NAD+ metabolism governs the proinflammatory senescence-associated secretome

https://www.nature.com/articles/s41556-019-0287-4

Nacarelli T, Lau L, Fukumoto T, Zundell J, Fatkhutdinov N, Wu S, Aird KM, Iwasaki O, Kossenkov AV, Schultz D, Noma KI, Baur JA, Schug Z, Tang HY, Speicher DW, David G, Zhang R

• THe authors show that the enzyme nicotinamide phosphoribosyltransferase (NAMPT), which is a rate-limiting enzyme of the NAD+ salvage pathway, is involved in the senesence-associated secretory phenotype (SASP), independent of the senesence-associated growth arrest.
• The signalling pathway the authors identify is promotes the SASP by enhancing glycolysis and mitochondrial respiration. 
• The tumour-promoting effects of SASP suggests that anti-ageing dietary NAD+ augmentation should be administered with care.

The NAD-Booster Nicotinamide Riboside Potently Stimulates Hematopoiesis through Increased Mitochondrial Clearance

https://www.sciencedirect.com/science/article/pii/S1934590919300621

Vannini N, Campos V, Girotra M, Trachsel V, Rojas-Sutterlin S, Tratwal J, Ragusa S, Stefanidis E, Ryu D, Rainer PY, Nikitin G, Giger S, Li TY, Semilietof A, Oggier A, Yersin Y, Tauzin L, Pirinen E, Cheng WC, Ratajczak J, Canto C, Ehrbar M, Sizzano F, Petrova TV, Vanhecke D, Zhang L, Romero P, Nahimana A, Cherix S, Duchosal MA, Ho PC, Deplancke B, Coukos G, Auwerx J, Lutolf MP, Naveiras O

• Boosting NAD+ via dietary supplementation of nicotinamide riboside (NR) in mice
• Reduces mitochondrial activity in hematopoetic stem cells 
• Increases mitophagy in HSCs (determined in the mitoQC mouse)
• Reduces mitochondrial membrane potential (perhaps related to the increased mitophagy)
• Leading to increased asymmetric division of mitochondria (quantified by TMRM/mitochondrial mass) in HSCs, resulting in expansion of the hematopoetic progenitor compartment.
• NR supplementation enhanced survival and blood cell production following HSC transplantation.

Nuclear genetic regulation of the human mitochondrial transcriptome

https://elifesciences.org/articles/41927

Aminah T Ali, Lena Boehme, Guillermo Carbajosa, Vlad C Seitan, Kerrin S Small, Alan Hodgkinson

• The authors analyse >11k RNA sequencing libraries across 36 tissue types and investigate the variability in transcription of the mitochondrial genome
• The authors identify 64 nuclear genetic loci associated with expression of mitochondrially-encoded genes. 
• The authors replicate ~21% of associations with independent tissue-matched datasets.

Systems biology identifies preserved integrity but impaired metabolism of mitochondria due to a glycolytic defect in Alzheimer’s disease neurons

https://onlinelibrary.wiley.com/doi/full/10.1111/acel.12924

Pierre Theurey, Niamh M. C. Connolly, Ilaria Fortunati, Emy Basso, Susette Lauwen, Camilla Ferrante, Catarina Moreira Pinho, Alvin Joselin,  Anna Gioran, Daniele Bano, David S. Park,  Maria Ankarcrona, Paola Pizzo,  Jochen H. M. Prehn

• Many studies have focussed on the role of mitochondrial dysfunctions in Alzheimer's disease (AD), but most of them cannot tell whether mitochondrial defects are a cause or a consequence of AD.
• The authors use a combined experimental and computational approach to study mitochondrial function in the neurons of a transgenic mouse model.
• Experiments show that AD neurons have limited respiratory capacity. The computational model predicted that this could not be explained by any defect in the respiratory chain (RC),  but could be observed by simulating an impairment in the NADH flux to the RC.
• The authors used NAD(P)H autofluorescence measurements to validate the computationally predicted mitochondrial NADH defect in transgenic AD neurons.

Additionally, the authors investigated the cause of these reduced NADH flux and the resulting  mitochondrial NAD(P)H dyshomeostasis.

• Extracellular acidification experiments  measure the rate of excretion of lactic acid after its conversion from pyruvate, a product of glycolysis. These experiments  showed an impaired glycolytic flux in the transgenic AD neurons of the study.
• The authors supplemented neurons with pyruvate (therefore bypassing glycolysis), and this suppressed the NAD(P)H  impairment and the mitochondrial defects.
• This supports the hypothesis that a glycolytic defect is responsible for the unbalance of NAD(P)H observed in AD neurons.

The study shows that defects in glucose metabolism in vitro are detectable in neurons before the onset of any sign of pathology in transgenic AD mice.

Cardiolipin remodeling by ALCAT1 links mitochondrial dysfunction to Parkinson’s diseases

https://onlinelibrary.wiley.com/doi/full/10.1111/acel.12941

Chengjie Song  Jun Zhang  Shasha Qi  Zhen Liu  Xiaoyang Zhang  Yue Zheng  John‐Paul Andersen  Weiping Zhang  Randy Strong  Paul Anthony Martinez  Nicolas Musi  Jia Nie Yuguang Shi

• Parkinson's disease's (PD) causes remain elusive, but oxidative stress, mitochondrial dysfunction, and defective mitophagy are all considered as the primary pathogenic mechanisms.
• Cardiolipin (CL) is a phospholipid which is almost exclusively located in the inner mitochondrial membrane, where it is biosynthesized.
• ROS-induced damage of CL  is implicated in the pathogenesis of PD, but the mechanism remains unclear.
• The authors induced PD in a mouse model, induced by MPTP (a chemical that caused PD when injected, and has been used to study disease models in various animal studies). They oxidative stress, mtDNA mutations, and mitochondrial dysfunction in the midbrain.
• Then, they ablated  the ALCAT1 gene and treated mice with MPTP. This prevented MPTP‐induced neurotoxicity, apoptosis, and motor deficits and mitigated mitochondrial dysfunction.
• Mitophagy, which removes dysfunctional mitochondria, is also compromised in PD. The pharmacological inhibition of ALCAT1 significantly improved mitophagy, by stimulating the recruitment of Parkin to dysfunctional mitochondria and their association.
• These results show that ALCAT1 may be a promising drug target in the treatment of PD.

Alternative assembly of respiratory complex II connects energy stress to metabolic checkpoints

https://www.nature.com/articles/s41467-018-04603-z

Ayenachew Bezawork-Geleta, He Wen, LanFeng Dong, Bing Yan, Jelena Vider, Stepana Boukalova, Linda Krobova, Katerina Vanova, Renata Zobalova, Margarita Sobol, Pavel Hozak, Silvia Magalhaes Novais, Veronika Caisova, Pavel Abaffy, Ravindra Naraine, Ying Pang, Thiri Zaw, Ping Zhang, Radek Sindelka, Mikael Kubista, Steven Zuryn, Mark P. Molloy, Michael V. Berridge, Karel Pacak, Jakub Rohlena, Sunghyouk Park & Jiri Neuzil

• The authors show that depletion of mtDNA causes a shift in CII assembly from its full tetrameric form to an alternative 100 kDa form
• The authors suggest that cells may modulate their energy consumption by altering DNA synthesis and cell cycle progression. This modulation is mediated by the alternative form of CII

Lineage Tracing in Humans Enabled by Mitochondrial Mutations and Single-Cell Genomics

https://www.cell.com/cell/pdf/S0092-8674(19)30055-8.pdf

Leif S. Ludwig, Caleb A. Lareau, Jacob C. Ulirsch, Elena Christian, Christoph Muus, Lauren H. Li, Karin Pelka, Will Ge, Yaara Oren, Alison Brack, Travis Law, Christopher Rodman, Jonathan H. Chen, Genevieve M. Boland, Nir Hacohen, Orit Rozenblatt-Rosen, Martin J. Aryee, Jason D. Buenrostro, Aviv Regev, and Vijay G. Sankaran

INTRODUCTION

• Lineage tracing involves inferring the developmental history of an organism, with respect to its ancestors. Since single cells divide and proliferate, an emerging field is the inference of lineages of single-cells.
• In model organisms, this can be achieved through engineered genetic labels and single-cell RNA sequencing. These two approaches cannot be used together in humans, because of the genetic manipulations required to tag cells with heritable marks.
• Therefore, to date lineage tracing studies in humans have relied on the detection of naturally occurring somatic mutations in the nuclear genome. However, these mutations have high error rates and their detection is costly and difficult to perform at scale.
• The mitochondrial genome provides an attractive target for inferring cellular lineages for several reasons: 
• MtDNA is large enough to show substantial levels of variation
• It is short enough to be cost-effective for targetted sequencing: 18,000 mitochondrial genomes (17k bases) can be sequenced at 100-fold coverage for the same cost as a single nuclear genome (3.2bn bases) at 10-fold coverage. 
• Its mutation rate is reported to be 10-100 times larger than the nuclear genome.
• MtDNA is held in high copy number per cell (100-1000s), therefore less amplification is necessary.
• Mutations in mtDNA often reach a variant allele fraction of ~100% due to partitioning noise, random genetic drift, and faster replication relative to nuclear DNA.
• Existing methods (ATAC-seq and single-cell RNA-seq) can be used to detect mtDNA sequences and genetic variation.

MAIN FACTS OF THE PAPER

• The authors established 65 individual sub-clonal populations, over 8 generation, in an immortalised cell line. They derived subclones (populations of cells derived from a single cell) from the parental colony at each generation, and performed bulk mitochondria individual cells’ l genome sequencing  through ATAC-seq. The authors used high-confidence mtDNA mutations to reconstruct clonal relations between the subpopulations, allowing them to predict the most recent common ancestor with >80% accuracy (See Fig 1C, 1E and 1F).
• Since mtDNA is almost entirely transcribed, the authors hypothesized that single-cell RNA-seq would also be able to detect heteroplasmic mutations in mtDNA. The authors tested 6 protocols and found that full-length scRNA-seq methods showed better coverage of the mitochondrial genome than 3'-end-directed methods, with Smart-Seq2 having the best performance. 
• The authors performed whole-genome sequencing and single-cell RNA-seq simultaneously for single cells using SIDR, finding that several mutations were highly heteroplasmic in RNA, but not present in the genome, suggesting: RNA editing, transcription errors or technical errors in sc-RNA seq (Fig 2B). 
• To investigate inter- and intra-individual heterogeneity in mtDNA mutations, the authors analysed bulk RNA-seq data from 8.8k samples, spanning 49 tissues from at least 25 donors, as well as 426 donors with at least 10 tissues (GTEx project). 
• The authors found 2.7k mutations that were tissue-specific within an individual donor at a minimum of 3% heteroplasmy 
• Typically, ~25% of total mRNA originates from the mitochondrial genome across tissues, although this can be much larger in tissues such as the brain and heart. Tissues with a large proportion of mitochondrial mRNA tend to show very large variability -- see Fig 4B.
• Mitochondrial mutations around 10% are not uncommon across the whole mitochondrial genome (Fig 4D) and somatic mtDNA mutations with levels as low as 5% heteroplasmy can be stably propagated and serve as clonal markers in primary human cells.
• Every tissue had at least one tissue-specific mtDNA mutation across all individual donors, which likely arose via somatic mutation in a tissue-specific manner
• The authors used primary hematopoietic stem cells from two individual donors, and found that the mtDNA mutation profile separates single cells according to their donor of origin, as well as their single-cell-derived colony of origin via highly heteroplasmic mtDNA mutations.
• The authors performed bulk ATAC-seq and scRNA-seq on cells from colorectal adenocarcinoma primary tumor resection. Upon sequencing 238 cells, the authors found 12 distinct clusters of mtDNA mutations, suggesting clonal heterogeneity. 
• The authors provide an improved mutation detection framework, where mutation are first identified through bulk sequencing, and then called in scRNA-seq data. 

CONCLUSION AND OBSERVATIONS

• A potential limitation of inferring cell lineage from mtDNA sequence data comes from horizontal transfer of mtDNA between cells. However, the authors show that horizontal transfer would have to be relatively large to confound their analysis.
• Mapping the phenotypic impact of such genotypic diversity remains an open challenge.
• The authors use techniques for which reads mapping to the  mitochondrial genome are usually considered an unwanted by-product. Using assays focussed on the mitochondrial genomescan reduce costs and increase coverage.