Periodic Reporting for period 5 - NeuroInCellNMR (In-cell NMR monitoring of alpha-Synuclein aggregation in neuronal cells)
Periodo di rendicontazione: 2022-05-01 al 2022-10-31
The overarching goal of the NeuroInCellNMR project was to derive novel, high-resolution insights into early stages of the aggregation process of the Parkinson's disease protein alpha-synuclein (aSyn). In Parkinson's disease (PD), insoluble aggregates of aSyn in midbrain dopaminergic neurons are thought to exert strong cytotoxic effects and contribute to the demise of these cells during early disease stages, giving rise to the cardinal clinical symptoms of PD. Given the prevalence of PD as the second most common neurodegenerative disorder (after Alzheimer's disease) with over 10 million people affected worldwide and no curative treatments available thus far, efforts to delineate new angles on possible molecular causes of disease development and progression are urgently needed.
In our approach, we sought to investigate primary conformational rearrangements of aSyn that initiate the formation of toxic oligomers under particular cellular stress conditions, especially those that relate to the impairment of mitochondrial function, the production of increased levels of reactive oxygen species and general oxidative stress, which constitute hallmark features of many age-related, neurodegenerative disorders. One unique aspect of our approach was to use atomic-resolution methods such as in-cell nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopies to directly follow these events in live cells of different origins, including in human dopaminergic neurons that we derived from induced pluripotent stem cells (iPSCs). Within the initial part of our project, we set out to expose different cell types to oxidative stress conditions and to compare the phenotypic similarities or differences of induced aSyn aggregation with low-resolution light microscopy. Based on this information, we aimed at deriving high-resolution insights into the respective aggregation processes in the most disease-relevant cell types and under the most representative pathophysiological stress conditions in the later part of the project.
Thus, our main objectives were to gain a comprehensive structural understanding of conformational transitions that initiate aSyn aggregation and to determine their relevance for PD pathology. By studying these conformations directly in various cell types and with tools that allowed us to attain the highest possible levels of structural resolution, we hoped to generate information of unprecedented detail in native biological settings.
In our approach, we sought to investigate primary conformational rearrangements of aSyn that initiate the formation of toxic oligomers under particular cellular stress conditions, especially those that relate to the impairment of mitochondrial function, the production of increased levels of reactive oxygen species and general oxidative stress, which constitute hallmark features of many age-related, neurodegenerative disorders. One unique aspect of our approach was to use atomic-resolution methods such as in-cell nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopies to directly follow these events in live cells of different origins, including in human dopaminergic neurons that we derived from induced pluripotent stem cells (iPSCs). Within the initial part of our project, we set out to expose different cell types to oxidative stress conditions and to compare the phenotypic similarities or differences of induced aSyn aggregation with low-resolution light microscopy. Based on this information, we aimed at deriving high-resolution insights into the respective aggregation processes in the most disease-relevant cell types and under the most representative pathophysiological stress conditions in the later part of the project.
Thus, our main objectives were to gain a comprehensive structural understanding of conformational transitions that initiate aSyn aggregation and to determine their relevance for PD pathology. By studying these conformations directly in various cell types and with tools that allowed us to attain the highest possible levels of structural resolution, we hoped to generate information of unprecedented detail in native biological settings.
In the course of our project, we discovered a striking and altogether unexpected interaction of aSyn with a certain cellular organelle in a few selected cell types. These aSyn-lipid droplet (LD) interactions were strongly dependent on cellular identity, individual metabolic cell states and vastly exacerbated under cellular stress. Upon further investigating aSyn-LD interactions, we realised that binding to this organelle was prominently observed in all the cell types that we were investigating under appropriate metabolic conditions, including in iPCS-derived, human dopaminergic neurons. Eventually, we identified the molecular determinant that drove aSyn-LD binding and delineated that aSyn acted as a cholesteryl-ester (CE) sensor on LDs. We further showed that enhanced cholesterol uptake in neuronal and non-neuronal cells led to the accumulation of CE-rich LDs, which were efficiently bound by cellular aSyn.
Moreover, LD interactions led to the formation of membrane-induced aSyn oligomers, although without the characteristics of mature amyloid fibrils and clearly reversible in their abilities to disassemble and reassemble. Given the striking uniqueness of this behaviour and its implicative relevance for Parkinson's disease as a possible scenario that may 'wear out' over time and turn irreversible and toxic, we focused on aSyn-LD binding as a possible route to cellular toxicity in PD. This decision was further supported by cumulating evidence in the wider PD community that dysfunctional lipid homeostasis forms the basis of many aspects of PD pathology and that cellular aSyn lipid and membrane interactions may hold the key to its true biological function, which is still unknown. Tantalised by the prospects of being able to add a possible 'loss-of-physiological-function' angle to the widespread 'gain-of-toxic-function' aspect of aSyn's role in PD development, we embarked on deciphering the structural and functional details of aSyn-LD interactions in various cell types.
aSyn-LD interactions proved invaluably rich in providing numerous insights into the possible biological function(s) of aSyn (too many to summarise here) and they also formed the basis for new structural approaches at the interface of protein and membrane biology. Given the vastly different dynamic properties of soluble versus membrane-bound proteins, we faced the challenge to experimentally bridge this gap through the integrated use of different tools and methods. While we are making good progress in delineating the first experimentally derived, atomic-resolution model of aSyn bound to a truly native, cellular membrane compartment, we face persisting challenges to resolve the oligomeric state(s) of aSyn on LDs, both in terms of compositions of assemblies and their resulting architectures. Because these difficulties are technical in nature, we hope to resolve them within the near future.
Overall, NeuroInCellNMR provided us with a wealth of new information about physiological and pathological aspects about aSyn biology, with clear implications for strategies of novel therapeutic interventions. Given that the bulk of this work is still unpublished and, hence, not disseminated amongst the wider PD community, we are eagerly awaiting the general response to our findings and what colleagues will draw from our conclusions. Having worked in this field for many years, I am confident that our results will stir considerable excitement and offer an altogether new angle on a broadly relevant and utterly debilitating human disease.
Moreover, LD interactions led to the formation of membrane-induced aSyn oligomers, although without the characteristics of mature amyloid fibrils and clearly reversible in their abilities to disassemble and reassemble. Given the striking uniqueness of this behaviour and its implicative relevance for Parkinson's disease as a possible scenario that may 'wear out' over time and turn irreversible and toxic, we focused on aSyn-LD binding as a possible route to cellular toxicity in PD. This decision was further supported by cumulating evidence in the wider PD community that dysfunctional lipid homeostasis forms the basis of many aspects of PD pathology and that cellular aSyn lipid and membrane interactions may hold the key to its true biological function, which is still unknown. Tantalised by the prospects of being able to add a possible 'loss-of-physiological-function' angle to the widespread 'gain-of-toxic-function' aspect of aSyn's role in PD development, we embarked on deciphering the structural and functional details of aSyn-LD interactions in various cell types.
aSyn-LD interactions proved invaluably rich in providing numerous insights into the possible biological function(s) of aSyn (too many to summarise here) and they also formed the basis for new structural approaches at the interface of protein and membrane biology. Given the vastly different dynamic properties of soluble versus membrane-bound proteins, we faced the challenge to experimentally bridge this gap through the integrated use of different tools and methods. While we are making good progress in delineating the first experimentally derived, atomic-resolution model of aSyn bound to a truly native, cellular membrane compartment, we face persisting challenges to resolve the oligomeric state(s) of aSyn on LDs, both in terms of compositions of assemblies and their resulting architectures. Because these difficulties are technical in nature, we hope to resolve them within the near future.
Overall, NeuroInCellNMR provided us with a wealth of new information about physiological and pathological aspects about aSyn biology, with clear implications for strategies of novel therapeutic interventions. Given that the bulk of this work is still unpublished and, hence, not disseminated amongst the wider PD community, we are eagerly awaiting the general response to our findings and what colleagues will draw from our conclusions. Having worked in this field for many years, I am confident that our results will stir considerable excitement and offer an altogether new angle on a broadly relevant and utterly debilitating human disease.
Having completed this analysis within the framework of NeuroInCellNMR, we are proud of having uncovered novel aspects of aSyn biology that we believe are of profound and far-reaching impact. Reaching beyond the immediate relevance of our findings, we highlight a direct link between aSyn pathology and lipid metabolism, which currently emerges as an underlying theme in PD and related synucleinopathies, and is commonly supported by a great number of recent, independent publications pointing in the very same direction. In this light, our results perfectly align with current trends in the field and offer yet another argument for the importance of lipids and lipid metabolism for healthy neuronal function. Specifically, out data reconcile many previous observations in the field and, strikingly, raise the possibility that similar lipid-related defects may underlie general dementia symptoms in other neurodegenerative disorders. We are looking forward to communicating our findings with teh broader public and are excited to hear the response from colleagues in the field.