Compartment-Specific Regulation of Autophagy in Primary Neurons

Abstract

Autophagy is an essential degradative pathway that maintains neuronal homeostasis and prevents axon degeneration. Initial observations suggest that autophagy is spatially regulated in neurons, but how autophagy is regulated in distinct neuronal compartments is unclear. Using live-cell imaging in mouse hippocampal neurons, we establish the compartment-specific mechanisms of constitutive autophagy under basal conditions, as well as in response to stress induced by nutrient deprivation. We find that at steady state, the cell soma contains populations of autophagosomes derived from distinct neuronal compartments and defined by differences in maturation state and dynamics. Axonal autophagosomes enter the soma and remain confined within the somatodendritic domain. This compartmentalization likely facilitates cargo degradation by enabling fusion with proteolytically active lysosomes enriched in the soma. In contrast, autophagosomes generated within the soma are less mobile and tend to cluster. Surprisingly, starvation did not induce autophagy in either the axonal or somatodendritic compartment. While starvation robustly decreased mTORC1 signaling in neurons, this decrease was not sufficient to activate autophagy. Furthermore, pharmacological inhibition of mammalian target of rapamycin with Torin1 also was not sufficient to markedly upregulate neuronal autophagy. These observations suggest that the primary physiological function of autophagy in neurons may not be to mobilize amino acids and other biosynthetic building blocks in response to starvation, in contrast to findings in other cell types. Rather, constitutive autophagy in neurons may function to maintain cellular homeostasis by balancing synthesis and degradation, especially within distal axonal processes far removed from the soma.

Significance statement: Autophagy is an essential homeostatic process in neurons, but neuron-specific mechanisms are poorly understood. Here, we compare autophagosome dynamics within neuronal compartments. Axonal autophagy is a vectorial process that delivers cargo from the distal axon to the soma. The soma, however, contains autophagosomes at different maturation states, including input received from the axon combined with locally generated autophagosomes. Once in the soma, autophagosomes are confined to the somatodendritic domain, facilitating cargo degradation and recycling of biosynthetic building blocks to primary sites of protein synthesis. Neuronal autophagy is not robustly upregulated in response to starvation or mammalian target of rapamycin inhibition, suggesting that constitutive autophagy in neurons maintains homeostasis by playing an integral role in regulating the quality of the neuronal proteome.

Keywords: LC3; autophagy; axonal transport; hippocampal neuron; mTOR; soma.

Figure 1.

Axonal, LAMP1-positive autophagosomes enter the soma through the AIS and become restricted to the somatodendritic domain. A, Hippocampal neuron, grown 8 DIV, develops axon and dendritic processes. Neurons were transfected with mCherry-LC3 and YFP-NaVII-II, a marker for the AIS. Arrowheads denote autophagic vesicles. B, Kymographs of mCherry-LC3 motility through the AIS. For clarity, the mCherry-LC3 kymograph was traced and shown below; the gray box denotes the AIS region. C, Kymograph analysis of GFP-LC3 and LAMP1-RFP motility in the mid-axon and proximal axon. Closed arrowheads denote retrogradely moving autophagosomes that comigrate along the mid-axon with LAMP1-RFP, and enter the soma positive for LAMP1-RFP. Open arrowheads denote LAMP1-RFP-positive organelles that are negative for GFP-LC3. D, Still images along the mid-axon generated from the movie analyzed in C. Closed arrowheads denote autophagosomes positive for LAMP1-RFP. Open purple arrowheads denote organelles positive for LAMP1-RFP only. E, GFP-LC3 transgenic hippocampal neurons 10 DIV were immunostained for LAMP1. Closed arrowheads denote autophagosomes in the axon that are positive for endogenous LAMP1. Open arrowheads denote endogenous LAMP1-positive organelles that are negative for GFP-LC3. F, Time series of autophagosome motility at the base of an axon in a hippocampal neuron grown 10 DIV. Arrowheads denote a GFP-LC3-positive autophagosome rejected from entry into the axon. G, Time series of autophagosome motility into a dendrite of a hippocampal neuron grown 8 DIV. Arrowheads denote a GFP-LC3-positive autophagosome that robustly enters into the dendritic shaft. H, Frequency distribution of distances that autophagosomes enter from the base of each neurite population (axons, n = 12 events from 6 neurons from 3 experiments; dendrites, n = 51 events from 15 neurons from 5 experiments; 5–10 DIV). Values for dendrites are under-represented since autophagosomes move beyond the field of view. Scale bars: horizontal, 5 μm; vertical, 1 min.

Figure 2.

The soma contains autophagosomes from distinct compartments at different stages of maturation. A, Time series of autophagosome biogenesis in the soma of a hippocampal neuron grown 6 DIV. Arrowheads denote a newly forming autophagosome. Scale bar, 1 μm. B, GFP-LC3 transgenic hippocampal neurons grown 10 DIV were fixed and immunostained for GFP and p62. Arrowheads denote cells with GFP-LC3-positive structures that are copositive for p62. Lower panels are GFP-LC3 transgenic neurons cocultured with nontransgenic neurons (10 DIV); the presence of p62-positive clusters is not dependent on expression of GFP-LC3. C, GFP-LC3 transgenic hippocampal neurons grown 10 DIV were immunostained for GFP, p62, and LAMP1. Corresponding line scans are shown on the right; arrowheads denote overlapping peaks of fluorescence intensity. D, Stills from live-cell imaging of hippocampal neurons on 8 DIV expressing GFP-LC3 and LAMP1-RFP. Shown is an apical and basal plane of two individual cells and corresponding line-scan analysis. Closed arrowheads on basal images denote organelles positive for both GFP-LC3 and LAMP1-RFP. Colocalized puncta may appear slightly offset due to the sequential capture of images, green followed by purple. Arrowheads on line scans denote peaks of overlapping fluorescence. Open white arrowheads on GFP-LC3 basal image denote autophagosomes in neighboring axons that are not transfected with LAMP1-RFP. E, First frames and corresponding maximum projections of the entire frames of GFP-LC3 movies acquired in the apical or basal plane of two different hippocampal neurons grown 8 DIV. Individual autophagosomes are color-coded to visualize the entire trajectory in the maximum projection. Scale bars: B–E, 5 μm.

Figure 3.

Autophagy is not upregulated along the axon in response to amino acid starvation. A, Kymographs from live-cell imaging of GFP-LC3 transgenic hippocampal neurons (8 DIV). For clarity, GFP-LC3 tracks are traced and shown below each corresponding kymograph. Scale bars: horizontal, 5 μm; vertical, 1 min. B, Quantitation of autophagosome density (number of autophagosomes per 100 μm axon in the first frame of movie) in the mid-axon (mean ± SEM; n = 23–32 neurons from 5–6 experiments; 8–10 DIV; one-way ANOVA with Dunnett's post hoc test). C, Quantitation of autophagosome area flux (number of autophagosomes per 100 μm per minute) in the mid-axon (mean ± SEM; n = 23–33 neurons from 5–6 experiments; 8–10 DIV; one-way ANOVA with Dunnett's post hoc test). D, Quantitation of autophagosomes undergoing retrograde transport in the mid-axon (mean ± SEM; n = 23–33 neurons from 5–6 experiments; 8–10 DIV). *p ≤ 0.05; ***p ≤ 0.001 (one-way ANOVA with Dunnett's post hoc test). E, Quantitation of autophagosome motility along the mid-axon of hippocampal neurons (mean ± SEM; n = 23–33 neurons from 5–6 experiments; 8–10 DIV). *p ≤ 0.05; ***p ≤ 0.001 (one-way ANOVA with Dunnett's post hoc test within each grouping designated by x-axis breaks). ns, Not significant; AVs, autophagic vacuoles.

Figure 4.

Glucose deprivation does not induce autophagosome formation but impairs retrograde transport along the axon. A, Straightened en face images and corresponding kymographs of DsRed2-mito along the mid-axon of hippocampal neurons 8 DIV. The retrograde direction is toward the right. Scale bars: horizontal, 5 μm; vertical, 1 min. B, Quantitation of mitochondrial motility along the axon of hippocampal neurons grown 8 DIV (mean ± SEM; n = 13–17 neurons from 2 experiments; two-way ANOVA with Bonferroni's post hoc test). C, Quantitation of autophagosome density (number of autophagosomes per 100 μm axon in the first frame of movie) in the mid-axon (mean ± SEM; n = 27–32 neurons from 3–4 experiments; 8–9 DIV; one-way ANOVA with Dunnett's post hoc test). D, Quantitation of autophagosome area flux (number of autophagosomes per 100 μm per minute) in the mid-axon (mean ± SEM; n = 27–33 neurons from 3–4 experiments; 8–9 DIV; one-way ANOVA with Dunnett's post hoc test). E, Quantitation of autophagosomes undergoing retrograde transport in the mid-axon (mean ± SEM; n = 12–18 neurons from 3–4 experiments; 8–9 DIV). ***p ≤ 0.001 (two-way ANOVA with Bonferroni's post hoc test). ns, Not significant; AVs, autophagic vacuoles.

Figure 5.

Autophagosomes accumulate in the soma with bafilomycin A1 treatment. A, Maximum projections of z-stacks of the soma of GFP-LC3 transgenic hippocampal neurons (8–10 DIV). Inset, hippocampal neurons also display larger clusters of GFP-LC3. Scale bar, 5 μm. B, GFP-LC3-expressing hippocampal neurons were immunostained for p62. Scale bar, 5 μm. C, Quantitation of GFP-LC3 area normalized to the soma area (mean ± SEM; n = 24–36 neurons from 5–6 experiments; 8–10 DIV). ***p ≤ 0.001 (one-way ANOVA with Dunnett's post hoc test). D, Quantitation of GFP-LC3 area normalized to the soma area (mean ± SEM; n = 30–33 neurons from 3–4 experiments; 8–9 DIV; one-way ANOVA with Dunnett's post hoc test). E, Quantitation of immunoblotting of lysates from cultured hippocampal neurons. LC3-II levels were normalized to GAPDH (mean ± SEM; n = 3 experiments; SDS-PAGE and immunoblotting were performed in duplicate for each experiment, 8–10 DIV). ***p ≤ 0.001 (one-way ANOVA with Dunnett's post hoc test). F, Immunoblot analysis of lysates from cultured hippocampal neurons for endogenous LC3 and GAPDH (loading control). G, Immunoblot analysis of lysates from cultured wild-type hippocampal neurons for mTORC1 activity. Alpha-tubulin loading controls are shown below each corresponding immunoblot. H, I, Immunoblot analysis and corresponding quantitation of LC3-II levels in lysates from cultured wild-type hippocampal neurons treated with Torin1. LC3-II levels were normalized to GAPDH (mean ± SEM; n = 3 experiments; 8–10 DIV). *p ≤ 0.05; **p ≤ 0.01 (one-way ANOVA with Dunnett's post hoc test). Calnexin and α-tubulin serve as loading controls for pS6 and total S6 immunoblots, respectively. J, Model schematic of the compartment-specific regulation of autophagy in neurons. ns, Not significant.