m6 A mRNA methylation in human brain is disrupted in Lewy body disorders.

AIMS: N6 -methyladenosine modification of RNA (m6 A) regulates translational control, which may influence neuronal dysfunction underlying neurodegenerative diseases. METHODS: Using microscopy and a machine learning approach, we performed cellular profiling of m6 A-RNA abundance and YTHDF1/YTHDF3 m6 A reader expression within four regions of the human brain from non-affected individuals and individuals with Parkinson's disease, dementia with Lewy bodies or mild cognitive impairment (MCI). RESULTS: In non-diseased tissue, we found that m6 A-modified RNAs showed cell-type and sub-compartment-specific variation. YTHDF1 and YTHDF3 showed opposing expression patterns in the cerebellum and the frontal and cingulate cortices. Machine learning quantitative image analysis revealed that m6 A-modified transcripts were significantly altered in localisation and abundance in disease tissue with significant decreases in m6 A-RNAs in Parkinson's disease, and significant increases in m6 A-RNA abundance in dementia with Lewy bodies. MCI tissue showed variability across regions but similar to DLB; in brain areas with an overall significant increase in m6 A-RNAs, modified RNAs within dendritic processes were reduced. Using mass spectrometry proteomic datasets to corroborate our findings, we found significant changes in YTHDF3 and m6 A anti-reader protein abundance in Alzheimer's disease (AD) and asymptomatic AD/MCI tissue and correlation with cognitive resilience. CONCLUSIONS: These results provide evidence for disrupted m6 A regulation in Lewy body diseases and a plausible mechanism through which RNA processing could contribute to the formation of Lewy bodies and other dementia-associated pathological aggregates. The findings suggest that manipulation of epitranscriptomic processes influencing translational control may lead to new therapeutic approaches for neurodegenerative diseases.

Modifying the m6A brain methylome by ALKBH5-mediated demethylation: a new contender for synaptic tagging.

Synaptic plasticity processes, which underlie learning and memory formation, require RNA to be translated local to synapses. The synaptic tagging hypothesis has previously been proposed to explain how mRNAs are available at specific activated synapses. However how RNA is regulated, and which transcripts are silenced or processed as part of the tagging process is still unknown. Modification of RNA by N6-methyladenosine (m6A/m) influences the cellular fate of mRNA. Here, by advanced microscopy, we showed that m6A demethylation by the eraser protein ALKBH5 occurs at active synaptic ribosomes and at synapses during short term plasticity. We demonstrated that at activated glutamatergic post-synaptic sites, both the YTHDF1 and YTHDF3 reader and the ALKBH5 eraser proteins increase in co-localisation to m6A-modified RNAs; but only the readers showed high co-localisation to modified RNAs during late-stage plasticity. The YTHDF1 and YTHFDF3 readers also exhibited differential roles during synaptic maturation suggesting that temporal and subcellular abundance may determine specific function. m6A-sequencing of human parahippocampus brain tissue revealed distinct white and grey matter m6A methylome profiles indicating that cellular context is a fundamental factor dictating regulated pathways. However, in both neuronal and glial cell-rich tissue, m6A effector proteins are themselves modified and m6A epitranscriptional and posttranslational modification processes coregulate protein cascades. We hypothesise that the availability m6A effector protein machinery in conjunction with RNA modification, may be important in the formation of condensed synaptic nanodomain assemblies through liquid-liquid phase separation. Our findings support that m6A demethylation by ALKBH5 is an intrinsic component of the synaptic tagging hypothesis and a molecular switch which leads to alterations in the RNA methylome, synaptic dysfunction and potentially reversible disease states.

Carbohydrates from Pseudomonas aeruginosa biofilms interact with immune C-type lectins and interfere with their receptor function.

Bacterial biofilms represent a challenge to the healthcare system because of their resilience against antimicrobials and immune attack. Biofilms consist of bacterial aggregates embedded in an extracellular polymeric substance (EPS) composed of polysaccharides, nucleic acids and proteins. We hypothesised that carbohydrates could contribute to immune recognition of Pseudomonas aeruginosa biofilms by engaging C-type lectins. Here we show binding of Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209), mannose receptor (MR, CD206) and Dectin-2 to P. aeruginosa biofilms. We also demonstrate that DC-SIGN, unlike MR and Dectin-2, recognises planktonic P. aeruginosa cultures and this interaction depends on the presence of the common polysaccharide antigen. Within biofilms DC-SIGN, Dectin-2 and MR ligands appear as discrete clusters with dispersed DC-SIGN ligands also found among bacterial aggregates. DC-SIGN, MR and Dectin-2 bind to carbohydrates purified from P. aeruginosa biofilms, particularly the high molecular weight fraction (HMW; >132,000 Da), with KDs in the nM range. These HMW carbohydrates contain 74.9-80.9% mannose, display α-mannan segments, interfere with the endocytic activity of cell-associated DC-SIGN and MR and inhibit Dectin-2-mediated cellular activation. In addition, biofilm carbohydrates reduce the association of the DC-SIGN ligand Lewisx, but not fucose, to human monocyte-derived dendritic cells (moDCs), and alter moDC morphology without affecting early cytokine production in response to lipopolysaccharide or P. aeruginosa cultures. This work identifies the presence of ligands for three important C-type lectins within P. aeruginosa biofilm structures and purified biofilm carbohydrates and highlights the potential for these receptors to impact immunity to P. aeruginosa infection.

Fluorescence imaging of single Kinesin motors on immobilized microtubules.

Recent developments in optical microscopy and nanometer tracking have greatly improved our understanding of cytoskeletal motor proteins. Using fluorescence microscopy, dynamic interactions are now routinely observed in vitro on the level of single molecules mainly using a geometry, where fluorescently labeled motors move on surface-immobilized filaments. In this chapter, we review recent methods related to single-molecule kinesin motility assays. In particular, we aim to provide practical advice on: how to set up the assays, how to acquire high-precision data from fluorescently labeled kinesin motors and attached quantum dots, and how to analyze data by nanometer tracking.

Urea-induced unfolding of the immunity protein Im9 monitored by spFRET.

We have studied the urea-induced unfolding of the E colicin immunity protein Im9 using diffusion single-pair fluorescence resonance energy transfer. Detailed examination of the proximity ratio of the native and denatured molecules over a wide range of urea concentrations suggests that the conformational properties of both species are denaturant-dependent. Whereas native molecules become gradually more expanded as urea concentration increases, denatured molecules show a dramatic dependence of the relationship between proximity ratio and denaturant concentration, consistent with substantial compaction of the denatured ensemble at low denaturant concentrations. Analysis of the widths of the proximity ratio distributions for each state suggests that whereas the native state ensemble is relatively narrow and homogeneous, the denatured state may possess heterogeneity in mildly denaturing conditions.