Biology subjects

Eva, R.

Publications and source records attributed to Eva, R..

4 recordsLinked to original sources

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↗

bioRxiv2020

Protrudin functions as a scaffold in the endoplasmic reticulum to support axon regeneration in the adult central nervous system

Adult mammalian central nervous system axons have intrinsically poor regenerative capacity, so axonal injury has permanent consequences. One approach to enhancing regeneration is to increase the axonal supply of growth molecules and organelles. We achieved this by expressing the adaptor molecule Protrudin which is normally found at low levels in non-regenerative neurons. Elevated Protrudin expression enabled robust central nervous system regeneration both in vitro in primary cortical neurons and in vivo in the injured adult optic nerve. Protrudin overexpression facilitated the accumulation of endoplasmic reticulum, integrins and Rab11 endosomes in the distal axon, whilst removing Protrudins endoplasmic reticulum localization, kinesin-binding or phosphoinositide-binding properties abrogated the regenerative effects. These results demonstrate that Protrudin promotes regeneration by functioning as a scaffold to link axonal organelles, motors and membranes, establishing important roles for these cellular components in mediating regeneration in the adult central nervous system.

neuroscience↗

bioRxiv2019

PI 3-kinase delta enhances axonal PIP3 to support axon regeneration in the adult CNS

Peripheral nervous system (PNS) neurons support axon regeneration into adulthood, whereas central nervous system (CNS) neurons lose regenerative ability after development. To better understand this decline whilst aiming to improve regeneration, we focused on phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol(3,4,5)-trisphosphate (PIP3). We found that neuronal PIP3 decreases with maturity in line with regenerative competence, firstly in the cell body and subsequently in the axon. We show that adult PNS neurons utilise two catalytic subunits of PI3K for efficient regeneration: p110 and p110{delta}. Overexpressing p110 in CNS neurons had no effect, however expression of p110{delta} restored axonal PIP3 and enhanced CNS regeneration in rat and human neurons and in transgenic mice, functioning in the same way as the hyperactivating H1047R mutation of p110. Furthermore, viral delivery of p110{delta} promoted robust regeneration after optic nerve injury. These findings demonstrate a deficit of axonal PIP3 as a reason for intrinsic regeneration failure and show that native p110{delta} facilitates axon regeneration by functioning in a hyperactive fashion.

neuroscience↗

bioRxiv2017

EFA6 regulates selective polarised transport and axon regeneration from the axon initial segment

It is not clear why central nervous system (CNS) axons lose their intrinsic ability to regenerate with maturity, whilst peripheral (PNS) axons do not. A key difference between these neuronal types is their ability to transport integrins into axons. Integrins can mediate PNS regeneration, but are excluded from adult CNS axons along with their rab11 positive carriers. We reasoned that this exclusion might contribute to the intrinsic inability of CNS neurons to regenerate, and investigated this hypothesis using laser axotomy. We identify a novel regulator of selective axon transport and regeneration, the ARF6 GEF EFA6. EFA6 exerts its effects from a previously unreported location within the axon initial segment (AIS). EFA6 does not localise here in DRG axons, and in these neurons, ARF activation is counteracted by an ARF-GAP which is absent from the CNS, ACAP1. Depleting EFA6 from cortical neurons permits endosomal integrin transport and enhances regeneration, whilst overexpressing EFA6 prevents DRG regeneration. Our results demonstrate that ARF6 is an intrinsic regulator of regenerative capacity, implicating EFA6 as a focal molecule linking the axon initial segment, signalling and transport.\n\nSummary StatementWe identify a novel resident of the axon initial segment, EFA6. This functions to prevent growth-promoting molecules from entering mature CNS axons. Removing EFA6 elevates the axons regenerative potential.

cell biology↗