Identification of Intrinsic Axon Growth Modulators in Intact CNS Neurons After Injury [electronic resource].

9781369619386

Ann Arbor : ProQuest Dissertations & Theses, 2016.

1 online resource (200 p.)

Access is available to the Yale community.

Neurons in the adult central nervous system (CNS) are unable to regenerate after spinal cord injury (SCI) as a result of an inhibitory environment and a decreased intrinsic growth capacity. Modulating environmental inhibitors and their neuronal receptors such as Nogo Receptor 1 (NgR1) results in increased regeneration and sprouting of intact neurons spared by injury, which has been correlated with enhanced functional recovery in model systems. Cell intrinsic factors have also been identified that increase regeneration and sprouting, but side effects and lack of functional improvements suggest these factors do not recruit endogenous sprouting mechanisms. We sought to identify the mechanism underlying spontaneous sprouting of intact neurons after incomplete SCI. To achieve this, we differentially labeled intact corticospinal motor neurons (CSMNs) in an active or quiescent growth state after unilateral corticospinal tract (CST) lesion (pyramidotomy, PyX) for transcriptomics analysis. For comprehensive labeling of the corticospinal tract (CST), we utilized a transgenic mouse line that expresses GFP under the p-crystallin (crym) promoter (crym-GFP). We show that crym-GFP comprehensively and specifically labels CSMNs and their axonal projections in the CST throughout the neuraxis. We performed a unilateral PyX in transgenic wild type and plasticity-sensitized crym-GFP mice lacking NgR1 (ngrl-/- mice). Two weeks post-lesion, mice received intraspinal infusion of the retrograde tracer fast blue (FB) into the denervated spinal cord to label sprouting CSMNs. Two weeks later, we used laser capture microdissection to isolate the CST neurons that were in a quiescent (GFP+FB-) or active (GFP+FB+) growth state. With enhanced sprouting in ngrl-/- mice, an abundance of FB+ sprouting neurons allowed us to perform RNAseq and conduct transcriptomic analysis. We used comprehensive crym-GFP CST labeling and differential gene expression analysis to confirm that increased sprouting in ngrl-/- mice is a result of a decreased brake on plasticity and not a result of anatomical or genomic differences in ngrl-/- mice. Comparing intact CSMNs four weeks after PyX, we identified 1174 genes that are differentially regulated between sprouting and quiescent neurons, with lysophosphatidic acid (LPA) receptor 1 (lparl) being the most downregulated gene in sprouting neurons. Lpar1 interactors, including a negative regulator of Lpar1, lipid phosphate phosphatase related protein 1 (lppr1), were also significantly differentially expressed in sprouting neurons, suggesting a role for the LPA pathway in intrinsic CNS axon growth. Overexpressing Lppr1 in cortical neurons in vitro resulted in an increase in neurite growth in an acute outgrowth assay and an increase in axon growth in more mature cultures using an in vitro scrape injury model. We next sought to determine if modulation of the LPA pathway in vivo would enhance functional sprouting. Adult wild type mice received PyX or sham lesion and either cortical infusion of AAV-Lppr1, oral treatment with an Lpar1 antagonist AM095, or vehicle control. Lppr1-expressing and AM095-treated mice had significantly enhanced sprouting of CST neurons into the denervated ventral horn and AM095-treated mice recovered greater fore and hind limb function in a grid walking task. Taken together, our findings demonstrate that bidirectional modulation of the LPA pathway is beneficial for axon growth with therapeutic potential for restoring function after SCI. In sum, we utilized a novel screening approach to identify pro-axon growth pathways within intact CNS neurons after injury. Furthermore, these data provide a platform to comprehensively dissect the molecular mechanisms that drive plasticity mediated functional recovery after injury that can be exploited to maximally restore function after CNS trauma.

Books / Online / Dissertations & Theses

English

August 03, 2017

Thesis (Ph.D.)--Yale University, 2016.

Yale University.