Trans Species RNA Activity: Sperm RNA of the Father of an Autistic Child Programs Glial Cells and Behavioral Disorders in Mice.

Recently, we described the alteration of six miRNAs in the serum of autistic children, their fathers, mothers, siblings, and in the sperm of autistic mouse models. Studies in model organisms suggest that noncoding RNAs participate in transcriptional modulation pathways. Using mice, approaches to alter the amount of RNA in fertilized eggs enable in vivo intervention at an early stage of development. Noncoding RNAs are very numerous in spermatozoa. Our study addresses a fundamental question: can the transfer of RNA content from sperm to eggs result in changes in phenotypic traits, such as autism? To explore this, we used sperm RNA from a normal father but with autistic children to create mouse models for autism. Here, we induced, in a single step by microinjecting sperm RNA into fertilized mouse eggs, a transcriptional alteration with the transformation in adults of glial cells into cells affected by astrogliosis and microgliosis developing deficiency disorders of the 'autism-like' type in mice born following these manipulations. Human sperm RNA alters gene expression in mice, and validates the possibility of non-Mendelian inheritance in autism.

Alterations in Serum miR-126-3p Levels over Time: A Marker of Pituitary Insufficiency following Head Trauma.

INTRODUCTION: Traumatic brain injuries (TBIs) pose a high risk of pituitary insufficiency development in patients. We have previously reported alterations in miR-126-3p levels in sera from patients with TBI-induced pituitary deficiency. METHODS: To investigate why TBI-induced pituitary deficiency develops only in some patients and to reveal the relationship between miR-126-3p with hormone axes, we used mice that were epigenetically modified with miR-126-3p at the embryonic stage. These modified mice were subjected to mild TBI (mTBI) according to the Marmarou's weight-drop model at 2 months of age. The levels of miR-126-3p were assessed at 1 and 30 days in serum after mTBI. Changes in miR-126-3p levels after mTBI of wild-type and miR-126-3p* modified mouse lines validated our human results. Additionally, hypothalamus, pituitary, and adrenal tissues were analyzed for transcripts and associated serum hormone levels. RESULTS: We report that miR-126-3p directly affects hypothalamus-pituitary-adrenal (HPA) axis upregulation and ACTH secretion in the acute phase after mTBI. We also demonstrated that miR-126-3p suppresses Gnrh transcripts in the hypothalamus and pituitary, but this is not reflected in serum FSH/LH levels. The increase in ACTH levels in the acute phase may indicate that upregulation of miR-126-3p at the embryonic stage has a protective effect on the HPA axis after TBI. Notably, the most prominent transcriptional response is found in the adrenals, highlighting their role in the pathophysiology of TBI. CONCLUSION: Our study revealed the role of miR-126-3p in TBI and pituitary deficiency developing after TBI, and the obtained data will significantly contribute to elucidating the mechanism of pituitary deficiency development after TBI and development of new diagnostic and treatment strategies.

From Data to Insights: Machine Learning Empowers Prognostic Biomarker Prediction in Autism.

Autism Spectrum Disorder (ASD) poses significant challenges to society and science due to its impact on communication, social interaction, and repetitive behavior patterns in affected children. The Autism and Developmental Disabilities Monitoring (ADDM) Network continuously monitors ASD prevalence and characteristics. In 2020, ASD prevalence was estimated at 1 in 36 children, with higher rates than previous estimates. This study focuses on ongoing ASD research conducted by Erciyes University. Serum samples from 45 ASD patients and 21 unrelated control participants were analyzed to assess the expression of 372 microRNAs (miRNAs). Six miRNAs (miR-19a-3p, miR-361-5p, miR-3613-3p, miR-150-5p, miR-126-3p, and miR-499a-5p) exhibited significant downregulation in all ASD patients compared to healthy controls. The current study endeavors to identify dependable diagnostic biomarkers for ASD, addressing the pressing need for non-invasive, accurate, and cost-effective diagnostic tools, as current methods are subjective and time-intensive. A pivotal discovery in this study is the potential diagnostic value of miR-126-3p, offering the promise of earlier and more accurate ASD diagnoses, potentially leading to improved intervention outcomes. Leveraging machine learning, such as the K-nearest neighbors (KNN) model, presents a promising avenue for precise ASD diagnosis using miRNA biomarkers.

Heterozygous Cc2d1a mice show sex-dependent changes in the Beclin-1/p62 ratio with impaired prefrontal cortex and hippocampal autophagy.

Autism Spectrum Disorders (ASD) are a group of neurodevelopmental disorders characterized by repetitive behaviors, lack of social interaction and communication. CC2D1A is identified in patients as an autism risk gene. Recently, we suggested that heterozygous Cc2d1a mice exhibit impaired autophagy in the hippocampus. We now report the analysis of autophagy markers (Lc3, Beclin and p62) in different regions hippocampus, prefrontal cortex, hypothalamus and cerebellum, with an overall decrease in autophagy and changes in Beclin-1/p62 ratio in the hippocampus. We observed sex-dependent variations in transcripts and protein expression levels. Moreover, our analyses suggest that alterations in autophagy initiated in Cc2d1a heterozygous parents are variably transmitted to offspring, even when the offspring's genotype is wild type. Aberration in the autophagy mechanism may indirectly contribute to induce synapse alteration in the ASD brain.

Partial changes in apoptotic pathways in hippocampus and hypothalamus of Cc2d1a heterozygous.

Alterations in the apoptosis pathway have been linked to changes in serotonin levels seen in autistic patients. Cc2d1a is a repressor of the HTR1A gene involved in the serotonin pathway. The hippocampus and hypothalamus of Cc2d1a ± mice were analyzed for the expression of apoptosis markers (caspase 3, 8 and 9). Gender differences were observed in the expression levels of the three caspases consistent with some altered activity in the open-field assay. The number of apoptotic cells was significantly increased. We concluded that apoptotic pathways are only partially affected in the pathogenesis of the Cc2d1a heterozygous mouse model. A) Apoptosis is suppressed because the cell does not receive a death signal, or the receptor cannot activate the caspase 8 pathway despite the death signal. B) Since Caspase 8 and Caspase 3 expression is downregulated in our mouse model, the mechanism of apoptosis is not activated.

The Role of Apoptosis and Autophagy in the Hypothalamic-Pituitary-Adrenal (HPA) Axis after Traumatic Brain Injury (TBI).

Traumatic brain injury (TBI) is a major health problem affecting millions of people worldwide and leading to death or permanent damage. TBI affects the hypothalamic-pituitary-adrenal (HPA) axis either by primary injury to the hypothalamic-hypophyseal region or by secondary vascular damage, brain, and/or pituitary edema, vasospasm, and inflammation. Neuroendocrine dysfunctions after TBI have been clinically described in all hypothalamic-pituitary axes. We established a mild TBI (mTBI) in rats by using the controlled cortical impact (CCI) model. The hypothalamus, pituitary, and adrenals were collected in the acute (24 h) and chronic (30 days) groups after TBI, and we investigated transcripts and protein-related autophagy (Lc3, Bcln1, P150, Ulk, and Atg5) and apoptosis (pro-caspase-3, cleaved caspase-3). Transcripts related to autophagy were reduced in the hypothalamus, pituitary, and adrenals after TBI, however, this was not reflected in autophagy-related protein levels. In contrast, protein markers related to apoptosis increased in the adrenals during the acute phase and in the pituitary during the chronic phase. TBI stresses induce a variation of autophagy-related transcripts without modifying the levels of their proteins in the HPA axis. In contrast, protein markers related to apoptosis are increased in the acute phase in the adrenals, which could lead to impaired communication via the hypothalamus, pituitary, and adrenals. This may then explain the permanent pituitary damage with increased apoptosis and inflammation in the chronic phase. These results contribute to the elucidation of the mechanisms underlying endocrine dysfunctions such as pituitary and adrenal insufficiency that occur after TBI. Although the adrenals are not directly affected by TBI, we suggest that the role of the adrenals along with the hypothalamus and pituitary should not be ignored in the acute phase after TBI.

Predicting Progression of Autosomal Dominant Polycystic Kidney Disease by Changes in the Telomeric Epigenome.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited cause of chronic kidney disease with Polycystin (PKD) 1 and 2 gene mutation. However, the intra-familial variability in symptoms further suggests a non-Mendelian contribution to the disease. Our goal was to find a marker to track the epigenetic changes common to rapidly progressing forms of the disease. The risk of ADPKD increases with age, and aging shortens the telomere length (TL). Telomeres are a nucleoprotein structure composed mainly of three complexes, shelterin, CST and RNA-containing telomere repeat(TERRA), which protects the ends of chromosomes from degradation and fusion, and plays a role in maintaining cellular stability and in the repair of telomeric damage. TERRAs are transcribed from telomeric regions and a part of them is engaged in a DNA/RNA hybrid (R-loop) at each chromosome end. We tracked TL and TERRA levels in blood samples of 78 patients and 20 healthy control. Our study demonstrates that TL was shortened and TERRA expression levels in the DNA-attached fraction increased in autosomal dominant polycystic kidney patients with mutations in PKD1 and PKD2 compared to the control group. Moreover, it was observed that the expression of TERRA engaged in the R-loop was higher and the length of telomeres shorter in patients with ADPKD who showed rapid disease progression. Intrafamilial variation in TL and TERRA levels with the same mutation would indicate reliable epigenetic potential biomarkers in disease monitoring.

Mouse Paternal RNAs Initiate a Pattern of Metabolic Disorders in a Line-Dependent Manner.

A wide range of diseases result from environmental effects, and the levels of many native transcripts are altered. The alteration of non-coding RNAs (ncRNAs) and transmission of the variation to the next generation is increasingly recognized as a marker of disease. However, the determining signals and mechanisms of RNA-induced heritability remain unclear. We performed functional tests with four different genotypes of mice maintained on a high-fat diet to trace the transfer of the obesity/diabetes phenotype to the next generation in order to detect common signals. Two founders of four mouse lines (B6/D2 hybrid and Dnmt2 -/-C57BL/6 ) resist and do not change their phenotype while their sperm RNAs after microinjection into fertilized mouse eggs transfer the newly acquired phenotypes in a susceptible inbred line (C57BL/6 or Balb/c). Unexpectedly, in the same line of experiments, sperm RNA from animals raised on a normal diet when mixed with the sperm RNA from animals raised on a diet high in fat or synthetic miR-19b (inducer of obesity) affects or prevents the development of obesity and diabetes. However, it remains unclear what happens to ncRNA signaling under diet. With a comprehensive new analysis of the transcripts maintained as an RNA/DNA hybrid in sperm, we suggest that a fraction of the RNAs are stably attached to the genome. Thus, we propose that changes in the dynamics of ncRNA retention on DNA by factors such as transcriptional variations or lack of adequate methylation could serve as molecular markers to trace these epigenetics events.

Decrease in RNase HII and Accumulation of lncRNAs/DNA Hybrids: A Causal Implication in Psoriasis?

Functional long non-coding RNAs (lncRNAs) have been in the limelight in aging research because short telomeres are associated with higher levels of TERRA (Telomeric Repeat containing RNA). The genomic instability, which leads to short telomeres, is a mechanism observed in cell aging and in a class of cancer cells. Psoriasis, a skin disease, is a disorder of epidermal keratinocytes, with altered telomerase activity. Research on the fraction of nascent RNAs in hybrid with DNA offers avenues for new strategies. Skin and blood samples from patients were fractionated to obtain the RNA associated with DNA as a R-loop structure. The higher amount of TERRA levels attached with each chromosome end was found with psoriasis patients in blood and skin. In addition to telomeric TERRA, we evidenced accumulation of others non-coding RNA, such as non-telomeric TERRA and centromeric transcripts. Increased levels of non-coding RNAs attached to DNA correlates with a decreased in Ribonuclease HII (RNase-HII) transcript which means that overall unresolved DNA-RNA hybrids can ultimately weaken DNA and cause skin lesions. Since the genome is actively transcribed, cellular RNase-HII is essential for removing RNA from the DNA-RNA hybrid in controls of genome stability and epigenome shaping and can be used as a causal prognostic marker in patients with psoriasis.

The Characterization of Sex Differences in Hypoglycemia-Induced Activation of HPA Axis on the Transcriptomic Level.

Activation of the hypothalamic-pituitary-adrenal (HPA) axis using an insulin tolerance test (ITT) is a medical diagnostic procedure that is frequently used in humans to assess the HPA and growth-hormone (GH) axes. Whether sex differences exist in the response to ITT stress is unknown. Thus, investigations into the analysis of transcripts during activation of the HPA axis in response to hypoglycemia have revealed the underlying influences of sex in signaling pathways that stimulate the HPA axis. We assessed four time points of ITT application in Balb/c mice. After insulin injection, expression levels of 192 microRNAs and 41 mRNAs associated with the HPA, GH and hypothalamic-pituitary-gonadal (HPG) axes were determined by real-time RT-PCR in the hypothalamus, pituitary and adrenal tissues, as well as blood samples (Raw data accession: https://drive.google.com/drive/folders/10qI00NAtjxOepcNKxSJnQbJeBFa6zgHK?usp=sharing ). Although the ITT is commonly used as a gold standard for evaluating the HPA axis, we found completely different responses between males and females with respect to activation of the HPA axis. While activation of several transcripts in the hypothalamus and pituitary was observed after performing the ITT in males within 10 min, females responded via the pituitary and adrenal immediately and durably over 40 min. Additionally, we found that microRNA alterations precede mRNA responses in the HPA axis. Furthermore, robust changes in the levels of several transcripts including Avpr1b and Avpr2 observed at all time points strongly suggest that transcriptional control of these genes occurs mostly via differential signaling in pituitary and blood between males and females. Male and female HPA axis responses to ITT involve a number of sophisticated regulatory signaling pathways of miRNAs and mRNAs. Our results highlight the first robust markers in several layers of HPA, HPG and GH axis involved in ITT/hypoglycemia stress-induced dynamics.

DNA-RNA Hybrid (R-Loop): From a Unified Picture of the Mammalian Telomere to the Genome-Wide Profile.

Local three-stranded DNA/RNA hybrid regions of genomes (R-loops) have been detected either by binding of a monoclonal antibody (DRIP assay) or by enzymatic recognition by RNaseH. Such a structure has been postulated for mouse and human telomeres, clearly suggested by the identification of the complementary RNA Telomeric repeat-containing RNA "TERRA". However, the tremendous disparity in the information obtained with antibody-based technology drove us to investigate a new strategy. Based on the observation that DNA/RNA hybrids in a triplex complex genome co-purify with the double-stranded chromosomal DNA fraction, we developed a direct preparative approach from total protein-free cellular extract without antibody that allows their physical isolation and determination of their RNA nucleotide sequence. We then define in the normal mouse and human sperm genomes the notion of stable DNA associated RNA terminal R-loop complexes, including TERRA molecules synthesized from local promoters of every chromosome. Furthermore, the first strong evidence of all telomeric structures, applied additionally to the whole murine sperm genome compared to the testes, showed reproducible R-loop complexes of the whole genome and suggesting a defined profile in the sperm genome for the next generation.

Implications of the Wilms' Tumor Suppressor Wt1 in Cardiomyocyte Differentiation.

The Wilms' tumor suppressor Wt1 is involved in multiple developmental processes and adult tissue homeostasis. The first phenotypes recognized in Wt1 knockout mice were developmental cardiac and kidney defects. Wt1 expression in the heart has been described in epicardial, endothelial, smooth muscle cells, and fibroblasts. Expression of Wt1 in cardiomyocytes has been suggested but remained a controversial issue, as well as the role of Wt1 in cardiomyocyte development and regeneration after injury. We determined cardiac Wt1 expression during embryonic development, in the adult, and after cardiac injury by quantitative RT-PCR and immunohistochemistry. As in vitro model, phenotypic cardiomyocyte differentiation, i.e., the appearance of rhythmically beating clones from mouse embryonic stem cells (mESCs) and associated changes in gene expression were analyzed. We detected Wt1 in cardiomyocytes from embryonic day (E10.5), the first time point investigated, until adult age. Cardiac Wt1 mRNA levels decreased during embryonic development. In the adult, Wt1 was reactivated in cardiomyocytes 48 h and 3 weeks following myocardial infarction. Wt1 mRNA levels were increased in differentiating mESCs. Overexpression of Wt1(-KTS) and Wt1(+KTS) isoforms in ES cells reduced the fraction of phenotypically cardiomyocyte differentiated clones, which was preceded by a temporary increase in c-kit expression in Wt1(-KTS) transfected ES cell clones and induction of some cardiomyocyte markers. Taken together, Wt1 shows a dynamic expression pattern during cardiomyocyte differentiation and overexpression in ES cells reduces their phenotypical cardiomyocyte differentiation.

A heritable profile of six miRNAs in autistic patients and mouse models.

Autism spectrum disorder (ASD) is a group of developmental pathologies that impair social communication and cause repetitive behaviors. The suggested roles of noncoding RNAs in pathology led us to perform a comparative analysis of the microRNAs expressed in the serum of human ASD patients. The analysis of a cohort of 45 children with ASD revealed that six microRNAs (miR-19a-3p, miR-361-5p, miR-3613-3p, miR-150-5p, miR-126-3p, and miR-499a-5p) were expressed at low to very low levels compared to those in healthy controls. A similar but less pronounced decrease was registered in the clinically unaffected parents of the sick children and in their siblings but never in any genetically unrelated control. Results consistent with these observations were obtained in the blood, hypothalamus and sperm of two of the established mouse models of ASD: valproic acid-treated animals and Cc2d1a+/- heterozygotes. In both instances, the same characteristic miRNA profile was evidenced in the affected individuals and inherited together with disease symptoms in the progeny of crosses with healthy animals. The consistent association of these genetic regulatory changes with the disease provides a starting point for evaluating the changes in the activity of the target genes and, thus, the underlying mechanism(s). From the applied societal and medical perspectives, once properly confirmed in large cohorts, these observations provide tools for the very early identification of affected children and progenitors.

Sperm RNA: Quo vadis?

Various different classes of RNAs contained in sperm cells have emerged as causal vectors for the transmission of acquired information from father to offspring. This has invigorated research and raised many new questions concerning the heritability of disease risk and the ability to adapt to novel environments. Here, we will focus on recent advances in the field of epigenetic germline inheritance in mammals, with a particular focus on the following three questions: (1) What is the current evidence for an involvement of sperm RNAs in the transmission of acquired information from father to offspring? (2) How can acquired changes in the sperm-RNA payload be induced in the male germline? (3) How can acquired changes be transferred from sperm to oocyte? We propose a novel mechanism for transfer of sperm RNA to the oocyte in a DNA/RNA-hybrid, possibly interacting with DNA-bound proteins, and suggest experiments that should advance our understanding of epigenetic germline inheritance.

Genome-Wide Distribution of Nascent Transcripts in Sperm DNA, Products of a Late Wave of General Transcription.

Mature spermatozoa contain a whole repertoire of the various classes of cellular RNAs, both coding and non-coding. It was hypothesized that after fertilization they might impact development, a claim supported by experimental evidence in various systems. Despite the current increasing interest in the transgenerational maintenance of epigenetic traits and their possible determination by RNAs, little remains known about conservation in sperm and across generations and the specificities and mechanisms involved in transgenerational maintenance. We identified two distinct fractions of RNAs in mature mouse sperm, one readily extracted in the aqueous phase of the classical TRIzol procedure and a distinct fraction hybridized with homologous DNA in DNA-RNA complexes recovered from the interface, purified after DNase hydrolysis and analyzed by RNA-seq methodology. This DNA-associated RNA (D RNA) was found to represent as much as half of the cell contents in differentiated sperm, in which a major part of the cytoplasmic material has been discarded. Stable complexes were purified free of proteins and identified as hybrids (R-loops) on the basis of their sensitivity to RNase H hydrolysis. Further analysis by RNA-seq identified transcripts from all the coding and non-coding regions of the genome, thus revealing an extensive wave of transcription, prior to or concomitant with the terminal compaction of the chromatin.

Sperm RNA code programmes the metabolic health of offspring.

Mammalian sperm RNA is increasingly recognized as an additional source of paternal hereditary information beyond DNA. Environmental inputs, including an unhealthy diet, mental stresses and toxin exposure, can reshape the sperm RNA signature and induce offspring phenotypes that relate to paternal environmental stressors. Our understanding of the categories of sperm RNAs (such as tRNA-derived small RNAs, microRNAs, ribosomal RNA-derived small RNAs and long non-coding RNAs) and associated RNA modifications is expanding and has begun to reveal the functional diversity and information capacity of these molecules. However, the coding mechanism endowed by sperm RNA structures and by RNA interactions with DNA and other epigenetic factors remains unknown. How sperm RNA-encoded information is decoded in early embryos to control offspring phenotypes also remains unclear. Complete deciphering of the 'sperm RNA code' with regard to metabolic control could move the field towards translational applications and precision medicine, and this may lead to prevention of intergenerational transmission of obesity and type 2 diabetes mellitus susceptibility.

Pipeline for the generation of gene knockout mice using dual sgRNA CRISPR/Cas9-mediated gene editing.

Animal models possess undeniable utility for progress on biomedical research projects and developmental and disease studies. Transgenic mouse models recreating specific disease phenotypes associated with ß-hemoglobinopathies have been developed previously. However, traditional methods for gene targeting in mouse using embryonic stem cells (ESCs) are laborious and time consuming. Recently, CRISPR has been developed to facilitate and improve genomic modifications in mouse or isogenic cell lines. Applying CRISPR to gene modification eliminates the time consuming steps of traditional approach including selection of targeted ESC clones and production of chimeric mouse. This study shows that microinjection of a plasmid DNA encoding Cas9 protein along with dual sgRNAs specific to Hbb-bs gene (hemoglobin, beta adult s chain) enables breaking target sequences at exons 2 and 3 positions. The injections led to a knockout allele with efficiency around 10% for deletion of exons 2 and 3 and 20% for indel mutation.

Nutrition meets heredity: a case of RNA-mediated transmission of acquired characters.

RNA-based inheritance provides a reasonable hypothesis to explain multigenerational maintenance of the disease in the progeny of either a male or female parent suffering from the metabolic syndrome (obesity and type 2 diabetes) induced by abnormal diet. Although, it is still difficult to formulate a complete rational mechanism, study of inheritance is a most direct way to learn about the epigenetic control of gene expression and we wished to summarised our current approach along this line.