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The Role of Human Endogenous Retroviruses (HERVs) in the Pathologies of the Nervous System
CHAPTER 22
The Role of Human Endogenous Retroviruses (HERVs) in the Pathologies of the Nervous System ~ ks1, 2, 3, Gea Ko ~ ks1, 3 Sulev Ko
€ Tartu, Estonia University of Tartu, Tartu, Estonia; 2Estonian University of Life Sciences, Tartu, Estonia; 3Prion OU,
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INTRODUCTION The human genome contains almost half of its size, 45%, repetitive retroelements or transposable elements (TEs). TEs are derived from ancient viruses and the result of viral genome integration that occurred a long time ago. TEs can be divided into two major classes (Table 22.1): transposons (3% of the genome) and retrotransposons or retroelements (42% of the genome).1 Retroelements are divided into long terminal repeat (LTR) and non-LTR elements. Non-LTR elements are the most abundant and are divided into short interspersed elements (SINE) and long interspersed elements (LINE). SINEs are Alu and MIR repeats without independent amplification and protein coding capacity, and they are also considered nonautonomous TEs.2 Alu repeats are the most common elements in the human genome, and they make up around 15% of the human genome.3 LINE elements contain autonomous L1 and L2 elements (LINE repeats). The LTR class consists of elements of the retroviral origin, and this class is more heterogenous (Table 22.2). One group of LTR elements are human endogenous retroviruses (HERVs), and they occupy around 8% of our genome.4 These viruses are derived from ancient infections of retroviruses that were reverse transcribed
Table 22.1 Overview of the transposable elements (TEs) in the human genome2 TEs (45% of genome) DNA-transposons 2.8%
Retrotransposons or retroelements 42.2%
Non-LTR, 33.9%
LTR, 8.3%
SINE (Alu, MIR) LINE (L1, L2) Pseudogenes Class I ERV þ MER4 Class II ERV Class II ERV Others (MST, MLT)
Percentage of the elements in the genome is given in brackets.
Molecular-Genetic and Statistical Techniques for Behavioral and Neural Research ISBN 978-0-12-804078-2, https://doi.org/10.1016/B978-0-12-804078-2.00022-2
© 2018 Elsevier Inc. All rights reserved.
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Table 22.2 Characteristics of subgroups of LTR retroelements2 Element Characteristics
Class I ERV Class II ERV Class III ERV MER4 MST MLT
Similar to type C or g retroviruses Similar to type B or b retroviruses Also called ERV-L, limited similarity to spumaviruses Nonautonomous class I ERV Named after restriction enzyme site MstII, MaLR Mammalian LTR transposon, MaLR
and integrated into the genome of ancestral host animals. These viral genomes were fixed and inherited tens of millions of years ago.1,5 Almost all of the HERVs have lost their infectivity. However, presence of the env gene in HERVs confers their potential to spread between cells. LTR class also contains HERV-like repeat elements that are derived from HERVs (SINE-R) and contain one-copy of LTR element. Finally, there are sequences with LTR-like features but without any homologous proviral structure. Repbase is a central curated database for repetitive elements in which more than 200 families of LTR-containing retroelements are defined and grouped into six superfamilies.6 These families are class I ERVs (similar to type C retroviruses), class II ERV (similar to type B retroviruses), class III ERV (distantly similar to spuma retroviruses), MER4 (nonautonomous class I-related ERVs), MST (common MstII restriction site), and MLT (mammalian LTR transposon). MST and MLT are both part of the larger nonautonomous mammalian apparent LTR retrotransposon (MaLR) superfamily.2 TEs form the basis of the “junk DNA” that has been without any understandable function in the recent past. Generally, it has been suggested that about half of our genome is derived from the TEs, and they do not perform any specific function. New analyses have estimated that more than two-thirds of our genome has resulted from incorporation of viral DNA.7 However, during evolution, our genome has “domesticated” several TEs to fulfill different physiological functions, like V(D)J recombination in the immune system or cellular fusion during placental development and even cellular protection against retroviral infections.8e10 Taken together, it is obvious that many TEs have had substantial impact on shaping our genomes and changing our phenotypes. Analysis of human genes has found that TEs are overrepresented in the mRNAs of rapidly evolving mammalian genes and potentially have gene regulation function.11 On the other hand, HERVs and other LTRs are normally suppressed, but they can potentially act as transcriptional regulators and this way activate malignant disorders or autoimmune responses.5,12 The importance of HERVs and LTRs in gene regulatory networks has been verified in several studies.13 The role of HERVs has been shown in embryonic development, in immune regulation, and in the pathogenesis of complex disorders.14e16 Most remarkably, HERVs are involved in different diseases including melanoma,
The Role of Human Endogenous Retroviruses (HERVs) in the Pathologies of the Nervous System
amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), schizophrenia (SCH), bipolar disorder, autism spectrum disorder (ASD), and attention deficit hyperactivity disorder (ADHD).17e19 The present chapter gives an overview of the role of HERVs in the neurological and psychiatric disorders.
HUMAN ENDOGENOUS RETROVIRUSES IN NEUROLOGICAL DISEASES To appreciate the potential impact of repetitive elements on the genome function, it is important to mention that roughly one-quarter of human promoter regions contain TEs.20 TEs produce negative impact by causing mutations in genomes, but it is also recognized that TEs are involved in the regulation of gene expression.21,22 In the latter case, TEs should have significant impact in the pathogenesis of diseases. The first study where TEs were described as causal agents for human diseases was published in 1988. In this paper, two separate insertions of L1s were described to cause hemophilia A by disrupting the factor VII gene.23 Thereafter, TEs were found to cause muscular dystrophy, neurofibromatosis, and colon cancer.24e26 LINE-1-induced insertional mutagenesis in the myc locus has been shown to cause breast cancer.25 In a more recent study, aberrantly activated LTR of the MaLR family (THE1B) was found to induce the expression of CSF1R in Hodgkin lymphoma.27 Therefore, TEs can contribute to the pathogenesis of diseases directly via mutagenesis or by the regulation of the gene expression networks. From this perspective, two of the most well-studied neurological disorders are MS and ALS. Summarized information is given in Table 22.3.
Multiple Sclerosis MS is associated with demyelination in the CNS leading to disturbed sensory, motor, autonomic, and cognitive functions and fatigue. The etiology of MS remains debated, but pathogenesis involves chronic inflammation and autoimmune mechanisms.28 The role of viruses on the pathogenesis of this disease has been suspected for a long time.28,29 The first discovery of reverse transcriptase activity of the cell line isolated Table 22.3 Neurological disorders with the involvement of transposable elements (TEs) and type of evidence Disorder TEs Evidence
Multiple sclerosis
Amyotrophic lateral sclerosis
HERV-W (MSRV) HERV-H (Fc1) HERV-K (K13, K18, K113) HERV-K HERV-W
Induction of immune response, expression, experimental Association study Association study, expression Expression, experimental Controversial evidence
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from cerebrospinal fluid (CSF) of a patient with MS indicated that this activity was not related to the common “suspects” HTLV-I or HIV-1 and HIV-2.30 Later, the viral particles were isolated and designated as “MS-associated” retrovirus (MSRV).31 Additional molecular characterization of the MSRV identified its similarity to the endogenous retroviral sequence ERV9.32 MSRV was found to be expressed not in all, but in many MS patients, and it was found in noncellular RNA from the plasma and CSF of the patients.32 The MSRV-related transcript contains gag, pol, and env genes, and it is expressed in the placenta. This new HERV family was named HERV-W.33 Subsequently, the role of HERV-W in MS was repeatedly confirmed in different independent studies.34e37 More importantly, HERV-W (MSRV) is not only associated with the presence of MS, but in the CSF, it correlates with the clinical progression of the disease.35,36,38 Presence of the HERV-W sequences in the CSF is a marker for rapid disease progression.36e38 The molecular mechanism of the infectivity of HERV-W has been studied for more than a decade. HERV-W has been shown to activate innate immunity with subsequent release of proinflammatory cytokines by using a Toll-like receptor 4 (TLR4) agonist effect.39 TLR4-activated macrophages introduce T-lymphocytes that are required to turn HERV-W envelope (ENV) protein into superantigen, and to ignite the downstream immune cascade leading to production of the IL-1b, IL-6, or TNF-a.39 In addition, dendritic cells are activated and Th1-like responses are developed. Both TLR4 and CD14 receptors are involved in the proinflammatory effects of ENV protein.39 The pathogenicity of HERV-W was further analyzed in relation to the T-lymphocyte-mediated immunopathology.40 HERV-W particles induced polyclonal T-lymphocyte response with a bias in the Vb16 chain usage as a surface receptor. This means that HERV-W triggers an abnormal immune response that is characteristic of superantigens.40 In addition to the peripheral immune system, further studies indicated the role of HERV-W in the differentiation of the oligodendroglial precursor cells. More precisely, HERV-W particles induced the proinflammatory cytokines and nitric oxide synthase in oligodendroglial precursors cells expressing TLR4.41 In addition to the in vitro studies, animal experiments also confirmed the impact of HERV-W in the induction of immune response.40 More precisely, mice immunized with the HERV-W showed clear experimental allergic encephalomyelitis.42 In vivo experiments confirmed that HERV-W is a highly potent agonist for the TLR4, and this interaction leads to the proinflammatory activity and impairs differentiation of the oligodendroglial precursor cells to mature myelinating cells.43 Taken together, the role of HERV-W in pathophysiology of MS has been demonstrated in clinical observational studies, as well as in vitro and in vivo laboratory experimental studies. Based on these findings, a humanized antibody, GNbAC1, against HERV-W ENV extracellular domain was developed to treat patients with MS.43e45 This antibody has been successful in preclinical and clinical studies, showing low toxicity and good
The Role of Human Endogenous Retroviruses (HERVs) in the Pathologies of the Nervous System
tolerability.45 Preclinical studies indicated no safety risks, so Phase I, the first-in-human randomized, double-blind, placebo-controlled, dose-escalation study was performed.44 In 33 male subjects, only minor and nonspecific adverse effects were recorded, and this allowed launching of the Phase II program.44 A Phase 2a study was performed as a single-blind, placebo-controlled, randomized study in 10 MS patients.45e47 All patients tolerated the GNbAC1, and treatment significantly reduced transcription of the MSRV. Moreover, a recent study indicated that HERV-W is also involved in chronic inflammatory demyelinating polyradiculoneuropathy and that it induces inflammatory damage of Schwann cells.48 The pathogenic effect of HERV-W was inhibited by the GNbAC1, suggesting a broad utility for the antibody.48 The story of HERV-W is an elegant example of how discovery may progress from the identification of a potentially infectious agent to the development of a promising treatment option for a disease that has been without any possible cure. This is probably the first endogenous retrovirus that has been used as a specific therapeutic target for the development of antibodies. In addition to the HERV-W, genetic association studies have found increased odds ratio in the locus for the HERV-Fc1 gene.49 HERV-Fc1 is closely related to the HERV-H, and more distantly to the HERV-W and HERV-K.49 The association of HERV-Fc1 with MS was repeated in another independent study.50 Moreover, highly significant genetic interaction between HERV-Fc1 and HERV-K13 loci was described.50 In one study using postmortem tissues, increased mRNA level of HERV-K and HERV-W was found.51 Taken together, the case of MS excellently exemplifies the pathogenetic potential and impact of HERVs in our genomes. While the involvement of HERV-W in MS is clearly demonstrated, the role of HERVs in other diseases needs further analysis.
Amyotrophic Lateral Sclerosis ALS is a progressive and fatal neurodegenerative disease that is characterized by the loss of upper (in the cerebral cortex or brain stem) and lower (in the spinal cord or cranial nerves) motor neurons, leading to increasing weakness.52 ALS is sporadic disease with the prevalence of five in 100,000, and viral involvement in its aetiology has been suspected for many years, mainly on the basis of similarities with poliomyelitis.53 The cause of ALS is not known, but the role of HERVs has been suggested recently. The first pioneering studies analyzed peripheral blood sera from patients with motor neuron diseases. In one study, sera from the 56 patients were analyzed for reverse transcriptase activity by using product-enhanced reverse transcriptase assay (PERT).53 Indeed, in 33 patients (59%) cell-free reverse transcriptase activity was detected compared to only three of the controls.53 This activity was not caused by the HIV-1, HIV-2, HTLV-I, HTLV-II, HRV-5, or human foamy virus. In another study, 30 patients with sporadic ALS were analyzed for reverse transcriptase activity
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in blood sera.54 The authors applied a similar serum purification protocol combined with a sensitive PERT assay. Fourteen patients were PERT positive (47%), a statistically significant difference compared to the genetically related and unrelated controls.54 Increased RT activity on sera of HIV-negative ALS patients was further confirmed by an additional study.55 In this study, RT activity was measured also in CSF, but the difference (39% vs. 19%) was not statistically significant.55 All these early studies used biochemical detection of RT activity, so the identity of retroviruses was not discoverable. Application of RNA analysis with RT-PCR subsequently answered this question and identified increased expression of the HERV-K in the autopsy brains.56 Authors also found that HERV-K is expressed in cortical neurons of the ALS patients. All these studies were descriptive and were designed to show association between RT activity and ALS. A recent study identified causative connection between HERV-K and ALS.18 First, authors found increased expression of the HERV-K in cortical and spinal neurons of ALS patients. Afterward, they showed causal relationship between the expression of HERV-K and neuronal pathologies in cellular models as well as in mice using transgenic technology.18 Transgenic mice expressing the HERV-K developed progressive motor dysfunction accompanied by selective loss of volume of the motor cortex and decreased synaptic activity. Transgenic mice had injuries in the anterior horn of the spinal cord, and this was associated with muscle atrophy.18 Moreover, authors identified that expression of HERV-K is regulated by TDP-43, a protein that is overexpressed in ALS and is known to form cytoplasmic aggregates. HERV-K has five binding sites for the TDP-43, and as TDP-43 seems to be involved in other neurodegenerative disorders (e.g., frontotemporal dementia), the potential pathophysiological impact of HERV-K is likely not limited to ALS.18
HUMAN ENDOGENOUS RETROVIRUSES IN PSYCHIATRIC DISEASES Out of all psychiatric diseases, SCH and bipolar disease are the most frequently studied disorders with regard to endogenous retroviruses. But some information is also available on autism. A general overview is given in Table 22.4.
Schizophrenia In the past, it has been suggested that exogenous viruses and retroviral infections are responsible for SCH, but there were no studies proposing endogenous retroviruses to play any role. Some early studies suggested that one of the pathogenic factors for SCH could be retroviral infection, or that the disease is induced by quantal changes in “virogene” or “schizovirus” within our genome.57e59 These early studies proposed that endogenous retroviral integration to the human genome near the gene critical for SCH leads to the disruption of this gene and development of disease.57 These hypotheses were too complex to challenge during their time, but recent developments in molecular
The Role of Human Endogenous Retroviruses (HERVs) in the Pathologies of the Nervous System
Table 22.4 Psychiatric disorders with the involvement of transposable elements (TEs) and type of evidence Disorder TEs Evidence
Schizophrenia
Bipolar disorder
Autism
ADHD
SZRV-1 (schizovirus, HERV-W, MSRV) SZRV-2 HERV-K (K115) L1 elements HERV-I (IP-T47D) HERV-9 (Seq63) HERV-L NMWV7 HERV-K (K10, KC4) HERV-W HERV-Fb HERV-H HERV-K HERV-W L1 HERV-H
DNA cloning, expression Differential cloning Variations, expression More copies Expression Expression Expression Expression Expression, treatment Expression, treatment Expression, treatment Expression Expression Expression Expression Expression, treatment
technologies allowed performing better experimental testing. One of the first studies used representational difference analysis (RDA) of blood DNA from three pairs (two male and one female) of monozygotic twins discordant for SCH.60 Each affected member met the DSM-III-R criteria for SCH. The unaffected twins had no psychotic or affective illness. For the independent testing and analysis of the isolated DNA fragments, DNA from five additional pairs of affected individuals and their unrelated matched controls was extracted. This study gave very complex results. Authors identified 128 sequences in the genomes of the affected persons that were grouped in four contigs.60 The first contig was designated SZRV-1, and this sequence was similar to the sequence found to be implicated in MS (MSRV, ERV-9, or HERV-W).32 The identified sequence (HERV-W) was later found to be differentially expressed in cell-free CSF of individuals with SCH.61 Analysis of CSFs and postmortem brain tissue from the SCH patients revealed significant increase of transcription of HERV-W family of endogenous retroviruses.61,62 The results from the second RDA of DNA revealed a sequence named SZRV-2.63 SZRV-2 is confined to 12q13, and this site was also shared with the SZRV1. Southern blot analysis indicated that this region contains tandem repeats and is known to harbor other viral elements.63 Interestingly, this locus contains several genes potentially important for the pathogenesis of SCH, thus supporting the original hypothesis of retroviral integration near the “SCH gene.”63 Further supporting the initial findings, HERV-W was also found in the plasma of the individuals with recent-onset SCH.64
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Another study analyzed sera from 58 SCH patients and identified the ERV9 family retrovirus in 20 patient samples.65 No ERV9 was detected in control persons. This result was further confirmed with Western blot analysis and also by positive immunoreactivity in the sera of patients.65 In addition to the HERV-W, the expression of HERV-K has been identified in the cDNA libraries of the postmortem brains of the SCH patients.66 HERV-K belongs to an interesting group of HERVs, because it is a full-length provirus, and is probably capable for reinfections.67 Transcriptional activity of HERV-K has been found in the brains of SCH and bipolar patients.68 HERV-K115 is mapped to the 8p23.1 region, which is related to SCH susceptibility.69 A case-control study did not find any significant associations between SCH patients and variations in the HERK-K115 gene.70 However, it was concluded that variations in the HERV-K115 could modify the onset of the disease.70 Variations in the HERV-K18 were found to be associated with type 2 diabetes (T2D) in SCH patients.71 More precisely, the haplotype defined by two polymorphisms (7086 and 8146 C/T) in the envelope region was highly associated with T2D in SCH patients. The established odds ratio was 9.0 (95% CI 2.3e34.7, P < .001) after adjustment for age, gender, race, and current therapy with clozapine.71 This finding was partially confirmed in a Danish population with 750 cases.72 They did not find any association between HERV-K18 and SCH or T2D in patients with SCH. However, in younger SCH patients, there was a trend for association with T2D, a finding that was in agreement with previous results.71,72 Genetic interaction between SCH and T2D based on HERVs is plausible, as several studies have found comorbidity between SCH and T2D.73,74 Peripheral activation of endogenous viruses has also been studied in several investigations. For instance, HERV-W antigenemia was described in SCH patients.75 In addition, activation of HERV-W sequences has been analyzed by several additional research papers.76,77 In the plasma of 42 out of 118 individuals suffering from recent-onset SCH, activation of expression of the sequences homologous to HERV-W env gene was found. Overexpression of the HERV-W in glioma cell line was found to induce upregulation of brain-derived neurotrophic factor, neurotrophic tyrosine kinase receptors type 2 (NTRK2 or TrkB), and dopamine receptor D3 genes.77 Authors concluded that transcriptional activation of HERV-W causes upregulation of the genes implicated in SCH. Similarly, HERV-W LTR has been described in the regulatory region of the GABA receptor B1 gene (GABBR1).76 Similar insertion of the human ERV in the enhancer has been shown for the PRODH gene.78 The PRODH gene encodes proline dehydrogenase, and it is involved in the synthesis of neurotransmitters. Hypomethylation of this enhancer induced higher expression of the PRODH gene, and this could have an impact in brain development.78 While all these previous studies were more focused on specific HERVs or a specific insertion locus, recent technologies enable a much wider approach. Comprehensive
The Role of Human Endogenous Retroviruses (HERVs) in the Pathologies of the Nervous System
analysis of L1 retrotransposition was performed in neuronal genome in postmortem samples from different mental diseases.79 Clear increase in the L1 retrotransposition was found in SCH. In major depression and bipolar disorder samples, the increased L1 content was borderline in terms of statistical significance.79 SCH brains showed robust differences, and these differences were confirmed in an independent sample of additional 34 brains. Authors used whole-genome sequencing to identify genomic regions for the L1 insertions, and they found that it localized preferentially to synapse- and SCH-related genes. This study also showed that hyperactive L1 retrotransposition can be triggered after immune activation, so environmental factors can contribute to the susceptibility of SCH.79 In one metagenomics study, 23 drug-naïve, first-onset SCH patients were analyzed. The authors did not find any evidence for the activation of endogenous retroviruses in the sera of these patients.80 Taken together, HERV-W and HERV-K seem to be the most common HERVs found to be associated with SCH.
Bipolar Disorder Bipolar disorder is studied less intensively compared to the previously described diseases. Samples from bipolar disorder patients are usually studied along with SCH samples. In one large-scale study, 50 members of 20 HERV families were analyzed in 215 brain samples.68 The authors of this study found HERV-IP-T47D (HERV-I family) to be reduced in bipolar samples compared to those of SCH. Seq63 from the ERV9 family was reduced in bipolar samples compared to controls. HERV-K10 from the HML-2 family was upregulated in both SCH and bipolar samples compared to the control. And finally, NMWV7 from the HML-7 subclass and HERV-L were downregulated compared to controls and SCH.68 Interestingly, some HERVs had similar expression profiles between diseases, but other HERVs exhibited disease-specific idiosyncratic profiles. In another study using immunohistochemistry, significant downregulation of the HERV-W in different brain areas of bipolar patients was described.81 This reduced expression in immunohistochemical staining could indicate neuronal loss in the brains of bipolar patients, potentially resulting from altered expression of HERV during the illness.81 However, this hypothesis needs further clarification. Some studies have analyzed the effect of treatment on the expression of HERVs. Valproic acid (VLP) is one of the most commonly used drugs for bipolar disorder. The effect of VLP on the expression of different HERV families in different human cell lines (glioblastoma, neuroblastoma, neural stem cell line) has been studied.82 VLP dose-dependently upregulated transcription of several HERVs from different subclasses. VLP upregulated HERVs from class I (HERV-E, HERV-H, HERV-W, ERV9, and HERV-F) and from class II (HML-3, HML-4, HML-6, HML-9, HML-10). The stronger influence of VLP was established for HERV-Fb, ERV9, Seq26 (HML-3),
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and HERV-KC4/HML-10.82 In postmortem brain samples, HML-2 and ERV9 were upregulated in patients with bipolar disease without treatment compared to the control samples, and VLP treatment did not affect this result.82 In summary, existing evidence suggests that HERVs do play roles in bipolar disorder, and traditional pharmacotherapies may affect expression of HERVs.
Other Psychiatric Diseases Autism or ASD is a complex disorder associated with altered brain development. ASD is clinically variable. However, an often observed feature is impaired social interaction with peers, impaired communication, and stereotypical and rigid patterns of behavior.83 The prevalence of ASD is increasing, and the increasing societal burden necessitates more research.84 Despite decades of studies, the etiology of ASD is still unknown. Accumulating evidence suggests that ASD is a multifactorial disease with a strong genetic basis.84 Twin studies have found high heritability (more than 90%). Nevertheless, the exact genetic loci and mechanisms underlying the disease have still not been identified. As a new development, an increasing number of studies have described positive association between copy number variants (CNVs) and ASD.85 In the light of the emerging role of CNVs, analysis of TEs in ASD becomes feasible. RNA from peripheral blood mononuclear cells (PBMCs) of 28 Caucasian children was analyzed for the presence of retroviral RNA from four HERV families (HERV-E, HERV-H, HERV-K, HERV-W).83 The expression of HERV-H was higher in ASD patients, and expression of HERV-W was lower in ASD patients than in controls. Expression on HERV-K was similar, and the expression of HERV-E was absent.83 In vitro stimulation of the PBMCs from the ASD patients resulted in higher upregulation of the HERV-H expression compared to controls.83 Moreover, authors described negative correlation between the HERV-H expression and the Psychoeducational Profile-3 score.83 In an additional analysis performed by the same group, an independent sample of Albanian ASD patients was studied.86 HERV-W was found upregulated, while HERV-K and HERV-W were downregulated in ASD patients compared to controls.86 Elevated HERV-W expression in autistic children suggests that infection or inflammation during pregnancy could be an etiological risk factor for the development of ASD.86 Another recent study was performed on frozen postmortem cerebellar cortex samples from 13 autistic patients and 13 control individuals.87 Expression analysis of L1 ORF1 and ORF2 was performed with QRT-PCR. L1 expression was found to be significantly increased in the brains of autistic patients, which was inversely correlated with GSH/GSSG ratio.87 Positive correlation between L1 expression and FOXO3 was also established.87 However, the impact of L1 expression to neuronal
The Role of Human Endogenous Retroviruses (HERVs) in the Pathologies of the Nervous System
CNVs and genomic instability requires additional research with innovative genomic technologies. Similarly to ASD, HERVs may impact the development of ADHD. The expression of retroviral RNAs of three HERV families (H, K, and W) were analyzed in 30 patients with ADHD and 30 healthy controls using PBMCs.88 The only significantly increased expression found in ADHD patients was for HERV-H. Expression of HERV-K and HERV-W remained unchanged. ADHD has a complex etiology, and the authors concluded that HERVs may present a possible link between the environmental, biologic, and genetic factors contributing to the development of this disease. In another study with a single ADHD patient, HERV-H expression in PBMCs was evaluated before and after treatment with methylphenidate (MPH).89 Compared to healthy controls, HERV-H was highly and significantly overexpressed in the patients before treatment, but after 6 months of treatment with MPH the expression of HERV-H was normalized to the level of that of controls.89 This study gives support to the idea that expression changes in HERVs can be pathogenic, and treatments can modify HERV expression and relieve the symptoms.
CONCLUSIONS Almost half of the human genome contains TEs, and the impact of these elements on physiology and pathophysiology is not understood yet. However, growing data suggest that TEs play significant roles in a number of CNS disorders. The best studied example is MS, where a clear role of HERV-W in disease development has been demonstrated. Moreover, treatment with antibodies against HERV-W aborts the destruction of neurons and stops progression of the disease.45 Another elegant example is the involvement of HERV-K in ALS.18 This chapter summarized existing evidence on how HERVs and other TEs could impact our health, and how understanding this impact may help us develop treatment either by development of new drugs or by repurposing already existing drugs to target the activity of endogenous retroviruses. Taking together accumulated existing evidence, I suggest that HERV elements represent a missing link between environmental and genetic factors underlying numerous pathogenic processes of complex human diseases. Notably, for the majority of complex diseases, we do not have an obvious and validated etiological explanation. Activation of dormant retroviruses in our genome is “the enemy within.”90 Endogenous retroviruses constitute a new class of pathogens different from classical environmental and infectious microbes, arising from the host’s genome itself.90 Understanding their function and effects thus may open entire new research avenues and therapeutic applications.
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REFERENCES 1. Bannert N, Kurth R. Retroelements and the human genome: new perspectives on an old relation. Proc Natl Acad Sci USA. 2004;101(suppl 2):14572e14579. 2. Medstrand P, van de Lagemaat LN, Mager DL. Retroelement distributions in the human genome: variations associated with age and proximity to genes. Genome Res. 2002;12(10):1483e1495. 3. Kazazian Jr HH, Moran JV. The impact of L1 retrotransposons on the human genome. Nat Genet. 1998;19(1):19e24. 4. Hughes JF, Coffin JM. Evidence for genomic rearrangements mediated by human endogenous retroviruses during primate evolution. Nat Genet. 2001;29(4):487e489. 5. Katoh I, Kurata S. Association of endogenous retroviruses and long terminal repeats with human disorders. Front Oncol. 2013;3:234. 6. Jurka J. Repbase update: a database and an electronic journal of repetitive elements. Trends Genet. 2000; 16(9):418e420. 7. Solyom S, Kazazian Jr HH. Mobile elements in the human genome: implications for disease. Genome Med. 2012;4(2):12. 8. Zhou L, Mitra R, Atkinson PW, Hickman AB, Dyda F, Craig NL. Transposition of hAT elements links transposable elements and V(D)J recombination. Nature. 2004;432(7020):995e1001. 9. Ponferrada VG, Mauck BS, Wooley DP. The envelope glycoprotein of human endogenous retrovirus HERV-W induces cellular resistance to spleen necrosis virus. Arch Virol. 2003;148(4): 659e675. 10. Blond JL, Lavillette D, Cheynet V, et al. An envelope glycoprotein of the human endogenous retrovirus HERV-W is expressed in the human placenta and fuses cells expressing the type D mammalian retrovirus receptor. J Virol. 2000;74(7):3321e3329. 11. van de Lagemaat LN, Landry JR, Mager DL, Medstrand P. Transposable elements in mammals promote regulatory variation and diversification of genes with specialized functions. Trends Genet. 2003; 19(10):530e536. 12. Dolei A. Endogenous retroviruses and human disease. Expet Rev Clin Immunol. 2006;2(1):149e167. 13. Chuong EB, Elde NC, Feschotte C. Regulatory evolution of innate immunity through co-option of endogenous retroviruses. Science. 2016;351(6277):1083e1087. 14. Goke J, Lu X, Chan YS, et al. Dynamic transcription of distinct classes of endogenous retroviral elements marks specific populations of early human embryonic cells. Cell Stem Cell. 2015;16(2): 135e141. 15. Suntsova M, Garazha A, Ivanova A, Kaminsky D, Zhavoronkov A, Buzdin A. Molecular functions of human endogenous retroviruses in health and disease. Cell Mol Life Sci. 2015;72(19):3653e3675. 16. Wang J, Xie G, Singh M, et al. Primate-specific endogenous retrovirus-driven transcription defines naive-like stem cells. Nature. 2014;516(7531):405e409. 17. Singh S, Kaye S, Francis N, et al. Human endogenous retrovirus K (HERV-K) rec mRNA is expressed in primary melanoma but not in benign naevi or normal skin. Pigm Cell Melanoma Res. 2013;26(3): 426e428. 18. Li W, Lee MH, Henderson L, et al. Human endogenous retrovirus-K contributes to motor neuron disease. Sci Transl Med. 2015;7(307), 307ra153. 19. Morandi E, Tarlinton RE, Gran B. Multiple sclerosis between genetics and infections: human endogenous retroviruses in monocytes and macrophages. Front Immunol. 2015;6:647. 20. Jordan IK, Rogozin IB, Glazko GV, Koonin EV. Origin of a substantial fraction of human regulatory sequences from transposable elements. Trends Genet. 2003;19(2):68e72. 21. Kashkush K, Feldman M, Levy AA. Transcriptional activation of retrotransposons alters the expression of adjacent genes in wheat. Nat Genet. 2003;33(1):102e106. 22. Hamdi HK, Nishio H, Tavis J, Zielinski R, Dugaiczyk A. Alu-mediated phylogenetic novelties in gene regulation and development. J Mol Biol. 2000;299(4):931e939. 23. Kazazian Jr HH, Wong C, Youssoufian H, Scott AF, Phillips DG, Antonarakis SE. Haemophilia A resulting from de novo insertion of L1 sequences represents a novel mechanism for mutation in man. Nature. 1988;332(6160):164e166.
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24. Holmes SE, Dombroski BA, Krebs CM, Boehm CD, Kazazian Jr HH. A new retrotransposable human L1 element from the LRE2 locus on chromosome 1q produces a chimaeric insertion. Nat Genet. 1994; 7(2):143e148. 25. Morse B, Rotherg PG, South VJ, Spandorfer JM, Astrin SM. Insertional mutagenesis of the myc locus by a LINE-1 sequence in a human breast carcinoma. Nature. 1988;333(6168):87e90. 26. Miki Y, Nishisho I, Horii A, et al. Disruption of the APC gene by a retrotransposal insertion of L1 sequence in a colon cancer. Cancer Res. 1992;52(3):643e645. 27. Lamprecht B, Walter K, Kreher S, et al. Derepression of an endogenous long terminal repeat activates the CSF1R proto-oncogene in human lymphoma. Nat Med. 2010;16(5):571e579, 571 pp. following 579. 28. Christensen T. HERVs in neuropathogenesis. J Neuroimmune Pharmacol. 2010;5(3):326e335. 29. Koprowski H, DeFreitas EC, Harper ME, et al. Multiple sclerosis and human T-cell lymphotropic retroviruses. Nature. 1985;318(6042):154e160. 30. Perron H, Geny C, Laurent A, et al. Leptomeningeal cell line from multiple sclerosis with reverse transcriptase activity and viral particles. Res Virol. 1989;140(6):551e561. 31. Perron H, Lalande B, Gratacap B, et al. Isolation of retrovirus from patients with multiple sclerosis. Lancet. 1991;337(8745):862e863. 32. Perron H, Garson JA, Bedin F, et al. Molecular identification of a novel retrovirus repeatedly isolated from patients with multiple sclerosis. The Collaborative Research Group on multiple sclerosis. Proc Natl Acad Sci USA. 1997;94(14):7583e7588. 33. Blond JL, Beseme F, Duret L, et al. Molecular characterization and placental expression of HERV-W, a new human endogenous retrovirus family. J Virol. 1999;73(2):1175e1185. 34. Mameli G, Astone V, Arru G, et al. Brains and peripheral blood mononuclear cells of multiple sclerosis (MS) patients hyperexpress MS-associated retrovirus/HERV-W endogenous retrovirus, but not Human herpesvirus 6. J Gen Virol. 2007;88(Pt 1):264e274. 35. Arru G, Mameli G, Astone V, et al. Multiple sclerosis and HERV-W/MSRV: a multicentric study. Int J Biomed Sci. 2007;3(4):292e297. 36. Sotgiu S, Arru G, Mameli G, et al. Multiple sclerosis-associated retrovirus in early multiple sclerosis: a six-year follow-up of a Sardinian cohort. Mult Scler. 2006;12(6):698e703. 37. Serra C, Sotgiu S, Mameli G, Pugliatti M, Rosati G, Dolei A. Multiple sclerosis and multiple sclerosisassociated retrovirus in Sardinia. Neurol Sci. 2001;22(2):171e173. 38. Sotgiu S, Serra C, Mameli G, et al. Multiple sclerosis-associated retrovirus and MS prognosis: an observational study. Neurology. 2002;59(7):1071e1073. 39. Rolland A, Jouvin-Marche E, Viret C, Faure M, Perron H, Marche PN. The envelope protein of a human endogenous retrovirus-W family activates innate immunity through CD14/TLR4 and promotes Th1-like responses. J Immunol. 2006;176(12):7636e7644. 40. Perron H, Jouvin-Marche E, Michel M, et al. Multiple sclerosis retrovirus particles and recombinant envelope trigger an abnormal immune response in vitro, by inducing polyclonal Vbeta16 T-lymphocyte activation. Virology. 2001;287(2):321e332. 41. Kremer D, Schichel T, Forster M, et al. Human endogenous retrovirus type W envelope protein inhibits oligodendroglial precursor cell differentiation. Ann Neurol. 2013;74(5):721e732. 42. Perron H, Dougier-Reynaud HL, Lomparski C, et al. Human endogenous retrovirus protein activates innate immunity and promotes experimental allergic encephalomyelitis in mice. PLoS One. 2013;8(12): e80128. 43. Madeira A, Burgelin I, Perron H, Curtin F, Lang AB, Faucard R. MSRV envelope protein is a potent, endogenous and pathogenic agonist of human toll-like receptor 4: relevance of GNbAC1 in multiple sclerosis treatment. J Neuroimmunol. 2016;291:29e38. 44. Curtin F, Lang AB, Perron H, et al. GNbAC1, a humanized monoclonal antibody against the envelope protein of multiple sclerosis-associated endogenous retrovirus: a first-in-humans randomized clinical study. Clin Ther. 2012;34(12):2268e2278. 45. Curtin F, Perron H, Kromminga A, Porchet H, Lang AB. Preclinical and early clinical development of GNbAC1, a humanized IgG4 monoclonal antibody targeting endogenous retroviral MSRV-Env protein. MAbs. 2015;7(1):265e275.
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46. Derfuss T, Curtin F, Guebelin C, et al. A phase IIa randomized clinical study testing GNbAC1, a humanized monoclonal antibody against the envelope protein of multiple sclerosis associated endogenous retrovirus in multiple sclerosis patients - a twelve month follow-up. J Neuroimmunol. 2015;285:68e70. 47. Derfuss T, Curtin F, Guebelin C, et al. A phase IIa randomised clinical study of GNbAC1, a humanised monoclonal antibody against the envelope protein of multiple sclerosis-associated endogenous retrovirus in multiple sclerosis patients. Mult Scler. 2015;21(7):885e893. 48. Faucard R, Madeira A, Gehin N, et al. Human endogenous retrovirus and neuroinflammation in chronic inflammatory demyelinating polyradiculoneuropathy. EBioMedicine. 2016;6:190e198. 49. Nexo BA, Christensen T, Frederiksen J, et al. The etiology of multiple sclerosis: genetic evidence for the involvement of the human endogenous retrovirus HERV-Fc1. PLoS One. 2011;6(2):e16652. 50. Nexo BA, Villesen P, Nissen KK, et al. Are human endogenous retroviruses triggers of autoimmune diseases? Unveiling associations of three diseases and viral loci. Immunol Res. 2016;64(1):55e63. 51. Johnston JB, Silva C, Holden J, Warren KG, Clark AW, Power C. Monocyte activation and differentiation augment human endogenous retrovirus expression: implications for inflammatory brain diseases. Ann Neurol. 2001;50(4):434e442. 52. Christensen T. Human endogenous retroviruses in neurologic disease. Acta Pathol Microbiol Immunol Scand. 2016;124(1e2):116e126. 53. Andrews WD, Tuke PW, Al-Chalabi A, et al. Detection of reverse transcriptase activity in the serum of patients with motor neurone disease. J Med Virol. 2000;61(4):527e532. 54. Steele AJ, Al-Chalabi A, Ferrante K, Cudkowicz ME, Brown Jr RH, Garson JA. Detection of serum reverse transcriptase activity in patients with ALS and unaffected blood relatives. Neurology. 2005;64(3): 454e458. 55. MacGowan DJ, Scelsa SN, Imperato TE, Liu KN, Baron P, Polsky B. A controlled study of reverse transcriptase in serum and CSF of HIV-negative patients with ALS. Neurology. 2007;68(22):1944e1946. 56. Douville R, Liu J, Rothstein J, Nath A. Identification of active loci of a human endogenous retrovirus in neurons of patients with amyotrophic lateral sclerosis. Ann Neurol. 2011;69(1):141e151. 57. Crow TJ. The continuum of psychosis and its implication for the structure of the gene. Br J Psychiatry. 1986;149:419e429. 58. O’Reilly RL, Singh SM. Retroviruses and schizophrenia revisited. Am J Med Genet. 1996;67(1):19e24. 59. Fuller Torrey E, Yolken RH. Familial and genetic mechanisms in schizophrenia. Brain Res Brain Res Rev. 2000;31(2e3):113e117. 60. Deb-Rinker P, Klempan TA, O’Reilly RL, Torrey EF, Singh SM. Molecular characterization of a MSRV-like sequence identified by RDA from monozygotic twin pairs discordant for schizophrenia. Genomics. 1999;61(2):133e144. 61. Karlsson H, Bachmann S, Schroder J, McArthur J, Torrey EF, Yolken RH. Retroviral RNA identified in the cerebrospinal fluids and brains of individuals with schizophrenia. Proc Natl Acad Sci USA. 2001; 98(8):4634e4639. 62. Yolken RH, Karlsson H, Yee F, Johnston-Wilson NL, Torrey EF. Endogenous retroviruses and schizophrenia. Brain Res Brain Res Rev. 2000;31(2e3):193e199. 63. Deb-Rinker P, O’Reilly RL, Torrey EF, Singh SM. Molecular characterization of a 2.7-kb, 12q13specific, retroviral-related sequence isolated by RDA from monozygotic twin pairs discordant for schizophrenia. Genome. 2002;45(2):381e390. 64. Karlsson H, Schroder J, Bachmann S, Bottmer C, Yolken RH. HERV-W-related RNA detected in plasma from individuals with recent-onset schizophrenia or schizoaffective disorder. Mol Psychiatry. 2004;9(1):12e13. 65. Huang W-J, Liu Z-C, Wei W, Wang G-H, Wu J-G, Zhu F. Human endogenous retroviral pol RNA and protein detected and identified in the blood of individuals with schizophrenia. Schizophr Res. 2006; 83(2e3):193e199. 66. Kim HS, Wadekar RV, Takenaka O, et al. SINE-R.C2 (a Homo sapiens specific retroposon) is homologous to CDNA from postmortem brain in schizophrenia and to two loci in the Xq21.3/Yp block linked to handedness and psychosis. Am J Med Genet. 1999;88(5):560e566. 67. Belshaw R, Dawson AL, Woolven-Allen J, Redding J, Burt A, Tristem M. Genomewide screening reveals high levels of insertional polymorphism in the human endogenous retrovirus family HERV-K(HML2): implications for present-day activity. J Virol. 2005;79(19):12507e12514.
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68. Frank O, Giehl M, Zheng C, Hehlmann R, Leib-Mosch C, Seifarth W. Human endogenous retrovirus expression profiles in samples from brains of patients with schizophrenia and bipolar disorders. J Virol. 2005;79(17):10890e10901. 69. Suarez BK, Duan J, Sanders AR, et al. Genomewide linkage scan of 409 European-ancestry and African American families with schizophrenia: suggestive evidence of linkage at 8p23.3-p21.2 and 11p13.1-q14.1 in the combined sample. Am J Hum Genet. 2006;78(2):315e333. 70. Otowa T, Tochigi M, Rogers M, Umekage T, Kato N, Sasaki T. Insertional polymorphism of endogenous retrovirus HERV-K115 in schizophrenia. Neurosci Lett. 2006;408(3):226e229. 71. Dickerson F, Rubalcaba E, Viscidi R, et al. Polymorphisms in human endogenous retrovirus K-18 and risk of type 2 diabetes in individuals with schizophrenia. Schizophr Res. 2008;104(1e3):121e126. 72. Nyegaard M, Demontis D, Thestrup BB, et al. No association of polymorphisms in human endogenous retrovirus K18 and CD48 with schizophrenia. Psychiatr Genet. 2012;22(3):146e148. 73. Gragnoli C, Reeves GM, Reazer J, Postolache TT. Dopamine-prolactin pathway potentially contributes to the schizophrenia and type 2 diabetes comorbidity. Transl Psychiatry. 2016;6:e785. 74. Foley DL, Mackinnon A, Morgan VA, et al. Common familial risk factors for schizophrenia and diabetes mellitus. Aust NZ J Psychiatry. 2016;50(5):488e494. 75. Perron H, Mekaoui L, Bernard C, Veas F, Stefas I, Leboyer M. Endogenous retrovirus type W GAG and envelope protein antigenemia in serum of schizophrenic patients. Biol Psychiatry. 2008;64(12): 1019e1023. 76. Hegyi H. GABBR1 has a HERV-W LTR in its regulatory regionea possible implication for schizophrenia. Biol Direct. 2013;8:5. 77. Huang W, Li S, Hu Y, et al. Implication of the env gene of the human endogenous retrovirus W family in the expression of BDNF and DRD3 and development of recent-onset schizophrenia. Schizophr Bull. 2011;37(5):988e1000. 78. Suntsova M, Gogvadze EV, Salozhin S, et al. Human-specific endogenous retroviral insert serves as an enhancer for the schizophrenia-linked gene PRODH. Proc Natl Acad Sci USA. 2013;110(48): 19472e19477. 79. Bundo M, Toyoshima M, Okada Y, et al. Increased l1 retrotransposition in the neuronal genome in schizophrenia. Neuron. 2014;81(2):306e313. 80. Canuti M, van Beveren NJM, Jazaeri Farsani SM, et al. Viral metagenomics in drug-naive, firstonset schizophrenia patients with prominent negative symptoms. Psychiatry Res. 2015;229(3): 678e684. 81. Weis S, Llenos IC, Sabunciyan S, et al. Reduced expression of human endogenous retrovirus (HERV)-W GAG protein in the cingulate gyrus and hippocampus in schizophrenia, bipolar disorder, and depression. J Neural Transm. 2007;114(5):645e655. 82. Diem O, Schaffner M, Seifarth W, Leib-Mosch C. Influence of antipsychotic drugs on human endogenous retrovirus (HERV) transcription in brain cells. PLoS One. 2012;7(1):e30054. 83. Balestrieri E, Arpino C, Matteucci C, et al. HERVs expression in autism spectrum disorders. PLoS One. 2012;7(11):e48831. 84. Aldinger KA, Plummer JT, Qiu S, Levitt P. SnapShot: genetics of autism. Neuron. 2011;72(2), 418e418.e411. 85. Salyakina D, Cukier HN, Lee JM, et al. Copy number variants in extended autism spectrum disorder families reveal candidates potentially involved in autism risk. PLoS One. 2011;6(10):e26049. 86. Balestrieri E, Cipriani C, Matteucci C, et al. Transcriptional activity of human endogenous retrovirus in Albanian children with autism spectrum disorders. N Microbiol. 2016;39(3):228e231. 87. Shpyleva S, Melnyk S, Pavliv O, Pogribny I, Jill James S. Overexpression of LINE-1 retrotransposons in autism brain. Mol Neurobiol. 2017. 88. Balestrieri E, Pitzianti M, Matteucci C, et al. Human endogenous retroviruses and ADHD. World J Biol Psychiatry. 2014;15(6):499e504. 89. D’Agati E, Pitzianti M, Balestrieri E, Matteucci C, Sinibaldi Vallebona P, Pasini A. First evidence of HERV-H transcriptional activity reduction after methylphenidate treatment in a young boy with ADHD. N Microbiol. 2016;39(3):237e239. 90. Engel ME, Hiebert SW. The enemy within: dormant retroviruses awaken. Nat Med. 2010;16(5): 517e518.
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The Role of Human Endogenous Retroviruses (HERVs) in the Pathologies of the Nervous System ~ ks1, 2, 3, Gea Ko ~ ks1, 3 Sulev Ko
€ Tartu, Estonia University of Tartu, Tartu, Estonia; 2Estonian University of Life Sciences, Tartu, Estonia; 3Prion OU,
1
INTRODUCTION The human genome contains almost half of its size, 45%, repetitive retroelements or transposable elements (TEs). TEs are derived from ancient viruses and the result of viral genome integration that occurred a long time ago. TEs can be divided into two major classes (Table 22.1): transposons (3% of the genome) and retrotransposons or retroelements (42% of the genome).1 Retroelements are divided into long terminal repeat (LTR) and non-LTR elements. Non-LTR elements are the most abundant and are divided into short interspersed elements (SINE) and long interspersed elements (LINE). SINEs are Alu and MIR repeats without independent amplification and protein coding capacity, and they are also considered nonautonomous TEs.2 Alu repeats are the most common elements in the human genome, and they make up around 15% of the human genome.3 LINE elements contain autonomous L1 and L2 elements (LINE repeats). The LTR class consists of elements of the retroviral origin, and this class is more heterogenous (Table 22.2). One group of LTR elements are human endogenous retroviruses (HERVs), and they occupy around 8% of our genome.4 These viruses are derived from ancient infections of retroviruses that were reverse transcribed
Table 22.1 Overview of the transposable elements (TEs) in the human genome2 TEs (45% of genome) DNA-transposons 2.8%
Retrotransposons or retroelements 42.2%
Non-LTR, 33.9%
LTR, 8.3%
SINE (Alu, MIR) LINE (L1, L2) Pseudogenes Class I ERV þ MER4 Class II ERV Class II ERV Others (MST, MLT)
Percentage of the elements in the genome is given in brackets.
Molecular-Genetic and Statistical Techniques for Behavioral and Neural Research ISBN 978-0-12-804078-2, https://doi.org/10.1016/B978-0-12-804078-2.00022-2
© 2018 Elsevier Inc. All rights reserved.
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Table 22.2 Characteristics of subgroups of LTR retroelements2 Element Characteristics
Class I ERV Class II ERV Class III ERV MER4 MST MLT
Similar to type C or g retroviruses Similar to type B or b retroviruses Also called ERV-L, limited similarity to spumaviruses Nonautonomous class I ERV Named after restriction enzyme site MstII, MaLR Mammalian LTR transposon, MaLR
and integrated into the genome of ancestral host animals. These viral genomes were fixed and inherited tens of millions of years ago.1,5 Almost all of the HERVs have lost their infectivity. However, presence of the env gene in HERVs confers their potential to spread between cells. LTR class also contains HERV-like repeat elements that are derived from HERVs (SINE-R) and contain one-copy of LTR element. Finally, there are sequences with LTR-like features but without any homologous proviral structure. Repbase is a central curated database for repetitive elements in which more than 200 families of LTR-containing retroelements are defined and grouped into six superfamilies.6 These families are class I ERVs (similar to type C retroviruses), class II ERV (similar to type B retroviruses), class III ERV (distantly similar to spuma retroviruses), MER4 (nonautonomous class I-related ERVs), MST (common MstII restriction site), and MLT (mammalian LTR transposon). MST and MLT are both part of the larger nonautonomous mammalian apparent LTR retrotransposon (MaLR) superfamily.2 TEs form the basis of the “junk DNA” that has been without any understandable function in the recent past. Generally, it has been suggested that about half of our genome is derived from the TEs, and they do not perform any specific function. New analyses have estimated that more than two-thirds of our genome has resulted from incorporation of viral DNA.7 However, during evolution, our genome has “domesticated” several TEs to fulfill different physiological functions, like V(D)J recombination in the immune system or cellular fusion during placental development and even cellular protection against retroviral infections.8e10 Taken together, it is obvious that many TEs have had substantial impact on shaping our genomes and changing our phenotypes. Analysis of human genes has found that TEs are overrepresented in the mRNAs of rapidly evolving mammalian genes and potentially have gene regulation function.11 On the other hand, HERVs and other LTRs are normally suppressed, but they can potentially act as transcriptional regulators and this way activate malignant disorders or autoimmune responses.5,12 The importance of HERVs and LTRs in gene regulatory networks has been verified in several studies.13 The role of HERVs has been shown in embryonic development, in immune regulation, and in the pathogenesis of complex disorders.14e16 Most remarkably, HERVs are involved in different diseases including melanoma,
The Role of Human Endogenous Retroviruses (HERVs) in the Pathologies of the Nervous System
amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), schizophrenia (SCH), bipolar disorder, autism spectrum disorder (ASD), and attention deficit hyperactivity disorder (ADHD).17e19 The present chapter gives an overview of the role of HERVs in the neurological and psychiatric disorders.
HUMAN ENDOGENOUS RETROVIRUSES IN NEUROLOGICAL DISEASES To appreciate the potential impact of repetitive elements on the genome function, it is important to mention that roughly one-quarter of human promoter regions contain TEs.20 TEs produce negative impact by causing mutations in genomes, but it is also recognized that TEs are involved in the regulation of gene expression.21,22 In the latter case, TEs should have significant impact in the pathogenesis of diseases. The first study where TEs were described as causal agents for human diseases was published in 1988. In this paper, two separate insertions of L1s were described to cause hemophilia A by disrupting the factor VII gene.23 Thereafter, TEs were found to cause muscular dystrophy, neurofibromatosis, and colon cancer.24e26 LINE-1-induced insertional mutagenesis in the myc locus has been shown to cause breast cancer.25 In a more recent study, aberrantly activated LTR of the MaLR family (THE1B) was found to induce the expression of CSF1R in Hodgkin lymphoma.27 Therefore, TEs can contribute to the pathogenesis of diseases directly via mutagenesis or by the regulation of the gene expression networks. From this perspective, two of the most well-studied neurological disorders are MS and ALS. Summarized information is given in Table 22.3.
Multiple Sclerosis MS is associated with demyelination in the CNS leading to disturbed sensory, motor, autonomic, and cognitive functions and fatigue. The etiology of MS remains debated, but pathogenesis involves chronic inflammation and autoimmune mechanisms.28 The role of viruses on the pathogenesis of this disease has been suspected for a long time.28,29 The first discovery of reverse transcriptase activity of the cell line isolated Table 22.3 Neurological disorders with the involvement of transposable elements (TEs) and type of evidence Disorder TEs Evidence
Multiple sclerosis
Amyotrophic lateral sclerosis
HERV-W (MSRV) HERV-H (Fc1) HERV-K (K13, K18, K113) HERV-K HERV-W
Induction of immune response, expression, experimental Association study Association study, expression Expression, experimental Controversial evidence
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from cerebrospinal fluid (CSF) of a patient with MS indicated that this activity was not related to the common “suspects” HTLV-I or HIV-1 and HIV-2.30 Later, the viral particles were isolated and designated as “MS-associated” retrovirus (MSRV).31 Additional molecular characterization of the MSRV identified its similarity to the endogenous retroviral sequence ERV9.32 MSRV was found to be expressed not in all, but in many MS patients, and it was found in noncellular RNA from the plasma and CSF of the patients.32 The MSRV-related transcript contains gag, pol, and env genes, and it is expressed in the placenta. This new HERV family was named HERV-W.33 Subsequently, the role of HERV-W in MS was repeatedly confirmed in different independent studies.34e37 More importantly, HERV-W (MSRV) is not only associated with the presence of MS, but in the CSF, it correlates with the clinical progression of the disease.35,36,38 Presence of the HERV-W sequences in the CSF is a marker for rapid disease progression.36e38 The molecular mechanism of the infectivity of HERV-W has been studied for more than a decade. HERV-W has been shown to activate innate immunity with subsequent release of proinflammatory cytokines by using a Toll-like receptor 4 (TLR4) agonist effect.39 TLR4-activated macrophages introduce T-lymphocytes that are required to turn HERV-W envelope (ENV) protein into superantigen, and to ignite the downstream immune cascade leading to production of the IL-1b, IL-6, or TNF-a.39 In addition, dendritic cells are activated and Th1-like responses are developed. Both TLR4 and CD14 receptors are involved in the proinflammatory effects of ENV protein.39 The pathogenicity of HERV-W was further analyzed in relation to the T-lymphocyte-mediated immunopathology.40 HERV-W particles induced polyclonal T-lymphocyte response with a bias in the Vb16 chain usage as a surface receptor. This means that HERV-W triggers an abnormal immune response that is characteristic of superantigens.40 In addition to the peripheral immune system, further studies indicated the role of HERV-W in the differentiation of the oligodendroglial precursor cells. More precisely, HERV-W particles induced the proinflammatory cytokines and nitric oxide synthase in oligodendroglial precursors cells expressing TLR4.41 In addition to the in vitro studies, animal experiments also confirmed the impact of HERV-W in the induction of immune response.40 More precisely, mice immunized with the HERV-W showed clear experimental allergic encephalomyelitis.42 In vivo experiments confirmed that HERV-W is a highly potent agonist for the TLR4, and this interaction leads to the proinflammatory activity and impairs differentiation of the oligodendroglial precursor cells to mature myelinating cells.43 Taken together, the role of HERV-W in pathophysiology of MS has been demonstrated in clinical observational studies, as well as in vitro and in vivo laboratory experimental studies. Based on these findings, a humanized antibody, GNbAC1, against HERV-W ENV extracellular domain was developed to treat patients with MS.43e45 This antibody has been successful in preclinical and clinical studies, showing low toxicity and good
The Role of Human Endogenous Retroviruses (HERVs) in the Pathologies of the Nervous System
tolerability.45 Preclinical studies indicated no safety risks, so Phase I, the first-in-human randomized, double-blind, placebo-controlled, dose-escalation study was performed.44 In 33 male subjects, only minor and nonspecific adverse effects were recorded, and this allowed launching of the Phase II program.44 A Phase 2a study was performed as a single-blind, placebo-controlled, randomized study in 10 MS patients.45e47 All patients tolerated the GNbAC1, and treatment significantly reduced transcription of the MSRV. Moreover, a recent study indicated that HERV-W is also involved in chronic inflammatory demyelinating polyradiculoneuropathy and that it induces inflammatory damage of Schwann cells.48 The pathogenic effect of HERV-W was inhibited by the GNbAC1, suggesting a broad utility for the antibody.48 The story of HERV-W is an elegant example of how discovery may progress from the identification of a potentially infectious agent to the development of a promising treatment option for a disease that has been without any possible cure. This is probably the first endogenous retrovirus that has been used as a specific therapeutic target for the development of antibodies. In addition to the HERV-W, genetic association studies have found increased odds ratio in the locus for the HERV-Fc1 gene.49 HERV-Fc1 is closely related to the HERV-H, and more distantly to the HERV-W and HERV-K.49 The association of HERV-Fc1 with MS was repeated in another independent study.50 Moreover, highly significant genetic interaction between HERV-Fc1 and HERV-K13 loci was described.50 In one study using postmortem tissues, increased mRNA level of HERV-K and HERV-W was found.51 Taken together, the case of MS excellently exemplifies the pathogenetic potential and impact of HERVs in our genomes. While the involvement of HERV-W in MS is clearly demonstrated, the role of HERVs in other diseases needs further analysis.
Amyotrophic Lateral Sclerosis ALS is a progressive and fatal neurodegenerative disease that is characterized by the loss of upper (in the cerebral cortex or brain stem) and lower (in the spinal cord or cranial nerves) motor neurons, leading to increasing weakness.52 ALS is sporadic disease with the prevalence of five in 100,000, and viral involvement in its aetiology has been suspected for many years, mainly on the basis of similarities with poliomyelitis.53 The cause of ALS is not known, but the role of HERVs has been suggested recently. The first pioneering studies analyzed peripheral blood sera from patients with motor neuron diseases. In one study, sera from the 56 patients were analyzed for reverse transcriptase activity by using product-enhanced reverse transcriptase assay (PERT).53 Indeed, in 33 patients (59%) cell-free reverse transcriptase activity was detected compared to only three of the controls.53 This activity was not caused by the HIV-1, HIV-2, HTLV-I, HTLV-II, HRV-5, or human foamy virus. In another study, 30 patients with sporadic ALS were analyzed for reverse transcriptase activity
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in blood sera.54 The authors applied a similar serum purification protocol combined with a sensitive PERT assay. Fourteen patients were PERT positive (47%), a statistically significant difference compared to the genetically related and unrelated controls.54 Increased RT activity on sera of HIV-negative ALS patients was further confirmed by an additional study.55 In this study, RT activity was measured also in CSF, but the difference (39% vs. 19%) was not statistically significant.55 All these early studies used biochemical detection of RT activity, so the identity of retroviruses was not discoverable. Application of RNA analysis with RT-PCR subsequently answered this question and identified increased expression of the HERV-K in the autopsy brains.56 Authors also found that HERV-K is expressed in cortical neurons of the ALS patients. All these studies were descriptive and were designed to show association between RT activity and ALS. A recent study identified causative connection between HERV-K and ALS.18 First, authors found increased expression of the HERV-K in cortical and spinal neurons of ALS patients. Afterward, they showed causal relationship between the expression of HERV-K and neuronal pathologies in cellular models as well as in mice using transgenic technology.18 Transgenic mice expressing the HERV-K developed progressive motor dysfunction accompanied by selective loss of volume of the motor cortex and decreased synaptic activity. Transgenic mice had injuries in the anterior horn of the spinal cord, and this was associated with muscle atrophy.18 Moreover, authors identified that expression of HERV-K is regulated by TDP-43, a protein that is overexpressed in ALS and is known to form cytoplasmic aggregates. HERV-K has five binding sites for the TDP-43, and as TDP-43 seems to be involved in other neurodegenerative disorders (e.g., frontotemporal dementia), the potential pathophysiological impact of HERV-K is likely not limited to ALS.18
HUMAN ENDOGENOUS RETROVIRUSES IN PSYCHIATRIC DISEASES Out of all psychiatric diseases, SCH and bipolar disease are the most frequently studied disorders with regard to endogenous retroviruses. But some information is also available on autism. A general overview is given in Table 22.4.
Schizophrenia In the past, it has been suggested that exogenous viruses and retroviral infections are responsible for SCH, but there were no studies proposing endogenous retroviruses to play any role. Some early studies suggested that one of the pathogenic factors for SCH could be retroviral infection, or that the disease is induced by quantal changes in “virogene” or “schizovirus” within our genome.57e59 These early studies proposed that endogenous retroviral integration to the human genome near the gene critical for SCH leads to the disruption of this gene and development of disease.57 These hypotheses were too complex to challenge during their time, but recent developments in molecular
The Role of Human Endogenous Retroviruses (HERVs) in the Pathologies of the Nervous System
Table 22.4 Psychiatric disorders with the involvement of transposable elements (TEs) and type of evidence Disorder TEs Evidence
Schizophrenia
Bipolar disorder
Autism
ADHD
SZRV-1 (schizovirus, HERV-W, MSRV) SZRV-2 HERV-K (K115) L1 elements HERV-I (IP-T47D) HERV-9 (Seq63) HERV-L NMWV7 HERV-K (K10, KC4) HERV-W HERV-Fb HERV-H HERV-K HERV-W L1 HERV-H
DNA cloning, expression Differential cloning Variations, expression More copies Expression Expression Expression Expression Expression, treatment Expression, treatment Expression, treatment Expression Expression Expression Expression Expression, treatment
technologies allowed performing better experimental testing. One of the first studies used representational difference analysis (RDA) of blood DNA from three pairs (two male and one female) of monozygotic twins discordant for SCH.60 Each affected member met the DSM-III-R criteria for SCH. The unaffected twins had no psychotic or affective illness. For the independent testing and analysis of the isolated DNA fragments, DNA from five additional pairs of affected individuals and their unrelated matched controls was extracted. This study gave very complex results. Authors identified 128 sequences in the genomes of the affected persons that were grouped in four contigs.60 The first contig was designated SZRV-1, and this sequence was similar to the sequence found to be implicated in MS (MSRV, ERV-9, or HERV-W).32 The identified sequence (HERV-W) was later found to be differentially expressed in cell-free CSF of individuals with SCH.61 Analysis of CSFs and postmortem brain tissue from the SCH patients revealed significant increase of transcription of HERV-W family of endogenous retroviruses.61,62 The results from the second RDA of DNA revealed a sequence named SZRV-2.63 SZRV-2 is confined to 12q13, and this site was also shared with the SZRV1. Southern blot analysis indicated that this region contains tandem repeats and is known to harbor other viral elements.63 Interestingly, this locus contains several genes potentially important for the pathogenesis of SCH, thus supporting the original hypothesis of retroviral integration near the “SCH gene.”63 Further supporting the initial findings, HERV-W was also found in the plasma of the individuals with recent-onset SCH.64
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Another study analyzed sera from 58 SCH patients and identified the ERV9 family retrovirus in 20 patient samples.65 No ERV9 was detected in control persons. This result was further confirmed with Western blot analysis and also by positive immunoreactivity in the sera of patients.65 In addition to the HERV-W, the expression of HERV-K has been identified in the cDNA libraries of the postmortem brains of the SCH patients.66 HERV-K belongs to an interesting group of HERVs, because it is a full-length provirus, and is probably capable for reinfections.67 Transcriptional activity of HERV-K has been found in the brains of SCH and bipolar patients.68 HERV-K115 is mapped to the 8p23.1 region, which is related to SCH susceptibility.69 A case-control study did not find any significant associations between SCH patients and variations in the HERK-K115 gene.70 However, it was concluded that variations in the HERV-K115 could modify the onset of the disease.70 Variations in the HERV-K18 were found to be associated with type 2 diabetes (T2D) in SCH patients.71 More precisely, the haplotype defined by two polymorphisms (7086 and 8146 C/T) in the envelope region was highly associated with T2D in SCH patients. The established odds ratio was 9.0 (95% CI 2.3e34.7, P < .001) after adjustment for age, gender, race, and current therapy with clozapine.71 This finding was partially confirmed in a Danish population with 750 cases.72 They did not find any association between HERV-K18 and SCH or T2D in patients with SCH. However, in younger SCH patients, there was a trend for association with T2D, a finding that was in agreement with previous results.71,72 Genetic interaction between SCH and T2D based on HERVs is plausible, as several studies have found comorbidity between SCH and T2D.73,74 Peripheral activation of endogenous viruses has also been studied in several investigations. For instance, HERV-W antigenemia was described in SCH patients.75 In addition, activation of HERV-W sequences has been analyzed by several additional research papers.76,77 In the plasma of 42 out of 118 individuals suffering from recent-onset SCH, activation of expression of the sequences homologous to HERV-W env gene was found. Overexpression of the HERV-W in glioma cell line was found to induce upregulation of brain-derived neurotrophic factor, neurotrophic tyrosine kinase receptors type 2 (NTRK2 or TrkB), and dopamine receptor D3 genes.77 Authors concluded that transcriptional activation of HERV-W causes upregulation of the genes implicated in SCH. Similarly, HERV-W LTR has been described in the regulatory region of the GABA receptor B1 gene (GABBR1).76 Similar insertion of the human ERV in the enhancer has been shown for the PRODH gene.78 The PRODH gene encodes proline dehydrogenase, and it is involved in the synthesis of neurotransmitters. Hypomethylation of this enhancer induced higher expression of the PRODH gene, and this could have an impact in brain development.78 While all these previous studies were more focused on specific HERVs or a specific insertion locus, recent technologies enable a much wider approach. Comprehensive
The Role of Human Endogenous Retroviruses (HERVs) in the Pathologies of the Nervous System
analysis of L1 retrotransposition was performed in neuronal genome in postmortem samples from different mental diseases.79 Clear increase in the L1 retrotransposition was found in SCH. In major depression and bipolar disorder samples, the increased L1 content was borderline in terms of statistical significance.79 SCH brains showed robust differences, and these differences were confirmed in an independent sample of additional 34 brains. Authors used whole-genome sequencing to identify genomic regions for the L1 insertions, and they found that it localized preferentially to synapse- and SCH-related genes. This study also showed that hyperactive L1 retrotransposition can be triggered after immune activation, so environmental factors can contribute to the susceptibility of SCH.79 In one metagenomics study, 23 drug-naïve, first-onset SCH patients were analyzed. The authors did not find any evidence for the activation of endogenous retroviruses in the sera of these patients.80 Taken together, HERV-W and HERV-K seem to be the most common HERVs found to be associated with SCH.
Bipolar Disorder Bipolar disorder is studied less intensively compared to the previously described diseases. Samples from bipolar disorder patients are usually studied along with SCH samples. In one large-scale study, 50 members of 20 HERV families were analyzed in 215 brain samples.68 The authors of this study found HERV-IP-T47D (HERV-I family) to be reduced in bipolar samples compared to those of SCH. Seq63 from the ERV9 family was reduced in bipolar samples compared to controls. HERV-K10 from the HML-2 family was upregulated in both SCH and bipolar samples compared to the control. And finally, NMWV7 from the HML-7 subclass and HERV-L were downregulated compared to controls and SCH.68 Interestingly, some HERVs had similar expression profiles between diseases, but other HERVs exhibited disease-specific idiosyncratic profiles. In another study using immunohistochemistry, significant downregulation of the HERV-W in different brain areas of bipolar patients was described.81 This reduced expression in immunohistochemical staining could indicate neuronal loss in the brains of bipolar patients, potentially resulting from altered expression of HERV during the illness.81 However, this hypothesis needs further clarification. Some studies have analyzed the effect of treatment on the expression of HERVs. Valproic acid (VLP) is one of the most commonly used drugs for bipolar disorder. The effect of VLP on the expression of different HERV families in different human cell lines (glioblastoma, neuroblastoma, neural stem cell line) has been studied.82 VLP dose-dependently upregulated transcription of several HERVs from different subclasses. VLP upregulated HERVs from class I (HERV-E, HERV-H, HERV-W, ERV9, and HERV-F) and from class II (HML-3, HML-4, HML-6, HML-9, HML-10). The stronger influence of VLP was established for HERV-Fb, ERV9, Seq26 (HML-3),
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and HERV-KC4/HML-10.82 In postmortem brain samples, HML-2 and ERV9 were upregulated in patients with bipolar disease without treatment compared to the control samples, and VLP treatment did not affect this result.82 In summary, existing evidence suggests that HERVs do play roles in bipolar disorder, and traditional pharmacotherapies may affect expression of HERVs.
Other Psychiatric Diseases Autism or ASD is a complex disorder associated with altered brain development. ASD is clinically variable. However, an often observed feature is impaired social interaction with peers, impaired communication, and stereotypical and rigid patterns of behavior.83 The prevalence of ASD is increasing, and the increasing societal burden necessitates more research.84 Despite decades of studies, the etiology of ASD is still unknown. Accumulating evidence suggests that ASD is a multifactorial disease with a strong genetic basis.84 Twin studies have found high heritability (more than 90%). Nevertheless, the exact genetic loci and mechanisms underlying the disease have still not been identified. As a new development, an increasing number of studies have described positive association between copy number variants (CNVs) and ASD.85 In the light of the emerging role of CNVs, analysis of TEs in ASD becomes feasible. RNA from peripheral blood mononuclear cells (PBMCs) of 28 Caucasian children was analyzed for the presence of retroviral RNA from four HERV families (HERV-E, HERV-H, HERV-K, HERV-W).83 The expression of HERV-H was higher in ASD patients, and expression of HERV-W was lower in ASD patients than in controls. Expression on HERV-K was similar, and the expression of HERV-E was absent.83 In vitro stimulation of the PBMCs from the ASD patients resulted in higher upregulation of the HERV-H expression compared to controls.83 Moreover, authors described negative correlation between the HERV-H expression and the Psychoeducational Profile-3 score.83 In an additional analysis performed by the same group, an independent sample of Albanian ASD patients was studied.86 HERV-W was found upregulated, while HERV-K and HERV-W were downregulated in ASD patients compared to controls.86 Elevated HERV-W expression in autistic children suggests that infection or inflammation during pregnancy could be an etiological risk factor for the development of ASD.86 Another recent study was performed on frozen postmortem cerebellar cortex samples from 13 autistic patients and 13 control individuals.87 Expression analysis of L1 ORF1 and ORF2 was performed with QRT-PCR. L1 expression was found to be significantly increased in the brains of autistic patients, which was inversely correlated with GSH/GSSG ratio.87 Positive correlation between L1 expression and FOXO3 was also established.87 However, the impact of L1 expression to neuronal
The Role of Human Endogenous Retroviruses (HERVs) in the Pathologies of the Nervous System
CNVs and genomic instability requires additional research with innovative genomic technologies. Similarly to ASD, HERVs may impact the development of ADHD. The expression of retroviral RNAs of three HERV families (H, K, and W) were analyzed in 30 patients with ADHD and 30 healthy controls using PBMCs.88 The only significantly increased expression found in ADHD patients was for HERV-H. Expression of HERV-K and HERV-W remained unchanged. ADHD has a complex etiology, and the authors concluded that HERVs may present a possible link between the environmental, biologic, and genetic factors contributing to the development of this disease. In another study with a single ADHD patient, HERV-H expression in PBMCs was evaluated before and after treatment with methylphenidate (MPH).89 Compared to healthy controls, HERV-H was highly and significantly overexpressed in the patients before treatment, but after 6 months of treatment with MPH the expression of HERV-H was normalized to the level of that of controls.89 This study gives support to the idea that expression changes in HERVs can be pathogenic, and treatments can modify HERV expression and relieve the symptoms.
CONCLUSIONS Almost half of the human genome contains TEs, and the impact of these elements on physiology and pathophysiology is not understood yet. However, growing data suggest that TEs play significant roles in a number of CNS disorders. The best studied example is MS, where a clear role of HERV-W in disease development has been demonstrated. Moreover, treatment with antibodies against HERV-W aborts the destruction of neurons and stops progression of the disease.45 Another elegant example is the involvement of HERV-K in ALS.18 This chapter summarized existing evidence on how HERVs and other TEs could impact our health, and how understanding this impact may help us develop treatment either by development of new drugs or by repurposing already existing drugs to target the activity of endogenous retroviruses. Taking together accumulated existing evidence, I suggest that HERV elements represent a missing link between environmental and genetic factors underlying numerous pathogenic processes of complex human diseases. Notably, for the majority of complex diseases, we do not have an obvious and validated etiological explanation. Activation of dormant retroviruses in our genome is “the enemy within.”90 Endogenous retroviruses constitute a new class of pathogens different from classical environmental and infectious microbes, arising from the host’s genome itself.90 Understanding their function and effects thus may open entire new research avenues and therapeutic applications.
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