Control of Autophagosome Axonal Retrograde Flux by Presynaptic Activity Unveiled Using Botulinum Neurotoxin Type A

Abstract

Botulinum neurotoxin type A (BoNT/A) is a highly potent neurotoxin that elicits flaccid paralysis by enzymatic cleavage of the exocytic machinery component SNAP25 in motor nerve terminals. However, recent evidence suggests that the neurotoxic activity of BoNT/A is not restricted to the periphery, but also reaches the CNS after retrograde axonal transport. Because BoNT/A is internalized in recycling synaptic vesicles, it is unclear which compartment facilitates this transport. Using live-cell confocal and single-molecule imaging of rat hippocampal neurons cultured in microfluidic devices, we show that the activity-dependent uptake of the binding domain of the BoNT/A heavy chain (BoNT/A-Hc) is followed by a delayed increase in retrograde axonal transport of BoNT/A-Hc carriers. Consistent with a role of presynaptic activity in initiating transport of the active toxin, activity-dependent uptake of BoNT/A in the terminal led to a significant increase in SNAP25 cleavage detected in the soma chamber compared with nonstimulated neurons. Surprisingly, most endocytosed BoNT/A-Hc was incorporated into LC3-positive autophagosomes generated in the nerve terminals, which then underwent retrograde transport to the cell soma, where they fused with lysosomes both in vitro and in vivo. Blocking autophagosome formation or acidification with wortmannin or bafilomycin A1, respectively, inhibited the activity-dependent retrograde trafficking of BoNT/A-Hc. Our data demonstrate that both the presynaptic formation of autophagosomes and the initiation of their retrograde trafficking are tightly regulated by presynaptic activity.

Interneuronal Transfer and Distal Action of Tetanus Toxin and Botulinum Neurotoxins A and D in Central Neurons

“…Microfluidic isolation is achieved and maintained by keeping the media volume in the soma chamber higher (300 μl) as compared to the axon chamber (200 μl). This volume difference prevents diffusion of molecules from the axon side to the soma side, as reported previously (Taylor et al, 2005, Wang et al, 2015, David et al, 2012); fluidic isolation is validated using fluorescent markers in Figure S1. Moreover, we routinely include fluorescent dyes in the microfluidics to ensure fluidic isolation is maintained and leak does not occur; when leak occurs these samples are excluded from further analysis (Figure S1; <5% of the devices fail to seal).…”

KIF1A/UNC-104 Transports ATG-9 to Regulate Neurodevelopment and Autophagy at Synapses

“…Autophagy has also been implicated in postdevelopmental events in neurons, such as synaptic transmission and vesicle recycling (Binotti et al, 2015; Hernandez et al, 2012; Wang et al, 2015). Although we focused on the characterization of developmental phenotypes, we note that autophagy protein expression persists in adults and hypothesize that the mechanisms reported here could influence synaptic physiology and function postdevelopmentally.…”

Mechanisms of neuronal homeostasis: Autophagy in the axon

“…2), driven by the microtubule-based molecular motor dynein (Cheng et al, 2015; Hollenbeck, 1993; Lee et al, 2011; Maday et al, 2012; Maday and Holzbaur, 2014; Wang et al, 2015; Yue, 2007). Retrogradely moving autophagosomes transport engulfed soluble and organelle cargoes such as ubiquitin and mitochondrial fragments (Maday et al, 2012).…”

“…Retrogradely moving autophagosomes transport engulfed soluble and organelle cargoes such as ubiquitin and mitochondrial fragments (Maday et al, 2012). As autophagosomes travel along the axon, they mature into degradative organelles (Lee et al, 2011; Maday et al, 2012; Wang et al, 2015). Autophagosomes along the mid-axon are positive for the late endosome/lysosome marker LAMP1, suggesting that exit from the distal axon is accompanied by fusion with late endosomes/lysosomes (Maday et al, 2012).…”