Biology subjects

Bradley, J.

Publications and source records attributed to Bradley, J..

4 recordsLinked to original sources

bioRxiv2018

Fast two-photon volumetric imaging of an improved voltage indicator reveals electrical activity in deeply located neurons in the awake brain

Imaging of transmembrane voltage deep in brain tissue with cellular resolution has the potential to reveal information processing by neuronal circuits in living animals with minimal perturbation. Multi-photon voltage imaging in vivo, however, is currently limited by speed and sensitivity of both indicators and imaging methods. Here, we report the engineering of an improved genetically encoded voltage indicator, ASAP3, which exhibits up to 51% fluorescence responses in the physiological voltage range, sub-millisecond activation kinetics, and full responsivity under two-photon illumination. We also introduce an ultrafast local volume excitation (ULOVE) two-photon scanning method to sample ASAP3 signals in awake mice at kilohertz rates with increased stability and sensitivity. ASAP3 and ULOVE allowed continuous single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution, and with repeated sampling over multiple days. By imaging voltage in visual cortex neurons, we found evidence for cell type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULOVE enable continuous high-speed high-resolution imaging of electrical activity in deeply located genetically defined neurons during awake behavior.

neuroscience↗

bioRxiv2018

The MS4A gene cluster is a key regulator of soluble TREM2 and Alzheimer disease risk

Soluble triggering receptor expressed on myeloid cells 2 (sTREM2) levels in the cerebrospinal fluid (CSF) have been associated with Alzheimer disease (AD) status. TREM2 plays a critical role in microglial activation, survival, and phagocytosis; however, the pathophysiological role of sTREM2 in AD is not well understood. Understanding the role of sTREM2 in AD may help reveal biological mechanisms underlying AD and identify novel therapeutic targets. We performed a genome-wide association study (GWAS) to identify genetic modifiers of CSF sTREM2 levels. Common variants in the membrane-spanning 4-domains subfamily A (MS4A) gene region were associated with higher CSF sTREM2 levels (rs1582763; P = 1.15x10-15) and replicated in independent datasets. The variants associated with increased levels of sTREM2 are also associated with reduced AD risk and delayed age-at-onset. Rs1582763 influences expression of MS4A4A and MS4A6A in multiple tissues, suggesting that one or both of these genes are important for regulating sTREM2. MS4A genes encode transmembrane proteins that may play a role in intracellular protein trafficking in microglia. We used human macrophages to begin to test the relationship between MS4A4A and TREM2 and found that they co-localize intracellularly and that antibody-mediated targeting of MS4A4A reduces sTREM2. Thus, genetic, molecular, and cellular findings suggest that MS4A4A regulates sTREM2. These findings also provide a mechanistic explanation of the original GWAS signal in the MS4A locus for AD risk and indicate that TREM2 is involved in sporadic AD risk in general, not only in TREM2 risk-variant carriers.

genomics↗

bioRxiv2018

Pulsed ELDOR Measurement of the Distance Between a Spin-Label and Copper (II) Centre in the Copper Loaded R48C Mutant of N. gonorrhoeae Ferric Binding Protein

Distance determination in proteins and biomolecules using pulsed EPR (electron paramagnetic resonance) techniques is becoming an increasingly popular and accessible technique. PELDOR (pulsed electron-electron double resonance), is a technique designed for distance determination over a nanoscopic scale. Here, ferric binding protein (Fbp) is used to demonstrate the practicability of this technique to Cu (II) Metalloproteins. PELDOR is usually applied to bi-radicals or endogenous radicals, and distance determination using pulsed EPR of metal containing centres in biomolecules has been restricted to relaxation experiments. PELDOR distance measurements between a Cu (II) ion and a nitroxide have previously only been reported for model compounds [1, 2].\n\nFbp as the name suggests usually, contains a Fe (III) ion centre. For the purposes of this investigation the Fe (III) ion was removed and replaced by a Cu (II) ion, after a nitroxide spin-label was added to the Fbp using of site directed spin-labelling (SDSL). PELDOR was then applied to measure the distance between the two centres.\n\nSimulation methods were then employed to fully investigate these data and allow a quantitative interpretation of the copper nitroxide PELDOR data. The observed PELDOR time traces were analysed using DEER analysis[3].

biochemistry↗

bioRxiv2017

Intrinsic specificity differences between transcription factor paralogs partly explain their differential in vivo binding

Members of transcription factor (TF) families, i.e. paralogous TFs, are oftentimes reported to have identical DNA-binding motifs, despite the fact that they perform distinct regulatory functions in the cell. Differential genomic targeting by paralogous TFs is generally assumed to be due to interactions with protein cofactors or the chromatin environment. Contrary to previous assumptions, we find that paralogous TFs have different intrinsic preferences for DNA, not captured by current motif models, and these differences partly explain differential genomic binding and functional specificity. Our finding was possible due to a unique combination of carefully designed high-throughput assays and rigorous computation modeling, integrated into a unified framework called iMADS. We used iMADS to quantify, model, and analyze specificity differences between 11 paralogous TFs from 4 distinct human TF families. Our finding of differential specificity between closely related TFs has important implications for the interpretation of the regulatory effects of non-coding genetic variants.

genomics↗