High-frequency variants in PKA signaling-related genes within a large pediatric cohort with obesity or metabolic abnormalities.

Introduction: Pediatric obesity has steadily increased in recent decades. Large-scale genome-wide association studies (GWAS) conducted primarily in Eurocentric adult populations have identified approximately 100 loci that predispose to obesity and type II diabetes. GWAS in children and individuals of non-European descent, both disproportionately affected by obesity, are fewer. Rare syndromic and monogenic obesities account for only a small portion of childhood obesity, so understanding the role of other genetic variants and their combinations in heritable obesities is key to developing targeted and personalized therapies. Tight and responsive regulation of the cAMP-dependent protein kinase (PKA) signaling pathway is crucial to maintaining healthy energy metabolism, and mutations in PKA-linked genes represent the most common cause of monogenic obesity. Methods: For this study, we performed targeted exome sequencing of 53 PKA signaling-related genes to identify variants in genomic DNA from a large, ethnically diverse cohort of obese or metabolically challenged youth. Results: We confirmed 49 high-frequency variants, including a novel variant in the PDE11A gene (c.152C>T). Several other variants were associated with metabolic characteristics within ethnic groups. Discussion: We conclude that a PKA pathway-specific variant search led to the identification of several new genetic associations with obesity in an ethnically diverse population.

The astroglial and stem cell functions of adult rat folliculostellate cells.

The mammalian pituitary gland is a complex organ consisting of hormone-producing cells, anterior lobe folliculostellate cells (FSCs), posterior lobe pituicytes, vascular pericytes and endothelial cells, and Sox2-expressing stem cells. We present single-cell RNA sequencing and immunohistofluorescence analyses of pituitary cells of adult female rats with a focus on the transcriptomic profiles of nonhormonal cell types. Samples obtained from whole pituitaries and separated anterior and posterior lobe cells contained all expected pituitary resident cell types and lobe-specific vascular cell subpopulations. FSCs and pituicytes expressed S100B, ALDOC, EAAT1, ALDH1A1, and VIM genes and proteins, as well as other astroglial marker genes, some common and some cell type-specific. We also found that the SOX2 gene and protein were expressed in ~15% of pituitary cells, including FSCs, pituicytes, and a fraction of hormone-producing cells, arguing against its stem cell specificity. FSCs comprised two Sox2-expressing subclusters; FS1 contained more cells but lower genetic diversity, while FS2 contained proliferative cells, shared genes with hormone-producing cells, and expressed genes consistent with stem cell niche formation, regulation of cell proliferation and stem cell pluripotency, including the Hippo and Wnt pathways. FS1 cells were randomly distributed in the anterior and intermediate lobes, while FS2 cells were localized exclusively in the marginal zone between the anterior and intermediate lobes. These data indicate the identity of the FSCs as anterior pituitary-specific astroglia, with FS1 cells representing differentiated cells equipped for classical FSC roles and FS2 cells exhibiting additional stem cell-like features.

A reference human induced pluripotent stem cell line for large-scale collaborative studies.

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.

Preparation of Single Pineal Cells.

The isolation of single cells from the pineal gland plays an essential role in understanding the complex nature of such processes as differentiation, metabolism, and cell-cell communication within the pineal gland. This procedure is the portal to single-cell RNA sequencing, which produces the transcriptome of individual cells. As such, single-cell RNA sequencing is critical to the continued development of knowledge of the pineal cell physiology. This chapter describes a simple procedure for isolating individual cells. Starting with the incubation of whole tissue in an enzyme preparation, which dissociates the pineal gland into small pieces, it continues with gentle trituration and then isolation of single cells through filtration. The procedure takes less than 2 h.

RNA Sequencing of Single Pineal Cells.

The pineal gland presents a powerful genetic tool to study a broad range of physiological processes. It has been instrumental as a model in understanding transduction processes and daily changes in gene expression and holds great promise in understanding development. Currently, the field is at an exciting point, with methods available for the isolation of individual cells and, as presented here, the preparation of these single cells for sequencing. The resulting cellular transcriptomes have played a role in categorizing cells in the pineal gland, with current estimates including two types of pinealocytes, three types of astrocytes, two types of microglia, and two types of endothelial cells, including the poorly understood vascular and meningeal cell. The methods described in this chapter will serve to support and advance cellular studies of the pineal gland in the twenty-first century.

The Pineal Transcriptome Webpage.

The pineal transcriptome webpage is described, which provides access to the transcript expression profile of the vertebrate pineal gland and, in many cases, the retina. Experimental material was obtained during the day and night, providing an opportunity to examine rhythmicity. The vertebrates represented include human, rhesus, rat, mouse, chicken, and zebrafish. In addition, data on the effects of surgical denervation and pharmacological treatments of the rat are included. Data are freely available to users.

Structural basis of 3'-end poly(A) RNA recognition by LARP1.

La-related proteins (LARPs) comprise a family of RNA-binding proteins involved in a wide range of posttranscriptional regulatory activities. LARPs share a unique tandem of two RNA-binding domains, La motif (LaM) and RNA recognition motif (RRM), together referred to as a La-module, but vary in member-specific regions. Prior structural studies of La-modules reveal they are pliable platforms for RNA recognition in diverse contexts. Here, we characterize the La-module of LARP1, which plays an important role in regulating synthesis of ribosomal proteins in response to mTOR signaling and mRNA stabilization. LARP1 has been well characterized functionally but no structural information exists for its La-module. We show that unlike other LARPs, the La-module in LARP1 does not contain an RRM domain. The LaM alone is sufficient for binding poly(A) RNA with submicromolar affinity and specificity. Multiple high-resolution crystal structures of the LARP1 LaM domain in complex with poly(A) show that it is highly specific for the RNA 3'-end, and identify LaM residues Q333, Y336 and F348 as the most critical for binding. Use of a quantitative mRNA stabilization assay and poly(A) tail-sequencing demonstrate functional relevance of LARP1 RNA binding in cells and provide novel insight into its poly(A) 3' protection activity.

Modeling SARS-CoV-2 and influenza infections and antiviral treatments in human lung epithelial tissue equivalents.

There is a critical need for physiologically relevant, robust, and ready-to-use in vitro cellular assay platforms to rapidly model the infectivity of emerging viruses and develop new antiviral treatments. Here we describe the cellular complexity of human alveolar and tracheobronchial air liquid interface (ALI) tissue models during SARS-CoV-2 and influenza A virus (IAV) infections. Our results showed that both SARS-CoV-2 and IAV effectively infect these ALI tissues, with SARS-CoV-2 exhibiting a slower replication peaking at later time-points compared to IAV. We detected tissue-specific chemokine and cytokine storms in response to viral infection, including well-defined biomarkers in severe SARS-CoV-2 and IAV infections such as CXCL10, IL-6, and IL-10. Our single-cell RNA sequencing analysis showed similar findings to that found in vivo for SARS-CoV-2 infection, including dampened IFN response, increased chemokine induction, and inhibition of MHC Class I presentation not observed for IAV infected tissues. Finally, we demonstrate the pharmacological validity of these ALI tissue models as antiviral drug screening assay platforms, with the potential to be easily adapted to include other cell types and increase the throughput to test relevant pathogens.

Regulation of Glucose Insulinotropic Peptide and Intestinal Glucose Transporters in the Diet-Induced Obese Mouse.

Our recent studies have shown that glucose-dependent insulinotropic polypeptide (GIP), but not glucagon-like peptide 1 (GLP-1), augments Na-glucose transporter 1- (SGLT1-) mediated glucose absorption in mouse jejunum. Na-dependent glucose absorption sharply rose and peaked in 3 months of high-fat (i.e., obese) compared to normal (i.e., normal weight) diet fed animals. Previous studies have shown that GIP-augmented SGLT1 and PEPT1 (peptide transporter 1) are regulated by protein kinase A (PKA) signaling in mouse jejunum. Additional studies have indicated that cAMP and PI3 kinase signaling augment PEPT1 through EPAC and AKT activation pathways, respectively, through increased apical PEPT1 trafficking in intestinal epithelial cells. However, little is known about how the signaling glucose transport paradigm is altered over a long period. Early on, increased glucose absorption occurs through SGLT1, but as the obesity and diabetes progress, there is a dramatic shift towards a Na-independent mechanism. Surprisingly, at the peak of glucose absorption during the fifth month of the progression of obesity, the SGLT1 activity was severely depressed, while a Na-independent glucose absorptive process begins to appear. Since glucose transporter 2 (GLUT2) is expressed on the apical membrane of the small intestine in obese patients and animal models of obesity, it was hypothesized to be the new more efficient route. Western blot analyses and biotinylation of the apical membrane revealed that the GIP expression increases in the obese animals and its trafficking to the apical membrane increases with the GIP treatment.

TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A.

Variants of UNC13A, a critical gene for synapse function, increase the risk of amyotrophic lateral sclerosis and frontotemporal dementia1-3, two related neurodegenerative diseases defined by mislocalization of the RNA-binding protein TDP-434,5. Here we show that TDP-43 depletion induces robust inclusion of a cryptic exon in UNC13A, resulting in nonsense-mediated decay and loss of UNC13A protein. Two common intronic UNC13A polymorphisms strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia risk overlap with TDP-43 binding sites. These polymorphisms potentiate cryptic exon inclusion, both in cultured cells and in brains and spinal cords from patients with these conditions. Our findings, which demonstrate a genetic link between loss of nuclear TDP-43 function and disease, reveal the mechanism by which UNC13A variants exacerbate the effects of decreased TDP-43 function. They further provide a promising therapeutic target for TDP-43 proteinopathies.

Morphologic and Molecular Characterization of Adrenals and Adrenal Rest Affected by Congenital Adrenal Hyperplasia.

Introduction: Adrenocortical hyperplasia and adrenal rest tumor (ART) formation are common in congenital adrenal hyperplasia (CAH). Although driven by excessive corticotropin, much is unknown regarding the morphology and transformation of these tissues. Our study objective was to characterize CAH-affected adrenals and ART and compare with control adrenal and gonadal tissues. Patients/Methods: CAH adrenals, ART and control tissues were analyzed by histology, immunohistochemistry, and transcriptome sequencing. We investigated protein expression of the ACTH receptor (MC2R), steroidogenic (CYP11B2, CYP11B1, CYB5A) and immune (CD20, CD3, CD68) biomarkers, and delta-like 1 homolog (DLK1), a membrane bound protein broadly expressed in fetal and many endocrine cells. RNA was isolated and gene expression was analyzed by RNA sequencing (RNA-seq) followed by principle component, and unsupervised clustering analyses. Results: Based on immunohistochemistry, CAH adrenals and ART demonstrated increased zona reticularis (ZR)-like CYB5A expression, compared to CYP11B1, and CYP11B2, markers of zona fasciculata and zona glomerulosa respectively. CYP11B2 was mostly absent in CAH adrenals and absent in ART. DLK1 was present in CAH adrenal, ART, and also control adrenal and testis, but was absent in control ovary. Increased expression of adrenocortical marker MC2R, was observed in CAH adrenals compared to control adrenal. Unlike control tissues, significant nodular lymphocytic infiltration was observed in CAH adrenals and ART, with CD20 (B-cell), CD3 (T-cell) and CD68 (macrophage/monocyte) markers of inflammation. RNA-seq data revealed co-expression of adrenal MC2R, and testis-specific INSL3, HSD17B3 in testicular ART indicating the presence of both gonadal and adrenal features, and high expression of DLK1 in ART, CAH adrenals and control adrenal. Principal component analysis indicated that the ART transcriptome was more similar to CAH adrenals and least similar to control testis tissue. Conclusions: CAH-affected adrenal glands and ART have similar expression profiles and morphology, demonstrating increased CYB5A with ZR characteristics and lymphocytic infiltration, suggesting a common origin that is similarly affected by the abnormal hormonal milieu. Immune system modulators may play a role in tumor formation of CAH.

The PRKAR1B p.R115K Variant is Associated with Lipoprotein Profile in African American Youth with Metabolic Challenges.

CONTEXT: High childhood obesity rates coincide with increased incidence of nonalcoholic fatty liver disease (NAFLD) and other comorbidities. Understanding the genetics of susceptibility to obesity and its comorbidities could guide intervention. The cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) signaling pathway regulates energy balance, glucose homeostasis, and lipid metabolism. OBJECTIVE: We hypothesized that PKA-related gene variants may be associated with obesity or associated metabolic conditions. METHODS: We included 457 youths from the Yale Obesity Clinic into the Pathogenesis of Youth-Onset Diabetes cohort (NCT01967849); a variety of clinical tests were performed to characterize NAFLD. Exon sequencing of 54 PKA pathway genes was performed. Variants were confirmed by Sanger sequencing. Clinical data were analyzed, correcting for NAFLD status and body mass index z-score with adjustments for multiple comparisons. Fluorescence resonance energy transfer (FRET) and PKA enzymatic assays were performed in HEK293 cells transfected with the PRKAR1B p.R115K construct. In silico structural analysis for this variant was done. RESULTS: We identified the variant PRKAR1B p.R115K in 4 unrelated, African American patients. Analyses compared this variant group to other African American patients in the cohort. PRKAR1B p.R115K was associated with favorable circulating lipoprotein levels. Analysis of FRET and PKA enzymatic assay showed stronger interaction between the R1ß mutant and PKA catalytic subunit Cα and decreased basal PKA activity compared with the wildtype (P < .0001). Structural analysis revealed that p.R115K may hinder conformational changes resulting from cAMP binding at cAMP binding domain A. CONCLUSION: Data suggest PRKAR1B p.R115K affects cAMP signaling and may favorably modulate lipoprotein profile in African American youth, protecting them from some adverse metabolic outcomes.

Ablation of microRNA-155 and neuroinflammation in a mouse model of CLN1-disease.

Infantile neuronal ceroid lipofuscinosis (INCL), also known as CLN1-disease, is a devastating neurodegenerative lysosomal storage disorder (LSD), caused by inactivating mutations in the CLN1 gene. The Cln1-/- mice, which mimic INCL, manifest progressive neuroinflammation contributing to neurodegeneration. However, the underlying mechanism of neuroinflammation in INCL and in Cln1-/- mice has remained elusive. Previously, it has been reported that microRNA-155 (miR-155) regulates inflammation and miR profiling in Cln1-/- mouse brain showed that the level of miR-155 was upregulated. Thus, we sought to determine whether ablation of miR-155 in Cln1-/- mice may suppress neuroinflammation in these mice. Towards this goal, we generated Cln1-/-/miR-155-/- double-knockout mice and evaluated the inflammatory signatures in the brain. We found that the brains of double-KO mice manifest progressive neuroinflammatory changes virtually identical to those found in Cln1-/- mice. We conclude that ablation of miR-155 in Cln1-/- mice does not alter the neuroinflammatory trajectory in INCL mouse model.

Single-molecule polyadenylated tail sequencing (SM-PAT-Seq) to measure polyA tail lengths transcriptome-wide.

Polyadenylation of the 3' end of mRNAs is an important mechanism for regulating their stability and translation. We developed a nucleotide-resolution, transcriptome-wide, single-molecule SM-PAT-Seq method to accurately measure the polyA tail lengths of individual transcripts using long-read sequencing. The method generates cDNA using a double stranded splint adaptor targeting the far 3' end of the polyA tail for first strand synthesis along with random hexamers for second strand synthesis. This straight-forward method yields accurate polyA tail sequence lengths, can identify non-A residues in those tails, and quantitate transcript abundance.

Single-Cell RNA Sequencing Analysis of the Drosophila Larval Ventral Cord.

Drosophila provides a powerful genetic system and an excellent model to study the development and function of the nervous system. The fly's small brain and complex behavior has been instrumental in mapping neuronal circuits and elucidating the neural basis of behavior. The fast pace of fly development and the wealth of genetic tools has enabled systematic studies on cell differentiation and fate specification, and has uncovered strategies for axon guidance and targeting. The accessibility of neuronal structures and the ability to edit and manipulate gene expression in selective cells and/or synaptic compartments has revealed mechanisms for synapse assembly and neuronal connectivity. Recent advances in single-cell RNA sequencing (scRNA-seq) have further enhanced our appreciation and understanding of neuronal diversity in a fly brain. However, due to the small size of the fly brain and its constituent cells, scRNA-seq methodologies require a few adaptations. Here, we describe a set of protocols optimized for scRNA-seq analysis of the Drosophila larval ventral nerve cord, starting from tissue dissection and cell dissociation to cDNA library preparation, sequencing, and data analysis. We apply this workflow to three separate samples and detail the technical challenges associated with successful application of scRNA-seq to studies on neuronal diversity. An accompanying article (Vicidomini, Nguyen, Choudhury, Brody, & Serpe, 2021) presents a custom multistage analysis pipeline that integrates modules contained in different R packages to ensure high-flexibility, high-quality RNA-seq data analysis. These protocols are developed for Drosophila larval ventral nerve cord, but could easily be adapted to other tissues and model organisms. © 2021 U.S. Government. Basic Protocol 1: Dissection of larval ventral nerve cords and preparation of single-cell suspensions Basic Protocol 2: Preparation and sequencing of single-cell transcriptome libraries Basic Protocol 3: Alignment of raw sequencing data to indexed genome and generation of count matrices.

Single molecule poly(A) tail-seq shows LARP4 opposes deadenylation throughout mRNA lifespan with most impact on short tails.

La-related protein 4 (LARP4) directly binds both poly(A) and poly(A)-binding protein (PABP). LARP4 was shown to promote poly(A) tail (PAT) lengthening and stabilization of individual mRNAs presumably by protection from deadenylation (Mattijssen et al., 2017). We developed a nucleotide resolution transcriptome-wide, single molecule SM-PAT-seq method. This revealed LARP4 effects on a wide range of PAT lengths for human mRNAs and mouse mRNAs from LARP4 knockout (KO) and control cells. LARP4 effects are clear on long PAT mRNAs but become more prominent at 30-75 nucleotides. We also analyzed time courses of PAT decay transcriptome-wide and for ~200 immune response mRNAs. This demonstrated accelerated deadenylation in KO cells on PATs < 75 nucleotides and phasing consistent with greater PABP dissociation in the absence of LARP4. Thus, LARP4 shapes PAT profiles throughout mRNA lifespan with impact on mRNA decay at short lengths known to sensitize PABP dissociation in response to deadenylation machinery.

Parallel intermediate conductance K+ and Cl- channel activity mediates electroneutral K+ exit across basolateral membranes in rat distal colon.

Transepithelial K+ absorption requires apical K+ uptake and basolateral K+ exit. In the colon, apical H+-K+-ATPase mediates cellular K+ uptake, and it has been suggested that electroneutral basolateral K+ exit reflects K+-Cl- cotransporter-1 (KCC1) operating in parallel with K+ and Cl- channels. The present study was designed to identify basolateral transporter(s) responsible for K+ exit in rat distal colon. Active K+ absorption was determined by measuring 86Rb+ (K+ surrogate) fluxes across colonic epithelia under voltage-clamp conditions. With zero Cl- in the mucosal solution, net K+ absorption was reduced by 38%, indicating that K+ absorption was partially Cl--dependent. Serosal addition of DIOA (KCC1 inhibitor) or Ba2+ (nonspecific K+ channel blocker) inhibited net K+ absorption by 21% or 61%, respectively, suggesting that both KCC1 and K+ channels contribute to basolateral K+ exit. Clotrimazole and TRAM34 (IK channel blockers) added serosally inhibited net K+ absorption, pointing to the involvement of IK channels in basolateral K+ exit. GaTx2 (CLC2 blocker) added serosally also inhibited net K+ absorption, suggesting that CLC2-mediated Cl- exit accompanies IK channel-mediated K+ exit across the basolateral membrane. Net K+ absorption was not inhibited by serosal addition of either IbTX (BK channel blocker), apamin (SK channel blocker), chromanol 293B (KV7 channel blocker), or CFTRinh172 (CFTR blocker). Immunofluorescence studies confirmed basolateral membrane colocalization of CLC2-like proteins and Na+-K+-ATPase α-subunits. We conclude that active K+ absorption in rat distal colon involves electroneutral basolateral K+ exit, which may reflect IK and CLC2 channels operating in parallel.NEW & NOTEWORTHY This study demonstrates that during active electroneutral K+ absorption in rat distal colon, K+ exit across the basolateral membrane mainly reflects intermediate conductance K+ channels operating in conjunction with chloride channel 2, with a smaller, but significant, contribution from K+-Cl- cotransporter-1 (KCC1) activity.

Resource: A multi-species multi-timepoint transcriptome database and webpage for the pineal gland and retina.

The website and database https://snengs.nichd.nih.gov provides RNA sequencing data from multi-species analysis of the pineal glands from zebrafish (Danio rerio), chicken (White Leghorn), rat (Rattus novegicus), mouse (Mus musculus), rhesus macaque (Macaca mulatta), and human (Homo sapiens); in most cases, retinal data are also included along with results of the analysis of a mixture of RNA from tissues. Studies cover day and night conditions; in addition, a time series over multiple hours, a developmental time series and pharmacological experiments on rats are included. The data have been uniformly re-processed using the latest methods and assemblies to allow for comparisons between experiments and to reduce processing differences. The website presents search functionality, graphical representations, Excel tables, and track hubs of all data for detailed visualization in the UCSC Genome Browser. As more data are collected from investigators and improved genomes become available in the future, the website will be updated. This database is in the public domain and elements can be reproduced by citing the URL and this report. This effort makes the results of 21st century transcriptome profiling widely available in a user-friendly format that is expected to broadly influence pineal research.

Role of Phosphorylated Gonadotropin-Regulated Testicular RNA Helicase (GRTH/DDX25) in the Regulation of Germ Cell Specific mRNAs in Chromatoid Bodies During Spermatogenesis.

GRTH/DDX25 is a member of the DEAD-box family of RNA helicases that play an essential role in spermatogenesis. GRTH knock-in (KI) mice with the human mutant GRTH gene (R242H) show loss of the phospho-species from cytoplasm with preservation of the non-phospho form in the cytoplasm and nucleus. GRTH KI mice are sterile and lack elongated spermatids and spermatozoa, with spermatogenic arrest at step 8 of round spermatids which contain chromatoid body (CB) markedly reduced in size. We observed an absence of phospho-GRTH in CB of GRTH KI mice. RNA-Seq analysis of mRNA isolated from CB revealed that 1,421 genes show differential abundance, of which 947 genes showed a decrease in abundance and 474 genes showed an increase in abundance in GRTH KI mice. The transcripts related to spermatid development, differentiation, and chromatin remodeling (Tnp1/2, Prm1/2/3, Spem1/2, Tssk 2/3/6, Grth, tAce, and Upf2) were reduced, and the transcripts encoding for factors involved in RNA transport, regulation, and surveillance and transcriptional and translational regulation (Eef1a1, Ppp1cc, Pabpc1, Ybx3, Tent5b, H2al1m, Dctn2, and Dync1h1) were increased in the CB of KI mice and were further validated by qPCR. In the round spermatids of wild-type mice, mRNAs of Tnp2, Prm2, and Grth were abundantly co-localized with MVH protein in the CB, while in GRTH KI mice these were minimally present. In addition, GRTH binding to Tnp1/2, Prm1/2, Grth, and Tssk6 mRNAs was found to be markedly decreased in KI. These results demonstrate the importance of phospho-GRTH in the maintenance of the structure of CB and its role in the storage and stability of germ cell-specific mRNAs during spermiogenesis.