Targeting the cellular metabolism to treat tissue-specific mitochondrial diseases

Lead Research Organisation: University of Cambridge
Department Name: Clinical Neurosciences

Abstract

The mitochondria are specialised units (organelles) within cells that are responsible for transforming nutrients into energy. Mitochondria contain their own genetic material (mtDNA) which is replicated independently from the DNA in the nucleus. mtDNA is very small and only contains the information for 13 proteins; all other proteins that the mitochondria need to function are coded in the nuclear DNA. Changes in either mtDNA or nuclear DNA can cause mitochondrial diseases. These are disabling or fatal conditions, affecting the brain, liver, skeletal muscle, heart and other organs, and currently there are no effective cures. Although all mitochondrial diseases have a similar mechanism, they can affect the body in strikingly different ways. To date, the reasons for this are poorly understood.
We study an unusual mitochondrial disease named reversible infantile respiratory chain deficiency (RIRCD). RIRCD is characterised by severe muscle weakness before 3 months of age, followed by a spontaneous recovery after 6 months of age in surviving children. RIRCD is caused by a spelling error(=mutation) in the mtDNA. Interestingly, many more people carry this mutation without getting ill, however only around 100 are affected by RIRCD worldwide. We demonstrate that a second change in the nuclear DNA in addition to the mtDNA mutation is needed to cause RIRCD.
The mutation that underlies RIRCD is situated in a part of the mtDNA molecule called transfer-RNA (tRNA). tRNAs deliver the appropriate amino acids to a machinery called ribosome, which puts the amino acids together into a protein; this process is called translation. If there are not enough amino acids available or if the tRNA is modified by a mutation, the tRNA could stay empty. Empty tRNAs are a negative sign and can be detected by a protein called GCN2. GCN2 triggers a reaction of the cell called the integrated stress response (ISR). This stress response leads to changes that either help the cell adapt to the stress or cause its death if it lasts too long.
Our hypothesis is that the total amount of tRNAs and the empty tRNAs can differ in different cell types. A higher amount of empty tRNAs could trigger a stronger stress response, which could have a negative or positive impact on the cell. We will analyse skin cells obtained from patients with various mitochondrial diseases and healthy controls. The organs affected by the disease (brain, muscles, heart) are not easily accessible for analysis. Therefore, we will turn the skin cells into stem cells through a process called reprogramming. From the stem cells we can derive brain, heart and muscle cells. By looking at different cell types from the same person we are able to compare their reactions in stress situations. We will check if levels of empty tRNAs or ISR are different in the different cell types. We will add certain amino acids to see if this can reduce the amount of empty tRNA and the stress response.
Another model that we will use are zebrafish. We can introduce different mutations into the zebrafish DNA and look at how the different organs (such as brain, heart and skeletal muscle) are affected. We will look at tRNA amounts and ISR in different organs of the fish. These experiments will help to explain why tissues are affected in a different way despite carrying the same mutations.
The spontaneous recovery of patients with RIRCD is very unusual. We will compare the cells from RIRCD patients with cells from other mitochondrial diseases caused by changes affecting tRNAs but the patients do not recover. We believe that the changes induced by ISR are helping the RIRCD cells to change their way of functioning and mobilise different energy sources, eventually leading to the recovery. We don't know, however why this does not happen in other mitochondrial diseases. If we understand the differences between RIRCD and other mitochondrial diseases we might be able to find a way to treat other forms of mitochondrial disease.

Technical Summary

Although mitochondria are present in almost all eukaryotic cell types, mitochondrial diseases have distinct tissue-specific phenotypes that are poorly understood. Our project focuses on mitochondrial diseases characterised by impaired mitochondrial translation, which can be caused either by mtDNA mutations (often in mt-tRNAs) or in nuclear encoded proteins that play a role in mitochondrial translation. Mt-tRNA or nuclear gene mutations affect tRNA levels and structure directly or indirectly, leading to a defect of mitochondrial protein synthesis. These mutations can also lead to the presence of free tRNAs without their cognate amino acid ('uncharged tRNA'), which constitutes a stress signal for cells.
We will investigate whether different tissues have different levels of uncharged tRNAs and whether this correlates with the vulnerability for mitochondrial defects. Uncharged tRNA activates the integrated stress response (ISR), a major signalling pathway that allows eukaryotic cells to sense stress and adapt to it. We obtained fibroblasts from patients with different mitochondrial translation defects to determine tRNA and ISR levels. We will reprogram the fibroblasts to iPSCs and differentiate those to neurons, muscle cells and cardiomyocytes. Next, we will examine what metabolic changes are induced by the activated ISR in these cells.
In one disease studied by our laboratory, reversible infantile respiratory chain deficiency (RIRCD), the metabolic changes induced by the ISR in the muscle of patients allowed a recovery of the patients from severe disease. Our results indicate that the ISR is protective to some cells and damaging to others. We would like to exploit the knowledge gained in RIRCD to induce similar metabolic changes in mitochondrial conditions that are currently not reversible. Modifying the ISR or manipulating key metabolic factors (amino acids, FGF21) may enable us to devise therapeutic options for mitochondrial translation defects in the future.

Publications

10 25 50
publication icon
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

publication icon
Gangfuß A (2021) NEW GENES AND DISEASES in Neuromuscular Disorders

 
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 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 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 TEFM 
Organisation Monash University
Country Australia 
Sector Academic/University 
PI Contribution We are working together on proving the pathogenicity of novel genes causing mitochondrial protein synthesis defect.
Collaborator Contribution Exchanging cell lines, collecting data.
Impact not yet, paper in progress
Start Year 2019
 
Description joint work on a new gene TEFM 
Organisation Children's Hospital of Eastern Ontario
Country Canada 
Sector Hospitals 
PI Contribution Joint molecular work on characterizing TEFM as a new mitochondrial disease gene
Collaborator Contribution One out of 5 families were from CHEO. CHEO scientist Emily O`Connor made a zebrafish model of TEFM deficiency.
Impact Paper under review in Nature Communications This is a multi-disciplinary partnership including clinicians and scientists
Start Year 2020
 
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 Cure_ARS 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Cure-ARS symposium was organised by the Cure-ARS charity. I gave a presentation and I have discussions with the Charity for joint research
Year(s) Of Engagement Activity 2022
URL https://www.curears.org/event-details/mt-aars-scientific-symposium-2022
 
Description HNF 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Patients, carers and/or patient groups
Results and Impact We discussed and initiated a natural history study on C12orf65 mutations
Year(s) Of Engagement Activity 2021
URL https://www.hnf-cure.org/
 
Description HNF 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Patients, carers and/or patient groups
Results and Impact We discussed and initiated a natural history study on C12orf65 mutations
Year(s) Of Engagement Activity 2021
URL https://www.hnf-cure.org/