Herynek, V. | Search bioRxiv

bioRxiv2023

Hyperactive PI3Kinase delta enables long distance regeneration of the rat corticospinal tract

Maturation of central nervous system neurons leads to loss of their intrinsic regeneration potential. In particular after injury of the adult spinal cord there is minimal regeneration of corticospinal axons, which control gait and fine movement. Previous work has shown that knockdown of PTEN to increase PIP3 levels can promote regeneration in young animals, but the effect is much less in adults probably due to low PIP3 production. Here, we have transduced sensorimotor cortex neurons with a hyperactive form of PI3K, PI3K{delta}, which increases PIP3 in mature neurons. This enables cortical neurons to regenerate corticospinal axons and improve behavioural outcomes. We used a C4 dorsal column lesion model in adult rats and injected the right motor cortex at 4 sites concurrently with a mixture AAV1-PIK3CD and AAV1-eGFP or titre matched AAV1-eGFP only. We allowed rats to survive for 6, 9, 12 or 16 weeks. Immunostaining showed 70 - 80% co-expression in cortical neurons which remained stable at both 12 and 16 weeks. We counted GFP labelled axons in 20 m spinal cord sections. In PI3KCD-treated animals many axons were seen to have regenerated around the margins of lesions, collecting into a knot of axons with the typical appearance of regeneration at the caudal end. Tracing down the cord, and excluding axons and neurites that could have come from unlesioned ventral CST, we found axons extending up to 1 cm below lesions, numbers decreasing with distance from the lesion. After 16 weeks there were circa 200 axons at the caudal end of lesions with a regeneration index of 0.2, with half this number at 12 weeks. Behavioural testing for 16 weeks revealed functional improvements in skilled paw reaching, grip strength and ladder rung walking in rats treated with PIK3CD compared to GFP only controls. In addition to behavioural testing, functional recovery of PIK3CD treated rats was confirmed with electrophysiological recordings during which we stimulated the right pyramid. Cord dorsum potentials (CDPs) above and below lesion and EMG forepaw distal flexor muscles showed greatly increased connectivity compared with GFP only controls, lesion only controls and uninjured shams. We conclude that forcing upregulation of PI3K{delta} in cortical neurons leads to robust regeneration after spinal cord injury that results in functional restoration.

neuroscience↗

bioRxiv2023

Alpha 9 integrin expression enables reconstruction of the spinal cord sensory pathway.

Full recovery from spinal cord injury can only occur if the axon pathways connecting the brain and spinal cord regenerate and restore motor and sensory connections. Neither sensory nor motor axons can regenerate spontaneously in the spinal cord in mammals. This failure is partly due to the lack of suitable adhesion molecules on the sensory axons that allows them to interact with the environment of the damaged spinal cord. In this rat study, an integrin adhesion molecule along with its activator was expressed in sensory neurons using an adeno-associated viral (AAV) vector. Expression of these adhesion molecules allowed sensory axons to regenerate through the spinal cord injury and all the way back to the brainstem, restoring the sensory pathway. Treated animals regained touch sensation and sensory behaviours. The integrin ligands in the injured spinal cord are tenascin-C and osteopontin, but adult PNS and CNS neurons lack receptors to them. Sensory neurons were transduced with 9 integrin, which combines with endogenous {beta}1 as 9{beta}1 (which is a tenascin/osteopontin receptor) together with the integrin activator kindlin-1. Regeneration from sensory neurons transduced with 9integrin and kindlin-1 was examined after C4 and after T10 dorsal column lesions with C6,7 and L4.5 sensory ganglia injected with AAV1 vectors. In animals treated with 9 integrin and kindlin-1, sensory axons regenerated through tenascin-C-expressing connective tissue strands and bridges across the lesion and then re-entered the CNS tissue. Many axons regenerated rostrally to the level of the medulla. Regenerated axons were particularly visible at the border between white and grey matter in the dorsal cord. Stimulation of the median/sciatic nerve caused many neurons rostral to the injury to activate and express cFos. VGLUT1/2 staining indicated newly formed functional synapses above the lesion. Behavioural recovery was seen in heat, mechanical sensation and tape removal tests. Many axons regenerated from the thoracic lesions to the brainstem, a distance of 4-5 cm, equivalent to the length of 1 or 2 spinal segments in humans.

neuroscience↗