Developmental Cell Article Autophagosome Biogenesis in Primary Neurons Follows an Ordered and Spatially Regulated Pathway Sandra Maday1 and Erika L.F. Holzbaur1,* 1Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2014.06.001 SUMMARY Despite the evidence that autophagy is critical in maintaining neuronal homeostasis, little is understood about the mecha- Autophagy is an essential degradative pathway in nisms driving this process in neurons. Much of the work dissect- neurons, yet little is known about mechanisms ing the autophagic pathway has been performed in yeast and driving autophagy in highly polarized cells. Here, nonpolarized mammalian cells (Mizushima et al., 2011; Weid- we use dual-color live-cell imaging to investigate berg et al., 2011). However, neurons exhibit a highly polarized the neuron-specific mechanisms of constitutive and elongated morphology that poses a unique challenge to autophagosome biogenesis in primary dorsal root cellular trafficking and transport pathways. Many neurodegener- ative disease-associated mutations have been identified in the ganglion (DRG) and hippocampal cultures. Under machinery that transports organelles and proteins across the basal conditions, autophagosomes are continuously extended distance of the axon (Millecamps and Julien, 2013; generated in the axon tip. There is an ordered assem- Perlson et al., 2010), emphasizing the unique vulnerability of bly of proteins recruited with stereotypical kinetics the neuronal system. Furthermore, the majority of studies to onto the developing autophagosome. Plasma- or date have focused on stress-induced autophagy as a result of mitochondrial-derived membranes were not incor- nutrient deprivation. However, knockout mouse models have porated into nascent autophagosomes in the distal demonstrated that basal levels of autophagy are essential for axon. Rather, autophagosomes are generated at neuronal survival (Hara et al., 2006; Komatsu et al., 2006, double FYVE-containing protein 1 (DFCP1)-positive 2007). Thus, we set out to determine the spatiotemporal dy- subdomains of the endoplasmic reticulum (ER), namics of autophagy in primary neurons under basal growth distinct from ER exit sites. Biogenesis events are conditions. Here, we describe the assembly of an autophagosome in real enriched distally; autophagosomes form infrequently time in primary dorsal root ganglion (DRG) and hippocampal in dendrites, the soma, or midaxon, consistent with a neurons. We find that autophagosome formation in primary compartmentalized pathway for constitutive auto- neurons is a constitutive process under basal growth conditions. phagy in primary neurons. Distal biogenesis may Autophagosomes are continuously generated at the axon tip; facilitate degradation of damaged mitochondria this biogenesis involves an ordered recruitment of proteins that and long-lived cytoplasmic proteins reaching the assemble onto the nascent autophagosome with stereotypical axon tip via slow axonal transport. kinetics. Under basal conditions, we find that neuronal autopha- gosomes are not generated from plasma- or mitochondrial- derived membrane but, rather, from specific subdomains of INTRODUCTION the endoplasmic reticulum (ER). Most strikingly, autophago- somes are preferentially generated at the distal end of the Macroautophagy (autophagy) is an evolutionarily conserved axon, emphasizing the high degree of compartmentalization lysosomal degradation pathway that maintains the homeostasis and spatial regulation controlling autophagosome biogenesis of the cellular environment by eliminating damaged organelles in primary neurons. We propose that this pathway provides a and aggregated proteins (Xie and Klionsky, 2007). This pathway mechanism to recycle distally localized aged mitochondria as is particularly important in postmitotic cells such as neurons well as long-lived proteins transported to the distal axon via that are unable to dilute out proteotoxins by cell division. CNS- slow axonal transport. specific or neuron-specific knockout of genes required for auto- phagy induces axonal degeneration and neuron cell death (Hara et al., 2006; Komatsu et al., 2006, 2007). Furthermore, mutations RESULTS in the mitophagy machinery PINK1 and Parkin lead to early-onset Parkinson’s disease (Kitada et al., 1998; Narendra et al., 2008, Autophagosome Biogenesis in Primary Neurons Follows 2010; Valente et al., 2004), potentially linking defective autophagy an Ordered and Spatially Regulated Pathway with the progression of neurodegenerative disease. Autophagy is Autophagy is initiated when components of the cytosol are thus a protective mechanism against neuronal dysfunction and enveloped within a membrane cisterna termed an ‘‘isolation degeneration (Marin˜ o et al., 2011; Rubinsztein et al., 2005). membrane’’ or ‘‘phagophore’’ (Xie and Klionsky, 2007). The Developmental Cell 30, 71–85, July 14, 2014 ª2014 Elsevier Inc. 71 Developmental Cell Autophagosome Biogenesis in Primary Neurons A 0s 1m 30s 2m 2s 4m 12s 4m 44s Atg13 LC3 Merge B 0s 1m 16s 1m 40s 2m 54s 4m 12s Atg5 LC3 Merge 120 120 100 C D Atg13 Atg13 LC3 LC3 100 100 80 80 80 60 60 60 40 40 40 Intensity (A.U.) Intensity (A.U.) 20 20 Intensity (A.U.) 20 0 0 0 0 0 0 0 0 0 -8 -40 40 80 -8 -40 40 80 -80 -60 -4 -20 20 40 60 80 -160 -120 120 160 200 240 280 -160 -120 120 160 200 240 280 -100 100 120 Time (sec) Time (sec) Time (sec) 120 120 100 E F Atg5 Atg5 LC3 LC3 100 100 80 80 80 60 60 60 40 40 40 Intensity (A.U.) Intensity (A.U.) 20 Intensity (A.U.) 20 20 0 0 0 0 0 0 0 0 0 0 0 -80 -60 -40 -20 20 40 60 80 -80 -60 -40 -20 20 40 60 80 -80 -60 -40 -20 20 40 60 80 -100 100 120 140 16 18 20 -100 100 12 140 160 18 200 -100 100 120 140 160 180 Time (sec) Time (sec) Time (sec) G H I J 100 Atg13 First frame Last frame LC3 (13) 2.5 2.5 80 Atg5 Atg5 LC3 (5) 2.0 2.0 60 1.5 1.5 Atg5 decay initiated 40 LC3 1.0 1.0 Atg5 decay 0.5 complete 0.5 Y coordinate (µm) Y Atg5 decay initiated coordinate (µm) Y Intensity (A.U.) 20 Atg5 decay complete 0 0 0 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0 Merge X coordinate (µm) X coordinate (µm) 0 -80 -60 -40 -20 20 40 60 80 -100 100 120 Time (sec) Figure 1. Autophagosome Biogenesis in DRG Neurons Involves an Ordered Recruitment of Assembly Factors (A) Time series of mCherry-Atg13 and GFP-LC3 in the distal axon of DRG neurons. Red and green arrowheads indicate the ordered appearance of Atg13 followed by LC3, respectively. Yellow arrowheads denote colocalization between Atg13 and LC3. The retrograde direction is toward the right. Scale bar, 1 mm. (B) Time series of mCherry-Atg5 and GFP-LC3 in the distal axon. Red and green arrowheads indicate the ordered appearance of Atg5 followed by LC3, respectively. Yellow arrowheads denote colocalization between Atg5 and LC3. The retrograde direction is toward the right. Scale bar, 1 mm. See also Movie S1. (legend continued on next page) 72 Developmental Cell 30, 71–85, July 14, 2014 ª2014 Elsevier Inc. Developmental Cell Autophagosome Biogenesis in Primary Neurons edges of the isolation membrane fuse to form a closed double- proceeds with highly consistent kinetics. On average, Atg13 and membrane autophagosome. These autophagosomes are shut- Atg5 reached a maximum intensity within 1.3 and 1.0 min tled toward lysosomes for fusion, and the contents are degraded after initial appearance, respectively, and sustained that signal by lysosomal hydrolases and recycled back into the cytosol. for 50 s before decay was initiated (Figures 1C–1F). Alignment Seminal work in yeast identified proteins important for the forma- of Atg13 and Atg5 mean intensity profiles indicated that they are tion of an autophagosome (Harding et al., 1995; Klionsky et al., recruited to nascent autophagosomes with kinetics that cannot 2003; Thumm et al., 1994; Tsukada and Ohsumi, 1993), and be distinguished at this time resolution (one frame every 2 s; many are conserved in mammals (Mizushima et al., 2011; Figure 1J). However, Atg5 decayed from the nascent autopha- Weidberg et al., 2011). During autophagosome formation, these gosome prior to Atg13 (Figure 1J). This order of disassembly proteins are recruited to the assembly site in an ordered fashion from the nascent autophagosome was also observed with direct (Itakura and Mizushima, 2010; Suzuki et al., 2001, 2007). We set pairwise imaging of GFP-Atg13 and mCherry-Atg5 (data not out to determine whether neurons employ this conserved mech- shown). The dynamics of LC3 intensity changes over time anism for autophagosome formation and define the temporal measured across all experiments were extraordinarily consis- relationship between assembly proteins as they arrive at the tent. Following a lag phase, LC3 was robustly recruited, rising nascent autophagosome. from initial signal to maximum intensity within 1.5 min (Figures We performed live-cell imaging on DRG neurons isolated from 1C–1F and 1J). Decay of Atg5 was always initiated prior to obser- transgenic mice expressing GFP-LC3. To measure autophago- vation of maximum LC3 intensity at the developing autophago- some assembly, neurons were transfected with either Atg13 or some (Figures 1F and 1J). Atg5 labeled with mCherry to allow pairwise imaging with GFP- Next, we asked whether there is a relationship between the LC3. Atg13 is a component of the Ulk1 complex that regulates stage of autophagosome formation and the mobility of the initial formation of the autophagosome (Mizushima et al., 2011) structure. We hypothesized that perhaps during the Atg5-posi- and Atg5 localizes to the isolation membrane and regulates its tive phase, the autophagosome is confined due to tethering to elongation (Mizushima et al., 2001). Thus, both Atg13 and Atg5 the membrane source.