Identification of novel genes in synaptic communication

Our brain consists of over a 100 billion neurons, organized into neuronal circuits that transmit electrical pulses. Neurons in these circuits communicate by releasing transmitters from vesicles and to maintain communication, neurons dependent 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 these molecular mechanisms of neuronal communication using morphological and functional assays.

Given the experimental advantages, we are using Drosophila as a model.  Flies are ideally suited for our studies as genes implicated in synaptic transmission are very well conserved across species.  As a general strategy, we employ different types of genome-wide genetic screens in Drosophila to identify components affecting synaptic function and subsequently we analyze the function of these genes in detail at the synapse. 

We are following several complementary methodologies to gain further insight in the processes of synaptic development and neuronal communication. In one such approach we hypothesize that based on phenotypes in human, neurological disease genes may affect synaptic transmission. To identify such genes, we are testing a large number of genes implicated in neurological disease for defects in synaptic function. Here we are taking advantage of a collection of RNAi lines to knock down the genes only in the nervous system (VDRC) and then test the function of the genes using a variety of functional assays, such as live imaging of vesicles or electrophysiology. The ability to combine human disease phenotypes with genetic screening using simple assays is relatively unique and will most likely provide new insights into common processes that underlie neurological disease progression and synaptic transmission. 

In another approach we are screening the Drosophila genome by feeding flies chemical mutagens (EMS). Flies that carry individual mutations in their genome are then tested using simple  phenotypic assays, including behaviour and electrophysiological recordings of the photoreceptors in the eye.  Using this strategy we have already isolated several genes, but we are continuously devising novel strategies to identify additional components.  Classical mapping strategies combined with whole genome sequencing then allow us to quickly identify the genes affected.    

 

 

Functional regulation of synaptic vesicle release sites

One of the proteins we identified in our EMS screens is Elongator Protein 3, that is involved in histone acetylation in the nucleus to regulate chromatin structure and gene expression.  Interestingly, we found that Elp3 in neurons is present in the cytoplasm and concentrates at synapses. Using morphological and biochemical studies, our data indicate that Elp3 is necessary and sufficient to acetylate Bruchpilot, a protein that is present at synaptic vesicle release sites.  Bruchpilot is a large protein and individual Bruchpilot protein strands join at their N-terminal ends near the synaptic membrane and send their strands into the cytoplasm to contact synaptic vesicles.  The assembled structure thus has the appearance of a mushroom.  We now find that reduced acetylation of the cytoplasmic ends of Bruchpilot results in the ‘mushroom cap’ to be larger, where the protein sends extensive tentacles into the cytoplasm, contacting more synaptic vesicles, resulting in more neurotransmitter release.  Thus, Elp3 dependent acetylation not only orchestrates chromatin structure  in the nucleus, but in neurons, by acetylating Bruchpilot, Elp3 is also a critical regulator of synaptic transmission (Miskiewicz et al., 2011).  Interestingly, in collaboration we recently found Elp3 polymorphisms in human are associated with Amyotrophic Lateral Sclerosis (Simpson et al., 2009) and we are now analyzing how this function of Elp3 is implicated in this devastating neurological disease

 

 

Skywalker and the regulation of vesicle protein rejuvenation

During intense activity, neurons can release massive amounts of neurotransmitters and to ensure continuous neuronal communication the majority of synaptic vesicles appear to recycle through a pathway involving clathrin. Using genetics in combination with imaging, super-resolution imaging, electron microscopy, electrophysiology and biochemistry, but also acute protein inactivation called FlAsH-FALI (Kasprowicz et al., 2008; Venken et al., 2008), we are studying the molecular mechanisms of vesicle formation and transport at the synapse.  Our focus is mostly on novel proteins we identify in our genome wide screen approaches.

While we are pursuing several endocytic factors, one such novel protein is Skywalker.  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 we are using fruit flies to study this feature in detail.

We find fly Sky harbours GTPase stimulatory activity allowing it to inactivate specific Rab proteins.  At the neuromuscular synapse, we find that this activity is needed to inhibit trafficking of synaptic vesicles via endosomes that are Rab5 and 2xFYVE positive, suggesting they are sorting stations.  Indeed, using genetic analyses in combination with chimeric synaptic vesicle proteins that we expressed, we find that at these endosomes, dysfunctional ubiquitinatied synaptic vesicle proteins are sorted for degradation, resulting in synaptic vesicles with more functional proteins.  This in turn results in a larger readily releasable pool and increased neurotransmitter release (Uytterhoeven et al., 2011).  This novel regulatory mechanism endows neurons to control synaptic plasticity and our future studies are aimed at understanding the regulatory mechanisms that govern Sky function and the sorting of synaptic vesicle components at synaptic endosomal compartments.  

 

 

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, in direct effects of disease genes on synaptic processes are not well characterized. For genes implicated in Parkinson’s disease evidence for a role in synaptic vesicle trafficking is starting to accumulate. We previously found that Pink1, via a role in mitochondria, controls the mobilization of synaptic vesicles that reside in the reserve pool (Morais et al., 2009) and also Parkin is implicated in synaptic function.  Furthermore, different groups found that both LRRK2 and alpha-synuclein have been implicated in regulating endocytosis of synaptic vesicles.   Hence, analysis of Parkinson related genes may yield additional valuable insight into the mechanisms of synaptic vesicle recycling.

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.  We are studying the role of LRRK at the synapse and are analyzing how the kinase activity of this protein is controlling synaptic transmission. We are also analysing the function of the E3 ubiquitin ligase Parkin at the synapse and we are studying the kinase Pink1 and Parkin in the control of synaptic mitochondrial integrity. Our work is not only revealing the cellular function of these disease related proteins but our studies are also probing into the molecular pathways of synaptic function.

 

 

Icelandic boiling mud pools, where mud bubbles represent new synaptic vesicles forming at the plasma membrane

Similar to a bat propelling a baseball in the air, mitochondrial ATP propels synaptic vesicles from a reserve pool to synaptic release sites.

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Elp3 dependent acetylation of Bruchpilot regulates the morphology and function of synaptic release sites that have the appearance of mushrooms in Drosophila.

Soap bubbles are reminiscent of synaptic vesicles that form at the plasma membrane during synaptic vesicle endocytosis.