Aberrant miR-29 is a predictive feature of severe phenotypes in pediatric Crohn's disease.

Crohn's disease (CD) is a chronic inflammatory gut disorder. Molecular mechanisms underlying the clinical heterogeneity of CD remain poorly understood. MicroRNAs (miRNAs) are important regulators of gut physiology, and several have been implicated in the pathogenesis of adult CD. However, there is a dearth of large-scale miRNA studies for pediatric CD. We hypothesized that specific miRNAs uniquely mark pediatric CD. We performed small RNA-Seq of patient-matched colon and ileum biopsies from treatment-naive pediatric patients with CD (n = 169) and a control cohort (n = 108). Comprehensive miRNA analysis revealed 58 miRNAs altered in pediatric CD. Notably, multinomial logistic regression analysis revealed that index levels of ileal miR-29 are strongly predictive of severe inflammation and stricturing. Transcriptomic analyses of transgenic mice overexpressing miR-29 show a significant reduction of the tight junction protein gene Pmp22 and classic Paneth cell markers. The dramatic loss of Paneth cells was confirmed by histologic assays. Moreover, we found that pediatric patients with CD with elevated miR-29 exhibit significantly lower Paneth cell counts, increased inflammation scores, and reduced levels of PMP22. These findings strongly indicate that miR-29 upregulation is a distinguishing feature of pediatric CD, highly predictive of severe phenotypes, and associated with inflammation and Paneth cell loss.

Discriminating Between Apoptosis, Necrosis, Necroptosis, and Ferroptosis by Microscopy and Flow Cytometry.

Apoptosis is a mode of programmed cell death that plays important roles in tissue sculpting during development, in the maintenance of tissue homeostasis in the adult, and in the eradication of injured or infected cells during pathological processes. Numerous physiological as well as pathological stimuli trigger apoptosis, such as engagement of plasma-membrane-associated Fas, TRAIL, or TNF receptors, growth factor deprivation, hypoxia, radiation, and exposure to diverse cytotoxic drugs. Apoptosis is coordinated by members of the caspase family of cysteine proteases, which, upon activation, trigger a series of dramatic morphological and biochemical changes including retraction from the substratum, cell shrinkage, extensive and protracted plasma membrane blebbing, chromatin condensation, DNA hydrolysis, nuclear fragmentation, and proteolytic cleavage of numerous caspase substrates. These dramatic structural and biochemical alterations result not only in the controlled dismantling of the cell, but also in the rapid recognition and removal of apoptotic cells by phagocytes through the cell surface display of phagocytotic triggers such as phosphatidylserine. Necrosis, which is typically nonprogrammed or imposed upon the cell by overwhelming membrane or organelle damage, is characterized by high-amplitude cell swelling, followed by rapid plasma membrane rupture and release of cellular contents into the extracellular space. Necrosis is often provoked by infectious agents or severe departure from physiological conditions due to toxins, temperature extremes, or physical injury. However, forms of programmed necrosis (necroptosis, pyroptosis, ferroptosis) can also occur in specific circumstances. Nonprogrammed and programmed necrosis can be distinguished from apoptosis by morphological features, based on the rapid uptake of vital dyes, and through the application of specific inhibitors of key molecules associated with the latter modes of cell death. This unit describes protocols for the measurement of apoptosis and necrosis and for distinguishing apoptosis from programmed as well as conventional necrosis. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Analysis of cell morphology by phase-contrast microscopy Alternative Protocol 1: Assessment of morphological changes using eosin-methylene blue staining Alternative Protocol 2: Analysis of nuclear morphology by fluorescence microscopy Support Protocol: Preparation of cytospins Basic Protocol 2: Measurement of plasma membrane composition with annexin V and propidium iodide Basic Protocol 3: Measurement of DNA fragmentation by flow cytometry Alternative Protocol 3: Analysis of DNA fragmentation by the TUNEL assay Basic Protocol 4: Measurement of caspase activation by flow cytometry Basic Protocol 5: Discriminating between apoptosis, necrosis, necroptosis, and ferroptosis.

Constitutive High Expression of NOXA Sensitizes Human Embryonic Stem Cells for Rapid Cell Death.

Human embryonic stem (hES) cells are highly sensitive to apoptotic stimuli such as DNA damage, which allows for the rapid elimination of mutated cells during development. However, the mechanisms that maintain hES cells in the primed apoptotic state are not completely known. Key activators of apoptosis, the BH3-only proteins, are present at low levels in most cell types. In contrast, hES cells have constitutive high levels of the BH3-only protein, NOXA. We examined the importance of NOXA for enabling apoptosis in hES cells. hES cells deleted for NOXA showed remarkable protection against multiple apoptotic stimuli. NOXA was constitutively localized to the mitochondria, where it interacted with MCL1. Strikingly, inhibition of MCL1 in NOXA knockout cells was sufficient to sensitize these cells to DNA damage-induced cell death. Our study demonstrates that an essential function of constitutive high levels of NOXA in hES cells is to effectively antagonize MCL1 to permit rapid apoptosis.

MicroRNA-29 is an essential regulator of brain maturation through regulation of CH methylation.

Although embryonic brain development and neurodegeneration have received considerable attention, the events that govern postnatal brain maturation are less understood. Here, we identify the miR-29 family to be strikingly induced during the late stages of brain maturation. Brain maturation is associated with a transient, postnatal period of de novo non-CG (CH) DNA methylation mediated by DNMT3A. We examine whether an important function of miR-29 during brain maturation is to restrict the period of CH methylation via its targeting of Dnmt3a. Deletion of miR-29 in the brain, or knockin mutations preventing miR-29 to specifically target Dnmt3a, result in increased DNMT3A expression, higher CH methylation, and repression of genes associated with neuronal activity and neuropsychiatric disorders. These mouse models also develop neurological deficits and premature lethality. Our results identify an essential role for miR-29 in restricting CH methylation in the brain and illustrate the importance of CH methylation regulation for normal brain maturation.

Characterization of a Cul9-Parkin double knockout mouse model for Parkinson's disease.

Mitochondrial quality control is essential for the long-term survival of postmitotic neurons. The E3 ubiquitin ligase Parkin promotes the degradation of damaged mitochondria via mitophagy and mutations in Parkin are a major cause of early-onset Parkinson's disease (PD). Surprisingly however, mice deleted for Parkin alone are rather asymptomatic for PD-related pathology, suggesting that other complementary or redundant mitochondrial quality control pathways may exist in neurons. Mitochondrial damage is often accompanied by the release of toxic proteins such as cytochrome c. We have reported that once in the cytosol, cytochrome c is targeted for degradation by the E3 ligase CUL9 in neurons. Here we examined whether CUL9 and Parkin cooperate to promote optimal neuronal survival in vivo. We generated mice deficient for both Cul9 and Parkin and examined them for PD-related phenotypes. Specifically, we conducted assays to examine behavioural deficits (locomotor, sensory, memory and learning) and loss of dopaminergic neurons in both males and females. Our results show that the loss of Cul9 and Parkin together did not enhance the effect of Parkin deficiency alone. These results indicate that while both Parkin and CUL9 participate in mitochondrial quality control, neurons likely have multiple redundant mechanisms to ensure their long-term survival.

Apoptotic cell death regulation in neurons.

Apoptosis plays a major role in shaping the developing nervous system during embryogenesis as neuronal precursors differentiate to become post-mitotic neurons. However, once neurons are incorporated into functional circuits and become mature, they greatly restrict their capacity to die via apoptosis, thus allowing the mature nervous system to persist in a healthy and functional state throughout life. This robust restriction of the apoptotic pathway during neuronal differentiation and maturation is defined by multiple unique mechanisms that function to more precisely control and restrict the intrinsic apoptotic pathway. However, while these mechanisms are necessary for neuronal survival, mature neurons are still capable of activating the apoptotic pathway in certain pathological contexts. In this review, we highlight key mechanisms governing the survival of post-mitotic neurons, while also detailing the physiological and pathological contexts in which neurons are capable of overcoming this high apoptotic threshold.

Differential role of FL-BID and t-BID during verotoxin-1-induced apoptosis in Burkitt's lymphoma cells.

The globotriaosylceramide Gb3 is a glycosphingolipid expressed on a subpopulation of germinal center B lymphocytes which has been recognized as the B cell differentiation antigen CD77. Among tumoral cell types, Gb3/CD77 is strongly expressed in Burkitt's lymphoma (BL) cells as well as other solid tumors including breast, testicular and ovarian carcinomas. One known ligand of Gb3/CD77 is Verotoxin-1 (VT-1), a Shiga toxin produced in specific E. coli strains. Previously, we have reported that in BL cells, VT-1 induces apoptosis via a caspase-dependent and mitochondria-dependent pathway. Yet, the respective roles of various apoptogenic factors remained to be deciphered. Here, this apoptotic pathway was found to require cleavage of the BID protein by caspase-8 as well as activation of two other apoptogenic proteins, BAK and BAX. Surprisingly however, t-BID, the truncated form of BID resulting from caspase-8 cleavage, played no role in the conformational changes of BAK and BAX. Rather, their activation occurred under the control of full length BID (FL-BID). Indeed, introducing a non-cleavable form of BID (BID-D59A) into BID-deficient BL cells restored BAK and BAX activation following VT-1 treatment. Still, t-BID was involved along with FL-BID in the BAK-dependent and BAX-dependent cytosolic release of CYT C and SMAC/DIABLO from the mitochondrial intermembrane space: FL-BID was found to control the homo-oligomerization of both BAK and BAX, likely contributing to the initial release of CYT C and SMAC/DIABLO, while t-BID was needed for their hetero-oligomerization and ensuing release amplification. Together, our results reveal a functional cooperation between BAK and BAX during VT-1-induced apoptosis and, unexpectedly, that activation of caspase-8 and production of t-BID were not mandatory for initiation of the cell death process.

Physiological functions of non-apoptotic caspase activity in the nervous system.

Caspases are cysteine proteases that play important and well-defined roles in apoptosis and inflammation. Increasing evidence point to alternative functions of caspases where restricted and localized caspase activation within neurons allows for a variety of non-apoptotic and non-inflammatory processes required for brain development and function. In this review, we highlight sublethal caspase functions in axon and dendrite pruning, neurite outgrowth and dendrite branches formation, as well as in long-term depression and synaptic plasticity. Importantly, as non-apoptotic activity of caspases is often confined in space and time in neurons, we also discuss the mechanisms that restrict caspase activity in order to maintain the neuronal networks in a healthy and functional state.

Measuring Apoptosis by Microscopy and Flow Cytometry.

Apoptosis is a mode of programmed cell death that plays an important role during development and in the maintenance of tissue homeostasis. Numerous physiological as well as pathological stimuli trigger apoptosis such as engagement of Fas, TRAIL, or TNF receptors, growth factor deprivation, hypoxia, or exposure to cytotoxic drugs. Apoptosis is coordinated from within by members of the caspase family of cysteine proteases that, upon activation, trigger a series of morphological changes including cell shrinkage, extensive plasma membrane blebbing, chromatin condensation, DNA hydrolysis, and nuclear fragmentation. These dramatic structural and biochemical alterations result not only in the controlled dismantling of the cell, but also in the efficient recognition and removal of apoptotic cells by phagocytes. Necrosis, which is typically nonprogrammed or imposed upon the cell by overwhelming membrane or organelle damage, is characterized by rapid plasma membrane rupture followed by organelle and cell swelling. Necrosis is often provoked by infectious agents or a severe departure from physiological conditions. This unit describes protocols for the measurement of apoptosis and for distinguishing apoptosis from necrosis.

Parkin sensitizes toward apoptosis induced by mitochondrial depolarization through promoting degradation of Mcl-1.

Mitochondrial depolarization promotes Parkin- and PTEN-induced kinase 1 (PINK1)-dependent polyubiquitination of multiple proteins on mitochondrial outer membranes, resulting in the removal of defective mitochondria via mitophagy. Because Parkin mutations occur in Parkinson's disease, a condition associated with the death of dopaminergic neurons in the midbrain, wild-type Parkin is thought to promote neuronal survival. However, here we show that wild-type Parkin greatly sensitized toward apoptosis induced by mitochondrial depolarization but not by proapoptotic stimuli that failed to activate Parkin. Parkin-dependent apoptosis required PINK1 and was efficiently blocked by prosurvival members of the Bcl-2 family or knockdown of Bax and Bak. Upon mitochondrial depolarization, the Bcl-2 family member Mcl-1 underwent rapid Parkin- and PINK1-dependent polyubiquitination and degradation, which sensitized toward apoptosis via opening of the Bax/Bak channel. These data suggest that similar to other sensors of cell stress, such as p53, Parkin has cytoprotective (mitophagy) or cytotoxic modes (apoptosis), depending on the degree of mitochondrial damage.

Bcl-2 family proteins participate in mitochondrial quality control by regulating Parkin/PINK1-dependent mitophagy.

Mitophagy facilitates the selective elimination of impaired or depolarized mitochondria through targeting the latter to autophagosomes. Parkin becomes localized to depolarized mitochondria in a PINK1-dependent manner and polyubiquitinates multiple mitochondrial outer membrane proteins. This permits ubiquitin-binding proteins (e.g., p62 and NBR1) to target impaired mitochondria to autophagosomes via Atg8/LC3II. Bcl-2 family proteins regulate mitochondrial outer membrane permeabilization during apoptosis and can also influence macroautophagy via interactions with Beclin-1. Here, we show that Parkin-dependent mitophagy is antagonized by prosurvival members of the Bcl-2 family (e.g., Bcl-xL and Mcl-1) in a Beclin-1-independent manner. Bcl-2 proteins suppressed mitophagy through inhibition of Parkin translocation to depolarized mitochondria. Consistent with this, Parkin translocation to mitochondria was enhanced by BH3-only proteins or a BH3-only mimetic. Taken together with their role as regulators of apoptosis-associated mitochondrial permeabilization, as well as mitochondrial fission/fusion dynamics, this suggests that Bcl-2 family proteins act as global regulators of mitochondrial homeostasis.

Measuring apoptosis by microscopy and flow cytometry.

Apoptosis is a programmed mode of cell death that is accompanied by numerous morphological as well as biochemical changes to the cellular architecture. This results not only in cell death but also in the efficient removal of apoptotic cells by phagocytes. Apoptotic cells display a range of common characteristics that include cell shrinkage, plasma membrane blebbing, cell detachment, nuclear condensation, DNA fragmentation, externalization of phosphatidylserine (PS) and activation of caspases. In contrast, necrotic cell death is characterised by rapid plasma membrane, organelle swelling and plasma membrane rupture with none of the features of apoptosis. Apart from severe physical stresses, necrotic cell death often betrays the activities of viral infection and the activities of bacterial toxins. While necrotic cell death is characterized by the release of endogenous 'danger signals' and subsequent inflammation, apoptosis is largely tolergenic. Therefore, care must be taken when assessing whether cells are dying via apoptosis or necrosis. Here, we highlight a number of assays, utilizing both microscopy and flow cytometry, to determine whether cells have undergone apoptosis or alternative modes of cell death.

Greasing the path to BAX/BAK activation.

BAX/BAK activation leading to mitochondrial outer-membrane permeabilization is a key commitment point in apoptosis. Chipuk et al. now identify two sphingolipids as specific cofactors for BAX/BAK activation that lower the threshold for apoptosis-associated cytochrome c release. Association of mitochondria with other cellular membrane compartments is required for BAK/BAX exposure to these sphingolipids.

Skin tumors induced by sorafenib; paradoxic RAS-RAF pathway activation and oncogenic mutations of HRAS, TP53, and TGFBR1.

PURPOSE: The emergence of skin tumors in patients treated with sorafenib or with more recent BRAF inhibitors is an intriguing and potentially serious event. We carried out a clinical, pathologic, and molecular study of skin lesions occurring in patients receiving sorafenib. EXPERIMENTAL DESIGN: Thirty-one skin lesions from patients receiving sorafenib were characterized clinically and pathologically. DNA extracted from the lesions was screened for mutation hot spots of HRAS, NRAS, KiRAS, TP53, EGFR, BRAF, AKT1, PI3KCA, TGFBR1, and PTEN. Biological effect of sorafenib was studied in vivo in normal skin specimen and in vitro on cultured keratinocytes. RESULTS: We observed a continuous spectrum of lesions: from benign to more inflammatory and proliferative lesions, all seemingly initiated in the hair follicles. Eight oncogenic HRAS, TGFBR1, and TP53 mutations were found in 2 benign lesions, 3 keratoacanthomas (KA) and 3 KA-like squamous cell carcinoma (SCC). Six of them correspond to the typical UV signature. Treatment with sorafenib led to an increased keratinocyte proliferation and a tendency toward increased mitogen-activated protein kinase (MAPK) pathway activation in normal skin. Sorafenib induced BRAF-CRAF dimerization in cultured keratinocytes and activated CRAF with a dose-dependent effect on MAP-kinase pathway activation and on keratinocyte proliferation. CONCLUSION: Sorafenib induces keratinocyte proliferation in vivo and a time- and dose-dependent activation of the MAP kinase pathway in vitro. It is associated with a spectrum of lesions ranging from benign follicular cystic lesions to KA-like SCC. Additional and potentially preexisting somatic genetic events, like UV-induced mutations, might influence the evolution of benign lesions to more proliferative and malignant tumors.

Caspase-8-mediated cleavage of Bid and protein phosphatase 2A-mediated activation of Bax are necessary for Verotoxin-1-induced apoptosis in Burkitt's lymphoma cells.

Verotoxin (VT-1) is a cytotoxin, produced by Shigella dysenteriae type 1 or by Shiga toxin-producing Escherichia coli, which binds specifically to globotriaosylceramide (Gb3). This glycosphingolipid is a B cell differentiation antigen (Gb3/CD77) strongly expressed on Burkitt's lymphoma cells. We have previously shown that, in these cells, VT-1 induces apoptosis via a caspase- and mitochondria-dependent pathway. In this report, we provide new insights into this signal transduction pathway. First, we demonstrate that VT-1-induced apoptosis requires degradation of the caspase-8 inhibitory molecule c-FLIPL and that this degradation occurs through the ubiquitin-proteasome pathway. Furthermore, we show that mitochondrial activation is mainly due to i) cleavage and activation of the pro-apoptotic Bcl-2 family member Bid by caspase-8 and ii) Bax relocalization to mitochondrial membranes which lead to cytochrome c release. However, tBid is not involved in Bax relocalization, and relocalization is most likely controlled by the extent of Bax phosphorylation: in non-treated BL cells, p38 MAPK participates in the retention of Bax in the cytoplasm in an inactive form whereas in VT-1 treated cells, protein phosphatase 2A is activated and induces Bax relocalization to mitochondria.

Structural basis of the preferential binding for globo-series glycosphingolipids displayed by Pseudomonas aeruginosa lectin I.

The opportunistic pathogen Pseudomonas aeruginosa contains several carbohydrate-binding proteins, among which is the P. aeruginosa lectin I (PA-IL), which displays affinity for alpha-galactosylated glycans. Glycan arrays were screened and demonstrated stronger binding of PA-IL toward alphaGal1-4betaGal-terminating structures and weaker binding to alphaGal1-3betaGal ones in order to determine which human glycoconjugates could play a role in the carbohydrate-mediated adhesion of the bacteria. This was confirmed in vivo by testing the binding of the lectin to Burkitt lymphoma cells that present large amounts of globotriaosylceramide antigen Gb3/CD77/P(k). Trisaccharide moieties of Gb3 (alphaGal1-4betaGal1-4Glc) and isoglobotriaosylceramide (alphaGal1-3betaGal1-4Glc) were tested by titration microcalorimetry, and both displayed similar affinity to PA-IL in solution. The crystal structure of PA-IL complexed to alphaGal1-3betaGal1-4Glc trisaccharide has been solved at 1.9-A resolution and revealed how the second galactose residue makes specific contacts with the protein surface. Molecular modeling studies were performed in order to compare the binding mode of PA-IL toward alphaGal1-3Gal with that toward alphaGal1-4Gal. Docking studies demonstrated that alphaGal1-4Gal creates another network of contacts for achieving a very similar affinity, and 10-ns molecular dynamics in explicit water allowed for analyzing the flexibility of each disaccharide ligand in the protein binding site. The higher affinity observed for binding to Gb3 epitope, both in vivo and on glycan array, is likely related to the presentation effect of the oligosaccharide on a surface, since only the Gb3 glycosphingolipid geometry is fully compatible with parallel insertion of neighboring trisaccharide heads in two binding sites of the same tetramer of PA-IL.

Truncated form of the Epstein-Barr virus protein EBNA-LP protects against caspase-dependent apoptosis by inhibiting protein phosphatase 2A.

The Epstein-Barr virus (EBV)-encoded leader protein, EBNA-LP, strongly activates the EBNA2-mediated transcriptional activation of cellular and viral genes and is therefore important for EBV-induced B-cell transformation. However, a truncated form of EBNA-LP is produced in cells infected with variant EBV strains lacking EBNA2 due to a genetic deletion. The function of this truncated form is unknown. We show here that some Burkitt's lymphoma cells harboring defective EBV strains are specifically resistant to the caspase-dependent apoptosis induced by verotoxin 1 (VT-1) or staurosporine. These cells produced low-molecular-weight Y1Y2-truncated isoforms of EBNA-LP, which were partly localized in the cytoplasm. The transfection of sensitive cells with constructs encoding truncated EBNA-LP isoforms, but not full-length EBNA-LP, induced resistance to caspase-mediated apoptosis. Furthermore, VT-1 induced protein phosphatase 2A (PP2A) activation in sensitive cells but not in resistant cells, in which the truncated EBNA-LP interacted with this protein. Thus, the resistance to apoptosis observed in cells harboring defective EBV strains most probably results from the inactivation of PP2A via interactions with low-molecular-weight Y1Y2-truncated EBNA-LP isoforms.