Report
Teaser, summary, work performed and final results
Periodic Reporting for period 2 - RobustSynapses (Maintaining synaptic function for a healthy brain: Membrane trafficking and autophagy in neurodegeneration)
Teaser
Neurodegeneration is characterized by misfolded proteins and dysfunctional synapses. Synapses are often located very far away from their cell bodies and they must therefore largely independently cope with the unfolded, dysfunctional proteins that form as a result of synaptic...
Summary
Neurodegeneration is characterized by misfolded proteins and dysfunctional synapses. Synapses are often located very far away from their cell bodies and they must therefore largely independently cope with the unfolded, dysfunctional proteins that form as a result of synaptic activity and stress. My hypothesis is that synaptic terminals have adopted specific mechanisms to maintain robustness over their long lives and that these may become disrupted in neurodegenerative diseases. Recent evidence indicates an intriguing relationship between several Parkinson disease genes, synaptic vesicle trafficking and autophagy, providing an excellent entry point to study key molecular mechanisms and interactions in synaptic membrane trafficking and synaptic autophagy. We are using novel genome editing methodologies enabling fast in vivo structure-function studies in fruit flies and we are using differentiated human neurons to assess the conservation of mechanisms across evolution. In a complementary approach we are capitalizing on innovative in vitro liposome-based proteome-wide screening methods as well as in vivo genetic screens in fruit flies to find novel membrane-associated machines that mediate synaptic autophagy with the ultimate aim to reveal how these mechanisms regulate the maintenance of synaptic health. Our work not only has the capacity to uncover novel aspects in the regulation of presynaptic autophagy and function, but it will also reveal mechanisms of synaptic dysfunction in models of neuronal demise and open new research lines on mechanisms of synaptic plasticity.
The basic cell biology of neurotransmission has been studied since long, but how synaptic transmission is maintained over the lifetime of a postmitotic neuron is not well understood. The polarized nature of neurons necessitates synapses to locally cope with cellular debris while maintaining robust transmission. Understanding the maintenance of synaptic function will also have significant implications for human health. Synaptic transmission defects are linked to the early stages of neurodegenerative disorders. Neurodegeneration is extremely debilitating and can affect motoric (ALS, PD) and cognitive systems (Alzheimer\'s, Frontotemporal dementia, late stage PD). A cure for these disorders does not exist, and only symptomatic treatment based on neurotransmitter substitution is available for PD, and to a limited extend for Alzheimer\'s. Although more than 65 million people in the world suffer from a neurodegenerative disorder, the grim perspective for the advanced stages of such diseases is that only palliative help can currently be offered. The work we propose will help in starting to change this outlook and open avenues to new therapeutic inroads to combat these disorders. We aim to reveal and characterize novel targets to modulate synaptic vesicle trafficking and autophagy, and based on our fly and human neuron models we will be able to make predictions about the specific synaptic pathways and proteins that are affected in disease. With the great strides in human brain imaging and early risk determination that are being made, an increasing number of more young, functional and healthier people will be diagnosed with these scourges. As a result, the focus given to therapy must increase, changing society\'s fatalistic attitude.
The overarching goal of this project is to unveil mechanisms that synapses use to rejuvenate, to study the importance of these mechanisms in relation to synaptic protein recycling and synaptic robustness, and to understand how these processes are connected to disease.
Work performed
Neurodegeneration, including PD, is characterized by synaptic dysfunction. Different proteins that are implicated in PD are linked to vesicle trafficking and autophagy, including ATG5, ATG7, Synaptojanin, Parkin and LRRK2. Some of these genes, ATG5 and ATG7, are members of the core-autophagy machinery while others, Synaptojanin, Parkin and LRRK2 are connected to endocytosis and membrane trafficking at the synapse. We uncovered that that these PD genes are also involved in the regulation of autophagy at synapses. In addition, we uncovered intriguing connections between several PD genes and EndophilinA (EndoA), found to be essential for synaptic endocytosis and neuronal survival and variation at the EndoA locus is also a risk factor for PD. We found that EndoA is also critical for synaptic autophagy, suggesting endocytosis and vesicle trafficking may play an important role in autophagy. In confusion, our studies of these PD-relevant proteins has yielded invaluable insights into the mechanisms involved in autophagy specifically at the synapse. Our studies included standard molecular biology and imaging approaches, but we also developed new imaging strategies based on correlative light and electron microscopy, allowing us to study autophagosome formation at synapses at unprecedented resolution. In addition, to assess if the synaptic autophagic mechanisms we uncovered in fruit flies were evolutionary conserved, we also generated human neurons and confirmed out findings there as well.
Autophagy requires extensive membrane shaping as to engulf cytoplasmic regions for degradation and we have deployed a novel in vitro screen approach aimed at identifying proteins involved in these processes. We expressed all fly proteins in E. coli using standard expression procedures, cracked the cells open, applied the supernatant to labeled giant unilamellar vesicles (GUVs), and used fluorescence-microscopy-screening to determine if the membranes of the GUVs are deformed. There was no need to purify the proteins as protein lysate from wild-type E. coli does not display GUV membrane deformation activity. We have prepared special protein expression libraries and found 204 novel membrane deforming proteins. Interestingly, we find several proteins linked to protein homeostasis and autophagy, including a significant enrichment of 7 chaperones; in our work we are now elucidating how these chaperones deform membranes and how this process is connected to protein homeostasis at synapses. We were successful in this approach as we were able to show that Hsc70, one of the most abundant synaptic chaperones, is involved in the invagination of the endosomal membrane to mediate endosomal microautophagy. This is exciting as we were able to show that more than 50% of the synaptic proteins have the capacity of being turned over by this process, including major disease released proteins such as alpha-synuclein and Tau. For our studies we developed several new fluorescent markers that allow us to follow microautophagy at synapses as well as follow protein turn-over at synapses.
Final results
If we succeed in finalising the work proposed in this project, we will have significantly advanced the understanding of the in vivo mechanisms of synaptic demise and elucidate pathways of autophagy at synapses in healthy but also diseased neurons, including PD, the most common movement disorder. Our work will reveal novel components and mechanisms of synaptic protein turn-over and rejuvenation with a link to synapse robustness. Finally, we will identify molecular mechanisms by which the neurological disease genes impinge on the process of vesicle recycling, with the ultimate goal to identify targets for novel therapeutic strategies.