Neurons in the adult central nervous system (CNS) are unable to regenerate after spinal cord injury (SCI) due to 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 correlates with enhanced functional recovery. Cell intrinsic factors also increase regeneration and sprouting, but side effects and limited functional improvements suggest these factors do not activate endogenous sprouting mechanisms. We sought to identify the mechanisms underlying spontaneous sprouting of intact neurons after incomplete SCI. We completed a unilateral corticospinal tract (CST) lesion (pyramidotomy, PyX) in transgenic wild type (n=6) and NgR1 knockout mice (ngr1-/-, n=6) expressing GFP under the µ-crystallin (crym) promoter (crym-GFP) for intrinsic corticospinal tract (CST) labeling. Two weeks post-lesion, mice received infusion of the retrograde tracer fast blue (FB) into the denervated spinal cord to label sprouting CST neurons. Two weeks later, we used laser capture microdissection to isolate CST neurons in a quiescent (GFP+FB-) or active (GFP+FB+) growth state. With enhanced sprouting in ngr1-/- mice, an abundance of FB+ sprouting neurons allowed us to complete RNAseq and conduct a transcriptomic analysis. 1174 genes were significantly differentially expressed (SDE) between sprouting and quiescent neurons, with lysophosphatidic acid (LPA) receptor 1 (lpar1) the most downregulated gene in sprouting neurons. Lpar1 interactors, including a negative regulator of Lpar1, lipid phosphate phosphatase related protein 1 (lppr1), were also SDE in sprouting neurons, suggesting a role for the LPA pathway in regulating intrinsic CNS axon growth. Overexpressing Lppr1 in cortical neurons in vitro resulted in an increase in neurite outgrowth and an increase in growth in an in vitro injury model. Next we sought to determine if modulating 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 (n=21), oral treatment with an Lpar1 antagonist AM095 (n=15), or vehicle control (n=19). 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. With these data, we have demonstrated that bidirectional modulation of the LPA pathway is beneficial for axon growth with therapeutic potential for restoring function after SCI.
*K. FINK, S. STRITTMATTER, W. CAFFERTY;
Yale Univ., New Haven, CT
K. Fink: None. S. Strittmatter: None. W. Cafferty: None.