Exosomal protein deficiencies: how abnormal RNA metabolism results in childhood-onset neurological diseases

All genes are copied into short-lived RNA molecules, which are then translated to protein, forming the building box of the cells in the body. The transcription of DNA, the processing of pre-mRNA into mature mRNA (splicing), the degradation of mRNA and the translation into proteins are all tightly regulated. Both too much and too little of a certain RNA species could be dangerous for a cell, but our understanding of the mechanisms to fine-tune the RNA amounts is still limited. The regulation of gene expression (RNA metabolism) is of utmost importance for normal cell function, nerve cells, however, seem less able to cope with errors. This is illustrated by an increasing number of severe inherited neurological diseases of infancy or childhood caused by a defect in the RNA production machinery; these disorders are characterized by abnormalities of the development and/or the structure of the brain and the central nervous system.

The aim of this proposal is to study and characterize a novel form of RNA related neurological disorder due to dysfunction of a multi-protein complex called the human exosome. The role of the exosome consists of degradation and maturation of RNA. Mutations in the exosome subunit gene EXOSC3 were reported in patients with infantile-onset degeneration of the brainstem and cerebellum and muscle weakness due to spinal motor neuron dysfunction. We have recently identified mutations in EXOSC8, encoding another component of the human exosome, in infants with a similar severe neurological disease. In addition, these children also lose myelin in the brain and spinal cord, which normally coats and insulates nerves and serves to speed up signalling. We also identified a homozygous mutation in a novel gene RBM7 in a child with severe spinal motor neuron dysfunction. RBM7 is an interacting partner of the human exosome. The severe and isolated neurological symptoms in these children raise the possibility that the exosome is particularly important in neurons; however, there are still many open questions.

The aim of this proposal is to further characterize the role of the exosome in nerve cells and will address the following aspects:
1. Neuronal cells from patients: We will analyse RNA levels in patient derived skin cells and will transform them into neuronal cells with recently published methods. We will determine which RNA species are modified by changes in the exosome in the different cell types.
2. Genetically modified zebrafish: In parallel, we will create zebrafish models of exosomal protein deficiencies. By artificially removing single exosome components and by introducing mutations in zebrafish embryos we will explore its effect on the development and function of the brain and different types of neurons. In addition, we will also study the effect of modifying the exosome on myelin formation in zebrafish.

Our results will identify the most important roles of the exosome in regulating gene expression and why is it more damaging for neurons. Combining the data in human cells and zebrafish will enable us to better define the important genes and interacting partners of the exosome. By modifying exosome components or some of the here identified interacting factors we may discover potential pathways to alter RNA degradation or processing in neurons, which can be further developed as a therapeutic approach in exosomal diseases or in other types of neurodegenerative diseases caused by abnormal RNA function.

The degradation of mRNAs is an important regulatory step which controls gene expression. Three major degradation systems are responsible for the destruction of transcripts: the 5'-3' exoribonucleases, the nonsense-mediated mRNA decay machinery and the exosome. The exosome degrades and processes a variety of RNA species in the nucleus and in the cytoplasm of eukaryotic cells. The versatility and specificity of the exosome and its associated complexes regulate and maintain the fidelity of gene expression.

A novel group of RNA-associated neurological disorders has been identified with mutations in genes encoding components of the human exosome. Mutations in EXOSC3 were reported in pontocerebellar hypoplasia and spinal motor neuron abnormalities (PCH1). Our group identified EXOSC8 mutations in hypomyelination, spinal muscular atrophy and cerebellar hypoplasia. We showed that the hypomyelination is caused by imbalanced supply of myelin proteins due to disturbed degradation of AU-rich element containing mRNA.
The prominent neurological presentation in these cases raises the possibility that the exosome is particularly important in developing neurons. However, there are many open questions, which will be the focus of this project.

We will investigate which RNA types are affected in their expression or degradation by mutations in the exosome and whether this depends on the cell type and on which exosomal subunit is mutated. We will study human fibroblasts and neuronal cells, converted and differentiated from fibroblasts by recently published methods. Zebrafish will be used as in vivo animal model to analyse how alterations of the exosome affect the development of the nervous system and the myelination of neurons. By combining our results we will identify and further explore novel targets involved in neurodegenerative diseases affecting spinal motor neurons, cerebellar Purkinje cells and oligodendroglia.

Despite major advances in diagnosing inherited neurological diseases, our understanding of neuronal death pathways in the majority of disorders is still limited and there are very few effective therapies. The importance of RNA processing in neurodegeneration is highlighted with a rapidly increasing number of inherited human neurological diseases caused by mutations in proteins involved in mRNA metabolism including spinal muscular atrophy (SMA) and pontocerebellar hypoplasias (PCH).

A novel mechanism of RNA-associated neurodegeneration has been suggested by the identification of mutations in genes encoding human exosome components. Mutations in EXOSC3 were reported in pontocerebellar hypoplasia and spinal motor neuron abnormalities (PCH1), and our group recently identified mutations in a novel gene EXOSC8, encoding a core component of the human exosome in children with overlapping symptoms of cerebellar hypoplasia, spinal muscular atrophy and hypomyelination. We also detected mutations in another exosome related gene RBM7 in a child with the clinical presentation of spinal muscular atrophy.

We will investigate in this project how abnormal RNA metabolism due to defect of exosomal proteins (EXOSC8, EXOSC3, RBM7) affect different human cells (fibroblasts, induced neuronal cells, neurons) in vitro. We will explore gene expression (RNASeq) and RNA-protein interactions (CLIP) within the exosome. In parallel we will study different zebrafish models and will investigate whether the primary effect of exosome dysfunction alters neurodevelopment, or triggers neurodegeneration and which structures are most affected by deficient function of components of the exosome. Integration of the data in human cells and zebrafish will facilitate the recognition of important genes and proteins which lead to neuronal dysfunction. Modifications of the exosome and its interacting partners will be further studied with the aim to develop novel, RNA-based therapies for neurodegenerative conditions, such as SMA, pontocerebellar hypoplasias or demyelination, which represent a major cause of infantile mortality.

Expected main benefits
1. Our research will help to diagnose patients with exosomal protein deficiencies. Obtaining the genetic cause enables genetic counselling and prenatal or pre-implantation diagnostic testing.
2. Understanding the disease pathomechanism will provide important information on the role of RNA metabolism in neurons.
3. Our results may be applied to develop novel therapies in exosomal diseases but also potentially in other neurological conditions.
4. Patient organisations, national and international patient registries can be formed as a further benefit of our results.
5. For the broader UK economy appropriate diagnosis, prevention and therapy for disabling and life-threatening disorders will reduce the clinical burden of these disorders.
6. This research fits with the aims of Rare Disorders research, which has become increasingly important and receives priority for national, European and international organisations and funding bodies (NIHR, RD-CONNECT, IRDIRC).

Impact in research
1. Better characterization of developmental and degenerative aspects of exosomal protein deficiencies.
2. Defining whether defective RNA degradation or other effect of the exosome on regulating gene expression (splicing, RNA toxicity) is the main disease mechanisms in human exosome disease.
3. Document the potential power of combining RNASeq and CLIP to unveil abnormal RNA metabolism.
4. Evaluate a novel technology to directly transform and (without iPSCs) differentiate neurons from human fibroblasts.
5. Develop transgenic zebrafish lines with CRISPR/Cas9 technology.
6. Integration of human and zebrafish data to explore deficiencies of neuronal development.
7. Exploring whether manipulation of the exosome may be a feasible approach to alter the mechanism of neurodegenerative disease (SMA, PCH).