GAK and PRKCD kinases regulate basal mitophagy.

The removal of mitochondria in a programmed or stress-induced manner is essential for maintaining cellular homeostasis. To date, much research has focused upon stress-induced mitophagy that is largely regulated by the E3 ligase PRKN, with limited insight into the mechanisms regulating basal "housekeeping" mitophagy levels in different model organisms. Using iron chelation as an inducer of PRKN-independent mitophagy, we recently screened an siRNA library of lipid-binding proteins and determined that two kinases, GAK and PRKCD, act as positive regulators of PRKN-independent mitophagy. We demonstrate that PRKCD is localized to mitochondria and regulates recruitment of ULK1-ATG13 upon induction of mitophagy. GAK activity, by contrast, modifies the mitochondrial network and lysosomal morphology that compromise efficient transport of mitochondria for degradation. Impairment of either kinase in vivo blocks basal mitophagy, demonstrating the biological relevance of our findings.Abbreviations: CCCP: carbonyl cyanide-m-chlorophenyl hydrazone; DFP: deferiprone; GAK: cyclin G associated kinase; HIF1A: hypoxia inducible factor 1 subunit alpha; PRKC/PKC: protein kinase C; PRKCD: protein kinase C delta; PRKN: parkin RBR E3 ubiquitin protein ligase.

The cholesterol transport protein GRAMD1C regulates autophagy initiation and mitochondrial bioenergetics.

During autophagy, cytosolic cargo is sequestered into double-membrane vesicles called autophagosomes. The contributions of specific lipids, such as cholesterol, to the membranes that form the autophagosome, remain to be fully characterized. Here, we demonstrate that short term cholesterol depletion leads to a rapid induction of autophagy and a corresponding increase in autophagy initiation events. We further show that the ER-localized cholesterol transport protein GRAMD1C functions as a negative regulator of starvation-induced autophagy and that both its cholesterol transport VASt domain and membrane binding GRAM domain are required for GRAMD1C-mediated suppression of autophagy initiation. Similar to its yeast orthologue, GRAMD1C associates with mitochondria through its GRAM domain. Cells lacking GRAMD1C or its VASt domain show increased mitochondrial cholesterol levels and mitochondrial oxidative phosphorylation, suggesting that GRAMD1C may facilitate cholesterol transfer at ER-mitochondria contact sites. Finally, we demonstrate that expression of GRAMD family proteins is linked to clear cell renal carcinoma survival, highlighting the pathophysiological relevance of cholesterol transport proteins.

GAK and PRKCD are positive regulators of PRKN-independent mitophagy.

The mechanisms involved in programmed or damage-induced removal of mitochondria by mitophagy remains elusive. Here, we have screened for regulators of PRKN-independent mitophagy using an siRNA library targeting 197 proteins containing lipid interacting domains. We identify Cyclin G-associated kinase (GAK) and Protein Kinase C Delta (PRKCD) as regulators of PRKN-independent mitophagy, with both being dispensable for PRKN-dependent mitophagy and starvation-induced autophagy. We demonstrate that the kinase activity of both GAK and PRKCD are required for efficient mitophagy in vitro, that PRKCD is present on mitochondria, and that PRKCD facilitates recruitment of ULK1/ATG13 to early autophagic structures. Importantly, we demonstrate in vivo relevance for both kinases in the regulation of basal mitophagy. Knockdown of GAK homologue (gakh-1) in C. elegans or knockout of PRKCD homologues in zebrafish led to significant inhibition of basal mitophagy, highlighting the evolutionary relevance of these kinases in mitophagy regulation.

AXL Targeting Abrogates Autophagic Flux and Induces Immunogenic Cell Death in Drug-Resistant Cancer Cells.

INTRODUCTION: Acquired cancer therapy resistance evolves under selection pressure of immune surveillance and favors mechanisms that promote drug resistance through cell survival and immune evasion. AXL receptor tyrosine kinase is a mediator of cancer cell phenotypic plasticity and suppression of tumor immunity, and AXL expression is associated with drug resistance and diminished long-term survival in a wide range of malignancies, including NSCLC. METHODS: We aimed to investigate the mechanisms underlying AXL-mediated acquired resistance to first- and third-generation small molecule EGFR tyrosine kinase inhibitors (EGFRi) in NSCLC. RESULTS: We found that EGFRi resistance was mediated by up-regulation of AXL, and targeting AXL reduced reactivation of the MAPK pathway and blocked onset of acquired resistance to long-term EGFRi treatment in vivo. AXL-expressing EGFRi-resistant cells revealed phenotypic and cell signaling heterogeneity incompatible with a simple bypass signaling mechanism, and were characterized by an increased autophagic flux. AXL kinase inhibition by the small molecule inhibitor bemcentinib or siRNA mediated AXL gene silencing was reported to inhibit the autophagic flux in vitro, bemcentinib treatment blocked clonogenicity and induced immunogenic cell death in drug-resistant NSCLC in vitro, and abrogated the transcription of autophagy-associated genes in vivo. Furthermore, we found a positive correlation between AXL expression and autophagy-associated gene signatures in a large cohort of human NSCLC (n = 1018). CONCLUSION: Our results indicate that AXL signaling supports a drug-resistant persister cell phenotype through a novel autophagy-dependent mechanism and reveals a unique immunogenic effect of AXL inhibition on drug-resistant NSCLC cells.

Progesterone anti-inflammatory properties in hereditary retinal degeneration.

The interactions between steroid gonadal hormones and the retina (a part of the visual system and the central nervous system (CNS)) have received limited attention and beneficial effects of these hormones in retinal diseases is controversial. Retinitis pigmentosa (RP) is the most common cause of retinal hereditary blindness and to date no treatment is available. However, results regarding the effects of progesterone on the progression of RP are promising. With the idea of demonstrating if the progesterone retinal protection in RP is related to its possible anti-inflammatory properties, we have administered orally progesterone to rd10 mice, an animal model of RP. We observed that progesterone decreased photoreceptors cell death, reactive gliosis and the increase in microglial cells caused by RP. We also examined the expression of neuronal and inducible nitric oxide synthase (nNOS and iNOS), the enzyme responsible for NO production. The results demonstrated a decrease in nNOS expression only in control mice treated with progesterone. Inflammation has been related with an increase in lipid peroxidation. Noticeably progesterone administration was able to diminish retinal malondialdehyde (MDA, a lipid peroxidation product) concentrations in rd10 mice. Altogether, we can conclude that progesterone could be a good therapeutic option not only in RP but also for other retinal diseases that have been associated with inflammation and lipid peroxidation.

Pretreatment of Glioblastoma with Bortezomib Potentiates Natural Killer Cell Cytotoxicity through TRAIL/DR5 Mediated Apoptosis and Prolongs Animal Survival.

Background: Natural killer (NK) cells are potential effectors in anti-cancer immunotherapy; however only a subset potently kills cancer cells. Here, we examined whether pretreatment of glioblastoma (GBM) with the proteasome inhibitor, bortezomib (BTZ), might sensitize tumour cells to NK cell lysis by inducing stress antigens recognized by NK-activating receptors. Methods: Combination immunotherapy of NK cells with BTZ was studied in vitro against GBM cells and in a GBM-bearing mouse model. Tumour cells were derived from primary GBMs and NK cells from donors or patients. Flow cytometry was used for viability/cytotoxicity evaluation as well as in vitro and ex vivo phenotyping. We performed a Seahorse assay to assess oxygen consumption rates and mitochondrial function, Luminex ELISA to determine NK cell secretion, protein chemistry and LC-MS/MS to detect BTZ in brain tissue. MRI was used to monitor therapeutic efficacy in mice orthotopically implanted with GBM spheroids. Results: NK cells released IFNγ, perforin and granzyme A cytolytic granules upon recognition of stress-ligand expressing GBM cells, disrupted mitochondrial function and killed 24-46% of cells by apoptosis. Pretreatment with BTZ further increased stress-ligands, induced TRAIL-R2 expression and enhanced GBM lysis to 33-76% through augmented IFNγ release (p < 0.05). Blocking NKG2D, TRAIL and TRAIL-R2 rescued GBM cells treated with BTZ from NK cells, p = 0.01. Adoptively transferred autologous NK-cells persisted in vivo (p < 0.05), diminished tumour proliferation and prolonged survival alone (Log Rank10.19, p = 0.0014, 95%CI 0.252-0.523) or when combined with BTZ (Log Rank5.25, p = 0.0219, 95%CI 0.295-0.408), or either compared to vehicle controls (median 98 vs. 68 days and 80 vs. 68 days, respectively). BTZ crossed the blood-brain barrier, attenuated proteasomal activity in vivo (p < 0.0001; p < 0.01 compared to vehicle control or NK cells only, respectively) and diminished tumour angiogenesis to promote survival compared to vehicle-treated controls (Log Rank6.57, p = 0.0104, 95%CI 0.284-0.424, median 83 vs. 68 days). However, NK ablation with anti-asialo-GM1 abrogated the therapeutic efficacy. Conclusions: NK cells alone or in combination with BTZ inhibit tumour growth, but the scheduling of BTZ in vivo requires further investigation to maximize its contribution to the efficacy of the combination regimen.

Oxidative stress and autophagy-related changes during retinal degeneration and development.

Retinitis pigmentosa (RP) is an inherited retinopathy that leads to photoreceptor loss. RP has been related to oxidative stress, autophagy, and inflammation. This study aimed to identify changes in the levels of oxidative stress and autophagy markers in the retina of control and rd10 mice during different phases of retinal development. Changes in the retinal oxidation system were investigated by measuring the levels of oxidized and reduced glutathione (GSH/GSSG), retinal avidin-positive cells, and 4-hydroxynonenal (4-HNE) staining intensity. Autophagy characterization was explored by measuring the levels of microtubule-associated protein 1 light chain 3 (LC3), beclin, autophagy-related proteins 5 and 7 (Atg5 and Atg7), and lysosomal associated membrane protein-2A (LAMP-2A). At P28 retinal GSH concentrations decreased in rd10 mice compared to the controls. No differences were found in retinal GSSG concentrations between the control and rd10 mice. There was an increase in retinal GSSG concentrations and a decrease in the GSH/GSSG ratio in the control and rd10 mice at P21 and P28 compared to P13. We observed an increase in avidin-positive cells in rd10 retinas. 4-HNE was increased in rd10 retinas at P13, and it also increased in control mice with age. We did not observe any differences in the retinal levels of LC3II/I ratio, Beclin, Atg5, or Atg7 in the rd10 mice compared to the controls. There was an increase in the LAMP-2A concentrations in the control and rd10 mice with development age (P28 concentrations vs. P13). Although only slight differences were found in the oxidative stress and autophagy markers between the control and rd10 mice, there were increases in the GSSG, 4-HNE, and LAMP-2A with age. This increase in the oxidative stress and chaperone-mediated autophagy has not been described before and occurred just after the mice opened their eyes, potentially indicating a retinal response to light exposure.

Alterations in glutamate cysteine ligase content in the retina of two retinitis pigmentosa animal models.

Retinitis Pigmentosa (RP) comprises a group of rare genetic retinal disorders in which one of several different mutations induces photoreceptor death. Oxidative stress and glutathione (GSH) alterations may be related to the pathogenesis of RP. GSH has been shown to be present in high concentrations in the retina. In addition, the retina has the capability to synthesize GSH. In this study, we tested whether the two subunits of glutamate cysteine ligase, the rate-limiting enzyme in GSH synthesis, and the concentrations of retinal GSH, oxidized glutathione (GSSG), cysteine (Cys) and glutamate are altered in the retina of two different RP mice models. Retinas from C3H and rd1 mice at different postnatal days (P7, P11, P15, P19, P21 and P28) and from C57BL/6 and rd10 mice at P21 were obtained. Western blot analysis was performed to determine the protein content of catalytic and modulatory subunits from glutamate cysteine ligase (GCLC and GCLM, respectively). In another set of experiments, control and rd1 mice were administered buthinine sulfoximine, a glutathione synthase inhibitor, or paraquat. GSH, GSSG, glutamate and Cys concentrations were determined, by HPLC. A decrease in retinal GCLC content was observed in C3H and rd1 mice with age, nevertheless, there was an increase in retinal GCLC in rd1 mice compared to control retinas at P19. No modifications in GCLM content with age and no difference between GCLM content in rd1 and control retinas were observed. The GSH concentration decreased in the rd1 retinas compared with control ones at P15, it increased at P19, and was again similar at P21 and P28. No changes in GSSG concentration in control retinas with age were observed; the GSSG levels in rd1 retinas were similar from P7 to P19 and then increased significantly at P21 and P28. Glutamate concentration was increased in the rd1 retinas compared to control mice from P7 to P15 and were comparable at P21 and P28. The Cys concentrations was measured in control and rd1 retinas, but no significant changes were observed between them. BSO administration decreases GSH retinal concentration in control and rd1 mice, while paraquat administration induced an increase in GSH retinal concentration in control mice and a decrease in GSH in rd1 mice retina. Retinal GCLC was significantly increased in rd10 mice at P21 as well as GSSG. Our results suggest alterations in retinal GCLC content and GSH and/or its precursors in these two RP animal models. Regulation of the enzymes related to GSH metabolism and the retinal concentration of glutamate may be a possible target to delay especially cone death in RP.

Organotypic retinal explant cultures as in vitro alternative for diabetic retinopathy studies.

Diabetic retinopathy (DR) is a major cause of vision loss and one of the most common and debilitating complications of diabetes. Research to prevent DR is hindered by a lack of experimental model systems that faithfully reproduce the disease pathology, in particular for type 2 diabetes, which requires prolonged disease progression in animals to develop some hallmarks of DR. Here, we introduce an alternative in vitro model system for DR, based on serum-free, organotypic rodent retinal explant cultures, which allow physiological and pharmacological manipulation of the retina for up to two weeks under tightly controlled conditions. Retinal explant cultures have the advantage of isolating direct neuronal consequences of diabetic conditions from indirect systemic effects mediated via the retinal vasculature or the immune system. Exposed to conditions emulating type 1 or type 2 diabetes, retinal explants displayed elevated cell death rates among inner retinal neurons as well as photoreceptors, with a particularly strong loss of cone photoreceptors. Our results support a direct impact of diabetic conditions on retinal neurons and may help explain color vision defects observed in DR patients. This serum-free in vitro DR model avoids the animal suffering of established DR models and reduces the overall number of animals needed for such research. It should prove useful to study the mechanisms of neuronal cell death caused by DR and to screen for potential future DR treatments.