PTEN Deletion Promotes Regenerative Sprouting in the Aged Rubrospinal Tract

Background: The paralysis that occurs following spinal cord injury (SCI) is largely the result of transected central nervous system (CNS) axons failing to regenerate. Previous research has indicated that a major contributor to this regenerative failure is a diminished intrinsic capacity of adult CNS axons to grow, based largely on inactivity in the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway which is negatively regulated by phosphatase and tensin homolog deleted on chromosome ten (PTEN). By targeting PTEN, it may be possible to functionally regenerate CNS neurons following SCI and re-establish connections between the brain and the rest of the body. The rubrospinal tract (RST), which originates in the red nucleus of the midbrain and travels contralaterally in the dorsolateral funiculus of the spinal cord, functions in movement and fails to regenerate after transection. Here, we assessed whether PTEN deletion would promote axon regeneration in the RST with deletion occurring in aged (7-8 month old) mice. Methods: Floxed PTEN mice were injected with adeno-associated virus serotype 2 expressing Cre and GFP (AAV2-Cre) or GFP alone for control (AAV2-GFP) into the right red nucleus. Four weeks later, mice underwent a left dorsolateral crush at cervical level C4/C5. Six weeks later, mice were injected with biotinylated dextran amine (BDA) into the right red nucleus to anterogradely trace the RST. Two weeks later, mice were sacrificed, transcardially perfused with fixative, and their spinal cord tissue cryoprotected and cut longitudinally into 20 µM sections. Following immunocytochemical staining of cervical spinal cord tissue, axon regeneration was assessed via analysis of images collected using a laser scanning confocal microscope under 40x objective.  Results: Based on independent analyses of BDA and GFP labelling, AAV2-Cre injected animals showed significantly decreased dieback and increased regenerative sprouting of rubrospinal axons through the lesion site in comparison to AAV2-GFP injected animals, for up to 100µm caudal measured from the middle of the lesion site. Conclusions: Our findings suggest that PI3K/Akt/mTOR activity is a significant determinant of rubrospinal regenerative potential, yet advanced age may play an important role in decreasing the ability of RST axons to regenerate long distances.