AAV-mediated inhibition of ULK1 promotes axonal regeneration in the central nervous system in vitro and in vivo.

Axonal damage is an early step in traumatic and neurodegenerative disorders of the central nervous system (CNS). Damaged axons are not able to regenerate sufficiently in the adult mammalian CNS, leading to permanent neurological deficits. Recently, we showed that inhibition of the autophagic protein ULK1 promotes neuroprotection in different models of neurodegeneration. Moreover, we demonstrated previously that axonal protection improves regeneration of lesioned axons. However, whether axonal protection mediated by ULK1 inhibition could also improve axonal regeneration is unknown. Here, we used an adeno-associated viral (AAV) vector to express a dominant-negative form of ULK1 (AAV.ULK1.DN) and investigated its effects on axonal regeneration in the CNS. We show that AAV.ULK1.DN fosters axonal regeneration and enhances neurite outgrowth in vitro. In addition, AAV.ULK1.DN increases neuronal survival and enhances axonal regeneration after optic nerve lesion, and promotes long-term axonal protection after spinal cord injury (SCI) in vivo. Interestingly, AAV.ULK1.DN also increases serotonergic and dopaminergic axon sprouting after SCI. Mechanistically, AAV.ULK1.DN leads to increased ERK1 activation and reduced expression of RhoA and ROCK2. Our findings outline ULK1 as a key regulator of axonal degeneration and regeneration, and define ULK1 as a promising target to promote neuroprotection and regeneration in the CNS.

Effects of a combinatorial treatment with gene and cell therapy on retinal ganglion cell survival and axonal outgrowth after optic nerve injury.

After an injury, axons in the central nervous system do not regenerate over large distances and permanently lose their connections to the brain. Two promising approaches to correct this condition are cell and gene therapies. In the present work, we evaluated the neuroprotective and neuroregenerative potential of pigment epithelium-derived factor (PEDF) gene therapy alone and combined with human mesenchymal stem cell (hMSC) therapy after optic nerve injury by analysis of retinal ganglion cell survival and axonal outgrowth. Overexpression of PEDF by intravitreal delivery of AAV2 vector significantly increased Tuj1-positive cells survival and modulated FGF-2, IL-1ß, Iba-1, and GFAP immunostaining in the ganglion cell layer (GCL) at 4 weeks after optic nerve crush, although it could not promote axonal outgrowth. The combination of AAV2.PEDF and hMSC therapy showed a higher number of Tuj1-positive cells and a pronounced axonal outgrowth than unimodal therapy after optic nerve crush. In summary, our results highlight a synergistic effect of combined gene and cell therapy relevant for future therapeutic interventions regarding optic nerve injury.

Long-term neuronal survival, regeneration, and transient target reconnection after optic nerve crush and mesenchymal stem cell transplantation.

BACKGROUND: Retina and/or optic nerve injury may cause irreversible blindness, due to degeneration of retinal ganglion cells. We and others have previously shown that the intravitreal injection of mesenchymal stem cells (MSCs) protects injured retinal ganglion cells and stimulates their regeneration after optic nerve injury, but the long-term effects of this therapy are still unknown. METHODS: We injected rat MSC (rMSC) intravitreally in adult (3-5 months) Lister Hooded rats of either sex after optic nerve crush. Retinal ganglion cell survival, axonal regeneration, and reconnection were analyzed 60 and 240 days after crush by immunohistochemistry for Tuj1, anterograde labeling with cholera-toxin B and by immunohistochemistry for nerve growth factor-induced gene A (NGFI-A, driven by light stimulation) in the superior colliculus after a cycle of light deprivation-stimulation. Visual behaviors (optokinetic reflex, looming response, and preference for dark) were analyzed 70 days after crush. RESULTS: rMSC treatment doubled the number of surviving retinal ganglion cells, preferentially of a larger subtype, and of axons regenerating up to 0.5 mm. Some axons regenerated to the lateral geniculate nucleus and superior colliculus. NGFI-A+ cells were doubled in rMSC-treated animals 60 days after crush, but equivalent to vehicle-injected animals 240 days after crush, suggesting that newly formed synapses degenerated. Animals did not recover visual behaviors. CONCLUSIONS: We conclude that rMSC-induced neuroprotection is sustained at longer time points. Although rMSCs promoted long-term neuroprotection and long-distance axon regeneration, the reconnection of retinal ganglion cells with their targets was transitory, indicating that they need additional stimuli to make stable reconnections.

Hypothermia Prevents Retinal Damage Generated by Optic Nerve Trauma in the Rat.

Ocular and periocular traumatisms may result in loss of vision. Hypothermia provides a beneficial intervention for brain and heart conditions and, here, we study whether hypothermia can prevent retinal damage caused by traumatic neuropathy. Intraorbital optic nerve crush (IONC) or sham manipulation was applied to male rats. Some animals were subjected to hypothermia (8 °C) for 3 h following surgery. Thirty days later, animals were subjected to electroretinography and behavioral tests. IONC treatment resulted in amplitude reduction of the b-wave and oscillatory potentials of the electroretinogram, whereas the hypothermic treatment significantly (p < 0.05) reversed this process. Using a descending method of limits in a two-choice visual task apparatus, we demonstrated that hypothermia significantly (p < 0.001) preserved visual acuity. Furthermore, IONC-treated rats had a lower (p < 0.0001) number of retinal ganglion cells and a higher (p < 0.0001) number of TUNEL-positive cells than sham-operated controls. These numbers were significantly (p < 0.0001) corrected by hypothermic treatment. There was a significant (p < 0.001) increase of RNA-binding motif protein 3 (RBM3) and of BCL2 (p < 0.01) mRNA expression in the eyes exposed to hypothermia. In conclusion, hypothermia constitutes an efficacious treatment for traumatic vision-impairing conditions, and the cold-shock protein pathway may be involved in mediating the beneficial effects shown in the retina.

Distribution of mesenchymal stem cells and effects on neuronal survival and axon regeneration after optic nerve crush and cell therapy.

Bone marrow-derived cells have been used in different animal models of neurological diseases. We investigated the therapeutic potential of mesenchymal stem cells (MSC) injected into the vitreous body in a model of optic nerve injury. Adult (3-5 months old) Lister Hooded rats underwent unilateral optic nerve crush followed by injection of MSC or the vehicle into the vitreous body. Before they were injected, MSC were labeled with a fluorescent dye or with superparamagnetic iron oxide nanoparticles, which allowed us to track the cells in vivo by magnetic resonance imaging. Sixteen and 28 days after injury, the survival of retinal ganglion cells was evaluated by assessing the number of Tuj1- or Brn3a-positive cells in flat-mounted retinas, and optic nerve regeneration was investigated after anterograde labeling of the optic axons with cholera toxin B conjugated to Alexa 488. Transplanted MSC remained in the vitreous body and were found in the eye for several weeks. Cell therapy significantly increased the number of Tuj1- and Brn3a-positive cells in the retina and the number of axons distal to the crush site at 16 and 28 days after optic nerve crush, although the RGC number decreased over time. MSC therapy was associated with an increase in the FGF-2 expression in the retinal ganglion cells layer, suggesting a beneficial outcome mediated by trophic factors. Interleukin-1ß expression was also increased by MSC transplantation. In summary, MSC protected RGC and stimulated axon regeneration after optic nerve crush. The long period when the transplanted cells remained in the eye may account for the effect observed. However, further studies are needed to overcome eventually undesirable consequences of MSC transplantation and to potentiate the beneficial ones in order to sustain the neuroprotective effect overtime.