Cell Death & Disease

Cellular material in apoptotic cells must be efficiently cleared by phagocytes to maintain tissue homeostasis. Defects in this process can lead to the onset of secondary necrosis and the release of intracellular contents such as damage associated molecular patterns (DAMPs) and autoantigens that are often derived from the nucleus. Therefore, appropriate handling and clearance of apoptotic material is vital to prevent unwanted inflammatory response and the onset of autoimmune disorders. However, how nuclear material is packaged by apoptotic cells for effective clearance by phagocytes is not well understood. By utilising murine models of apoptosis, we observed that a distinct subset of large extracellular vesicles generated from apoptotic thymocytes, known as apoptotic bodies (ApoBDs), can harbour the majority of nuclear contents. Mechanistically, we discovered that apoptotic cells can asymmetrically partition the nucleus into a single large membrane bleb located at one side of the cell, with other cellular contents such as mitochondria and acid organelles distributed to the opposite side. Whilst this newly observed apoptotic morphology, coined as asymmetric cell death morphology (AsyCDM), is morphologically similar to the process of erythroblast enucleation, pharmacological compounds that could interfere with erythroblast enucleation did not block the establishment of AsyCDM during apoptosis. Notably, AsyCDM was reliant on the contractile forces generated by ROCK1-dependent plasma membrane blebbing. Taken together, this study suggests that intracellular contents are partitioned into different ApoBD subsets during apoptosis through a regulated process driven by ROCK1-dependent actomyosin contraction.

Autosomal recessive Stargardt disease type 1 (STGD1) is the most prevalent inherited juvenile macular degeneration, ultimately leading to irreversible blindness through photoreceptor cell loss, yet the underlying cell death mechanisms remain poorly defined. Receptor-interacting protein kinase 3 (RIPK3) is a well-established mediator of necroptosis and, under certain circumstances, apoptosis downstream of TNF family ligands, and it was found to be progressively upregulated in the retina of a STGD1 abca4-/-rdh8-/- (DKO) mouse model coinciding with the onset and progression of photoreceptor degeneration. Despite elevated RIPK3 expression, necroptosis was not detectable in this model, as evidenced by the absence of phosphorylated MLKL and unaltered photoreceptor degeneration in abca4-/- rdh8-/-mlkl-/- mice. Intriguingly, genetic ablation of Ripk3 exacerbated photoreceptor loss in DKO mice in both chronic (age-dependent) and acute (light-induced) retinal degeneration paradigms. This detrimental effect was partially ameliorated by pan-caspase inhibition in the acute degeneration model, indicating caspase-dependent apoptosis as the primary executioner. Mechanistically, we demonstrated that RIPK3 suppressed extrinsic apoptosis by attenuating caspase-8 activation downstream of TNF family ligands. Collectively, our findings reveal a non-canonical, protective role of RIPK3 in photoreceptors, as a brake on apoptotic signaling rather than a necroptotic executor, in the context of STGD1. These findings redefine the role of RIPK3 in retinal degeneration and emphasize the contextual plasticity of cell death regulators in neurodegenerative diseases.

Ferroptosis, an intracellular iron-catalyzed form of programmed cell death (PCD) driven by lipid reactive oxygen species induced membrane damage, is mechanistically uncharacterized in its execution process. Here, we investigated ferroptosis execution in mesenchymal-like ovarian cancer cells treated with ferroptosis inducers ML162 and erastin. We showed that YVAD (a pyroptosis-associated inflammatory caspase inhibitor) and disulfiram (preventing gasdermin pore formation on plasma membrane) deterred ferroptotic cell death. Moreover, we also observed LDH release and IL-1{beta} secretion from ferroptotic cells, suggesting that ferroptosis involves a pore-forming process. Intriguingly, ferroptosis is independent of the canonical inflammasome pathway because caspase-1 is dispensable and not activated upon ferroptosis induction. In contrast, we found that caspase-5 was activated while caspase-4 was not during ferroptosis. In addition, depletion of caspase-5 rendered cells not responding to ferroptosis inducers. Also intriguingly, GSDMD, the well-established caspase-5 substrate, was not involved in ferroptosis. We instead detected GSDME cleavage upon ferroptosis induction and knockdown of GSDME reduced cell death induced by ferroptosis inducers. As caspase-5 activity was necessary for ferroptosis and caspase-5 directly cleaved GSDME, we conclude that the axis of caspase-5/GSDME executes ferroptosis in ovarian cancer cells.

Bacterial genotoxins, Cytolethal Distending Toxin (CDT) and colibactin, cause DNA damage in intoxicated epithelial cells of the host. DNA damage influences {beta}-catenin signaling, altering its stability and nuclear translocation, potentially contributing to cancer development. Using non-transformed hepatocytes and cancer-derived intestinal and hepatic epithelial cell lines, we showed that CDT/CdtB induces the phosphorylation of {beta}-catenin at serine 552, along with the loss of {beta}-catenin from adherens junctions. This leads to the subsequent cytoplasmic accumulation and nuclear translocation of {beta}-catenin, ultimately driving TCF/LEF transcription, the crucial downstream event of Wnt/{beta}-catenin signaling, as well as the transcription of some {beta}-catenin target genes. Colibactin induces similar effects. MK-2206, a direct AKT inhibitor, and metformin, an AMP-activated protein kinase activator that indirectly inhibits AKT, both protected cells against various effects induced by CdtB exposure. These effects include {beta}-catenin phosphorylation at Ser552, the disassembly of cell-cell junctions and the subsequent nuclear accumulation of phosphorylated {beta}-catenin, leading to reduced TCF/LEF-mediated transcription. Additionally, MK-2206 and metformin protected from CdtB-induced epithelial-mesenchymal transition (EMT), including increased nuclear accumulation of SNAIL, enhanced matrix degradation and motility. Overall, these data show that infection with genotoxin-producing bacteria controls some EMT features through {beta}-catenin and AKT-dependent signaling.

Hepatocytes undergo extensive proliferation to facilitate liver repair after injury, yet early adaptive changes prior to proliferation remain unclear. Here, we report that during early acetaminophen (APAP)-induced liver injury, hepatocytes exhibit transient proliferation suppression, most pronounced in mid-zone hepatocytes due to zonal APAP metabolism. Using spatial transcriptomics (ST), immunohistochemistry, and functional studies, we identified a unique mid-zone stress-response program. Central to this adaptation is the Atf4-Chop axis, which actively suppresses proliferation via the cell cycle inhibitor Btg2, prioritizing cytoprotection over cell division. This transient arrest is a critical survival strategy: halting energy-intensive proliferation during peak injury allows mid-zone hepatocytes to redirect resources towards protection, enhancing their survival in early APAP-induced liver injury. Thus, Atf4-Chop-mediated quiescence preserves a hepatocyte reservoir necessary for subsequent regenerative proliferation and effective repair. Our findings reveal a key adaptive trade-off in mid-zone hepatocytes where transient proliferation arrest promotes early survival to enable repair.

Colorectal cancer (CRC) poses a severe global health threat with high incidence, mortality, and poor 5-year survival rates for advanced cases despite existing treatments. This study aims to explore the role of STRIP2 in CRC progression and its underlying mechanisms. Impact of STRIP2 on CRC in vitro was investigated via CRC cell proliferation, migration, invasion, and apoptosis. The in vivo impact was investigated via nude mice models. The role of STRIP2 in CRC was investigated via transcriptomic analysis, Western blot, Co-immunoprecipitation assays and ferroptosis validations. STRIP2 is overexpressed in CRC, driving malignant phenotypes in vitro and in vivo. Mechanically, STRIP2 stabilizes the IL17 downstream effector LCN2 by blocking its K48-linked ubiquitination and degradation, enhances anti-ferroptosis of CRC cells. Oe-STRIP2 suppresses ferroptosis, boosting proliferation and reducing oxidative stress; while si-STRIP2 induces the opposite effect. This study suggests STRIP2-mediated stabilization of LCN2 and enhances CRC cells ferroptosis resistance, thus promoting CRC cell survival and mediates malignant progression in CRC, which provides a novel link between STRIP2 and ferroptosis regulation in CRC. HighlightO_LISTRIP2 is overexpressed in CRC tissues and cells C_LIO_LISTRIP2 blocks LCN2 Ubiquitination and stabilizes LCN2 C_LIO_LISTRIP2 suppresses CRC ferroptosis C_LIO_LISTRIP2 drives CRC malignant phenotypes both in vitro & in vivo C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/725308v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1baf7baorg.highwire.dtl.DTLVardef@1de15d9org.highwire.dtl.DTLVardef@16c8078org.highwire.dtl.DTLVardef@667840_HPS_FORMAT_FIGEXP M_FIG C_FIG

The tight regulation of iron homeostasis is of great importance for cellular health. An increase in intracellular iron levels results in the formation of free radicals, which damages macromolecules and membranes, eventually resulting in cell death by Ferroptosis. Recently, we showed that patients with mutations in VPS41 display a severe neurodegenerative phenotype with iron deposition in the brain. VPS41 is well known as subunit of the HOPS complex required for fusion of late endosomes and autophagosomes with lysosomes. However, VPS41 has also been identified as inhibitor of Ferroptosis and regulator of redox homeostasis. How VPS41 exerts these functions and if these are dependent on the HOPS complex is unknown. Here we show that depletion of VPS41 results in increased intracellular iron levels, ROS formation and mitochondrial fission. Our findings indicate an important role for VPS41 in the regulation of iron homeostasis and mitochondrial fission and suggest Ferroptosis as a possible cause for neurodegeneration in VPS41 patients.

Mitochondrial dysfunction is a central driver of neonatal hypoxic-ischaemic encephalopathy (HIE), yet the specific vulnerabilities of mitochondrial fusion machinery in the neonatal brain remain poorly defined. Here, we investigate Optic Atrophy (OPA)1 as a critical determinant of mitochondrial resilience during hypoxia-ischaemia (HI). Human developmental transcriptomics showed stable perinatal expression of mitochondrial dynamics genes, supporting their potential utility as therapeutic targets at birth. In a neonatal mouse model, HI induced rapid proteolytic processing of OPA1 in whole brain. In vitro, exposure of primary astrocytes to oxygen-glucose deprivation (OGD) mimicked the OPA1 sensitivity observed in whole brain, with aberrant processing and loss of expression. We genetically replicated this observation by knocking down OPA1 expression in primary astrocytes. The predicted mitochondrial fragmentation and impaired bioenergetics was also accompanied by increased vulnerability to hypoxia, revealing an OPA1dependent susceptibility under moderate metabolic stress. Transcriptomics analyses of these cells highlighted an OPA1-mediated depletion of mitochondrial DNA. This mtDNA depletion was also evident in OGD-treated astrocytes and ex vivo brain samples at 24h after HI in our rodent model. In contrast, mild OPA1 overexpression enhanced astrocyte survival following OGD and OPA1 overexpression in vivo markedly reduced tissue damage after neonatal HI. MtDNA levels in OPA1-overexpressing mice before and at 7 days after HI were significantly higher than in wild-type mice. These findings position OPA1 as a key mediator of mitochondrial impairment after HI and to our knowledge, is the first study showing that loss of mtDNA is a consequence of neonatal HI. Our data highlight that maintaining OPA1 expression is a promising therapeutic strategy for protecting the neonatal brain following birth asphyxia.

Programmed cell death is essential for organismal development. When dysregulated, it leads to neurodegenerative diseases and cancer. Although apoptotic pathways are well studied, the role of mitochondrial import translocases in regulating cell death remains unclear. Our study reveals a unique apoptotic pathway controlled by the TIM22 complex, an inner mitochondrial membrane translocase. This pathway involves a multiprotein complex formed by Tim22 and Nde1, a part of the respiratory electron transport chain. Under stress, the cytosol-exposed Nde1 isoform, a pro-apoptotic factor, is stabilised by the TIM22 complex, which includes the Tim18 subunit and Tim22s transmembrane segments. Notably, impairing the TIM22 complex and deleting Nde1 suppresses apoptosis and restores mitochondrial health. Beyond its role in import, our study uncovers a moonlighting function of the TIM22 complex in regulating mitochondria-dependent apoptotic cell death.

Danger-associated molecular patterns (DAMPs), such as high mobility group protein B1 (HMGB1), are released from dying cells, yet the kinetics and functional consequences of their release remain unclear. Using Hmgb1-mCherry transgenic mice and live-cell imaging, we visualize Hmgb1 and interleukin-1{beta} (IL-1{beta}) secretion at single-cell resolution. Hmgb1 exhibits two distinct release kinetics: short-duration completed within 1 min and long duration spanning several minutes. Mathematical modeling demonstrates that short-duration release generates substantially higher local Hmgb1 concentrations and steeper gradients near the dying cell than long-duration release. Such rapid release may enable one or a few dying cells to produce sufficient Hmgb1 to stimulate neighboring cells, consistent with its role as an alarmin. In vivo imaging of cisplatin-induced kidney injury reveals an inverse correlation between intracellular Hmgb1 levels and monocyte infiltration. Together, these findings suggest that Hmgb1 release kinetics shape its extracellular functions during regulated cell death.

TP53 (also called TRP53 or p53) is a critical tumour suppressor that prevents cancer development by inducing a transcriptional program which can lead to diverse cellular responses, most prominently, cell proliferation arrest/senescence with survival of cells or cell death by apoptosis. Why distinct cell types undergo different outcomes after p53 activation remains unclear. Using integrated RNA-sequencing, proteomic and functional analyses across a diverse range of murine primary cell types, we demonstrate that cell fate is governed by the balance between pro-survival BCL-2 and pro-apoptotic BH3-only proteins. Cells resistant to apoptosis displays a higher starting ratio of pro-survival BCL-2 to pro-apoptotic BH3-only proteins, along with transcriptional upregulation of the pro-survival gene Bcl2l1, encoding BCL-XL. This control of cell fate is also seen in human wild-type p53 cancer cell lines. These findings reveal the mechanism for understanding p53-driven cell fate decisions, suggest therapeutic strategies to shift p53-induced cell proliferation arrest/senescence toward apoptotic cell death and allowed generation of an RNAseq data-based predictor of outcome for cancer cells after p53 activation.

Cell replacement represents a potential treatment modality for retinal disorders characterized by photoreceptor loss. However, photoreceptor replacement approaches have not been clinically established. To take this forward, the main goal of this study was to systematically compare human photoreceptors of different ages and identify those that enable functional integration into the degenerative retina. Donor cells were isolated from iPSC-derived retinal organoids generated by a GMP-compliant protocol at differentiation days 120, 150, or 200 and transplanted subretinally into cone photoreceptor function loss 1 (Cpfl1) recipients, an inherited mouse model of cone degeneration. While younger photoreceptors showed slightly improved transplantation outcomes, donor photoreceptors of all culture stages displayed long-term survival, cone identity, structural integration into the host retina, and tight interactions with host Mueller glia, including formation of a continuous outer limiting membrane. Transplanted photoreceptors showed signs of advanced maturation, including correct polarization with generation of apical inner- and outer segments, while basal synapses were formed with host bipolar cells. Electrophysiological assessment of host retinal ganglion cells revealed light-evoked responses in transplant-containing regions, providing evidence for functional incorporation of human photoreceptors into the mouse neuro-retinal circuitry. Thus, GMP-compliant human iPSC-derived photoreceptors are stable over a wide range of differentiation stages and constitute a robust cell source for retinal transplantation and functional repair. The findings provide important prerequisites for the development of standardized procedures towards clinical translation of photoreceptor replacement in the retina.

Mouse models have historically been central to studies of TNF-induced cell death and guided pharmaceutical translation into clinic, based on the assumption that TNF signaling is conserved between human and mouse. Here, our work uncovers critical species-specific differences between the two. By systematically dissecting the roles of RIPK1, TRADD, and sensitivity to RIPK1 inhibitors in TNF signaling--including RIPK1 kinase-dependent and-independent apoptosis--we found that both apoptosis modalities diverge between human and mouse cells. In mouse cells, RIPK1 suppresses TRADD-mediated kinase-independent apoptosis, whereas in human cells, RIPK1 and TRADD act redundantly. Moreover, RIPK1 inhibitors block kinase-dependent apoptosis in mouse but not human cells, despite effectively inhibiting RIPK1 S166 phosphorylation. Cross-species complementation revealed that these discrepancies stem not from RIPK1 itself but from cell-context differences. These findings echo the clinical failures of RIPK1 inhibitors despite efficacy in mouse models and underscore the need for humanized models and therapeutics that more faithfully predict clinical outcomes.

Photoreceptor transplantation is considered a disease-agnostic therapeutic strategy for retinal degenerative diseases with highly heterogenous genetic, molecular, and cellular pathologies. While integration of human photoreceptors enriched from stem cell-derived retinal organoids was noted in previous preclinical studies, the potential influence of retinal degeneration severity on transplantation efficiency has not been systematically assessed. Here, we employed mice presenting mild or severe retinal degeneration as recipients for human induced pluripotent stem cell-derived photoreceptors. Donor cells formed multi-cellular clusters that structurally integrated from 3 weeks post-transplantation (wpt) in mildly degenerated retinas, closely interacting with host Muller glia, resulting in proper maturation characterized by inner/outer segment and synapse formation by 26 wpt. In contrast, in severely degenerated hosts, donor photoreceptors remained mainly singularized and scattered in the subretinal space, showing limited structural integration or signs of maturation. Differential maturation of donor cells in mild vs. severe hosts was confirmed by single-cell RNA-sequencing analysis. However, transplantation at the beginning of the degeneration process of the severe model allowed structural integration and maturation of donor photoreceptors, despite complete loss of endogenous photoreceptors over time. The study thus shows that survival, integration, and maturation of donor photoreceptors depend on the degenerative retinal microenvironment shaping significantly transplantation efficiency.

In cancer cells, septins assemble into enigmatic higher-order structures of 300-700 nanometers, including long needle-like filaments, thick perinuclear rings, and cytoplasmic bundles or aggregates. The absence of genetic or pharmacological tools to recapitulate these architectures in-vitro has impeded mechanistic studies of their formation, function, and therapeutic targeting. Here, first, determining the overexpression of septin-2 in epithelial ovarian cancer (EOC) and its association with increased mortalities and dependencies, we select SKOV-3 ovarian cancer cells as a tractable model in which septin supramolecular assemblies can be recreated in-vitro and interrogated. This system shows that the forchlorfenuron (FCF) analog UR214-9 remodels septin architecture, converting co-expressed human septin octamers (SEPT2-SEPT6-SEPT7-SEPT9-SEPT9-SEPT7-SEPT6-SEPT2) into large cytoplasmic aggregates. In parallel, transiently expressed SEPT2 is reorganized into septin-rich noodle-like filaments, perinuclear rings, and web-like networks encircling the nucleus upon UR214-9 treatment. Mechanistically, UR214-9 disrupts the incorporation of SEPT2, SEPT7, and SEPT9 into canonical septin hetero-octamers, resulting in assembly-defective or imperfect oligomers that preferentially reorganize into these aberrant higher-order structures. This aggregation likely prevents septin-2 migration during interphase-to-cleavage furrow transition in NRK-49F-SEPT2-EGFP homozygous cells and impacts SKOV-3 cytokinesis, cell proliferation, adhesion and invasion and migration while sparing ceramide transport to the Golgi, preserving ER and cis-Golgi structure. These effects manifested in reduced growth of ovarian, endometrial and breast cancer xenografts without attracting significant off-target engagements per the global transcriptomic analysis of JIMT1 breast cancer and PANC-1 pancreatic cells. UR214-9 treated animals showed observable safety in animals. Thus, a tool to recreate aberrant septin structures and identification of septins as a druggable cytoskeletal target for ovarian, endometrial, breast and pancreatic cancer by perturbing their hetero-octamerization assembly is presented. SignificanceWe provide a method to reconstruct the higher-order septin architecture observed in cancer cells, to study their assembly and functions. Intriguingly, cancer cells tolerate hetero-oligomeric septins lacking specific subunits, suggesting that compositionally deficient oligomers are not efficiently targeted for degradation, unlike unincorporated septin monomers in normal cells. This tolerance may enable accumulation of structurally aberrant septin complexes acquiring long-needles, rings or thick-aggregates in disease cells. We further show that septin oligomerization can be pharmacologically perturbed. By integrating structural, cellular, and energetic readouts using in-silico techniques, we establish a quantitative framework for septin-targeted modulation, generating UR214-9 as a new chemotype that disrupts septin oligomeric assembly via preventing incorporation of SEPT2/7/9, into canonical hetero-octamers, causes defects in cytokinesis, altered cell migration, viability, and remodels septin-actin architectures, ultimately impairing tumor cell growth. Thus, pharmacological targeting of septin assembly represents a tractable strategy to perturb septin-dependent cellular processes in cancer and neurodegenerative diseases with reported septin dysregulation.

Astrocytes directly influence neuronal survival and increasingly are understood to contribute to the progression of neurodegenerative diseases including Parkinsons disease (PD). Mitochondrial damage is a hallmark of PD pathology in both neurons and astrocytes. Damaged mitochondria are cleared by PINK1/Parkin-mediated mitophagy; loss-of-function mutations in either PINK1 or Parkin are sufficient to cause PD. Neuronal mitophagy is well-studied, but far less is known about how mitochondrial dysfunction in astrocytes affects neural health. While microglial release of pro-inflammatory cytokines has been shown to induce astrocytes to mount their own inflammatory response, we hypothesize that a more direct pathway is involved, and that mitochondrial damage to astrocytes directly triggers release of proinflammatory cytokines. To address these questions, we treated primary murine cortical astrocytes with oxidative phosphorylation (OXPHOS) inhibitors antimycin A (AA) and oligomycin A (OA) and observed the PINK1-dependent accumulation of Parkin on damaged mitochondria, leading to phospho-ubiquitination of proteins in the outer mitochondrial membrane and the recruitment of the autophagy receptor SQSTM1/p62. To identify transcriptional changes caused by mitochondrial damage and the resulting activation of mitophagic machinery, we performed bulk RNA-sequencing on astrocytes isolated from WT, PINK1-/-, or Parkin-/- mice treated with AA/OA or a vehicle control. In WT astrocytes, TNF- signaling via NF-{kappa}B was the most significantly upregulated pathway following OXPHOS inhibition. OXPHOS inhibitor treatment also stimulated p62 expression, while NF-{kappa}B inhibition prevented this upregulation. Astrocytic secretion of cytokines, including TNF-, was increased following mitochondrial damage; this secretion was dependent on NF-{kappa}B activation and occurred at levels sufficient to induce mitochondrial depolarization in hippocampal neurons. Compared to WT astrocytes, PINK1-/- astrocytes showed a significant reduction in transcriptional signatures associated with TNF- signaling following mitochondrial damage, while Parkin-/- astrocytes exhibited upregulation of both IFN-{gamma} and IFN- signaling. These findings indicate altered inflammatory responses to mitochondrial damage in the absence of functional PINK1 or Parkin. Finally, we analyzed scRNA-sequencing data from substantia nigra astrocytes harvested from human brain tissue from PD-positive or control samples. Distinct clusters comprised predominantly of PD-positive or control astrocytes emerged. Astrocytes in the PD-positive cluster were enriched for NF-{kappa}B, IFN- and IFN-{gamma} responses, consistent with the signaling observed in vitro post-OXPHOS inhibition. Together, these findings identify inflammatory signatures activated by mitochondrial damage in astrocytes, and establish this pathway as a potential contributor to neuroinflammation in PD.

Retinopathy of prematurity (ROP) is caused by hyperoxic exposure of prematurely born infants. The mouse model of oxygen-induced retinopathy (OIR) recapitulates pathological features of both phase I and phase II ROP. We here looked at the retinal proteins that change in response to hyperoxia in phase I of the mouse model of OIR. Using tandem mass tag labeled proteomics, we found several differentially expressed proteins (DEPs) in phase I of OIR. Of all the DEPs, we investigated the role of previously unknown protein NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4-like 2 (NDUFA4L2). NDUFA4L2 protein and its paralog NDUFA4 are both mitochondrial complex I proteins; however, here we demonstrate that NDUFA4L2 changes in both phases of OIR, with no changes in its paralog NDUFA4, implying its unique function in pathophysiology of the disease. We demonstrate that NDUFA4L2 is an oxygen-sensitive protein and regulates retinal endothelial cell migration by rescuing isocitrate dehydrogenase flux impaired by hyperoxia in phase I of OIR.

Proteostatic imbalance caused by CRAF (Raf1) kinase mutation alters MAPK signaling and contributes to RASopathies. CRAF proteostasis is maintained by chaperones, Hsp90 which aids in folding, maturation, and activation; whereas Hsp70 recruits ubiquitin ligase CHIP for proteasomal degradation. However, the proteostasis of disease-associated CRAF mutants remains undefined. Here, we identify HECTD3, a HECT-ubiquitin ligase in promoting degradation of hypertrophic CRAF mutants. Interestingly, increased Hsp90 binding to CRAF plays a determinant factor in degradation via HECTD3. Transient increase in Hsp90-CRAF association upon Hsp90 inhibition promote ubiquitination, whereas prolonged inhibition stabilizes mutant CRAF, indicating temporally distinct chaperone functions. Notably, HECTD3 undergoes proteosomal degradation under stress via Cullin-RING ubiquitin ligase Cul5 in a neddylation-dependent manner. Overexpression of Cul5 thus reduces HECTD3 level resulted in stabilization of CRAFD486N, while promoting degradation of wild-type CRAF. Therefore, modulation of the Cul5-HECTD3 axis alters hypertrophic signaling in cardiomyocyte cells. These results establish a hierarchical proteostasis mechanism in which Hsp90 binding primes substrate for HECTD3-mediated ubiquitination, and Cul5-controlled ligase turnover collectively determine mutant CRAF fate.

Oxidative stress and the progressive degeneration of dopaminergic neurons are key features of Parkinsons disease (PD). The intrinsically disordered structure of the transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2), which coordinates the main cellular antioxidant response of the body, makes it highly susceptible to misfolding and aggregation under severe oxidative stress, compromising cellular survival. Cannabidiol (CBD) has potent neuroprotective properties, but its exact molecular mechanism within the dopaminergic redox environment remains unclear. In this study, we investigated the protective effects of CBD against 6-hydroxydopamine (6-OHDA)-induced toxicity in both undifferentiated and mature, post-mitotic differentiated SH-SY5Y cells. We found that CBD confers robust Nrf2-dependent neuroprotection against 6-OHDA. Importantly, we uncover a previously unexplored mechanism of neuroprotection by which CBD actively prevents the stress-induced sequestration of Nrf2 into insoluble cytoplasmic inclusions under oxidative stress. We find that CBD keeps Nrf2 in a soluble, functional state, increases Ser40 phosphorylation, restores nuclear localization, and drives the robust transcriptional upregulation of antioxidant enzymes. This targeted activation of Nrf2 effectively reduces intracellular ROS, significantly attenuates mitochondrial fragmentation, and decreases aberrant mitophagic activity. Overall, our results show that rather than merely scavenging reactive oxygen species, CBD directly increases Nrf2 activity during oxidative stress, enabling a sustained cytoprotective response. We thus identify CBD as a highly specific, targeted molecule with a high potential for neuroprotective therapy in PD.

Leydig cells (LCs) are essential for male reproductive function due to their role in testosterone synthesis, a process critically dependent on mitochondrial cholesterol transport mediated by the Steroidogenic Acute Regulatory protein (StAR). Despite their importance, LCs are highly sensitive to metabolic and exogenous stressors. Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, has emerged as a key link between cellular metabolism and cell fate; however, its role in LCs and steroidogenesis remains poorly understood. In this study, we investigated the induction of ferroptosis in LCs and its impact on their steroidogenic capacity. We evaluated cellular responses to canonical ferroptosis inducers (Erastin and RSL3) alongside the transcriptional regulation of key genes. Our results demonstrate that LCs are vulnerable to ferroptotic stress, which significantly downregulates Star expression. Notably, we uncovered a novel endocrine-metabolic crosstalk: hormonal stimulation via hCG effectively rescues LCs from Erastin-induced toxicity and fully sustains maximal steroidogenesis. However, this hormone-driven cytoprotection fails against direct GPX4 inhibition by RSL3, indicating an absolute reliance on functional GPX4. These mechanistic findings highlight the paradoxical dual role of ACSL4 in Leydig cell biology and are further supported by bioinformatic analysis of public transcriptomic profiles from infertile patients, which reveal a detrimental imbalance in the ACSL4/GPX4 axis. Together, our data position ferroptosis as a critical disruptor of male endocrine function and reveal a hormone-mediated metabolic adaptation that could inform novel therapeutic strategies against oxidative stress in the testis. Highlights-Leydig cells exhibit a strong vulnerability to ferroptotic cell death. -Ferroptosis disrupts StAR expression and halts Leydig cell steroidogenesis. -hCG signaling promotes metabolic adaptation against Erastin-induced ferroptosis.