We are interested in the factors that drive cell death susceptibility in Alzheimer’s disease and Brain Cancer, as well as ROS damage in order to exploit mechanisms for neuronal protection. We know that a number of pathways related to autophagic flux, protein degradation, axonal transport, mitochondrial fusion & respiration as well as tubulin stability are implicated. Central is ATP availability, especially in the compartmentalized regions where the ATP demand is highest. Pathways for neuronal migration and plasticity rely on a highly functional actin, tubulin and mitochondrial network interplay, which allow us to better understand the susceptibility for undergoing a network collapse and metabolic failure, changing the cell’s matrices for apoptosis or necrosis onset. Lastly, we aim to exploit the relationship between ATP availability and apoptosis onset in context of astrocytomas, in order to maximize cell death through sensitization. Central to all of the above is the accurate and robust quantification of autophagic flux, the rate of protein degradation through autophagy, in order to control it with highest degree of precision.
Therefore, the focus of our current research projects concern both 1) the role of autophagy in disease pathogenesis, 2) the regulation and control of autophagic flux, 3) the relationship between mitochondria and neurodegenerative diseases, 4) Mitochondrial fission and fusion, 5) Reactive oxygen species and mitochondrial dynamics 6) Cancer and autophagy
1. THE ROLE OF AUTOPHAGY IN DISEASE PATHOGENESIS
Given the involvement of autophagy dysfunction in both neurodegeneration and tumourigenesis, we are interested in the following ares:
The role of autophagic flux in the context of neurotoxicity.
How autophagic activity alters substrate metabolism and mitochondrial function.
How to beter utilise intervention strategies such as autopaghy modulators, nutrient starvation or ketone bodies to protect neurons from cell death or to induce it in gliomas.
2. REGULATION AND CONTROL OF AUTOPHAGIC FLUX
Systems biology is uniquely equipped to address questions central to cellular physiology, especially in terms of complex network regulatory mechanisms. Our group includes therefore a systems biology approach, where suitable, to the autophagy regulatory network. In particular, we currently focus here on:
Flux assessment and control analysis of protein degradation through autophagy.
Constructing robust mathematical models and high throughput measurement tools of autopaghic flux.
The relationship between autophagic flux and cell death prediction.
3. RELATIONSHIP BETWEEN MITOCHONDRIA AND NEURODEGENERATIVE DISEASES
Dementia is defined as severe cognitive decline that leads to the loss of thinking abilities such as, memory, language and problem-solving, which is significant enough to interfere with independent daily functioning (Dave et al., 2020). Common causes include genetic pre-disposition, traumatic brain injury and brain cancer in young adults and neurodegenerative diseases (NDDs) amongst the elderly. (Gale, Acar & Daffner, 2018.) However, mitochondrial dysfunction has been proved to be a central causative factor in many of these pathological conditions (Lin & Beal, 2006; Tapias et al.,2019). Mitochondria are crucial for the maintenance of cellular and metabolic homeostasis through the regulation of numerous biological processes (Ni, Williams & Ding, 2015). Therefore, they are vital for the maintenance of the health and survival of all eukaryotic organisms especially human beings (Loos et al., 2013). As such, the maintenance of healthy mitochondria is beneficial for our well-being. Conveniently, there are multiple mechanisms by which mitochondria maintain their homeostasis also known as mitochondrial quality control (MQC) (Karbowski & Youle, 2011). These include; mitochondrial biogenesis, where by, new mitochondria are synthesized from pre-existing
mitochondria to increase their quantity, mass and volume ( Boland, Chourasia & Macleod, 2013); the regulation of mitochondrial dynamics, which includes mitochondrial transport and the regulation of mitochondrial conformation through regulated cycles of fission and fusion (Youle et al,.2012); and finally mitophagy, where by, damaged mitochondria are be enveloped by autophagosomes to trigger their degradation in the lysosome. (Narendra et al. 2010; Ding, Chen & Yin 2012; Lemasters, 2014). Malfunctions in any of these regulatory processes can lead to mitochondrial dysfunction and therefore, they have become targets for the therapeutic treatment of these NDDs (Liu et al., 2019).
4. MITOCHONDRIAL FISSION AND FUSION
Mitochondrial fission and fusion can be measured as it happens in the cells, either by Western blotting or by MEL. We do this because more fission events will mean that the mitochondrial network is more fragmented and therefore we can deduce that the cell is probably under a lot of stress. More fusion on the other hand is a bit of a double-edged sword as it may be beneficial, as the cell makes more energy, but also that the cell may be making more energy because it is in the early stages of stress. Ideally, cells should be in an intermediate state. Western blotting can only inform so much and that is why Rensu developed MEL and why he and I are looking into new ways of measuring mitochondrial morphology at a single cell level. The output measurables from MEL inform us on the state of the mitochondrial network and should be taken together to get an idea of what state the cell is in. One example of this might be that the cell could be so fragmented that there aren't may fission events to measure, but in this case we expect there to be an increase in the number of mitochondria present vs the control.
In conclusion, we measure fission and fusion events as a means of understanding the likelihood of mitophagy and overall mitochondrial network maintenance.
5. REACTIVE OXYGEN SPECIES AND MITOCHONDRIAL DYNAMICS
The highly dynamic mitochondrial network is able to readily respond to the metabolic needs of the cell. Mitochondria can be classified into one of three phenotypes; punctate mitochondria where there has been increased fission events, filamentous mitochondria where the number of fusion events has increased, and an intermediate state with both punctate and elongated mitochondrial structures present (Chaudhry et al., 2020).
The role of ROS in regulating mitochondrial dynamics has recently been described, which suggests that there is an association between the morphology of mitochondria and the redox homeostasis of the cell (Ježek et al., 2018). The fragmentation, swelling, and shortening of mitochondria occurs from elevated levels of ROS, whereas the filamentation of mitochondria occurs when there are lowered levels of ROS within the cell (Cid-Castro et al., 2018). In human umbilical vein endothelial cells, exogenous concentrations of H2O2 resulted in the fragmentation of mitochondria and the expression of numerous fission and fusion related genes that occurred in a dose dependent manner. Moreover, in C2C12 myocytes H2O2 resulted in the depolarisation of the mitochondrial membrane and the subsequent fragmentation of the mitochondria where increased Drp1 activity was observed (Brillo et al., 2021).
In contrast, decreased levels of ROS in fibroblasts resulted in the filamentation of mitochondria, which was Mfn2 dependent. At the transcriptional level ROS can stimulate the expression of factors that are involved in mitochondrial dynamics as well as in redox regulation (Cid-Castro et al., 2018). AMPK plays an important role in the relationship between ROS and mitochondrial dynamics. Once AMPK is activated it is able to phosphorylate mitochondrial fission factor and Drp1, which is essential for mitochondrial fission (Brillo et al., 2021).
6. CANCER AND AUTOPHAGY
Autophagy is an intracellular degradative process which occurs at a basal level but it can also be regulated accordingly depending on stressors such as nutrient deprivation or organelle damage. Thus, autophagy maintains homeostasis within an organism (Russel et al., 2014 ). Cancer can be defined as the uncontrollable growth and spread to damage normal body tissue. Autophagy modulation plays a dual role in tumour suppression and growth promotion. Autophagy is also able to regulate properties of cancer stem cells which maintains recurrence induction, stemness and its ability to resist cancer treatment reagents. The relationship between autophagy and cancer is complex and by understanding the mechanisms, we can take advantage of the mechanisms to help to treat cancers, especially the resistant cancers. There are recent studies which report that deficits in autophagy control are associated which metabolic stress, tumourigenesis and metabolic stress. Furthermore, studies have shown that autophagy is actively involved in the development or inhibition of cancer as well as the promotion of tumourigenesis (Yun & Lee, 2018). Depending on the type and stage of cancer, autophagy enhancement or inhibition will decrease stemness and survival of cancer cells. Autophagy inhibition prevents the degradation of damaged organelles or oxidative stressed cells, which may lead to cancer development. BIF-1, an important autophagic protein that seems to be abnormal or absent in various cancer types such as gastric cancer and colorectal cancer (Takayashi et al., 2007 ). This is one of many examples to show the link between autophagy components and cancer promotion. Autophagy as previously mentioned, can regulate tumour suppression, such as ATG5, when this is deficient, there is oncogenesis. Autophagy is able to prevent tumour formation through managing excess ROS which can be induced when there are damaged mitochondria. Hence, autophagy is at the centre and can inhibit tumour generation in certain cases. Since, we know that the relationship between autophagy and cancer is multifaceted, our research group focusses on glioblastoma multiforme specifically and how autophagy can be manipulated to result in a favourable outcome for the patient.