In-vivo fast non-linear microscopy reveals impairment of fast axonal transport induced by molecular motor imbalances in the brain of zebrafish larvae

Abstract

Cargo transport by molecular motors along microtubules is essential for the function of eucaryotic cells, in particular neurons in which axonal transport defects constitute the early pathological features of neurodegenerative diseases. Mainly studied in motor and sensory neurons, axonal transport is still difficult to characterize in neurons of the brain in absence of appropriate in vivo tools. Here, we measured fast axonal transport by tracing the second harmonic generation (SHG) signal of potassium titanyl phosphate (KTP) nanocrystals endocytosed by brain neurons of zebrafish (Zf) larvae. Thanks to the optical translucency of Zf larvae and of the perfect photostability of nanoKTP SHG, we achieved a high scanning speed of 20 frames (of ≈90 μm×60 μm size) per second in Zf brain. We focused our study on endosomal transport in axons of known polarization, separately analyzing kinesin and dynein motor-driven displacements. To validate our assay, we used either loss-of-function mutations of dynein or kinesin 1 or the dynein inhibitor dynapyrazole, and quantified several transport parameters. We successfully demonstrated that dynapyrazole reduces nanoKTP mobile fraction and retrograde run length consistently, while the retrograde run length increased in kinesin 1 mutants. Taking advantage of nanoKTP SHG directional emission, we also quantified fluctua- tions of endosome orientation. Thus, by combining endocytosis of nanocrystals having non-linear response, fast two-photon microscopy, and high-throughput analysis, we are able to finely monitor fast axonal transport in vivo in the brain of a vertebrate, and re- veal subtle axonal transport alterations. The high spatiotemporal resolution achieved in our model may be relevant to precisely investigate axonal transport impairment associated to disease models.

In Vivo Fast Nonlinear Microscopy Reveals Impairment of Fast Axonal Transport Induced by Molecular Motor Imbalances in the Brain of Zebrafish Larvae

“…Right: in green, the SHG radiating pattern of one nanoKTP. (D) Analysis pipeline with the Python program MINT we developed, consisting, from left to right, in two parts of analysis. The first part includes (i) trajectory extraction, (ii) trajectory regularization (filtering out nanoparticle position noise), and (iii) parsing in fast (“Go”) and slow/pause (“Stop”) phases of motion.…”

“…Videos were first exported from the Leica LIF proprietary format as stacks of 8-bit TIFF files with a custom Fiji macro relying on the import function of the Bio-Formats plugin . The subsequent data analysis process is automated, using a set of Python scripts that we developed and made freely accessible online . It consists of two main parts: part 1 aims at extracting valid trajectories directly from the video files and part 2 calculates transport parameter observables from the trajectories.…”