ER-mitochondria interactions and neurodegeneration

Abstract

Physical membrane contact sites between the ER and mitochondria play a critical role in regulating a variety of processes including calcium signalling, lipid exchange, controlling mitochondrial dynamics and cell death signalling. These contact sites are formed at specialised regions of membrane, termed mitochondrial associated membranes or MAMs, that are enriched for a group of proteins acting as tethers to hold the ER and mitochondria at appropriate distances from one another. The distance of these junctions is usually defined between 10-30 nm but can vary in response to certain cellular conditions and it is believed that heterogeneity in the distance of the contact sites may be reflective of different protein compositions or activities at these sites. Abnormal alterations to ER-mitochondria contacts are observed in numerous neurodegenerative disorders including Alzheimer’s, Parkinson’s and amyotrophic lateral sclerosis (ALS) and therefore, it is believed that a dysfunction to the MAMs may be a common pathogenic mechanism underlying neuronal cell death. Due to the associated dysfunction of organelle contact sites in neurodegenerative disorders, the ability to detect these structures could provide critical information on the pathogenic mechanisms of neuronal cell death. Existing techniques for detecting MAMs have numerous limitations or are restricted to fixed samples. The aim of this thesis was to develop a fluorescent-based method for the visualisation of contact sites that can also be applied to living systems to study organelle contact dynamics. Here, we have generated split fluorescent Venus fragments targeted to the ER and outer mitochondrial membrane respectively, as the basis for our bi-fluorescence complementation (BiFC) system for the detection and quantification of MAMs in living cells. The principle of this technique relies on close spatial proximity of the reporter probes for the restoration of the fluorescent protein and the emission of a detectable signal. Validation of this method highlighted several advantages over existing methods of detecting ER-mitochondria contacts and we were able to report on changes in agreement with previously published, high-resolution electron microscopy studies. Adaptations to the technique allow for the detection of other organelle contact sites by varying the targeting sequence of the complementary Venus fragments. As these reporter proteins detect junctions of a maximum distance of around 6-10 nm, we could use the BiFC system to correlate the Venus signal with specific functions of the MAMs to try and elucidate the functional significance of these particularly tight contact sites. Our results suggest that some of these contact sites may represent sites of actively dividing mitochondria. Furthermore, our results indicated that these tight ER-mitochondria contacts are formed on a sub-population of mitochondria of higher than average resting membrane potential and mitochondrial calcium levels, which may indicate differences in the bioenergetic state or the health of mitochondria with tight ER-mitochondria contact sites. Finally, this technique was used to investigate the role of a MAM-enriched protein, VAPB, of which a proline to serine missense mutation is associated with a dominantly inherited form of ALS termed ALS8. The data shows that expression of the mutant disease-linked VAPBP56S significantly increases the mean number of contact sites per cell whereas altering the levels of wild-type VAPB has no significant effect. This finding suggests that the expression of VAPBP56S induces abnormalities in ER-mitochondria tethering but it is still unclear whether this is through direct binding properties of mutant VAPB or through an indirect secondary mechanism. As the MAMs regulate many of the pathways that are commonly perturbed in neurodegenerative disorders, alterations in ER-mitochondria contact sites may represent a key early pathogenic event in ALS.