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

Lead Research Organisation: Newcastle University
Department Name: Institute of Genetic Medicine


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.

Technical Summary

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.

Planned Impact

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).


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100,000 Genomes Project Pilot Investigators (2021) 100,000 Genomes Pilot on Rare-Disease Diagnosis in Health Care - Preliminary Report. in The New England journal of medicine

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Bansagi B (2017) Genetic heterogeneity of motor neuropathies. in Neurology

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Bansagi B (2016) Phenotypic convergence of Menkes and Wilson disease. in Neurology. Genetics

Description as Chair of the mitochondrial group within the ERN-NMD I participate in endorsing and making guidelines for mitochondrial diseases
Geographic Reach Europe 
Policy Influence Type Influenced training of practitioners or researchers
Impact As the Chair of the mitochondrial group within the EURO-NMD reference network I participate in making diagnosis and management of patients with mitochondrial diseases harmonised in Europe
URL https://ern-euro-nmd.eu/
Description Horizon 2020
Amount € 15,000,000 (EUR)
Organisation European Union 
Sector Public
Country European Union (EU)
Start 01/2018 
End 12/2023
Description Investigate new treatment options in zebrafish models of mtDNA depletion syndromes
Amount £62,432 (GBP)
Organisation The Lily Foundation 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2019 
End 07/2021
Title BN-PAGE 
Description functional analysis of human patient cell lines 
Type Of Material Cell line 
Provided To Others? No  
Impact we revealed the pathomechanism of mitochondrial disease in 20 patients 
Title TRMU cells 
Description we have obtained cells (fibrobalsts and myoblasts) from a patient and established a special technique to study 2-thiolation of mt-tRNAs 
Type Of Material Cell line 
Provided To Others? No  
Impact We are currently investigating the possible role of 2-thiolation as a possible disease mechanism in reversible COX deficiency as part of the project 
Title iPSC neuronal conversion 
Description We established the conversion of neurons from iPSCs of patients with mitochondrial disease. 
Type Of Material Cell line 
Year Produced 2020 
Provided To Others? No  
Impact We are currently working on these cells and we will publish our results. 
Title induced neuronal progenitor cells 
Description We can successfully convert human finroblasts into induced neuronal progenitor cells. 
Type Of Material Model of mechanisms or symptoms - human 
Provided To Others? No  
Impact We have already converted 4 patient and 2 contol cell lines into induced neuronal progenitor cells. Currently the analysis of mitochondrial function is in progress in these cells. 
Title studying the neuromuscular junction 
Description co-investigator on a multi-user equipment funded by Wellcome to study electrophysiology of the neuromuscular junction 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2018 
Provided To Others? Yes  
Impact no impact yet, equipment is currently being set up 
Title zebrafish 
Description I used zebrafish to model human disease. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact Published a paper (Boczonadi et al. 2014) 
Title bioinformatic analysis of RNAseq 
Description performed RNAseq in several human cell and muscle samples and analysed different parameters to gain understanding of the metabolic signature of neurogenetic diseases 
Type Of Material Data analysis technique 
Year Produced 2017 
Provided To Others? No  
Impact papers are currently in progress 
Title proteomic analysis of cells/tissues 
Description performed proteomic analysis of paatient cells and skeletal muscle samples 
Type Of Material Data analysis technique 
Year Produced 2017 
Provided To Others? Yes  
Impact papers in progress 
Description Consequitur - cohort of patients from Turkey for WES 
Organisation Dokuz Eylül University
Country Turkey 
Sector Academic/University 
PI Contribution We collaborate with Dr. Yavuz Oktay and Dr. Semra Hiz on identiying new disease genes in consanguineous Turkish families with various neurogenetic diseases.
Collaborator Contribution Collected 400 families and DNA samples, perfomred phenotyping
Impact We are currently writing abstracts for conferences from the first results and drafting papers.
Start Year 2016
Description Identifying novel disease genes in hereditary motor neuropathies 
Organisation University of Miami
Country United States 
Sector Academic/University 
PI Contribution We have identified mutations in a novel disease gene in a family with autosomal dominant hereditary motor neuropathy.
Collaborator Contribution The collaborators also had one family with another mutation in the same gene.
Impact We have published a paper together in AJHG.
Start Year 2014
Description Metablic testing of serum and lymphoblastoid cells of patients with motor neuropathy 
Organisation University of Antwerp
Country Belgium 
Sector Academic/University 
PI Contribution We collected serum and blood samples of patients with hereditary motor neuropathies and Prof. Vincent Timmermann`s group converted them to lymphoblastoid cells and conduct metabolomics studies
Collaborator Contribution Prof. Timmermann`s group convert the blood cells to lymphoblastoid cells.
Impact samples are currently being analysed
Start Year 2016
Description Metabolic measurements in mitochondrial carrier protein deficiency 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution We have patient samples for metabolic measurements to Dr. Christian Frezza`s laboratory.
Collaborator Contribution We will receive the results soon and will have a joint publication.
Impact no output yet
Start Year 2013
Description Next Generation Sequencing 
Organisation Broad Institute
Country United States 
Sector Charity/Non Profit 
PI Contribution Prof. Daniel McArthur`s group in the Broad Institute agreed to perform WES in >300 Turkish families with neurogenetic disease.
Collaborator Contribution Performed WES for free.
Impact currently writing up conference abstracts and papers.
Start Year 2016
Description Search for modifyers in reversible COX deficiency 
Organisation Columbia University Medical Center
Department Neurological Institute of New York
Country United States 
Sector Academic/University 
PI Contribution I contribute a large family and performed exome sequencing
Collaborator Contribution contributing further families
Impact currently being worked up
Start Year 2011
Description Studying 2-thiolation of mt-tRNA Glu, Lys, Gln 
Organisation McGill University
Department Department of Molecular Neurogenetics
Country Canada 
Sector Academic/University 
PI Contribution I have started to collaborate on the function of TRMU
Collaborator Contribution common publication
Impact There is a Hom Mol Genet paper (Sasarman et al. 2011) already out of this collaboration.
Start Year 2011
Description Studying a novel mitochondrial carriers in a patient mitochondrial disease 
Organisation Medical Research Council (MRC)
Department MRC Mitochondrial Biology Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution We identified a patient with mutations in a novel mitochondrial carrier protein.
Collaborator Contribution Dr. Edmund Kunji`s laboratory performed functional analysis of the carrier to prove that the mutation is pathogenic.
Impact We are currently drafting a manuscript.
Start Year 2014
Description Studying the function of the exosome in human disease. 
Organisation Hebrew University of Jerusalem
Department Hebrew University Hadassah Medical School
Country Israel 
Sector Academic/University 
PI Contribution We have identified a novel disease gene and performed functional studies.
Collaborator Contribution The partner had another family with mutations in the same gene.
Impact We published a nice paper together (Boczonadi et al. 2014)
Start Year 2014
Description mitochondrial fusion/fission 
Organisation Pontifical Catholic University of Chile
Country Chile 
Sector Academic/University 
PI Contribution I have sent cell lines to Dr. Veronica Eisner for studiying mitochondrial fusion/fission.
Collaborator Contribution studying mitochondrial fission in cells with a special technique
Impact A novel mechanism causing imbalance of mitochondrial fusion and fission in human myopathies. Bartsakoulia M, Pyle A, Troncosco D, Vial J, Paz-Fiblas MV, Duff J, Griffin H, Boczonadi V, Lochmüller H, Kleinle S, Chinnery PF, Grünert S, Kirschner J, Eisner V, Horvath R. Hum Mol Genet. 2018 Jan 19. doi: 10.1093/hmg/ddy033. [Epub ahead of print] PMID: 29361167
Start Year 2015
Description mitochondrial tRNA synthetase related diseases 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution I started a collaboration with Prof. William Newman on mt tRNA synthetase diseases. I sent him DNA samples of patients with potential Perrault syndrome.
Collaborator Contribution Dr. Newmn is sequencing with a NGS panel novel genes which could cause Perrault syndrorme.
Impact no output yet
Start Year 2015
Description search for biomarkers in CMT 
Organisation University of Antwerp
Country Belgium 
Sector Academic/University 
PI Contribution We have performed targeted proteomics on serum of patients with CMT. We extended the analysis on mouse models of CMT.
Collaborator Contribution We have received serum from mouse models of CMT from Prof. Vincent Timmermann`s team. We search for biomarkers in CMT in this collaboration.
Impact submitted an abstract to the UK MRC Translational Research Conference (22-23 April, UCL)
Start Year 2019
Title A Study of Bezafibrate in Mitochondrial Myopathy" (NUTH NHS Trust, 2015) 
Description We are testing the feasibility of bezafibrate supplementation in MELAS. Trial has been finished. We published the paper very recently in EMBO Mol Med 
Type Therapeutic Intervention - Drug
Current Stage Of Development Early clinical assessment
Year Development Stage Completed 2019
Development Status Under active development/distribution
Impact trial has not shown clinical benefit 
Description Genetic Matters 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Patients, carers and/or patient groups
Results and Impact Postdoctoral research associates from my group participated in the organisation of the "Genetic Matters" event in the Centre for Life. Patients, students and general public attended the event or rare genetic diseases.
Year(s) Of Engagement Activity 2017
Description Organizing SHORT VIDEO AND a lightshow for the Mitochondrial Awareness week 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Supporters
Results and Impact We organized a lightshow and produced a short video for the Mitochondrial Awareness Week.
Year(s) Of Engagement Activity 2020
Description Participating with a demonstration of zebrafish research at the Newcastle High School for Girls research day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact We had a table at the STEM Research Day of Newcastle High School for Girls.
Year(s) Of Engagement Activity 2017
Description creating video about patient journey for European Joint Program 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact We actively participated in making a video about a patient journey of a Turkish patient who we dignosed within our research for the European Joint Program activities.
Year(s) Of Engagement Activity 2019
URL https://twitter.com/GA4GH/status/1186993739991900165?s=17