Carl Ward

Autophagy, lipophagy and lysosomal lipid storage disorders.

Autophagy is a catabolic process with an essential function in the maintenance of cellular and tissue homeostasis. It is primarily recognised for its role in the degradation of dysfunctional proteins and unwanted organelles, however in recent years the range of autophagy substrates has also been extended to lipids. Degradation of lipids via autophagy is termed lipophagy. The ability of autophagy to contribute to the maintenance of lipo-homeostasis becomes particularly relevant in the context of genetic lysosomal storage disorders where perturbations of autophagic flux have been suggested to contribute to the disease aetiology. Here we review recent discoveries of the molecular mechanisms mediating lipid turnover by the autophagy pathways. We further focus on the relevance of autophagy, and specifically lipophagy, to the disease mechanisms. Moreover, autophagy is also discussed as a potential therapeutic target in several key lysosomal storage disorders.

Resistance exercise initiates mechanistic target of rapamycin (mTOR) translocation and protein complex co-localisation in human skeletal muscle

The mechanistic target of rapamycin (mTOR) is a central mediator of protein synthesis in skeletal muscle. We utilized immunofluorescence approaches to study mTOR cellular distribution and protein-protein co-localisation in human skeletal muscle in the basal state as well as immediately, 1 and 3 h after an acute bout of resistance exercise in a fed (FED; 20 g Protein/40 g carbohydrate/1 g fat) or energy-free control (CON) state. mTOR and the lysosomal protein LAMP2 were highly co-localised in basal samples. Resistance exercise resulted in rapid translocation of mTOR/LAMP2 towards the cell membrane. Concurrently, resistance exercise led to the dissociation of TSC2 from Rheb and increased in the co-localisation of mTOR and Rheb post exercise in both FED and CON. In addition, mTOR co-localised with Eukaryotic translation initiation factor 3 subunit F (eIF3F) at the cell membrane post-exercise in both groups, with the response significantly greater at 1 h of recovery in the FED compared to CON. Collectively our data demonstrate that cellular trafficking of mTOR occurs in human muscle in response to an anabolic stimulus, events that appear to be primarily influenced by muscle contraction. The translocation and association of mTOR with positive regulators (i.e. Rheb and eIF3F) is consistent with an enhanced mRNA translational capacity after resistance exercise.

PEG-lipid micelles enable cholesterol efflux in Niemann-Pick Type C1 disease-based lysosomal storage disorder

2-Hydroxy-propyl-β-cyclodextrin (HPβCD), a cholesterol scavenger, is currently undergoing Phase 2b/3 clinical trial for treatment of Niemann Pick Type C-1 (NPC1), a fatal neurodegenerative disorder that stems from abnormal cholesterol accumulation in the endo/lysosomes. Unfortunately, the extremely high doses of HPβCD required to prevent progressive neurodegeneration exacerbates ototoxicity, pulmonary toxicity and autophagy-based cellular defects. We present unexpected evidence that a poly (ethylene glycol) (PEG)-lipid conjugate enables cholesterol clearance from endo/lysosomes of Npc1 mutant (Npc1−/−) cells. Herein, we show that distearyl-phosphatidylethanolamine-PEG (DSPE-PEG), which forms 12-nm micelles above the critical micelle concentration, accumulates heavily inside cholesterol-rich late endosomes in Npc1−/− cells. This potentially results in cholesterol solubilization and leakage from lysosomes. High-throughput screening revealed that DSPE-PEG, in combination with HPβCD, acts synergistically to efflux cholesterol without significantly aggravating autophagy defects. These well-known excipients can be used as admixtures to treat NPC1 disorder. Increasing PEG chain lengths from 350 Da-30 kDa in DSPE-PEG micelles, or increasing DSPE-PEG content in an array of liposomes packaged with HPβCD, improved cholesterol egress, while Pluronic block copolymers capable of micelle formation showed slight effects at high concentrations. We postulate that PEG-lipid based nanocarriers can serve as bioactive drug delivery systems for effective treatment of lysosomal storage disorders.

Origin‐Specific Adhesive Interactions of Mesenchymal Stem Cells with Platelets Influence Their Behavior After Infusion

We investigated the adhesive behavior of mesenchymal stem cells (MSC) in blood, which might influence their fate when infused as therapy. Isolated human bone marrow MSC (BMMSC) or umbilical cord MSC (UCMSC) adhered efficiently from flow to the matrix proteins, collagen, or fibronectin, but did not adhere to endothelial selectins. However, when suspended in blood, BMMSC no longer adhered to collagen, while UCMSC adhered along with many aggregated platelets. Neither MSC adhered to fibronectin from flowing blood, although the fibronectin surface did become coated with a platelet monolayer. UCMSC induced platelet aggregation in platelet rich plasma, and caused a marked drop in platelet count when mixed with whole human or mouse blood in vitro, or when infused into mice. In contrast, BMMSC did not activate platelets or induce changes in platelet count. Interestingly, isolated UCMSC and BMMSC both adhered to predeposited platelets. The differences in behavior in blood were attributable to expression of podoplanin (an activating ligand for the platelet receptor CLEC‐2), which was detected on UCMSC, but not BMMSC. Thus, platelets were activated when bound to UCMSC, but not BMMSC. Platelet aggregation by UCMSC was inhibited by recombinant soluble CLEC‐2, and UCMSC did not cause a reduction in platelet count when mixed with blood from mice deficient in CLEC‐2. We predict that both MSC would carry platelets in the blood, but their interaction with vascular endothelium would depend on podoplanin‐induced activation of the bound platelets. Such interactions with platelets might target MSC to damaged tissue, but could also be thrombotic. Stem Cells 2018;36:1062–1074

Discovery of pan autophagy inhibitors through a high-throughput screen highlights macroautophagy as an evolutionarily conserved process across 3 eukaryotic kingdoms

ABSTRACT Due to the involvement of macroautophagy/autophagy in different pathophysiological conditions such as infections, neurodegeneration and cancer, identification of novel small molecules that modulate the process is of current research and clinical interest. In this work, we developed a luciferase-based sensitive and robust kinetic high-throughput screen (HTS) of small molecules that modulate autophagic degradation of peroxisomes in the budding yeast Saccharomyces cerevisiae. Being a pathway-specific rather than a target-driven assay, we identified small molecule modulators that acted at key steps of autophagic flux. Two of the inhibitors, Bay11 and ZPCK, obtained from the screen were further characterized using secondary assays in yeast. Bay11 inhibited autophagy at a step before fusion with the vacuole whereas ZPCK inhibited the cargo degradation inside the vacuole. Furthermore, we demonstrated that these molecules altered the process of autophagy in mammalian cells as well. Strikingly, these molecules also modulated autophagic flux in a novel model plant, Aponogeton madagascariensis. Thus, using small molecule modulators identified by using a newly developed HTS autophagy assay, our results support that macroautophagy is a conserved process across fungal, animal and plant kingdoms.

Fine-Tuning Mybl2 Is Required for Proper Mesenchymal-to-Epithelial Transition during Somatic Reprogramming

Summary During somatic reprogramming, Yamanaka’s pioneer factors regulate a complex sequence of molecular events leading to the activation of a network of pluripotency factors, ultimately resulting in the acquisition and maintenance of a pluripotent state. Here, we show that, contrary to the pluripotency factors studied so far, overexpression of Mybl2 inhibits somatic reprogramming. Our results demonstrate that Mybl2 levels are crucial to the dynamics of the reprogramming process. Mybl2 overexpression changes chromatin conformation, affecting the accessibility of pioneer factors to the chromatin and promoting accessibility for early immediate response genes known to be reprogramming blockers. These changes in the chromatin landscape ultimately lead to a deregulation of key genes that are important for the mesenchymal-to-epithelial transition. This work defines Mybl2 level as a gatekeeper for the initiation of reprogramming, providing further insights into the tight regulation and required coordination of molecular events that are necessary for changes in cell fate identity during the reprogramming process.

MYBL2 Supports DNA Double Strand Break Repair in Hematopoietic Stem Cells

Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases characterized by blood cytopenias that occur as a result of somatic mutations in hematopoietic stem cells (HSC). MDS leads to ineffective hematopoiesis, and as many as 30% of patients progress to acute myeloid leukemia (AML). The mechanisms by which mutations accumulate in HSC during aging remain poorly understood. Here we identify a novel role for MYBL2 in DNA double-strand break (DSB) repair in HSC. In patients with MDS, low MYBL2 levels associated with and preceded transcriptional deregulation of DNA repair genes. Stem/progenitor cells from these patients display dysfunctional DSB repair kinetics after exposure to ionizing radiation (IR). Haploinsufficiency of Mybl2 in mice also led to a defect in the repair of DSBs induced by IR in HSC and was characterized by unsustained phosphorylation of the ATM substrate KAP1 and telomere fragility. Our study identifies MYBL2 as a crucial regulator of DSB repair and identifies MYBL2 expression levels as a potential biomarker to predict cellular response to genotoxic treatments in MDS and to identify patients with defects in DNA repair. Such patients with worse prognosis may require a different therapeutic regimen to prevent progression to AML.Significance: These findings suggest MYBL2 levels may be used as a biological biomarker to determine the DNA repair capacity of hematopoietic stem cells from patients with MDS and as a clinical biomarker to inform decisions regarding patient selection for treatments that target DNA repair.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/20/5767/F1.large.jpg Cancer Res; 78(20); 5767-79. ©2018 AACR.

Prognostic significance of high GFI1 expression in AML of normal karyotype and its association with a FLT3-ITD signature

Growth Factor Independence 1 (GFI1) is a transcriptional repressor that plays a critical role during both myeloid and lymphoid haematopoietic lineage commitment. Several studies have demonstrated the involvement of GFI1 in haematological malignancies and have suggested that low expression of GFI1 is a negative indicator of disease progression for both myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). In this study, we have stratified AML patients into those defined as having a normal karyotype (CN-AML). Unlike the overall pattern in AML, those patients with CN-AML have a poorer survival rate when GFI1 expression is high. In this group, high GFI1 expression is paralleled by higher FLT3 expression, and, even when the FLT3 gene is not mutated, exhibit a FLT3-ITD signature of gene expression. Knock-down of GFI1 expression in the human AML Fujioka cell line led to a decrease in the level of FLT3 RNA and protein and to the down regulation of FLT3-ITD signature genes, thus linking two major prognostic indicators for AML.