Mutations in genes coding for mitochondrial (mt)-tRNAs (MTTs) are responsible for a wide range of currently untreatable pathologies. We recently demonstrated that the carboxy-terminal domain (Cterm) of
human mt leucyl-tRNA synthetase (mt-LeuRS), and two peptides derived from it (ß30_31 and ß32_33), are able to rescue the defects caused by the two mt-tRNA mutations most frequently causing severe syndromes: the m.3243A>G in mt-tRNALeu(UUR) and the m.8344A>G in mt-tRNALys. We further demonstrated that these molecules directly interact with both mutant tRNAs and stabilize a wild-type like conformation of human mt-tRNALeu(UUR) in vitro. The rescuing activity appears therefore to be mediated by a chaperonic mechanism. These peptides are therefore attractive lead molecules for the development of therapeutic compounds against mt-tRNA mutations-related syndromes. In the frame of the global effort to translate these highly promising results into therapeutic applications, we aim to:
Develop strategies to deliver our chaperonic rescuing molecules, i.e., LeuRS-Cterm and ß32_33 peptide, which is more active than ß30_31, to the mt matrix, where they are to exert their therapeutic activity;
Analyze in detail the tissue-specific rescuing effect of the above molecules on cell models developed by our group (i.e. neurons and myotubes differentiated from induced pluripotent stem cells-iPSCs)
One of the major challenges in mt-tRNAs related disease is to understand the molecular link between disease phenotypes and genotypes. For example, the heteroplasmic m.3243A>G mutation in MTTL1, which is the most common among MTTs mutations can occur in association with a number of clinical syndromes such as encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome; chronic progressive external ophthalmoplegia (CPEO); maternally inherited diabetes and deafness (MIDD) and cardiomyopathy. The mechanisms underlying the heterogeneity of clinical phenotypes are not fully understood. Moreover, recent evidence suggests that the effects of MTTs mutations on respiratory chain (RC) activity may be modulated by the specific cellular context. Current transgenic technologies are not efficient in manipulating mitochondrial DNA, limiting the targeted engineering of mutations in mtDNA. Thus, due to the relative lack of animal models of mt-tRNA disease, the most widely used model is still the transmitochondrial cybrid. This is an undifferentiated neoplastic cell, lacking tissue-specific features. The development of iPSC technology has opened up new possibilities for the use of patient material.We expect that iPSC-derived neurons and myocytes will be reliable cellular model useful to better define the molecular consequences of mt tRNAs mutations and to test the effects of therapeutic molecules. This will have a significant impact on the development of therapeutic strategies for mt-tRNA related disease. In addition the set up of a system for the efficient delivery in the mitochondrial matrix of peptides able to rescue the effect of mitochondrial tRNA mutations will represent a key achievement towards the development of a pharmacological therapy with
significant technological ,economic and social impact.