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ENeuro Jun 2024Acetylcholine (ACh) neurons in the central nervous system are required for the coordination of neural network activity during higher brain functions, such as attention,...
Acetylcholine (ACh) neurons in the central nervous system are required for the coordination of neural network activity during higher brain functions, such as attention, learning, and memory, as well as locomotion. Disturbed cholinergic signaling has been described in many neurodevelopmental and neurodegenerative disorders. Furthermore, co-transmission of other signaling molecules, such as glutamate and GABA, with ACh has been associated with essential roles in brain function or disease. However, it is unknown when ACh neurons become cholinergic during development. Thus, understanding the timeline of how the cholinergic system develops and becomes active in the healthy brain is a crucial part of understanding brain development. To study this, we used transgenic mice to selectively label ACh neurons with tdTomato. We imaged serial sectioned brains and generated whole-brain reconstructions at different time points during pre- and postnatal development. We found three crucial time windows - two in the prenatal and one in the postnatal brain - during which most ACh neuron populations become cholinergic in the brain. We also found that cholinergic gene expression is initiated in cortical ACh interneurons, while the cerebral cortex is innervated by cholinergic projection neurons from the basal forebrain. Taken together, we show that ACh neuron populations are present and become cholinergic before postnatal day 12, which is the onset of major sensory processes, such as hearing and vision. We conclude that birth of ACh neurons and initiation of cholinergic gene expression are temporally separated during development but highly coordinated by brain anatomical structure. Acetylcholine (ACh) neurons are required for higher brain functions and locomotion. Disturbed cholinergic signaling was observed in neurodevelopmental disorders and intellectual disability. While the role of ACh release in neural circuit function is well understood, it is unknown when ACh neurons become cholinergic. We labelled ACh neurons to investigate when ACh neurons become cholinergic in the developing brain and performed reconstructions of serial sectioned brains. Here, we show that ACh neuron populations become cholinergic during three time windows pre- and postnatally. ACh neurons become cholinergic following the caudorostral direction of brain formation. In cortex and hippocampus, activation of cholinergic gene expression in ACh interneurons coincides with cholinergic innervation from the basal forebrain. We highlight that brain ACh neurons are cholinergic before P12, the onset of major sensory functions, such as hearing and vision.
PubMed: 38942474
DOI: 10.1523/ENEURO.0542-23.2024 -
Stem Cell Reports Jun 2024Genetic differences between pluripotent stem cell lines cause variable activity of extracellular signaling pathways, limiting reproducibility of directed differentiation...
Genetic differences between pluripotent stem cell lines cause variable activity of extracellular signaling pathways, limiting reproducibility of directed differentiation protocols. Here we used human embryonic stem cells (hESCs) to interrogate how exogenous factors modulate endogenous signaling events during specification of foregut endoderm lineages. We find that transforming growth factor β1 (TGF-β1) activates a putative human OTX2/LHX1 gene regulatory network which promotes anterior fate by antagonizing endogenous Wnt signaling. In contrast to Porcupine inhibition, TGF-β1 effects cannot be reversed by exogenous Wnt ligands, suggesting that induction of SHISA proteins and intracellular accumulation of Fzd receptors render TGF-β1-treated cells refractory to Wnt signaling. Subsequently, TGF-β1-mediated inhibition of BMP and Wnt signaling suppresses liver fate and promotes pancreas fate. Furthermore, combined TGF-β1 treatment and Wnt inhibition during pancreatic specification reproducibly and robustly enhance INSULIN cell yield across hESC lines. This modification of widely used differentiation protocols will enhance pancreatic β cell yield for cell-based therapeutic applications.
PubMed: 38942030
DOI: 10.1016/j.stemcr.2024.05.010 -
Cell Genomics Jun 2024Mycena s.s. is a ubiquitous mushroom genus whose members degrade multiple dead plant substrates and opportunistically invade living plant roots. Having sequenced the...
Mycena s.s. is a ubiquitous mushroom genus whose members degrade multiple dead plant substrates and opportunistically invade living plant roots. Having sequenced the nuclear genomes of 24 Mycena species, we find them to defy the expected patterns for fungi based on both their traditionally perceived saprotrophic ecology and substrate specializations. Mycena displayed massive genome expansions overall affecting all gene families, driven by novel gene family emergence, gene duplications, enlarged secretomes encoding polysaccharide degradation enzymes, transposable element (TE) proliferation, and horizontal gene transfers. Mainly due to TE proliferation, Arctic Mycena species display genomes of up to 502 Mbp (2-8× the temperate Mycena), the largest among mushroom-forming Agaricomycetes, indicating a possible evolutionary convergence to genomic expansions sometimes seen in Arctic plants. Overall, Mycena show highly unusual, varied mosaic-like genomic structures adaptable to multiple lifestyles, providing genomic illustration for the growing realization that fungal niche adaptations can be far more fluid than traditionally believed.
PubMed: 38942024
DOI: 10.1016/j.xgen.2024.100586 -
EBioMedicine Jun 2024Drug development for atrial fibrillation (AF) has failed to yield new approved compounds. We sought to identify and prioritise potential druggable targets with support...
BACKGROUND
Drug development for atrial fibrillation (AF) has failed to yield new approved compounds. We sought to identify and prioritise potential druggable targets with support from human genetics, by integrating the available evidence with bioinformatics sources relevant for AF drug development.
METHODS
Genetic hits for AF and related traits were identified through structured search of MEDLINE. Genes derived from each paper were cross-referenced with the OpenTargets platform for drug interactions. Confirmation/validation was demonstrated through structured searches and review of evidence on MEDLINE and ClinialTrials.gov for each drug and its association with AF.
FINDINGS
613 unique drugs were identified, with 21 already included in AF Guidelines. Cardiovascular drugs from classes not currently used for AF (e.g. ranolazine and carperitide) and anti-inflammatory drugs (e.g. dexamethasone and mehylprednisolone) had evidence of potential benefit. Further targets were considered druggable but remain open for drug development.
INTERPRETATION
Our systematic approach, combining evidence from different bioinformatics platforms, identified drug repurposing opportunities and druggable targets for AF.
FUNDING
KK is supported by Barts Charity grant G-002089 and is mentored on the AFGen 2023-24 Fellowship funded by the AFGen NIH/NHLBI grant R01HL092577. RP is supported by the UCL BHF Research Accelerator AA/18/6/34223 and NIHR grant NIHR129463. AFS is supported by the BHF grants PG/18/5033837, PG/22/10989 and UCL BHF Accelerator AA/18/6/34223 as well as the UK Research and Innovation (UKRI) under the UK government's Horizon Europe funding guarantee EP/Z000211/1 and by the UKRI-NIHR grant MR/V033867/1 for the Multimorbidity Mechanism and Therapeutics Research Collaboration. AF is supported by UCL BHF Accelerator AA/18/6/34223. CF is supported by UCL BHF Accelerator AA/18/6/34223.
PubMed: 38941956
DOI: 10.1016/j.ebiom.2024.105194 -
Current Opinion in Plant Biology Jun 2024Plant hormones activate receptors, initiating intracellular signaling pathways. Eventually, hormone-specific transcription factors become active in the nucleus,... (Review)
Review
Plant hormones activate receptors, initiating intracellular signaling pathways. Eventually, hormone-specific transcription factors become active in the nucleus, facilitating hormone-induced transcriptional regulation. Chromatin plays a fundamental role in the regulation of transcription, the process by which genetic information encoded in DNA is converted into RNA. The structure of chromatin, a complex of DNA and proteins, directly influences the accessibility of genes to the transcriptional machinery. The different signaling pathways and transcription factors involved in the transmission of information from the receptors to the nucleus have been readily explored, but not so much for the specific mechanisms employed by the cell to ultimately instruct the chromatin changes necessary for a fast and robust transcription activation, specifically for plant hormone responses. In this review, we will focus on the advancements in understanding how chromatin receives plant hormones, facilitating the changes necessary for fast, robust, and specific transcriptional regulation.
PubMed: 38941723
DOI: 10.1016/j.pbi.2024.102592 -
Science Advances Jun 2024The blood-brain barrier (BBB) acts as the crucial physical filtration structure in the central nervous system. Here, we investigate the role of a specific subset of...
The blood-brain barrier (BBB) acts as the crucial physical filtration structure in the central nervous system. Here, we investigate the role of a specific subset of astrocytes in the regulation of BBB integrity. We showed that expressing astrocytes transfer mitochondria to endothelial cells via their endfeet for maintaining BBB integrity. Deletion of the Mitofusin 2 () gene in -expressing astrocytes inhibited the mitochondrial transfer and caused BBB leakage. In addition, the decrease of MFN2 in astrocytes contributes to the age-associated reduction of mitochondrial transfer efficiency and thus compromises the integrity of BBB. Together, we describe a mechanism in which astrocytes regulate BBB integrity through mitochondrial transfer. Our findings provide innnovative insights into the cellular framework that underpins the progressive breakdown of BBB associated with aging and disease.
Topics: Astrocytes; Blood-Brain Barrier; Animals; Mitochondria; Mice; Endothelial Cells; GTP Phosphohydrolases
PubMed: 38941455
DOI: 10.1126/sciadv.adk2913 -
PloS One 2024Prenatal alcohol exposure (PAE) causes cognitive impairment and a distinctive craniofacial dysmorphology, due in part to apoptotic losses of the pluripotent cranial...
Prenatal alcohol exposure (PAE) causes cognitive impairment and a distinctive craniofacial dysmorphology, due in part to apoptotic losses of the pluripotent cranial neural crest cells (CNCs) that form facial bones and cartilage. We previously reported that PAE rapidly represses expression of >70 ribosomal proteins (padj = 10-E47). Ribosome dysbiogenesis causes nucleolar stress and activates p53-MDM2-mediated apoptosis. Using primary avian CNCs and the murine CNC line O9-1, we tested whether nucleolar stress and p53-MDM2 signaling mediates this apoptosis. We further tested whether haploinsufficiency in genes that govern ribosome biogenesis, using a blocking morpholino approach, synergizes with alcohol to worsen craniofacial outcomes in a zebrafish model. In both avian and murine CNCs, pharmacologically relevant alcohol exposure (20mM, 2hr) causes the dissolution of nucleolar structures and the loss of rRNA synthesis; this nucleolar stress persisted for 18-24hr. This was followed by reduced proliferation, stabilization of nuclear p53, and apoptosis that was prevented by overexpression of MDM2 or dominant-negative p53. In zebrafish embryos, low-dose alcohol or morpholinos directed against ribosomal proteins Rpl5a, Rpl11, and Rps3a, the Tcof homolog Nolc1, or mdm2 separately caused modest craniofacial malformations, whereas these blocking morpholinos synergized with low-dose alcohol to reduce and even eliminate facial elements. Similar results were obtained using a small molecule inhibitor of RNA Polymerase 1, CX5461, whereas p53-blocking morpholinos normalized craniofacial outcomes under high-dose alcohol. Transcriptome analysis affirmed that alcohol suppressed the expression of >150 genes essential for ribosome biogenesis. We conclude that alcohol causes the apoptosis of CNCs, at least in part, by suppressing ribosome biogenesis and invoking a nucleolar stress that initiates their p53-MDM2 mediated apoptosis. We further note that the facial deficits that typify PAE and some ribosomopathies share features including reduced philtrum, upper lip, and epicanthal distance, suggesting the facial deficits of PAE represent, in part, a ribosomopathy.
Topics: Animals; Neural Crest; Zebrafish; Ribosomes; Ethanol; Tumor Suppressor Protein p53; Apoptosis; Mice; Proto-Oncogene Proteins c-mdm2; Cell Nucleolus; Ribosomal Proteins; Skull; Zebrafish Proteins
PubMed: 38941348
DOI: 10.1371/journal.pone.0304557 -
ELife Jun 2024Genetic diversity is a hallmark of RNA viruses and the basis for their evolutionary success. Taking advantage of the uniquely large genomic database of SARS-CoV-2, we...
Genetic diversity is a hallmark of RNA viruses and the basis for their evolutionary success. Taking advantage of the uniquely large genomic database of SARS-CoV-2, we examine the impact of mutations across the spectrum of viable amino acid sequences on the biophysical phenotypes of the highly expressed and multifunctional nucleocapsid protein. We find variation in the physicochemical parameters of its extended intrinsically disordered regions (IDRs) sufficient to allow local plasticity, but also observe functional constraints that similarly occur in related coronaviruses. In biophysical experiments with several N-protein species carrying mutations associated with major variants, we find that point mutations in the IDRs can have nonlocal impact and modulate thermodynamic stability, secondary structure, protein oligomeric state, particle formation, and liquid-liquid phase separation. In the Omicron variant, distant mutations in different IDRs have compensatory effects in shifting a delicate balance of interactions controlling protein assembly properties, and include the creation of a new protein-protein interaction interface in the N-terminal IDR through the defining P13L mutation. A picture emerges where genetic diversity is accompanied by significant variation in biophysical characteristics of functional N-protein species, in particular in the IDRs.
Topics: SARS-CoV-2; Coronavirus Nucleocapsid Proteins; Mutation; COVID-19; Humans; Intrinsically Disordered Proteins; Phosphoproteins; Nucleocapsid Proteins; Thermodynamics; Protein Stability
PubMed: 38941236
DOI: 10.7554/eLife.94836 -
Cell Biology and Toxicology Jun 2024Structural cardiotoxicity (SCT) presents a high-impact risk that is poorly tolerated in drug discovery unless significant benefit is anticipated. Therefore, we aimed to...
Structural cardiotoxicity (SCT) presents a high-impact risk that is poorly tolerated in drug discovery unless significant benefit is anticipated. Therefore, we aimed to improve the mechanistic understanding of SCT. First, we combined machine learning methods with a modified calcium transient assay in human-induced pluripotent stem cell-derived cardiomyocytes to identify nine parameters that could predict SCT. Next, we applied transcriptomic profiling to human cardiac microtissues exposed to structural and non-structural cardiotoxins. Fifty-two genes expressed across the three main cell types in the heart (cardiomyocytes, endothelial cells, and fibroblasts) were prioritised in differential expression and network clustering analyses and could be linked to known mechanisms of SCT. This transcriptomic fingerprint may prove useful for generating strategies to mitigate SCT risk in early drug discovery.
Topics: Humans; Cardiotoxicity; Transcriptome; Myocytes, Cardiac; Induced Pluripotent Stem Cells; Gene Expression Profiling; Computational Biology; Machine Learning; Cardiotoxins; Fibroblasts; Endothelial Cells
PubMed: 38940987
DOI: 10.1007/s10565-024-09880-7 -
MBio Jun 2024Conjugative type 4 secretion systems (T4SSs) are the main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. To deliver the DNA...
Conjugative type 4 secretion systems (T4SSs) are the main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. To deliver the DNA substrate to recipient cells, it must cross the cell envelopes of both donor and recipient bacteria. In the T4SS from the enterococcal conjugative plasmid pCF10, PrgK is known to be the active cell wall degrading enzyme. It has three predicted extracellular hydrolase domains: metallo-peptidase (LytM), soluble lytic transglycosylase (SLT), and cysteine, histidine-dependent amidohydrolases/peptidases (CHAP). Here, we report the structure of the LytM domain and show that its active site is degenerate and lacks the active site metal. Furthermore, we show that only the predicted SLT domain is functional and that it unexpectedly has a muramidase instead of a lytic transglycosylase activity. While we did not observe any peptidoglycan hydrolytic activity for the LytM or CHAP domain, we found that these domains downregulated the SLT muramidase activity. The CHAP domain was also found to be involved in PrgK dimer formation. Furthermore, we show that PrgK interacts with PrgL, which likely targets PrgK to the rest of the T4SS. The presented data provides important information for understanding the function of Gram-positive T4SSs.IMPORTANCEAntibiotic resistance is a large threat to human health and is getting more prevalent. One of the major contributors to the spread of antibiotic resistance among different bacteria is type 4 secretion systems (T4SS). However, mainly T4SSs from Gram-negative bacteria have been studied in detail. T4SSs from Gram-positive bacteria, which stand for more than half of all hospital-acquired infections, are much less understood. The significance of our research is in identifying the function and regulation of a cell wall hydrolase, a key component of the pCF10 T4SS from . This system is one of the best-studied Gram-positive T4SSs, and this added knowledge aids in our understanding of horizontal gene transfer in as well as other medically relevant Gram-positive bacteria.
PubMed: 38940556
DOI: 10.1128/mbio.00488-24