Impact of α-Synuclein Fibrillar Strains and β-Amyloid Assemblies on Mouse Cortical Neurons Endo-Lysosomal Logistics

Abstract

Endosomal transport and positioning cooperate in the establishment of neuronal compartment architecture, dynamics, and function, contributing to neuronal intracellular logistics. Furthermore, dysfunction of endo-lysosomal has been identified as a common mechanism in neurodegenerative diseases. Here, we analyzed endo-lysosomal transport when α-synuclein (α-syn) fibrillar polymorphs, β-amyloid (Aβ) fibrils, and oligomers were externally applied on primary cultures of mouse cortical neurons. To measure this transport, we used a simple readout based on the spontaneous endocytosis in cultured neurons of fluorescent nanodiamonds (FNDs), a perfectly stable nano-emitter, and the subsequent automatic extraction and quantification of their directed motions at high-throughput. α-Syn fibrillar polymorphs, Aβ fibrils, and oligomers induce a 2-fold decrease of the fraction of nanodiamonds transported along microtubules, while only slightly reducing their interaction with cortical neurons. This important decrease in moving endosomes is expected to have a huge impact on neuronal homeostasis. We next assessed lysosomes dynamics, using LysoTracker. Neurons exposure to Aβ oligomers led to an increase in the number of lysosomes, a decrease in the fraction of moving lysosome and an increase in their size, reminiscent of that found in APP transgenic model of Alzheimer's disease. We then analyzed the effect of α-syn fibrillar polymorphs, Aβ fibrils, and oligomers on endosomal and lysosomal transport and quantified directed transport of those assemblies within cortical neurons. We report different impacts on endosomal and lysosomal transport parameters and differences in the trajectory lengths of cargoes loaded with pathogenic protein assemblies. Our results suggest that intraneuronal pathogenic protein aggregates internalization and transport may represent a target for novel neuroprotective therapeutic strategies.

Keywords: endosome; intraneuronal transport; lysosome; mouse cortical neuron; α-synuclein; β-amyloid assemblies.

Graphical abstract

Figure 1.

Recording FND trajectories in mouse cortical neurons at DIC21. A, Schematic representation of the cargoes that are tracked thanks to FND, at different stages after their endocytosis. We previously showed (Haziza et al., 2017) that FND are present in cargoes at different stage of their lifetime after endocytosis, as shown by colocalization measurements with specific membrane protein markers: Rab5 for early endosome; Rab7 for late endosome; Rab11 for recycling endosome; LysoTracker for the lysosome. B, Illustration of FND trajectories with go (green) and stop (red) phases. C, Differential interference contrast images of cortical neurons overlapped with 10 representative trajectories. Scale bar: 10 μm.

Figure 2.

Effect of α-synF or R on the mobility of endosomes and their transport as measured by tracking FND-containing cargoes in mouse cortical neurons at DIC21; 24-h exposure to α-synF or R at 0.2 μm concentration, compared with nothing added control (Ctrl). A, Number of FNDs detected per FoV of 40 × 80 μm size during 2 min of observation. B, Fraction of FNDs-containing cargoes having a directed motion. C, Length of FND trajectories. D, Examples of FND trajectories. Scale bar: 10 μm. E–H, Comparison of four transport parameters: (E) curvilinear velocity, (F) run length, (G) pausing time, and (H) pausing frequency. The number within each bar represents the total number of FoVs (A, B) or trajectories (C, E–H) analyzed from n = 8 coverslips (4 independent cultures). Insets, Box-plots representation of the same dataset. * and *** mean p<0.05 and p<0.001 respectively; ns: non significant. See also Extended Data Figures 2-1, 2-2.

Figure 3.

Effect of α-synF or α-synR on the mobility of LysoTracker-labeled lysosomes and their transport in mouse cortical neurons at DIC21; 24-h exposure to α-synF or α-synR at 0.2 μm concentration, compared with nothing added control (Ctrl). A, Number of lysosomes detected per FoV of 40 × 80 μm size during 2 min of observation. B, Fraction of lysosomes having a directed motion. C, Length of lysosome trajectories. D, Examples of lysosome trajectories. Scale bar: 10 μm. E–H, Comparison of four transport parameters: curvilinear velocity (E), run length (F), pausing time (G), and pausing frequency (H). The number within each bar represents the total number of FoVs (A, B) or trajectories (C, E–H) analyzed from n = 2 coverslips (from one culture). Insets, Box-plots representation of the same dataset. *, ** and *** mean p<0.05, p<0.01 and p<0.001 respectively; ns: non significant.

Figure 4.

Effect of AβF and AβO on the mobility of endosomes and their transport as measured by tracking FND-containing cargoes in mouse cortical neurons at DIC21; 24-h exposure to AβF and AβO at 1 μm concentration, compared with nothing added control (Ctrl). A, Number of FNDs detected per FoV of 40 × 80 μm size during 2 min of observation. B, Fraction of FNDs-containing cargoes having a directed motion. C, Length of FND trajectories. D, Examples of FND trajectories. Scale bar: 10 μm. E–H, Comparison of four transport parameters: curvilinear velocity (E), run length (F), pausing time (G), and pausing frequency (H). The number inside the bar represents the total number of FoVs (A, B) or trajectories (C, E–H) analyzed from n = 6 coverslips (3 independent cultures). Insets, Box-plots representation of the same dataset. *, ** and *** mean p<0.05, p<0.01 and p<0.001 respectively; ns: non significant. See also Extended Data Figure 4-1.

Figure 5.

Effect of AβF and AβO on the mobility of LysoTracker-labeled lysosomes and their transport in mouse cortical neurons at DIC21; 24-h exposure to AβF and AβO at 1 μm concentration, compared with nothing added control. A, Number of lysosomes detected per FoV of 40 × 80 μm size during 2 min of observation. B, Fraction of lysosomes having a directed motion. C, Length of lysosome trajectories. D, Examples of lysosome trajectories. Scale bar: 10 μm. E–H, Comparison of four transport parameters: curvilinear velocity (E), run length (F), pausing time (G), and pausing frequency (H). I, J, Comparison of lysosome size. The number inside the bar represents the total number of FoVs (A, B), trajectories (E–H), and lysosomes (I, J) analyzed from n = 2 coverslips (from one culture). Insets, Box-plots representation of the same dataset. * and *** mean p<0.05 and p<0.001 respectively. See also Extended Data Figures 5-1, 5-2, 5-3.

Figure 6.

Intraneuronal transport of ATTO 488-labeled α-synF and R in mouse cortical neurons at DIC21. DIC20 cortical neurons were exposed to α-synF and R during 24 h, at concentration of 0.2 μm. A, B, Examples of α-synF and R, and FND trajectories (in the presence of α-synF and R). Scale bar: 10 μm. C, Length of α-synF, α-synR, and FND trajectories. D–G, Comparison of four transport parameters: curvilinear velocity (D), run length (E), pausing time (F), and pausing frequency (G). The number inside the bar represents the total number of trajectories analyzed from n = 8 coverslips (4 independent cultures) is indicated in each bar. Insets, Box-plots representation of the same dataset. * and *** mean p<0.05 and p<0.001 respectively; ns: non significant.

Figure 7.

Intraneuronal transport of ATTO 488-labeled AβF and AβO in mouse cortical neurons at DIC21. DIC20 cortical neurons were exposed to AβF and AβO (1 μm) during 24 h. A, B, Examples of trajectories. Scale bar: 10 μm. C, Length of AβF, AβO and FND trajectories. D–G, Comparison of four transport parameters: curvilinear velocity (D), run length (E), pausing time (F), and pausing frequency (G). The number inside the bar represents the total number of trajectories analyzed from n = 6 coverslips (from 3 independent cultures) is indicated in each bar. Insets: box plots representation of the same dataset. * and *** mean p<0.05 and p<0.001 respectively; ns: non significant. See also Extended Data Figure 7-1.

Figure 8.

Colocalized events between moving neurodegenerative disease-related molecular species and moving lysosomes at different time points. A, α-synF and α-synR were incubated for 24 and 48 h, at concentration of 0.2 μm. B, AβF and AβO were incubated for 24 and 48 h, at concentration of 1 μm. The number inside the donut plot represents the percentage of moving α-syn or Aβ assemblies colocalized with lysosomes (LysoTracker labeled). The table on the right panel indicates the number of neurodegenerative-related molecular species trajectories colocalized with lysosomes. n represents the total number of trajectories. The percentage and number of trajectories in each time point were analyzed from two coverslips (from one culture). See also Extended Data Figure 8-1.