Christopher V Gabel’s research while affiliated with Boston University and other places

Cellular metabolism plays an essential role in the regrowth and regeneration of a neuron following physical injury. Yet, our knowledge of the specific metabolic pathways that are beneficial to neuron regeneration remains sparse. Previously, we have shown that modulation of O-linked β-N-acetylglucosamine (O-GlcNAc) signaling, a ubiquitous post-translational modification that acts as a cellular nutrient sensor, can significantly enhance in vivo neuron regeneration. Here, we define the specific metabolic pathway by which O-GlcNAc transferase ( ogt-1 ) loss of function mediates increased regenerative outgrowth. Performing in vivo laser axotomy and measuring subsequent regeneration of individual neurons in C. elegans , we find that glycolysis, serine synthesis pathway (SSP), one-carbon metabolism (OCM), and the downstream transsulfuration metabolic pathway (TSP) are all essential in this process. The regenerative effects of ogt-1 mutation are abrogated by genetic and/or pharmacological disruption of OCM and the SSP linking OCM to glycolysis. Testing downstream branches of this pathway, we find that enhanced regeneration is dependent only on the vitamin B12 independent shunt pathway. These results are further supported by RNA sequencing that reveals dramatic transcriptional changes by the ogt-1 mutation, in the genes involved in glycolysis, OCM, TSP, and ATP metabolism. Strikingly, the beneficial effects of the ogt-1 mutation can be recapitulated by simple metabolic supplementation of the OCM metabolite methionine in wild-type animals. Taken together, these data unearth the metabolic pathways involved in the increased regenerative capacity of a damaged neuron in ogt-1 animals and highlight the therapeutic possibilities of OCM and its related pathways in the treatment of neuronal injury.

Cellular metabolism plays an essential role in the regrowth and regeneration of a neuron following physical injury. Yet, our knowledge of the specific metabolic pathways that are beneficial to neuron regeneration remains sparse. Previously, we have shown that modulation of O-linked β-N-acetylglucosamine (O-GlcNAc), a ubiquitous post-translational modification that acts as a cellular nutrient sensor, can significantly enhance in vivo neuron regeneration. Here we define the specific metabolic pathway by which mutation of the O-GlcNAc transferase ( ogt-1) increases regenerative outgrowth. Performing in vivo laser axotomy and measuring subsequent regeneration of individual neurons in C. elegans , we find that the ogt-1 mutation increases regeneration by diverting the metabolic flux of enhanced glycolysis towards one carbon metabolism (OCM) and the downstream transsulfuration metabolic pathway (TSP). These effects are abrogated by genetic and/or pharmacological disruption of OCM or the serine synthesis pathway (SSP) that links OCM to glycolysis. Testing downstream branches of this pathway, we find that enhanced regeneration is dependent only on the vitamin B12 independent shunt pathway. These results are further supported by RNA-sequencing that reveals dramatic transcriptional changes, by the ogt-1 mutation, in the genes involved in glycolysis, OCM, TSP and ATP metabolism. Strikingly, the beneficial effects of the ogt-1 mutation can be recapitulated by simple metabolic supplementation of the OCM metabolite methionine in wild-type animals. Taken together, these data unearth the metabolic pathways involved in the increased regenerative capacity of a damaged neuron in ogt-1 animals and highlight the therapeutic possibilities of OCM and its related pathways in the treatment of neuronal injury. Abstarct Figure. Metabolic pathways involved in the enhanced neuronal regeneration in ogt-1 animals The green highlighted pathway illustrates the metabolic rewiring in ogt-1 mutant animals supporting enhanced axonal regeneration of injured neurons in vivo .

CNS neurons do not regenerate after injury, leading to permanent functional deficits. Although sensory and motor neuron axons do regrow after peripheral nerve injury, functional outcome is limited due to the incomplete and slow regrowth. The lack of human-relevant assays suitable for large-scale drug screens has limited neuro-repair therapy discovery. To address this we developed a phenotypic screening strategy using human induced pluripotent stem cell-derived motor neurons to identify axon-regeneration promoting compounds and targets. The screens involve both re-plating human motor neurons on chondroitin sulfate proteoglycans and measuring regeneration responses to laser axotomy in spot cultures, and from them we identified multiple hits that promote injured axon regrowth. The top hit blebbistatin, a non-muscle myosin II inhibitor, accelerated axon regeneration and functional recovery after sciatic nerve injury in vivo. Human injury in a dish assays are suitable, therefore, to screen for therapeutic interventions that can induce or accelerate axon regeneration.

The toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease1, 2. Accordingly, neurons invest considerable cellular resources in chaperones, protein degradation, autophagy and mitophagy to maintain proteostasis and mitochondrial quality3, 4. Complicating the challenges of neuroprotection, misfolded human disease proteins and mitochondria can move into neighbouring cells via unknown mechanisms, which may promote pathological spread5, 6. Here we show that adult neurons from Caenorhabditis elegans extrude large (approximately 4 μm) membrane-surrounded vesicles called exophers that can contain protein aggregates and organelles. Inhibition of chaperone expression, autophagy or the proteasome, in addition to compromising mitochondrial quality, enhances the production of exophers. Proteotoxically stressed neurons that generate exophers subsequently function better than similarly stressed neurons that did not produce exophers. The extruded exopher transits through surrounding tissue in which some contents appear degraded, but some non-degradable materials can subsequently be found in more remote cells, suggesting secondary release. Our observations suggest that exopher-genesis is a potential response to rid cells of neurotoxic components when proteostasis and organelle function are challenged. We propose that exophers are components of a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, which, when dysfunctional or diminished with age, might actively contribute to pathogenesis in human neurodegenerative disease and brain ageing.

Mitochondrial transport is crucial for neuronal and axonal physiology. However, whether and how it impacts neuronal injury responses, such as neuronal survival and axon regeneration, remain largely unknown. In an established mouse model with robust axon regeneration, we show that Armcx1, a mammalian-specific gene encoding a mitochondria-localized protein, is upregulated after axotomy in this high regeneration condition. Armcx1 overexpression enhances mitochondrial transport in adult retinal ganglion cells (RGCs). Importantly, Armcx1 also promotes both neuronal survival and axon regeneration after injury, and these effects depend on its mitochondrial localization. Furthermore, Armcx1 knockdown undermines both neuronal survival and axon regeneration in the high regenerative capacity model, further supporting a key role of Armcx1 in regulating neuronal injury responses in the adult central nervous system (CNS). Our findings suggest that Armcx1 controls mitochondrial transport during neuronal repair.