SUMMARYMitochondrial DNA (mtDNA) mutations frequently cause neurological diseases. Modeling of these defects has been difficult because of the challenges associated with engineering mtDNA. We show here that neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) retain the parental mtDNA profile and exhibit a metabolic switch toward oxidative phosphorylation. NPCs derived in this way from patients carrying a deleterious homoplasmic mutation in the mitochondrial gene MT-ATP6 (m.9185T>C) showed defective ATP production and abnormally high mitochondrial membrane potential (MMP), plus altered calcium homeostasis, which represents a potential cause of neural impairment. High-content screening of FDA-approved drugs using the MMP phenotype highlighted avanafil, which we found was able to partially rescue the calcium defect in patient NPCs and differentiated neurons. Overall, our results show that iPSC-derived NPCs provide an effective model for drug screening to target mtDNA disorders that affect the nervous system.

Leigh syndrome (LS) is a severe manifestation of mitochondrial disease in children and is currently incurable. The lack of effective models hampers our understanding of the mechanisms underlying the neuronal pathology of LS. Using patient-derived induced pluripotent stem cells and CRISPR/Cas9 engineering, we developed a human model of LS caused by mutations in the complex IV assembly gene SURF1. Single-cell RNA-sequencing and multi-omics analysis revealed compromised neuronal morphogenesis in mutant neural cultures and brain organoids. The defects emerged at the level of neural progenitor cells (NPCs), which retained a glycolytic proliferative state that failed to instruct neuronal morphogenesis. LS NPCs carrying mutations in the complex I gene NDUFS4 recapitulated morphogenesis defects. SURF1 gene augmentation and PGC1A induction via bezafibrate treatment supported the metabolic programming of LS NPCs, leading to restored neuronal morphogenesis. Our findings provide mechanistic insights and suggest potential interventional strategies for a rare mitochondrial disease.

Regeneration of injured central nervous system axons is highly restricted, causing neurological impairment. To date, although the lack of intrinsic regenerative potential is well described, a key regulatory molecular mechanism for the enhancement of both axonal regrowth and functional recovery after central nervous system injury remains elusive. While ubiquitin ligases coordinate neuronal morphogenesis and connectivity during development as well as after axonal injury, their role specifically in axonal regeneration is unknown. Following a bioinformatics network analysis combining ubiquitin ligases with previously defined axonal regenerative proteins, we found a triad composed of the ubiquitin ligases MDM4, MDM2 and the transcription factor p53 (encoded by TP53) as a putative central signalling complex restricting the regeneration program. Indeed, conditional deletion of MDM4 or pharmacological inhibition of MDM2/p53 interaction in the eye and spinal cord promote axonal regeneration and sprouting of the optic nerve after crush and of supraspinal tracts after spinal cord injury. The double conditional deletion of MDM4-p53 as well as MDM2 inhibition in p53-deficient mice blocks this regenerative phenotype, showing its dependence upon p53. Genome-wide gene expression analysis from ex vivo fluorescence-activated cell sorting in MDM4-deficient retinal ganglion cells identifies the downstream target IGF1R, whose activity and expression was found to be required for the regeneration elicited by MDM4 deletion. Importantly, we demonstrate that pharmacological enhancement of the MDM2/p53-IGF1R axis enhances axonal sprouting as well as functional recovery after spinal cord injury. Thus, our results show MDM4-MDM2/p53-IGF1R as an original regulatory mechanism for CNS regeneration and offer novel targets to enhance neurological recovery.media-1vid110.1093/brain/awv125_video_abstractawv125_video_abstract.

High attrition rates and loss of capital plague the drug discovery process. This is particularly evident for mitochondrial disease that typically involves neurological manifestations and is caused by nuclear or mitochondrial DNA defects. This group of heterogeneous disorders is difficult to target because of the variability of the symptoms among individual patients and the lack of viable modeling systems. The use of induced pluripotent stem cells (iPSCs) might significantly improve the search for effective therapies for mitochondrial disease. iPSCs can be used to generate patientspecific neural cell models in which innovative compounds can be identified or validated. Here we discuss the promises and challenges of iPSC-based drug discovery for mitochondrial disease with a specific focus on neurological conditions. We anticipate that a proper use of the potent iPSC technology will provide critical support for the development of innovative therapies against these untreatable and detrimental disorders. STEM CELLS 2017;35:1655-1662 SIGNIFICANCE STATEMENTMitochondrial disease is an untreatable condition caused by mutations in nuclear or mitochondrial DNA. This review describes the application of patient-derived induced pluripotent stem cells (iPSCs) in the drug discovery process of mitochondrial disease. iPSCs allow the development of innovative and effective cellular model systems in a personalized approach. Their use may significantly benefit the search for treatments against debilitating mitochondrial disease.

Mutations in the mitochondrial complex IV assembly factor SURF1 represent a major cause of Leigh syndrome (LS), a rare fatal neurological disorder. SURF1-deficient animals have failed to recapitulate the neuronal pathology of human LS, hindering our understanding of the disease mechanisms. We generated induced pluripotent stem cells from LS patients carrying homozygous SURF1 mutations (SURF1 iPS) and performed biallelic correction via CRISPR/Cas9. In contrast to corrected cells, SURF1 iPS showed impaired neuronal differentiation. Aberrant bioenergetics in SURF1 iPS occurred already in neural progenitor cells (NPCs), disrupting their neurogenic potency. Cerebral organoids from SURF1 iPS were smaller and recapitulated the neurogenesis defects. Our data imply that SURF1 mutations cause a failure in the development of maturing neurons. Using NPC function as an interventional target, we identified SURF1 gene augmentation as a potential strategy for restoring neurogenesis in LS patients carrying SURF1 mutations.

The metabolic switch associated with the reprogramming of somatic cells to pluripotency has received increasing attention in recent years. However, the impact of mitochondrial and metabolic modulation on stem cell differentiation into neuronal/glial cells and related brain disease modeling still remains to be fully addressed. Here, we seek to focus on this aspect by first addressing brain energy metabolism and its inter-cellular metabolic compartmentalization. We then review the findings related to the mitochondrial and metabolic reconfiguration occurring upon neuronal/glial specification from pluripotent stem cells (PSCs). Finally, we provide an update of the PSC-based models of mitochondria-related brain disorders and discuss the challenges and opportunities that may exist on the road to develop a new era of brain disease modeling and therapy.3 Mitochondrial remodeling in cell fate specificationThe majority of cellular energy in form of ATP is provided through oxidative phosphorylation (OXPHOS) by mitochondria. Mitochondria are also involved in the metabolism of amino acids, fatty acids, and steroids, and contribute to cell signaling through the modulation of reactive oxygen species (ROS), calcium homeostasis, and apoptosis [1].Furthermore, intermediate metabolites can cross-talk to the nucleus acting as epigenetic regulators [2].In low oxygen environments, a typical conversion process of 1 molecule of glucose results into 2 molecules of ATP through glycolysis in the cytosol, which terminates with the secretion of lactate into the extracellular environment. Under normoxic conditions, the 2 molecules of pyruvate, generated through glycolysis, can enter the mitochondria and undergo further oxidation in the tricarboxylic acid (TCA) cycle, leading to the production of additional 34 molecules of ATP [3]. However, under conditions requiring high proliferative rates, this mitochondrial-based energy generation may be shut-down despite the presence of normal oxygen concentration [4]. This situation, known as aerobic glycolysis or Warburg effect, was first described by Otto Warburg in the context of cancer [5]. Recent studies demonstrated that a Warburg-like effect may also represent a defining feature of stem cells [6][7][8].Since distinct cell types have different energy demands, regulation of mitochondria may represent an essential process allowing the cells to meet their biological requirements.The metabolic identity of cells may in fact be influenced not only by changes in the expression of metabolic genes, but also by modulation of mitochondrial dynamics and mitochondrial DNA (mtDNA) copy number [9,10]. In particular, energy metabolism is shifted towards glycolysis in stem cells, whose mitochondria appear round-shaped with poorlydeveloped cristae [11,12]. This is in sharp contrast to cells with high energy demands, like muscle cells and neurons, where mitochondria are abundant in number and exhibit tubularlike morphology and cristae-rich structures [13]. 4As a consequence, acquisition of a distinct metabot...

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The evolution and functional integration of new genes, especially those that become core to key functions, remains enigmatic. We consider the mammal-specific gene, piggyBac transposable element derived 1 (PGBD1), implicated in neuronal disorders. While it no longer recognises piggyBac transposon-like inverted repeats and transposase functionality having been lost, it has evolved a core role in neural homeostasis. Depletion of PGBD1 triggers accumulation of mammal-specific paraspeckles and neural differentiation. It acts by two modalities, DNA binding and protein-protein interaction. As a transcriptional repressor of (lnc)NEAT1, the backbone of paraspeckles, it inhibits paraspeckle formation in neural progenitor cells (NPCs). At the protein level it is associated with the stress response system, a function partially shared with (lnc)NEAT1. PGBD1 thus presents as an unusual exemplar of new gene creation, being a recently acquired multi-function, multi-modal gene. Mammalian specificity associated with control of a mammal-specific structure implies coevolution of new genes with new functions.