Examining the coupling of small GTPase activation and metabolism of the phosphoinositide lipid PI(3,5)P2 in Charcot Marie Tooth Type 4 Neuropathies

Lead Research Organisation: University of Liverpool
Department Name: Institute of Translational Medicine


Charcot-Marie Tooth type 4 (CMT4) neuropathies are a group of diseases characterized by a failure of myelin, the "insulation" for the nerves sending signals to muscles in the body. Patients who have CMT4 generally develop it early in life, meaning that they face a lifetime of needing assistance with mobility and basic tasks. It is a progressive disorder, where the myelin insulation slowly stops wrapping properly around the nerves it is meant to insulate. This stops the nerve from sending signals properly to the limbs, leading to both sensory and motor impairment (particularly in the more distant limbs). As this disease slowly gets worse over time, there is a possibility that if we understand what is going wrong, the disease process could be arrested or even reversed by the appropriate therapy.
We wish to study several related genes which all cause CMT4. These have different functions, but work together to move proteins to and from the surface of the cell (we call this trafficking). CMT4 pathologies occur because of the failure of proper trafficking in the cells that make myelin, meaning proteins that should be at the surface of the myelin-making cells don't arrive properly or at the wrong time.

CMT4 pathologies are caused by mutation any of 11 different genes. 5 of these genes (the ones we want to study) make enzymes. 3 of these enzymes (called Frabin, MTMR13, and MTMR5) switch on GTPases. 4 enzymes (called MTMR2, MTMR13, MTMR5 and Fig4) act on a rare lipid (PI(3,5)P2) which helps conduct trafficking in the cell by adding a 'tag' which tells you where inside the cell you are during the trafficking process. Small GTPases are another kind of 'tag'- when they are switched on, they instruct certain groups of proteins to collect on the membrane. Together, lipids like PI(3,5)P2 and proteins like small GTPases can specify a time and a place for the process of moving proteins to the cell surface. For CMT4 patients we still don't know where the important pool of PI(3,5)P2 lipid is in the cell, and what actions that should be co-ordinated by small GTPases are failing to happen in CMT4 patients with mutations in these genes.
The enzymes mentioned above are the most promising targets for therapies, as they are potentially druggable, or can have their enzymatic activities replaced by other means. This project aims to understand what is going wrong in the cells making myelin and identify the best targets for therapy, preliminary to studies for therapeutic strategies.

Technical Summary

This project aims to understand the function of a group of related enzymes in membrane trafficking, and how their loss may cause Charcot Marie Tooth Type 4 neuropathies. We aim to identify critical points in the exo-endocytic cycle which may be open to therapeutic manipulation to arrest or reverse this progressive disorder. We will use proteins produced in human cell suspension cultures as material to identify small GTPase interactors of CMT4 genes using label-free differential mass spectrometry and then characterize these interactions in model cells, human patient fibroblasts (where available) and in in vitro Schwann cell- neuron co-cultures. We will use lentiviral treatments of Schwann cells to establish cell-autonomous effects of CMT4 gene manipulations in myelinating cells in these co-cultures. Our second aim will establish tools to acutely manipulate the lipid PI(3,5)P2 in cell culture and in vitro myelinating culture models using the CRY2-CIBN optogenetic system, so as to establish the location and function of the pool of this lipid in (dis)myelination.

Planned Impact

Apart from the Academic beneficiaries outlined above, our research will have long-term relevance for several groups:

Directly (2-5 years)- patients and families suffering from Charcot Marie Tooth Type 4 Neuropathies (CMT4). CMT4 is an early-onset disease, where patients often need considerable care and adjustments made to their living situation over the course of a long life. The economic and social benefits of even small improvements in mobility in these patients are considerable. This project aims to find where therapeutic interventions could be made, and to build in vitro assays that could subsequently be used for screens for therapies. Moreover, we will produce results that will help interested parties understand how CMT4 develops, and to help patients and families engage with the process of discovering therapies.

This project will also be training researchers in a novel and growing field of cell biology, and in the use of cutting-edge techniques for transient manipulation of cell biology. The PDRA employed by this project will have an unique and valuable set of experimental tools with which to develop their further career.

In the case of other human pathologies- our data has relevance for neurodegenerative disorders (ALS and PLS) through our studies and manipulation of Fig4 activity with the attendant financial and social benefit if these studies can point the way to therapeutic interventions in these disorders.

In the longer term (5-15 years): It is becoming rapidly apparent that the rare lipid PI(3,5)P2 is at the nexus of several critical signalling pathways (please see academic beneficiaries for a fuller description of the processes so far found to be regulated by PI(3,5)P2). Some of these pathways, such as cell-survival and aging signalling regulated by mTOR, or insulin signaling are pathways that if we can understand and manipulate, will have great implications for public health, given our aging and increasingly sedentary population. Our work here will produce new tools to manipulate this lipid and to understand functionally what is happening to cells where PI(3,5)P2 in specific cellular compartments, and will contribute to ongoing efforts to manipulate cell signaling for improving health and quality of life in the general population.

Economically- this study is aimed towards establishing phenotypes which can be subsequently used for drug discovery screens. This has a direct commercial relevance for biotechnology in the UK, not least because we are studying processes which may be relevant to significantly more than the one set of diseases we are focused on (as I have outlined above). The generation of further basic knowledge and intellectual property centred around this membrane trafficking pathway has the potential to be an unexplored and commercially significant area in biomedical/pharmaceutical healthcare.


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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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Boczonadi V (2018) Mitochondrial DNA transcription and translation: clinical syndromes. in Essays in biochemistry

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Boczonadi V (2018) Mitochondrial oxodicarboxylate carrier deficiency is associated with mitochondrial DNA depletion and spinal muscular atrophy-like disease. in Genetics in medicine : official journal of the American College of Medical 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 Protein processing and secretion in a new cause of CMD, INPP5K mutation.
Amount £224,962 (GBP)
Funding ID 18GRO-PG36-0270 
Organisation Muscular Dystrophy UK 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2019 
End 01/2022
Description Research Grant
Amount £15,000 (GBP)
Funding ID RG170076 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2017 
End 11/2018
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 genetically encoded sensor of lysosomal pH 
Description we built a ratiometric reporter of lysosomal pH coupled to a purification tag which allows correlated live measurements of lysosomal physiology with mass spectroscopic analysis of lysosomal protein recruitment. The sensor is simpler to use than similar approaches using dyes/endocytic probes and stable for days of continuous imaging in culture. 
Type Of Material Biological samples 
Year Produced 2020 
Provided To Others? Yes  
Impact the probe is one of the highly-requested probes on Addgene, is simpler to use than other approaches, and offers more information (in the form of proteomic analysis of lysosomes with a known physiological state) than other techniques. 
URL https://www.addgene.org/browse/article/28211549/
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 collecting new variants of INPP5K mutations in patients 
Organisation University of Southampton
Department Clinical and Experimental Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution enzymology, creation of cell lines and cell biological analysis of new INPP5K patient variants
Collaborator Contribution partners are clinicians.
Impact Identification of a novel disease mechanism caused by INPP5K mutation (ongoing)
Start Year 2019
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 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