A genome-wide screen uncovers multiple roles for mitochondrial nucleoside diphosphate kinase D in inflammasome activation

Noncanonical inflammasome activation by cytosolic lipopolysaccharide (LPS) is a critical component of the host response to Gram-negative bacteria. Cytosolic LPS recognition in macrophages is preceded by a Toll-like receptor (TLR) priming signal required to induce transcription of inflammasome components and facilitate the metabolic reprograming that fuels the inflammatory response. Using a genome-scale arrayed siRNA screen to find inflammasome regulators in mouse macrophages, we identified the mitochondrial enzyme nucleoside diphosphate kinase D (NDPK-D) as a regulator of both noncanonical and canonical inflammasomes. NDPK-D was required for both mitochondrial DNA synthesis and cardiolipin exposure on the mitochondrial surface in response to inflammasome priming signals mediated by TLRs, and macrophages deficient in NDPK-D had multiple defects in LPS-induced inflammasome activation. In addition, NDPK-D was required for the recruitment of TNF receptor–associated factor 6 (TRAF6) to mitochondria, which was critical for reactive oxygen species (ROS) production and the metabolic reprogramming that supported the TLR-induced gene program. NDPK-D knockout mice were protected from LPS-induced shock, consistent with decreased ROS production and attenuated glycolytic commitment during priming. Our findings suggest that, in response to microbial challenge, NDPK-D–dependent TRAF6 mitochondrial recruitment triggers an energetic fitness checkpoint required to engage and maintain the transcriptional program necessary for inflammasome activation.

VPS13D interacts with VCP/p97 and negatively regulates ER- mitochondrial interactions

Membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and mitochondria are emerging as critical hubs for diverse cellular events, and alterations in the extent of these contacts are linked to neurodegenerative diseases. However, the mechanisms that control ER-mitochondrial interactions are so far elusive. Here, we demonstrate a key role of vacuolar protein sorting-associated protein 13D (VPS13D) in the negative regulation of ER-mitochondrial MCSs. VPS13D suppression results in extensive ER-mitochondrial tethering, a phenotype that can be substantially rescued by suppression of the tethering proteins VAPB and PTPIP51. VPS13D interacts with valosin-containing protein (VCP/p97) to control the level of ER-resident VAPB at contacts. VPS13D is required for the stability of p97. Functionally, VPS13D suppression leads to severe defects in the mitochondrial morphology, mitochondrial cellular distribution and mitochondrial DNA synthesis. Together our results suggest that VPS13D negatively regulates the ER-mitochondrial MCSs partially through its interactions with VCP/p97. [Media: see text]

Mitochondrial DNA enables AIM2 inflammasome activation and hepatocyte pyroptosis in non-alcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is typified by accumulating excess liver triacylglycerol, inflammation, and liver dysfunction. This study was aimed to investigate the role of mitochondrial DNA synthesis-induced activation of Absent in melanoma 2 (AIM2) inflammasome and pyroptosis in NAFLD. Mice were raised on a high-fat diet for 24 weeks to establish NAFLD models. F4/80 immunofluorescence was performed to reflect the inflammatory response in the liver of mice. Western blot, ELISA, and immunofluorescence were adopted to determine the expression of AIM2 inflammasome-related proteins and factors. EdU immunofluorescence was applied for the examination of mitochondrial DNA expression and flow cytometry for cell pyroptosis. Agarose gel electrophoresis was used to detect the integrity of extracted mouse mitochondrial DNA (mtDNA). The levels of AIM2 inflammasome-related proteins in the liver and the levels of IL-1β and IL-18 in serum were elevated in high-fat diet-induced NAFLD mice. AIM2 inflammasome activation and pyroptosis were triggered, and suppressed activation of AIM2 inflammasome alleviated the inflammation and pyroptosis in the liver of NAFLD mice. Mitochondria were severely damaged and mtDNA was synthesized after NAFLD modeling. Further, mtDNA treatment could promote palmitate (PA)-induced activation of AIM2 inflammasome and pyroptosis. Moreover, inhibition of IRF1 gene alleviated PA-induced AIM2 inflammasome activation and pyroptosis. In conclusion, mitochondrial DNA synthesis could enable AIM2 inflammasome activation and induce the hepatocyte pyroptosis, thereby exacerbating NAFLD.

The Roles of Mitochondrial Folate Metabolism in Supporting Mitochondrial DNA Synthesis, Oxidative Phosphorylation, and Cellular Function

ABSTRACT Folate-mediated one-carbon metabolism (FOCM) is compartmentalized within human cells to the cytosol, nucleus, and mitochondria. The recent identifications of mitochondria-specific, folate-dependent thymidylate [deoxythymidine monophosphate (dTMP)] synthesis together with discoveries indicating the critical role of mitochondrial FOCM in cancer progression have renewed interest in understanding this metabolic pathway. The goal of this narrative review is to summarize recent advances in the field of one-carbon metabolism, with an emphasis on the biological importance of mitochondrial FOCM in maintaining mitochondrial DNA integrity and mitochondrial function, as well as the reprogramming of mitochondrial FOCM in cancer. Elucidation of the roles and regulation of mitochondrial FOCM will contribute to a better understanding of the mechanisms underlying folate-associated pathologies.

Epigallocatechin-3-Gallate Prevents Acute Gout by Suppressing NLRP3 Inflammasome Activation and Mitochondrial DNA Synthesis

Gout is a chronic inflammatory disease evoked by the deposition of monosodium urate (MSU) crystals in joint tissues. The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome is responsible for the gout inflammatory symptoms induced by MSU crystals. We investigated whether epigallocatechin-3-gallate (EGCG) suppresses the activation of the NLRP3 inflammasome, thereby effectively preventing gouty inflammation. EGCG blocked MSU crystal-induced production of caspase-1(p10) and interleukin-1β in primary mouse macrophages, indicating its suppressive effect on the NLRP3 inflammasome. In an acute gout mouse model, oral administration of EGCG to mice effectively alleviated gout inflammatory symptoms in mouse foot tissue injected with MSU crystals. The in vivo suppressive effects of EGCG correlated well with the suppression of the NLRP3 inflammasome in mouse foot tissue. EGCG inhibited the de novo synthesis of mitochondrial DNA as well as the production of reactive oxygen species in primary mouse macrophages, contributing to the suppression of the NLRP3 inflammasome. These results show that EGCG suppresses the activation of the NLRP3 inflammasome in macrophages via the blockade of mitochondrial DNA synthesis, contributing to the prevention of gouty inflammation. The inhibitory effects of EGCG on the NLRP3 inflammasome make EGCG a promising therapeutic option for NLRP3-dependent diseases such as gout.