Quantification of mRNA ribosomal engagement in human neurons using parallel translating ribosome affinity purification (TRAP) and RNA sequencing.

Translation regulation is a fundamental step in gene regulation with critical roles in neurodevelopment. Here, we describe three protocols to calculate the ribosomal-engagement levels of the transcriptome from in vitro-derived neuronal cells. The protocols described here include enrichment of in vitro-generated pluripotent-derived neurons, immunoaffinity purification of ribosome-bound RNAs, and calculation of the fraction of ribosome-engaged mRNAs. The ribosome-engaged RNA fraction is a measurement of the translation activity, and differences between genotype or growth conditions report change in translational regulation. For complete details on the use and execution of this protocol, please refer to Rodrigues et al. (2020).

Regulation, diversity and function of MECP2 exon and 3'UTR isoforms.

The methyl-CpG-binding protein 2 (MECP2) is a critical global regulator of gene expression. Mutations in MECP2 cause neurodevelopmental disorders including Rett syndrome (RTT). MECP2 exon 2 is spliced into two alternative messenger ribonucleic acid (mRNA) isoforms encoding MECP2-E1 or MECP2-E2 protein isoforms that differ in their N-termini. MECP2-E2, isolated first, was used to define the general roles of MECP2 in methyl-deoxyribonucleic acid (DNA) binding, targeting of transcriptional regulatory complexes, and its disease-causing impact in RTT. It was later found that MECP2-E1 is the most abundant isoform in the brain and its exon 1 is also mutated in RTT. MECP2 transcripts undergo alternative polyadenylation generating mRNAs with four possible 3'untranslated region (UTR) lengths ranging from 130 to 8600 nt. Together, the exon and 3'UTR isoforms display remarkable abundance disparity across cell types and tissues during development. These findings indicate discrete means of regulation and suggest that protein isoforms perform non-overlapping roles. Multiple regulatory programs have been explored to explain these disparities. DNA methylation patterns of the MECP2 promoter and first intron impact MECP2-E1 and E2 isoform levels. Networks of microRNAs and RNA-binding proteins also post-transcriptionally regulate the stability and translation efficiency of MECP2 3'UTR isoforms. Finally, distinctions in biophysical properties in the N-termini between MECP2-E1 and E2 lead to variable protein stabilities and DNA binding dynamics. This review describes the steps taken from the discovery of MECP2, the description of its key functions, and its association with RTT, to the emergence of evidence revealing how MECP2 isoforms are differentially regulated at the transcriptional, post-transcriptional and post-translational levels.

Methylglyoxal couples metabolic and translational control of Notch signalling in mammalian neural stem cells.

Gene regulation and metabolism are two fundamental processes that coordinate the self-renewal and differentiation of neural precursor cells (NPCs) in the developing mammalian brain. However, little is known about how metabolic signals instruct gene expression to control NPC homeostasis. Here, we show that methylglyoxal, a glycolytic intermediate metabolite, modulates Notch signalling to regulate NPC fate decision. We find that increased methylglyoxal suppresses the translation of Notch1 receptor mRNA in mouse and human NPCs, which is mediated by binding of the glycolytic enzyme GAPDH to an AU-rich region within Notch1 3'UTR. Interestingly, methylglyoxal inhibits the enzymatic activity of GAPDH and engages it as an RNA-binding protein to suppress Notch1 translation. Reducing GAPDH levels or restoring Notch signalling rescues methylglyoxal-induced NPC depletion and premature differentiation in the developing mouse cortex. Taken together, our data indicates that methylglyoxal couples the metabolic and translational control of Notch signalling to control NPC homeostasis.

Everolimus Rescues the Phenotype of Elastin Insufficiency in Patient Induced Pluripotent Stem Cell-Derived Vascular Smooth Muscle Cells.

OBJECTIVE: Elastin gene deletion or mutation leads to arterial stenoses due to vascular smooth muscle cell (SMC) proliferation. Human induced pluripotent stem cells-derived SMCs can model the elastin insufficiency phenotype in vitro but show only partial rescue with rapamycin. Our objective was to identify drug candidates with superior efficacy in rescuing the SMC phenotype in elastin insufficiency patients. Approach and Results: SMCs generated from induced pluripotent stem cells from 5 elastin insufficiency patients with severe recurrent vascular stenoses (3 Williams syndrome and 2 elastin mutations) were phenotypically immature, hyperproliferative, poorly responsive to endothelin, and exerted reduced tension in 3-dimensional smooth muscle biowires. Elastin mRNA and protein were reduced in SMCs from patients compared to healthy control SMCs. Fourteen drug candidates were tested on patient SMCs. Of the mammalian target of rapamycin inhibitors studied, everolimus restored differentiation, rescued proliferation, and improved endothelin-induced calcium flux in all patient SMCs except one Williams syndrome. Of the calcium channel blockers, verapamil increased SMC differentiation and reduced proliferation in Williams syndrome patient cells but not in elastin mutation patients and had no effect on endothelin response. Combination treatment with everolimus and verapamil was not superior to everolimus alone. Other drug candidates had limited efficacy. CONCLUSIONS: Everolimus caused the most consistent improvement in SMC differentiation, proliferation and in SMC function in patients with both syndromic and nonsyndromic elastin insufficiency, and offers the best candidate for drug repurposing for treatment of elastin insufficiency associated vasculopathy.

Generation of infant- and pediatric-derived urinary induced pluripotent stem cells competent to form kidney organoids.

BACKGROUND: Human induced pluripotent stem cells (iPSCs) are a promising tool to investigate pathogenic mechanisms underlying human genetic conditions, such as congenital anomalies of the kidney and urinary tract (CAKUT). Currently, iPSC-based research in pediatrics is limited by the invasiveness of cell collection. METHODS: Urine cells (UCs) were isolated from pediatric urine specimens, including bag collections, and reprogrammed using episomal vectors into urinary iPSCs (UiPSCs). Following iPSC-quality assessment, human kidney organoids were generated. RESULTS: UCs were isolated from 71% (12/17) of single, remnant urine samples obtained in an outpatient setting (patients 1 month-17 years, volumes 10-75 ml). Three independent UCs were reprogrammed to UiPSCs with early episome loss, confirmed pluripotency and normal karyotyping. Subsequently, these UiPSCs were successfully differentiated into kidney organoids, closely resembling organoids generated from control fibroblast-derived iPSCs. Importantly, under research conditions with immediate sample processing, UC isolation was successful 100% for target pediatric CAKUT patients and controls (11/11) after at most two urine collections. CONCLUSIONS: Urine in small volumes or collected in bags is a reliable source for reprogrammable somatic cells that can be utilized to generate kidney organoids. This constitutes an attractive approach for patient-specific iPSC research involving infants and children with wide applicability and a low threshold for participation.

AT1 and aldosterone receptors blockade prevents the chronic effect of nandrolone on the exercise-induced cardioprotection in perfused rat heart subjected to ischemia and reperfusion.

PURPOSE: Myocardial tolerance to ischaemia/reperfusion (I/R) injury is improved by exercise training, but this cardioprotection is impaired by the chronic use of anabolic androgenic steroids (AAS). The present study evaluated whether blockade of angiotensin II receptor (AT1-R) with losartan and aldosterone receptor (mineralocorticoid receptor, MR) with spironolactone could prevent the deleterious effect of AAS on the exercise-induced cardioprotection. METHODS AND RESULTS: Male Wistar rats were exercised and treated with either vehicle, nandrolone decanoate (10 mg/kg/week i.m.) or the same dose of nandrolone plus losartan or spironolactone (20 mg/kg/day orally) for 8 weeks. Langendorff-perfused hearts were subjected to I/R and evaluated for the postischaemic recovery of left ventricle (LV) function and infarct size. mRNA and protein expression of angiotensin II type 1 receptor (AT1-R), mineralocorticoid receptor (MR), and KATP channels were determined by reverse-transcriptase polymerase chain reaction and Western blotting. Postischaemic recovery of LV function was better and infarct size was smaller in the exercised rat hearts than in the sedentary rat hearts. Nandrolone impaired the exercise-induced cardioprotection, but this effect was prevented by losartan (AT1-R antagonist) and spironolactone (MR antagonist) treatments. Myocardial AT1-R and MR expression levels were increased, and the expression of the KATP channel subunits SUR2a and Kir6.1 was decreased and Kir6.2 increased in the nandrolone-treated rat hearts. The nandrolone-induced changes of AT1-R, MR, and KATP subunits expression was normalized by the losartan and spironolactone treatments. CONCLUSION: The chronic nandrolone treatment impairs the exercise-induced cardioprotection against ischaemia/reperfusion injury by activating the cardiac renin-angiotensin-aldosterone system and downregulating KATP channel expression.