Large-scale in vivo femtosecond laser neurosurgery screen reveals small-molecule enhancer of regeneration

Abstract

Discovery of molecular mechanisms and chemical compounds that enhance neuronal regeneration can lead to development of therapeutics to combat nervous system injuries and neurodegenerative diseases. By combining high-throughput microfluidics and femtosecond laser microsurgery, we demonstrate for the first time largescale in vivo screens for identification of compounds that affect neurite regeneration. We performed thousands of microsurgeries at single-axon precision in the nematode Caenorhabditis elegans at a rate of 20 seconds per animal. Following surgeries, we exposed the animals to a hand-curated library of approximately one hundred small molecules and identified chemicals that significantly alter neurite regeneration. In particular, we found that the PKC kinase inhibitor staurosporine strongly modulates regeneration in a concentration-and neuronal type-specific manner. Two structurally unrelated PKC inhibitors produce similar effects. We further show that regeneration is significantly enhanced by the PKC activator prostratin. C. elegans | chemical screen | microfluidicsT he ability of neurons in the adult mammalian central nervous system to regenerate their axons after injury is extremely limited, which has been attributed to both extrinsic signals of the inhibitory glial environment (1) as well as intrinsic neuronal factors (2-4). The discovery of cell-permeable small molecules that modulate axon regrowth can potentiate the development of efficient therapeutic treatments for spinal cord injuries, brain trauma, stroke, and neurodegenerative diseases. Identification of such molecules can also provide valuable tools for fundamental investigations of the mechanisms involved in the regeneration process. Currently, small-molecule screens for neuronal regeneration are performed in simple in vitro cell culture systems. Such screens have already revealed large numbers of chemicals that enhance regeneration and/or affect cellular morphogenesis, yet many of these hits still remain untested in vivo. In addition, most in vitro studies do not translate to animal models and also fail to reveal off-target, toxic, or lethal effects. Thus, a thorough investigation of neuronal regeneration mechanisms requires in vivo neuronal injury models.In vivo investigation of neuronal regeneration has been performed mainly in mice and rats. However, their long developmental periods, complicated genetics and biology, and expensive maintenance prevent large-scale studies on these animals. The nematode Caenorhabditis elegans is a simple, well-studied, invertebrate model-organism with a fully mapped neuronal network comprising 302 neurons. Its short developmental cycle, simple and low-cost laboratory maintenance, and genetic amenability make it an ideal model for large-scale screens, rapid identification of the molecular targets of screened compounds, and discovery of novel signaling pathways implicated in regeneration.Until recently however, the small size of C. elegans (∼50 μm in diameter) prevented its use for investigation of neuronal re...

Regeneration ofDrosophilasensory neuron axons and dendrites is regulated by the Akt pathway involvingPtenand microRNAbantam

“…These findings show that class IV da neurons regenerate their dendrites in an all-or-none fashion; an injured dendritic branch either initiates regeneration or completely fails to regenerate. This is consistent with the stochastic nature of regeneration (Ghosh-Roy and Chisholm 2010; Samara et al 2010).…”

<em>In vivo</em> Laser Axotomy in <em>C. elegans</em>

“…An alternative anesthetic-free method for immobilization is the use of microfluidic devices. The use of microfluidics for axotomy has been extensively described [17][18][19][20][21][22] .…”

Axon regeneration requires coordinate activation of p38 and JNK MAPK pathways

“…Subsequent studies by several laboratories have described the similar features of axon regeneration in C. elegans compared with mammals, and provided some insights into the genetic basis of axon regeneration (16)(17)(18)(19)(20)(21). The development of microfluidic chambers to automate the immobilization of worms, combined with laser axotomy, raises the hope for high throughput axon regeneration screening assays (22)(23)(24)(25). We made an observation that makes it possible to screen for genes that affect axon regeneration in C. elegans without laser axotomy (26).…”