Regulation Of M Tor And Neural Activity Promote Axon Regeneration After Spinal Cord Injury

As one of the leading causes of disability among young people in the world,spinal cord injury(SCI)has always been the focus in the field of neuroscience research.The basic pathology of SCI is the rupture of descending and ascending axons which send information between the brain and the spinal cord.Therefore,to promote axonal regeneration is a possiblebreakthrough point in the treatment of SCI.Numerous studies have shown that axonal regeneration is mainly affected by the following two factors:(1)the inhibitory microenvironment,such as myelin-associated inhibitors(MAIs),chondroitin sulfate proteoglycans(CSPGs),glial scar and so on.(2)the reduced intrinsic regeneration ability of adult neurons.By regulating exogenous inhibitory factors,such as knocking out Nogo,promoting the degradation of CSPGs,etc.,it was found that the regenerative(regeneration and sprouting)capacity of SCI animals increased.However,the regulation of exogenous factors has a limited effect on the regeneration of the corticospinal tract(CST),whichcontrolling the voluntary movements.The above studies indicate that enhancingintrinsic regeneration capacity may be more important.Studies have shown that knockout Pten(phosphatase and tensin homolog)enhance intrinsic axonal regeneration ability.Pten is a tumor suppressor gene and negatively regulate PI3 K / AKT / m TOR(mammalian rapamycin target protein,m TOR)pathway.The activation of the m TOR could alter metabolic conditions,promoting the synthesis of structural units and proteins which is necessary forcellular growth.m TOR down regulated rapidly during the development of CNS.Pten KOof the retinal ganglion cells(RGCs)could not only promote axon regeneration but also reduce the death rate of RGCs caused by injury.Similarly,Pten KO incortical neurons was found to promote axonal regeneration of the CST in a SCI model.This was then repeated by several labs.Accordingly,regulating m TOR has become a typical way to promote intinsic regeneration ability.However,most of those Pten KOregeneration studiesused transgenic mice(Pten was inserted in to two Lox P site which is facilitating the conditional knockout).And,the Adeno-associated virus(AAV)carrying recombinase(Cre)needs to be injected into the sensorimotor cortex several weeks before the injury.Plus,consistent knockout of Pten increase the risk of tumorigenesis and epilepsy.All these problems limit the possibility of clinical translation.We developeda novel method for protein degradation,coined Degron peptides,that carry a Tat domain consisting of a 10 or 11-amino acid proteintransduction sequence from the HIV-1 Tat protein(GRKKRRQRRR)which renders them highly membrane permeable.This Tat domaincan be linked to a targeting peptide that can specifically bind tothe protein of interest(PTEN)to direct them tolysosomal/proteasomal degradation.This TAT-peptide approachcould be clinically translatable and has been used with promisingresults in a clinical trial on stroke patients with 92 subjects,evaluating neuroprotection using a PSD-95 interacting peptidetermed NA-1.A phase 3 trial has recently been initiated for strokein Canada(Frontier Trial).We tested the effects of the PTEN Degron in cultured neurons and SCI model.We further explored the effects of Pten KO and neural activity in axon regeneration and behavior recovery.Main method:1.Cervical 5 SCI crush model was made by a costumer designed clamp;Rearing cylinder,horizontal ladder,staircase pellets reaching,BMS score were used for behavior testing.The expression of GFAP,CSPGs,Iba-1,MAG and Nogo-A were detected by immunofluorescence.Anterograde tracing was used to observe CST and the rubrospinal tract(RST).Retrograde tracing was used to analyze the down regulation of m TOR pathway in cortical neurons and red nucleusafter SCI.2.PTEN Degron peptide was design and synthesized.The effects of PTEN Degron was tested in cultured cortical neurons and dorsal ganglion cells(DRGs).WB was employed to test the PTEN protein level and immunofluorescence was used to test the neurite outgrowth.CSPGs artificial inhibitory environment was also used to test the neurite outgrowth ability.3.Local cortex injection and osmatic pump implantation were used to test the effects of PTEN Degron on m TOR pathwayin vivo.Iba-1,GFAP,Neu N staining were employed to verify the safety of PTEN Degron peptide.C5 crush injury model were used to test the effects of PTEN Degron on axon regeneration.4.Designer Receptors Exclusively Activated by Designer Drugs(DREADDs)were used to elevated the cortical neural activity.This was added by Pten knockout using Ptenfl/fl mice.We tested the effects of combination of neural activity and m TOR activation on axon regeneration in SCI mice as well as the behavior recovery.Main results: 1.C5 crush injury model had little effectson hind limb as the locomotor keeps normal.However,injured animals showed a bad performance of skilled movements(P < 0.05).Both the CST and RST were ruptured due to the injury.There is no sign of regeneration 8 weeks post injury,instead axonal retractionwas obvious.Inhibitory proteins such as Nogo-A,CSPGs,MAGs were upregulated(P < 0.05)and the m TOR activity was downregulated(P < 0.05)within 2 weeks after injury.2.PTEN Degron intervention rapidly knockdown the PTEN protein in cortical neurons,P < 0.05.Neurite outgrowth of DRGs and expression of p-S6 were promoted by PTEN Degron intervention,both P < 0.05 and this could be blocked by MG132(proteasome inhibitor)and rapamycin(m TOR inhibitor).PTEN Degron induced neurite outgrowth could also be observed in CSPGs coated dishes.3.Four hours after a single injection,PTEN Degron induced upregulation of p-S6 level in the cortex,and last at least 12 hours(both P < 0.05).This was further verified by osmatic pump implantation for 2 weeks.PTEN Degron lateral ventricle infusion for two weeks promote the axon sprouting ipsilateral to the infusion side but have no effects on axon regeneration over the lesion.No obvious behavior recovery was observed by PTEN Degron treatment,P > 0.05.4.Knockout of Pten or stimulate the neurological activity promotes the CST axon regeneration.This effects would be further enhanced by combination of the two methods,P < 0.05.Combined use of m TOR activation andneuralactivity would also benefit toskilled motorrecovery of the upper limbs,P < 0.05.Main conclusions:1.C5 spinal cord crush injury is closely related to clinic and is suitable for axonal regeneration as a research model.SCI not only induce the formation of variousinhibitory microenvironment,but also led to decrease of intrinsic axon regeneration capacity.2.PTEN Degron quickly induces the degradation of PTEN and activating m TOR pathway.PTEN Degron promotes the outgrowth of DRG processes.The degradation of PTEN is related to the proteasome function.3.PTEN Degron in vivo applications quickly activate the m TOR pathway which further play a limited role in promoting axonal sprouting.Lateral ventricle infusion for two weeks has no affects on the recovery of skilled motor function in SCI mice.4.Combined regulation of m TOR and neural activity promotes axonal regeneration in SCI mice and this regeneration brings forelimb skilled motor function.