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Communication Dans Un Congrès Année : 2022

Measurement of intraneuronal transport in vivo in zebrafish larvae brain by tracking nanocrystal-labelled endosomes with fast non-linear microscopy

Karine Duroure
Filippo del Bene

Résumé

Motor proteins are responsible for the intracellular transport of critical cargoes such as organelles, vesicles, protein complexes and other materials within the cell, along the cytoskeleton. This transport is an essential molecular process participating in cell homeostasis, especially in neurons [1]. Axonal transport deficits are found in several neurological disorders and are a hallmark of neurodegenerative diseases [2]. Over the past few decades, much progress has been made in developing tools and methods to visualize and measure intracellular transport, by tracking the movement of molecular motors or the transported cargoes. In particular, fluorescent proteins (FP) fused to motors or cargo proteins [3] is largely used to investigate axonal transport, as it is a versatile tool with target specificity that is used in vivo in small organisms. These include Drosophila and Danio rerio (Zebrafish, Zf) larvae, which offers the advantage for optical microscopy of being transparent, and which are also amenable to genetic modifications. For instance, the motion of mitochondria was investigated in Zf larvae with genetically engineered fluorescent reporters [4] in the context of neurodegenerative disease [5] or axonal regeneration [6], using wide-field microscopy and more recently using a real-time 3D single particle tracking setup [7]. Such studies were also conducted in axons of motor-neurons of live mice, but these observations require a complex and invasive preparation consisting in surgically exposing the nerves. These various methods have reached a high degree of sophistication but to date the vast majority of axonal transport studies have only been conducted in sensory-motor neurons. Measurements in central nervous system neurons, where this transport also plays a key role, especially in disease conditions, are still lacking. Takihara et al [8] developed a low invasive surgical procedure allowing them to record by two-photon fluorescence microscopy bidirectional mitochondria motions in axons of retinal ganglion cells of a live mouse. Using two-photon microscopy imaging of the motor cortex, Knabbe et al [9] measured axonal transport properties of dense core vesicles labeled by a virally induced fluorescent reporter. While they addressed more complex systems, these live mouse observations [8,9] were carried out at moderate temporal resolutions of a maximum of one frame per second (corresponding to a dwell time of a few μs) with a spatial resolution of about 200 nm and for a maximum field-of-view size of 200 μm. Such a time resolution prevents the observation of transient events of short duration (<1 s) like short pauses induced by microtubule associated proteins obstacles. These limitations stem from the photobleaching of the FP reporter that prevents using the more intense laser excitation necessary to lower the dwell time. Therefore, in order to measure with a higher sensitivity axonal transport parameters in the brain of live organisms, there is a need for novel methodologies capable of achieving larger spatiotemporal resolution while maintaining a high throughput recording of ≈100 μm large field-of-views. To this aim, photostable optically active nanocrystals were used to measure the intraneuronal transport of endosomal compartments that they labeled from inside after their spontaneous internalization by endocytosis. Following the seminal work of Cui et al [10] using semiconductor nanocrystals as fluorescent labels to measure nerve growth factor retrograde transport, we used fluorescent diamond nanocrystals and evidenced subtle changes in endosomal transport in cultured neurons of transgenic mouse bearing a genetic risk factor of a neuropsychiatric disease [11]. These experiments were conducted in cultured neurons. Here we extend such nanoparticle-based assay to measure the endosomal transport parameters in neurons of the brain of zebrafish larvae. To this aim we use size ≈ 120 nm nanocrystals exhibiting large second-order non-linear optical properties, composed of potassium titanate phosphate (KTiOPO4, KTP), injected in Zf larvae optical tectum (OT) where they are subsequently endocyted by neurons. Indeed, in a previous work, we showed that these KTP nanocrystals (nanoKTP) are spontaneously internalized in 2D cultured primary neuron and can be imaged by collecting their second harmonic generation (SHG) signal un- der infrared (IR) pulsed laser excitation [13]. Key advantages of SHG over fluorescence are non-saturation and non-bleaching. In the present in vivo work, we harnessed these properties combined with fast raster scanning of the IR laser beam to achieve a large, up to 20 frames/s, frame rate identical to the one employed in our in vitro intraneuronal transport assay [11]. Such large frame rate allows the detection of short pausing duration underpinning complex molecular environments otherwise smeared out by too low temporal resolutions. Moreover, due to a dominant coefficient of the nonlinear suceptibility tensor [13], SHG from KTP behaves like a dipole and therefore has a direction emission, that we can further exploit to reveal rotational dynamics. Incidentally, the ability to image nanoKTP in live Zf larvae blood circulation at high frame rate was recently reported in a wide-field configuration with light-sheet illumination [14]. In our experiment, we showed strong evidence that the nanoKTP move within axons of periventricular neurons (PVN), which cell bodies are located between the ventricle and the neuropil. These axons project radially inside the neuropil where they establish synaptic contacts with retinal ganglion cells dendrites. As microtubule orientation is polarized far enough from the axonal initial segment, we are then able to separate the retrograde phases of motion from the anterograde ones. To this aim, we developed a novel pipeline of video analysis, which identifies the direction of transport and directly yields as the outputs of a set of data, the statistical distributions of various intraneuronal transport metrics for anterograde and retrograde motions, in normal and perturbed situations. While in our previous in vitro study we investigated the impact of microtubule disruption or of concentration in associated proteins, here we address the complementary aspects of the modifications of specific molecular motor concentrations, either by applying dynapyrazole [15], a recently developed retrograde motor dynein inhibiting drug, or by using transgenic Zf engineered to bear loss-of-function alleles of the anterograde motor protein Kif5aa [16]. Dynapyrazole induces a reduction of 41% of the retrograde runlength, accompanied with a trend of 31% reduction of the mobile fraction of nanoKTP. In kif5aa mutant we confirm the previously reported increase of the mobile fraction of vesicles (45% increase for synaptophysin vesicles in moto-neuron axons [16]), here of +25%. This “release” of motion upon decrease of Kif5aa motor concentration is consistent with the tug-of-war model of axonal transport [21]. This interpretation is further supported by dose-dependent increases of retrograde motion directionality within individual trajectories, and of retrograde run length (+42% compared to wild-type) that could not be detected in the previous work [16]. The high sensitivity of our nonlinear nanoparticle-based axonal transport measurement assay in revealing small molecular concentration changes opens prospects in screening the functional impacts of neurodegenerative disease genetic factors in the whole animal model of zebrafish larvae for which genetic tools are largely available. References: 1.1. Hirokawa, N.; Niwa, S.; Tanaka, Y. Molecular Motors in Neurons: Transport Mechanisms and Roles in Brain Function, Development, and Disease. Neuron 2010, 68, 610–638. 2.Millecamps, S.; Julien, J.-P. Axonal transport deficits and neurodegenerative diseases. Nature Reviews Neuroscience 2013, 14, 161–176. 3.Surana, S.; Villarroel-Campos, D.; Lazo, O. M.; Moretto, E.; Tosolini, A. P.; Rhymes, E. R.; Richter, S.; Sleigh, J. N.; Schiavo, G. The evolution of the axonal transport toolkit. Traffic 2020, 21, 13–33. 4.Mandal, A.; Pinter, K.; Drerup, C. M. Analyzing Neuronal Mitochondria in vivo Using Fluorescent Reporters in Zebrafish. Frontiers in Cell and Developmental Biology 2018, 6, 144. 5.Plucinska, G.; Paquet, D.; Hruscha, A.; Godinho, L.; Haass, C.; Schmid, B.; Misgeld, T. In Vivo Imaging of Disease-Related Mitochondrial Dynamics in a Vertebrate Model System. Journal of Neuroscience 2012, 32, 16203–16212. 6.Xu, Y.; Chen, M.; Hu, B.; Huang, R.; Hu, B. In vivo Imaging of Mitochondrial Trans- port in Single-Axon Regeneration of Zebrafish Mauthner Cells. Frontiers in Cellular Neuroscience 2017, 11, 4. 7.Wehnekamp, F.; Plucińska, G.; Thong, R.; Misgeld, T.; Lamb, D. C. Nanoresolution real-time 3D orbital tracking for studying mitochondrial trafficking in vertebrate axons in vivo. eLife 2019, 8, e46059. 8.Takihara, Y.; Inatani, M.; Eto, K.; Inoue, T.; Kreymerman, A.; Miyake, S.; Ueno, S.; Na- gaya, M.; Nakanishi, A.; Iwao, K. et al. In vivo imaging of axonal transport of mitochondria in the diseased and aged mammalian CNS. Proceedings of the National Academy of Sciences 2015, 112, 10515–10520. 9.Knabbe, J.; Nassal, J. P.; Verhage, M.; Kuner, T. Secretory vesicle trafficking in awake and anaesthetized mice: differential speeds in axons versus synapses. The Journal of Physiology 2018, 596, 3759–3773. 10.Cui, B.; Wu, C.; Chen, L.; Ramirez, A.; Bearer, E. L.; Li, W.-P.; Mobley, W. C.; Chu, S. One at a time, live tracking of NGF axonal transport using quantum dots. Proceedings of the National Academy of Sciences 2007, 104, 13666–13671. 11.Haziza, S.; Mohan, N.; Loe-Mie, Y.; Lepagnol-Bestel, A.-M.; Massou, S.; Adam, M.-P.; Le, X. L.; Viard, J.; Plancon, C.; Daudin, R. et al. Fluorescent nanodiamond tracking reveals intraneuronal transport abnormalities induced by brain-disease-related genetic risk factors. Nature nanotechnology 2016, 12, 322–328. 12.Mayer, L.; Slablab, A.; Dantelle, G.; Jacques, V.; Lepagnol-Bestel, A.-M.; Perruchas, S.; Spinicelli, P.; Thomas, A.; Chauvat, D.; Simonneau, M. et al. Single KTP nanocrystals as second-harmonic generation biolabels in cortical neurons. Nanoscale 2013, 5, 8466 8471. 13.Xuan, L. L.; Zhou, C.; Slablab, A.; Chauvat, D.; Tard, C.; Perruchas, S.; Gacoin, T.; Villeval, P.; Roch, J. Photostable Second-Harmonic Generation from a Single KTiOPO4 Nanocrystal for Nonlinear Microscopy. Small 2008-09, 4, 1332–1336. 14.Malkinson, G.; Mahou, P.; Chaudan, E.; Gacoin, T.; Sonay, A. Y.; Pantazis, P.; Beau- repaire, E.; Supatto, W. Fast In Vivo Imaging of SHG Nanoprobes with Multiphoton Light-Sheet Microscopy. ACS Photonics 2020, 7, 1036–1049. 15.Steinman, J. B.; Santarossa, C. C.; Miller, R. M.; Yu, L. S.; Serpinskaya, A. S.; Furukawa, H.; Morimoto, S.; Tanaka, Y.; Nishitani, M.; Asano, M. et al. Chemical structure-guided design of dynapyrazoles, cell-permeable dynein inhibitors with a unique mode of action. eLife 2017, 6, e25174. 16.Auer, T. O.; Xiao, T.; Bercier, V.; Gebhardt, C.; Duroure, K.; Concordet, J.-P.; Wyart, C.; Suster, M.; Kawakami, K.; Wittbrodt, J. et al. Deletion of a kinesin I motor unmasks a mechanism of homeostatic branching control by neurotrophin-3. eLife 2015, 4, e05061. 17.Encalada, S. E.; Goldstein, L. S. B. Biophysical Challenges to Axonal Transport: Motor- Cargo Deficiencies and Neurodegeneration. Annual Review of Biophysics 2014, 43, 141– 169.

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hal-04442807 , version 1 (06-02-2024)

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Baptiste Grimaud, Maxence Frétaud, Feriel Terras, Karine Duroure, Valerie Bercier, et al.. Measurement of intraneuronal transport in vivo in zebrafish larvae brain by tracking nanocrystal-labelled endosomes with fast non-linear microscopy. SPIE Photonics Europe - Neurophotonics I Symposium, Apr 2022, Strasbourg, France. ⟨10.1117/12.2621637⟩. ⟨hal-04442807⟩
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