Live Imaging of Calcium Dynamics during Axon Degeneration Reveals Two Functionally Distinct Phases of Calcium Influx

Abstract

Calcium is a key regulator of axon degeneration caused by trauma and disease, but its specific spatial and temporal dynamics in injured axons remain unclear. To clarify the function of calcium in axon degeneration, we observed calcium dynamics in single injured neurons in live zebrafish larvae and tested the temporal requirement for calcium in zebrafish neurons and cultured mouse DRG neurons. Using laser axotomy to induce Wallerian degeneration (WD) in zebrafish peripheral sensory axons, we monitored calcium dynamics from injury to fragmentation, revealing two stereotyped phases of axonal calcium influx. First, axotomy triggered a transient local calcium wave originating at the injury site. This initial calcium wave only disrupted mitochondria near the injury site and was not altered by expression of the protective WD slow (WldS) protein. Inducing multiple waves with additional axotomies did not change the kinetics of degeneration. In contrast, a second phase of calcium influx occurring minutes before fragmentation spread as a wave throughout the axon, entered mitochondria, and was abolished by WldS expression. In live zebrafish, chelating calcium after the first wave, but before the second wave, delayed the progress of fragmentation. In cultured DRG neurons, chelating calcium early in the process of WD did not alter degeneration, but chelating calcium late in WD delayed fragmentation. We propose that a terminal calcium wave is a key instructive component of the axon degeneration program.

In vivo Imaging of Mitochondrial Transport in Single-Axon Regeneration of Zebrafish Mauthner Cells

“…Laser axotomy has been widely used in zebrafish, especially in the PNS (O’Brien et al, 2009; O’Donnell et al, 2013; Vargas et al, 2015). Different from mechanical lesioning (Bhatt et al, 2004; Feng et al, 2010), we utilized a two-photon laser to transect M axons over cloacal pores at 6 dpf ( Figure 3A ).…”

“…There are many models for studying mitochondrial transport in living animal axons, including Drosophila (Pilling et al, 2006) mammals (Misgeld et al, 2007; Sorbara et al, 2014; Takihara et al, 2015; Zhou et al, 2016), and others. Because of their optical transparency and small size, researchers have come to realize that zebrafish are useful organisms for exploring mitochondria from transport to function without intricate surgery before direct microscope imaging (Plucinska et al, 2012; O’Donnell et al, 2013; Vargas et al, 2015; Bergamin et al, 2016; Dukes et al, 2016). However, none of those models allowed the observation of mitochondrial transport in the vertebrate spinal cord at a single-axon level through non-invasive in vivo time-lapse imaging until now, making it impossible to explore the correlation between axon regeneration and internal mitochondrial transport in the living vertebrate CNS.…”

Microtubule‐dependent processes precede pathological calcium influx in excitotoxin‐induced axon degeneration

“…Our data are in line with previous studies that have investigated calcium alterations in other forms of axonal injury, particularly Wallerian degeneration. Vargus et al () investigated calcium alterations in zebrafish sensory axons undergoing Wallerian degeneration and demonstrated an initial localized benign calcium influx, which was followed by a terminal calcium rise prior to fragmentation. Similar effects have been reported in cultured neurons following transection (Villegas et al, ), where expression of the wlds protein did not affect initial calcium increases but prevented the later calcium rise and downstream degeneration (Adalbert et al, ).…”

Axon degeneration: context defines distinct pathways

“…Exactly how these signaling pathways induce axon degeneration is not fully understood, but this involves additional mediators of Wallerian degeneration which include the ubiquitin-proteasome system [20], the ubiquitin ligase Phr1/Highwire [21,22], and SCG10 [23,24]. Furthermore, Ca 2+ signaling and calpain activation are also important for mediating late events in axon degeneration [25,26,27]. …”