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Ferroptosis is a form of programmed cell death that is pathogenic to several acute and chronic diseases and executed via oxygenation of polyunsaturated phosphatidylethanolamines (PE) by 15-lipoxygenases (15-LO) that normally use free polyunsaturated fatty acids as substrates. Mechanisms of the altered 15-LO substrate specificity are enigmatic. We sought a common ferroptosis regulator for 15LO. We discovered that PEBP1, a scaffold protein inhibitor of protein kinase cascades, complexes with two 15LO isoforms, 15LO1 and 15LO2, and changes their substrate competence to generate hydroperoxy-PE. Inadequate reduction of hydroperoxy-PE due to insufficiency or dysfunction of a selenoperoxidase, GPX4, leads to ferroptosis. We demonstrated the importance of PEBP1-dependent regulatory mechanisms of ferroptotic death in airway epithelial cells in asthma, kidney epithelial cells in renal failure, and cortical and hippocampal neurons in brain trauma. As master regulators of ferroptotic cell death with profound implications for human disease, PEBP1/15LO complexes represent a new target for drug discovery.

Abstract The Receptor for Advanced Glycation Endproducts [RAGE] is an evolutionarily recent member of the immunoglobulin super-family, encoded in the Class III region of the major histocompatability complex. RAGE is highly expressed only in the lung at readily measurable levels but increases quickly at sites of inflammation, largely on inflammatory and epithelial cells. It is found either as a membrane-bound or soluble protein that is markedly upregulated by stress in epithelial cells, thereby regulating their metabolism and enhancing their central barrier functionality. Activation and upregulation of RAGE by its ligands leads to enhanced survival. Perpetual signaling through RAGE-induced survival pathways in the setting of limited nutrients or oxygenation results in enhanced autophagy, diminished apoptosis, and (with ATP depletion) necrosis. This results in chronic inflammation and in many instances is the setting in which epithelial malignancies arise. RAGE and its isoforms sit in a pivotal role, regulating metabolism, inflammation, and epithelial survival in the setting of stress. Understanding the molecular structure and function of it and its ligands in the setting of inflammation is critically important in understanding the role of this receptor in tumor biology.

Gas cluster ion beam-secondary ion mass spectrometry (GCIB-SIMS) has shown the full potential of mapping intact lipids in biological systems with better than 10 μm lateral resolution. This study investigated further the capability of GCIB-SIMS in imaging high-mass signals from intact cardiolipin (CL) and gangliosides in normal brain and the effect of a controlled cortical impact model (CCI) of traumatic brain injury (TBI) on their distribution. A combination of enzymatic and chemical treatments was employed to suppress the signals from the most abundant phospholipids (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)) and enhance the signals from the low-abundance CLs and gangliosides to allow their GCIB-SIMS detection at 8 and 16 μm spatial resolution. Brain CLs have not been observed previously using other contemporary imaging mass spectrometry techniques at better than 50 μm spatial resolution. High-resolution images of naive and injured brain tissue facilitated the comparison of CL species across three multicell layers in the CA1, CA3, and DG regions of the hippocampus. GCIB-SIMS also reliably mapped losses of oxidizable polyunsaturated CL species (but not the oxidation-resistant saturated and monounsaturated gangliosides) to regions including the CA1 and CA3 of the hippocampus after CCI. This work extends the detection range for SIMS measurements of intact lipids to above m/z 2000, bridging the mass range gap compared with MALDI. Further advances in high-resolution SIMS of CLs, with the potential for single cell or supra-cellular imaging, will be essential for the understanding of CL's functional and structural organization in normal and injured brain. [ABSTRACT FROM AUTHOR]

Lamin B1 is a component of the nuclear lamina and plays a critical role in maintaining nuclear architecture, regulating gene expression and modulating chromatin positioning. We have previously shown that LMNB1 gene duplications cause autosomal dominant leukodystrophy (ADLD), a fatal adult onset demyelinating disease. The mechanisms by which increasedLMNB1levels causeADLDare unclear. To address this, we used a transgenic mouse model where Lamin B1 overexpression is targeted to oligodendrocytes. These mice showed severe vacuolar degeneration of the spinal cord white matter together with marked astrogliosis, microglial infiltration, and secondary axonal damage. Oligodendrocytes in the transgenic mice revealed alterations in histone modifications favoring a transcriptionally repressed state. Chromatin changes were accompanied by reduced expression of genes involved in lipid synthesis pathways, many of which are known to play important roles in myelin regulation and are preferentially expressed in oligodendrocytes. Decreased lipogenic gene expression resulted in a significant reduction in multiple classes of lipids involved in myelin formation. Many of these gene expression changes and lipid alterations were observed even before the onset of the phenotype, suggesting a causal role. Our findings establish, for the first time, a link between LMNB1 and lipid synthesis in oligodendrocytes, and provide a mechanistic framework to explain the age dependence and white matter involvement of the disease phenotype. These results have implications for disease pathogenesis and may also shed light on the regulation of lipid synthesis pathways in myelin maintenance and turnover. [ABSTRACT FROM AUTHOR]

Dysregulations of epithelial-immune interactions frequently culminate in chronic inflammatory diseases of the skin, lungs, kidneys, and gastrointestinal tract. Yet, the intraepithelial processes which initiate and perpetuate inflammation in these organs are poorly understood. Here, by utilizing redox lipidomics we identified ferroptosis-associated peroxidation of polyunsaturated phosphatidylethanolamines in the epithelia of patients with asthma, cystic fibrosis, psoriasis and renal failure. Focusing on psoriasis as a disease model, we used high-resolution mass spectrometry imaging and identified keratin 14 (K14)-expressing keratinocytes executing a ferroptotic death program in human psoriatic skin. Psoriatic phenotype with characteristic Th1/Th17 skin and extracutaneous immune responses was initiated and maintained in a murine model designed to actuate ferroptosis in a fraction of K14+ glutathione peroxidase 4 (Gpx4)-deficient epidermal keratinocytes. Importantly, an anti-ferroptotic agent, Liproxstatin-1, was as effective as clinically relevant biologic IL-12/IL-23/TNFα-targeting therapies or the depletion of T cells in completely abrogating molecular, biochemical and morphologic features of psoriasis. As ferroptosis in select epidermal keratinocytes triggers and sustains a pathologic psoriatic multi-organ inflammatory circuit, we suggest that strategies targeting ferroptosis, or its causes, may be effective in preventing or ameliorating a variety of chronic inflammatory diseases.

The temporo-spatial organization of different cells in the tumor microenvironment (TME) is the key to understanding their complex communication networks and the immune landscape that exists within compromised tissues. Multi-omics profiling of single-interacting cells in the native TME is critical for providing further information regarding the reprograming mechanisms leading to immunosuppression and tumor progression. This requires new technologies for biomolecular profiling of phenotypically heterogeneous cells on the same tissue sample. Here, we developed a new methodology for comprehensive lipidomic and metabolomic profiling of individual cells on frozen-hydrated tissue sections using water gas cluster ion beam secondary ion mass spectrometry ((H 2 O) n -GCIB-SIMS) (at 1.6 μm beam spot size), followed by profiling cell-type specific lanthanide antibodies on the same tissue section using C 60 -SIMS (at 1.1 μm beam spot size). We revealed distinct variations of distribution and intensities of >150 key ions (e.g., lipids and important metabolites) in different types of the TME individual cells, such as actively proliferating tumor cells as well as infiltrating immune cells. The demonstrated feasibility of SIMS imaging to integrate the multi-omics profiling in the same tissue section at the single-cell level will lead to new insights into the role of lipid reprogramming and metabolic response in normal regulation or pathogenic discoordination of cell-cell interactions in a variety of tissue microenvironments.

Millions of diverse molecules constituting the lipidome act as important signals within cells. Of these, cardiolipin (CL) and phosphatidylethanolamine (PE) participate in apoptosis and ferroptosis, respectively. Their subcellular distribution is largely unknown. Imaging mass spectrometry is capable of deciphering the spatial distribution of multiple lipids at subcellular levels. Here we report the development of a unique 70 keV gas-cluster ion beam that consists of (CO 2 ) n + (n>10 000) projectiles. Coupled with direct current beam buncher-time-of-flight secondary-ion mass spectrometry, it is optimized for sensitivity towards high-mass species (up to m/z 3000) at high spatial resolution (1 μm). In combination with immunohistochemistry, phospholipids, including PE and CL, have been assessed in subcellular compartments of mouse hippocampal neuronal cells and rat brain tissue., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Damage of presynaptic mitochondria could result in release of proapoptotic factors that threaten the integrity of the entire neuron. We discovered that alpha-synuclein (Syn) forms a triple complex with anionic lipids (such as cardiolipin) and cytochrome c, which exerts a peroxidase activity. The latter catalyzes covalent hetero-oligomerization of Syn with cytochrome c into high molecular weight aggregates. Syn is a preferred substrate of this reaction and is oxidized more readily than cardiolipin, dopamine, and other phenolic substrates. Co-localization of Syn with cytochrome c was detected in aggregates formed upon proapoptotic stimulation of SH-SY5Y and HeLa cells and in dopaminergic substantia nigra neurons of rotenone-treated rats. Syn-cardiolipin exerted protection against cytochrome c-induced caspase-3 activation in a cell-free system, particularly in the presence of H(2)O(2). Direct delivery of Syn into mouse embryonic cells conferred resistance to proapoptotic caspase-3 activation. Conversely, small interfering RNA depletion of Syn in HeLa cells made them more sensitive to dopamine-induced apoptosis. In human Parkinson disease substantia nigra neurons, two-thirds of co-localized Syn-cytochrome c complexes occurred in Lewy neurites. Taken together, these results indicate that Syn may prevent execution of apoptosis in neurons through covalent hetero-oligomerization of cytochrome c. This immediate protective function of Syn is associated with the formation of the peroxidase complex representing a source of oxidative stress and postponed damage.

Bone marrow-derived dendritic cells engineered using recombinant adenovirus to secrete high levels of IL-12p70 dramatically inhibited the growth of established CMS4 sarcomas in BALB/c mice after intratumoral administration. An analysis of splenic CD8(+) T cells in regressor mice revealed a strong, complex reactivity pattern against high-performance liquid chromatography (HPLC)-resolved peptides isolated by acid elution from single-cell suspensions of surgically resected CMS4 lesions. Mass spectrometry analyses defined two major overlapping peptide species that derive from the murine hemoglobin-beta (HBB) protein within the most stimulatory HPLC fractions. Although cultured CMS4 tumor cells failed to express HBB mRNA based on reverse transcription-PCR analyses, prophylactic vaccination of BALB/c mice with vaccines containing HBB peptides promoted specific CD8(+) T-cell responses that protected mice against a subsequent challenge with CMS4 or unrelated syngeneic (HBB(neg)) tumors of divergent histology (sarcoma, carcinomas of the breast or colon). In situ imaging suggested that vaccines limit or destabilize tumor-associated vascular structures, potentially by promoting immunity against HBB+ vascular pericytes. Importantly, there were no untoward effects of vaccination with the HBB peptide on peripheral RBC numbers, RBC hemoglobin content, or vascular structures in the brain or eye.