Introduction Macroautophagy (hereafter referred to as autophagy) is a lysosomal degradation process that removes and degrades damaged proteins and organelles. Autophagy is initiated when a portion of the cytoplasm is enclosed within a double-membrane organelle, termed an autophagosome (Xie and Klionsky, 2007). Autophagosomes then fuse with degradative compartments in the endosomal–lysosomal pathway. Autophagy occurs at a basal, constitutive level and functions as a quality control system that can be up-regulated in response to cellular stresses, such as starvation (Mizushima et al., 2008). Autophagy is an essential pathway in postmitotic cells, such as neurons, cells that are particularly susceptible to the accumulation of defective proteins and organelles. Neuron-specific disruption of autophagy results in neurodegeneration (Hara et al., 2006; Komatsu et al., 2006). Defects in autophagy have been observed in multiple models of neurodegenerative disease, including Alzheimer’s, Huntington’s, and amyotrophic lateral sclerosis (ALS; Rubinsztein et al., 2005; Ventruti and Cuervo, 2007; Mariño et al., 2011). However, the biogenesis, maturation, and dynamics of autophagosomes in neurons are only poorly understood. Most studies to date have focused on model systems that lack the extended and highly polarized processes that characterize neurons (Jahreiss et al., 2008; Kimura et al., 2008). Here, we track the dynamics of autophagosomes in real time along the axons of primary neurons. We find that autophagosome initiation is a constitutive and spatially restricted process in the distal axon. Autophagosomes engulf both cytosolic and organelle cargo and are driven by dynein and kinesin motors. Once formed, autophagosomes undergo a spatially defined maturation as they move processively along the axon toward the cell body. We find that autophagosome dynamics remain robust in a mouse model of neurodegenerative disease, but autophagosome flux is not increased even as protein aggregates accumulate along the axon. Thus, in primary neurons, the dynamics of the autophagy pathway are robust and spatially specific but may not be efficiently up-regulated in response to increased protein aggregation. Results and discussion Distal initiation followed by robust retrograde transport of autophagosomes in primary neurons To monitor the dynamics of autophagosomes in primary neurons, we isolated dorsal root ganglion (DRG) neurons from transgenic mice expressing the autophagosome marker GFP-LC3 (Mizushima et al., 2004). After 2 d in vitro, DRG neurons extend neurites ∼1,000 µm in length that are tau positive (Perlson et al., 2009). Microtubules in the neurite are uniformly polarized with plus ends outward as indicated by the directionality of the plus-tip protein EB3 (Fig. S1 A). The linear separation provided by the extended length of the processes allows sufficient spatial resolution to examine the steps of autophagosome formation and maturation along the axon using live-cell imaging. Cytosolic GFP-LC3 is found throughout the neuron but becomes lipidated and incorporated into autophagosomes upon their formation (Xie and Klionsky, 2007). Imaging the distal tips of neurons revealed the initiation of autophagosome formation. Within bulbous regions near the distal end of the axon, ∼1 mm from the cell soma, we observed the incorporation of GFP-LC3 into ringlike structures ∼800 nm in diameter (Fig. 1, A and B). Often, multiple rings were observed to accumulate within a single bulbous region (Fig. 1 B). We identified these ringlike structures as autophagosomes based on their strong LC3-positive signals and the similarity in morphology to the LC3-positive structures seen in vivo in transgenic GFP-LC3 mice (Mizushima et al., 2004). 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. Real-time analysis of distal neurites revealed direct evidence for autophagosome biogenesis. GFP-LC3–positive puncta appeared in distal bulbs and grew progressively into ring structures (Fig. 1, C–E; and Video 1). This growth was further resolved using FRAP. Within ∼20 s of photobleaching, puncta began to recover their fluorescence and continued to grow into a ring, indicating continued recruitment of unbleached GFP-LC3 into the expanding organelle (Fig. 1 D). This process was rapid, taking 4–6 min for recovering puncta to develop into a ring. In contrast, preexisting rings did not recover their fluorescence after photobleaching, appearing as black holes within the GFP-LC3–labeled cytoplasm (Fig. 1 E). The lack of recovery suggests that GFP-LC3 incorporated into the autophagosome ring is relatively stable and not rapidly exchanged with the cytosolic pool. Autophagosome biogenesis appears to be spatially restricted to the distal neurite, as we performed FRAP along the axon and did not observe either formation or growth of autophagosomes; also, preexisting autophagosomes along the axon showed no recovery (Fig. S1 B). Thus, in primary neurons, autophagosome precursors are generated distally. We have not observed biogenesis along the axon but cannot rule out initiation in the cell body caused by high levels of autofluorescence that limit our resolution. This spatial regulation of autophagosome biogenesis in primary neurons is distinct from observations in smaller, less polarized cells in which autophagosomes form throughout the cytosol (Jahreiss et al., 2008). The origin of the membrane that forms the distal LC3-positive rings remains to be determined. Possible sources include endocytosed plasma membrane, ER, mitochondria, or Golgi membranes (Hollenbeck, 1993; Hayashi-Nishino et al., 2009; Hailey et al., 2010; Ravikumar et al., 2010; van der Vaart and Reggiori, 2010; Yen et al., 2010). GFP-LC3–positive rings in the distal process either remained stationary or displayed saltatory bidirectional motility in a constrained region within ∼150–200 µm of the neurite tip (Fig. 1 F). Occasionally, autophagosomes could be seen exiting this bidirectional pool to initiate travel toward the cell body (Fig. 1 F and Video 2). Along the axon shaft, autophagosomes exhibited a different type of movement. Within the axon (50–500 µm from the cell body), autophagosomes displayed robust and primarily unidirectional motility (Fig. 1, G–I; and Video 3), with 82 ± 2% (±SEM) moving a net distance of ≥10 µm in the retrograde direction. Anterograde movement was rarely observed ( 0.067 µm/s in the opposite direction as compared with the net displacement of the vesicle. The number of reversals within 100 µm was determined based on the net distance a vesicle traveled during the 3 min. Kymographs were generated using MetaMorph from neurites having at least one GFP-LC3–positive puncta that traveled a net distance of ≥10 µm. From each kymograph, the percentage of autophagosomes moving in the net retrograde direction (≥10 µm) versus net anterograde direction (≥10 µm) was determined. Nonprocessive vesicles that did not move a net distance of 10 µm exhibited bidirectional and stationary motility. From these kymographs, the total number of vesicles was determined and normalized by kymograph length (micrometers). Flux (number of vesicles moving within 100 µm/min) was determined by the sum of retrograde and anterograde vesicles (excluding bidirectional/stationary vesicles) and normalized by kymograph and video length. For mCherry-EGFP-LC3 analysis, the number of LC3 puncta positive for both GFP and mCherry fluorescence was counted based on kymographs. The proximal region of the neurite was defined as being within ∼200 µm of the cell body, and the distal region was within ∼100 µm of the end of the neurite. Biochemistry Fractions enriched for autophagosomes were prepared from brains of GFP-LC3 transgenic mice using protocols adapted from Morvan et al. (2009) and Strømhaug et al. (1998). Two brains were homogenized in 10 ml of 250 mM sucrose in 10 mM Hepes-KOH, pH 7.4 (with 1 mM EDTA for three-step gradient protocol) using a 30-ml homogenizer with a round-bottom Teflon pestle. Volumes of the gradient steps were scaled proportionately for a rotor (SW41; Beckman Coulter). The final gradient of the three-step fractionation protocol (Strømhaug et al., 1998) was spun in a rotor (TLS-55; Beckman Coulter). Equal total protein of low speed supernatant and the autophagosome-enriched fraction was separated by SDS-PAGE and subjected to immunoblot analysis. Immunofluorescence For immunofluorescence analysis, DRG neurons were plated on coverslips and cultured for 2 d. Cells were washed once in PBS (150 mM NaCl in 50 mM NaPO4, pH 7.4) and fixed in 3% PFA in PBS for 15 min at room temperature. Cells were washed twice in PBS and blocked and permeabilized in 2% (wt/vol) BSA and 0.1% (wt/vol) saponin in PBS for 1 h. All subsequent steps were performed in blocking/permeabilization buffer. Samples were incubated in primary antibody for 1 h, washed 3 × 5 min, incubated in secondary antibody for 1 h, washed 3 × 5 min, and mounted with ProLong gold. Online supplemental material Fig. S1 shows the anterograde movement of EB3 in DRG axons, FRAP of GFP-LC3 along the axon, and distributions of mean vesicle velocities and percentage of pausing for GFP-LC3 puncta along the axon. Fig. S2 shows that autophagosomes along the axon are positive for DIC2C-mCherry and mCherry-Kif3A but not the Golgi marker GPP130-mCherry. Fig. S2 also shows that LysoTracker red–positive compartments are positive for the late endosomal marker Rab7 but are largely negative for the early endosomal marker Rab5. In Fig. S3, we present data showing that despite the accumulation of SOD1G93A aggregates along the axon, autophagosome motility, direction, velocity, density, and flux are unaffected in a mouse model of fALS. Video 1 shows the appearance and growth of GFP-LC3–positive puncta in the neurite tip that grow into ringlike structures characteristic of autophagosomes. Video 2 shows an autophagosome escaping from the bidirectional pool at the neurite tip and moving processively toward the cell soma. Video 3 shows the robust retrograde motility of autophagosomes along the axon, and Videos 4 and 5 show that dynein co-migrates with these autophagosomes transfected with DIC1B-mCherry (Video 4) or DIC2C-mCherry (Video 5). Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201106120/DC1.
