Intrinsic and extrinsic regulation of injury-induced axonal growth in the CNS: a combinatorial approach with known pathways and exploring the unknown role of microRNAs
Final Report Abstract
Axon regeneration in the adult central nervous system (CNS) is very limited and spinal cord injury patients suffer from often permanent motoric and sensory disabilities. Recent spinal cord injury research indicated that stimulating axon intrinsic growth may be a promising pathway to finding treatment for patients in the future. The Funded project had two distinct projects with congregating laboratory techniques. In the first project the function of NgR1 (reticulon 4 receptor) and NgR3 (reticulon 4 receptor-like 1) for axon sprouting and regrowth was studied in mice conditionally deficient of the Pten gene. With the aim to generate consistent and reproducible axon tracing it was decided to breed double and triple knockout mice into the conditionally tdTomate expressing mouse line. AAV-mediated Cre expression results in tdTomato expression in cells that are conditionally deprived of PTEN and/or NgR3. The complicated breeding scheme significantly delayed progress in this project and the in vivo study could not be finished during the funding period. I focused on perusing the second aim of the project; studying the role of neuronal miRNAs for axonal growth after injury as well as on establishing a tissue clearing technique in the Zheng lab which allowed for three dimensional axon tracing in spinal cord. During my post-doctorate in Dr. Binhai Zheng’s laboratory at UC San Diego I learned and applied two mouse surgery techniques that a commonly used in the spinal cord injury research field. The pyramidotomy model is used to study sprouting of corticospinal axons. The dorsal hemi-section model is used to study axon regrowth after injury in vivo. Both models require a high technical skillset to achieve reliable and reproducible results and consistency in axon counting and quantification is crucial for reproducible results. To increase the reproducibility of quantifications and limit the number of animals required for a singe study, a CLARITY based tissue clearing technique that allows for three-dimensional imaging and axon tracing was established in the Zheng lab. This promising technology is currently being used in several labs including the Zheng lab at UCSD. In addition, I established an in vitro axotomy model based on a microfluidic chamber system in the Zheng lab that was used to study axon growth in primary neurons after axotomy. This technique was used to study expression levels of miRNAs before and after injury as well as to study the impact of candidate microRNA overexpression for axon growth in vitro. The microfluidic device-based system revealed that overexpressing miRNA124 and miR214 can significantly elicit axonal growth after injury in vitro. To study microRNA overexpression related increased axon growth in vivo, several AAV constructs expressing tdTomato or eGFP simultaneously to a given candidate miRNA were cloned and tested for efficiency of neuron transduction, expression level and sustained expression in vivo. The AAV based miRNA expression approach with simultaneous reporter gene expression was shown to be combinable with three-dimensional imaging of cleared tissue. In this study, I laid the groundwork to study the role of microRNAs in injury-induced axonal growth and their potential as therapeutics to stimulate CNS axon regeneration. This is to my knowledge the first study showing that miRNAs overexpression can promote axonal growth after injury.
Publications
- Leucine Zipper-bearing Kinase promotes axon growth in mammalian central nervous system neurons. Sci Rep. 2016;6:31482
Chen M, Geoffroy CG, Wong HN, Tress O, Nguyen MT, Holzman LB, Jin Y, Zheng B
(See online at https://doi.org/10.1038/srep31482)
