Aleksandra Trifunovic | Karolina Szczepanowska - C 15

Despite a steady increase in the number of novel genes implicated in the pathogenesis of mitochondrial diseases, an effective treatment for any mitochondrial disorder is still missing.

We recently discovered a novel respiratory complex I salvage pathway that maintains fully functional CI and thereby healthy mitochondria through a favourable mechanism requiring lower energetic expenditure. Our results also shed light in ClpXP activity as unexpected potential target for therapeutic interventions in the large group of mitochondrial disorders characterized by the CI instability that we will explore in this project.

Mitochondria are essential for maintaining numerous fundamental cell functions. Mutations in either mtDNA or nDNA genes coding for mitochondrial proteins are known to lead to major and catastrophic diseases in humans. They are one of the most common inborn errors of metabolism with a frequency of about 1 in 5000 and come with an impressive variability of symptoms, organ involvement, and clinical course, which considerably impact the quality of life and often shorten the lifespan expectancy.

Unfortunately, currently no treatment is available for myriad of diseases caused by mutations in mitochondrial genes and therapies are mainly aimed to alleviate symptoms and/or slow down the progression of the diseases.

Our preliminary and unpublished data show that by removing the major mitochondrial matrix protease CLPXP, and therefore stabilizing CI, we could ameliorate the symptoms of respiratory deficiency in different cellular models of mitochondrial dysfunction (Figure 1.A-D). The loss of CLPP in these models resulted not only in increased stability of CI (Figure 1D), but also normalized NAD+/NADH ratios. Remarkably, even partial loss of CLPXP activity in respiratory deficient cells led to mild increase in the CI levels, opening an exciting prospect for therapeutic interventions (Figure 1E).

Therefore, the overall goal of this project is to explore the possibility of targeting CLPP activity to ameliorate symptoms of mitochondrial diseases in in vivo models through genetic interventions and usage of specific protease inhibitors. To this end we will use a panel of patient derived cell lines with documented CI deficiency. We will further explore a possible beneficial effect of CLPP deficiency in mouse models for mitochondrial diseases through genetic interventions and usage of protease inhibitors in vivo.

1. Explore the therapeutical potential of CLPP depletion in cell lines derived from patients with mitochondrial diseases.
2. Investigate the effect of CLPP deficiency on in vivo murine models of mitochondrial deficiency.
3. Analyse molecular mechanism of specific CLPP inhibitors and test their efficiency in cello and in vivo models

We demonstrated that a strong mitochondrial cardiomyopathy and diminished respiration due to DARS2 deficiency can be alleviated by the loss of CLPP, leading to an increased de novo synthesis of individual OXPHOS subunits.

We discovered a novel CI salvage pathway that maintains highly functional CI through an energetically favourable mechanism that demands much lower cost than de novo synthesis and reassembly of the entire CI. 

In this pathway the NADH-oxidizing N-module of CI is turned over at a higher rate and largely independently of the rest of the complex by mitochondrial matrix protease ClpXP, which selectively removes and degrades damaged subunits.

The observed mechanism seems to be a safeguard against the accumulation of dysfunctional CI arising from the inactivation of the N-module subunits due to attrition caused by its constant activity under physiological conditions.

Our results also illuminate ClpXP activity as an unforeseen target for therapeutic interventions in the large group of mitochondrial diseases characterized by the CI instability.

1. Dogan SA, Pujol C, Maiti P, Kukat A, Hermans S, Senft K, Wibom R, Rugarli EI, Trifunovic A: Tissue-specific loss of DARS2 activates stress responses independently of respiratory chain deficiency in the heart. Cell Metab. 2014 Mar 4;19(3):458-69.    
2. Seiferling D, Szczepanowska K, Becker C, Senft K, Hermans S, König T, Kukat A, Trifunovic A: Loss of CLPP alleviates mitochondrial cardiomyopathy without affecting the mammalian UPRmt. EMBO Reports 2016 May 6. pii: e201642077.
3. Becker C, Kukat A, Szczepanowska K, Hermans S, Senft K, Brandscheid CP, Maiti P,  and Trifunovic A. (2018) CLPP deficiency protects against metabolic syndrome but hinders adaptive thermogenesis. EMBO Rep. 2018 Mar 27. pii: e45126. doi:10.15252/embr.201745126. PMID: 29588285
4. Szczepanowska K, Senft K, Heidler J, Herholz M, Kukat A, Höhne MN, Hofsetz E, Becker C, Kaspar S, Giese H, Zwicker K, Guerrero-Castillo S, Baumann L, Kauppila J, Rumyantseva A, Müller S, Frese C, Brandt U, Riemer J, Wittig, Trifunovic A: A salvage pathway maintains highly functional respiratory complex I. Nature Communications 2020 ; in press
5. Szczepanowska K, Maiti P, Kukat A, Hofetz E, Nolte H, Senft K, Becker C, Ruzzenente B, Hornig-Do HT, Wibom R, Wiesner RJ, Krüger M and Trifunovic A CLPP coordinates mitoribosomal assembly through the regulation of ERAL1 levels. EMBO J 2016 Dec 1;35(23):2566-2583