Center for Molecular Medicine Cologne

Aleksandra Trifunovic - C 15

Manipulation of CLPP protease to complement mitochondrial respiratory deficiency

Our project explored possible therapeutic intervention in a largest group of mitochondrial diseases that directly affect the function of mitochondrial tRNAs. We presented the first evidence that manipulation of the levels of mitochondrial matrix protease, CLPP could ameliorate mitochondrial encephalopathy, caused by loss of mitochondrial aspartate tRNA synthase (DARS2), holding a promise for further exploration of specific protease inhibitors in treatment of mitochondrial diseases. 


Mitochondria are essential organelles found in almost every eukaryotic cell, required to convert food into usable energy. Therefore, it is not surprising that mutations in genes encoding mitochondrial proteins cause devastating diseases whose prevalence has been estimated to be ≈1:5000. Despite this, it is surprising that our understanding of the mechanisms governing the mitochondrial gene expression and its associated pathologies remain superficial and therapeutic interventions largely unexplored.In the first part of this project we aimed to understand how deficiency of ubiquitously expressed mitochondrial aspartyl tRNA synthetase (Dars2) cause a disorder in which tracts of the central and peripheral nervous systems are selectively affected. Later, we explored therapeutic possibilities of CLPP deficiency induced by genetic manipulation in various cell lines derived from patients carrying either mutation in mitochondrial tRNA genes (MT-TRNA) or mitochondrial tRNA synthetase genes (aaRS2), as well as in mouse models for two different models for mitochondrial encephalopathy caused by Dars2deficiency.

DARS2 protects against neuronal loss, but is dispensable for myelin producing cells 

Although mitochondria are ubiquitous, each mitochondrial disease has surprisingly distinctly different pattern of tissue and organ involvement. Congruently, mutations in genes encoding for different mitochondrial tRNA synthetases result in the development of a very flamboyant group of diseases (Theisen et al., 2017). 

Puzzled by the white matter disease phenotypes caused by DARS2 deficiency when numerous other mutations in the genes encoding proteins involved in mitochondrial translation have a detrimental effect predominantly on neurons, we generated transgenic mice in which DARS2 was specifically depleted in forebrain-hippocampal neurons or myelin-producing cells. Our results provided the first evidence that loss of DARS2 in adult neurons leads to strong mitochondrial dysfunction and progressive loss of cells(Aradjanski et al., 2017). 

In contrast, myelin-producing cells seemed to be resistant to cell death induced by DARS2 depletion despite robust respiratory chain deficiency arguing that LBSL might originate from the primary neuronal and axonal defect (Aradjanski et al., 2017). Remarkably, our results also suggested a role for early neuroinflammation in the disease progression, highlighting the possibility for therapeutic interventions of this process (Aradjanski et al., 2017).

Remarkably, loss of CLPP caused mild respiratory chain deficiency in various tissues, with general improvement of glucose metabolism and normal life span (Becker et al., 2018). Upon CLPP depletion,we detected some improvements of phenotypes in cell lines derived mainly from primary skin fibroblast from patientswith different aaRS2mutations. Unfortunately, these cell models only partially recapitulate phenotypes seen in mitochondrial patients that present very strong tissue and organ specificity. Therefore, we focused most of our attention on in vivo models. Remarkably, we observed a strong positive effect of CLPP depletion in vivoin DARS2NeuKOmice, as the onset of mitochondrial dysfunction was strongly delayed (Figure 1). 

Loss of CLPP ameliorates neurodegeneration in DARS2 deficient models 

We have previously shown depletion of mitochondrial matrix protease, CLPP could ameliorate mitochondrial cardiomyopathy caused by loss of mitochondrial aspartate tRNA synthase (DARS2) (Seiferling et al., 2016). 

WhileDARS2NeuKO mice developed strong mitochondrial deficiency around 20 weeks of age, DKO mice (Dars2L/L,ClppL/LCamK2-cre) where largely protected (Fig. 1). The beneficial effect of CLPP deficiency resulted in better performance on the composite tests, longer health and lifespan. This was likely caused by lower levels of neuronal cell loss in both cortex and hippocampus area (Fig. 2). The observed positive effect was accompanied by strong decreased in markers of neuroinflammation. Importantly, even the Clpphaploinsufficiency (Dars2L/L,Clpp+/Lled to a significantdecrease in neuroinflammation. 

To confirm that the beneficial effect of CLPP deficiency is not specific for forebrain neurons, wealsocreated DARS2 deficient mice specifically inPurkinje cells (DARS2PCKO-Dars2L/L, L7-cre). We first investigated the effect of primary mitochondrial dysfunction on PCs dendritic outgrowth, spine formation and survival to be able to further address the effect of CLPP deficiency in this model. DARS2PCKOmice were born at the expected ratio, they were viable and displayed no symptoms until 15 weeks of age, when they started showing unsteady gait, and mildly disturbed balance. A loss of Purkinje neurons preceded dramatic aggravation of symptoms at 18 weeks of age that ultimately forced us to sacrifice these mice at this point. As in the case of DARS2NeuKOCLPP deficiency, delayed the observed symptoms, including loss of Purkinje neurons and increased neuroinflammation, adding evidence that the observed positive effect of CLPP depletion is not cell type specific. To gain more mechanistic insight behind observed beneficial effect of CLPP depletion we recently isolated primary neurons from Dars2L/L,ClppL/Lmice and proceeded to induce Cre-mediated knockout of both genes in culture. 


Our exciting results hold a promise that even partial CLPP deficiency might be used as a therapeutic option for mitochondrial patients with diseases caused by mutations inaaRS2genes. 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 (Szczepanowska et al., 2019). They also shed light in ClpXP activity as unexpected potential target for therapeutic interventions in the even larger group of mitochondrial disorders characterized by the CI instability that we will intend to explore in the next funding period. 

Selected publications 

Aradjanski, M., et al.(2017). DARS2 protects against neuroinflammation and apoptotic neuronal loss, but is dispensable for myelin producing cells. Hum Mol Genet26, 4181-4189.

Becker, C., et al.(2018). CLPP deficiency protects against metabolic syndrome but hinders adaptive thermogenesis. EMBO Rep19, e45126.

Seiferling, D., et al.(2016). Loss of CLPP alleviates mitochondrial cardiomyopathy without affecting the mammalian UPRmt. EMBO Rep17, 953-964.

Szczepanowska, K., et al.(2019). Salvage pathway maintains functional respiratory complex I. submitted.

Theisen, B.E., et al.(2017). Deficiency of WARS2, encoding mitochondrial tryptophanyl tRNA synthetase, causes severe infantile onset leukoencephalopathy. Am J Med Genet A173, 2505-2510.

Prof. Dr. Aleksandra Trifunovic CMMC Cologne
Prof. Dr. Aleksandra Trifunovic

Inst. for Mitochondrial Diseases and Ageing / RG location - CECAD Building

Principal Investigator C 15

+49 221 478 84291

+49 221 478 32400

Inst. for Mitochondrial Diseases and Ageing / RG location - CECAD Building

Joseph-Stelzmann-Str. 26

50931 Cologne

CMMC Profile Page

Curriculum Vitae (CV)

Publications - Aleksandra Trifunovic

Link to PubMed

Group Members

Karolina Szczepanowska (PostDoc)
Anastasia Rumyantseva (doctoral student)
Katarina Senft (technitian)

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