Periodic Reporting for period 1 - RARE MAPS (Dynamic proteomic maps of stem cell-derived neurons as a mechanistic discovery pipeline for rare neurological disease)
Periodo di rendicontazione: 2021-02-01 al 2023-01-31
‘RARE MAPS’ aimed to address the challenge of investigating disease mechanisms for rare genetic neurological disorders. In Europe, rare diseases are those that affect less than 1 in 2000 people. However, the existence of up to 8000 different rare (mostly genetic) diseases means that collectively they are anything but rare; around 30 million people are estimated to suffer from a rare disease in Europe alone. This creates a huge medical burden, as most rare diseases have no known effective treatment. The bottleneck in developing targeted therapies is in understanding the underlying mechanisms of diseases at a cellular level. This is particularly challenging for neurological diseases, because the affected tissue (the nervous system) is inaccessible and difficult to model in the lab. Thus, it is important to develop new approaches to systematically study rare disease pathology.
The overarching goal of RARE MAPS is to develop a mechanistic discovery pipeline that can be widely applied to rare neurological disorders. To do this, RARE MAPS proposed to combine disease modelling using human induced pluripotent stem cells (hiPSCs) with a spatial proteomics method called ‘Dynamic Organellar Maps’ (DOMs), to understand how proteins within neurons are altered during disease. hiPSCs are cells that can differentiate into any cell type of the human body, including neurons. Gene editing technology can be used to introduce genetic mutations into hiPSCs, which can then be differentiated into neurons, providing a model of neurological disease in a dish. The DOMs method is then applied to reveal differences between healthy and diseased neurons, by providing information on the identity, quantity and localisation of proteins within the cell. Protein localisation is critical for protein function; cells consist of different membrane-bound compartments called organelles and proteins must be in the correct place to perform their function. Protein trafficking pathways make sure that proteins get to the right destinations. The importance of these pathways is highlighted by the fact that common neurological diseases, e.g. Parkinson’s disease, involve defects in protein trafficking.
Many rare genetic neurological disorders are also caused by problems with protein trafficking, for example, the childhood neurodegenerative disease, AP-4 deficiency syndrome. AP-4 deficiency syndrome is a form of hereditary spastic paraplegia, caused by mutations in a set of genes that create a protein complex called AP-4, which is required for protein trafficking. Using AP-4 deficiency syndrome as a test-case, the main objectives of RARE MAPS were: 1) to establish the DOMs approach in hiPSC-derived neurons; 2) to apply DOMs to study protein trafficking defects in whole brain tissue; 3) to investigate the mechanisms leading to AP-4 deficiency syndrome. The action was successful in meeting its objectives, leading both to the development of methods that can be widely used to study neurological disease, as well as to an increased understanding of the protein mislocalisation events that contribute to disease caused by AP-4 deficiency.
The overarching goal of RARE MAPS is to develop a mechanistic discovery pipeline that can be widely applied to rare neurological disorders. To do this, RARE MAPS proposed to combine disease modelling using human induced pluripotent stem cells (hiPSCs) with a spatial proteomics method called ‘Dynamic Organellar Maps’ (DOMs), to understand how proteins within neurons are altered during disease. hiPSCs are cells that can differentiate into any cell type of the human body, including neurons. Gene editing technology can be used to introduce genetic mutations into hiPSCs, which can then be differentiated into neurons, providing a model of neurological disease in a dish. The DOMs method is then applied to reveal differences between healthy and diseased neurons, by providing information on the identity, quantity and localisation of proteins within the cell. Protein localisation is critical for protein function; cells consist of different membrane-bound compartments called organelles and proteins must be in the correct place to perform their function. Protein trafficking pathways make sure that proteins get to the right destinations. The importance of these pathways is highlighted by the fact that common neurological diseases, e.g. Parkinson’s disease, involve defects in protein trafficking.
Many rare genetic neurological disorders are also caused by problems with protein trafficking, for example, the childhood neurodegenerative disease, AP-4 deficiency syndrome. AP-4 deficiency syndrome is a form of hereditary spastic paraplegia, caused by mutations in a set of genes that create a protein complex called AP-4, which is required for protein trafficking. Using AP-4 deficiency syndrome as a test-case, the main objectives of RARE MAPS were: 1) to establish the DOMs approach in hiPSC-derived neurons; 2) to apply DOMs to study protein trafficking defects in whole brain tissue; 3) to investigate the mechanisms leading to AP-4 deficiency syndrome. The action was successful in meeting its objectives, leading both to the development of methods that can be widely used to study neurological disease, as well as to an increased understanding of the protein mislocalisation events that contribute to disease caused by AP-4 deficiency.
The project was divided into three work packages (WP). WP1 focused on the development of a workflow for applying the DOMs method to hiPSC-derived neurons. DOMs is a proteomics method that uses mass spectrometry (MS) to identify and quantify proteins in different parts of the cell. The data can be used to create a so-called ‘map of the cell’ that displays the localisation of proteins in different organelles. By comparing maps made from healthy and diseased cells, we can identify proteins that are found in the wrong part of the cell during disease, which may contribute to cellular pathology. As part of WP1, the fellow worked as a team with other members of the Borner Lab to greatly improve the performance of DOMs using an MS technique called data-independent acquisition (DIA). The optimised method can now provide localisation data for around 6000 proteins in a single experiment. This work is currently under review at an Open Access journal. The method was then tested on a well-characterised HeLa cell model of AP-4 deficiency, which demonstrated excellent sensitivity and specificity for detecting known protein mislocalisation events. Finally, the method was applied to generate maps from control and AP-4-deficienct hiPSC-derived neurons. Data analysis is still ongoing, but preliminary analysis demonstrated proof-of-principle by detecting the mislocalisation of a known AP-4 cargo protein, ATG9A. Full analysis of the dataset is expected to reveal candidates for neuron-specific AP-4 cargo proteins. In addition to DOMs, DIA MS was used to quantify thousands of proteins in healthy and AP-4-deficient neurons, revealing protein abundance changes that may be relevant to disease.
In WP2 MS-based proteomics was used to investigate the effects of AP-4 deficiency on proteins in the whole brain. The study made use of a mouse model of AP-4 deficiency syndrome. These mice can be used as a model of human disease because they develop brain abnormalities and movement deficits, which are consistent with the symptoms of human AP-4 deficiency syndrome. The study used whole brain proteomics, DOMs and proteomic analysis of vesicles (transport carriers within the cell), to generate a complete picture of the dysregulation of protein abundance and localisation that occurs in AP-4-deficient brains. Cross-comparison to the data from hiPSC neurons revealed consistent changes between both model systems, providing strong evidence for their relevance to disease.
WP3 focused on investigating the mechanisms of AP-4 deficiency syndrome, in particular examining the functional role of novel and known AP-4-associated proteins. A major success here was the identification of an enzyme called DAGLB as a novel cargo protein of AP-4 vesicles. In AP-4-deficient neurons there is disruption of the transport pathway that delivers DAGLB to the axon, where it is required to produce a signalling molecule called 2-AG (an endocannabinoid), which promotes axon growth. These findings suggested that neurodevelopmental defects in AP-4-deficient patients may result from defects in endocannabinoid signalling. In support of this, treatment of AP-4-deficient neurons with a drug that increases 2-AG levels led to improvements in axon growth, suggesting a possible therapeutic avenue for AP-4 deficiency. This work was published in the Open Access journal Nature Communications.
In WP2 MS-based proteomics was used to investigate the effects of AP-4 deficiency on proteins in the whole brain. The study made use of a mouse model of AP-4 deficiency syndrome. These mice can be used as a model of human disease because they develop brain abnormalities and movement deficits, which are consistent with the symptoms of human AP-4 deficiency syndrome. The study used whole brain proteomics, DOMs and proteomic analysis of vesicles (transport carriers within the cell), to generate a complete picture of the dysregulation of protein abundance and localisation that occurs in AP-4-deficient brains. Cross-comparison to the data from hiPSC neurons revealed consistent changes between both model systems, providing strong evidence for their relevance to disease.
WP3 focused on investigating the mechanisms of AP-4 deficiency syndrome, in particular examining the functional role of novel and known AP-4-associated proteins. A major success here was the identification of an enzyme called DAGLB as a novel cargo protein of AP-4 vesicles. In AP-4-deficient neurons there is disruption of the transport pathway that delivers DAGLB to the axon, where it is required to produce a signalling molecule called 2-AG (an endocannabinoid), which promotes axon growth. These findings suggested that neurodevelopmental defects in AP-4-deficient patients may result from defects in endocannabinoid signalling. In support of this, treatment of AP-4-deficient neurons with a drug that increases 2-AG levels led to improvements in axon growth, suggesting a possible therapeutic avenue for AP-4 deficiency. This work was published in the Open Access journal Nature Communications.
RARE MAPS provides a new approach for investigating the molecular mechanisms of neurological disorders, with a focus on protein mislocalisation events, which are often overlooked. While the action focused on the major unmet medical need of rare diseases, the approach could also be applied to investigate common neurodegenerative diseases like Alzheimer’s disease. The project has provided the first comparative application of DOMs to hiPSC neurons for the study of disease mechanisms, as well as the first application of DOMs to whole tissues. Analysis of these large datasets is still ongoing but expected to yield many insights into the molecular basis of the childhood neurological disease AP-4 deficiency syndrome. The discovery of a new AP-4 cargo protein, DAGLB, has already been used to inform drug discovery studies and suggests that modulation of endocannabinoid signalling could provide a new therapeutic avenue for AP-4 deficiency syndrome.