Figure 1

<p>A Mechanism through which PINK1 Mutations under Conditions of Increased Oxidative Stress May Sensitize Dopaminergic Neurons to Cytochrome c Release and Subsequent Cell Death, Eventually Causing PD</p

The anticancer peptide RT53 induces immunogenic cell death

<div><p>In recent years, immunogenic cell death (ICD) has emerged as a revolutionary concept in the development of novel anticancer therapies. This particular form of cell death is able, through the spatiotemporally defined emission of danger signals by the dying cell, to induce an effective antitumor immune response, allowing the immune system to recognize and eradicate malignant cells. To date, only a restricted number of chemotherapeutics can trigger ICD of cancer cells. We previously reported that a peptide, called RT53, spanning the heptad leucine repeat region of the survival protein AAC-11 fused to a penetrating sequence, selectively induces cancer cell death <i>in vitro</i> and <i>in vivo</i>. Interestingly, B16F10 melanoma cells treated by RT53 were able to mediate anticancer effects in a tumor vaccination model. Stimulated by this observation, we investigated whether RT53 might mediate ICD of cancer cells. Here, we report that RT53 treatment induces all the hallmarks of immunogenic cell death, as defined by the plasma membrane exposure of calreticulin, release of ATP and the exodus of high-mobility group box 1 protein (HMGB1) from dying cancer cells, through a non-regulated, membranolytic mode of action. In a prophylactic mouse model, vaccination with RT53-treated fibrosarcomas prevented tumor growth at the challenge site. Finally, local intratumoral injection of RT53 into established cancers led to tumor regression together with T-cell infiltration and the mounting of an inflammatory response in the treated animals. Collectively, our results strongly suggest that RT53 can induce <i>bona fide</i> ICD of cancer cells and illustrate its potential use as a novel antitumor and immunotherapeutic strategy.</p></div

RT53 induces unregulated necrotic cell death.

<p>(A) Ultrastructural analysis of RT53-mediated cell death. U2OS cells were left untreated (Control) or exposed to 15 μM of RT53 for 30 min. Cells were then analyzed by transmission electron microscopy following osmium tetroxide staining. (B) U2OS cells were exposed to 20 μM of RT53 in the presence or absence of 50 μM zVAD-fmk, 50 μM Necrostatin-1 (Nec-1) or 100 mM cyclosporin A (CsA) for 1 h. Necrotic cell death was monitored by lactate dehydrogenase (LDH) release from cells into the culture medium. The obtained values were normalized to those of the maximum LDH released (completely lysed) control. Data are means±s.e.m. (<i>n</i> = 3).</p

Intratumoral administration of RT53 induces tumor necrosis, immune cells infiltration and inflammation.

<p>(A) Plasma was harvested 4, 24, 48 or 96 h following single intratumoral injection of normal saline (control) or 300 μg RT53 in normal saline and IL-1β (left panel) as well as IL-6 (right panel) levels were estimated using ELISA. Data from 3 animals are presented for each time point as mean±SEM. (B) Established MCA205 fibrosarcomas were surgically excised 24 h or 96 h post intratumoral injection with normal saline (control) or 300 μg RT53 in normal saline and sections subjected to H&E staining (top) or stained for CD3 or rabbit IgG isotype control (middle and bottom, respectively). (C) Number of infiltrating CD3-positive cells per view field following CD3 staining. Data from 3 animals are presented as mean±SEM. (D) Relative transcription of CCL2 and CXCL10 (normalized to GAPDH) as determined by real-time RT-PCR on samples of tumors 24 h after RT53 or normal saline injection. Values of CCL2 and CXCL10 are represented as fold change relative to untreated tumors, set to 1 (mean±SEM; n = 3).</p

RT53 triggers calreticulin exposure as well as the release of HMGB1 and ATP.

<p>(A) U2OS cells stably expressing CRT-GFP were treated with 10 μM of RT53 or 1 μM mitoxantrone for 6 h, in the presence or absence of zVAD-fmk. Cells were then, fixed, stained for DNA, and examined by fluorescence microscopy. (B) U2OS cells were left untreated or treated with 10 μM of RT53 or 200 nM thapsigargin (TG) as a positive control for 6 h. Cell lysates were analyzed by Western blot for phosphorylated and total protein eIF2α. (C) U2OS cells were left untreated or exposed to increasing concentrations of RT53 for 3h. Extracellular HMGB1 was then measured in the culture supernatant. Data are means±s.e.m. (<i>n</i> = 3). (D) U2OS cells were left untreated or exposed to increasing concentrations of RT53 for 3h. Extracellular ATP was then measured in the culture supernatant. Data are means±s.e.m. (<i>n</i> = 3).</p

Abrogation of respiration suppresses apoptosis and ROS production in a maximal proliferating solid cell population.

<p>(A) Isolated colonies were grown from single cells, manipulated onto agar plates. Clonogenic assay of cells removed from colonies of the wild-type strain, rho⁰ (no mitochondrial DNA) and two single gene-deletion strains (<i>mgm1</i> and <i>oxa1</i>), grown on SCGlu. After 3 days 500 cells of whole colonies, after 5 days 500 cells of the central colony-region were analysed (mean±SEM, <i>n</i> = 2). Statistical significance of p<0.006 compares colony forming units (cfu) of mutant strains to wild-type strain at respective time-points. (B) Cells from the whole colony (3 days) as well as from the central region (5 days) were stained for reactive oxygen species (ROS) with dihydroethidium and analysed by FACSAria flow cytometry (mean±SEM, <i>n</i> = 2; **p<0.01, ***p<0.001 compared to deletion strains at respective time-points). (C) Approximately 50 (2 days) and 10 (3 days) colonies grown on SCGlu plates were washed off and clonogenic assays were performed with the collected cells (mean±SEM, <i>n</i> = 5; **p<0.01, ***p<0.001). (D) Approximately 50 colonies grown on SCGlu plates for 2 days were stained for phosphatidylserine exposition (AnnV) and DNA breakage (TUNEL) and analyzed by FACSAria flow cytometry (mean±SEM, <i>n</i> = 3; **p<0.01, ***p<0.001). (E) Fluorescence microscopy from central and outer region of 5 days old wild-type colony cells, harbouring plasmid dsRED-MLS.</p

An <i>oxa1</i> deletion does not influence the strains growth rate.

<p>(A) Growth curves of an <i>oxa1</i> deletion strain and the corresponding wild-type strain. Experiment was performed in triplicate. The y-axis is scaled logarithmically. (B) Size of isolated colonies of the indicated strains grown for 3 and 5 days on SCGlu plates, respectively. Diameters were evaluated by processing the photos with Metamorph Imaging (mean±SEM, <i>n</i> = 2).</p

Metabolomic analyses reveal that anti-aging metabolites are depleted by palmitate but increased by oleate <i>in vivo</i>

<p>Recently, we reported that saturated and unsaturated fatty acids trigger autophagy through distinct signal transduction pathways. Saturated fatty acids like palmitate (PA) induce autophagic responses that rely on phosphatidylinositol 3-kinase, catalytic subunit type 3 (PIK3C3, best known as VPS34) and beclin 1 (BECN1). Conversely, unsaturated fatty acids like oleate (OL) promote non-canonical, PIK3C3- and BECN1-independent autophagy. Here, we explored the metabolic effects of autophagy-inducing doses of PA and OL in mice. Mass spectrometry coupled to principal component analysis revealed that PA and OL induce well distinguishable changes in circulating metabolites as well as in the metabolic profile of the liver, heart, and skeletal muscle. Importantly, PA (but not OL) causes the depletion of multiple autophagy-inhibitory amino acids in the liver. Conversely, OL (but not PA) increased the hepatic levels of nicotinamide adenine dinucleotide (NAD), an obligate co-factor for autophagy-stimulatory enzymes of the sirtuin family. Moreover, PA (but not OL) raised the concentrations of acyl-carnitines in the heart, a phenomenon that perhaps is linked to its cardiotoxicity. PA also depleted the liver from spermine and spermidine, 2 polyamines have been ascribed with lifespan-extending activity. The metabolic changes imposed by unsaturated and saturated fatty acids may contribute to their health-promoting and health-deteriorating effects, respectively.</p

Neuroprotection by selective neuronal deletion of <i>Atg7</i> in neonatal brain injury

<p>Perinatal asphyxia induces neuronal cell death and brain injury, and is often associated with irreversible neurological deficits in children. There is an urgent need to elucidate the neuronal death mechanisms occurring after neonatal hypoxia-ischemia (HI). We here investigated the selective neuronal deletion of the <i>Atg7</i> (autophagy related 7) gene on neuronal cell death and brain injury in a mouse model of severe neonatal hypoxia-ischemia. Neuronal deletion of <i>Atg</i>7 prevented HI-induced autophagy, resulted in 42% decrease of tissue loss compared to wild-type mice after the insult, and reduced cell death in multiple brain regions, including apoptosis, as shown by decreased caspase-dependent and -independent cell death. Moreover, we investigated the lentiform nucleus of human newborns who died after severe perinatal asphyxia and found increased neuronal autophagy after severe hypoxic-ischemic encephalopathy compared to control uninjured brains, as indicated by the numbers of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3)-, LAMP1 (lysosomal-associated membrane protein 1)-, and CTSD (cathepsin D)-positive cells. These findings reveal that selective neuronal deletion of <i>Atg7</i> is strongly protective against neuronal death and overall brain injury occurring after HI and suggest that inhibition of HI-enhanced autophagy should be considered as a potential therapeutic target for the treatment of human newborns developing severe hypoxic-ischemic encephalopathy.</p

Model of MetR-mediated longevity.

<p>MetR specifically enhances autophagy, either by interfering upstream of TOR-pathway or presumably by impinging on (metabolic) pathways that potentially target autophagy directly, downstream of the TOR-pathway. MetR-specific vacuolar acidification is dependent on autophagy and elongates CLS. High levels of methionine inhibit autophagy induction during early phases of chronological aging, enhancing ROS and diminishing acidic vacuoles in a cell population, which leads to cell death.</p