Probing intraneuronal transport with optically active photostable nanocrystals
Prof. Dr. Francois Treussart (Laboratoire Aimé Cotton, CNRS, Univ. Paris-Sud, ENS Paris-Saclay, Université Paris-Saclay, Orsay, France)

Neurodegenerative disorders such as Alzheimer’s disease (affecting 18% of >75 years old population) involve a large network of genes displaying subtle changes in their expression. Abnormalities in intraneuronal transport have been linked to genetic risk factors found in patients, suggesting the relevance of measuring this key biological process. However, current techniques are not sensitive enough to detect minor abnormalities.
In 2017, we reported a sensitive method to measure changes in intraneuronal endosomal transport induced by brain disease-related genetic risk factors using fluorescent nanodiamonds (FNDs)[1]. We showed that the high brightness, photostability and absence of cytotoxicity allow FNDs to be spontaneously internalized inside the endosomes neurons in cultures and subsequently tracked with up to 12 nm spatial and 50 ms time resolutions. As proof-of-principle, we applied the FND-tracking assay to transgenic mouse lines that mimic the slight changes in protein concentration (≈30%) found in brains of patients. In both cases, we showed that the FND assay is sufficiently sensitive to detect these changes trough modifications of transport parameters.
This nanoparticle tracking based-approach applies also to multiphoton microscopy (MPM), opening the possibility of intracellular transport measurement in vivo thanks to tissue transparency in the excitation wavelength range of MPM. To be able to keep a high framerate while raster scanning MPM infrared focused excitation beam, we use sized≈100 nm KTiOPO4 (KTP) nanocrystals possessing a large nonlinear second order optical response, that were identified as possible cell labels in an earlier work [2]. As a first step toward deep imaging of transport, we have tracked nanoKTP in axons of the periventricular neurons of the optical tectum of living zebrafish (Zf) larvae at the same 20 frames/s rate as in widefield imaging with FND, while keeping a subwavelength precision of localization of ≈150 nm. Surprisingly, in transgenic Zf with a reduced concentration of kinesin-1 family motor kif5a, we have observed improved transport parameters (increase of velocity and runlength, and lower rotational fluctuations) in the direction of motion driven by these family of motors. We are now testing the hypothesis that this results from an improved coordination of kinesin-2 motors when kif5a does not compete for the binding to the microtubule track. Indeed, kinesin-2 family motors are likely to dominate the driving of the late endosomes or lysosome we track.
This in vivo intraneuronal transport assay in Zf larvae is a first step toward measurement in mature brain of juvenile fishes (≈1 month old) and mouse brain sections.
References
[1] S. Haziza, et al. Nat. Nanotechnol. 12, 322 (2017).
[2] L. Mayer et al. Nanoscale 5, 8466 (2013)