A high order accurate space-time trajectory reconstruction technique for quantitative particle trafficking analysis

The study of moving particles (e.g. molecules, virus, vesicles, organelles, or whole cells) is crucial to decipher a plethora of cellular mechanisms within physiological and pathological conditions. Powerful live-imaging approaches enable life scientists to capture particle movements at different scale from cells to single molecules, that are collected in a series of frames. However, although these events can be captured, an accurate quantitative analysis of live-imaging experiments still remains a challenge. Two main approaches are currently used to study particle kinematics: kymographs, which are graphical representation of spatial motion over time, and single particle tracking (SPT) followed by linear linking. Both kymograph and SPT apply a space-time approximation in quantifying particle kinematics, considering the velocity constant either over several frames or between consecutive frames, respectively. Thus, both approaches intrinsically limit the analysis of complex motions with rapid changes in velocity. Therefore, we design, implement and validate a novel reconstruction algorithm aiming at supporting tracking particle trafficking analysis with mathematical foundations. Our method is based on polynomial reconstruction of 4D (3D+time) particle trajectories, enabling to assess particle instantaneous velocity and acceleration, at any time, over the entire trajectory. Here, the new algorithm is compared to state-of-the-art SPT followed by linear linking, demonstrating an increased accuracy in quantifying particle kinematics. Our approach is directly derived from the governing equations of motion, thus it arises from physical principles and, as such, it is a versatile and reliable numerical method for accurate particle kinematics analysis which can be applied to any live-imaging experiment where the space-time coordinates can be retrieved

Precursor miRNAs are locally processed to regulate growth cone steering

During the development of the nervous system, axons grow and generate a complex network of interconnected neurons. To establish these connections, the tip of the growing axon, the growth cone, is guided by chemotropic cues en route to its target with exquisite precision. Axons must sometimes navigate a significant distance before reaching their final destination. As an alternative to energy-expensive protein transport from distant cell bodies, seminal studies have revealed that growth cones rely on local mRNA translation to generate certain proteins acutely on demand. These cue-induced newly synthesized proteins contribute to fuel growth cone steering. Several groups reported the presence of Dicer at growth cones, and I observe the presence of endogenous Dicer in RGC axons of FLAG-HA2-Dicer transgenic mice. These observations raise the intriguing possibility that not only proteins but also miRNAs could be produced locally in this compartment. In my work, I have therefore explored whether miRNA biogenesis occurs locally within growth cone and if this is important for growth cone steering, using Xenopus laevis retinal ganglion cell (RGC) axons as a model. Specific precursor microRNAs (pre-miRNAs) are detected in pure Xenopus RGC axonal preparations by miRNA-seq and PCR, and endogenous pre-miR-181a-1 is actively tracked to distal axons by hitchhiking on vesicles. Upon exposure to Sema3A, but not Slit-2, pre-miR-181a-1/a-2 are processed within axons by Dicer into newly generated miRNAs (NGmiRNAs). In contrast, pre-miR-182 remains unprocessed upon Sema3A exposure, highlighting a mechanism that is not only cue-, but also pre-miRNA molecule specific. Inhibiting NGmiRNAs in axons abolishes growth cone responsiveness to cues ex vivo. miRNAs are thus locally produced and these newly generated miRNAs mediate cue-induced growth cone steering. To deepen mechanistic insights, I assess whether newly generated miRNAs silence the translation of specific mRNAs in response to cues using FRAP analysis with a Venus reporter. I observe that APP and TUBB3 are locally translated in axons in basal conditions and that are both silenced in response to Sema3A. I uncover that this cue-induced silencing of TUBB3 is mediated by newly generated miRNAs specifically in axons ex vivo and in vivo. Taken together, these results indicate that newly generated miRNAs gate cue-induced silencing of a specific subset of mRNAs in time and space, thereby regulating growth cone behavior. Local biogenesis of miRNAs in axons constitute an important additional regulatory layer in the complex mechanism of axon targeting

A high order accurate space-time trajectory reconstruction technique for quantitative particle trafficking analysis

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Plasma and White Blood Cells Show Different miRNA Expression Profiles in Parkinsonâ\u80\u99s Disease

Parkinsonâ\u80\u99s disease (PD) diagnosis is based on the assessment of motor symptoms, which manifest when more than 50% of dopaminergic neurons are degenerated. To date, no validated biomarkers are available for the diagnosis of PD. The aims of the present study are to evaluate whether plasma and white blood cells (WBCs) are interchangeable biomarker sources and to identify circulating plasma-based microRNA (miRNA) biomarkers for an early detection of PD. We profiled plasma miRNA levels in 99 l-dopa-treated PD patients from two independent data collections, in ten drug-naïve PD patients, and in unaffected controls matched by sex and age. We evaluated expression levels by reverse transcription and quantitative real-time PCR (RT-qPCR) and combined the results from treated PD patients using a fixed effect inverse-variance weighted meta-analysis. We revealed different expression profiles comparing plasma and WBCs and drug-naïve and l-dopa-treated PD patients. We observed an upregulation trend for miR-30a-5p in l-dopa-treated PD patients and investigated candidate target genes by integrated in silico analyses. We could not analyse miR-29b-3p, normally expressed in WBCs, due to the very low expression in plasma. We observed different expression profiles in WBCs and plasma, suggesting that they are both suitable but not interchangeable peripheral sources for biomarkers. We revealed miR-30a-5p as a potential biomarker for PD in plasma. In silico analyses suggest that miR-30a-5p might have a regulatory role in mitochondrial dynamics and autophagy. Further investigations are needed to confirm miR-30a-5p deregulation and targets and to investigate the influence of l-dopa treatment on miRNA expression levels