Holota, R. | bioRxiv Science

bioRxiv2025

Dibutyryl cyclic AMP downregulates tenascin-C in neurons and astrocytes and reduces AAV-mediated gene expression in DRG neurons

Functional recovery after spinal cord injury (SCI) is hindered by the limited ability of axons to regenerate in the adult mammalian central nervous system (CNS). Overcoming this barrier is critical for achieving effective recovery. Axonal regeneration depends on the activation of intracellular processes like transcription factor induction, protein and lipid trafficking, and cytoskeletal remodelling. Targeting these pathways offers a promising approach for promoting neuronal repair. This study examined the combined therapeutic effects of dibutyryl-cAMP (db-cAMP), which primes neurons for growth, and integrin 9 overexpression, which supports axonal extension. Using in vitro models with dorsal root ganglion (DRG) neurons and astrocytes, as well as an in vivo SCI model, we evaluated the potential of this approach. In vitro, the combination of db-cAMP and integrin 9 significantly enhanced neuronal growth. However, in vivo results were less consistent, with db-cAMP affecting AAV-mediated transcription and the expression of tenascin C (TnC) in neurons and astrocytes. These findings highlight the potential of modulating intracellular signalling and integrin activation but underscore the challenges posed by the complexity of the in vivo environment. Further studies are necessary to unravel these mechanisms and refine therapeutic strategies for effective SCI recovery. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/653846v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1473630org.highwire.dtl.DTLVardef@36a8beorg.highwire.dtl.DTLVardef@80593corg.highwire.dtl.DTLVardef@6285e3_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

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↗