Screening for Therapeutics against Untreatable Mitochondrial Disorders

Using Neural Progenitors from Patient - iPSCs

  • Fig. 1: Neural progenitor cells (NPCs). NPCs derived from patient iPSCs. Blue: nuclear staining with Hoechst; Green: neuronal marker MAP2; Red: neuronal marker TUJ1.Fig. 1: Neural progenitor cells (NPCs). NPCs derived from patient iPSCs. Blue: nuclear staining with Hoechst; Green: neuronal marker MAP2; Red: neuronal marker TUJ1.
  • Fig. 1: Neural progenitor cells (NPCs). NPCs derived from patient iPSCs. Blue: nuclear staining with Hoechst; Green: neuronal marker MAP2; Red: neuronal marker TUJ1.
  • Fig. 2: Proposed NPC-based drug discovery approach for mtDNA disease.  In our recent manuscript (Lorenz et al., 2017), we first demonstrated that iPSC-derived NPCs exhibit the correct genetic and metabolic properties for modeling neurological conditions caused by mtDNA mutations. We next applied this approach to iPSCs generated from patients carrying the mutation m.9185T>C in the mitochondrial gene MT-ATP6. We detected disease phenotypes in NPCs, including increased mitochondrial membrane potential (MMP) and calcium defects. Finally, we developed a high-content screening (HCS) method for MMP and used it for screening FDA-approved drugs.
Mitochondrial DNA (mtDNA) mutations cause untreatable disorders affecting the nervous system. These diseases suffer from a lack of viable modeling tools due to the challenges associated with mtDNA engineering. We recently showed that neural progenitor cells (NPCs) differentiated from patient-derived induced pluripotent stem cells (iPSCs) can be used to model mtDNA disorders and to set up phenotypic compound screenings. This strategy opens the way to the discovery of novel therapeutics against these so far untreatable human diseases.
Mitochondrial DNA Mutations Cause Untreatable Neurological Disorders
The major task of mitochondria is to produce energy in form of ATP which is used by every cell. While the functionality of mitochondria is under control of both the nuclear and the mitochondrial genome, the latter encodes for only 37 genes. Mitochondrial DNA (mtDNA) is exclusively maternally inherited and mutations in mtDNA are strongly linked to diseases affecting the nervous system and for which no effective treatment exists. 
With a minimum disease prevalence for mtDNA mutations of 1 per 5000 [1], these mutations mainly lead to defects of the electron transport chain (ETC) and ATP synthase located in the inner mitochondrial membrane therefore influencing the ATP production. Hence, especially cell types with high numbers of mitochondria and high energy demands, such as neurons or cardiac muscles, are susceptible to altered mitochondrial function. An important fact for understanding the pathogenesis of mitochondrial disorders is the multicopy nature of mtDNA meaning that each mitochondrion contains several mtDNA molecules. Therefore, mutations in the mitochondrial genome can be either homoplasmic, where all mtDNA molecules carry the mutation, or heteroplasmic, where there is a mix of mutant and wild-type mtDNA molecules. Deletions or point mutations have been reported in every mtDNA gene, and have been associated with clinical phenotypes ranging in severity from asymptomatic to fatal within the first years of life [2,3].  
Mitochondrial Diseases Lack Modeling Tools
It has been difficult to develop animal models for mitochondrial disease, due to challenges inherent in engineering mtDNA. 
Existing cellular models often lack the metabolic features of neural cells and do not provide the patient-specific match between mitochondrial and nuclear genomes.

But this is crucial in the study of mtDNA disorders, since specific characteristics of an individual patient’s nuclear DNA have been shown to influence the course of these diseases. 

A good example to understand the challenges of modeling mitochondrial diseases is to look at Leigh syndrome, which is the most severe mitochondrial disorder affecting children. This disease can be caused by nuclear or mitochondrial mutations. Therefore, it is difficult to untangle the genotype–phenotype correlation, since the same clinical phenotype can be caused by defects in several different genes. 
The development of appropriate model systems maintaining the patient-specific nuclear and mitochondrial genome and recapitulating the features of the neural cells affected in the patients is therefore critical in order to advance our understanding of the pathogenetic mechanisms of mitochondrial disorders. 
Induced Pluripotent Stem Cells (iPSCs) Allow the Generation of Patient Neural Cells in a Dish
A decade ago, Takahashi et al. established a novel method of reprogramming adult somatic cells using transcription factors related to pluripotency [4,5]. The resulting induced pluripotent stem cells (iPSCs) demonstrated the same pluripotency and self-renewal properties that are characteristic of embryonic stem cells (ESCs). The potential of iPSCs to differentiate into cell types from all three germ layers and hence to any cell type of the body makes them a powerful research tool. 
With the progress of iPSC technology applied to human cells, neuroscience research has taken a significant step forward. Modeling of neurological diseases is now possible with the help of patient-derived cells differentiated to disease-relevant tissue which was not accessible before. Even distinct neuronal subtypes, such as midbrain dopaminergic neurons [6,7,8], GABAergic neurons [9,10], or serotonergic neurons [11], can be generated in a dish.
Unfortunately, however, when using human iPSCs, long (up to 6 month) and complex protocols are necessary in order to reach neuronal maturation and expression of specific neuronal protein markers. The differentiation of iPSCs into neuronal-like cells in a dish is therefore very time-consuming and often results in heterogeneous cultures with a batch-dependent yield [12,13,14]. This may lead to problems when neuronal cells are applied to high-throughput compound screenings, since these are highly dependent on reproducibility and robustness of the cell model. 
NPCs as a Drug Discovery Model
Many of the differentiation protocols used for the generation of neural cells require the intermediate step of neural progenitor cells (NPCs) before further differentiation into desired cell types such as glia cells or neurons (fig. 1).  
In humans, NPCs exist in specific brain regions and enable neurogenesis even in the adult human brain [15,16]. More precisely, these multipotent neural progenitors reside in the subgranular zone of the dentate gyrus in the hippocampus and in the subventricular zone of the cortex [17,18].
In vitro, NPCs can be rapidly produced from PSCs by different strategies including the formation of embryoid bodies (EBs) followed by manual isolation of NPCs from neural rosettes [19,20]. As a rapid alternative a small molecule-based conversion of PSCs to NPCs [21,22] is possible and allows clonal expansion to reach homogeneous populations, while circumventing the time-consuming and operator-dependent EB selection step. 
NPCs may hold advantages for drug discovery applications in comparison to fully mature neurons. The neural progenitors have the advantage that their derivation from stem cells can be realized within one week and the resulting cell population is mainly homogeneous meaning that purification steps are not required. Their mild proliferative state makes cultivation easy and allows rapid application to assays. Therefore, NPCs,appear particularly well suited to scalability for the use in high-throughput drug screens. It is therefore conceivable that NPCs can be used for the high-throughput screenings, while mature post-mitotic neurons may be used for the validation and confirmation of the identified candidate drugs [23]. 
Mitochondrial Properties of iPSC-derived NPCs
Regarding mitochondrial structure and functionality, iPSC-derived NPCs appear similar to brain-derived NPCs [24]. Upon conversion of PSCs into NPCs, a mitochondrial maturation occurs, which leads to the acquisition of elongated mitochondrial structures with clearly defined cristae and dense matrices [24,25]. As a consequence, mitochondrial mass and mitochondrial DNA content increase [26]. Additionally, the remodeling of mitochondrial morphology during neural induction is associated with a metabolic shift towards a more oxidative phosphorylation (OXPHOS)-based metabolism, which is accompanied by a decreased lactate production and a reduced expression level of genes involved in glycolysis [25]. 
Increasing mitochondrial respiration in differentiating cells bears elevated production of reactive oxygen species (ROS) which are common by-products of OXPHOS and may lead to oxidative damage especially of the mitochondrial genome [27]. But mtDNA integrity represents an important aspect of neural differentiation in vitro and in vivo since failure to maintain mtDNA integrity may lead to impaired neuronal maturation [28]. Therefore, verification of mtDNA sequences appears inevitable when establishing model systems for neurological diseases.
In our recent work [25], we demonstrated that during generation of patient-derived iPSCs and subsequent neural induction to NPCs, the mitochondrial genome is entirely retained. This is of utter importance when studying mitochondrial disorders caused by mutations in the mitochondrial DNA. 
Compound Screenings of Mitochondrial Diseases Using NPCs
In order to establish a model system for mitochondrial diseases, we generated iPSC-derived NPCs from patients carrying a deleterious homoplasmic mutation in the mitochondrial gene MT-ATP6 (m.9185T>C) causing Leigh syndrome [29]. We found that these patient cells show defects in ATP production and abnormal increase in mitochondrial membrane potential (MMP). Patient NPCs also exhibited altered calcium homeostasis, which might represent a potential cause of neural impairment. 
We then used patient NPCs to carry out a high-content screening based on the MMP phenotype using FDA-approved drugs. The employment of FDA-approved libraries in such phenotype-based screenings may allow a fast translation to clinical application through drug repositioning. The screening highlighted the compound avanafil, which we found capable of partially rescuing the calcium defect in patient-derived NPCs as well as in differentiated neurons derived from patient iPSCs. 
Overall, NPCs display the genotype/metabotype match and the metabolic and functional features required for modeling neurological disorders. Therefore NPCs may allow phenotypic drug discovery for mitochondrial disorders affecting the nervous system (fig. 2). This promising iPSC-based strategy provides a useful in vitro platform for a better understanding of mechanisms in mitochondrial disorders, drug discovery, pre-clinical validation and future personalized therapeutic approaches for these so far incurable human diseases.
The authors declare no competing financial or commercial interests. We acknowledge financial support from the Bundesministerium für Bildung und Forschung (BMBF) (e:Bio Young Investigator grant AZ.031A318 to A.P.) and the Berlin Institute of Health (BIH).
Carmen Lorenz1,2 and Alessandro Prigione1,2
1 Max Delbrueck Center for Molecular Medicine (MDC), Berlin, Germany 
2 Berlin Institute of Health (BIH), Berlin, Germany
Max Delbrueck Center for Molecular Medicine
Berlin, Germany

1. Gorman GS, Schaefer AM, Ng Y et al. Prevalence of nuclear and mitochondrial DNA mutations related to adult mitochondrial disease. Ann Neurol 2015; 77: 753–759. DOI: 10.1002/ana.24362
2. Schon EA, DiMauro S and Hirano M. Human mitochondrial DNA: roles of inherited and somatic mutations. Nat Rev Genet 2012; 13: 878-890. DOI: 10.1038/nrg3275
3. Lieber DS, Calvo SE, Shanahan K et al. Targeted exome sequencing of suspected mitochondrial disorders. Neurology 2013; 80: 1762-1770. DOI: 10.1212/WNL.0b013e3182918c40
4. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126(4):663–676 DOI:10.1016/j.cell.2006.07.024
5. Takahashi K, Tanabe K, Ohnuki M et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007 Nov 30;131(5):861-72. DOI: 10.1016/j.cell.2007.11.019
6. Chambers SM, Fasano CA, Papapetrou EP et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nature Biotechnology 2009; 27(3), 275–280. DOI: 10.1038/nbt.1529
7. Kriks S, Shim JW, Piao J et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature 2011; 480, 547–551. DOI: 10.1038/nature10648
8. Reinhardt P, Glatza M, Hemmer K et al. Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling. PLoS One 2013; 8, e59252. DOI: 10.1371/journal.pone.0059252
9. Maroof AM, Keros S, Tyson JA et al. Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells. Cell Stem Cell 2013; 12, 559–572. DOI: 10.1016/j.stem.2013.04.008
10. Nicholas CR, Chen J, Tang Y et al. Functional maturation of hPSC-derived forebrain interneurons requires an extended timeline and mimics human neural development. Cell Stem Cell 2013; 12:573–586. DOI:10.1016/j.stem.2013.04.005
11. Erceg S, Laınez S, Ronaghi M et al. Differentiation of Human Embryonic Stem Cells to Regional Specific Neural Precursors in Chemically Defined Medium Conditions. PLoS ONE 2008; 3(5): e2122. DOI: 10.1371/journal.pone.0002122
12. Boulting GL, Kiskinis E, Croft GF et al. A functionally characterized test set of human induced pluripotent stem cells. Nat. Biotechnol. 2011; 29, 279–286 DOI: 10.1038/nbt.1783
13. Nguyen HN, Byers B, Cord B et al. LRRK2 mutant iPSC-derived DA neurons demonstrate increased susceptibility to oxidative stress. Cell Stem Cell 2011; 8, 267–280. DOI: 10.1016/j.stem.2011.01.013
14. Di Giorgio FP, Boulting GL, Bobrowicz S and Eggan KC. Human embryonic stem cell-derived motor neurons are sensitive to the toxic effect of glial cells carrying an ALS-causing mutation. Cell Stem Cell 2008; 3, 637-648. DOI: 10.1016/j.stem.2008.09.017
15. Kempermann G, New neurons for survival of the fittest, Nat. Rev. Neurosci. 2012; 13, 727–736, DOI: 10.1038/nrn3319
16. Ninkovic J, Götz M. How to make neurons–thoughts on the molecular logic of neurogenesis in the central nervous system. Cell Tissue Res. 2015; 3595–16, DOI: 10.1007/s00441-014-2048-9
17. Alvarez-Buylla A, Temple S. Stem cells in the developing and adult nervous system, J. Neurobiol. 1998; 36, 105–110.  PMID: 9712298

18. Zhao C, Deng W, Gage FH. Mechanisms and functional implications of adult neurogenesis, Cell 2008; 132, 645–660, DOI: 10.1016/j.cell.2008.01.033
19. Elkabetz Y, Panagiotakos G, Shamy G. et al. Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage. Genes & Development 2008; 22, 152–165. DOI: 10.1101/gad.1616208
20. Koch P, Opitz T, Steinbeck JA et al. A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration. Proc Natl Acad Sci U S A 2009; 106, 3225-3230. DOI: 10.1073/pnas.0808387106
21. Li W, Sun W, Zhang Y et al.Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors. Proc Natl Acad Sci U S A 2011; 108(20), 8299–304. DOI: 10.1073/pnas.1014041108
22. Reinhardt P, Glatza M, Hemmer K et al. Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling. PLoS One 2013; 8, e59252. DOI: 10.1371/journal.pone.0059252
23. Inak G, Lorenz C, Lisowski P, Zink A, Mlody B, Prigione A. Concise Review: Induced Pluripotent Stem Cell-Based Drug Discovery for Mitochondrial Disease. Stem Cells 2017;[Epub ahead of print] DOI: 10.1002/stem.2637
24. Choi HW, Kim JH, Chung MK et al. Mitochondrial and metabolic remodeling during reprogramming and differentiation of the reprogrammed cells. Stem Cells Dev 2015; 24, 1366–1373 DOI: 10.1089/scd.2014.0561          
25. Lorenz C, Lesimple P, Bukowiecki R, Zink A, Inak G….Prigione A. Human iPSC-derived neuronal progenitors are an effective drug discovery model for neurological mitochondrial DNA disorders. Cell Stem Cell 2017; 20(5):659-674 DOI: 10.1016/j.stem.2016.12.013
26. Cho YM, Kwon S, Pak YK et al. Dynamic changes in mitochondrial biogenesis and antioxidant enzymes during the spontaneous differentiation of human embryonic stem cells. Biochem. Biophys. Res. Commun. 2006; 348, 1472–1478, DOI: 10.1016/j.bbrc.2006.08.020
27. Balaban RS, Nemoto S, Finkel T. Mitochondria, oxidants, and aging, Cell 2005; 120, 483–495, DOI: 10.1016/j.cell.2005.02.001
28. Wang W, Osenbroch P, Skinnes R et al. Mitochondrial DNA integrity is essential for mitochondrial maturation during differentiation of neural stem cells, Stem Cells 2010; 28, 2195–2204, DOI: 10.1002/stem.542
29. Auré K, Jardel C, Clarysse L et al. Episodic weakness due to mitochondrial DNA MT-ATP6/8 mutations. American Academy of Neurology 2013; 81, 1810–1818. DOI:   10.1212/01.wnl.0000436067.43384.0b



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