Spencer Alexander Freeman

Lytic cell death pathways drive hepatic ischemia-reperfusion injury (IRI). The transmembrane protein ninjurin-1 (NINJ1) aggregates in the plasma membrane to permeabilize the cell during multiple cell death pathways implicated in hepatic IRI. We hypothesized that NINJ1 mediates liver IRI and that its inhibition would mitigate injury. We found that NINJ1 is highly expressed in human liver tissue and that its up-regulation and activation correlate with early allograft dysfunction in liver transplant patients. Using a segmental hepatic IRI model in mice and rats, Ninj1 genetic deletion or pharmacologic inhibition diminished acute injury. Mice with hepatocyte- or macrophage-specific Ninj1 knockout had reduced hepatocellular injury following IRI, suggesting that NINJ1 within both populations contributes to the resulting liver injury. Mechanistically, we found that hepatocytes and Kupffer cells are susceptible to hypoxia-induced NINJ1-mediated plasma membrane rupture, which can be pharmacologically prevented. We therefore position NINJ1 as a potential new therapeutic target to limit hepatic IRI, with important implications for liver transplantation.

Lysosomes are subject to perturbations that can cause damage to their limiting membrane. Osmotic shifts, pore-forming toxins, and the growth of luminal polymers or pathogens all stand to increase lysosomal membrane tension and/or disrupt the bilayer. In some contexts, this leads to lysosomal rupture and cell death. Here, we describe a mechanism that enables lysosomes to sense and respond to acute increases in tension of their limiting membrane. We report that the lysosome-resident nonselective cation channel, TMEM63A, can drive the directional flux of monovalent cations, major osmoticants, out of the lumen when gated by mechanical tension on the organelle. This results in the ability for lysosomes to relieve hydrostatic pressure and, proportionally, membrane tension, affording lysosomes the time to acquire additional lipids. Lysosomes without this mechanism—either because TMEM63A is deleted or in the case when cells express disease-causing variants of TMEM63A—are an order of magnitude more sensitive to lysis upon increases to their membrane tension when compared with their WT counterparts. These findings suggest that lysosomes are capable of regulating hydrostatic pressure and volume in response to high tension.

The ubiquitous and highly conserved programmed cell death pathways that are essential for tissue development and homeostasis are accompanied by distinct morphological alterations. Apoptotic cells undergo fragmentation that is concomitant with the exposure of phosphatidylserine (PS) on the membrane surface. Large fragments, called apoptotic bodies, as well as much smaller and more numerous vesicles, are released. While the molecular mechanisms underlying apoptotic body formation have been explored, much less is known about vesicle biogenesis. We used an inducible, active form of TMEM16F to determine the role of lipid scrambling in vesiculation, separately from other apoptotic signaling events. Plasmalemmal lipid scrambling sufficed to release apoptotic-like vesicles without causing changes in cytosolic calcium or the submembrane cytoskeleton. The scrambled bilayer showed pronounced segregation of exofacial lipids and redistribution of detectable cholesterol to the inner leaflet. The clustering of raft-associated components with bulky headgroups—typified by glycophosphatidylinositol-linked proteins—formed domains of outward (convex) curvature, while regions of accumulation of phosphatidylethanolamine (PE) generated inward (concave) curvature that facilitated the scission of vesicles. Thus, scrambling of plasma membrane lipids suffices to induce regions of acute membrane curvature and facilitates detachment of vesicles analogous to those released from the surface of apoptotic cells.

Hepatic ischemia-reperfusion injury (IRI) results from interrupted perfusion to the liver and contributes to acute liver dysfunction such as following liver transplantation. Lytic cell death pathways are major drivers of IRI and the subsequent inflammatory response. The transmembrane protein ninjurin-1 (NINJ1) was identified as the key executor of terminal plasma membrane rupture across multiple lytic cell death pathways implicated in hepatic IRI. We hypothesized that NINJ1-mediated lytic cell death drives IRI and that its therapeutic inhibition would mitigate liver IRI. Using human liver specimens, we found that NINJ1 is highly expressed in human liver tissue and that its activation correlates with early allograft dysfunction in patients undergoing liver transplantation. Utilizing a segmental hepatic IRI model in mice and rats, Ninj1 genetic deletion or pharmacologic inhibition diminished acute liver injury. Mice with hepatocyte- or macrophage-specific Ninj1 knockout both had reduced hepatocellular injury following IRI, suggesting that NINJ1 within both populations contributes to the resulting liver injury. Mechanistically, we found that hepatocytes and Kupffer cells are highly susceptible to hypoxia-induced NINJ1-mediated plasma membrane rupture, which can be pharmacologically prevented. These data position NINJ1 as a potential new therapeutic target to limit hepatic IRI, with important implications for organ preservation during liver transplantation.

The abnormal protein degradation implicated in the pathogenesis of Parkinson’s disease was previously attributed to defective H+ leakage from lysosomes via TMEM175 (https://doi.org/10.1016/j.cell.2022.05.021). In this issue, Riederer et al. (https://doi.org/10.1083/jcb.202501145) demonstrate that TMEM175 is instead a K+ channel, minimally permeable to H+.

Inflammasomes are nucleated by receptors that become activated upon cellular stresses, including ionic dyshomeostasis. Rather than forming in the cytosol, recent evidence suggests that inflammasomes are nucleated at specific sites in the cell, including on cytoskeletal polymers and the membrane surfaces of organelles. The NLRP3 inflammasome, which is formed upon the loss of cytosolic K⁺, had been proposed to form on intermediate filaments as well as on vesicles along the endocytic pathway. To determine the necessary requirement of either mechanism, we used vimentin knockout macrophages that do not have intermediate filaments and compared the formation and function of NLRP3 inflammasomes. We report that vimentin was dispensable for the activation of caspase-1, IL-1β cleavage and release, and inflammatory responses in mice attributed to the inflammasome. Instead, NLRP3 was recruited to PI(3,5)P2, PI(4)P- and LAMP1-positive compartments undergoing osmotic swelling. Swelling of these compartments was dependent on the V-ATPase, the inhibition of which curtailed NLRP3 recruitment and inflammasome activation. Similarly, decreasing the hydrostatic pressure on these vesicles prevented NLRP3 recruitment, IL-1β release and pyroptosis. The results suggest that NLRP3 is activated by biophysical features of acidic organelles in the endocytic pathway.

The ubiquitous and highly conserved programmed cell death pathways that are essential for tissue development and homeostasis are accompanied by distinct morphological alterations. Apoptotic cells undergo fragmentation that is concomitant with the exposure of phosphatidylserine (PS) on the membrane surface. Large fragments, called apoptotic bodies, as well as much smaller and more numerous vesicles are released. While the molecular mechanisms underlying apoptotic body formation have been explored, much less is known about vesicle biogenesis. We used an inducible, active form of TMEM16F to determine the role of lipid scrambling in vesiculation, separately from other apoptotic signaling events. Plasmalemmal lipid scrambling sufficed to release apoptotic-like vesicles without causing changes in cytosolic calcium or the submembrane cytoskeleton. The scrambled bilayer showed pronounced segregation of exofacial lipids and redistribution of apparent cholesterol to the inner leaflet. The clustering of raft-associated components with bulky headgroups –typified by glycophosphatidylinositol-linked proteins– formed domains of outward (convex) curvature, while regions of accumulation of phosphatidylethanolamine (PE) generated inward (concave) curvature that facilitated the scission of vesicles. Thus, scrambling of plasma membrane lipids suffices to induce regions of acute membrane curvature and facilitates detachment of vesicles analogous to those released from the surface of apoptotic cells.

Mutations or ablation of Snx10 are associated with neurodegeneration, blindness, and osteopetrosis. The similarities between osteoclasts and macrophages prompted us to analyze the role of Snx10 in phagocytosis. Deletion of Snx10 impaired phagosome resolution. Defective resolution was caused by reduced Cl− accumulation within (phago)lysosomes, replicating the phenotype reported in macrophages lacking ClC-7, a lysosomal 2Cl−/H+ antiporter. Delivery of ClC-7 to (phago)lysosomes was unaffected by ablation of Snx10, but its activity was markedly depressed. Snx10 was found to regulate ClC-7 activity indirectly by controlling the availability of phosphatidylinositol 3,5-bisphosphate (PI[3,5]P2), which inhibits ClC-7. By limiting the formation of PI(3,5)P2, Snx10 enables the accumulation of luminal Cl− in phagosomes and lysosomes, which is required for their optimal degradative function. Our data suggest that Snx10 regulates the delivery of PI 3-phosphate (PI[3]P), the precursor of PI(3,5)P2, from earlier endocytic compartments to (phago)lysosomes. By controlling the traffic of phosphoinositides, Snx10 regulates phagosomal resolution and possibly accounts for the impaired bone resorption in Snx10-deficient osteoclasts.