals

### **1. Introduction**

In 1943, the child psychiatrist Leo Kanner described children preferring loneliness with repetitive patterns of behaviour. Similar symptoms were reported by Hans Asperger, an Austrian paediatrician, in 1944 mainly in people of high intelligence [1]. Kanner, first spoke about 'childhood or early-onset schizophrenia', and later he called this condition 'infantile autism', and concerning the aetiology of autism, he attributed autism to a lack of maternal warmth and attachment. Following this hypothesis, Bruno Bettelheim with his book 'The Empty Fortress' popularized the theory of 'refrigerator mother' by stating that 'the infant that misreads the mother's actions or feelings, or correctly assesses her negative feelings, may retreat from her and the world'. This view was widely criticized and nowadays represents an obsolete thought [2]. From this time, many hypotheses and models emerged to explain this complex condition focusing on symptomatology, phenotype and pathogenesis [3, 4]. However, despite many promising hypotheses, the current literature is made up of controversial findings and lacking of definitive proof about the mechanism underlying the complex aethiopathogenesis of Autism Spectrum Disorder (ASD). ASD is currently referred to as a pervasive neurodevelopmental disorder with an impact on emotional and social behaviour that persists throughout life [5]. The clinical presentation is very heterogeneous, and its incidence is continuously increasing [6]. Despite the consolidated evidence that the main contribution to the increase in the incidence of autism comes from the improvement of the diagnostic process, it has also been hypothesized that at the basis of the onset of autism, there is not a single cause but a set of risk factors acting together to produce the phenotype. Decades of studies have indeed shown that autism is a complex pathology influenced by the combination of genetic, environmental and epigenetic factors, mainly acting during prenatal and/or perinatal phases [7–9]. The concordance rate of ASD in monozygotic twins much higher than in dizygotic twins seemed to indicate that genetic factors were more likely to contribute to ASD than environmental factors [10]. More than a thousand ASD-associated genes known to be involved in brain development have been identified to date [11], and many genomic copy number variants have been associated with neurodevelopmental disorders including ASD [12, 13]. Several epidemiological studies indicate that potential risk factors for ASD also include various determinants [14, 15], such as the age of the pregnant woman, advancing paternal age [14, 16, 17] and prematurity [18]. However, an ever increasing important role has been attributed to risk factors related to the early foetal environment, including toxicants, diet, air pollution, smoking or chemicals exposure, which have been suggested to induce a prenatal and/or perinatal brain insult able to contribute to the development of autism in genetically predisposed individuals [19]. These environmental insults share in common the activation of the maternal immune system (MIA), which has therefore been recognized as an additional risk factor for ASD [20]. MIA is an inflammatory response triggered by pathogenic infection and autoimmune diseases in the mother. It is known that several microorganisms, vertically transmitted to the foetus, affect its development resulting in severe complications such as miscarriage and malformations [21]. However, even non-vertically transmitted infections during pregnancy can cause harm to the offspring by producing inflammatory cytokines, which directly damage the foetal brain by crossing the placental and blood-brain barrier [22, 23].

Preclinical studies, using mouse model of MIA induced by prenatal exposure to polyinosinic:polycytidylic acid (Poly I:C), a synthetic double-stranded RNA molecule targeting TLR-3, mimicking viral maternal infection, demonstrated that the exposure to a prenatal insult induced derailed neurodevelopment in offspring. Particularly, in the mothers, the Poly I:C injection leads to the production of interleukin-17 that reaches the foetal brain *via* the placenta inducing cell death and decreasing synaptic density and expression levels of synapse formation-associated proteins and resulting in ASD-like behavioural and morphological brain abnormalities, also described in the pathophysiology of human ASD [24–26]. In line with the hypothesis that maternal immune response could impact on neurodevelopment in the newborn,

#### *Human Endogenous Retroviruses in Autism Spectrum Disorders: Recent Advances… DOI: http://dx.doi.org/10.5772/intechopen.108671*

epidemiological studies have reported that MIA, caused by autoimmune diseases, also increases the risk of ASD [27]. Altogether these studies suggest that MIA-induced inflammation and cytokines can impair placental function and lead to the disruption of its barrier function, resulting in exposure of the foetus to toxic substances. Accumulated evidence also shows an important role of epigenetic factors, such as DNA methylation, histone modification and noncoding RNA in predisposition to disease development. Epigenetic mechanisms regulate chromatin structure and gene expression without altering the DNA sequence. In consideration of this last characteristic, in the past it was believed to have no role in the growth and development of the individual [28] while the study of their interaction with environmental conditions has highlighted their important role in the development of genes related to brain development. In recent years, there have been rapid advances in the understanding of epigenetic mechanisms that ultimately regulate gene activity and expression during development and differentiation or in response to environmental stimuli. Instead, it is now known that the main function of epigenetic factors is to regulate development through cell differentiation processes, tissue specification and maintenance of cell lineages [29]. Therefore, environmental stimuli can alter the epigenome and consequently gene transcription, changing the phenotype [30].

Within this interplay among genetic susceptibility, MIA, epigenetic and environmental factors are placed the human endogenous retroviruses (HERVs), which we proposed as novel contributing factors involved in ASD.

#### **2. Human endogenous retroviruses and their co-evolution with the host**

In contrast to the prevailing early twentieth century conception of genetic material as fixed, in the 1940s, the Nobel Prize Barbara McClintock discovered in maize the 'mutable loci' which were capable to move between chromosomes. This pioneering study paved the way for future research into the role of these 'jumping genes' or transposable elements (TEs) in both health and disease conditions [31]. Indeed, it was later discovered that about 46% of the human genome consists of TEs [32]. They consist of repetitive sequences that are able to insert copies of themselves elsewhere in the genome [33]. They are divided, according to their size and functionally related structures, into short interspersed elements (SINEs), long interspersed elements (LINEs), long terminal repetition retrotransposons (LTR) and DNA transposons [34]. The major subset of LTR retrotransposons is represented by HERVs, which together with their derivative sequences comprise at least 8% of the human genome [32, 35].

These elements have their origin in the numerous environmental events that shaped the human genome during evolution, including the occasional infection of germ cells of our ancestors by exogenous retroviruses and the insertion of their RNA genome as proviruses into the cell's chromosomal DNA [36]. Hence, HERVs are transmitted in a Mendelian manner to all subsequent generations (**Figure 1a**). Retroviral proviruses share the canonical structure of retroviruses consisting of an internal region of four essential viral genes (gag, pro, pol and env), flanked at either side by long terminal repeats sequences (the 5′ and 3′ LTRs) that are identical at the time of integration and contain promoter, enhancer and polyadenylation signals that shape the cellular transcriptome (**Figure 1a**) [37].

A non-coding sequence containing a tRNA-specifc primer-binding site (PBS) is usually present between the end of the 5′ LTR and the first codon of the gag gene,

**Figure 1.** *HERV origin (a) and their activation in response to environmental stimuli (b) (created by BioRender).*

and HERVs are classified into families on the basis of the tRNA that binds to the viral primer-binding site to prime reverse transcription.

During human evolution, HERVs invaded the human genome undergoing amplification, retrotransposition and/or reinfection events, and resulting in the presence of multiple copies fixed in the DNA of all nucleated cells [36]. While a majority of these sequences have accumulated mutations and/or deletions and are mostly defective, several HERVs preserve the properties of the ancient viruses and, still being transcriptionally active and competent to produce some retroviral proteins, have been co-opted for physiological functions [36, 38, 39] (**Figure 1a**). In the light of the current knowledge, what Weiss stated in 2016 'If Charles Darwin reappeared today, he might be surprised to learn that humans are descended from viruses as well as from apes' [40] is still current.

#### **3. The role of human endogenous retroviruses in physiological conditions**

For a long time, HERVs have been considered as junk DNA with no impact on the host. However, during the last decades, the great efforts of the scientific community highlighted that some 'well preserved' ERV sequences influence different physiological properties being involved in a variety of biological pathways [41, 42]. The most intriguingly example of the co-option of HERVs during the host evolution comes from the well-studied properties of the syncytins, the products of the Envelope (Env) genes of HERV-W-1 and HERV-FRD. Specifically, Syncytin-1, encoded by the HERV-W-1

#### *Human Endogenous Retroviruses in Autism Spectrum Disorders: Recent Advances… DOI: http://dx.doi.org/10.5772/intechopen.108671*

gene, is the first retroviral protein found to have a defined physiological function mediating the cell-cell fusion as the terminal differentiation of the trophoblast lineage [43]. As emerging from different reports, the decrease of syncytin expression and the consequential fusion deficiency could contribute to placental anomalies including pre-eclampsia disorder [44]. Moreover, other studies suggested that syncytin-1 also possesses non-fusogenic activities, as the regulation of trophoblast proliferation and apoptosis and indicated a widely expression in different cell types such as granulocytes, T lymphocytes, monocytes, glial cell of the brain and cancer cells [45, 46]. In human, syncytin-1 interacts with the type D mammalian retrovirus receptor ASCT-1/ASCT-2 (sodium-dependent neutral amino acid transporter type 1/2) on cell membranes. Syncytin-2, encoded by the HERV-FRD gene, is also expressed in trophoblasts and able of mediating cell fusion by interacting with a different receptor known as MFSD2 (major facilitator superfamily domain containing 2) [47, 48]. Moreover, Syncytin-2 is involved in maternal immune tolerance towards the semi-allogenic foetus [49]. A similar functional domestication emerged for ERV-encoded GAG gene, involved in the memory consolidation in the mammalian brain, including long-term potentiation and depression [50]. Given the abundance of HERVs in the human genome, they represent an important source of genomic variability, also providing potential coding and regulatory elements for the acquisition of new cellular functions [51, 52]. In line, growing evidence has been obtained regarding the general expression of HERVs in normal tissues [53, 54], and in this context, we demonstrated an age-related transcriptional activity of HERV-H, HERV-K and HERV-W in peripheral blood mononuclear cells (PBMCs) from a large cohort of healthy human subjects aged between 1 and 80 years, reinforcing the hypothesis of a physiological correlation between HERV activity and the different stages of life in human [55]. Among the proposed mechanisms by which HERVs could contribute to the human physiology, it is recognized that various sequences, concentrated in the LTRs, are involved in the regulation of the expression of neighbouring genes acting as promoters, enhancers, polyadenylation signals, regulators of chromatin folding and binding sites for transcriptional factors [56, 57]. In the genome, most HERVs reside as solo-LTRs, resulting from homologous recombination between the LTRs of a full-length HERV [58] able to act as alternative tissue-specific promoters to drive the expression of host genes [59–61]. During embryo development, some HERV sequences are also engaged by the host for the regulation of gene expression [62]. In particular, non-coding RNA expressed by the HERV-H group and the recruitment of specific cellular transcriptional factors on HERV-H LTRs seem to be involved in the conservation of stem cell identity [57, 63]. Of note, the HERV-H loci seem to be more preserved in a full-length state than other HERV families, suggesting that the full-length elements rather than solo-LTRs are useful to the host and that the internal regions of HERV-H may be involved in the process of exaptation [64]. Similarly, an ancestral env gene named HEMO [human endogenous MER34 (medium reiteration-frequency-family-34) ORF] has been found highly expressed in embryos, already in the early stages of development and in all subsequent differentiation periods as well as in the placenta and in the blood of pregnant women [65]. Finally, in the regulation of stem cell function, HERV-K ENV was highly expressed in the cell membrane of pluripotent stem cells and signals *via* direct binding to CD98HC, leading to activation of signalling pathways that regulate stem cell function [66]. Moreover, the expression of HERVs has a direct key role for the maintenance of human embryonic stem cells and induced pluripotent stem cells (iPSCs), and their activation could be considered a marker of pluripotency [67].

To conclude, the more profound insight into the mechanisms explaining the roles of HERVs in various biological/physiological contexts will help to clarify the contribution of HERVs in pathological conditions.

## **4. The activation of human endogenous retroviruses**

In addition to their physiological role, HERVs have been also proposed as possible cofactors in the aetiology of several human diseases. Proviruses are known to remain dormant for long periods of time within the host, but they may occasionally be triggered by factors present in the environment. Indeed, one of the peculiar features of HERVs is their intrinsic responsiveness to microenvironmental stress and various stimuli likely *via* epigenetic mechanisms [68, 69]. Epigenetics represents a fine mechanism to control HERV activity to ensure genomic stability and integrity and, on the other side, it represents one of the mechanisms by which HERVs could modulate the gene expression. HERV sequences are epigenetically silenced by DNA methylation and histone modifications in addition to being located in chromosomal regions with heterochromatic chromatin architecture leading to a low transcriptional degree in most cell types [70, 71]. It is known that epigenetics is a driver of the embryonic development, contributing to global remodeling, cell commitment and tissue specification [72] and that ERVs are highly active during early embryogenesis and germline development [62, 73]. Indeed, they are involved in the pre-implantation transcription network although the mechanisms are still unclear [62, 74]. Thus, during a sensitive phase, as the embryogenesis, any environmental insults could have an impact on the development, and epigenetic modifications could directly link the environmental stimuli and the molecular regulatory pathways, explaining some aspects of complex pathologies including HERV activation. A multitude of environmental factors and xenobiotics can activate HERV expression causing DNA rearrangements, HERV reinsertions and HERV copy number variation, resulting in abnormal HERV activity that could, in turn, potentially affect crucial pathway such as inflammation [69, 75, 76]. Microorganisms, cytokines, hormones, vitamins, nutrients and drugs could represent triggers leading to HERV transactivation (**Figure 1b**). Of particular relevance is the role played by the interaction with microbes, including viruses, exogenous retroviruses, intestinal microbiota and protozoan. Among viruses, the herpesviruses, Hepatitis B virus, the human immunodeficiency virus-1 (HIV-1), Influenza A virus and more recently SARS-CoV-2 have been found able to deregulate HERV activity and although so far, an unequivocal pathogenic cause-effect relationship has not been established, their contribution to the development of viral diseases, including virusassociated tumors, has been suggested [77–83]. Moreover, several *in vitro* studies demonstrated that cells express HERVs at high levels in response to stimulation by using lipopolysaccharide or interferon-γ (IFN-γ), cytokines as Interleukine-1β (IL-1β) and tumor necrosis factor-α (TNF-α) or mitogens, such as phytohemagglutinin [84–86]. These observations could be due to the fact that HERVs showed various regulatory sequences that have been linked to the transcriptional modulation systems [87]. More recently, the hormonal regulation of HERVs has been investigated, and specifically, cross talks among the female sex hormones and HERVs in contributing to breast cancer tumorigenesis and proliferation have been elucidated providing useful knowledge for the development of novel cancer therapies. Specifically, the effect of progesterone on HERV-K expression is at least partly mediated by OCT4 known to be involved in

*Human Endogenous Retroviruses in Autism Spectrum Disorders: Recent Advances… DOI: http://dx.doi.org/10.5772/intechopen.108671*

embryogenesis and expressed in diverse cancer types as well [88]. These findings were in line with the peculiar expression of HERVs in peripheral leukocytes during the menstrual cycle suggesting a well-coordinated hormonal regulation of HERV activity [89]. Also, drugs are able to modulate HERV expression, both *in vitro* and *in vivo* with different proposed mechanisms, mainly linked to the epigenetic one. In particular, neuroleptics and antidepressants influence HERV activity in human brain cell lines and in post-mortem brain samples of patients with mental disorders in therapy during their lifetime [90].

Thus, HERVs have been found particularly responsive to environmental stimuli that can determine their dysregulation at transcriptional levels and/or encoded protein expression that could influence the onset of complex diseases.
