We study how the brain operates and how brain function is affected by neurodegenerative diseases, specifically Parkinson’s disease and Tauopathies. We use fruit flies and human neurons for our studies and resort to genetics, biochemistry, electron microscopy, imaging and electrophysiology to analyse neuronal communications and synaptic survival.
Synaptic communication in fly and human neurons
Neurons depend on evolutionary conserved mechanisms that ensure a continuous supply of synaptic vesicles to release sites; these include endocytosis, vesicle mobilization and trafficking, calcium signalling, etc. Our long term goal is to understand how the molecular mechanisms of neuronal communication are affected in the diseased brain using morphological and functional assays and to that end, we are studying a number of specific aspects of synaptic function, also in the context of major neurodegenerative conditions including Parkinson’s disease and Alzheimer’s disease.
Our studies involve genetic screening in fruit flies for novel pathways and interacting components as well as in depth analyses of neuronal function. We conduct these studies in flies but also in human neurons that we derive from embryonic stem cells. Flies are an ideal system for ‘gene discovery’ because the pathways we study are in general very well conserved. We complement our studies with elaborate neuronal cell biology in fly and human neurons, adding complexity and enabling us to assess the molecular mechanisms of the pathways we discovered in healthy and diseased situations.
Vesicle recycling at synapses
During intense activity, neurons can release massive amounts of neurotransmitters and to ensure continuous neuronal communications synaptic vesicles recycle. Using genetics in combination with imaging, super-resolution imaging, electron microscopy, electrophysiology and biochemistry, but also acute protein inactivation called FIASH-FALI, we are studying the molecular mechanisms of vesicle formations and transport at the synapse. Our focus is mostly on novel proteins we identify in our genome wide screen approaches, but we are also including detailed analyses of the ‘classical players’.
The regulation of protein turnover at synapses
When vesicles intensely cycle, the proteins resident at the synapse may become damaged. Mechanisms to deal with these damaged proteins at synapses must exist, and we are studying these mechanisms in the lab. One of the proteins we are studying is Skywalker, a protein we show regulates the trafficking of synaptic vesicles to synaptic endosomes. the Sky protein harbours homologues in nematodes and man (TBC1D24) but has not been studied in detail. Interestingly, in human, mutations in the gene cause epilepsy and DOOR syndrome, and we are using fruit flies to study this feature in detail.
While Skywalker and its interactor regulate the degradation of synaptic vesicle-associated proteins by directing the synaptic vesicles to endosomes and later to lysosomes, we are also studying additional mechanisms of protein turn over. Or example processes related to autophagy, where membranes engulf debris and send this junk to the lysosome for degradations. We are conducting innovative in vitro and in vivo screens to identify novel components in these pathways specifically at synapses, and we are also developing new tools to directly visualize the turnover of synaptic debris in vivo in addition we also developing new electron microscopy techniques to be able to follow the process at synapses. Finally, our goal is to not only assess protein turn over mechanisms at fly synapses but also at synapses of human neurons.
Parkinson’s disease and vesicle traffic
Neurological and psychiatric illness is thought to arise, at least in part, by imbalances in synaptic communication within neuronal circuits. However, the effects of disease genes on synaptic processes are not well characterized. For genes implicated in Parkinson’s disease, including pink1, parkin alpha-synuclein and LRRK2, evidence for a role in synaptic vesicle trafficking but also in the regulation of protein turn over is quickly accumulating. We previously found that Pink1, via a role in mitochondria, controls the mobilization of synaptic vesicles that reside in the reserve pool and also Parkin is implicated in synaptic function by ubiquitinating mitochondrial and synaptic proteins. Furthermore, different pieces of evidence indicate that LRRK2 and alpha-synuclein are implicated in specific aspects of the synaptic vesicle cycle. Hence, analysis of Parkinson related genes may yield additional valuable insight into the mechanisms of synaptic function.
We are therefore studying a role for Parkinson related genes in synaptic function using large scale biochemical and genetics approaches, including interaction screens and modifier screens. This strategy has allowed us to identify pathways and processes that have an impact on the defects caused by loss of function of Parkinson related genes. For example, we identified heix as a very strong enhancer of defects seen in pink1 mutants. Heix is a prenyltransferase involved in vitaminK2 synthesis in mitochondria, and we were able to show that vitaminK2 can act as a mitochondrial electron carrier molecule that can be used to improve mitochondrial function and ATP production particularly in mutant animals that suffer from mitochondrial defects (including pink1 mutants). This and several other genetic modifiers are currently under investigation, with the ultimate hope of testing these models also in patients. Furthermore, by performing additional genetic interaction screens and large scale biochemical approaches we are in the process of building a genetic and functional network of genes and pathways that interact with different Parkinson disease-relevant genes.
In recent years several new Parkinson releated genes have been identified, and we are conducting systematic studies to assess if and how the proteins encoded by these genes are affecting the synapse, both in flies and in human neurons. For example, we have studied the role of LRRK, the fruit fly orthologue of human LRRK2, at the synapse and we have analyzed how the kinase activity of this protein controls synaptic function. We found that EndophilinA, a protein essential for synaptic vesicle endocytosis, is directly phosphorylated by LRRK/LRRK2. Using complementary approaches, in vitro and in vivo, we show that this phosphorylation event directly regulates EndophilinA function at the synapse. We already showed that synaptic vesicle endocytosis is affected, but likely other processes are affected as well. Studies similar to this are also underway for other PD-related genes in the hope to further expand the synaptic PD-interactome to further define how these proteins impact on synaptic function and on dopaminergic neuron health.
Taken together, our work is not only revealing the cellular function of these Parkinson disease-related proteins but our studies are also probing into the molecular pathways of synaptic function.