Rapid degeneration of iPSC-derived motor neurons lacking Gdap1 engages a mitochondrial-sustained innate immune response.

Charcot-Marie-Tooth disease is a chronic hereditary motor and sensory polyneuropathy targeting Schwann cells and/or motor neurons. Its multifactorial and polygenic origin portrays a complex clinical phenotype of the disease with a wide range of genetic inheritance patterns. The disease-associated gene GDAP1 encodes for a mitochondrial outer membrane protein. Mouse and insect models with mutations in Gdap1 have reproduced several traits of the human disease. However, the precise function in the cell types affected by the disease remains unknown. Here, we use induced-pluripotent stem cells derived from a Gdap1 knockout mouse model to better understand the molecular and cellular phenotypes of the disease caused by the loss-of-function of this gene. Gdap1-null motor neurons display a fragile cell phenotype prone to early degeneration showing (1) altered mitochondrial morphology, with an increase in the fragmentation of these organelles, (2) activation of autophagy and mitophagy, (3) abnormal metabolism, characterized by a downregulation of Hexokinase 2 and ATP5b proteins, (4) increased reactive oxygen species and elevated mitochondrial membrane potential, and (5) increased innate immune response and p38 MAP kinase activation. Our data reveals the existence of an underlying Redox-inflammatory axis fueled by altered mitochondrial metabolism in the absence of Gdap1. As this biochemical axis encompasses a wide variety of druggable targets, our results may have implications for developing therapies using combinatorial pharmacological approaches and improving therefore human welfare. A Redox-immune axis underlying motor neuron degeneration caused by the absence of Gdap1. Our results show that Gdap1-/- motor neurons have a fragile cellular phenotype that is prone to degeneration. Gdap1-/- iPSCs differentiated into motor neurons showed an altered metabolic state: decreased glycolysis and increased OXPHOS. These alterations may lead to hyperpolarization of mitochondria and increased ROS levels. Excessive amounts of ROS might be the cause of increased mitophagy, p38 activation and inflammation as a cellular response to oxidative stress. The p38 MAPK pathway and the immune response may, in turn, have feedback mechanisms, leading to the induction of apoptosis and senescence, respectively. CAC, citric acid cycle; ETC, electronic transport chain; Glc, glucose; Lac, lactate; Pyr, pyruvate.

c-MYC Triggers Lipid Remodelling During Early Somatic Cell Reprogramming to Pluripotency.

Metabolic rewiring and mitochondrial dynamics remodelling are hallmarks of cell reprogramming, but the roles of the reprogramming factors in these changes are not fully understood. Here we show that c-MYC induces biosynthesis of fatty acids and increases the rate of pentose phosphate pathway. Time-course profiling of fatty acids and complex lipids during cell reprogramming using lipidomics revealed a profound remodelling of the lipid content, as well as the saturation and length of their acyl chains, in a c-MYC-dependent manner. Pluripotent cells displayed abundant cardiolipins and scarce phosphatidylcholines, with a prevalence of monounsaturated acyl chains. Cells undergoing cell reprogramming showed an increase in mitochondrial membrane potential that paralleled that of mitochondrial-specific cardiolipins. We conclude that c-MYC controls the rewiring of somatic cell metabolism early in cell reprogramming by orchestrating cell proliferation, synthesis of macromolecular components and lipid remodelling, all necessary processes for a successful phenotypic transition to pluripotency. c-MYC promotes anabolic metabolism, mitochondrial fitness and lipid remodelling early in cell reprogramming. A high rate of aerobic glycolysis is crucial to provide intermediaries for biosynthetic pathways. To ensure the availability of nucleotides, amino acids and lipids for cell proliferation, cells must provide with a constant flux of the elemental building blocks for macromolecule assembly and fulfil the anabolic demands to reach the critical cellular mass levels to satisfactorily undergo cell division. A high rate of aerobic glycolysis is induced by c-MYC, increasing the amounts of intracellular Glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), and glyceraldehyde-3-phosphate (GA3P), which can all enter pentose phosphate pathway (PPP) to produce Ribose-5-Phosphate (R5P) and NADPH, which are necessary for the biosynthesis of biomolecules such as proteins, nucleic acids, or lipids. C-MYC-dependent activation of glucose-6-phosphate dehydrogenase (G6PD) may play a critical role in the shunting of G6P to PPP and generation of NADPH. High glycolytic flux increases the amounts of dihydroxyacetone phosphate (DHAP), which is crucial for biosynthesis of phospholipids and triacylglycerols, and pyruvate (Pyr), which can be converted to citrate (Cit) in the mitochondria and enter the biosynthesis of fatty acids (FA). During cell reprogramming, c-MYC-dependent lipid remodelling leads to Polyunsaturated Fatty Acid (PUFA) downregulation and Monounsaturated Fatty Acid (MUFA) upregulation, which may play critical roles in cytoarchitectural remodelling of cell membrane or non-canonical autophagy, respectively. Cardiolipin (pink dots) rise early in cell reprogramming correlates with an increase in mitochondrial fitness, suggesting that c-MYC may restore proper levels of cardiolipins and antioxidant proteins, such as UCP2, to guarantee an optimal mitochondrial function while upholding ROS levels, reinforcing the idea of cell rejuvenation early in cell reprogramming.

Generation of an iPSC line from a retinitis pigmentosa patient carrying a homozygous mutation in CERKL and a healthy sibling.

Dermal fibroblasts from an autosomal recessive retinitis pigmentosa (RP) patient, homozygous for the mutation c.769 C>T, p.Arg257Ter, in CERKL (Ceramide Kinase-Like) gene, and a healthy sibling were derived and reprogrammed by Sendai virus. The generated human induced pluripotent stem cell (hiPSC) lines RP3-FiPS4F1 and Ctrl3-FiPS4F1, were free of genomically integrated reprogramming genes, showed stable karyotypes, expressed pluripotency markers and could be differentiated towards the three germ layers in vitro. These hiPSC lines offer a useful resource to study RP pathomechanisms, drug testing and therapeutic opportunities.

Generation of gene-corrected human induced pluripotent stem cell lines derived from retinitis pigmentosa patient with Ser331Cysfs*5 mutation in MERTK.

The human induced pluripotent stem cell (hiPSC) line RP1-FiPS4F1 generated from the patient with autosomal recessive retinitis pigmentosa (arRP) caused by homozygous Ser331Cysfs*5 mutation in Mer tyrosine kinase receptor (MERTK) was genetically corrected using CRISPR/Cas9 system. Two isogenic hiPSCs lines, with heterozygous and homozygous correction of c.992_993delCA mutation in the MERTK gene were generated. These cell lines demonstrate normal karyotype, maintain a pluripotent state, and can differentiate toward three germ layers in vitro. These genetically corrected hiPSCs represent accurate controls to study the contribution of the specific genetic change to the disease, and potentially therapeutic material for cell-replacement therapy.

Near-Complete Genome Sequences of Streptomyces sp. Strains AC1-42T and AC1-42W, Isolated from Bat Guano from Cabalyorisa Cave, Mabini, Pangasinan, Philippines.

Streptomyces sp. strains AC1-42T and AC1-42W, isolated from bat guano from Cabalyorisa Cave, Mabini, Pangasinan, Philippines, are active against Bacillus subtilis subsp. subtilis KCTC 3135T. The near-complete genome sequences reported here represent a possible source of ribosomally synthesized, posttranslationally modified peptides, such as lantipeptides, bacteriocins, linaridin, and a lasso peptide.

MYC Induces a Hybrid Energetics Program Early in Cell Reprogramming.

Cell reprogramming is thought to be associated with a full metabolic switch from an oxidative- to a glycolytic-based metabolism. However, neither the dynamics nor the factors controlling this metabolic switch are fully understood. By using cellular, biochemical, protein array, metabolomic, and respirometry analyses, we found that c-MYC establishes a robust bivalent energetics program early in cell reprogramming. Cells prone to undergo reprogramming exhibit high mitochondrial membrane potential and display a hybrid metabolism. We conclude that MYC proteins orchestrate a rewiring of somatic cell metabolism early in cell reprogramming, whereby somatic cells acquire the phenotypic plasticity necessary for their transition to pluripotency in response to either intrinsic or external cues.

Generation of human induced pluripotent stem cell (iPSC) line from an unaffected female carrier of mutation in SACSIN gene.

The human iPSC cell line, CARS-FiPS4F1 (ESi064-A), derived from dermal fibroblast from the apparently healthy carrier of the mutation of the gene SACSIN, was generated by non-integrative reprogramming technology using OCT3/4, SOX2, CMYC and KLF4 reprogramming factors. The pluripotency was assessed by immunocytochemistry and RT-PCR. This iPSC line can be used as control for Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) disease.

Correction: Bacterial diversity of bat guano from Cabalyorisa Cave, Mabini, Pangasinan, Philippines: A first report on the metagenome of Philippine bat guano.

[This corrects the article DOI: 10.1371/journal.pone.0200095.].

Generation of a human iPSC line from a patient with Leber congenital amaurosis caused by mutation in AIPL1.

The human induced pluripotent stem cell (hiPSC) line, derived from dermal fibroblasts from Leber congenital amaurosis patient with homozygous mutation c.265 T > C, p.Cys89Arg in aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) was generated by Sendai virus reprogramming. The generated hiPSC line was free of Sendai virus genes, had stable karyotype, carried the homozygous mutation, was immunopositive to pluripotency markers and able to generate all three germ layers upon embryoid body formation. Resource table.

Bacterial diversity of bat guano from Cabalyorisa Cave, Mabini, Pangasinan, Philippines: A first report on the metagenome of Philippine bat guano.

Bats are highly diverse and ecologically valuable mammals. They serve as host to bacteria, viruses and fungi that are either beneficial or harmful to its colony as well as to other groups of cave organisms. The bacterial diversity of two bat guano samples, C1 and C2, from Cabalyorisa Cave, Mabini, Pangasinan, Philippines were investigated using 16S rRNA gene amplicon sequencing. V3-V4 hypervariable regions were amplified and then sequenced using Illumina MiSeq 250 PE system. Reads were processed using Mothur and QIIME pipelines and assigned 12,345 OTUs for C1 and 5,408 OTUs for C2. The most dominant OTUs in C1 belong to the Proteobacteria (61.7%), Actinobacteria (19.4%), Bacteroidetes (4.2%), Firmicutes (2.7%), Chloroflexi (2.5%), candidate phylum TM7 (2.3%) and Planctomycetes (1.9%) while Proteobacteria (61.7%) and Actinobacteria (34.9%) dominated C2. Large proportion of sequence reads mainly associated with unclassified bacteria indicated possible occurrence of novel bacteria in both samples. XRF spectrophotometric analyses of C1 and C2 guano revealed significant differences in the composition of both major and trace elements. C1 guano recorded high levels of Si, Fe, Mg, Al, Mn, Ti and Cu while C2 samples registered high concentrations of Ca, P, S, Zn and Cr. Community structure of the samples were compared with other published community profiling studies from Finland (SRR868695), Meghalaya, Northeast India (SRR1793374) and Maharashtra State, India (CGS). Core microbiome among samples were determined for comparison. Variations were observed among previously studied guano samples and the Cabalyorisa Cave samples were attributed to either bat sources or age of the guano. This is the first study on bacterial diversity of guano in the Philippines through high-throughput sequencing.

Generation of a human iPSC line by mRNA reprogramming.

The human iPSC cell line, derived from foreskin fibroblasts was generated by non-integrative, non-viral reprogramming technology using OCT4, SOX2, KLF4, LIN28, c-MYC mRNAs.

Generation of a human iPSC line from a patient with congenital glaucoma caused by mutation in CYP1B1 gene.

The human iPSC cell line, GLC-FiPS4F1 (ESi047-A), derived from dermal fibroblast from the patient with congenital glaucoma caused by the mutation of the gene CYP1B1, was generated by non-integrative reprogramming technology using OCT3/4, SOX2, CMYC and KLF4 reprogramming factors.

Generation of a disease-specific iPS cell line derived from a patient with Charcot-Marie-Tooth type 2K lacking functional GDAP1 gene.

Human CMT2-FiPS4F1 cell line was generated from fibroblasts of a patient with Charcot-Marie-Tooth disease harbouring the following mutations in the GDAP1 gene in heterozygosis: p.Q163X/p.T288NfsX3. This patient did not present mutations in the PM22, MPZ or GJB genes. Human reprogramming factors OCT3/4, KLF4, SOX2 and C-MYC were delivered using a non-integrative methodology that involves the use of Sendai virus.

Dysfunctional mitochondrial fission impairs cell reprogramming.

We have recently shown that mitochondrial fission is induced early in reprogramming in a Drp1-dependent manner; however, the identity of the factors controlling Drp1 recruitment to mitochondria was unexplored. To investigate this, we used a panel of RNAi targeting factors involved in the regulation of mitochondrial dynamics and we observed that MiD51, Gdap1 and, to a lesser extent, Mff were found to play key roles in this process. Cells derived from Gdap1-null mice were used to further explore the role of this factor in cell reprogramming. Microarray data revealed a prominent down-regulation of cell cycle pathways in Gdap1-null cells early in reprogramming and cell cycle profiling uncovered a G2/M growth arrest in Gdap1-null cells undergoing reprogramming. High-Content analysis showed that this growth arrest was DNA damage-independent. We propose that lack of efficient mitochondrial fission impairs cell reprogramming by interfering with cell cycle progression in a DNA damage-independent manner.

Early ERK1/2 activation promotes DRP1-dependent mitochondrial fission necessary for cell reprogramming.

During the process of reprogramming to induced pluripotent stem (iPS) cells, somatic cells switch from oxidative to glycolytic metabolism, a transition associated with profound mitochondrial reorganization. Neither the importance of mitochondrial remodelling for cell reprogramming, nor the molecular mechanisms controlling this process are well understood. Here, we show that an early wave of mitochondrial fragmentation occurs upon expression of reprogramming factors. Reprogramming-induced mitochondrial fission is associated with a minor decrease in mitochondrial mass but not with mitophagy. The pro-fission factor Drp1 is phosphorylated early in reprogramming, and its knockdown and inhibition impairs both mitochondrial fragmentation and generation of iPS cell colonies. Drp1 phosphorylation depends on Erk activation in early reprogramming, which occurs, at least in part, due to downregulation of the MAP kinase phosphatase Dusp6. Taken together, our data indicate that mitochondrial fission controlled by an Erk-Drp1 axis constitutes an early and necessary step in the reprogramming process to pluripotency.