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

• Published in: Theranostics Vol. 12; no. 12; pp. 5364 - 5388
• Main Authors: Li, Yun, Lei, Zhuofan, Ritzel, Rodney M, He, Junyun, Li, Hui, Choi, Harry M C, Lipinski, Marta M, Wu, Junfang
• Format: Journal Article
• Language: English
• Published: Australia Ivyspring International Publisher Pty Ltd 01.01.2022; Ivyspring International Publisher
• Subjects: Alzheimer's disease; Animals; Apoptosis; Autophagy; Contusions; Cytokines - metabolism; Drinking water; Laboratories; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia - metabolism; Neuroinflammatory Diseases; NLR Family, Pyrin Domain-Containing 3 Protein - metabolism; Research Paper; Spinal cord injuries; Spinal Cord Injuries - complications; Sucrose; Surgery; Trehalose - metabolism; Trehalose - pharmacology; Young adults; Beclin-1; Neuroinflammation; Spinal cord injury; Autophagy; Microglia
• ISSN: 1838-7640; 1838-7640
• DOI: 10.7150/thno.72713

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. Young adult male C57BL/6, CX3CR1-GFP, autophagy hypomorph 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. 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 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 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 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. 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.