ABSTRACT
Loss of glycogen myophosphorylase (PYGM) expression results in an inability to break down muscle glycogen, leading to McArdle disease-an autosomal recessive metabolic disorder characterized by exercise intolerance and muscle cramps. While previously considered relatively benign, this condition has recently been associated with pattern dystrophy in the retina, accompanied by variable sight impairment, secondary to retinal pigment epithelial (RPE) cell involvement. However, the pathomechanism of this condition remains unclear. In this study, we generated a PYGM-null induced pluripotent stem cell line and differentiated it into mature RPE to examine structural and functional defects, along with metabolite release into apical and basal media. Mutant RPE exhibited normal photoreceptor outer segment phagocytosis but displayed elevated glycogen levels, reduced transepithelial resistance, and increased cytokine secretion across the epithelial layer compared to isogenic WT controls. Additionally, decreased expression of the visual cycle component, RDH11, encoding 11-cis-retinol dehydrogenase, was observed in PYGM-null RPE. While glycolytic flux and oxidative phosphorylation levels in PYGM-null RPE were near normal, the basal oxygen consumption rate was increased. Oxygen consumption rate in response to physiological levels of lactate was significantly greater in WT than PYGM-null RPE. Inefficient lactate utilization by mutant RPE resulted in higher glucose dependence and increased glucose uptake from the apical medium in the presence of lactate, suggesting a reduced capacity to spare glucose for photoreceptor use. Metabolic tracing confirmed slower 13C-lactate utilization by PYGM-null RPE. These findings have key implications for retinal health since they likely underlie the vision impairment in individuals with McArdle disease.
ABSTRACT
Most mammalian cells have a single primary cilium that acts as a signalling hub in mediating cellular functions. However, little is known about the mechanisms that result in aberrant supernumerary primary cilia per cell. In this study, we re-analysed a previously published whole-genome siRNA-based reverse genetic screen for genes mediating ciliogenesis to identify knockdowns that permit multi-ciliation. We identified siRNA knockdowns that caused significant formation of supernumerary cilia, validated candidate hits in different cell-lines and confirmed that RACGAP1, a component of the centralspindlin complex, was the strongest candidate hit at the whole-genome level. Following loss of RACGAP1, mother centrioles were specified correctly prior to ciliogenesis and the cilia appeared normal. Live cell imaging revealed that increased cilia incidence was caused by cytokinesis failure which led to the formation of multinucleate cells with supernumerary cilia. This suggests that the signalling mechanisms for ciliogenesis are unable to identify supernumerary centrosomes and therefore allow ciliation of duplicated centrosomes as if they were in a new diploid daughter cell. These results, demonstrating that aberrant ciliogenesis is de-coupled from cell cycle regulation, have functional implications in diseases marked by centrosomal amplification.
Subject(s)
Cilia , Cytokinesis , GTPase-Activating Proteins , Animals , Humans , Centrioles/metabolism , Centrosome/metabolism , Cilia/genetics , Cilia/metabolism , Mammals/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , GTPase-Activating Proteins/metabolismABSTRACT
5-HT2A, a G-protein coupled receptor, is widely expressed in the human body, including in the gastrointestinal tract, platelets and the nervous system. It mediates various functions, for e.g. learning, memory, mood regulation, platelet aggregation and vasoconstriction, but its involvement in cell-adhesion remains largely unknown. Here we report a novel role for 5-HT2A in cell-matrix adhesion.In HEK293 cells, which are loosely adherent, expression and stimulation of human or rat 5-HT2A receptor by agonists such as serotonin or 2,5-dimethoxy-4-iodoamphetamine (DOI) led to a significant increase in adhesion, while inhibition of 5-HT2A by antipsychotics, such as risperidone, olanzapine or chlorpromazine prevented it. 5-HT2A activation gave rise to stress fibers in these cells and was also required for their maintenance. Mechanistically, the 5-HT2A-mediated adhesion was mediated by downstream PKC and Rho signaling. Since 5-HT2A is associated with many disorders such as dementia, depression and schizophrenia, its role in cell-matrix adhesion could have implications for neural circuits.
Subject(s)
Cell-Matrix Junctions/genetics , Cell-Matrix Junctions/metabolism , Receptor, Serotonin, 5-HT2A/physiology , Serotonin 5-HT2 Receptor Agonists/pharmacology , Serotonin 5-HT2 Receptor Antagonists/pharmacology , Stress Fibers/drug effects , Stress Fibers/genetics , Stress Fibers/metabolism , Amphetamines/pharmacology , Animals , Antipsychotic Agents/pharmacology , Cell-Matrix Junctions/drug effects , Chlorpromazine/pharmacology , HEK293 Cells , Humans , Mental Disorders/etiology , Mental Disorders/genetics , Olanzapine/pharmacology , Rats , Risperidone/pharmacology , Serotonin/pharmacologyABSTRACT
Mutations in pre-mRNA processing factors (PRPFs) cause autosomal-dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause non-syndromic retinal disease. Here, we generate transcriptome profiles from RP11 (PRPF31-mutated) patient-derived retinal organoids and retinal pigment epithelium (RPE), as well as Prpf31+/- mouse tissues, which revealed that disrupted alternative splicing occurred for specific splicing programmes. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and Prpf31+/- mouse retinae and RPE. Mis-splicing of genes implicated in ciliogenesis and cellular adhesion was associated with severe RPE defects that include disrupted apical - basal polarity, reduced trans-epithelial resistance and phagocytic capacity, and decreased cilia length and incidence. Disrupted cilia morphology also occurred in patient-derived photoreceptors, associated with progressive degeneration and cellular stress. In situ gene editing of a pathogenic mutation rescued protein expression and key cellular phenotypes in RPE and photoreceptors, providing proof of concept for future therapeutic strategies.
Subject(s)
Eye Proteins/metabolism , Retinitis Pigmentosa/etiology , Retinitis Pigmentosa/metabolism , Alternative Splicing/genetics , Alternative Splicing/physiology , Animals , Cell Adhesion/genetics , Cell Adhesion/physiology , Cell Differentiation/genetics , Cell Differentiation/physiology , Cilia/genetics , Cilia/metabolism , Cilia/physiology , Eye Proteins/genetics , Flow Cytometry , Humans , Immunohistochemistry , Induced Pluripotent Stem Cells/metabolism , Mice , Mutation/genetics , Organoids/cytology , Organoids/metabolism , RNA Splicing/genetics , RNA Splicing/physiology , Retina/cytology , Retina/metabolism , Retinitis Pigmentosa/geneticsABSTRACT
BACKGROUND: Ciliopathies are clinically diverse disorders of the primary cilium. Remarkable progress has been made in understanding the molecular basis of these genetically heterogeneous conditions; however, our knowledge of their morbid genome, pleiotropy, and variable expressivity remains incomplete. RESULTS: We applied genomic approaches on a large patient cohort of 371 affected individuals from 265 families, with phenotypes that span the entire ciliopathy spectrum. Likely causal mutations in previously described ciliopathy genes were identified in 85% (225/265) of the families, adding 32 novel alleles. Consistent with a fully penetrant model for these genes, we found no significant difference in their "mutation load" beyond the causal variants between our ciliopathy cohort and a control non-ciliopathy cohort. Genomic analysis of our cohort further identified mutations in a novel morbid gene TXNDC15, encoding a thiol isomerase, based on independent loss of function mutations in individuals with a consistent ciliopathy phenotype (Meckel-Gruber syndrome) and a functional effect of its deficiency on ciliary signaling. Our study also highlighted seven novel candidate genes (TRAPPC3, EXOC3L2, FAM98C, C17orf61, LRRCC1, NEK4, and CELSR2) some of which have established links to ciliogenesis. Finally, we show that the morbid genome of ciliopathies encompasses many founder mutations, the combined carrier frequency of which accounts for a high disease burden in the study population. CONCLUSIONS: Our study increases our understanding of the morbid genome of ciliopathies. We also provide the strongest evidence, to date, in support of the classical Mendelian inheritance of Bardet-Biedl syndrome and other ciliopathies.
Subject(s)
Cilia/genetics , Ciliary Motility Disorders/genetics , Ciliopathies/genetics , Encephalocele/genetics , Mutation/genetics , Polycystic Kidney Diseases/genetics , Alleles , Cilia/pathology , Ciliary Motility Disorders/pathology , Ciliopathies/pathology , DNA Mutational Analysis , Encephalocele/pathology , Genetic Association Studies , Genetic Heterogeneity , Genetic Predisposition to Disease , Humans , Phenotype , Polycystic Kidney Diseases/pathology , Retina/metabolism , Retina/pathology , Retinitis PigmentosaABSTRACT
Aberrant embryonic development of the hypothalamus and/or pituitary gland in humans results in congenital hypopituitarism (CH). Transcription factor 7-like 1 (TCF7L1), an important regulator of the WNT/ß-catenin signaling pathway, is expressed in the developing forebrain and pituitary gland, but its role during hypothalamo-pituitary (HP) axis formation or involvement in human CH remains elusive. Using a conditional genetic approach in the mouse, we first demonstrate that TCF7L1 is required in the prospective hypothalamus to maintain normal expression of the hypothalamic signals involved in the induction and subsequent expansion of Rathke's pouch progenitors. Next, we reveal that the function of TCF7L1 during HP axis development depends exclusively on the repressing activity of TCF7L1 and does not require its interaction with ß-catenin. Finally, we report the identification of two independent missense variants in human TCF7L1, p.R92P and p.R400Q, in a cohort of patients with forebrain and/or pituitary defects. We demonstrate that these variants exhibit reduced repressing activity in vitro and in vivo relative to wild-type TCF7L1. Together, our data provide support for a conserved molecular function of TCF7L1 as a transcriptional repressor during HP axis development in mammals and identify variants in this transcription factor that are likely to contribute to the etiology of CH.
Subject(s)
Hypothalamo-Hypophyseal System , Transcription Factor 7-Like 1 Protein/physiology , Animals , Cohort Studies , Humans , Mice , Pituitary Gland/abnormalities , Pituitary Gland/metabolism , Pituitary Gland/physiopathology , Prosencephalon/abnormalities , Prosencephalon/metabolismABSTRACT
Vertebrate left-right (LR) asymmetry originates at a transient left-right organizer (LRO), a ciliated structure where cilia play a crucial role in breaking symmetry. However, much remains unknown about the choreography of cilia biogenesis and resorption at this organ. We recently identified a mutation affecting NEK2, a member of the NIMA-like serine-threonine kinase family, in a patient with congenital heart disease associated with abnormal LR development. Here, we report how Nek2 acts through cilia to influence LR patterning. Both overexpression and knockdown of nek2 in Xenopus result in abnormal LR development and reduction of LRO cilia count and motility, phenotypes that are modified by interaction with the Hippo signaling pathway. nek2 knockdown leads to a centriole defect at the LRO, consistent with the known role of Nek2 in centriole separation. Nek2 overexpression results in premature ciliary resorption in cultured cells dependent on function of the tubulin deacetylase Hdac6. Finally, we provide evidence that the known interaction between Nek2 and Nup98, a nucleoporin that localizes to the ciliary base, is important for regulating cilium resorption. Together, these data show that Nek2 is a switch balancing ciliogenesis and resorption in the development of LR asymmetry.
Subject(s)
Body Patterning , Cilia/physiology , Gene Expression Regulation, Developmental , Protein Serine-Threonine Kinases/physiology , Xenopus Proteins/physiology , Animals , Centrioles/metabolism , Gene Knockdown Techniques , Histone Deacetylase 6 , Histone Deacetylases/metabolism , Homeodomain Proteins/metabolism , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Mice , Microscopy, Fluorescence , Mutation , NIMA-Related Kinases , Nuclear Pore Complex Proteins , Protein Serine-Threonine Kinases/genetics , RNA Interference , Signal Transduction , Transcription Factors/metabolism , Xenopus , Xenopus Proteins/genetics , Homeobox Protein PITX2ABSTRACT
Although fibroblast growth factor (FGF) signaling is critical for many developmental processes, the cellular mechanism(s) by which FGF exerts its effects remains obscure. In recent papers, Neugebauer et al. and Hong and Dawid focus on the role of FGF signaling on left-right axis patterning, showing that FGF functions at least in part via an effect on ciliogenesis.
Subject(s)
Body Patterning , Cilia/metabolism , Fibroblasts/metabolism , Animals , Fibroblast Growth Factors/metabolism , Signal TransductionABSTRACT
Serotonin is reported to be present in early embryos of many species and plays an important role in early patterning. Since it is a fluorophore, it can be directly visualized using fluorescence microscopy. Here, we use three-photon microscopy to image serotonin in live pre-implantation mouse embryos. We find that it is present as puncta averaging 1.3 square microns and in concentrations as high as 442 mM. The observed serotonin puncta were found to co-localize with mitochondria. Live embryos pre-incubated with serotonin showed a higher mitochondrial potential, indicating that it can modulate mitochondrial potential. Pre-implantation mouse embryos were also examined at various developmental stages for the presence of transcripts of the peripheral and neuronal forms of tryptophan hydroxylase (Tph1 and Tph2 respectively) and the classical serotonin transporter (Slc6a4). Transcripts of Tph2 were seen in oocytes and in two-cell stages, whereas transcripts of Tph1 were not detected at any stage. Transcripts of the transporter, Slc6a4, were present in all pre-implantation stages investigated. These results suggest that serotonin in embryos can arise from a combination of synthesis and uptake from the surrounding milieu.
Subject(s)
Blastocyst/chemistry , Mitochondria/chemistry , Serotonin/analysis , Animals , Female , Gene Expression Regulation, Developmental , Male , Mass Spectrometry , Mice , Mice, Inbred Strains , Microscopy, Fluorescence , Morula/chemistry , Oocytes/chemistry , Pregnancy , Reverse Transcriptase Polymerase Chain Reaction , Serotonin Plasma Membrane Transport Proteins/analysis , Tryptophan Hydroxylase/analysisABSTRACT
Cilia establish the vertebrate left-right (LR) axis and are integral to the development and function of the kidney, liver, and brain. Left-right asymmetry is established in the ciliated ventral node cells of the mouse. The chiral structure of the cilium provides a reference asymmetry to impose handed LR asymmetric development on the bilaterally symmetric vertebrate embryo. A ciliary mechanism of LR development is evolutionarily conserved, as ciliated organs essential to LR axis formation, called LR organizers, are found in other vertebrates, including rabbit, fish, and Xenopus. Mice with mutations affecting ciliary biogenesis, motility, or sensory function have abnormal LR development and abnormal development of the heart. The axonemal dynein heavy chain left-right dynein (lrd) localizes to the LR organizer and drives counterclockwise movement of node primary cilia. Node primary cilia are an admixture of 9 + 2 and 9 + 0 cilia. Mutations in lrd result in structurally normal, immotile node monocilia. In the mouse, coordinated, directional beating of motile node monocilia at the neural fold stage generates leftward flow of extraembryonic fluid surrounding the node (nodal flow). Nodal flow triggers a rise in intracellular calcium in cells at the left side of the node. The perinodal asymmetric rise in intracellular calcium generated by nodal flow subsequently leads to asymmetric gene expression and morphogenesis.