Molecular mechanisms of autophagy and the effect of autophagy dysfunction on mitochondrial function

PhD ThesisLong lifespan of evolutionary higher organisms including humans is associated with the challenge to maintain viability of post mitotic cells, such as neurons, for decades. Autophagy is increasingly recognized as an important prosurvival pathway in oxidative and proteotoxic stress conditions. Autophagy degrades cytosolic macromolecules in response to starvation and is involved in the selective degradation of damaged/toxic organelles, such as mitochondria. With age autophagy function declines, and is also compromised in several neurodegenerative diseases. We identified a novel role for autophagy in the maintenance of mitochondrial health, specifically respiratory complex I. Intriguingly, galactose-induced cell death of autophagy deficient cells was rescued by preventing ROS production at complex I or bypassing complex I-linked respiration. We propose that aberrant ROS production via complex I in response to autophagy deficiency could be pathogenic and result in neurodegeneration and preventing this could be an interesting therapeutic target. Furthermore, we found that vertebrates have evolved mechanisms to induce autophagy in response to oxidative stress. This involves the oxidation of the autophagy receptor p62, which promotes autophagy flux and the clearance of autophagy cargo, resulting in increased stress resistance in mammalian cells and survival under stress in flies. In addition, we obtained data revealing an important role for redox-regulated cysteines in NDP52 for the degradation of mitochondria via mitophagy and tools were created to study the role of other autophagy receptors in autophagy initiation and selective autophagy.Biotechnology and Biological Sciences Research Counci

Oxidation of p62 as an evolutionary adaptation to promote autophagy in stress conditions

Ageing and age-related diseases are characterised by increased oxidative and proteotoxic stress, which results in negative effects on cell function and survival. The cell possesses several mechanisms to deal with damaged proteins, including degradation via macroautophagy (hereafter called autophagy). This essential cellular pathway is conserved from yeast to humans and it is well established that its impairment reduces lifespan in multiple model organisms, including worms, flies and mice. In our study, recently published in Nature Communications, we asked if longer lifespan characteristic of higher organisms is the result of evolutionary adaptations to the autophagy machinery. We found that the autophagy receptor p62 can be oxidised leading to its oligomerisation which ultimately promotes autophagy. However this mechanism, present in vertebrates, has been acquired late in evolution. We propose that the ability of p62 to sense reactive oxygen species (ROS) via oxidation, and potentially other similar modifications, may have evolved in higher organisms and contributed to their increased lifespan. Indeed, impairment of this process could result in age-related neurodegeneration in humans

Autophagy, lipophagy and lysosomal lipid storage disorders

AbstractAutophagy 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

An iPSC Patient Specific Model of CFH (Y402H) Polymorphism Displays Characteristic Features of AMD and Indicates a Beneficial Role for UV Light Exposure

Age related macular degeneration (AMD) is the most common cause of blindness, accounting for 8.7% of all blindness globally. Vision loss is caused ultimately by apoptosis of the retinal pigment epithelium (RPE) and overlying photoreceptors. Treatments are evolving for the wet form of the disease, however these do not exist for the dry form. Complement factor H (CFH) polymorphism in exon 9 (Y402H) has shown a strong association with susceptibility to AMD resulting in complement activation, recruitment of phagocytes, retinal pigment epithelium (RPE) damage and visual decline. We have derived and characterised induced pluripotent stem cell (iPSCs) lines from two patients without AMD and low risk genotype and two patients with advanced AMD and high risk genotype and generated RPE cells that show local secretion of several proteins involved in the complement pathway including factor H (FH), factor I (FI) and factor H like 1 (FHL-1). The iPSC RPE cells derived from high risk patients mimic several key features of AMD including increased inflammation and cellular stress, accumulation of lipid droplets, impaired autophagy and deposition of “drüsen” like deposits. The low and high risk RPE cells respond differently to intermittent exposure to UV light which leads to an improvement in cellular and functional phenotype only in the high risk AMD-RPE cells. Taken together our data indicate that the patient specific iPSC model provides a robust platform for understanding the role of complement activation in AMD, evaluating new therapies based on complement modulation and drug testing

Oxidation of SQSTM1/p62 mediates the link between redox state and protein homeostasis.

Cellular homoeostatic pathways such as macroautophagy (hereinafter autophagy) are regulated by basic mechanisms that are conserved throughout the eukaryotic kingdom. However, it remains poorly understood how these mechanisms further evolved in higher organisms. Here we describe a modification in the autophagy pathway in vertebrates, which promotes its activity in response to oxidative stress. We have identified two oxidation-sensitive cysteine residues in a prototypic autophagy receptor SQSTM1/p62, which allow activation of pro-survival autophagy in stress conditions. The Drosophila p62 homologue, Ref(2)P, lacks these oxidation-sensitive cysteine residues and their introduction into the protein increases protein turnover and stress resistance of flies, whereas perturbation of p62 oxidation in humans may result in age-related pathology. We propose that the redox-sensitivity of p62 may have evolved in vertebrates as a mechanism that allows activation of autophagy in response to oxidative stress to maintain cellular homoeostasis and increase cell survival

Dynamic modelling of pathways to cellular senescence reveals strategies for targeted interventions.

Cellular senescence, a state of irreversible cell cycle arrest, is thought to help protect an organism from cancer, yet also contributes to ageing. The changes which occur in senescence are controlled by networks of multiple signalling and feedback pathways at the cellular level, and the interplay between these is difficult to predict and understand. To unravel the intrinsic challenges of understanding such a highly networked system, we have taken a systems biology approach to cellular senescence. We report a detailed analysis of senescence signalling via DNA damage, insulin-TOR, FoxO3a transcription factors, oxidative stress response, mitochondrial regulation and mitophagy. We show in silico and in vitro that inhibition of reactive oxygen species can prevent loss of mitochondrial membrane potential, whilst inhibition of mTOR shows a partial rescue of mitochondrial mass changes during establishment of senescence. Dual inhibition of ROS and mTOR in vitro confirmed computational model predictions that it was possible to further reduce senescence-induced mitochondrial dysfunction and DNA double-strand breaks. However, these interventions were unable to abrogate the senescence-induced mitochondrial dysfunction completely, and we identified decreased mitochondrial fission as the potential driving force for increased mitochondrial mass via prevention of mitophagy. Dynamic sensitivity analysis of the model showed the network stabilised at a new late state of cellular senescence. This was characterised by poor network sensitivity, high signalling noise, low cellular energy, high inflammation and permanent cell cycle arrest suggesting an unsatisfactory outcome for treatments aiming to delay or reverse cellular senescence at late time points. Combinatorial targeted interventions are therefore possible for intervening in the cellular pathway to senescence, but in the cases identified here, are only capable of delaying senescence onset

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

Model Lyapunov exponents.

<p>The Lyapunov exponents computed for this model were all negative, indicating that the model was asymptotically Lyapunov stable and therefore the trajectories eventually converged to an equilibrium.</p><p>Model Lyapunov exponents.</p

A dynamic model for cellular senescence.

<p>(A) Graphical model integrating the insulin-TOR (IIS-TOR) signalling pathway (left), the DDR-oxidative stress responses (top-right) and mitochondria (centre). The insulin-TOR signalling pathway regulated the anabolic process of mitochondrial biogenesis, FoxO3a translocation to the cytoplasm followed by ubiquitination, and inhibition of cell cycle arrest (black reactions). AMPK signalling promoted the catabolic pathway of mitophagy, but was also modelled to promote mitochondrial biogenesis (magenta reactions). Mitochondrial membrane potential (ψm) increased cellular energy levels (red reactions) deregulating AMPK, and enhanced intracellular ROS (blue reactions). DNA damage and ROS triggered a stress response and consolidated cell cycle arrest. (B) Representative western blot data used for calibrating the model. (C) Representative imaging data used for measuring co-localisation between mitochondria (COXIV) and lysosomes (LC3) at day 0 and 12. These data were used for creating a time course for mitophagy activation as indicated in panel E.(D) Additional controls for mitophagy data using Pink 1 and mitochondrial biogenesis using PGC-1α indicating correlation between mTOR-pS2448 and PGC-1α. (E) <i>In silico</i> versus <i>in vitro</i> time courses. The model (red lines) was calibrated over experimental time course data (blue points) collected for 14 readouts in the network up to 21 days. The inputs are amino acids/insulin (constant inputs) and irradiation (pulse input of 5 min which simulates 20 Gy X ray irradiation over 5 min). Experimental time points (blue points) are mean +/−1 standard deviation collected from 5 independent repetitions. N = number of data points used for fitting each readout time course. The goodness-of-fit statistical measures χ²<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003728#pcbi.1003728-Maiwald1" target="_blank">[61]</a> and AIC <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003728#pcbi.1003728-Akaike1" target="_blank">[68]</a> for this model are 70.4278 and 381.838, respectively. (F) Simulated time-courses of the internal states for mitochondrial mass, ψm and turnover.</p