Inhibition of the autophagic protein ULK1 attenuates axonal degeneration in vitro and in vivo, enhances translation, and modulates splicing

Abstract

Axonal degeneration is a key and early pathological feature in traumatic and neurodegenerative disorders of the CNS. Following a focal lesion to axons, extended axonal disintegration by acute axonal degeneration (AAD) occurs within several hours. During AAD, the accumulation of autophagic proteins including Unc-51 like autophagy activating kinase 1 (ULK1) has been demonstrated, but its role is incompletely understood. Here, we study the effect of ULK1 inhibition in different models of lesion-induced axonal degeneration in vitro and in vivo. Overexpression of a dominant negative of ULK1 (ULK1.DN) in primary rat cortical neurons attenuates axotomy-induced AAD in vitro. Both ULK1.DN and the ULK1 inhibitor SBI-0206965 protect against AAD after rat optic nerve crush in vivo. ULK1.DN additionally attenuates long-term axonal degeneration after rat spinal cord injury in vivo. Mechanistically, ULK1.DN decreases autophagy and leads to an mTOR-mediated increase in translational proteins. Consistently, treatment with SBI-0206965 results in enhanced mTOR activation. ULK1.DN additionally modulates the differential splicing of the degeneration-associated genes Kif1b and Ddit3. These findings uncover ULK1 as an important mediator of axonal degeneration in vitro and in vivo, and elucidate its function in splicing, defining it as a putative therapeutic target.

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

“…We previously generated and characterized an AAV vector expressing ULK1.DN (AAV.ULK1.DN), resulting in the reduction of endogenous ULK1 levels by ~50%, and analyzed the effects of ULK1 inhibition on neuroprotection 13 , 14 . AAV.ULK1.DN also expresses the fluorescent protein mCherry, which is used to visualize transduced neurons.…”

“…Furthermore, we showed that ULK1.DN-mediated ULK1 inhibition promoted axonal protection in different models of traumatic axonal lesion, in vitro and in vivo, and that application of the ULK1 inhibitor SBI-0206965 protected axons from degeneration induced by optic nerve crush (ONC) in vivo. Mechanistically, ULK1.DN attenuated autophagy, modulated the differential splicing of degeneration-associated genes and increased translation via an mTOR-mediated mechanism 14 . These findings suggest that ULK1 is a key mediator of axonal degeneration.…”

Systemic Administration of Fibroblast Growth Factor 21 Improves the Recovery of Spinal Cord Injury (SCI) in Rats and Attenuates SCI-Induced Autophagy

“…Inhibition of excessive autophagy activation by pharmacological intervention has been suggested to be a new tactic to reduce neuronal death ( Lee et al, 2020 ). Compared with the short-term stress responsees such as oxidative stress and apoptosis, autophagy exists in the whole process of SCI ( Cortes et al, 2014 ; He et al, 2016 ; Vahsen et al, 2020 ). Consistently, we found that autophagy-related protein level LC3 II was significantly induced in SCI, and whereas FGF21 attenuated SCI-induced autophagy and improves the functional recovery of SCI.…”

Post-Translational Modifications of the Energy Guardian AMP-Activated Protein Kinase

“…Furthermore, many studies use α-T172 as a marker for AMPK activity, which can be misleading. For example, the dual AMPK and autophagy initiator ULK1 inhibitor SBI-0206965 counterintuitively induces an increase in α-T172 phosphorylation, possibly due to de-repression of ULK1 negative feedback through this site [ 61 ], while suppressing AMPK signalling [ 81 , 82 ]. This highlights the importance of using downstream AMPK substrates as markers of AMPK activity instead of α-T172 phosphorylation.…”