Inhibition of the autophagic protein ULK1 attenuates axonal degeneration in vitro and in vivo and modulates differential splicing
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ABSTRACT:
PROVIDER: PRJNA509593 | ENA |
REPOSITORIES: ENA
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Project description:Several traumatic and neurodegenerative disorders of the CNS show axonal degeneration as a key and early pathological feature. Following a focal axonal lesion, an extended axonal disintegration by acute axonal degeneration (AAD) occurs within several hours. During AAD, activation of autophagic proteins including Unc-51 like autophagy activating kinase 1 (ULK1) has been demonstrated, but its role is incompletely understood. Here, we overexpressed a dominant negative of ULK1 (ULK1.DN) in primary rat cortical neurons in vitro and in the spinal cord (SC) and optic nerve (ON) in vivo. We found a significant inhibitory effect on autophagy in vitro and in vivo. While no effect on cell survival could be observed, ULK1.DN attenuated AAD after axotomy in microfluidic chambers in vitro. Models of SC injury and ON crush in vivo confirmed an attenuation of axonal degeneration by ULK1.DN. In a translational approach, we employed the selective ULK1 inhibitor SBI-0206965 (SBI) and found an attenuation of AAD after ON crush in vivo mediated by a reduction of autophagy by SBI. Quantitative proteomic profiling after ULK1.DN overexpression in vitro revealed a regulation of proteins associated with translation and splicing. A differential exon expression analysis identified the genes kinesin family member 1B (Kif1b), DNA damage inducible transcript 3 (Ddit3) and WD repeat domain 6 (Wdr6) as targets of ULK1.DN and potential mediators of its degeneration-attenuating effect. These findings reveal ULK1 as an important mediator of axonal degeneration and elucidate its function in splicing, defining it as a putative therapeutic target.
2020-09-23 | GSE123687 | GEO
Project description:Several traumatic and neurodegenerative disorders of the CNS show axonal degeneration as a key and early pathological feature. Following a focal axonal lesion, an extended axonal disintegration by acute axonal degeneration (AAD) occurs within several hours. During AAD, activation of autophagic proteins, including Unc-51 like autophagy activating kinase 1 (ULK1), has been shown but its role is incompletely understood. Here, we overexpressed a dominant negative of ULK1 (ULK1.DN) in primary rat cortical neurons in vitro and in the spinal cord (SC) and optic nerve (ON) in vivo. We found a significant inhibitory effect on autophagy in vitro and in vivo. While no effect on cell survival could be observed, ULK1.DN attenuated AAD after axotomy in microfluidic chambers in vitro. Models of SC injury and ON crush in vivo confirmed an attenuation of axonal degeneration by ULK1.DN. In a translational approach, we employed the selective ULK1 inhibitor SBI-0206965 (SBI) and found an attenuation of AAD after ON crush in vivo mediated by a reduction of autophagy by SBI. Quantitative proteomic profiling after ULK1.DN overexpression in vitro revealed a regulation of proteins associated with translation and splicing. A differential exon expression analysis then identified the genes kinesin family member 1B (Kif1b), DNA damage inducible transcript 3 (Ddit3) and WD repeat domain 6 (Wdr6) as targets of ULK1.DN and potential mediators of its degeneration-attenuating effect. These findings reveal ULK1 as an important mediator of axonal degeneration and elucidate its function in splicing, defining it as a putative therapeutic target.
2020-04-30 | PXD011862 | Pride
Project description: Not available
| S-EPMC7493890 | biostudies-literature
Project description:Axonal degeneration determines the clinical outcome of multiple sclerosis (MS), and is thought to result from exposure of denuded axons to immune-mediated damage. We challenge this view after finding in MS and its mouse models that myelin itself increases the risk of axons to degenerate under inflammatory conditions. We propose a model for demyelinating diseases in which for axons that remain myelinated, and thus shielded from the extracellular milieu, dependence from oligodendroglial support turns fatal in an autoimmune disease environment.
2023-05-02 | GSE222063 | GEO
Project description:VAMP7 is involved in autophagy and exocytosis mediating neurite growth, two yet unconnected cellular pathways. Here we show the occurrence of combined VAMP7/ATG9 secretory events. VAMP7 localized, together with LC3 and ATG9, in vesicles moving anterogradely along the axon towards growth cones. VAMP7 knockout disrupted the autophagy response to drugs and starvation. Release of extracellular vesicles triggered by autophagy was impaired in VAMP7-knockout cells and autophagy-deficient cells were impaired in VAMP7 exocytosis. Secretomics showed that VAMP7-knockout cells were impaired in unconventional secretion of cytoplasmic, exosomal and mitochondrial proteins. We further found that autophagy stimulated neurite growth in a VAMP7-dependent manner. Furthermore, neurons still grew long axons in nutrient-restricted conditions and when treated with autophagy-inducing drugs. A nanobody directed against VAMP7 inhibited the effect of nutrient restriction. We propose that VAMP7-dependent autophagic secretion contributes to a resilience mechanism to preserve axonal growth in restriction conditions, as part of brain sparing occurring in growth restriction.
2020-12-10 | PXD014231 | Pride
Project description:MFN2 is a mitochondrial outer membrane protein that plays a pivotal role in mitochondrial dynamics in most tissues, yet mutations in MFN2 (which cause Charcot-Marie-Tooth disease type 2A) primarily affect the nervous system. We generated a transgenic mouse model of CMT2A which developed severe early-onset vision loss and neurological deficits, axonal degeneration without cell body loss, and cytoplasmic and axonal accumulations of fragmented mitochondria labeled for degradation. Mutant MFN2R94Q had a dominant negative effect on mitochondrial fusion if MFN1 was absent or at low levels, as in neurons. Finally we found that augmenting the level of MFN1 in the nervous system in vivo led to rescue of all phenotypes in mutant MFN2R94Q expressing mice. These data demonstrate that the MFN1:MFN2 ratio is a key determinant of tissue specificity in CMT2A, and indicates that augmentation of MFN1 in the nervous system is a viable therapeutic strategy for the disease.
2019-04-03 | GSE116863 | GEO
Project description:Axon degeneration and neurological dysfunction in myelin diseases is often attributed to loss of myelin. Perturbed myelinating glia can instigate chronic neuroinflammation and contribute to demyelination and axonal damage. We have previously shown in mice that distinct defects in the proteolipid protein 1 gene result in axonal damage which is largely driven by cytotoxic T cells targeting myelinating oligodendrocytes. Here we show in these mutants that persistent ensheathment with perturbed myelin poses a risk for axon degeneration, neuron loss, and behavioral decline. We demonstrate that CD8+ T cell-driven axonal damage is less likely to progress towards degeneration when axons are efficiently demyelinated by activated microglia. Mechanistically, we show that cytotoxic T cell effector molecules induce aberrant cytoskeletal plasticity within myelinating glial processes and constriction of axons at paranodal domains. Our study identifies detrimental axon-glia interactions which promote neurodegeneration and possible therapeutic targets for disorders associated with myelin defects and neuroinflammation.
2023-09-29 | GSE224032 | GEO
Project description:Restoring Mitofusin balance in the nervous system prevents axonal degeneration in a CMT2A mouse model
| PRJNA480457 | ENA
Project description:During nutrient stress, macroautophagy is employed to degrade cellular macromolecules, thereby providing biosynthetic building blocks while simultaneously remodeling the proteome. While the machinery responsible for initiation of macroautophagy is well characterized, our understanding of the extent to which individual proteins, protein complexes and organelles are selected for autophagic degradation, and the underlying targeting mechanisms is limited. Here, we use orthogonal proteomic strategies to provide a global molecular inventory of autophagic cargo during nutrient stress in mammalian cell lines. Through prioritization of autophagic cargo, we identify a heterodimeric pair of membrane-embedded proteins, YIPF3 and YIPF4, as receptors for Golgiphagy. During nutrient stress, YIPF4 is mobilized into ATG8-positive vesicles that traffic to lysosomes as measured via Golgiphagy flux reporters in a process that requires the VPS34 and ULK1-FIP200 arms of the autophagy system. Cells lacking YIPF3 or YIPF4 are selectively defective in elimination of Golgi membrane proteins during nutrient stress. By merging absolute protein abundance with autophagic turnover, we create a global protein census describing how autophagic degradation maps onto protein abundance and subcellular localization. Our results, available via an interactive web tool, reveal that autophagic turnover prioritizes membrane-bound organelles (principally Golgi and ER) for proteome remodeling during nutrient stress.
2023-09-06 | PXD038358 | Pride
Project description:During nutrient stress, macroautophagy is employed to degrade cellular macromolecules, thereby providing biosynthetic building blocks while simultaneously remodeling the proteome. While the machinery responsible for initiation of macroautophagy is well characterized, our understanding of the extent to which individual proteins, protein complexes and organelles are selected for autophagic degradation, and the underlying targeting mechanisms is limited. Here, we use orthogonal proteomic strategies to provide a global molecular inventory of autophagic cargo during nutrient stress in mammalian cell lines. Through prioritization of autophagic cargo, we identify a heterodimeric pair of membrane-embedded proteins, YIPF3 and YIPF4, as receptors for Golgiphagy. During nutrient stress, YIPF4 is mobilized into ATG8-positive vesicles that traffic to lysosomes as measured via Golgiphagy flux reporters in a process that requires the VPS34 and ULK1-FIP200 arms of the autophagy system. Cells lacking YIPF3 or YIPF4 are selectively defective in elimination of Golgi membrane proteins during nutrient stress. By merging absolute protein abundance with autophagic turnover, we create a global protein census describing how autophagic degradation maps onto protein abundance and subcellular localization. Our results, available via an interactive web tool, reveal that autophagic turnover prioritizes membrane-bound organelles (principally Golgi and ER) for proteome remodeling during nutrient stress.
2023-09-13 | PXD043923 | Pride