New Insights into Endogenous Retrovirus-K Transcripts in Amyotrophic Lateral Sclerosis.

Retroviral reverse transcriptase activity and the increased expression of human endogenous retroviruses (HERVs) are associated with amyotrophic lateral sclerosis (ALS). We were interested in confirming HERVK overexpression in the ALS brain, its use as an accessory diagnostic marker for ALS, and its potential interplay with neuroinflammation. Using qPCR to analyze HERVK expression in peripheral blood mononuclear cells (PBMCs) and in postmortem brain samples from ALS patients, no significant differences were observed between patients and control subjects. By contrast, we report alterations in the expression patterns of specific HERVK copies, especially in the brainstem. Out of 27 HERVK copies sampled, the relative expression of 17 loci was >1.2-fold changed in samples from ALS patients. In particular, the relative expression of two HERVK copies (Chr3-3 and Chr3-5) was significantly different in brainstem samples from ALS patients compared with controls. Further qPCR analysis of inflammation markers in brain samples revealed a significant increase in NLRP3 levels, while TNFA, IL6, and GZMB showed slight decreases. We cannot confirm global HERVK overexpression in ALS, but we can report the ALS-specific overexpression of selected HERVK copies in the ALS brain. Our data are compatible with the requirement for better patient stratification and support the potential importance of particular HERVK copies in ALS.

Placental Infection Associated with SARS-CoV-2 Wildtype Variant and Variants of Concern.

The original SARS-CoV-2 lineages have been replaced by successive variants of concern (VOCs) over time. The aim of this study was to perform an assessment of the placental infection by SARS-CoV-2 according to the predominant variant at the moment of COVID-19 diagnosis. This was a prospective study of SARS-CoV-2-positive pregnant women between March 2020 and March 2022. The population was divided into pregnancies affected by COVID-19 disease during 2020 (Pre-VOC group) and pregnancies affected after December 2020 by SARS-CoV-2 variants of concern (VOC group). The presence of virus was assessed by RT-PCR, and the viral variant was determined by whole genome sequencing. A total of 104 placentas were examined, among which 54 cases belonged to the Pre-VOC group and 50 cases belonged to the VOC group. Sixteen positive placental RT-PCR tests for SARS-CoV-2 were reported. The NGS analysis confirmed the SARS-CoV-2 lineage in placenta tissue. All samples corresponded to the Pre-VOC group, whereas no placental presence of SARS-CoV-2 was detected in the VOC group (16, 29.6% vs. 0, 0.0% p = 0.000). Preterm birth (9, 16.7% vs. 2, 4%; p = 0.036) and hypertensive disorders of pregnancy (14, 25.9% vs. 3, 6%; p = 0.003) were more frequent in the Pre-VOC group than in the VOC group. Finally, the VOC group was composed of 23 unvaccinated and 27 vaccinated pregnant women; no differences were observed in the sub-analysis focused on vaccination status. In summary, SARS-CoV-2-positive placentas were observed only in pregnancies infected by SARS-CoV-2 wildtype. Thus, placental SARS-CoV-2 presence could be influenced by SARS-CoV-2 variants, infection timing, or vaccination status. According to our data, the current risk of SARS-CoV-2 placental infection after maternal COVID disease during pregnancy should be updated.

SARS-CoV-2 awakens ancient retroviral genes and the expression of proinflammatory HERV-W envelope protein in COVID-19 patients.

Patients with COVID-19 may develop abnormal inflammatory response, followed in some cases by severe disease and long-lasting syndromes. We show here that in vitro exposure to SARS-CoV-2 activates the expression of the human endogenous retrovirus (HERV) HERV-W proinflammatory envelope protein (ENV) in peripheral blood mononuclear cells from a subset of healthy donors, in ACE2 receptor and infection-independent manner. Plasma and/or sera of 221 COVID-19 patients from different cohorts, infected with successive SARS-CoV-2 variants including the Omicron, had detectable HERV-W ENV, which correlated with ENV expression in T lymphocytes and peaked with the disease severity. HERV-W ENV was also found in postmortem tissues of lungs, heart, gastrointestinal tract, brain olfactory bulb, and nasal mucosa from COVID-19 patients. Altogether, these results demonstrate that SARS-CoV-2 could induce HERV-W envelope protein expression and suggest its involvement in the immunopathogenesis of certain COVID-19-associated syndromes and thereby its relevance in the development of personalized treatment of patients.

Frequent Placental SARS-CoV-2 in Patients with COVID-19-Associated Hypertensive Disorders of Pregnancy.

INTRODUCTION: Studies described an increased frequency of hypertensive disorders of pregnancy (HDP) after a COVID-19 episode. There is limited evidence about SARS-CoV-2 viral load in placenta. This study aimed to investigate the relationship between SARS-CoV-2 viral load in the placenta and clinical development of HDP after COVID-19 throughout different periods of gestation. METHODS: This is a case-control study in women with and without gestational hypertensive disorders after SARS-CoV-2 infection diagnosed by RT-PCR during pregnancy. Patients were matched by gestational age at the moment of COVID-19 diagnosis. We performed an analysis of SARS-CoV-2 RNA levels in placenta. RESULTS: A total of 28 women were enrolled. Sixteen patients were diagnosed with COVID-19 during the third trimester and the remaining 12 patients in the other trimesters. Ten placentas (35.7%) were positive for SARS-CoV-2, 9 of them (9/14, 64.3%) belonged to the HDP group versus 1 (1/14, 7.2%) in the control group (p = 0.009). Those cases with the highest loads of viral RNA developed severe preeclampsia (PE). CONCLUSION: Among women diagnosed with COVID-19 during pregnancy, the presence of SARS-CoV-2 in the placenta was more frequent among women suffering from PE or gestational hypertension. Furthermore, the most severe cases of HDP were associated with high placental viral load, not necessarily associated with a positive nasopharyngeal RT-PCR at delivery. Our data suggest that SARS-CoV-2 infection during pregnancy could trigger gestational hypertensive disorders through persistent placental infection and resulting placental damage.

YY2 in Mouse Preimplantation Embryos and in Embryonic Stem Cells.

Yin Yang 2 encodes a mammalian-specific transcription factor (YY2) that shares high homology in the zinc finger region with both YY1 and REX1/ZFP42, encoded by the Yin Yang 1 and Reduced Expression Protein 1/Zinc Finger Protein 42 gene, respectively. In contrast to the well-established roles of the latter two in gene regulation, X chromosome inactivation and binding to specific transposable elements (TEs), much less is known about YY2, and its presence during mouse preimplantation development has not been described. As it has been reported that mouse embryonic stem cells (mESC) cannot be propagated in the absence of Yy2, the mechanistic understanding of how Yy2 contributes to mESC maintenance remains only very partially characterized. We describe Yy2 expression studies using RT-PCR and staining with a high-affinity polyclonal serum in mouse embryos and mESC. Although YY2 is expressed during preimplantation development, its presence appears dispensable for developmental progress in vitro until formation of the blastocyst. Attenuation of Yy2 levels failed to alter either Zscan4 levels in two-cell embryos or IAP and MERVL levels at later preimplantation stages. In contrast to previous claims that constitutively expressed shRNA against Yy2 in mESC prohibited the propagation of mESC in culture, we obtained colonies generated from mESC with attenuated Yy2 levels. Concomitant with a decreased number of undifferentiated colonies, Yy2-depleted mESC expressed higher levels of Zscan4 but no differences in the expression of TEs or other pluripotency markers including Sox2, Oct4, Nanog and Esrrb were observed. These results confirm the contribution of Yy2 to the maintenance of mouse embryonic stem cells and show the preimplantation expression of YY2. These functions are discussed in relation to mammalian-specific functions of YY1 and REX1.

Altered DNA methylation in human placenta after (suspected) preterm labor.

Aim: The aim of this study was to determine if alterations in DNA methylation in the human placenta would support suspected preterm labor as a pathologic insult associated with diminished placental health. Methods: We evaluated placental DNA methylation at seven loci differentially methylated in placental pathologies using targeted bisulfite sequencing, in placentas associated with preterm labor (term birth after suspected preterm labor [n = 15] and preterm birth [n = 15]), and controls (n = 15). Results: DNA methylation levels at the NCAM1 and PLAGL1 loci in placentas associated with preterm labor did differ significantly (p < 0.05) from controls. Discussion: Specific alterations in methylation patterns indicative of an unfavourable placental environment are associated with preterm labor per se and not restricted to preterm birth.

Study protocol for a randomised controlled trial: treatment of early intrauterine growth restriction with low molecular weight heparin (TRACIP).

INTRODUCTION: The incidence of intrauterine growth restriction (IUGR) is estimated at about 3% of pregnancies, and it is associated with 30% of all perinatal mortality and severe morbidity with adverse neurodevelopmental and cardiovascular health consequences in adult life. Early onset IUGR represents 20%-30% of all cases and is highly associated with severe placental insufficiency. The existing evidence suggests that low molecular weight heparin (LMWH) has effects beyond its antithrombotic action, improving placental microvessel structure and function of pregnant women with vascular obstetric complications by normalising proangiogenic and antiapoptotic protein levels, cytokines and inflammatory factors. The objective of our study is to demonstrate the effectiveness of LMWH in prolonging gestation in pregnancies with early-onset IUGR. METHODS AND ANALYSIS: This is a multicentre, triple-blind, parallel-arm randomised clinical trial. Singleton pregnancies qualifying for early (20-32 weeks at diagnosis) placental IUGR (according to Delphi criteria) will be randomised to subcutaneous treatment with bemiparin 3500 IU/0.2 mL/day or placebo from inclusion at diagnosis to the time of delivery. Analyses will be based on originally assigned groups (intention-to-treat). The primary objective will be analysed by comparing gestational age and prolongation of pregnancy (days) in each group with Student's t-tests for independent samples and by comparing Kaplan-Maier survival curves (from inclusion to delivery, log-rank test). A linear regression model for gestational age at birth will consider the following covariates: gestational age at inclusion (continuous) and pre-eclampsia (binary). ETHICS AND DISSEMINATION: The study will be conducted in accordance with the principles of Good Clinical Practice. This study was approved by the Clinical Research Ethics Committee (CEIC) of Sant Joan de Déu Hospital, on 13 July 2017. The trial is registered in the public registry www.clinicaltrial.gov. according to Science Law 14/2011, and the results will be published in an open access journal. TRIAL REGISTRATION NUMBER: NCT03324139; Pre-results.

In Vivo Chromatin Targets of the Transcription Factor Yin Yang 2 in Trophoblast Stem Cells.

BACKGROUND: Yin Yang 2 (YY2) is a zinc finger protein closely related to the well-characterized Yin Yang 1 (YY1). YY1 is a DNA-binding transcription factor, with defined functions in multiple developmental processes, such as implantation, cell differentiation, X inactivation, imprinting and organogenesis. Yy2 has been treated as a largely immaterial duplication of Yy1, as they share high homology in the Zinc Finger-region and similar if not identical in vitro binding sites. In contrast to these similarities, gene expression alterations in HeLa cells with attenuated levels of either Yy1 or Yy2 were to some extent gene-specific. Moreover, the chromatin binding sites for YY2, except for its association with transposable retroviral elements (RE) and Endogenous Retroviral Elements (ERVs), remain to be identified. As a first step towards defining potential Yy2 functions matching or complementary to Yy1, we considered in vivo DNA binding sites of YY2 in trophoblast stem (TS) cells. RESULTS: We report the presence of YY2 protein in mouse-derived embryonic stem (ES) and TS cell lines. Following up on our previous report on ERV binding by YY2 in TS cells, we investigated the tissue-specificity of REX1 and YY2 binding and confirm binding to RE/ERV targets in both ES cells and TS cells. Because of the higher levels of expression, we chose TS cells to understand the role of Yy2 in gene and chromatin regulation. We used in vivo YY2 association as a measure to identify potential target genes. Sequencing of chromatin obtained in chromatin-immunoprecipitation (ChIP) assays carried out with αYY2 serum allowed us to identify a limited number of chromatin targets for YY2. Some putative binding sites were validated in regular ChIP assays and gene expression of genes nearby was altered in the absence of Yy2. CONCLUSIONS: YY2 binding to ERVs is not confined to TS cells. In vivo binding sites share the presence of a consensus binding motif. Selected sites were uniquely bound by YY2 as opposed to YY1, suggesting that YY2 exerts unique contributions to gene regulation. YY2 binding was not generally associated with gene promoters. However, several YY2 binding sites are linked to long noncoding RNA (lncRNA) genes and we show that the expression levels of a few of those are Yy2-dependent.

Regulation of Mouse Retroelement MuERV-L/MERVL Expression by REX1 and Epigenetic Control of Stem Cell Potency.

About half of the mammalian genome is occupied by DNA sequences that originate from transposable elements. Retrotransposons can modulate gene expression in different ways and, particularly retrotransposon-derived long terminal repeats, profoundly shape expression of both surrounding and distant genomic loci. This is especially important in pre-implantation development, during which extensive reprograming of the genome takes place and cells pass through totipotent and pluripotent states. At this stage, the main mechanism responsible for retrotransposon silencing, i.e., DNA methylation, is inoperative. A particular retrotransposon called muERV-L/MERVL is expressed during pre-implantation stages and contributes to the plasticity of mouse embryonic stem cells. This review will focus on the role of MERVL-derived sequences as controlling elements of gene expression specific for pre-implantation development, two-cell stage-specific gene expression, and stem cell pluripotency, the epigenetic mechanisms that control their expression, and the contributions of the pluripotency marker REX1 and the related Yin Yang 1 family of transcription factors to this regulation process.

Functional analysis of Rex1 during preimplantation development.

Rex1/Zfp42 is a nuclear protein that is highly conserved in mammals, and widely used as an embryonic stem (ES) cell marker. Although Rex1 expression is associated with enhanced pluripotency, loss-of-function models recently described do not exhibit major phenotypes, and both preimplantation development and ES cell derivation appear normal in the absence of Rex1. To better understand the functional role of Rex1, we examined the expression and localization of Rex1 during preimplantation development. Our studies indicated that REX1 is expressed at all stages during mouse preimplantation development, with a mixed pattern of nuclear, perinuclear, and cytoplasmic localization. Chromatin association seemed to be altered in 8-cell embryos, and in the blastocyst, we found REX1 localized almost exclusively in the nucleus. A functional role for Rex1 in vivo was assessed by gain- and loss-of-function approaches. Embryos with attenuated levels of Rex1 after injection of zygotes with siRNAs did not exhibit defects in preimplantation development in vitro. In contrast, overexpression of Rex1 interfered with cleavage divisions and with proper blastocyst development, although we failed to detect alterations in the expression of lineage and pluripotency markers. Rex1 gain- and loss-of-function did alter the expression levels of Zscan4, an important regulator of preimplantation development and pluripotency. Our results suggest that Rex1 plays a role during preimplantation development. They are compatible with a role for Rex1 during acquisition of pluripotency in the blastocyst.

Molecular coupling of Tsix regulation and pluripotency.

The reprogramming of X-chromosome inactivation during the acquisition of pluripotency in vivo and in vitro is accompanied by the repression of Xist, the trigger of X-inactivation, and the upregulation of its antisense counterpart Tsix. We have shown that key factors supporting pluripotency-Nanog, Oct4 and Sox2-bind within Xist intron 1 in undifferentiated embryonic stem cells (ESC) to repress Xist transcription. However, the relationship between transcription factors of the pluripotency network and Tsix regulation has remained unclear. Here we show that Tsix upregulation in embryonic stem cells depends on the recruitment of the pluripotent marker Rex1, and of the reprogramming-associated factors Klf4 and c-Myc, by the DXPas34 minisatellite associated with the Tsix promoter. Upon deletion of DXPas34, binding of the three factors is abrogated and the transcriptional machinery is no longer efficiently recruited to the Tsix promoter. Additional analyses including knockdown experiments further demonstrate that Rex1 is critically important for efficient transcription elongation of Tsix. Hence, distinct embryonic-stem-cell-specific complexes couple X-inactivation reprogramming and pluripotency, with Nanog, Oct4 and Sox2 repressing Xist to facilitate the reactivation of the inactive X, and Klf4, c-Myc and Rex1 activating Tsix to remodel Xist chromatin and ensure random X-inactivation upon differentiation. The holistic pattern of Xist/Tsix regulation by pluripotent factors that we have identified suggests a general direct governance of complex epigenetic processes by the machinery dedicated to pluripotency.

Fibroblast growth factor homologous factors control neuronal excitability through modulation of voltage-gated sodium channels.

Neurons integrate and encode complex synaptic inputs into action potential outputs through a process termed "intrinsic excitability." Here, we report the essential contribution of fibroblast growth factor homologous factors (FHFs), a family of voltage-gated sodium channel binding proteins, to this process. Fhf1-/-Fhf4-/- mice suffer from severe ataxia and other neurological deficits. In mouse cerebellar slice recordings, WT granule neurons can be induced to fire action potentials repetitively (approximately 60 Hz), whereas Fhf1-/-Fhf4-/- neurons often fire only once and at an elevated voltage spike threshold. Sodium channels in Fhf1-/-Fhf4-/- granule neurons inactivate at more negative membrane potential, inactivate more rapidly, and are slower to recover from the inactivated state. Altered sodium channel physiology is sufficient to explain excitation deficits, as tested in a granule cell computer model. These findings offer a physiological mechanism underlying human spinocerebellar ataxia induced by Fhf4 mutation and suggest a broad role for FHFs in the control of excitability throughout the CNS.

Deafness disrupts chloride transporter function and inhibitory synaptic transmission.

Loss of sensory function leads to atrophy or death within the developing CNS, yet little is known about the physiology of remaining synapses. After bilateral deafening, gramicidin-perforated-patch recordings were obtained from gerbil inferior colliculus neurons in a brain slice preparation. Afferent-evoked IPSPs had a diminished ability to block current-evoked action potentials in deafened neurons. This change could be attributed, in part, to a loss of potassium-dependent chloride transport function, with little change in K-Cl cotransporter expression. Treatments that suppressed chloride cotransport (bumetanide, cesium, and genistein) had little or no effect on neurons from deafened animals. These same treatments depolarized the E(IPSC) of control neurons. Semiquantitative RT-PCR and immunohistochemical staining indicated no change in the expression of chloride cotransporter mRNA or protein after deafness. Therefore, profound hearing loss leads rapidly to the disruption of chloride homeostasis, which is likely attributable to the dysfunction of the potassium-dependent chloride cotransport mechanism, rather than a downregulation of its expression. This results in inhibitory synapses that are less able to block excitatory events.

Fibroblast growth factor homologous factors and the islet brain-2 scaffold protein regulate activation of a stress-activated protein kinase.

Fibroblast growth factor homologous factors (FHFs) form native intracellular complexes with the mitogen-activated protein kinase (MAPK) scaffold protein islet-brain 2 (IB2) in adult brain. FHF binding to IB2 facilitates recruitment of the MAPK p38delta (SAPK4), while failing to stimulate binding of JNK, the preferred kinase of the related scaffold IB1 (JIP-1). We now report further biochemical evidence supporting FHFs as regulators of IB2 scaffold activity. Mixed lineage kinase 3 (MLK3) and IB2 synergistically activate p38delta but not the MAPKs JNK-1 and p38alpha. Binding of p38delta to IB2 is mediated by the carboxyl-terminal half of the scaffold (IB2(Delta1-436)). FHF2 also binds weakly to IB2(Delta1-436) and can thereby increase p38delta interaction with IB2(Delta1-436). FHF-induced recruitment of p38delta to IB2 is accompanied by increased levels of activated p38delta, and synergistic activation of p38delta by MLK3 and IB2 is further enhanced by FHF2. Consistent with a role for FHFs as signaling molecules, FHF2 isolated from rat brain is serine/threonine-phosphorylated, and FHF can serve as a substrate for p38delta in vitro. These results support the existence of a signaling module in which IB2 scaffolds a MLK3/MKK/p38delta kinase cascade. FHFs aid in recruitment of p38 to IB2 and may serve as kinase substrates.