Short Hairpin RNA against PTEN Enhances Regenerative Growth of Corticospinal Tract Axons after Spinal Cord Injury

Abstract

Developing approaches to promote the regeneration of descending supraspinal axons represents an ideal strategy for rebuilding neuronal circuits to improve functional recovery after spinal cord injury (SCI). Our previous studies demonstrated that genetic deletion of phosphatase and tensin homolog (PTEN) in mouse corticospinal neurons reactivates their regenerative capacity, resulting in significant regeneration of corticospinal tract (CST) axons after SCI. However, it is unknown whether nongenetic methods of suppressing PTEN have similar effects and how regenerating axons interact with the extrinsic environment. Herein, we show that suppressing PTEN expression with short-hairpin RNA (shRNA) promotes the regeneration of injured CST axons, and these axons form anatomical synapses in appropriate areas of the cord caudal to the lesion. Importantly, this model of increased CST regrowth enables the analysis of extrinsic regulators of CST regeneration in vivo. We find that regenerating axons avoid dense clusters of fibroblasts and macrophages in the lesion, suggesting that these cell types might be key inhibitors of axon regeneration. Furthermore, most regenerating axons cross the lesion in association with astrocytes, indicating that these cells might be important for providing a permissive bridge for axon regeneration. Lineage analysis reveals that these bridge-forming astrocytes are not derived from ependymal stem cells within the spinal cord, suggesting that they are more likely derived from a subset of mature astrocytes. Overall, this study reveals insights into the critical extrinsic and intrinsic regulators of axon regeneration and establishes shRNA as a viable means to manipulate these regulators and translate findings into other mammalian models.

Optogenetic Interrogation of Functional Synapse Formation by Corticospinal Tract Axons in the Injured Spinal Cord

“…Consistent with our previous results, we find that forced Sox11 expression promotes sprouting of CST axons into denervated tissue in cervical spinal cord but that forelimb function is not detectably improved . In contrast, examples of spontaneous CST sprouting in injured primates and neonatal rodents show positive correlation with functional recovery, strongly implying successful functional connectivity (Z'Graggen et al, 2000;Rosenzweig et al, 2010). What might explain the difference?…”

“…Notably, because various approaches have succeeded in promoting axon regeneration and sprouting after CNS injury, effects on animal behavior are at best partially beneficial Zou et al, 2015) and can also be neutral (Lu et al, 2012a,b;Geoffroy et al, 2015) or even negative (Takeoka et al, 2011;Wang et al, 2015). Thus, the ability of growth-stimulated axons to synaptically integrate into target tissue is emerging as a key question in the field (Pernet and Schwab, 2014;Ramer et al, 2014).…”

“…Similarly, previous work has used optogenetic stimulation of cortical cell bodies to track injury-induced shifts in motor maps of forelimb movement (Harrison et al, 2012(Harrison et al, , 2013. Because cortical neurons can communicate with the spinal cord through a variety of relay circuits in the midbrain and brainstem (Z'Graggen et al, 2000;Alstermark et al, 2004;Nielsen et al, 2007;Krajacic et al, 2010;, it is challenging for these techniques to distinguish the contributions of the diverse inputs to the spinal cord or definitively assign function to sprouting cortical axons (but see Hunanyan et al, 2013). This makes it difficult to resolve the key question of the degree to which regenerated axons are effective in driving postsynaptic activity.…”

“…Functional recovery requires that axon growth be accompanied by the formation of effective synapses in appropriate target fields. In some cases, behavioral improvements have been observed, hinting at synaptic integration (Houle and Côté, 2013;Liu et al, 2015;Zou et al, 2015). However, behavioral gains are often modest, and it can be unclear whether they result from direct synaptic input from newly grown axons, as opposed to plasticity in the target field or in upstream relays (Onifer et al, 2011).…”

Cell biology of spinal cord injury and repair

“…Scar-forming astrocytes express the axon growth-supporting matrix protein laminin (19,56), and antibody blockade of laminin-integrin binding attenuates stimulated axon regeneration after SCI (19). Mature injured CNS axons regrow along astrocytes after CNS injury when stimulated by appropriate growth factors (57) or by genetic activation (58). Grafts of progenitor-derived astrocytes support axon regeneration though SCI lesion cores (59)(60)(61).…”