Femtosecond Laser Microdissection to Isolate Single Intact Neurons for Nerve Regeneration Studies

University Of Texas At Austin, Austin TX

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Abstract

Axon regeneration in adult central nervous system is limited due to loss of intrinsic regeneration capacity and extrinsic inhibitory activities. Interventions so far have only induced substantial regrowth in a small subset of neurons, while resulting in diverse, even opposite response among different neuronal subtypes. More promising outcome has been attained by simultaneous manipulation of multiple regeneration regulators, yet mechanisms underlying their synergistic activity remains unclear. Evidence has implicated a role for context-dependent activation of neuron intrinsic pathways in encoding diverse response to axon injury. Nevertheless, our knowledge on how the collective activities of such pathways encode the heterogeneous response to axon injury is still fragmentary. In seeking such knowledge, Caenorhabditis elegans provides a versatile model system. Since our group pioneered femtosecond laser ablation as laser injury mechanism and demonstrated spontaneous axon regeneration in the nematode, a myriad of conserved regeneration regulators has been discovered in C. elegans. Existing studies of axon regeneration in C. elegans have relied heavily on reverse genetic methods. We reason that molecular profiling of regrowing neurons would be a powerful complement to existing works and facilitate high-throughput discovery of novel conserved mechanisms of axon regeneration. Unfortunately, there is no feasible method to isolate regrowing neurons from C. elegans. In this proposal, we develop a new method, femtosecond laser microdissection (fs-LM), for precise and rapid isolation of intact neurons from living tissue or animals. In preliminary studies, we have successfully isolated single neurons from living C. elegans and fresh mice brain slices with fs-LM. Single cell RNA-seq of the collected C. elegans neurons detected 6,455 ± 693 genes in single neurons and 921 differentially expressed genes between regrowing and uninjured neurons. In this proposal, we aim to extensively validate fs-LM and perform the very first transcriptomic profiling of regrowing C. elegans neurons. We will accomplish our goals in 2 specific aims. In Aim 1, we will extensively validate fs-LM's ability to produce biologically relevant data and capture the time-lapse transcriptomic profile of axon regeneration in C. elegans, which we currently know very little of. We will validate our results with hybridization chain reaction, which allows quantification of mRNA molecules with in situ fluorescence microscopy. In Aim 2, we will delineate regeneration-associated gene signatures of two highly conserved mechanisms, aging and DLK signaling. We will validate potential regulators of axon regeneration using mutant strains and laser axotomy. The combination of fs-LM and laser axotomy in C. elegans will facilitate investigation of the molecular underpinnings of axon regrowth at single neuron resolution. Our dataset will likely spark new hypotheses that can be rapidly tested and potentially translated to mammals. The fs-LM method is not limited by tissue type, and can be broadly applied to acquire arbitrary cells from other models like mice.