Aberrant protein phosphorylation in Down Syndrome and activity-dependent bulk endocytosis - a causal link?
Lead Research Organisation:
University of Edinburgh
Department Name: Centre for Integrative Physiology
Abstract
Brain cells (neurones) communicate by releasing chemical neurotransmitters. Neurotransmitters are stored in small spherical compartments within neurones called synaptic vesicles (SVs). When neurones communicate, SVs fuse with the outer surface of the neurone causing neurotransmitter release. After this occurs, SVs reform and are refilled with neurotransmitter. There are multiple mechanisms by which SV can reform and one of them is called activity-dependent bulk endocytosis (ADBE). We have identified some of the molecules that control ADBE inside neurones, one of them being calcineurin. Calcineurin modifies an enzyme called dynamin I to stimulate ADBE, whereas ADBE is switched off by other enzymes that reverse this modification.
The genetic disorder Down Syndrome (DS) results from the overproduction of a number of key molecules. Two of these molecules, RCAN1 and DYRK1A are predicted to interfere with the calcineurin-dependent modification of dynamin I. This means that they might also perturb ADBE. In agreement, the brains of both DS patients and mice that overproduce a similar complement of molecules to DS have a very similar appearance to neurones that have had ADBE inhibited by stopping calcineurin activity.
Therefore the overall hypothesis of the application is that the symptoms of DS are a result of a dysregulation of dynamin I function which leads to defects in ADBE.
The project outlined in the application will test this hypothesis by monitoring the effect of increasing or decreasing the level of either RCAN1 and DYRK1A in neurones on both dynamin I function and ADBE. In addition to looking at these molecules in isolation, we will determine if ADBE and dynamin I function normally in neurones taken from mice that have an increased complement of all DS related molecules.
This application will systematically investigate the link between dynamin I function, ADBE and DS. If it can be proven that some or all of the downstream defects in brain function seen in DS are a result of dysfunctional dynamin I or ADBE this will provide an exciting new research opportunity to combat the symptoms of the disease. Furthermore, since drugs that influence ADBE have already been identified, a range of new and potentially key therapeutics could be manipulated to treat the downstream consequences of the disorder.
The genetic disorder Down Syndrome (DS) results from the overproduction of a number of key molecules. Two of these molecules, RCAN1 and DYRK1A are predicted to interfere with the calcineurin-dependent modification of dynamin I. This means that they might also perturb ADBE. In agreement, the brains of both DS patients and mice that overproduce a similar complement of molecules to DS have a very similar appearance to neurones that have had ADBE inhibited by stopping calcineurin activity.
Therefore the overall hypothesis of the application is that the symptoms of DS are a result of a dysregulation of dynamin I function which leads to defects in ADBE.
The project outlined in the application will test this hypothesis by monitoring the effect of increasing or decreasing the level of either RCAN1 and DYRK1A in neurones on both dynamin I function and ADBE. In addition to looking at these molecules in isolation, we will determine if ADBE and dynamin I function normally in neurones taken from mice that have an increased complement of all DS related molecules.
This application will systematically investigate the link between dynamin I function, ADBE and DS. If it can be proven that some or all of the downstream defects in brain function seen in DS are a result of dysfunctional dynamin I or ADBE this will provide an exciting new research opportunity to combat the symptoms of the disease. Furthermore, since drugs that influence ADBE have already been identified, a range of new and potentially key therapeutics could be manipulated to treat the downstream consequences of the disorder.
Technical Summary
Synaptic vesicle (SV) recycling is essential for neurotransmission, the fidelity of which is maintained across a wide range of stimuli by multiple modes of SV recycling in nerve terminals. Activity-dependent bulk endocytosis (ADBE) is triggered during high intensity stimulation via dynamin I dephosphorylation by calcineurin (CaN). Dynamin I rephosphorylation is also essential for the continuation of ADBE.
Down syndrome (DS) is a genetic disease caused by a trisomy in chromosome 21. Two genes upregulated in DS are regulator of calcineurin 1 (RCAN1) and dual-specificity Yak1-related kinase 1A (DYRK1A). We predict that overexpression of these enzymes in DS will result in dysfunctional dynamin I phosphorylation, and defective ADBE. In agreement, multiple DS models and patients display neuronal defects consistent with dysfunctional SV recycling.
We hypothesise that trisomy causes a dysregulation of dynamin I phosphoryation, leading to defects in ADBE, that contributes towards the symptoms of DS.
This hypothesis is supported by key pilot data generated in our laboratory. First, a vesicle trafficking phenotype observed in DS (enlarged endosomes) can be replicated in central nerve terminals by inhibition of CaN. Second, RCAN1 interacts with multiple targets (including CaN) in nerve terminals. Third, manipulation of DRYK1A dynamin I phosphorylation sites alter interactions with nerve terminal proteins.
The project has three major aims
1) To establish the role of RCAN1 in dynamin I phosphorylation and ADBE
2) To establish the role of DYRK1A in ADBE
3) To determine a causal link between DS trisomy and ADBE
We will integrate state of the art live cell fluorescence imaging of nerve terminal function with protein biochemistry / cell biology techniques in concert with genetic models of DS. This will involve loss/gain of function strategies using overexpression and shRNA silencing of RCAN1 and DYRK1A in primary neuronal cultures to determine their role in both dynamin I phosphorylation and SV turnover. To determine the role of these enzymes in the appropriate disease context, we will monitor the same parameters using an established mouse model of DS trisomy. Establishing a causal link between DS and downstream defects in SV turnover will provide a key conceptual advance in understanding the symptoms associated with the disease. Furthermore, a demonstration that specific ADBE defects contribute towards the DS outcome will potentially provide a range of new and important tools with which investigate and treat the downstream consequences of the disorder.
Down syndrome (DS) is a genetic disease caused by a trisomy in chromosome 21. Two genes upregulated in DS are regulator of calcineurin 1 (RCAN1) and dual-specificity Yak1-related kinase 1A (DYRK1A). We predict that overexpression of these enzymes in DS will result in dysfunctional dynamin I phosphorylation, and defective ADBE. In agreement, multiple DS models and patients display neuronal defects consistent with dysfunctional SV recycling.
We hypothesise that trisomy causes a dysregulation of dynamin I phosphoryation, leading to defects in ADBE, that contributes towards the symptoms of DS.
This hypothesis is supported by key pilot data generated in our laboratory. First, a vesicle trafficking phenotype observed in DS (enlarged endosomes) can be replicated in central nerve terminals by inhibition of CaN. Second, RCAN1 interacts with multiple targets (including CaN) in nerve terminals. Third, manipulation of DRYK1A dynamin I phosphorylation sites alter interactions with nerve terminal proteins.
The project has three major aims
1) To establish the role of RCAN1 in dynamin I phosphorylation and ADBE
2) To establish the role of DYRK1A in ADBE
3) To determine a causal link between DS trisomy and ADBE
We will integrate state of the art live cell fluorescence imaging of nerve terminal function with protein biochemistry / cell biology techniques in concert with genetic models of DS. This will involve loss/gain of function strategies using overexpression and shRNA silencing of RCAN1 and DYRK1A in primary neuronal cultures to determine their role in both dynamin I phosphorylation and SV turnover. To determine the role of these enzymes in the appropriate disease context, we will monitor the same parameters using an established mouse model of DS trisomy. Establishing a causal link between DS and downstream defects in SV turnover will provide a key conceptual advance in understanding the symptoms associated with the disease. Furthermore, a demonstration that specific ADBE defects contribute towards the DS outcome will potentially provide a range of new and important tools with which investigate and treat the downstream consequences of the disorder.
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| Michael Cousin (Principal Investigator) |