Structural and functional studies of the VAPB-PTPIP51 ER-mitochondria tethering proteins in neurodegenerative diseases
Lead Research Organisation:
King's College London
Department Name: Neuroscience
Abstract
Alzheimer's disease and related dementias, and amyotrophic lateral sclerosis (ALS) are major neurodegenerative diseases that afflict large proportions of our population and exert huge burdens on our healthcare system and economy. There are no cures for any of these diseases and for Alzheimer's disease and ALS, there are not even effective disease modifying treatments. There is therefore an urgent need to identify new therapeutic targets for treating these diseases.
Alzheimer's disease and ALS share several pathological features. These include damage to calcium concentrations in neurons, fat metabolism, mitochondria (the energy producers of the cell), transport of proteins and mitochondria within neurons, activation of cellular stress responses, alterations to a process termed autophagy which removes damaged proteins from neurons and damage to synapses which are the connections neurons make with each other to compute and relay signals. Finally, inflammation is seen in both diseases. The biological conundrum is how so many apparently disparate physiological processes are damaged collectively. The therapeutic challenge is selecting which of these different damaged processes to prioritize for drug discovery.
Recently, attention has focused on how two components of neurons, the endoplasmic reticulum (ER) and mitochondria, communicate and signal to each other in neurodegenerative diseases. This is because ER-mitochondria signaling regulates all of the functions described above that are damaged in these diseases. We and others have shown that ER-mitochondria signaling is perturbed in Alzheimer's disease and ALS, and we have identified a mechanism that facilitates this communication. It involves binding of the ER protein VAPB to the mitochondrial protein PTPIP51; this binding acts to "tether" ER with mitochondria to facilitate signaling. We have gone on to show that the VAPB-PTPIP51 tethers are disrupted in some neurodegenerative diseases and that this involves activation of a kinase enzyme termed GSK3beta. We have also identified another kinase enzyme (AMP kinase) that stimulates VAPB-PTPIP51 binding. These kinases modify proteins to influence their function and interactions via a process termed phosphorylation. One hypothesis is that GSK3beta and AMP kinase phosphorylate VAPB and/or PTPIP51 to regulate their binding and that this is perturbed in neurodegenerative diseases. Disruption of VAPB-PTPIP51 binding and ER-mitochondria signaling then leads to synapse damage, neuronal cell death and neurodegenerative disease. The aims of this project are to test aspects of this hypothesis. The objectives are:
1. To gain information on the structure of VAPB-PTPIP51 complexes since this knowledge will facilitate the design of drugs that can correct damaged tethers in neurodegenerative diseases.
2. To identify GSK3beta/AMP kinase phosphorylation sites in VAPB and PTPIP51 and to determine how this effects their binding.
3. To determine whether changes in VAPB and PTPIP51 phosphorylation occur in human post-mortem Alzheimer's disease and ALS tissues and in animal models of ALS.
4. To determine whether experimental disruption of the VAPB-PTPIP51 tethers in animals leads to neurodegenerative disease.
These findings will help us understand the role that the VAPB-PTPIP51 tethers play in Alzheimer's disease and ALS, and they will facilitate the design of potential therapeutics for neurodegenerative disease that target the VAPB-PTPIP51 interaction.
Alzheimer's disease and ALS share several pathological features. These include damage to calcium concentrations in neurons, fat metabolism, mitochondria (the energy producers of the cell), transport of proteins and mitochondria within neurons, activation of cellular stress responses, alterations to a process termed autophagy which removes damaged proteins from neurons and damage to synapses which are the connections neurons make with each other to compute and relay signals. Finally, inflammation is seen in both diseases. The biological conundrum is how so many apparently disparate physiological processes are damaged collectively. The therapeutic challenge is selecting which of these different damaged processes to prioritize for drug discovery.
Recently, attention has focused on how two components of neurons, the endoplasmic reticulum (ER) and mitochondria, communicate and signal to each other in neurodegenerative diseases. This is because ER-mitochondria signaling regulates all of the functions described above that are damaged in these diseases. We and others have shown that ER-mitochondria signaling is perturbed in Alzheimer's disease and ALS, and we have identified a mechanism that facilitates this communication. It involves binding of the ER protein VAPB to the mitochondrial protein PTPIP51; this binding acts to "tether" ER with mitochondria to facilitate signaling. We have gone on to show that the VAPB-PTPIP51 tethers are disrupted in some neurodegenerative diseases and that this involves activation of a kinase enzyme termed GSK3beta. We have also identified another kinase enzyme (AMP kinase) that stimulates VAPB-PTPIP51 binding. These kinases modify proteins to influence their function and interactions via a process termed phosphorylation. One hypothesis is that GSK3beta and AMP kinase phosphorylate VAPB and/or PTPIP51 to regulate their binding and that this is perturbed in neurodegenerative diseases. Disruption of VAPB-PTPIP51 binding and ER-mitochondria signaling then leads to synapse damage, neuronal cell death and neurodegenerative disease. The aims of this project are to test aspects of this hypothesis. The objectives are:
1. To gain information on the structure of VAPB-PTPIP51 complexes since this knowledge will facilitate the design of drugs that can correct damaged tethers in neurodegenerative diseases.
2. To identify GSK3beta/AMP kinase phosphorylation sites in VAPB and PTPIP51 and to determine how this effects their binding.
3. To determine whether changes in VAPB and PTPIP51 phosphorylation occur in human post-mortem Alzheimer's disease and ALS tissues and in animal models of ALS.
4. To determine whether experimental disruption of the VAPB-PTPIP51 tethers in animals leads to neurodegenerative disease.
These findings will help us understand the role that the VAPB-PTPIP51 tethers play in Alzheimer's disease and ALS, and they will facilitate the design of potential therapeutics for neurodegenerative disease that target the VAPB-PTPIP51 interaction.
Technical Summary
Alzheimer's disease and amyotrophic lateral sclerosis (ALS) are major neurodegenerative diseases, The both display damage to a number of disparate physiological functions that are regulated by ER-mitochondria signaling. This signaling is mediated by the VAPB-PTPIP51 ER-mitochondria "tethers". We have shown that the VAPB-PTPIP51 tethers are disrupted in Alzheimer's disease and ALS and that for three ALS genetic insults, this involves activation of the kinase GSK3beta. We also have evidence that AMP kinase stimulates VAPB-PTPIP51 binding. This project is to test two related hypotheses. Firstly, that GSK3beta and AMP kinase phosphorylate VAPB and/or PTPIP51 to regulate their binding, and secondly, that disruption of the VAPB-PTPIP51 interaction induces neurodegenerative disease in vivo. The objectives are:
1. To gain structural information of VAPB-PTPIP51 complexes using cryo-electron microscopy.
2. To identify GSK3beta/AMPK phosphorylation sites in VAPB and PTPIP51 and to determine their role in VAPB-PTPIP51 binding. This will involve i. quantitative mass spectrometry sequencing of VAPB and PTPIP51 immunopurified from HEK293 cells in which GSK3beta/AMPK activities are experimentally modulated; ii. monitoring VAPB-PTPIP51 binding in cellular assays in which identified sites are mutated and GSK3beta/AMPK activities are modulated
3. To create phospho-specific antibodies to these sites and use them to probe for changes in human post-mortem Alzheimer's disease and ALS tissues, and in TDP43 transgenic mice that develop ALS features using immunoblotting.
4. To analyse novel PTPIP51 knockout mice we have created that display neurodegenerative disease linked changes. This will involve motor tests and immunostaining for ALS pathologies.
The findings will help us understand the role that the VAPB-PTPIP51 tethers play in Alzheimer's disease and ALS, and facilitate the design of potential therapeutics that target the VAPB-PTPIP51 interaction.
1. To gain structural information of VAPB-PTPIP51 complexes using cryo-electron microscopy.
2. To identify GSK3beta/AMPK phosphorylation sites in VAPB and PTPIP51 and to determine their role in VAPB-PTPIP51 binding. This will involve i. quantitative mass spectrometry sequencing of VAPB and PTPIP51 immunopurified from HEK293 cells in which GSK3beta/AMPK activities are experimentally modulated; ii. monitoring VAPB-PTPIP51 binding in cellular assays in which identified sites are mutated and GSK3beta/AMPK activities are modulated
3. To create phospho-specific antibodies to these sites and use them to probe for changes in human post-mortem Alzheimer's disease and ALS tissues, and in TDP43 transgenic mice that develop ALS features using immunoblotting.
4. To analyse novel PTPIP51 knockout mice we have created that display neurodegenerative disease linked changes. This will involve motor tests and immunostaining for ALS pathologies.
The findings will help us understand the role that the VAPB-PTPIP51 tethers play in Alzheimer's disease and ALS, and facilitate the design of potential therapeutics that target the VAPB-PTPIP51 interaction.