| Ⅰ.BackgroundSpinal cord injury(SCI)is a temporary or permanent dysfunction of the spinal cord caused by traumatic factors(e.g.car accident injuries,falls,sports injuries)and nontraumatic factors(e.g.tumours,infections,degenerative disc disease),leading to a range of complications such as lower limb paralysis,persistent pathological pain,pressure sores and lung infections.The World Health Organization(WHO)estimates that between 250,000 and500,000 people suffer from SCI each year.The pathology of SCI is characterised by a cascade of amplified inflammatory storms following initial mechanical injury,leading to nerve cell death and a sustained demyelination response,with lipid-rich cell membranes and myelin debris accumulating in the centre of the injury and creating a hyperlipidic microenvironment locally.The disruption of the blood-spinal cord barrier is accompanied by the infiltration of large numbers of peripheral immune cells.As the predominant phagocyte,peripheral macrophages phagocytose and remove myelin debris.During 3 days(starting infiltration)to 7 days(reaching a peak)post SCI,main biological process of macrophage switches from cell migration to lipid catabolism and exhibit foam cell characteristics.Despite the initial macrophage phagocytosis eliminates myelin components that prevents axonal regeneration,prolonged uptake and excessive myelin-derived lipids accumulation leads to the formation of "foamy-like" macrophages and pro-inflammatory phenotype,ultimately counteracting the salutary effect of phagocytes.My master’s studies have shown that knockdown of macrophage scavenger receptor 1(MSR1)can alleviate the excessive uptake of myelin debris by macrophages,reduce the formation of pro-inflammatory "foamy-like" macrophages and neuronal apoptosis,and promote functional recovery.However,simple inhibition of macrophage phagocytosis also impairs its capacity to remove apoptotic cells,inhibitory myelin components and persisting neutrophils,which can amplify the local inflammatory responses and impede axonal remyelination and regeneration.Since the foamy phenotype of lipid-laden macrophages due to the imbalance between lipid uptake,storage and efflux,specifically promoting lipid digestion and excretion may be a much better entry point to reduce the negative impacts of lipid-laden macrophages after SCI.Autophagy is a conserved intracellular degradation process that maintains cellular homeostasis by wrapping proteins and organelles in a bilayer membrane structure and delivering them to lysosomes for degradation.Autophagy can also selectively degrade specific organelles and is referred to as selective autophagy.2009 Singh et al.first discovered that autophagy can selectively degrade LDs in hepatocytes and named this process "lipophagy".Lipophagy is a selective autophagy process that specifically recognizes ubiquitinated LDs and delivers them to lysosomes for degradation.Existing literature reports suggest that lipophagy plays a crucial role in regulating lipid disorders in various diseases such as fatty liver,neurodegenerative diseases,and atherosclerosis,and thus contributes to disease progression.However,the mechanism behind lipophagy’s recognition of ubiquitination-labeled LDs and the regulatory role of the ubiquitination system in lipophagy is not yet fully understood.The role of lipophagy in SCI disorders of lipid metabolism is yet to be explored.Ⅱ.Objectives of the study.(1)To elucidate the formation process,as well as the spatial and temporal distribution of "foamy-like" macrophages following spinal cord injury.(2)To investigate whether the disruption of autophagic flow on macrophages after spinal cord injury is involved in the accumulation of LDs.(3)To identify the key lipophagic receptors that target LDs degradation and verify their role in the accumulation of LDs in "foamy-like" macrophages after spinal cord injury.(4)To explore the intrinsic link between the ubiquitination system and lipophagy.Ⅲ.MethodsChapter 1: The macroscopic and microscopic observation of macrophage formation characterized by a "foamy" appearance post SCI was accomplished using immunofluorescence(CD68,MBP),oil red staining,and transmission electron microscopy.The predominant cellular sources contributing to the accumulation of LDs following SCI were identified using macrophage fluorescein reporter mice.Further,The accumulation of LDs and the recovery of motor function after SCI were observed in mice after macrophage clearance by chlorophosphate-liposome.Chapter 2: The macrophage autophagy levels post-SCI were studied using Western Blot and immunofluorescence(LC3,SQSTM1).ATG7 macrophage conditional knockout mice were engineered to observe the effects of ATG7 CKO on the accumulation of LDs and motor function recovery following SCI in vivo and in vitro experiments.Chapter 3: Transcriptome sequencing and small interfering RNA(si RNA)knockdown assays were implemented to identify potential selective lipophagic receptors.The changes in OPTN expression and its cellular localization during the formation of "foamy" macrophages were determined using Western Blot and immunofluorescence techniques(OPTN,BODIPY).In vitro experiments were conducted to study the impact of OPTN knockout on LDs accumulation,inflammatory factor secretion,and the state of neuronal axon growth.Furthermore,in vivo experiments were carried out to observe the effects of OPTN CKO on LDs accumulation and motor function recovery following SCI.Chapter 4: Immunoprecipitation-Mass Spectrometry techniques were utilized to identify potential E3 ubiquitin ligases that could bind with OPTN.Constructs of His-OPTN and Flag-Nedd4 plasmids were generated and transfected into 293 T cells;immunoprecipitation techniques were then used to to clarify the binding of OPTN and Nedd4.With the aid of adeno-associated virus overexpressing the CD68 promoter(AAV9),both in vitro and in vivo experiments were conducted to study the impact of Nedd4 overexpression in macrophages on LDs accumulation and motor function recovery after SCI.Ⅳ.ResultsIn this study,we aimed to investigate the formation and accumulation of LDs after spinal cord injury from both macroscopic and microscopic perspectives.We confirmed that the accumulation of LDs primarily occurred in peripheral macrophages utilizing macrophage fluorescein reporter mice.Further studies revealed that autophagic flow was disrupted in "foamy-like" macrophages in the injury area.Inhibition of autophagy significantly promoted the accumulation of LDs in macrophages and inhibited the recovery of motor function,suggesting a crucial role of autophagy in the formation of "foamy-like" macrophages.Transcriptome sequencing identified the key receptor of lipophagy,OPTN,and we successfully constructed a mouse model of OPTN macrophage-specific knockout using CreLoxp technology.OPTN conditional knockout considerably increased the accumulation of LDs and the release of inflammatory factors in macrophages,hindering the re-establishment of the regenerative microenvironment.Immunoprecipitation/mass spectrometry(IP/MS)was used to screen the key E3 ubiquitin ligase Nedd4,which regulates OPTN.Overexpression of Nedd4 in macrophages promoted the recognition of LDs by OPTN,initiating the lipophagic degradation of LDs and reducing the formation of "foamy-like" macrophages post-spinal cord injury.Specific overexpression of Nedd4 in macrophages improved the inflammatory microenvironment,reduced neuronal death,promoted axonal regeneration,and improved lower limb motor function recovery.Ⅴ.ConclusionAfter SCI,peripheral macrophages infiltrate the injury’s core and develop a foam-like phenotype,exacerbating inflammatory damage and hindering the reconstruction of the regenerative microenvironment.In this study,we discovered that the blocked autophagic flux of macrophages after SCI intensified the accumulation of LDs.We identified OPTN,a key lipophagic receptor targeting LDs,and found that NEDD4 facilitated the recognition of LDs by OPTN,activating lipophagy and reducing the buildup of LDs in "foamy-like" macrophages,thereby promoting functional recovery.This study partially elucidates the molecular mechanism responsible for LDs accumulation in macrophages after spinal cord injury,enriching our understanding of the inflammatory microenvironment in the injury area and providing novel treatment approaches for SCI.The study holds significant theoretical value and potential clinical translation. |
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