Biology subjects

Davey, T.

Publications and source records attributed to Davey, T..

6 recordsLinked to original sources

bioRxiv2023

Mitochondrial morphology and function in mitochondrial disease

Mitochondria play a crucial role in maintaining cellular health. It is interesting that the shape of mitochondria can vary depending on the type of cell, mitochondrial function, and other cellular conditions. The morphology of this network is maintained by the balance between mitochondrial fusion and fission, necessary for the proper functioning of the mitochondrial energetics as well as the maintenance of mitochondria DNA (mtDNA) (Chan, 2006; Friedman and Nunnari, 2014; Glancy et al., 2015). If the balance between the two processes is slightly perturbed, dramatic changes in mitochondrial morphology can occur. However, there are limited studies that link functional assessment with mitochondrial morphology evaluation at high magnification, even fewer that do so in situ and none in human muscle biopsies. Therefore, we have developed a method which combines functional assessment of mitochondria through Cytochrome c Oxidase (COX) histochemistry, with a 3D electron microscopy (EM) technique, serial block-face scanning electron microscopy (SBFSEM). Using this technique, we examine the relationship between 3D mitochondrial morphology and mitochondrial activity in human skeletal muscle. Here we apply COX-SBFSEM to muscle samples from patients with single, large-scale mtDNA deletions, a cause of mitochondrial disease. These deletions cause oxidative phosphorylation deficiency, which can be observed through changes in COX activity, and a typical mosaic pattern of enzyme deficiency with both COX normal and deficient fibres present in the same biopsy. Using COX-SBFSEM, we can distinguish three distinct mitochondrial populations within muscle fibres: COX normal (+), COX intermediate ({+/-}) and COX deficient (-) mitochondria. COX+ mitochondria were larger and more complex than the COX- mitochondria, However, in fibres that were generally COX-, there was no apparent difference between the three types of mitochondria and all mitochondria in fibres that are COX- overall are fragmented and spherical. One of the main advantages of combining 3D-EM with the COX reaction is the ability to look at how per-mitochondrion oxidative phosphorylation status is spatially distributed within muscle fibres. Here we show a robust spatial pattern in fibres that are generally COX+ and that the spatial pattern is less clear in fibres that are predominantly COX{+/-} and COX- .

molecular biology↗

bioRxiv2023

Urolithin A induces cardioprotection and enhanced mitochondrial quality during natural aging and heart failure

Cardiovascular diseases remain the primary cause of global mortality, necessitating effective strategies to alleviate their burden. Mitochondrial dysfunction is a driving force behind aging and chronic conditions, including heart disease. Here, we investigate the potential of Urolithin A (UA), a gut microbiome-derived postbiotic that enhances mitophagy, to ameliorate both age-related decline in cardiac function and cardiac failure. We highlight the significance of targeting mitochondria, by comparing gene expression changes in aging human hearts and cardiomyopathies. UA oral administration successfully counteracts mitochondrial and cardiac dysfunctions in preclinical models of aging and heart failure. UA improves both systolic and diastolic heart functions, distinguishing it from other mitochondrial interventions. In cardiomyocytes, UA recovers mitochondrial ultrastructural defects and decline in mitochondrial biomarkers occurring with aging and disease. These findings extend UAs benefits to heart health, making UA a promising nutritional intervention to evaluate in the clinic to promote healthy cardiovascular function as we age.

cell biology↗

bioRxiv2021

Progressive protein aggregation in PRPF31 patient retinal pigment epithelium cells: the mechanism and its reversal through activation of autophagy

Mutations in pre-mRNA processing factor 31 (PRPF31), a core protein of the spliceosomal tri-snRNP complex, cause autosomal-dominant retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiquitously expressed tri-snRNP proteins result in retina-specific disorders, and so far, the underlying mechanism of splicing factors-related RP is poorly understood. Here, we used iPSC technology to generate retinal organoids and RPE models from three patients with severe and very severe PRPF31-adRP, normal individuals and a CRISPR/Cas9-corrected isogenic control. To fully assess the impacts of PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells was carried out showing RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways to be significantly affected. Strikingly, the patient-derived RPE and retinal cells were characterised by the presence of large amounts of cytoplasmic aggregates containing the mutant PRPF31 and misfolded, ubiquitin-conjugated proteins including key visual cycle proteins, which accumulated progressively with time. Mutant PRPF31 variant was not incorporated into splicing complexes, but reduction of PRPF31 wildtype levels led to tri-snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells with reduced U4/U6 snRNPs and accumulation of U5, smaller nuclear speckles and reduced formation of active spliceosomes giving rise to global splicing dysregulation. Moreover, the impaired waste disposal mechanisms further exacerbated aggregate formation, and targeting these by activating the autophagy pathway using Rapamycin resulted in reduction of cytoplasmic aggregates and improved cell survival. Our data demonstrate that it is the progressive aggregate accumulation that overburdens the waste disposal machinery rather than direct PRPF31-initiated mis-splicing, and thus relieving the RPE cells from insoluble cytoplasmic aggregates presents a novel therapeutic strategy that can be combined with gene therapy studies to fully restore RPE and retinal cell function in PRPF31-adRP patients. HighlightsO_LIPRPF31 RP mutations lead to formation of insoluble aggregates containing the mutant PRPF31 and misfolded, ubiquitin conjugated proteins including key visual cycle proteins (e.g. RLBP1) in RPE cells, which accumulate progressively with time and affect tight junctions and cell survival. C_LIO_LIMutant PRPF31 is predominantly localised in cytoplasmic aggregates of patient specific RPE and retinal cells and is not able to be incorporated into splicing complexes to cause direct mis-splicing. C_LIO_LIHigh-throughput quantitative proteomics identifies significantly altered RNA splicing, visual perception, retinoid metabolism, waste disposal and unfolded protein response pathways in patient RPE cells, and autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways in photoreceptor cells. C_LIO_LIAccumulation of PRPF31 mutant variant as cytoplasmic aggregates reduces wildtype PRPF31 in the nucleus leading to tri-snRNP assembly defects, characterised by accumulation of U5 and reduction of U4/U6 snRNPs in Cajal bodies, altered morphology of nuclear speckles and consequently downregulation of active spliceosomes (Bact and C complexes) in PRPF31 patient RPE and retinal cells. C_LIO_LIProteomic study of insoluble aggregates identifies other RP-linked splicing factors and multiple key retinal-specific proteins, whose variants are linked to retinitis pigmentosa, within the aggregates of patient RPE cells. C_LIO_LIPRPF31 patient RPE cells have impaired waste disposal and proteasome mediated degradation, which together with the impaired autophagy pathway, further exacerbate aggregate formation. C_LIO_LIPhagocytosis of photoreceptor outer segment fragments (POS) shed daily by RPE cells accelerates aggregation of key proteins indicating enhanced cytoplasmic aggregate formation under physiological conditions in patient RPE cells. C_LIO_LIActivation of autophagy via administration of rapamycin results in reduction of cytoplasmic aggregates in RPE cells, correct localisation of mislocated and misfolded proteins to the nucleus, thereby improving cell survival. C_LI

cell biology↗

bioRxiv2021

3D Neuronal Mitochondrial Morphology in Axons, Dendrites, and Somata of the Aging Mouse Hippocampus

The brains ability to process complex informations relies on the constant supply of energy through aerobic respiration by mitochondria. Neurons contain three anatomically distinct compartments - the soma, dendrites, and projecting axons - which have different energetic and biochemical requirements, as well as different mitochondrial morphologies in cultured systems. Here we apply a quantitative three-dimensional electron microscopy approach to map mitochondrial network morphology and complexity in the mouse brain. We examine three neuronal sub-compartments - the soma, dendrites, myelinated axons - in the dentate gyrus and CA1 of the mouse hippocampus, two subregions with distinct principal cell types and functions. We also establish compartment-specific differences in mitochondrial morphology across these cell types between young and old mice, highlighting differences in age-related morphological recalibrations. Overall, these data define the nature of the neuronal mitochondrial network in the mouse hippocampus, providing a foundation to examine the role of mitochondrial morpho-function in the aging brain.

neuroscience↗

bioRxiv2021

Delayed induction of type I and III interferons and nasal epithelial cell permissiveness to SARS-CoV-2

The nasal epithelium is a plausible entry point for SARS-CoV-2, a site of pathogenesis and transmission, and may initiate the host response to SARS-CoV-2. Antiviral interferon (IFN) responses are critical to outcome of SARS-CoV-2. Yet little is known about the interaction between SARS-CoV-2 and innate immunity in this tissue. Here we applied single-cell RNA sequencing and proteomics to a primary cell model of human nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrated widespread tropism for nasal epithelial cell types. The host response was dominated by type I and III IFNs and interferon-stimulated gene products. This response was notably delayed in onset relative to viral gene expression and compared to other respiratory viruses. Nevertheless, once established, the paracrine IFN response began to impact on SARS-CoV-2 replication. When provided prior to infection, recombinant IFN{beta} or IFN{lambda}1 induced an efficient antiviral state that potently restricted SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data suggest that the IFN-I/III response to SARS-CoV-2 initiates in the nasal airway and suggest nasal delivery of recombinant IFNs to be a potential chemoprophylactic strategy.

immunology↗

bioRxiv2019

Missense Mutations in the Human Nanophthalmos Gene TMEM98 Cause Retinal Defects in the Mouse

PURPOSEWe previously found a dominant mutation, Rwhs, causing white spots on the retina accompanied by retinal folds. Here we identify the mutant gene to be Tmem98. In humans, mutations in the orthologous gene cause nanophthalmos. We modelled these mutations in mice and characterised the mutant eye phenotypes of these and Rwhs.\n\nMETHODSThe Rwhs mutation was identified to be a missense mutation in Tmem98 by genetic mapping and sequencing. The human TMEM98 nanophthalmos missense mutations were made in the mouse gene by CRISPR-Cas9. Eyes were examined by indirect ophthalmoscopy and the retinas imaged using a retinal camera. Electroretinography was used to study retinal function. Histology, immunohistochemistry and electron microscopy techniques were used to study adult eyes.\n\nRESULTSAn I135T mutation of Tmem98 causes the dominant Rwhs phenotype and is perinatally lethal when homozygous. Two dominant missense mutations of TMEM98, A193P and H196P are associated with human nanophthalmos. In the mouse these mutations cause recessive retinal defects similar to the Rwhs phenotype, either alone or in combination with each other, but do not cause nanophthalmos. The retinal folds did not affect retinal function as assessed by electroretinography. Within the folds there was accumulation of disorganised outer segment material as demonstrated by immunohistochemistry and electron microscopy, and macrophages had infiltrated into these regions.\n\nCONCLUSIONSMutations in the mouse orthologue of the human nanophthalmos gene TMEM98 do not result in small eyes. Rather, there is localised disruption of the laminar structure of the photoreceptors.

molecular biology↗