Cargando…

Impairment of autophagy after spinal cord injury potentiates neuroinflammation and motor function deficit in mice

Autophagy is a catabolic process that degrades cytoplasmic constituents and organelles in the lysosome, thus serving an important role in cellular homeostasis and protection against insults. We previously reported that defects in autophagy contribute to neuronal cell damage in traumatic spinal cord...

Descripción completa

Detalles Bibliográficos
Autores principales: Li, Yun, Lei, Zhuofan, Ritzel, Rodney M., He, Junyun, Li, Hui, Choi, Harry M. C., Lipinski, Marta M., Wu, Junfang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Ivyspring International Publisher 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9330534/
https://www.ncbi.nlm.nih.gov/pubmed/35910787
http://dx.doi.org/10.7150/thno.72713
_version_ 1784758185393913856
author Li, Yun
Lei, Zhuofan
Ritzel, Rodney M.
He, Junyun
Li, Hui
Choi, Harry M. C.
Lipinski, Marta M.
Wu, Junfang
author_facet Li, Yun
Lei, Zhuofan
Ritzel, Rodney M.
He, Junyun
Li, Hui
Choi, Harry M. C.
Lipinski, Marta M.
Wu, Junfang
author_sort Li, Yun
collection PubMed
description Autophagy is a catabolic process that degrades cytoplasmic constituents and organelles in the lysosome, thus serving an important role in cellular homeostasis and protection against insults. We previously reported that defects in autophagy contribute to neuronal cell damage in traumatic spinal cord injury (SCI). Recent data from other inflammatory models implicate autophagy in regulation of immune and inflammatory responses, with low levels of autophagic flux associated with pro-inflammatory phenotypes. In the present study, we examined the effects of genetically or pharmacologically manipulating autophagy on posttraumatic neuroinflammation and motor function after SCI in mice. Methods: Young adult male C57BL/6, CX3CR1-GFP, autophagy hypomorph Becn1(+/-) mice, and their wildtype (WT) littermates were subjected to moderate thoracic spinal cord contusion. Neuroinflammation and autophagic flux in the injured spinal cord were assessed using flow cytometry, immunohistochemistry, and NanoString gene expression analysis. Motor function was evaluated with the Basso Mouse Scale and horizontal ladder test. Lesion volume and spared white matter were evaluated by unbiased stereology. To stimulate autophagy, disaccharide trehalose, or sucrose control, was administered in the drinking water immediately after injury and for up to 6 weeks after SCI. Results: Flow cytometry demonstrated dysregulation of autophagic function in both microglia and infiltrating myeloid cells from the injured spinal cord at 3 days post-injury. Transgenic CX3CR1-GFP mice revealed increased autophagosome formation and inhibition of autophagic flux specifically in activated microglia/macrophages. NanoString analysis using the neuroinflammation panel demonstrated increased expression of proinflammatory genes and decreased expression of genes related to neuroprotection in Becn1(+/-) mice as compared to WT controls at 3 days post-SCI. These findings were further validated by qPCR, wherein we observed significantly higher expression of proinflammatory cytokines. Western blot analysis confirmed higher protein expression of the microglia/macrophage marker IBA-1, inflammasome marker, NLRP3, and innate immune response markers cGAS and STING in Becn1(+/-) mice at 3 day after SCI. Flow cytometry demonstrated that autophagy deficit did not affect either microglial or myeloid counts at 3 days post-injury, instead resulting in increased microglial production of proinflammatory cytokines. Finally, locomotor function showed significantly worse impairments in Becn1(+/-) mice up to 6 weeks after SCI, which was accompanied by worsening tissue damage. Conversely, treatment with a naturally occurring autophagy inducer trehalose, reduced protein levels of p62, an adaptor protein targeting cargo to autophagosomes as well as the NLRP3, STING, and IBA-1 at 3 days post-injury. Six weeks of trehalose treatment after SCI led to improved motor function recovery as compared to control group, which was accompanied by reduced tissue damage. Conclusions: Our data indicate that inhibition of autophagy after SCI potentiates pro-inflammatory activation in microglia and is associated with worse functional outcomes. Conversely, increasing autophagy with trehalose, decreased inflammation and improved outcomes. These findings highlight the importance of autophagy in spinal cord microglia and its role in secondary injury after SCI.
format Online
Article
Text
id pubmed-9330534
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher Ivyspring International Publisher
record_format MEDLINE/PubMed
spelling pubmed-93305342022-07-30 Impairment of autophagy after spinal cord injury potentiates neuroinflammation and motor function deficit in mice Li, Yun Lei, Zhuofan Ritzel, Rodney M. He, Junyun Li, Hui Choi, Harry M. C. Lipinski, Marta M. Wu, Junfang Theranostics Research Paper Autophagy is a catabolic process that degrades cytoplasmic constituents and organelles in the lysosome, thus serving an important role in cellular homeostasis and protection against insults. We previously reported that defects in autophagy contribute to neuronal cell damage in traumatic spinal cord injury (SCI). Recent data from other inflammatory models implicate autophagy in regulation of immune and inflammatory responses, with low levels of autophagic flux associated with pro-inflammatory phenotypes. In the present study, we examined the effects of genetically or pharmacologically manipulating autophagy on posttraumatic neuroinflammation and motor function after SCI in mice. Methods: Young adult male C57BL/6, CX3CR1-GFP, autophagy hypomorph Becn1(+/-) mice, and their wildtype (WT) littermates were subjected to moderate thoracic spinal cord contusion. Neuroinflammation and autophagic flux in the injured spinal cord were assessed using flow cytometry, immunohistochemistry, and NanoString gene expression analysis. Motor function was evaluated with the Basso Mouse Scale and horizontal ladder test. Lesion volume and spared white matter were evaluated by unbiased stereology. To stimulate autophagy, disaccharide trehalose, or sucrose control, was administered in the drinking water immediately after injury and for up to 6 weeks after SCI. Results: Flow cytometry demonstrated dysregulation of autophagic function in both microglia and infiltrating myeloid cells from the injured spinal cord at 3 days post-injury. Transgenic CX3CR1-GFP mice revealed increased autophagosome formation and inhibition of autophagic flux specifically in activated microglia/macrophages. NanoString analysis using the neuroinflammation panel demonstrated increased expression of proinflammatory genes and decreased expression of genes related to neuroprotection in Becn1(+/-) mice as compared to WT controls at 3 days post-SCI. These findings were further validated by qPCR, wherein we observed significantly higher expression of proinflammatory cytokines. Western blot analysis confirmed higher protein expression of the microglia/macrophage marker IBA-1, inflammasome marker, NLRP3, and innate immune response markers cGAS and STING in Becn1(+/-) mice at 3 day after SCI. Flow cytometry demonstrated that autophagy deficit did not affect either microglial or myeloid counts at 3 days post-injury, instead resulting in increased microglial production of proinflammatory cytokines. Finally, locomotor function showed significantly worse impairments in Becn1(+/-) mice up to 6 weeks after SCI, which was accompanied by worsening tissue damage. Conversely, treatment with a naturally occurring autophagy inducer trehalose, reduced protein levels of p62, an adaptor protein targeting cargo to autophagosomes as well as the NLRP3, STING, and IBA-1 at 3 days post-injury. Six weeks of trehalose treatment after SCI led to improved motor function recovery as compared to control group, which was accompanied by reduced tissue damage. Conclusions: Our data indicate that inhibition of autophagy after SCI potentiates pro-inflammatory activation in microglia and is associated with worse functional outcomes. Conversely, increasing autophagy with trehalose, decreased inflammation and improved outcomes. These findings highlight the importance of autophagy in spinal cord microglia and its role in secondary injury after SCI. Ivyspring International Publisher 2022-07-11 /pmc/articles/PMC9330534/ /pubmed/35910787 http://dx.doi.org/10.7150/thno.72713 Text en © The author(s) https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.
spellingShingle Research Paper
Li, Yun
Lei, Zhuofan
Ritzel, Rodney M.
He, Junyun
Li, Hui
Choi, Harry M. C.
Lipinski, Marta M.
Wu, Junfang
Impairment of autophagy after spinal cord injury potentiates neuroinflammation and motor function deficit in mice
title Impairment of autophagy after spinal cord injury potentiates neuroinflammation and motor function deficit in mice
title_full Impairment of autophagy after spinal cord injury potentiates neuroinflammation and motor function deficit in mice
title_fullStr Impairment of autophagy after spinal cord injury potentiates neuroinflammation and motor function deficit in mice
title_full_unstemmed Impairment of autophagy after spinal cord injury potentiates neuroinflammation and motor function deficit in mice
title_short Impairment of autophagy after spinal cord injury potentiates neuroinflammation and motor function deficit in mice
title_sort impairment of autophagy after spinal cord injury potentiates neuroinflammation and motor function deficit in mice
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9330534/
https://www.ncbi.nlm.nih.gov/pubmed/35910787
http://dx.doi.org/10.7150/thno.72713
work_keys_str_mv AT liyun impairmentofautophagyafterspinalcordinjurypotentiatesneuroinflammationandmotorfunctiondeficitinmice
AT leizhuofan impairmentofautophagyafterspinalcordinjurypotentiatesneuroinflammationandmotorfunctiondeficitinmice
AT ritzelrodneym impairmentofautophagyafterspinalcordinjurypotentiatesneuroinflammationandmotorfunctiondeficitinmice
AT hejunyun impairmentofautophagyafterspinalcordinjurypotentiatesneuroinflammationandmotorfunctiondeficitinmice
AT lihui impairmentofautophagyafterspinalcordinjurypotentiatesneuroinflammationandmotorfunctiondeficitinmice
AT choiharrymc impairmentofautophagyafterspinalcordinjurypotentiatesneuroinflammationandmotorfunctiondeficitinmice
AT lipinskimartam impairmentofautophagyafterspinalcordinjurypotentiatesneuroinflammationandmotorfunctiondeficitinmice
AT wujunfang impairmentofautophagyafterspinalcordinjurypotentiatesneuroinflammationandmotorfunctiondeficitinmice