The review draws a sharp line between two phenomena that have often been lumped together under the vague heading of lysosomal damage. The first, lysosomal membrane permeabilization, or LMP, was defined between 1972 and 1999 through cytochemical experiments by Brunk and colleagues using cultured cells exposed to various stresses. In LMP, the lysosomal limiting membrane remains ultrastructurally intact when viewed under the electron microscope, yet tiny pores—far too small to be resolved—allow a gradual and selective leakage of lysosomal contents into the cytoplasm. The second phenomenon, lysosomal membrane rupture, or LMR, was first identified in 1996 by Yamashima and coworkers using electron microscopic analysis of hippocampal CA1 neurons in Japanese macaque monkeys after transient global brain ischemia. In LMR, the limiting membrane shows an apparent, visible disruption: larger holes that permit an acute and massive outpouring of hydrolytic cathepsin enzymes, leading to the rapid, almost explosive vanishing of the lysosomes themselves.

The distinction matters because the two modes of membrane failure appear to steer cells toward entirely different fates. According to the review, LMP acts as an amplifier of cell death signaling and participates in apoptosis, the orderly, programmed form of cell suicide. The selective release of lysosomal enzymes through submicroscopic pores triggers the proteolytic activation of caspases, the executioner enzymes of apoptosis. LMR, by contrast, unleashes a catastrophic flood of cathepsins that drives necrosis, the messy, inflammatory form of cell death in which cells essentially dissolve. Yamashima proposes that the calpain-cathepsin cascade—a sequence in which the calcium-activated protease calpain and the lysosomal cathepsins work in concert—executes necrotic death, whereas caspase activation through LMP executes apoptosis. Which of the two outcomes prevails depends on the size and number of membrane perforations, the amount of enzyme leakage, and the cellular context.

The historical thread runs back to 1966, when de Duve and Wattiaux first proposed that cell degeneration and necrosis occur through the leakage of hydrolytic enzymes from damaged lysosomes. In 1972, Brunk and Ericsson observed an extensive diffusion of the lysosomal enzyme acid phosphatase through membranes that looked structurally intact, although the improper fixation used in those experiments may itself have created artificial pores. It was not until 1999 that Brunk and Svensson, working with human Jurkat leukemia cells exposed to oxidative stress, confirmed LMP directly by tracking the leakage of the lysosome-accumulating dye acridine orange. Because the electron microscope showed nothing wrong with the membrane, the researchers concluded that pores too small to image must be responsible. For decades afterward, lysosomes were widely regarded as sturdy organelles whose membranes might become leaky but would not visibly rupture until the cell was already dead—a view that caused interest in lysosomal cell death to fade.

That view changed with the 1996 primate study. In hippocampal CA1 neurons three to five days after transient ischemia—neurons that were degenerating but still alive—Yamashima’s group captured electron micrographs showing lysosomes with unmistakable holes in their limiting membranes, spilling their contents before necrosis had run its course. Similar ruptures were later documented in cultured macrophages and in mouse alveolar macrophages exposed to nanomaterials in 2012 and 2016. In 2016, Li and colleagues reported that loss of the scav-3 gene in the roundworm Caenorhabditis elegans—the worm counterpart of the human lysosomal membrane protein LIMP-2, a key guardian of membrane integrity—also produced LMR. Yamashima’s team subsequently observed comparable membrane disruptions, often appearing as double contours or irregular lysosomal shapes near autophagolysosomes and damaged mitochondria, in degenerating cortical neurons from patients with Alzheimer’s disease.

Recent live-cell imaging has brought the two phenomena into even sharper contrast. In experiments on a human hepatocellular carcinoma cell line, HepG2, the researchers compared the effects of epirubicin, a chemotherapeutic agent, with those of 4-hydroxy-2-nonenal, or 4-HNE, a lipid peroxidation product. Time-lapse imaging with LysoTracker, a fluorescent dye retained in acidic lysosomes, showed that epirubicin caused cell shrinkage and membrane blebbing while most lysosomes remained visible until apoptosis was complete—consistent with gradual, selective leakage through invisible pores. The 4-HNE treatment, however, produced bursting cell death in which every lysosome vanished before the cytoplasm and nuclear chromatin disintegrated, reflecting rapid, massive leakage through ruptured membranes. Notably, the intensity of lysosomal staining proved to be a poor proxy for the degree of membrane damage, and lysosomes could still be detected after apoptosis but not after necrosis had been executed.

Why are lysosomes so vulnerable in the first place? Unlike mitochondria, they lack antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase. They are also rich in iron released from degraded iron-containing proteins. When reactive oxygen species flood the cell, ferrous iron inside the lysosome reacts with hydrogen peroxide in the Fenton reaction, generating hydroxyl radicals that attack the membrane’s lipids. Experiments with silica particles illustrate the point: macrophages that swallowed iron-laden particles suffered extensive lysosomal damage, while those given particles pretreated with an iron chelator showed only modest leakage. The review identifies 4-HNE as a central culprit. Generated both from dietary linoleic acid in vegetable oils and from cardiolipin in the inner mitochondrial membrane, 4-HNE forms adducts with the amino acids cysteine, histidine, lysine and arginine in proteins, including the heat-shock protein Hsp70.1, which normally stabilizes the lysosomal membrane.

The molecular story that emerges is intricate. Hsp70.1 performs a dual role: it ferries damaged proteins to lysosomes for degradation via chaperone-mediated autophagy, and it preserves membrane integrity by promoting the interaction between the lipid BMP and acid sphingomyelinase, an enzyme whose product, ceramide, reinforces the membrane against rupture. In the ischemic monkey hippocampus, two-dimensional oxyblot analysis revealed a three- to nine-fold upregulation of Hsp70.1 together with a drop in its molecular weight, indicating that oxidative carbonylation had struck the arginine at position 469 of the protein during reperfusion. Once carbonylated, Hsp70.1 becomes susceptible to cleavage by the calcium-activated protease μ-calpain. In vitro experiments showed that calpain alone could not cut intact Hsp70.1, but cleavage proceeded in a time-dependent manner once 4-HNE had modified the protein. In living monkeys injected with 4-HNE over 24 weeks, Western blotting of liver tissue confirmed the same calpain-mediated cleavage, and immunofluorescence revealed extensive overlap of Hsp70.1 and activated calpain in hepatocytes and CD68-positive Kupffer cells, whose lysosomes largely faded away through rupture.

The review also reaches beyond apoptosis and necrosis. Pyroptosis, a highly inflammatory, lytic form of programmed cell death first named in 2001, defends the body against intracellular pathogens by rupturing the plasma membrane and releasing pro-inflammatory cytokines. Ferroptosis, described in 2012, is an iron-dependent death driven by lipid peroxidation of membranes. Because the acidic lysosomal lumen, packed with reactive iron and hydrogen peroxide, is an ideal chemical reactor for generating reactive oxygen species, recent work by Saimoto and colleagues suggests that lipid peroxidation at the lysosomal membrane triggers LMP during ferroptosis, with intralysosomal iron leakage then fanning cell-wide lipid peroxidation. Yamashima argues that the overlap runs deeper: given the shared involvement of the Fenton reaction, lipid peroxidation, calcium signaling and lysosomal membrane failure, ferroptosis may well employ the same calpain-cathepsin cascade that drives necrosis.

The practical upshot is methodological as much as conceptual. Because LMP involves pores that electron microscopy cannot resolve, it must be detected cytochemically—for instance with the lysosomal galectin puncta assay, which registers the translocation of galectins 1 and 3 to damaged membranes earlier than cathepsin-release measurements can. LMR, by contrast, is fleeting: ruptures occur within minutes or hours and the lysosomes then disappear, so researchers have only a narrow window to catch them in degenerating tissue. The review’s summary point is unambiguous: distinguishing LMP from LMR by careful electron microscopy is indispensable for elucidating the mechanisms of cell death in organs stressed by ischemia, toxins or disease. If the calpain-cathepsin hypothesis holds across neurons, hepatocytes and immune cells alike, then protecting the lysosomal membrane—and shielding Hsp70.1 from carbonylation—may become a unified therapeutic strategy against some of medicine’s most stubborn degenerative conditions.

Subject of Research: Distinct roles of lysosomal membrane permeabilization and rupture in determining apoptotic versus necrotic cell death

Article Title: Lysosomal membrane-permeabilization (LMP) and -rupture (LMR) are distinct for cell death

Article References: Yamashima, T. (2025). Lysosomal membrane-permeabilization (LMP) and -rupture (LMR) are distinct for cell death. Frontiers in Cell Death, 4, Article 1669955. https://doi.org/10.3389/fceld.2025.1669955

Image Credits: AI Generated

DOI: 10.3389/fceld.2025.1669955

Keywords: lysosome, lysosomal membrane permeabilization, lysosomal membrane rupture, apoptosis, necrosis, cathepsins, calpain, Hsp70.1, 4-hydroxy-2-nonenal, ferroptosis, pyroptosis, cell death