12,634 research outputs found

    Complement mediated synapse elimination in schizophrenia

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    Schizophrenia (SCZ) is a devastating psychiatric disorder with a typically age of onset in late adolescence. The heritability is estimated to be in between 60-80% and large-scale genome-wide studies have revealed a prominent polygenic component to SCZ risk and identified more than three-hundred common risk variants. Despite a better understanding of which genetic risk variants that increases SCZ risk, it has been challenging to map out the pathophysiology of the disorder. This has stalled the development of target drugs and current treatment options display moderate efficacy and are prone to produce side-effects. SCZ is generally considered a neurodevelopmental disorder and it was proposed more than forty years ago that physiological removal of less active synapses in adolescence, i.e., synaptic pruning, is increased in SCZ and hereby causes the core symptoms of the disorder. This theory has then been supported by post-mortem brain tissue and imaging studies displaying decreased synapse density in SCZ. More recently, it was then shown that the most strongly associated risk loci can largely be explained by copy numbers of a gene coding for the complement factor 4A (C4A). As microglia prune synapses with the help of complement signalling, we therefore decided to use a recently developed human 2D in vitro assay to assess microglial uptake of synaptic structures in models based on cells from individuals with SCZ and healthy controls (study I). We observed excessive uptake of synaptic structures in SCZ models and by mixing synapses from healthy controls with microglia from SCZ patients, and vice versa, we showed the contribution of microglial and neuronal factors contributing to this excessive uptake of synaptic structures. We then developed an in vitro assay to study neuronal complement deposition dependent on copy numbers of C4A in the neuronal lines. Complement 3 (C3) deposition increased by C4A copy numbers but was independent of C4B copy numbers (also unrelated to SCZ risk). Similar C4A copy numbers correlated with the extent of microglial uptake of synapses. Microglial uptake of synaptic structures could also be inhibited by the tetracycline minocycline that also decreased risk of developing SCZ in an electronic health record cohort. In study II, we cerebrospinal fluid (CSF) from first-episode psychosis patients to measure protein levels of C4A. In two independent cohorts, we observed elevated C4A levels (although not C4B levels) in first-episode patients that later were to develop SCZ and could show correlations with markers of synapse density. However, elevated C4A levels could not fully be explained by more copy numbers of C4A in individuals with SCZ and in vitro experiments revealed that SCZ-associated cytokines can induce C4A mRNA expression while also correlating with C4A in patient-derived CSF. In study III, we set-up a 3D brain organoid models to more fully comprehensively capture processes in the developing human brain and then also included innately developing microglia. We display synaptic pruning within these models and use single cell RNA sequencing to validate them. In conclusion, this thesis uses patient-derived cellular modelling to uncover a disease mechanism in SCZ that link genetic risk variants with bona fide protein changes in living patients

    Reprogramming viral immune evasion for a rational design of next-generation vaccines for RNA viruses

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    Type I interferons (IFNs-α/β) are antiviral cytokines that constitute the innate immunity of hosts to fight against viral infections. Recent studies, however, have revealed the pleiotropic functions of IFNs, in addition to their antiviral activities, for the priming of activation and maturation of adaptive immunity. In turn, many viruses have developed various strategies to counteract the IFN response and to evade the host immune system for their benefits. The inefficient innate immunity and delayed adaptive response fail to clear of invading viruses and negatively affect the efficacy of vaccines. A better understanding of evasion strategies will provide opportunities to revert the viral IFN antagonism. Furthermore, IFN antagonism-deficient viruses can be generated by reverse genetics technology. Such viruses can potentially serve as next-generation vaccines that can induce effective and broad-spectrum responses for both innate and adaptive immunities for various pathogens. This review describes the recent advances in developing IFN antagonism-deficient viruses, their immune evasion and attenuated phenotypes in natural host animal species, and future potential as veterinary vaccines

    Sanfilippo syndrome: molecular basis, disease models and therapeutic approaches

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    Sanfilippo syndrome or mucopolysaccharidosis III is a lysosomal storage disorder caused by mutations in genes responsible for the degradation of heparan sulfate, a glycosaminoglycan located in the extracellular membrane. Undegraded heparan sulfate molecules accumulate within lysosomes leading to cellular dysfunction and pathology in several organs, with severe central nervous system degeneration as the main phenotypical feature. The exact molecular and cellular mechanisms by which impaired degradation and storage lead to cellular dysfunction and neuronal degeneration are still not fully understood. Here, we compile the knowledge on this issue and review all available animal and cellular models that can be used to contribute to increase our understanding of Sanfilippo syndrome disease mechanisms. Moreover, we provide an update in advances regarding the different and most successful therapeutic approaches that are currently under study to treat Sanfilippo syndrome patients and discuss the potential of new tools such as induced pluripotent stem cells to be used for disease modeling and therapy development

    RNA pull-down-confocal nanoscanning (RP-CONA), a novel method for studying RNA/protein interactions in cell extracts that detected potential drugs for Parkinson’s disease targeting RNA/HuR complexes

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    MicroRNAs (miRNAs, miRs) are a class of small non-coding RNAs that regulate gene expression through specific base-pair targeting. The functional mature miRNAs usually undergo a two-step cleavage from primary miRNAs (pri-miRs), then precursor miRNAs (pre-miRs). The biogenesis of miRNAs is tightly controlled by different RNA-binding proteins (RBPs). The dysregulation of miRNAs is closely related to a plethora of diseases. Targeting miRNA biogenesis is becoming a promising therapeutic strategy. HuR and MSI2 are both RBPs. MiR-7 is post-transcriptionally inhibited by the HuR/MSI2 complex, through a direct interaction between HuR and the conserved terminal loop (CTL) of pri-miR-7-1. Small molecules dissociating pri-miR-7/HuR interaction may induce miR-7 production. Importantly, the miR-7 levels are negatively correlated with Parkinson’s disease (PD). PD is a common, incurable neurodegenerative disease causing serious motor deficits. A hallmark of PD is the presence of Lewy bodies in the human brain, which are inclusion bodies mainly composed of an aberrantly aggregated protein named α-synuclein (α-syn). Decreasing α-syn levels or preventing α-syn aggregation are under investigation as PD treatments. Notably, α-syn is negatively regulated by several miRNAs, including miR-7, miR-153, miR-133b and others. One hypothesis is that elevating these miRNA levels can inhibit α-syn expression and ameliorate PD pathologies. In this project, we identified miR-7 as the most effective α-syn inhibitor, among the miRNAs that are downregulated in PD, and with α-syn targeting potentials. We also observed potential post-transcriptional inhibition on miR-153 biogenesis in neuroblastoma, which may help to uncover novel therapeutic targets towards PD. To identify miR-7 inducers that benefit PD treatment by repressing α-syn expression, we developed a novel technique RNA Pull-down Confocal Nanoscaning (RP-CONA) to monitor the binding events between pri-miR-7 and HuR. By attaching FITC-pri-miR-7-1-CTL-biotin to streptavidin-coated agarose beads and incubating them in human cultured cell lysates containing overexpressed mCherry-HuR, the bound RNA and protein can be visualised as quantifiable fluorescent rings in corresponding channels in a confocal high-content image system. A pri-miR-7/HuR inhibitor can decrease the relative mCherry/FITC intensity ratio in RP-CONA. With this technique, we performed several small-scale screenings and identified that a bioflavonoid, quercetin can largely dissociate the pri-miR-7/HuR interaction. Further studies proved that quercetin was an effective miR-7 inducer as well as α-syn inhibitor in HeLa cells. To understand the mechanism of quercetin mediated α-syn inhibition, we tested the effects of quercetin treatment with miR-7-1 and HuR knockout HeLa cells. We found that HuR was essential in this pathway, while miR-7 hardly contributed to the α-syn inhibition. HuR can directly bind an AU-rich element (ARE) at the 3’ untranslated region (3’-UTR) of α-syn mRNA and promote translation. We believe quercetin mainly disrupts the ARE/HuR interaction and disables the HuR-induced α-syn expression. In conclusion, we developed and optimised RP-CONA, an on-bead, lysate-based technique detecting RNA/protein interactions, as well as identifying RNA/protein modulators. With RP-CONA, we found quercetin inducing miR-7 biogenesis, and inhibiting α-syn expression. With these beneficial effects, quercetin has great potential to be applied in the clinic of PD treatment. Finally, RP-CONA can be used in many other RNA/protein interactions studies

    Mapping Super-Relaxed States of Myosin Heads in Sarcomeres using Oblique Angle Fluorescent Microscopy

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    We have utilised modern methods of super-resolution fluorescent microscopy to spatially map fluorescently labelled ATP molecules in relaxed rabbit psoas skeletal muscles. For our imaging process, we have labelled ATP molecules with Rhodamine and Z-lines with Alexa488. Data from imaging these fluorophores have been collected using oblique angle fluorescent microscopy and further analysed to map super relaxed states (SRX) of myosin heads on the thick filament. Our experiments have concluded that most SRX of myosin heads were found in the C-zone of the thick filament, while other zones of thick filament had smaller populations of SRX. Further introduction of mavacamten (MAVA) to our imaging system has revealed an increase in SRX in both D and P zones, while the C zone population of SRX had remained constant. Further experiments must be conducted to establish a clear pattern and further proof our findings

    Investigation of a Histidine-Based Probe for the Exploration of Proteomes

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    Leishmaniasis is a neglected tropical disease which affects 0.7-1 million people per year. Current chemotherapies for leishmaniasis are toxic with long treatment times and reports of increasing resistance, which stresses the importance of this research area. Inositol phosphorylceramide synthase is a membrane bound enzyme that has no direct human homologue, which converts ceramide to inositol phosphorylceramide through the action of a highly conserved HHD catalytic triad. An ideal method to study this enzyme further would be through activity-based protein profiling, however, there are currently no activity-based probes reported that reacts with this type of active site. Therefore, an activity-based probe was designed based on the structure of diethyl pyrocarbonate, a compound known to bind covalently to active site histidine residues. The synthesised activity-based probe was shown to inhibit Leishmania major inositol phosphorylceramide synthase in a simple assay. In addition, the probe was shown to selectively bind to the active site histidine residue in two pure enzyme models; one of which has the same catalytic triad as inositol phosphorylceramide synthase, and the other was an acid base active site histidine residue. Further, this activity-based probe was able to isolate an overexpressed enzyme in the lysate of Escherichia coli as well as bind to intrinsic proteins. Following the function validation of the activity-based probe, preliminary work was started in Leishmania to isolate proteins identify expressed enzymes

    Hunting Wildlife in the Tropics and Subtropics

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    The hunting of wild animals for their meat has been a crucial activity in the evolution of humans. It continues to be an essential source of food and a generator of income for millions of Indigenous and rural communities worldwide. Conservationists rightly fear that excessive hunting of many animal species will cause their demise, as has already happened throughout the Anthropocene. Many species of large mammals and birds have been decimated or annihilated due to overhunting by humans. If such pressures continue, many other species will meet the same fate. Equally, if the use of wildlife resources is to continue by those who depend on it, sustainable practices must be implemented. These communities need to remain or become custodians of the wildlife resources within their lands, for their own well-being as well as for biodiversity in general. This title is also available via Open Access on Cambridge Core

    Investigating PAX6 and SOX2 dynamic interactions at the single molecule level in live cells

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    The abundance of transcription factor (TF) molecules in the nuclei of eukaryotic cells are in the range of thousands. However, the functional binding sites of most TFs lie in the range of hundreds. This suggests that there is a surplus of the number of molecules for many TFs, relative to their binding sites at any given time. Nevertheless, precise TF levels are instrumental for normal development and maintenance, with haploinsufficiency (namely lowering the dosage of a TF by half) being a hallmark of many TF-related human developmental disorders. Qualitative methods assessing TF binding such as chromatin immunoprecipitation, provide static information, from fixed cell populations and so fail to provide insight into TF dynamic behaviour. Live-cell imaging methodologies such as Fluorescence Correlation Spectroscopy (FCS) offer the ability to measure kinetics of binding to chromatin, protein-protein interactions, absolute concentrations of molecules and the underlying cell-to-cell variability. SOX2 and PAX6 TFs exhibit haploinsufficiency in humans. Heterozygous point mutations, deletions or insertions in these genes can lead to a plethora of abnormal ocular developmental disorders (e.g. coloboma, aniridia, microphthalmia, anopthalmia). SOX2 encodes a high-mobility group (HMG) domain-containing TF, essential for maintaining self-renewal of embryonic stem cells and is expressed in proliferating central nervous system (CNS) progenitors. PAX6 contains two DNA binding domains; a PAIRED domain (PD) and a homeodomain (HD). Both DNA binding domains present in PAX6 (PD and HD) can function either jointly, or separately, to regulate a plethora of genes implicated in the development and maintenance of the CNS, the eye and the pancreas. Despite existing genetic and phenotypic evidence, it remains unclear how PAX6 and SOX2 influence each other at the molecular level and how sensitive their stoichiometry is during ocular development. In this thesis I investigated the dynamic interplay between PAX6/SOX2 and chromatin in live cells, at the molecular level. I compared wild-type protein function with pathogenic missense variants using advanced fluorescence microscopy techniques and assessed how these mutations quantitatively and qualitatively affected molecular behaviour. My results showed that both SOX2 and PAX6 pathogenic missense mutants display differential subnuclear localisation, as well as altered protein-protein and protein-chromatin interactions, linking molecular diffusion to pathogenic phenotype in humans. More importantly, I identified a novel role of SOX2 in stabilising PAX6- chromatin complexes in live cells, providing further insight into the complex and dynamic relation of PAX6 and SOX2 in ocular tissue specification, maintenance and development
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