Targeting the cellular metabolism to treat tissue-specific mitochondrial diseases

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.

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.