Autophagosomes initiate distally and mature during transport toward the cell soma in primary neurons

Abstract

Autophagy is an essential cellular degradation pathway in neurons; defects in autophagy are sufficient to induce neurodegeneration. In this paper, we investigate autophagosome dynamics in primary dorsal root ganglion neurons. Autophagosome biogenesis occurs distally in a constitutive process at the neurite tip. Autophagosomes initially move bidirectionally and then switch to unidirectional, processive movement toward the cell soma driven by dynein. Autophagosomes copurify with anterograde and retrograde motors, suggesting that the activity of bound kinesin motors is effectively down-regulated to yield robust retrograde motility driven by dynein. Both organelle and soluble cargoes are internalized into autophagosomes, including mitochondria and ubiquitin. As autophagosomes move distally to proximally, they undergo maturation and become increasingly acidified, consistent with the formation of an autolysosomal compartment that may more efficiently degrade cargo. This maturation is accompanied by a switch to bidirectional motility characteristic of lysosomes. Together, autophagosome biogenesis and maturation in primary neurons is a constitutive process that is spatially and temporally regulated along the axon.

Figure 1.

Autophagosomes initiate distally and undergo retrograde movement toward the cell soma in primary neurons. (A) GFP-LC3 localization at the distal end of DRG neurons. Arrowheads denote the accumulation of GFP-LC3–positive puncta. (B) Autophagosomes initiate in the distal tip of the neurite, where pronounced ringlike structures accumulate (arrowheads). (C) Time series of autophagosome biogenesis; arrowheads denote the appearance and growth of GFP-LC3–positive puncta into a ring (Video 1). (D and E) FRAP analysis of the distal neurite. Open arrowheads denote recovering puncta and growth into a ring, cyan and dark blue arrowheads denote two different examples of black holes of bleached preexisting rings, three different pink-shaded arrowheads denote the appearance of three different GFP-LC3–positive puncta that grow into rings. (F) Kymograph of autophagosome motility in the distal neurite showing bidirectional movement within a constrained region (arrowheads). Occasional autophagosomes escape from the tip and move processively toward the cell soma (retrograde track is pseudocolored green; Video 2). (G) Percentage of retrograde, anterograde, or bidirectional/stationary (Bi/Stat) vesicles (means ± SEM; n = 91 neurites). (H and I) Times series and corresponding kymographs showing processive movement of GFP-LC3–positive puncta along the axon (Video 3). Arrowheads denote an autophagosome traveling along the axon toward the cell soma. Retrograde motility is toward the right in all figures. Horizontal bars: (B–E) 1 µm; (A, F, H, and I) 10 µm. Vertical bars: (F) 2 min; (I) 1 min.

Figure 2.

Autophagosome dynamics in primary neurons are driven primarily by cytoplasmic dynein. (A) Individual GFP-LC3 puncta along the axon were tracked to yield a histogram of instantaneous velocities (n = 8,872 events). Shown are pooled data for 173 vesicles from 59 neurons from 12 mice analyzed in 12 separate experiments. (B) Reversals within 100 µm for each vesicle (median is 1.6 reversals/100 µm, n = 173 vesicles analyzed as in A). Median reversal length is 0.3 µm. (C) Time series and corresponding kymograph of GFP-LC3 and DIC1B-mCherry motility along the axon (Video 4). Open and cyan arrowheads denote two different examples of vesicles positive for both markers. Yellow arrowheads in the kymograph denote multiple examples of vesicles positive for both markers. The red signal may occasionally precede the green signal because of delays in acquiring consecutive images. (D) Time series and corresponding kymograph of GFP-LC3 and mCherry-Kif5C tail motility along the axon. Open arrowhead denotes a vesicle positive for both markers. Yellow arrowheads in the kymograph denote multiple examples of vesicles positive for both markers. (E) Fractions enriched for autophagosomes were prepared using a one-step gradient (Morvan et al., 2009) or a three-step gradient (Strømhaug et al., 1998) that more effectively selects for the lipidated form of LC3, LC3-II. Equal total protein from the low speed supernatant (LSS) and autophagosome-enriched fraction (AVs) were analyzed by immunoblotting. Autophagosome membrane-associated LC3-II is distinguished from cytosolic LC3-I by molecular mass (shown is endogenous LC3). Motors may not be quantitatively retained on autophagosome membranes through the three-step gradient. (F) Kymograph of GFP-LC3 motility from DRG neurons transfected with the dominant-negative CC1, which disrupts dynein/dynactin function. Arrested autophagosomes are marked with arrowheads. Horizontal bars, 10 µm. Vertical bars, 1 min.

Figure 3.

Autophagosomes contain engulfed cargo. (A) Time series and corresponding kymograph of GFP-LC3 and DsRed2-mito motility along the axon. Open arrowheads in times series and yellow arrowhead in kymograph denote a vesicle positive for both markers. (B) Time-lapse images and corresponding kymograph of GFP-LC3 and RFP-Ub motility along the axon. Open, cyan, and dark blue arrowheads denote three different examples of vesicles positive for both markers. Yellow arrowheads in kymograph denote multiple examples of vesicles positive for both markers. (C) FRAP analysis of RFP-Ub fluorescence inside autophagosome rings at the distal end of the neurite. Arrowheads denote enrichment of RFP-Ub inside autophagosome ring. (D) Time series and corresponding kymograph of GFP-LC3 and mCherry-SOD1^G93A motility along the axon. Open, cyan, and dark blue arrowheads denote three different examples of vesicles positive for both markers. Yellow arrowheads in kymograph denote two different examples of vesicles positive for both markers. (E) FRAP analysis of mCherry-SOD1^G93A fluorescence inside an autophagosome along the axon. Box denotes photobleached region, and arrowheads denote bleached vesicle. (F) FRAP analysis of mCherry-SOD1^G93A fluorescence inside autophagosomes at the neurite tip. Arrowheads denote enrichment of mCherry-SOD1^G93A inside the autophagosome ring. For distal FRAP images, the contrast enhancement applied to prebleach and bleach images was different from that applied to recovery images. Horizontal bars: (axon images) 10 µm; (distal images) 1 µm. Vertical bars, 1 min.

Figure 4.

Autophagosomes along the axon have fused with late endosomes or lysosomes. (A) GFP-LC3 and LAMP1-RFP distribution at the distal end of the neurite. Yellow, green, and red arrowheads designate vesicles positive for both markers, GFP-LC3 only, or LAMP1-RFP only, respectively. (B) Time series and corresponding kymograph of GFP-LC3 and LAMP1-RFP motility along the axon. Open, cyan, and dark blue arrowheads denote three different examples of vesicles positive for both markers. Yellow arrowheads in kymograph denote multiple examples of vesicles positive for both markers. (C) GFP-LC3 and LysoTracker (LysoT) red localization at the neurite tip. Yellow, green, and red arrowheads designate vesicles positive for both markers, GFP-LC3 only, or LysoTracker red only, respectively. (D) Time series and corresponding kymograph of GFP-LC3 and LysoTracker red motility along the axon. Open arrowheads denote a vesicle positive for both markers. Yellow arrowheads in kymograph denote multiple examples of vesicles positive for both markers. Horizontal bars: (distal images) 1 µm; (axon images) 10 µm. Vertical bars, 1 min.

Figure 5.

Autophagosomes mature as they move distally to proximally along the axon. (A) Wild-type neurons transfected with mCherry-EGFP-LC3. In acidic environments, the GFP moiety is preferentially quenched, and only the red fluorescence persists (yellow arrowheads show LC3 puncta positive for mCherry and EGFP; red arrowheads show LC3 puncta positive for mCherry only). (B) Corresponding kymograph of mCherry-EGFP-LC3 motility at the distal tip. (C and D) Images from live-cell analysis and corresponding kymographs from regions proximal to the cell soma. (E) Quantitation of LC3 puncta positive for mCherry and GFP in the distal versus proximal regions of the axon (means ± SEM; n = 9 neurites; **, P = 0.0043, t test). (F) Percentage of retrograde, anterograde, or bidirectional/stationary vesicles (means ± SEM; n = 9 neurites). (G) Model for autophagosome biogenesis and maturation along the axon in primary neurons. Horizontal bars, 10 µm. Vertical bars, 1 min.