Targeting the cellular metabolism to treat tissue-specific mitochondrial diseases

线粒体是细胞内专门的单位（细胞器），负责将营养物质转化为能量。线粒体含有自己的遗传物质（mtDNA），它独立于细胞核中的DNA进行复制。mtDNA非常小，只包含13种蛋白质的信息；线粒体所需的所有其他蛋白质都编码在核DNA中。无论是mtDNA还是核DNA的变化都可能导致线粒体疾病。这些都是致残或致命的疾病，影响大脑、肝脏、骨骼肌、心脏和其他器官，目前还没有有效的治疗方法。尽管所有线粒体疾病都有相似的机制，但它们对身体的影响方式却截然不同。迄今为止，人们对其原因知之甚少。我们研究了一种罕见的线粒体疾病，称为可逆性婴儿呼吸链缺乏症（rrcd）。rcd的特征是在3个月前出现严重的肌肉无力，存活的儿童在6个月后自发恢复。rcd是由mtDNA中的拼写错误（=突变）引起的。有趣的是，更多的人携带这种突变而没有生病，然而全世界只有大约100人受到rcd的影响。我们证明，除了mtDNA突变外，核DNA的第二个变化需要引起RIRCD。导致rcd的突变位于mtDNA分子中被称为转移rna （tRNA）的部分。trna将适当的氨基酸传递给一种叫做核糖体的机器，核糖体将这些氨基酸组合成蛋白质；这个过程叫做翻译。如果没有足够的氨基酸可用，或者tRNA被突变修饰，tRNA可能会保持空状态。空trna是一个负面信号，可以被一种叫做GCN2的蛋白质检测到。GCN2触发细胞的综合应激反应（integrated stress response， ISR）。这种应激反应会导致变化，这些变化要么有助于细胞适应压力，要么如果持续时间过长就会导致细胞死亡。我们的假设是，在不同的细胞类型中，trna的总量和空trna的数量可能不同。更高数量的空trna可能引发更强的应激反应，这可能对细胞产生消极或积极的影响。我们将分析从患有各种线粒体疾病和健康对照的患者身上获得的皮肤细胞。受疾病影响的器官（大脑、肌肉、心脏）不容易进行分析。因此，我们将通过一个被称为重编程的过程将皮肤细胞转化为干细胞。从干细胞中我们可以得到大脑、心脏和肌肉细胞。通过观察同一个人的不同细胞类型，我们可以比较他们在压力情况下的反应。我们将检查空trna或ISR的水平在不同的细胞类型中是否不同。我们将添加某些氨基酸，看看这是否能减少空tRNA的数量和应激反应。我们要用的另一个模型是斑马鱼。我们可以在斑马鱼的DNA中引入不同的突变，并观察不同的器官（如大脑、心脏和骨骼肌）是如何受到影响的。我们将观察鱼的不同器官中的tRNA数量和ISR。这些实验将有助于解释为什么尽管携带相同的突变，组织却以不同的方式受到影响。rcd患者的自发恢复是非常罕见的。我们将把来自RIRCD患者的细胞与来自其他线粒体疾病的细胞进行比较，这些疾病是由影响trna的变化引起的，但患者没有康复。我们认为，ISR诱导的变化正在帮助RIRCD细胞改变其功能方式并调动不同的能量来源，最终导致恢复。然而，我们不知道为什么这不会发生在其他线粒体疾病中。如果我们了解了RIRCD和其他线粒体疾病之间的区别，我们可能就能找到治疗其他形式线粒体疾病的方法。

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.