miR449 Protects Airway Regeneration by Controlling AURKA/HDAC6-Mediated Ciliary Disassembly.

Airway mucociliary regeneration and function are key players for airway defense and are impaired in chronic obstructive pulmonary disease (COPD). Using transcriptome analysis in COPD-derived bronchial biopsies, we observed a positive correlation between cilia-related genes and microRNA-449 (miR449). In vitro, miR449 was strongly increased during airway epithelial mucociliary differentiation. In vivo, miR449 was upregulated during recovery from chemical or infective insults. miR0449-/- mice (both alleles are deleted) showed impaired ciliated epithelial regeneration after naphthalene and Haemophilus influenzae exposure, accompanied by more intense inflammation and emphysematous manifestations of COPD. The latter occurred spontaneously in aged miR449-/- mice. We identified Aurora kinase A and its effector target HDAC6 as key mediators in miR449-regulated ciliary homeostasis and epithelial regeneration. Aurora kinase A is downregulated upon miR449 overexpression in vitro and upregulated in miR449-/- mouse lungs. Accordingly, imaging studies showed profoundly altered cilia length and morphology accompanied by reduced mucociliary clearance. Pharmacological inhibition of HDAC6 rescued cilia length and coverage in miR449-/- cells, consistent with its tubulin-deacetylating function. Altogether, our study establishes a link between miR449, ciliary dysfunction, and COPD pathogenesis.

Protein lifetimes in aged brains reveal a proteostatic adaptation linking physiological aging to neurodegeneration.

Aging is a prominent risk factor for neurodegenerative disorders (NDDs); however, the molecular mechanisms rendering the aged brain particularly susceptible to neurodegeneration remain unclear. Here, we aim to determine the link between physiological aging and NDDs by exploring protein turnover using metabolic labeling and quantitative pulse-SILAC proteomics. By comparing protein lifetimes between physiologically aged and young adult mice, we found that in aged brains protein lifetimes are increased by ~20% and that aging affects distinct pathways linked to NDDs. Specifically, a set of neuroprotective proteins are longer-lived in aged brains, while some mitochondrial proteins linked to neurodegeneration are shorter-lived. Strikingly, we observed a previously unknown alteration in proteostasis that correlates to parsimonious turnover of proteins with high biosynthetic costs, revealing an overall metabolic adaptation that preludes neurodegeneration. Our findings suggest that future therapeutic paradigms, aimed at addressing these metabolic adaptations, might be able to delay NDD onset.

Resolving different presynaptic activity patterns within single olfactory glomeruli of Xenopus laevis larvae.

Olfactory sensing is generally organized into groups of similarly sensing olfactory receptor neurons converging into their corresponding glomerulus, which is thought to behave as a uniform functional unit. It is however unclear to which degree axons within a glomerulus show identical activity, how many converge into a glomerulus, and to answer these questions, how it is possible to visually separate them in live imaging. Here we investigate activity of olfactory receptor neurons and their axon terminals throughout olfactory glomeruli using electrophysiological recordings and rapid 4D calcium imaging. While single olfactory receptor neurons responsive to the same odor stimulus show a diversity of responses in terms of sensitivity and spontaneous firing rate on the level of the somata, their pre-synaptic calcium activity in the glomerulus is homogeneous. In addition, we could not observe the correlated spontaneous calcium activity that is found on the post-synaptic side throughout mitral cell dendrites and has been used in activity correlation imaging. However, it is possible to induce spatio-temporal presynaptic response inhomogeneities by applying trains of olfactory stimuli with varying amino acid concentrations. Automated region-of-interest detection and correlation analysis then visually distinguishes at least two axon subgroups per glomerulus that differ in odor sensitivity.

Correction: Circumvention of common labelling artefacts using secondary nanobodies.

Correction for 'Circumvention of common labelling artefacts using secondary nanobodies' by Shama Sograte-Idrissi et al., Nanoscale, 2020, 12, 10226-10239, DOI: 10.1039/D0NR00227E.

Circumvention of common labelling artefacts using secondary nanobodies.

A standard procedure to study cellular elements is via immunostaining followed by optical imaging. This methodology typically requires target-specific primary antibodies (1.Abs), which are revealed by secondary antibodies (2.Abs). Unfortunately, the antibody bivalency, polyclonality, and large size can result in a series of artifacts. Alternatively, small, monovalent probes, such as single-domain antibodies (nanobodies) have been suggested to minimize these limitations. The discovery and validation of nanobodies against specific targets are challenging, thus only a minimal amount of them are currently available. Here, we used STED, DNA-PAINT, and light-sheet microscopy, to demonstrate that secondary nanobodies (1) increase localization accuracy compared to 2.Abs; (2) allow direct pre-mixing with 1.Abs before staining, reducing experimental time, and enabling the use of multiple 1.Abs from the same species; (3) penetrate thick tissues more efficiently; and (4) avoid probe-induced clustering of target molecules observed with conventional 2.Abs in living or poorly fixed samples. Altogether, we show how secondary nanobodies are a valuable alternative to 2.Abs.

A mass spectrometry workflow for measuring protein turnover rates in vivo.

Proteins are continually produced and degraded, to avoid the accumulation of old or damaged molecules and to maintain the efficiency of physiological processes. Despite its importance, protein turnover has been difficult to measure in vivo. Previous approaches to evaluating turnover in vivo have required custom labeling approaches, involved complex mass spectrometry (MS) analyses, or used comparative strategies that do not allow direct quantitative measurements. Here, we describe a robust protocol for quantitative proteome turnover analysis in mice that is based on a commercially available diet for stable isotope labeling of amino acids in mammals (SILAM). We start by discussing fundamental concepts of protein turnover, including different methodological approaches. We then cover in detail the practical aspects of metabolic labeling and explain both the experimental and computational steps that must be taken to obtain accurate in vivo results. Finally, we present a simple experimental workflow that enables measurement of precise turnover rates in a time frame of ~4-5 weeks, including the labeling time. We also provide all the scripts needed for the interpretation of the MS results and for comparing turnover across different conditions. Overall, the workflow presented here comprises several improvements in the determination of protein lifetimes with respect to other available methods, including a minimally invasive labeling strategy and a robust interpretation of MS results, thus enhancing reproducibility across laboratories.

Precisely measured protein lifetimes in the mouse brain reveal differences across tissues and subcellular fractions.

The turnover of brain proteins is critical for organism survival, and its perturbations are linked to pathology. Nevertheless, protein lifetimes have been difficult to obtain in vivo. They are readily measured in vitro by feeding cells with isotopically labeled amino acids, followed by mass spectrometry analyses. In vivo proteins are generated from at least two sources: labeled amino acids from the diet, and non-labeled amino acids from the degradation of pre-existing proteins. This renders measurements difficult. Here we solved this problem rigorously with a workflow that combines mouse in vivo isotopic labeling, mass spectrometry, and mathematical modeling. We also established several independent approaches to test and validate the results. This enabled us to measure the accurate lifetimes of ~3500 brain proteins. The high precision of our data provided a large set of biologically significant observations, including pathway-, organelle-, organ-, or cell-specific effects, along with a comprehensive catalog of extremely long-lived proteins (ELLPs).

TAp73 is a central transcriptional regulator of airway multiciliogenesis.

Motile multiciliated cells (MCCs) have critical roles in respiratory health and disease and are essential for cleaning inhaled pollutants and pathogens from airways. Despite their significance for human disease, the transcriptional control that governs multiciliogenesis remains poorly understood. Here we identify TP73, a p53 homolog, as governing the program for airway multiciliogenesis. Mice with TP73 deficiency suffer from chronic respiratory tract infections due to profound defects in ciliogenesis and complete loss of mucociliary clearance. Organotypic airway cultures pinpoint TAp73 as necessary and sufficient for basal body docking, axonemal extension, and motility during the differentiation of MCC progenitors. Mechanistically, cross-species genomic analyses and complete ciliary rescue of knockout MCCs identify TAp73 as the conserved central transcriptional integrator of multiciliogenesis. TAp73 directly activates the key regulators FoxJ1, Rfx2, Rfx3, and miR34bc plus nearly 50 structural and functional ciliary genes, some of which are associated with human ciliopathies. Our results position TAp73 as a novel central regulator of MCC differentiation.

Detection of silent cells, synchronization and modulatory activity in developing cellular networks.

Developing networks in the immature nervous system and in cellular cultures are characterized by waves of synchronous activity in restricted clusters of cells. Synchronized activity in immature networks is proposed to regulate many different developmental processes, from neuron growth and cell migration, to the refinement of synapses, topographic maps, and the mature composition of ion channels. These emergent activity patterns are not present in all cells simultaneously within the network and more immature "silent" cells, potentially correlated with the presence of silent synapses, are prominent in different networks during early developmental periods. Many current network analyses for detection of synchronous cellular activity utilize activity-based pixel correlations to identify cellular-based regions of interest (ROIs) and coincident cell activity. However, using activity-based correlations, these methods first underestimate or ignore the inactive silent cells within the developing network and second, are difficult to apply within cell-dense regions commonly found in developing brain networks. In addition, previous methods may ignore ROIs within a network that shows transient activity patterns comprising both inactive and active periods. We developed analysis software to semi-automatically detect cells within developing neuronal networks that were imaged using calcium-sensitive reporter dyes. Using an iterative threshold, modulation of activity was tracked within individual cells across the network. The distribution pattern of both inactive and active, including synchronous cells, could be determined based on distance measures to neighboring cells and according to different anatomical layers.

Ca(2+)-BK channel clusters in olfactory receptor neurons and their role in odour coding.

Olfactory receptor neurons (ORNs) have high-voltage-gated Ca(2+) channels whose physiological impact has remained enigmatic since the voltage-gated conductances in this cell type were first described in the 1980s. Here we show that in ORN somata of Xenopus laevis tadpoles these channels are clustered and co-expressed with large-conductance potassium (BK) channels. We found approximately five clusters per ORN and twelve Ca(2+) channels per cluster. The action potential-triggered activation of BK channels accelerates the repolarization of action potentials and shortens interspike intervals during odour responses. This increases the sensitivity of individual ORNs to odorants. At the level of mitral cells of the olfactory bulb, odour qualities have been shown to be coded by first-spike-latency patterns. The system of Ca(2+) and BK channels in ORNs appears to be important for correct odour coding because the blockage of BK channels not only affects ORN spiking patterns but also changes the latency pattern representation of odours in the olfactory bulb.

Direct measurement of diffusion in olfactory cilia using a modified FRAP approach.

The diffusion coefficient of fluorescein in detached cilia of Xenopus laevis olfactory receptor neurons was measured using spatially-resolved FRAP, where the dye along half of the ciliary length was photobleached and its spatiotemporal fluorescence redistribution recorded. Fitting a one-dimensional numerical simulation of diffusion and photobleaching for 35 cilia resulted in a mean value of the diffusion coefficient (1.20 ± 0.23) · 10(-10)m(2)/s and thus a reduction by a factor of 3.4 compared to free diffusion in aqueous solution.

Structure and composition of myelinated axons: a multimodal synchrotron spectro-microscopy study.

We report elemental mappings on the sub-cellular level of myelinated sciatic neurons isolated from wild type mice, with high spatial resolution. The distribution of P, S, Cl, Na, K, Fe, Mn, Cu was imaged in freeze-dried as well as cryo-preserved specimen, using the recently developed cryogenic sample environment at beamline ID21 at the European Synchrotron Radiation Facility (ESRF). In addition, synchrotron radiation based Fourier transform infrared (FTIR) spectromicroscopy was used as a chemically sensitive imaging method. Finally single fiber diffraction in highly focused hard X-ray beams, and soft X-ray microscopy and tomography in absorption contrast are demonstrated as novel techniques for the study of single nerve fibers.

Activity correlation imaging: visualizing function and structure of neuronal populations.

For the analysis of neuronal networks it is an important yet unresolved task to relate the neurons' activities to their morphology. Here we introduce activity correlation imaging to simultaneously visualize the activity and morphology of populations of neurons. To this end we first stain the network's neurons using a membrane-permeable [Ca(2+)] indicator (e.g., Fluo-4/AM) and record their activities. We then exploit the recorded temporal activity patterns as a means of intrinsic contrast to visualize individual neurons' dendritic morphology. The result is a high-contrast, multicolor visualization of the neuronal network. Taking the Xenopus olfactory bulb as an example we show the activities of the mitral/tufted cells of the olfactory bulb as well as their projections into the olfactory glomeruli. This method, yielding both functional and structural information of neuronal populations, will open up unprecedented possibilities for the investigation of neuronal networks.